Handbook of Food Toxicology (Food Science and Technology)

ISBN: 0-8247-0760-5 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

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To all those who appreciate my sole existence, especially my wife Usha and daughter Maithili and To Ratan, Shastry, Suresh, and Prakash, for being such wonderful friends through thick and thin

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Preface

Food toxicology is deeply rooted in the history of civilization. In their quest for food, our ancestors must have attempted to eat a variety of plants and animals and soon recognized that there were harmful as well as beneficial effects of their consumption. The selection by nomadic humans of only a handful of plant species for domestication and cultivation from the estimated 350,000 species of plants documented in the annals of botany and plant sciences is certainly not a chance occurrence. Gathering peoples perhaps accidentally found the very species that were the most predisposed to domestication, as well as the most well-suited to fulfill human nutritional requirements. Our experience throughout history has taught us much about how to avoid injury from consuming natural products. We now know which products not to eat under any circumstances, which can be eaten under some circumstances, and how to process other products to render them safe for consumption. History has thus taught us how to classify all substances into two classes: those that are safe and those that are harmful or poisonous. Such a classification, however, is not warranted in a strict scientific sense, primarily because a strict line of demarcation classifying and separating beneficial and harmful chemicals cannot be drawn, and because the degree of harmfulness of any compound is essentially related to the amount consumed. In fact, over 400 years ago Paracelsus pointed out that “all substances are poisons; there is none which is not a poison. The right does differentiates a poison and a remedy.” Indeed, the entire concept of toxicity needs to be evaluated from the viewpoint of a risk/benefit concept associated with the

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consumption of any given material. There is no such thing as absolute safety. Our daily lives are still shaped by our acceptance of numerous acceptable risks. Nearly everything we consume, including salt, sugar, starch, fat, protein, some minerals and vitamins, and even water, has a harmful effect when consumed in high enough concentration. Hazardous substances associated with food include toxic or antinutritive compounds that are naturally present in plants and animals; toxins that are produced ruing processing; incidental contaminants such as pesticides, antibiotic drug residues, and environmental pollutants; and foodborne pathogens. However, this does not mean that food is hazardous to human beings. Toxic components in foods—although they should indeed be minimized—are inevitable hazards of living. A substance that is considered to be toxic/antinutritive has a more or less pronounced capacity to induce deleterious effects on an organism when tested by itself in certain doses. This does not always happen under usual dietary conditions. We consume many toxic substances in our normal diet every day without showing any signs of intoxication. This is probably because natural toxicants usually exert their effects only when other potentiating substances are available. Also, the concentration of these compounds occurring naturally in food is often so low that the item must be consumed in extraordinarily large amounts daily over a prolonged period for intoxication to occur. Similarly, most toxic effects of potentially hazardous chemicals are not additive. In fact, antagonistic reactions that make some ingredients interfere and reduce the toxic

effects of other components are not unusual. Thus, many natural products that are common in the human diet have found wide acceptance not because they are free of toxic substances, but because they do not contain enough toxins to be harmful when consumed in reasonable quantities as part of a balanced diet—or because cooking or another process eliminates their toxic activity. In the vast majority of instances, our food supply is quite wholesome. In the unfortunate incidents when some link(s) in the food production, processing, and distribution scheme fail(s), such foods, when consumed, have produced adverse toxic responses that vary in severity from insignificant to fatal. As compared with naturally occurring toxic/antinutritive compounds in the human food chain, the situation is quite different with microbial contaminant of foods. In fact, perhaps the greatest damage, in terms of both mortality and morbidity worldwide, can be directly attributed to microbial contamination. Although changes and improvements in food processing operations as well as in sanitary practices have contributed to an important increase in the life span of humans in the last century, these significant improvements are now challenged by the appearance of microbes resistant to multiple antibiotics (e.g., Salmonella sp. and the emergence of new bacterial and fungal pathogens (e.g., Campylobacter, Listeria, E. Coli 0157:H7, fumonisins). In the United States alone, between 6.5 million and 81 million cases of foodborne illness and as many as 10,000 related deaths from seven major foodborne pathogens occur each year, costing $6.6 billion to $37.1 billion in economic losses. The situation is grim even in developing countries where water-borne and food-borne diseases such as cholera, jaundice, and diarrhea—which impair human health to a great degree, and therefore the body’s efficiency of food absorption—are perhaps more important factors affecting human health than many naturally occurring toxic/antinutritive compounds in the food chain. These effects are further magnified by a shortage of such basic commodities as a clean and safe supply of drinking water and adequate food for subsistence—this alone was good enough motivation for me to undertake this project. The primary aims for Handbook of Food Toxicology are twofold. (1) to provide basic coverage of the principles of toxicology relevant to food science and nutrition, and (2) to provide the latest information on various toxic and microbial hazards associated with modern-day foods. This book is divided into two parts that comprise a total of 18 chapters. The first part, consisting of Chapters 1–6, deals with the science and principles of toxicology, manifestations of toxic effects, biotransformations of toxicants relevant to food science and human nutrition, and some of the

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regulatory and QA/QC issues. Chapters 7 through 18 describe the basic aspects of toxicity associated with commonly occurring dietary components and substances (naturally occurring, intentionally added, or incidental), as well as those associated with microbial contamination of foods. A basic understanding of the principles behind the occurrence of microbes in the food chain and their toxicity or toxic mechanisms not only allows us to appreciate the complexity of our food supply but is essential for developing newer and safer food production, processing, handling, and distribution technologies. No single food toxicology book can cover all aspects of the toxicity and safety of the myriad of food used in many different ways by humans worldwide. Indeed, volumes and monographs are available on the topic of practically every chapter in this book, and even on those of many of the chapter sections. Every effort, however, was made to cover important toxic hazards associated with food consumption. For some toxins, only historical viewpoints are described, since research during the past decade on many of these compounds (e.g., flavonoids, phytates, antioxidants) has shown several positive health benefits associated with their consumption as part of a normal, wellbalanced diet. In contrast, in-depth coverage is provided on microbial toxins and food pathogens, since these appear to be the predominant causes of morbidity and mortality associated with our food supply. Hopefully, this book represents a compromise between the historical views associated with the traditional, well-known toxic components found in our food supply and the exciting new developments occurring on several other fronts, especially on foodborne infections and intoxications. It is my sincere hope that the information presented in this book will serve professionals in many disciplines, including agriculture, food science, nutrition, microbiology, toxicology, public health, medicine, and other health-related areas. Selected chapters can also be used as college-level teaching material. Finally, it is inevitable in a book of this breadth that omissions, occasional errors, and lapses in the accuracy of interpretation will have escaped the detection of even the most assiduous proofreaders. I hope that any such mistakes are both minor and minimal, and I accept full and exclusive responsibility for them. I welcome comments and suggestions for improvement and for correction of any errors. Sincere appreciation is extended to the editorial and production staff of Marcel Dekker, Inc., especially to Ms. Maria Allegra, Ms. Lila Harris, Ms. Katie Stence, Ms. Theresa Stockton, and Ms. Susan Thornton. Without their cooperation and tremendous patience, this book would

never have been written. I gratefully acknowledge the original treatise in this field: the late Professor Jose M. Concon’s groundbreaking two-volume Food Toxicology, published in 1988. In fact, the origin of this book can be traced back to his monumental work in the field. I am also greatly indebted to Professor D. K. Salunkhe of Utah State University, who first encouraged me to undertake this task.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Finally, no words will ever fully describe the untiring and continued support and encouragement provided by my wife Usha and daughter Maithili during the arduous task of putting together this book.

S. S. Deshpande

Contents

Preface SCIENCE AND PRINCIPLES OF TOXICOLOGY 1.

The Science of Toxicology

2.

Principles of Toxicology

3.

Manifestations of Organ Toxicity

4.

Carcinogenesis, Mutagenesis, and Teratogenesis

5.

Biotransformation of Xenobiotics

6.

Measurement of Toxicants and Toxicity TOXICITY IN FOODS

7.

Dietary Constituents

8.

Food Additives

9.

Toxicants Resulting from Food Processing

10.

Toxicants and Antinutrients in Plant Foods

11.

Fungal Toxins

12.

Food-Borne Infections

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13.

Bacterial Toxins

14.

Seafood Toxins and Poisoning

15.

Mushroom Toxins

16.

Toxic Metals, Radionuclides, and Food Packaging Contaminants

17.

Pesticides and Industrial Contaminants

18.

Drug Residues

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1 The Science of Toxicology

1.1

INTRODUCTION

The origins of toxicology appear to be deeply rooted in the history of human civilization. Our ancestors in their quest for food must have attempted to eat a variety of foods of both plant and animal origin and soon recognized that there were harmful as well as beneficial consequences associated with the consumption of such material. The rise of agricultural knowledge has been traced back to ancient times, when humankind made the transition from nomadic hunting/gathering tribes to more settled societies supported by domesticated animal herds and cultivated crops. In terms of archaeological findings, primitive agriculture may have developed as early as 9000–7000 B.C. in the Near East (Garfield, 1990). The selection of only a handful of plant species for domestication and cultivation by the nomadic human is certainly not a chance occurrence. It must predate agriculture by at least some thousands of years. Its enormous complexity is further illustrated by the 3000-plus species of the estimated 350,000 species of plants documented in the annals of botany and plant sciences that have been used historically in some form to feed humans (Deshpande, 1992; Borlaug, 1981; Wittwer, 1980). Fewer than 300 are used currently worldwide in organized agriculture. Among these, at least 150 different species are grown in sufficient quantities to enter the world trade. In contrast, Wittwer (1980) suggests, today some 24 crops essentially stand between people and starvation. In approximate order of importance these crops are rice, wheat, corn, potato, barley, sweet potato, cassava, soybean, oat, sorghum, millet, sugarcane, sugar beet, rye, peanut, field bean, chick-

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pea, pigeon pea, mung bean, cowpea, broad bean, yam, banana, and coconut. Although cereals with nine species and legumes with eight constitute the preponderance of the global food production, together they constitute only 0.005% of the available wealth in the plant kingdom. Present-day humans, no doubt, have diversified the uses of important economic plants greatly. They, however, have added relatively little to this list of basic staples. Domestication of only such a few plant species as human food sources is truly one of the most extraordinary stories of our history. Beginning only by collecting, the gathering peoples perhaps accidentally(?) chanced upon the very species that were the most predisposed to domestication and were well suited to fulfill human nutritional requirements. Our experience throughout history has thus taught us much about how to prevent injury from consuming natural products as foods. We now know which products not to eat under any circumstances, which can be eaten under some circumstances, and how to process certain other products to render them safe for consumption. History has thus taught us how to classify all substances in two classes: those that are safe and the others that are harmful. Traditionally, the term food was used for those materials that were beneficial and essential for the functioning of human body. Substances that were distinctly harmful to the body were classified as poisons. This concept involving the division of chemicals into two categories has persisted to the present day. It readily places certain biological and botanical and, in fact, all distinctly harmful chemicals into a category that is accorded due respect. Loomis (1978), however, suggested that such a classification, in a strictly scientific sense, is

not warranted, primarily because a strict line of demarcation classifying and separating the beneficial and harmful chemicals cannot be drawn and because the degree of harmfulness of any compound is essentially related to the amount consumed. Indeed, the entire concept of toxicity needs to be evaluated from the viewpoint of a risk/benefit concept associated with the consumption of any given material. In fact, Paracelsus (1493–1541) over 400 years ago pointed out that “all substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy.” Since all substances can produce injury or death under some exposure conditions, it is evident that there is no such thing as an absolute safe substance or chemical that will be free of injurious effects under all conditions of exposure. As a corollary, it is also true that there is no chemical that cannot be used safely by limiting the dose or exposure. Our daily lives are still shaped by our acceptance of numerous acceptable risks. Nearly everything we consume, including salt, sugar, starch, fat, protein, some minerals and vitamins, and even water, has a harmful effect when consumed in high enough concentration. However, this does not necessarily mean that the substance is hazardous to human beings. Toxic compounds in our foods, medicines, and environment—though they should indeed be minimized—are inevitable hazards of living. A substance that is considered to be toxic/antinutritive has a more or less pronounced capacity to induce deleterious effects on the organism when tested by itself in certain doses. This does not always happen under the usual dietary conditions. We consume many toxic substances in our normal diet every day without showing any signs of intoxification. This is probably because natural toxicants usually exert their effects only when they are consumed under special conditions or when there are other potentiating substances present. Also, the concentration of these compounds occurring naturally in the food is often so low that the item must be consumed in usually unrealistically large amounts every day for a prolonged period for intoxification to occur. Furthermore, humans can handle small amounts of various toxicants. Similarly, most toxic effects of various chemicals that are potentially hazardous do not have an additive effect. In fact, antagonistic reactions that make some ingredients interfere with and reduce the toxic effects of other components are not unusual. Thus, many natural products that are common articles of the diet have found wide acceptance, not because they are free of toxic substances, but because they do not contain enough to be harmful when consumed in reasonable quantities as a part of a balanced diet, or because cooking or some other process eliminates their toxic activity.

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It is thus the “dose” of any given substance that determines its degree of harmfulness. If a sufficient dose is ingested or is in contact with a biological mechanism, then a harmful or toxic effect will be the consequence in the sense that the ability of that biological mechanism to carry on a function is either seriously impaired or destroyed. Such harmful effects often do not occur suddenly as the dose is increased from minimal to maximal levels. Rather a graded response related to progressive changes in dose is observed. The basic premise of the science of toxicology with respect to any biological effects of a chemical agent is to study this relationship that exists between the dose or concentration and the response that is obtained. Furthermore, toxic chemicals may be selective or nonselective in exerting their harmful effects on biological systems. Thus selective chemicals may exert their harmful effects only in a few living species, primarily because some protective mechanisms may be present in the resistant species. In contrast, those that act nonselectively exert an undesirable or harmful effect on all living matter. Fortunately, such compounds seldom find their way into our food chain under normal conditions. The term toxicology is derived from Latin and Greek origins (Latin toxicum meaning “poison”; Greek toxikom meaning “arrow poison”; and Latin logia, meaning “science” or “study”) and literally means a study of poisons in relation to living organisms. The science of toxicology therefore can be best approached as the study of the effects of chemicals on biological systems, with emphasis on the mechanisms of harmful effects of chemicals and the conditions under which such harmful effects can occur. In its broadest sense, it also includes socioeconomic considerations as well as legal ramifications.

1.2

HISTORY OF TOXICOLOGY

Ironically, toxicology must rank as one of the oldest practical sciences because humans, from the very beginning, needed to avoid the numerous toxic plants and animals in their environment. The Egyptian Ebers Papyrus (circa 1500 B.C.) and the Hindu Vedas (circa 5000 B.C.) rank as two of the earliest surviving medical records that contain information on several poisons. The surviving medical works of Hippocrates, Aristotle, and Theophrastus, published during the period 400–250 B.C., all included some mention of poisons. Dioscorides, a Greek employed by the Roman emperor Nero in about A.D. 50, first attempted to classify plants according to their toxic and therapeutic effects. There appear to have been very few noteworthy advances in either medicine or toxicology during the Middle

Ages. The only significant contribution to toxicology during this period appears to be that of Moses ben Maimon, or Maimonides (1135–1204). He published a treatise, Poisons and Their Antidotes, a first-aid guide to the treatment of accidental or intentional poisonings and insect, snake, and mad dog bites. Maimonides recommended that suction can be applied to insect stings or animal bites as a means of extracting the poison and advised application of a tight bandage above a wound located on a limb. He also noted that the absorption of toxins from the stomach could be delayed by ingestion of oily substances such as milk, butter, or cream. On the basis of critical and cautious observations, he also rejected numerous popular remedies of the day after finding them to be ineffective and mentioned his doubts concerning the efficacy of others. During the later Middle Ages, Philippus Aureolus Theophrastus Bombastus von Hohenheim Paracelsus (1493–1541) made revolutionary contributions to this discipline we now call toxicology. He first proposed the toxic agent as a chemical entity and was clearly aware of the dose-response relationship. The following four principles he first laid out still form the core of toxicological sciences. 1. 2. 3. 4.

Experimentation is essential in examination of responses to chemicals. A distinction must be made between therapeutic and toxic properties of chemicals. These properties are sometimes, but not always, indistinguishable except by dose. One can ascertain a degree of specificity of chemicals and their therapeutic or toxic effects.

Mathieu Joseph Bonaventura Orfila (1787–1853), a Spanish physician to Louis XVIII of France and a teacher at the University of Paris, is often cited as the father of modern toxicology. Orfila was the first to attempt a systematic correlation between the chemical and biological information of the then well-known poisons. He also singled out toxicology as a discipline distinct from others and defined it as the study of poisons. He wrote in 1815 the first book of general toxicology that was exclusively devoted to adverse effects of chemicals. Concerned with legal implications of poisoning, Orfila also pointed out the importance of determining a chemical analysis to establish a definitive cause of poisoning. Some of the analytical procedures he developed are still in use today. Since Orfila’s pioneering work, developments in toxicology slowly evolved. Although toxicology’s origins predate those of most other biological sciences and perhaps even those of medicine, most of the useful information related to modern toxicology has only been learned since the turn of the 20th century. In fact, the emergence of

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toxicology as a distinct discipline is a much more recent phenomenon. There are many reasons for this, including the development of new analytical methods since the end of the Second World War, the emphasis on drug testing that followed the thalidomide tragedy, the focus on pesticide testing since the publication of Rachel Carson’s Silent Spring, public concerns about hazardous waste disposal, and, more recently, the increased incidence of food poisonings by microbial pathogens. Detailed descriptions of the historical developments in the field of toxicology can be found in several excellent reviews (Loomis, 1978; Holmstedt and Liljestrand, 1981; Doull and Bruce, 1986).

1.3

SCOPE/DIVERSITY OF TOXICOLOGY

Modern toxicology is a multidisciplinary field, which has extracted many of the principles and techniques from several basic biological and chemical sciences (Figure 1.1). It is primarily an applied science, dedicated to the enhancement of the quality of life and the protection of the environment. It draws heavily on tools of chemistry and biochemistry. Those of chemistry provide analytical methods for toxic compounds, particularly for forensic toxicology and residue analysis, and those of biochemistry provide the techniques to investigate the metabolism and mode of action of toxic compounds. Toxicology may also be considered an area of fundamental biology since the adaptation of organisms to toxic environments has important implications for ecology and evolution. Sciences such as immunology, biomathematics, and ecology are also important, but to a more limited extent. The science of toxicology contributes heavily in the field of medicine, especially forensic medicine, clinical toxicology, pharmacy and pharmacology, public health, and industrial hygiene. It also contributes in an important way to veterinary medicine and to such aspects of agriculture as the development and safe use of agrochemicals. Its contributions to the field of environmental studies are among the most rapidly expanding areas in the world today. Thus, any attempt to define the scope of toxicology must take into account the fact that the various subdisciplines shown in Figure 1.1 are not mutually exclusive and frequently are heavily interdependent. Because of the overlapping of mechanisms, chemical classes, and use classes and effects, clear division into distinct branches of toxicology is often not possible. However, for the sake of convenience, subdivisions of toxicology can be defined by following any of the several classifications suggested by

Figure 1.1

The relationship of toxicology to basic sciences, its evolution and applications.

Hodgson (1987). These are briefly described in the following sections. 1.3.1 Applied Toxicology Based on Disciplines Various aspects of applied toxicology can be defined as they occur in or relate to a particular field (Figure 1.1). These include the following.

Veterinary Toxicology Veterinary toxicology is to animals what clinical toxicology is to humans. It deals with the diagnosis and treatment of the poisoning of animals, particularly livestock and household pets. An important concern of veterinary toxicology is the possible transmission of toxins and pathogens to the human population via meat, fish, milk, and other foodstuffs. Environmental Toxicology

Clinical and Forensic Toxicology Clinical toxicology is concerned with the diagnosis and treatment of human poisoning. It encompasses the study of chemicals originating from any and all sources and is primarily concerned with all aspects of the interaction between these chemicals and people. Forensic toxicology, in contrast, combines analytical chemistry with essential toxicological principles in order to deal with the medicolegal aspects of the toxic effects of drugs and chemicals on humans. Its primary role is to aid in establishing cause-effect relationships between exposure to a drug or poison and the toxic or lethal effects of the compound. It relies heavily on specific and highly sensitive analytical methods, which can efficiently isolate, identify, and quantitate the toxic compound in question from clinical and other samples.

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The broad discipline of environmental toxicology encompasses the study of chemicals that are the contaminants of food, water, soil, or the atmosphere. It is primarily concerned with the movement of chemicals and toxicants and their metabolites in the environment and in food chain, and the effect of such toxicants on individuals and populations. Environmental toxicology is also concerned with toxic substances that may enter the lakes, streams, rivers, and oceans. The most common problems dealt with in this aspect of toxicology are water-borne viruses and bacteria, radioactive waste, sewage eutrophication, and industrial pollutants. Occupational/Industrial Toxicology Occupational/ industrial toxicology is a specific area of environmental toxicology that has grown out of a need to

protect the worker from poisonous substances and, in general, to make the working environment safe. The objective obviously is to prevent impairment of health of an individual while on the job. In the United States, the Occupational Safety and Health Act (OSHA) was passed in 1970 to protect the health of workers. Two agencies, the National Institute for Occupational Safety and Health (NIOSH), responsible for developing safety and health standards, and the American Conference of Governmental Industrial Hygienists (ACGIH), devoted to setting safety standards for chemicals in the working environment, are primarily responsible for the enactment of OSHA guidelines under the jurisdiction of the Department of Labor. 1.3.2 Classification Based on Measurement of Toxicants and Toxic Actions By using a variety of techniques derived from analytical chemistry, bioassays, and applied mathematics, toxicants and their toxic effects can be measured and quantitated. This aspect of toxicology includes a number of important fields (Figure 1.1) as follows: Analytical Toxicology Analytical toxicology is a branch of analytical chemistry that is concerned with methods for the identification and assay of toxic chemicals and their metabolites in biological and environmental samples. Toxicity Testing

Structure-Activity Studies Structure-activity studies are important to understanding of the relationship between the chemical and physical properties of xenobiotics and toxicity and, particularly, the use of such relationships for the prediction of toxicity. Epidemiology Epidemiology, as it applies to toxicology, is closely related to the biomathematical and statistical models and is of great importance since it deals with the study of toxicity as it occurs, rather than in an experimental laboratory setting. 1.3.3 Classification Based on Mechanisms of Toxic Action and Effects Toxicants can also be classified on the basis of all the events leading to exertion of their toxic effects in vivo. This involves studies at the fundamental level of organ, cell, and molecular functions, including their uptake, distribution, metabolism, mode of action, and excretion. Important disciplines include the following: Biochemical Toxicology Biochemical toxicology considers events at the biochemical and molecular level, including enzymes that metabolize xenobiotics, generation of reactive intermediates, and interaction of xenobiotics or their metabolites with macromolecules.

The branch of toxicity testing involves the use of living systems to estimate toxic effects of various chemicals. It covers the entire gamut from short-term tests for genotoxicity such as the Ames test and cell culture techniques to the use of live animals for acute toxicity tests and for lifetime or multigeneration chronic toxicity tests. The term bioassay is used to describe the use of living organisms to quantitate the amount of a particular toxicant present.

Behavioral Toxicology

Biomathematics and Statistics

Nutritional toxicology deals with the effects of diet on the expression of toxicity and the mechanisms for these effects.

Mathematical and statistical techniques are used in a number of areas of toxicology. They deal with data analysis, the determination of significance, and the formulation of risk estimates and predictive models. The latter is particularly important in epidemiology and environmental toxicology.

Behavioral toxicology deals with the effects of toxicants on animal and human behavior. This involves peripheral and central nervous systems as well as effects mediated by other organ systems such as the endocrine glands. Nutritional Toxicology

Genetic Toxicology Genetic toxicology or mutagenesis is concerned with toxic effects of chemicals on the genetic material and the inheritance of these defects.

Toxicological Pathology Toxicological pathology deals with the effects of toxic agents as manifested by changes in subcellular, cellular, tissue, or organ morphological characteristics.

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Teratogenesis Teratogenesis includes the chemical and biochemical events that lead to deleterious effects on the developmental

process; it involves multigenerational studies of animals to study the long-term toxic effects on the reproductive processes.

cology, the toxic side effects and testing for them fall within the science of toxicology. Drugs of Abuse

Carcinogenesis Carcinogenesis includes the study of chemical and biochemical events upon exposure to toxic chemicals that lead to the large number of effects on cell growth.

Drugs of abuse are chemicals, often illegal, taken for psychological or other effects that cause dependence and toxicity. Food Additives

Organ Toxicity Organ toxicity considers the effects at the level of organ functions, e.g., neurotoxicity, hepatotoxicity, and nephrotoxicity. Regulatory Toxicology Regulatory toxicology is concerned with the formulation of laws and regulations authorized by laws to minimize the effect of toxic chemicals on human health and the environment. In the United States, the risk assessment, which is the definition of risks, potential risks, and the risk-benefit equations necessary for regulation of toxic substances, is primarily under the control of several government agencies. These include the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and OSHA. 1.3.4 Classification Based on Chemical Use Classes

Food additives are of concern to toxicologists only when they are toxic or are being tested for possible toxicity. Industrial Chemicals Because of the sheer number of industrial chemicals used, testing for toxicity and controlling exposure to those known to be toxic constitute a large field of toxicology. Naturally Occurring Substances Naturally occurring substances include many phytotoxins, mycotoxins, and inorganic minerals that occur naturally in the environment and may find a way into the human food chain. Combustion Products Combustion products are generated primarily from fuels and other industrial chemicals.

Hodgson (1987) also classified toxicants on the basis of use classes. This classification includes the toxicological aspects of the development of new chemicals for commercial uses. In some of these use classes, toxicity, at least to some organisms, is a desirable trait; in others, it is an undesirable side effect. This category includes both synthetic and natural products. The following classes can be identified by the use criteria.

1.4

Agricultural Chemicals

1.5

Agricultural chemicals include herbicides, fungicides, pesticides, and rodenticides, in which toxicity to the target organism is a desired quality, whereas toxicity to nontarget species must be prevented. Development of such selectively toxic chemicals is one of the applied roles of comparative toxicology. Clinical Drugs Although the development of clinical drugs is largely the responsibility of the pharmaceutical industry and pharma-

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SOURCES OF TOXIC COMPOUNDS

Several natural as well as synthetic compounds are potent toxicants and can enter the human food chain as contaminants. Although toxic, many find important uses at therapeutic dose levels in clinical medicine. Representative sources of toxic compounds are summarized in Table 1.1.

CLASSIFICATION OF TOXICANTS

Toxic agents can be classified in a variety of ways (Klaassen, 1986; Sperling, 1984; Manahan, 1992; Gossel and Bricker, 1984; Hodgson and Levi, 1987). For example, they can be classified in terms of their target organ (liver, kidney, lungs, skin, nervous system, hematopoietic system, etc.), their use (pesticides, solvents, food additives, etc.), their source (animal, plant, microbial toxins), and their effects (cancer, mutation, liver injury, etc.). Toxicants can also be grouped in terms of their physical state (gas, dust, liquid), their labeling requirements (explosive, flam-

Table 1.1 Representative Sources and Examples of Toxic Compounds

Table 1.2

Synthetic organic compounds Air (transportation, industrial processes, electric power generation, heating processes) Carbon monoxide, oxides of nitrogen and sulfur, hydrocarbons, particulates Water (runoffs, sewage, waste products discharged from refineries, swelters, or chemical plants) Agrochemicals, hydrocarbons, detergents, heavy metals Food contaminants Bacterial, fungal, and animal toxins; pesticide residues; plant alkaloids, residues of animal feed additives (e.g., antibiotics, estrogens); industrial chemicals Food additives Nitrates, nitrites Chemicals in the workplace Inorganic metals, aliphatic and aromatic hydrocarbons, halogenated hydrocarbons, alcohols, esters, organometallics, pesticides Drugs of abuse Cocaine, methamphetamines, lysergic acid diethylamide (LSD), morphine, nicotine, barbiturates Therapeutic drugs Essentially all therapeutic drugs, which can be toxic at high doses Agrochemicals Pesticides, herbicides, nematicides, rodenticides Solvents Aliphatic and aromatic hydrocarbons, halogenated solvents, alcohols Polycyclic aromatic hydrocarbons (incomplete combustion of organic materials) Pyrenes, anthracenes Cosmetics Thioglycolates, thioglycerol Naturally occurring toxins Mycotoxins Aflatoxins, fumonisins, ergot alkaloids, tricothecenes, patulin Microbial toxins Tetanus, botulinum, diphtheria, Staphylococcus spp. toxins Plant toxins Ricinine, solanine, chaconine, safrole, quinones, estrogens, enzyme inhibitors, lectins, cyanogenic glycosides Inorganic chemicals Heavy metals, oxides of nitrogen and sulfur

Rating/class

mable, oxidizer), their chemical properties (aromatic amines, polycyclic hydrocarbons, halogenated hydrocarbons, etc.), or their toxic potential, as shown in Table 1.2. According to Klaassen (1986), classification on the basis of the biochemical mechanism of action of toxicants is usually more informative than classification by general

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Toxicity Rating Chart

Practically nontoxic Slightly toxic Moderately toxic Very toxic Extremely toxic Supertoxic

Probable lethal oral dose for average human > 15 g/kg 5–15 g/kg 0.5–5 g/kg 50–500 mg/kg 5–50 mg/kg < 5 mg/kg

Source: Gossel and Bricker (1984) and Klaassen (1986).

category, such as irritants and corrosives. Nonetheless, more general classifications, such as air pollutants, occupation-related agents, and acute and chronic poisons, can still provide a useful focus on a specific problem. It is quite evident that no single classification can be used to cover the entire spectrum of toxic agents. In general, classification systems that take into consideration both the chemical and the biological properties of the toxicant and the exposure characteristics tend to provide useful information for legislative or control purposes as well as for toxicology in general.

1.6

FOOD TOXICOLOGY AND THE SCOPE OF THE BOOK

Food toxicology can be defined as a systematic study of toxicants found in foods. These compounds can be of natural origin as products of the metabolic processes of animals, plants, and microorganisms from which the food is derived; as biological and chemical contaminants from the air, water, and soil; as intentionally introduced food additives; and as those formed during the processing of foods. Food toxicology is thus concerned with the toxic potential of food, the conditions and factors affecting the presence of these toxicants in food, their interactions with essential dietary nutrients, the response of the human body to these toxins, and the means of prevention or minimization of these toxic effects as they pertain to food safety and human nutrition. As compared to the presence of toxicants that are naturally present in various foods, biological contamination of our food supply presents grave food safety concerns. Food-borne diseases caused by bacteria and viruses have varying degrees of severity ranging from mild indisposition to chronic or life-threatening illness. Their importance as a vital public health problem is often overlooked because the true incidence is difficult to evaluate and the severity of the health and economic consequences is often

not fully appreciated. For most food-borne diseases, only a small proportion of cases reach the notice of health services, and even fewer are investigated (Kaferstein et al., 1999). It is believed that in industrialized countries less than 10% of the cases are reported, and in developing countries reported cases probably account for less than 1% of the total (WHO, 1984). Studies in some industrialized countries point to an underreporting factor of up to 350 for certain food-borne diseases (Todd, 1989; Norling, 1994). Despite these limitations in reporting, available data give evidence of a tremendous public health problem. Furthermore, even in industrialized countries, the data indicate an increasing trend. Although the situation regarding foodborne diseases is very serious in developing countries, even the industrialized countries have experienced a succession of major epidemics, of which the mad cow disease is the latest entry in a long list of such food-borne outbreaks. The estimated annual incidence of food-borne diseases in the United States ranges from 6.5 million to 80 million cases. Surveys in other countries suggest that up to 10% of the general population may annually suffer from a food-borne disease (Todd, 1989; WHO, 1994; Notermans and Hooenboom-Verdegaal, 1992; Notermans and van de Giessen, 1993). In the United States, typically 400 to 500 food-borne outbreaks are reported annually to the Centers for Disease Control (CDC), with an average of about 40 cases per outbreak, for an average of about 18,000 food-borne disease cases annually (Bean and Griffin, 1990; Bean et al., 1990a, 1990b). However, these data include many fewer cases than do laboratory surveillance data. For example, for 1983 to 1987, 6249 salmonellosis cases per year were reported in the outbreak data compared with 44,000 cases per year in the laboratory surveillance data (CAST, 1994). Bennett and coworkers (1987) estimated that 96% of salmonellosis cases were food-borne. In the developing countries, diarrheal diseases, especially infant diarrhea, are the dominant problem and indeed one of massive proportions. Annually, some 1.5 billion episodes of diarrhea occur in children below the age of 5, and of these over 3 million die as a result (WHO, 1994). Although traditionally contaminated water supplies were believed to be the main source of pathogens causing diarrhea, up to 70% of diarrheal episodes may actually be due to food-borne organisms (Esrey and Feachem, 1989; Motarjemi et al., 1993). In addition to the food-borne diseases, food contamination with mycotoxins, pesticide residues, drug residues, and industrial chemicals is a serious issue that affects human safety and well-being. It should, however, be noted that such contamination occurs on a sporadic basis. Furthermore, it can easily be prevented by using

Copyright 2002 by Marcel Dekker. All Rights Reserved.

careful food production, storage, handling, and preparation practices. The primary aim of writing this book is to provide comprehensive information on the chemical and toxicological characteristics of various toxicants that occur in the human food chain in a single volume. Part I presents information on the basic concepts of toxicology as a science. Here, general information is presented on the basic principles of toxicology, the chemical and biochemical basis of toxicity of chemicals, manifestations of toxic effects, carcinogenesis, and detoxification mechanisms. Information on food toxicants from various sources is presented in Part II. Although these food toxicants are described individually and may indeed appear to present grave dangers to human health when consumed, the readers should be aware of the fact that any treatment of food toxicology must be considered from the viewpoint of overall nutrition. This is primarily because, as described earlier, there is no such thing as “absolute safety,” and everything we consume, from water to salt, sugar, proteins, and fats, has some associated toxicity. Moreover, toxic effects of these compounds are generally not additive. We thus consume many toxic substances in our normal diet without showing any signs of intoxification. Furthermore, several compounds thought to be antinutritional or toxic in the 1970s and early 1980s have now shown to have beneficial effects on human nutrition and well-being. In fact, if it were not for the biological contamination of foods and food hygiene issues, we probably now enjoy a safer food supply in a wider variety of forms than at any other time in the history of human civilization. The author would like to bring out this important message in this book.

REFERENCES Bean, N.H. and Griffin, P.M. 1990. Foodborne disease outbreaks in the United States, 1973–1987: Pathogens, vehicles, and trends. J. Food Protect. 53:804–817. Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B. 1990a. Foodborne diseas e outbreaks, 5-year summa ry, 1983–1987. Mor. Mortal. Wkly. Rep. CDC Surveill. Summ. Morb. Mort. Weekly Rep. (MMWR) 39(SS1):15–59. Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B. 1990b. Foodborne diseas e outbreaks, 5-year summa ry, 1983–1987. J. Food Protect. 53:711–728. Bennett, J.V., Holmberg, S.D., Rogers, M.F., and Solomon, S.L. 1987. Infectious and parasitic diseases. In Closing the Gap: The Burden of Unnecessary Illness, eds. R.W. Amler and H.B. Dull, pp. 102–114. Oxford University Press, New York.

Borlaug, N.E. 1981. Using plants to meet world food needs. In Future Dimensions of World Food and Population, ed. R.G. Woods, pp. 101–153. Westview Press, Boulder, CO. CAST. 1994. Foodborne pathogens: Risks and consequences. Task Force Report No. l22. Council for Agricultural Science and Technology, Ames, IA. Deshpande, S.S. 1992. Food legumes in human nutrition: A personal perspective. CRC Crit. Rev. Food Sci. Nutr. 32:333–363. Doull, J. and Bruce, M.C. 1986. Origin and scope of toxicology. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 3–10. Macmillan, New York. Esrey, S.A. and Feachem, R.G. 1989. Interventions for the Control of Diarrheal Diseases Among Young Children. Promotion of Food Hygiene. World Health Organization, Geneva. Garfield, E. 1990. Journal citation studies. 53. Agricultural sciences: Most fruitful journals and high yield research fields. Curr. Contents 21(51):3–10. Gossel, T.A. and Bricker, J.D. 1984. Principles of Clinical Toxicology. Raven Press, New York. Hodgson, E. 1987. Introduction to toxicology. In Modern Toxicology, eds. E. Hodgson and P.E. Levi, pp. 1–22. Elsevier, New York. Hodgson, E. and Levi, P.E. 1987. Modern Toxicology. Elsevier, New York. Holmstedt, B. and Liljestrand, G. 1981. Readings in Pharmacology. Raven Press, New York. Kaferstein, F.K., Motarjemi, Y., Moy, G.G., and Quevado, F. 1999. Food safety: A worldwide public issue. In International Food Safety Handbook, eds. K. van der Heijden, M. Younes, L. Fishbein, and S. Miller, pp. 1–20. Marcel Dekker, New York.

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Klaassen, C.D. 1986. Principles of toxicology. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 11–32. Macmillan, New York. Loomis, T.A. 1978. Essentials of Toxicology. 3rd ed. Lea & Febiger, Philadelphia. Manahan, S.E. 1992. Toxicological Chemistry. Lewis Publishers, Boca Raton, FL. Motarjemi, Y., Kaferstein, F., Moy, G., and Quevado, F. 1993. Contaminated weaning food. A major risk factor for diarrhea and associated malnutrition. Bull. World Health Organ. 71(1):79–92. Norling, B. 1994. Food poisoning in Sweden: Results of a field study. Report No. 41/94. National Food Administration, Uppsala, Sweden. Notermans, S. and Hooenboom-Verdegaal, A.S. 1992. Existing and emerging foodborne diseases. Int. J. Food Microbiol. 15:197–205. Notermans, S. and van de Giessen, K. 1993. Foodborne diseases in the 1980s and 1990s. Food Control 4(3):122–124. Sperling, F. 1984. Toxicology: Principles and Practice. John Wiley, New York. Todd, E.C.D. 1989. Preliminary estimates of costs of foodborne diseases in Canada and costs to reduce salmonellosis. J. Food Protect. 52:586–594. WHO. 1984. The role of food safety in health and development: A report of a Joint FAO/WHO Expert Committee on Food Safety. Technical Report Series No. 705. World Health Organization, Geneva. WHO. 1994. Program for Control of Diarrheal Diseases. 9th Program Report 1992–1993. World Health Organization, Geneva. Wittwer, S.H. 1980. The shape of things to come. In The Biology of Crop Productivity, ed. P.S. Carlson, pp. 413–436. Academic Press, New York.

2 Principles of Toxicology

2.1

INTRODUCTION

A poison or toxicant is a chemical that is harmful to living organisms because of its detrimental effects on tissues, organs, or biological processes. Any chemical may be a poison at a given dose and route of administration. Three factors primarily influence the toxicity of any chemical to a given species: the toxic substance itself and the matrix in which it is present, the circumstances of exposure, and the organism and its environment. Usually, an experimentally determined acute oral toxicity expression, such as an LD50 value, which is the dose required to kill half of test subjects, is used to express the toxic potential of any given chemical. Such estimates, however, are not an absolute description of the compound’s toxicity in all individuals or across different species. They neither assess the inherent capacity of the compound to produce an injury nor reflect the victim’s ability to respond in a manner other than predicted. Hence, quantitative estimates of toxicity in terms of mortality are usually not good parameters for toxicity measurements. Much more widespread than fatal poisoning, and certainly more subtle, are various manifestations of morbidity or unhealthiness. Morbidity can be manifested in several ways. Whereas the effects on vital signs are obvious, it is the subtle effects that are not life threatening per se but nonetheless are responsible for minor health ailments that ultimately cost millions of dollars in terms of treatment expenses and loss of productivity. In some instances, a toxic response may not be observed for years. It is therefore essential to distinguish acute toxicity, which

Copyright 2002 by Marcel Dekker. All Rights Reserved.

has an effect soon after exposure, and chronic toxicity, which has a long latency period. In practical situations, therefore, the critical factor is not the intrinsic toxicity of a chemical, but rather the risk or hazard associated with its use. In food science and nutrition, it is especially important to understand the concepts of relative risks and safety, hazard, and toxicity associated with the consumption of foods. Risk is the probability that a substance will produce harm under specified conditions. Absolute safety, in contrast, is the assurance that damage or injury from use of a substance is impossible. However, as discussed in Chapter 1, absolute safety is virtually unattainable. Hence, the concept of relative safety has been proposed (Hall, 1988, 1991). Relative food safety then can be defined as the practical certainty that injury or damage will not result from the consumption of food or ingredients used in food processing in a reasonable and customary manner and quantity. Food safety, however, does not refer to the food itself, but also to the people consuming it. For example, foods considered safe for most people when used in a reasonable and customary manner and quantity can be extremely toxic, even lethal, to certain sensitive or allergic individuals. The concepts of toxicity and hazard are also relevant to any discussions of food safety. Toxicity, as defined earlier, is the capacity of a chemical to produce harm or injury of any kind (chronic or acute) under given conditions. Generally, for humans, any deviation from normal is considered as a possible negative effect, even though the change may seem to be positive, such as increased growth

rates or enhanced nutrient absorption. The change is assumed to be negative until proved beneficial. Hazard is the relative probability that such harm or injury will result when the substance is used in a proposed manner and quantity. Assessments of whether a food or ingredient is safe should not be based on its inherent toxicity alone, but on whether or not a hazard is created. The inability to distinguish between toxicity and hazard as associated with the consumption of foods, especially by the general public, often results in inaccurate assessments of the relative risks and safety of our food supply. Both the regulations and the way that they are applied by the regulatory agencies often reflect the public attitudes toward particular types of chemicals and specific kinds of risks. The way that people generally perceive risk is quite different from the way scientists analyze risk, and this dichotomy has led to conflicts in the public policy arena. Generally, people tend to view catastrophic risks, e.g., airplane crashes, as greater than ordinary ones, e.g., automobile accidents. Similarly, synthetic chemicals are often viewed as riskier than natural ones; voluntary risks, such as smoking, as less significant than involuntary ones, such as air pollution; and those with immediate effects as less of a risk than those with delayed effects. Some of these factors are summarized in Table 2.1. The question of what constitutes an acceptable risk is also a matter of judgment. Such decisions are multifaceted and complex and involve a balance of risk and benefit. High risks may be acceptable in the use of lifesaving drugs but be unacceptable for food additives. Klaassen (1986a) has suggested the following factors that must be considered in determining an acceptable risk: •

Table 2.1

Benefits gained from use of the substance

Public Perception of Risk

Criteria Origin Volition Effect manifestation Severity (number of people affected per incident) Controllability Benefit Familiarity Exposure Necessity

Characteristics perceived as lower risk

Characteristics perceived as higher risk

Natural Voluntary Immediate Ordinary

Synthetic Involuntary Delayed Catastrophic

Controllable Clear Familiar Continuous Necessary

Uncontrollable Unclear Unfamiliar Occasional Luxury

Source: Kamrin (1988).

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• • • • • •

Adequacy and availability of alternative substances to meet the identified use Anticipated extent of public use Employment considerations Economic considerations Effects on environmental quality Conservation of natural resources

Public perception apart, several biological factors modify the response of a species to the toxic agent. Realization of these factors is intrinsic to our fundamental understanding of the principles of toxicology. This forms the basis of this chapter. For a more detailed treatment of the subject, the readers are advised to consult several excellent textbooks and monographs (Loomis, 1978; Hodgson and Guthrie, 1980; Timbrell, 1982; Gossel and Bricker, 1984; Klaassen et al., 1986; Hodgson and Levi, 1987; Kamrin, 1988; Marquis 1989; Manahan, 1992; Stine and Brown, 1996; Niesink et al., 1996).

2.2

TYPES AND CIRCUMSTANCES OF EXPOSURE

The exposure of an organism to a toxic substance is of prime importance in toxicology. In this regard, both the duration of exposure per incident as well as the frequency of exposure need to be considered. The rate of exposure, inversely related to the duration per exposure, and the total period over which the organism is exposed are primarily situational variables. Other factors that affect the toxicity of a substance include the dose, toxicant concentration, the exposure site, and the route of absorption. It is important to understand the differences between acute and chronic poisonings or exposures and the acute and chronic effects or symptoms. Acute and chronic poisonings differ in the number and duration of exposures to the toxicant. The toxic effects manifested as a result of poisoning may be either local, i.e., confined to a specific tissue or organ, or systemic. Generally, acute local exposure occurs at a specific location as single exposure to the toxicant. It may occur over a period of few seconds to a few hours and may affect the exposure site, particularly skin, eyes, or mucous membranes. Similar tissues or organs can also be affected by chronic local exposure. However, the time span for the manifestation of toxic effects could be several months or even years. As compared to local exposures, systemic exposures are usually manifested in toxic symptoms or effects in tissues or organs that are remote from the entry site. Thus toxicants may enter the body by inhalation or ingestion and affect organs such as the liver. The acute and chronic

systemic poisonings primarily differ in that the exposure occurs over a prolonged period in the latter case. It is also likely that an acute exposure or poisoning may result in chronic symptoms. Thus, a single exposure to potent carcinogens, such as aflatoxins or nitrosamines, may result in the chronic symptom of cancer, whereas a chronic exposure to cyanide in sufficient dose always results in acute symptoms. Therefore, the terms acute and chronic, when used to describe symptoms, refer to the duration and reversibility of the symptoms. An acute symptom is of short duration, usually severe, and generally reversible after removal of the toxicant. Chronic symptoms, in contrast, are prolonged and persist even after removal of the toxicant. For example, some organophosphate pesticides can produce chronic paralysis (Hays, 1972). Liver carcinogens in small doses may result in hepatic cancer but acute hepatic damage in large doses. Generally, exposures to toxicants between 24 hr and 90 days are usually referred to as prolonged intoxication (Concon, 1988). The symptoms of exposure or poisoning may be immediate or delayed. They are influenced by factors such as dose, type of compound, and route of contact. Toxic compounds, which produce immediate symptoms, may be easily identified as the cause of intoxication and, therefore, can be avoided. In contrast, those with delayed symptoms, especially if the effects do not appear until after several months or years, are not easily identified. Many food poisons fall under the highly delayed category. These compounds, therefore, are of concern because of their possible role in modern epidemic diseases whose causes have been difficult to establish. Furthermore, when symptoms are delayed, antidotal therapy, assuming that it is known, may be difficult to administer unless the symptoms have been correlated with those of a particular toxicant. Even so, antidotes are of little value when the appearance of symptoms, such as cancer and other effects indicating structural tissue damage, is markedly delayed. Although chronic symptoms are not necessarily due to the accumulation of the poison or toxicant in the tissues, there are toxicants that accumulate in the tissues to toxic levels, resulting in chronic delayed toxic symptoms. Some well-known examples of such toxicants include heavy metals, such as lead and mercury, and organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT). Similarly, some toxicants do not elicit chronic poisoning. For example, many of the neurotoxins that are extremely powerful poisons, e.g., the botulinum exotoxin, may progress from the no effect level to the lethal level without passing through the chronic range. In contrast, several carcinogens at the lower doses initiate carcinogen-

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esis but produce acute hepatic or renal damage when administered at high doses. 2.2.1 Exposure Assessment Because the problem of chronicity has significant bearing on public health, it is essential that those compounds having this property be identified and their other toxicological properties established. Therefore, some quantitative measure of the chronicity of a compound is important. However, the determination of chronicity has the inherent weakness in that it is based on data obtained from experimental animals with short life spans. Therefore, a major problem of validly extrapolating such data to humans remains. Nevertheless, such indices of chronicity may be potentially useful in serving as a guide in identifying those compounds, which may behave similarly in humans. Defining the exposure to a toxic agent is thus essential in assessing the significance of toxicological tests as well as understanding the risks to both humans and other organisms. There is no one uniform, well-established procedure for expressing exposure. Some commonly used expressions are summarized in Table 2.2. For proper risk assessment, the units in which exposure is expressed must be compatible with those that toxicologists commonly use to report the results of their experiments. The toxic pattern of exposure is also critical. To interpret the toxicological significance of an exposure, the following factors need to be considered (Brown and Bomberger, 1982; Brown, 1987): 1. 2. 3. 4.

Table 2.2 Toxicants

Duration of each exposure, if not instantaneous Frequency with which the exposure is repeated Variation of exposure level within each period of exposure Elapsed time to the observation of effects from the last (or sometimes first) exposure period

Methods of Expressing Exposure to Chemical

Method Concentration in medium

Quantity available for absorption

Rate of intake or exposure Concentration in body tissue Organ dose

Units mg/kg Food mg/L Water mg/m3 Air mg Inhaled, total mg Inhaled/kg body weight mg Ingested, total mg Ingested/kg body weight mg/kg Body weight mg/ml Serum mg To liver

Several possible time patterns of exposure of an individual to a toxicant are shown in Figure 2.1. The shortterm averages are generally appropriate for assessing acute toxicity responses, whereas the long-term annual average might be more appropriate for assessing chronic toxicity, such as carcinogenic or mutagenic potential of a given toxicant. The latter is especially important when a linear dose-response relationship is seen. Finally, for the control of various sources of exposure for risk assessment analysis, one needs to determine how much exposure is derived from each source. In this regard, the following factors need to be defined for a complete characterization of exposure to any given toxicant: • • • • • •

Specification of levels of exposure Specification of route(s) of exposure Distribution of exposures over time Distribution of exposures over geographical region Distribution of exposure over segments of population at risk Distribution of exposures over various sources

Detailed information on the methodology used for assessing the exposure to toxicants and the accompanying risk-benefit/safety analysis is supplied in several reviews (Brown and Bomberger, 1982; Brown and Suta, 1982; Klaassen, 1986a; Brown, 1987; Kamrin, 1988). An excellent monograph is also available on the principles of data interpretation including the statistical techniques used for such purposes (Tardiff and Rodricks, 1987).

Figure 2.1 Time patterns of exposure to toxicants: A, continuous; B, intermittent; C, cyclic; D, random; E, concentrated. (Adapted from Brown and Bomberger [1982].)

Copyright 2002 by Marcel Dekker. All Rights Reserved.

2.3

ROUTES OF TOXICANT EXPOSURE AND ABSORPTION

To exert a toxic effect, a compound must have contact with the biological system under consideration. The toxicant may exert a local effect at the site on initial exposure, but it must penetrate the organism in order to have a systemic effect. In fact, one of the main factors that influence the dose at the site of action, or the effective dose, is the route of exposure or the way in which the individual was exposed. Furthermore, the manner by which a potentially toxic chemical is introduced into the body can influence the time of onset, intensity, and duration of the toxic effects. The route of exposure may also predict the degree of toxicity and possibly the target systems that are most readily affected. Chemicals may be introduced into the complex biological organisms by a variety of routes. The major routes of accidental or intentional exposure to toxicants of humans and other animals include the skin (percutaneous route), the lungs (inhalation, respiration, pulmonary route), and the mouth (ingestion, oral route) (Figure 2.2). Other minor routes of exposure include rectal, vaginal, and parenteral. The parenteral route, viz., intraperitoneal, intramuscular, intravenous, or subcutaneous) is primarily confined to the administration of therapeutic agents. The chemical and physical properties of each compound largely determine the route by which intentional or accidental exposure occurs. The pulmonary system is most likely to take in toxic gases or very fine, respirable solid or liquid particles. In other than a respirable form, a solid usually enters the body orally. The percutaneous or dermal route is important in the absorption of liquids, solutes in solution, and semisolids through the skin. The site of entry of a toxicant is an important factor in the manifestation of final toxic effects. Thus, compounds taken by the oral route may be hydrolyzed to less (or sometimes more) toxic metabolites when exposed to the acid conditions in the stomach. The intestinal microflora may also change the nature of the compound by metabolism and thereby affect the toxicity outcome. The site of entry is also important to the final disposition of the compound. Thus, absorption through the skin may be slow and result in initial absorption into the peripheral circulation. Absorption from lungs, in contrast, is generally rapid and exposes major organs very quickly. Compounds absorbed from the gastrointestinal (GI) tract after oral exposure first pass through the liver, where they may be extensively metabolized. Irrespective of the route of exposure and absorption, there is always a potential for the toxicant to be absorbed into the bloodstream. If this occurs, the toxicant is then transported throughout the body,

Figure 2.2

Major sites of exposure, metabolism and storage, routes of distribution, and elimination of toxic substances in humans.

thereby potentially exposing all the organs and tissues. If one or more of these parts of the body are more sensitive than the site of entry, more severe toxic effects may occur to that organ or tissue. Furthermore, if the toxicant or its metabolites remain in the blood circulation for a long period, the tissues and organs are exposed to them repeatedly. Thus a single external exposure may lead to repeated internal exposures and possibly toxicity to a number of organs and tissues. Once the toxicant is absorbed and enters the bloodstream, it may undergo several metabolic changes. For example, it may be excreted or stored in body tissues as is, or it may interact with other body chemicals and be altered in some way. In the latter instance, since the metabolism is not 100% efficient, one or more metabolites may be generated from the parent toxic compound. All of the metabolites then undergo fates similar to that of the original absorbed material. Thus exposure to one chemical may result in the excretion or storage of several different chemicals as well as the potential for a variety of toxic effects. It should be emphasized that these are usually not either/or possibilities—the absorbed substances and their metabolites are often partially excreted, partially stored, and partially available to produce adverse effects (Kamrin, 1988). As a general rule, a chemical or toxicant injected by the intravenous parenteral route would be expected to be the most toxic. Administered by other routes, the approximate descending order of toxicity would be inhalation > intraperitoneal > subcutaneous > intramuscular > intrader-

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mal > oral > topical. Of course, not all of these routes are important in food toxicology. Nonetheless, the salient features of various routes of toxicant exposure and absorption are briefly described in the following sections. However, because of its obvious importance in food toxicology, a major emphasis is placed on the oral route of toxicant absorption involving the GI tract. 2.3.1 Percutaneous Exposure The simplest and most common exposure of humans and animals to exogenous chemicals of all types is exposure through accidental or intentional contact of the chemical with the skin. Toxicants can enter the skin through epidermal cells, sebaceous gland cells, or hair follicles. By far the greatest area of the skin is composed of the epidermal cell layers, and most toxicants absorbed through the skin enter through epidermal cells. Despite their much smaller total areas, however, the cells in the follicular walls and in sebaceous glands are much more permeable than epidermal cells (Manahan, 1992). The skin is a complex, multilayered tissue comprising approximately 19,000 cm2 of surface in an average human and contributing approximately 10% of the body weight (Guthrie and Hodgson, 1987a). It is a membrane that is relative impermeable to most ions as well as aqueous solutions. However, it is permeable to a large number of toxicants in the solid, liquid, or gaseous phase.

Although skin is the most readily accessible organ to all forms of chemicals, it is also an efficient barrier to most environmental toxins. The major barrier to dermal absorption of toxicants is the stratum corneum, or the outermost horny layer composed of highly keratinized cells. The permeability of the skin is inversely proportional to the thickness of this layer. It varies by location in the body in the order soles and palms > abdomen, back, legs, arms > genital (perineal) area. Disruption of the stratum corneum essentially removes all but a superficial deterrent to penetration, since the two other main areas of skin, viz., the dermis and the subcutaneous tissue, offer little resistance to penetration. Therefore, breaks in epidermis due to laceration, abrasion, or irritation increase the permeability, as do inflammation and higher degrees of skin hydration. However, in order to reach systemic circulation, the toxic compound still has to traverse several layers of cells; in contrast, in the GI tract, only two cell layers separate the toxicant from the bloodstream. Compounds, which are well absorbed percutaneously, are generally very lipid-soluble. In general, gases penetrate quite freely through the epidermal tissues, liquids less freely, and solids that are insoluble in water probably are incapable of penetrating to a significant degree. Penetration of a toxicant via the percutaneous route is also time dependent and a function of concentration gradient.

2.3.3 Oral Route The oral route is a major site of entry into the body for many toxic compounds. Food additives, food toxins, licking or rubbing, and airborne particles excluded from passage to the alveoli and returned to the glottis are among potential avenues for accidental ingestion. The GI tract can be regarded as a tube through the body from the mouth to the anus (Figure 2.3). Although it is within the body, its contents are essentially exterior to the body fluids. Therefore, toxicants in the GI tract can produce an effect only on the surface of the mucosal cells that line the tract. Any systemic effect of toxicant ingested by the oral route, therefore, requires its absorption through the mucosal cells that line the inside of the GI tract. Toxicants can be absorbed throughout the GI tract, including the buccal cavity and rectum. However, because of short residence times, most substances are not readily absorbed in the mouth or esophagus. The stomach is the first part of the GI tract where substantial absorption and translocation to other parts of the body may take place. Because of the stomach’s low pH (about 1.0), absorption through stomach is dependent on the amount of nonionized form available. Therefore, substances that are weak

2.3.2 Pulmonary/Inhalation Route The pulmonary system is the site of entry for numerous toxicants. Absorption via the lungs is an important route for toxic gases, volatile solvents, and aerosols and, in some cases, airborne particles. The pulmonary route can greatly accentuate the expected onset of toxicity for a given compound for two reasons. The rich capillary exchange at the deeper lung recesses causes a toxicant at the lung surface to be separated by only 1–2 µm from the circulation, enabling exchange of gases to occur in seconds or less. In addition, the surface area of lungs (about 50–100 m2) is some 50 times the area of skin. Because of its unpreventable contact with contaminated air, the respiratory system has also developed numerous mechanisms to avoid many airborne substances. Particles can be trapped in the upper respiratory or nasopharyngeal region. Those deposited in the tracheobronchiolar region are cleared upward by the mucus blanket. In addition to upper pathway clearance, lung phagocytosis is very active in both upper and lower pathways of the respiratory tract and may be coupled to the mucus cilia. Phagocytosis may also direct engulfed toxicants into the lymph.

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Figure 2.3 Schematic diagram of the alimentary canal and associated structures in the human. A, parotid gland (salivary gland); B, pharynx; C, sublingual gland (salivary gland); D, submandibular gland (salivary gland); E, esophagus; F, cardiac sphincter; G, stomach; H, liver; I, gallbladder; J, pancreas; K, duodenum; L, splenic flexure of transverse colon; M, hepatic flexure; N, transverse colon; O, descending colon; P, ascending colon; Q, jejunum; R, caecum; S, appendix; T, ileum; U, sigmoid colon; V, rectum; W, anus.

acids (i.e., ionic at pH near 7.0 and above) are neutral in stomach so that they readily traverse the stomach walls. In some cases, absorption is affected by stomach contents other than HCl. These include food particles, gastric mucin, gastric lipase, and pepsin. Compared to gastric absorption, intestinal absorption is extensive because of the presence of microvilli, which provide an extremely large surface area (Figure 2.4). The pH of the contents of the small intestine is close to neutral, so that weak bases that are charged in the acidic environment of the stomach are uncharged and absorbable in the intestine. Intestinal contents are moved through the intestinal tract by peristalsis, which has a mixing action on the contents and enables absorption to occur along the length of the intestine. There is also significant absorption of compounds in the colon. The epithelium of the colonal lumen behaves much as the small intestine does. The colon is also the major site of water absorption. Thus, the dragging effect is probably highly operative in this part of the GI tract. Furthermore, the metabolic activity of the colon microflora may change the absorbability as well as the toxicological properties of a compound. Their effects in transforming the acidity of this organ’s secretion as well as the acid-base and lipid solubility properties of the compound itself by metabolic degradation may transform the toxicological characteristics of the food altogether. Toxicants ingested by the oral route can also enter the bloodstream via the enterohepatic circulation system (Figure 2.2), which comprises the intestine-blood-liverbile loop. A toxicant absorbed through the intestine goes either directly to the lymphatic system or to the portal circulatory system. The latter carries blood to the portal vein

Figure 2.4 Schematic drawing of the lining of the small intestine. LP, lamina propria.

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that goes directly to the liver. The liver serves as a screening organ for xenobiotics, subjecting them to metabolic processes that usually reduce their toxicities, and secretes these substances or their metabolites back to the intestine. For some toxicants, there are mechanisms of active excretion into the bile in which the substances are concentrated by one to three orders of magnitude over their levels in the blood. Other substances enter the bile from blood simply by diffusion. The importance of enterohepatic circulation is discussed in greater detail in the section dealing with the excretion of toxicants in this chapter. The presence and type of food in the stomach can modify the absorption of a toxicant. A meal rich in protein or fat usually delays absorption. Carbonated beverages increase the rate of intestinal absorption by increasing gastric emptying time, with evolution of carbon dioxide. Ingestion of a concentrated chemical frequently causes a decrease in absorption as a result of gastric irritation and constriction of the pyloric sphincter. On the positive side, the oral route of intoxication may provide the body with a chance to metabolize the ingested toxicant readily.

2.4

MECHANISMS OF TOXICANT ABSORPTION

The toxicity of a chemical is dependent on the dose administered. However, it refers not to the dose administered, but rather to the concentration of the toxic chemical in the target organ. To exert its toxic action, a chemical must be absorbed in the biological tissue or organ. If the fraction of the chemical absorbed is low or the rate of absorption is low, then only a low concentration of the chemical in the target organ may be obtained, resulting in little or no toxicity. In this section, various mechanisms of toxicant absorption are briefly described. Again, a special emphasis is placed on the mechanisms of intestinal absorption. A toxicant may pass through a number of barriers before achieving a sufficient concentration in the organ where it produces its characteristic toxic effects. These include membranes of a number of cells. In all cases, the membranes of tissue, cell, and cell organelle are basically similar. They comprise a bimolecular layer of lipid molecules coated on each side with a protein layer. Certain branches of the protein layer appear to penetrate the lipid bilayer, and others extend through the membrane. The lipid portion of the membrane consists primarily of phosphatidylcholine, phosphatidylethanolamine, and cholesterol. The fatty acids of the membranes do not have a rigid crystalline structure and at physiological temperatures are quasi-fluid in character. The fluidity of

the membranes is largely determined by the structure and relative proportion of unsaturated fatty acids. When the membranes contain more unsaturated fatty acids, they are more fluidlike, and active transport (defined later in this section) is more rapid (Klaassen, 1986a; Guthrie and Hodgson, 1987a). The mechanism of the movement of toxicants across membranes is a poorly researched area. A toxicant may pass through a membrane by one of two general processes (Klaassen, 1986a): diffusion or passive transport of the chemical, in which the cell expends no energy in its transfer, and specialized transport, in which the cell takes an active part in the transfer of the toxicant through the cell membranes. In spite of these processes, the intestinal mucosae are relatively impermeable tissues to many substances, including various electrolytes, many organic compounds, and water-soluble macromolecules such as starches, pectins, and other heteropolysaccharides and hydrocarbons (Crane, 1979; Henning, 1979; Concon, 1988). 2.4.1 Passive Transport Most toxicants cross membranes by simple diffusion. Simple diffusion of compounds with appropriate water/lipid partition coefficients largely determines the rate of toxicant movement. However, the nature of the mucosal membrane is such that even passive or simple diffusion of compounds is selective. Six factors primarily govern the passive diffusion of substances: (a) Fick’s law, (b) molecular size, (c) lipid solubility, (d) degree of ionization, (e) “drag” effect or bulk flow of the absorption of water, and (f) the Donnan distribution effect. Their relationship to the movement of toxicants across the membrane barriers is briefly described in the following sections. Fick’s Law Many small hydrophilic compounds diffuse across cell membranes through aqueous channels. For molecules that can easily pass through these pores, Fick’s law can predict their rate of diffusion J = KA (Ce – Ci)/h = PA (Ce – Ci) Where

P

= K/h, permeability coefficient

Fick’s law holds quite well for nonelectrolytes such as urea. However, an additional mechanism is also operative with substances such as fructose, mannose, and xylose, even though their absorption also appears to follow Fick’s law. These sugars appear to be absorbed by facilitated diffusion. Simple diffusion through the cell membrane obeying Fick’s law generally cannot take place against a concentration gradient and is not inhibited by metabolic inhibitors. Furthermore, there is no competitive absorption with other substances (Schanker, 1961; Levine, 1970; Concon, 1988). Molecular Size The molecular size of a chemical also influences the rate of intestinal absorption. Generally, an inverse relationship is observed as the rate of absorption decreases with increasing molecular size. However, several factors influence the effect of molecular size on the absorption of these compounds. In this regard, lipid solubility and ionization effects are more important in regulating the passive absorption of compounds through the intestinal tract. Lipid Solubility Some substances seem to be absorbed through the intestinal tract by passive diffusion even though their molecular size greatly exceeds the postulated average pore size of the intestinal epithelium. Thus a route of entry through the cell membrane other than through the aqueous channels must be available to these molecules. These substances enter the cell by “dissolving” into the cellular membrane material, which is highly lipophilic in nature. Thus, their rate of absorption can be correlated to their solubility in oil or lipid solvents. Often, a linear relationship between lipid solubility and absorbability is seen for compounds with similar chemical properties. However, such correlation is often poor for compounds that, in addition to their lipid solubility characteristics, also behave as weak electrolytes in aqueous medium. The rate of absorption of such compounds through the cell membranes can be markedly affected by a change in pH as described later. Degree of Ionization

J

= rate of diffusion

k

= diffusion coefficient

A

= area of surface diffusion

h

= membrane thickness

Ce, Ci = concentrations of the solute outside and inside the cell, respectively

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Many toxic chemicals exist in solution in both ionized and nonionized forms. The ionized form is often unable to penetrate the cell membrane because of its low lipid solubility, whereas the nonionized form may be sufficiently lipid-soluble to diffuse across the cell membrane. Diffusion is thus primarily dependent on the lipid solubility of the nonionized form of the compound. This phenomenon

is observed with a wide range of chemicals, including weak acids and ammonium salts, and dyes. This is particularly true of chemicals for which absorption is not also mediated by mechanisms other than simple diffusion. The degree of ionization of a compound, i.e., the ratio of ionized and nonionized species (A–/HA), can be calculated from the Henderson-Hasselbalch equation pH = pKa + log (A–/HA) where A– and HA are the ionized and nonionized species, respectively. The amount of weak organic acid or base in the nonionized form is dependent on its dissociation constant. The pH at which an acid is 50% dissociated (ionized = nonionized) is called its pKa. It is defined as the negative logarithm of the acid dissociation constant. Conventionally, the dissociation constant for both acids and bases can be expressed as a pKa. The degree of dissociation and ionization of a weak acid or base is dependent on the pH of the medium. This relationship for a weak acid, benzoic acid, and a base, aniline, is shown in Figure 2.5. As the pH decreases, more of the acid becomes nonionized. The converse is true as the pH is increased. For an organic base like aniline, the opposite occurs. Thus, weak acids with pK a values of around 2 are ionized in the small intestine at pH 6 to the extent of approximately 10,000:1, and with weak bases with pKa of around 9, the degree of dissociation is around 1000:1. Since the effective mucosal cell pH is around 5, Schanker and coworkers (1958) postulated that for weak

Figure 2.5 Effect of pH on the ionization of a weak organic acid, benzoic acid (pKa = 4, curve A), and a weak organic base, aniline (pKa = 5, curve B).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

acids with a pKa of 2, the degree of dissociation in the cell is around 1000:1, whereas that of weak bases with pKa values around 9 is about 10,000:1. These values appear to be the minimal ratios that must be present for effective absorption of these compounds in the GI tract. Since the lipid soluble form (nonionized) of a weak electrolyte is the species that crosses cell membranes, organic acids are more likely to diffuse across membranes when they are in an acidic environment, whereas a basic environment favors diffusion of bases across membranes. Thus, on the basis of the effect of pH in the absorption of compounds, the degree of toxicity of a weak acid or base may be predicted from the pH of the GI tract. However, when the total area of absorptive surface and the rate of passage are considered, the pH effect may not have a significant influence on the total amount absorbed. For example, the absorption of a weakly acidic substance may be highly favored in the stomach as the pH of its contents decreases, but more of the substance may be absorbed in the intestines, assuming a normal rate of emptying occurs. This is because even though greater dissociation occurs in the intestines, the pH here permits the existence of a significant proportion of nonionized species, and the large surface area compared to that of stomach results in greater total absorption. Thus, even though the rate of absorption in the intestines per unit area may be less than that in the stomach, the greater total surface area of the former results in greater total absorption. Therefore, the large surface area of the intestines may obviate any advantage or disadvantage that the ionization effect may have in the absorption of chemicals. The ionizing effects also explain the poor absorbability by passive diffusion in the small intestines of salts, strong acids, bases, and organic and essential cations such as Fe2+, Fe3+, Mg2+, Ca2+, Zn2+, Mn2+, Co2+, and other trace metallic ions, and the anions, such as PO43– and Cl–. Examples of organic anions that are poorly absorbed are citrates, lactates, and tartrates. The poor absorbability of these substances, in fact, forms the physiological basis for their cathartic effects. Similarly, nonabsorbable sulfates and phosphates are also cathartics. Therefore, for nutritionally essential minerals, special mechanisms are necessary for absorption in order to provide adequate amounts for the animal’s nutritional requirements. However, even with these special mechanisms, absorption of these minerals is limited and is controlled by the body’s physiological requirements. These limitations afford a measure of protection for the organism because above a certain level, these substances are quite toxic (Ulmer, 1977; Concon, 1988). The mechanisms involving active or carrier-mediated transport are described later in this section.

“Drag” Effect and Bulk Flow of the Absorption of Water

Donnan Distribution Effect

When water flows in bulk across a porous membrane, any solute that is small enough to pass through the pores flows with it. Passage through these channels is called filtration, since it involves bulk flow of water due to a hydrostatic or osmotic force. Because such aqueous channels in most cells are relatively small (4–10 nm), only chemicals with molecular weight of 100 to 200 daltons can pass through (Schanker, 1961, 1964). Larger molecules are excluded except in more highly porous tissues (approximately 70-nm pore size) such as kidneys and liver. These allow molecules smaller than albumin (molecular weight 68,000) to pass through. Because many toxicants are relatively large molecules, this pathway is often of limited importance. The bulk flow of absorption of water follows Poiseuille’s law:

A pH differential on each side of a membrane allows only undissociated particles to diffuse out. The process, termed the Donnan distribution effect, causes a net transfer of a chemical to another compartment. Thus, an ionizable chemical that is free to diffuse through a membrane dissociates in the compartment where such dissociation is favored. This ionization reduces the concentration of the diffusible chemical so that a concentration differential is established. More of the diffusible chemical then migrates to the compartment where it can be dissociated more favorably until equilibrium between the two compartments with respect to this chemical is attained. The Donnan distribution effect may be a significant factor governing the flow of a weak acid from the stomach (pH 2 to 4) or the small intestine (pH 4.5 to 6.5) to the blood (pH 7.35 to 7.45). The degree to which the Donnan effect enhances the net transfer of a substance across the GI mucosa is highly dependent on the pH differential between the intestinal medium and the blood and the pKa of the substance. An example of this type of distribution is shown by the study of Shore and associates (1957), who injected acidic and basic drugs intravenously into dogs. In the gastric juice, where their ionization is favored, the concentration of basic drugs at equilibrium increased to as much as 40 times that in the plasma. In contrast, acidic drugs, whose ionization is not favored, either were absent or increased in concentrations to no more than six-tenths of those in the plasma. Zawoiski and colleagues (1958) also observed that the concentration in the gastric juice of an organic base injected into the dog increased as the pH decreased. The Donnan distribution effect is apparently less effective in the transfer of weak bases, such as some alkaloids, from the GI tract to the blood. This is because ionization in the blood is less favorable and, hence, there is a comparatively small, if any, concentration differential. The Donnan effect also occurs if one of the charged components in one compartment is a macromolecule too large to diffuse through the membrane. When this molecule dissociates, and one of the particles so formed is freely diffusible, the movement of this particle to the opposite side must be accompanied by a movement in the opposite direction of a particle of the same charge to maintain electrical neutrality. Thus, this process may cause the transfer of a different substance to the compartment containing the macromolecule. If, on the other hand, the compartment opposite that containing the macromolecule has freely diffusible positive and negative ions and the ions are the same as those associated with the macromolecule, then at equilibrium a net increase of these ions is found in the macromolecule compartment. This type of transfer of

V = 8πr4Phl where V = rate of flow r = radius of pore P = hydrostatic pressure or osmotic pressure h = viscosity of solution flowing through pore l = length of pore The extent of hydrodynamic flow across the membrane of intestinal epithelium differs from what can result from the osmotic gradients across the membrane of the small intestines. This difference is explained on the basis of the concept of bulk flow (Concon, 1988). The bulk flow can increase transport of solutes through the pore by “dragging” the molecules in the moving stream. The dragging effect may be expected to be greater at the time when absorption by the various mechanisms is at full capacity, so much so that the total osmolar concentration in the plasma is much greater than that in the intestinal lumen. In this case, the greater the total rate of absorption, the higher the total osmolar concentration of the plasma. Consequently, the dragging effect is also greater. The dragging effect will obviously be expected to be greatly increased when the amount of fluid ingested is large, since, in addition to osmotic effects, the hydrostatic pressure is also greater. However, because of the very nature of dragging and because of the relatively large molecules of many toxicants the contribution of the dragging phenomenon to the total absorption of toxicants is probably much less than that from other mechanisms, such as the active transport processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

charged particles, to a large extent, may not be applicable in the case of the GI tract because charged particles are not freely diffusible across the GI membrane. Thus, it is essential that the charged substances that are important to life are transported by special mechanisms. A net transfer of a substance can also result from the Donnan distribution effect, if in one compartment, a nondiffusible substance, such as a protein, can bind the diffusible molecule. The movement of the diffusible substance bound by the protein follows Fick’s law, since in effect, a large concentration gradient is present as long as the binding capacity of the protein is not exhausted. 2.4.2 Special Transport There are a number of instances in which the movement of a compound across a membrane cannot be explained by simple diffusion or filtration because the compound is too water-soluble to dissolve in the cell membranes and too large to flow through the channels. Thus, if the GI absorption were to rely solely on passive transport, the limitations imposed on this process would exclude many compounds, even those essential to the survival of the organism. Those compounds that meet the conditions necessary for passive absorption in many cases cannot be absorbed at a rate commensurate with the needs of the body. Furthermore, the requirement of these substances by the body may be such that their concentration in the lumen of the GI tract may be lower than that in the blood. Therefore, even though the GI epithelium is an “open” membrane, absorption under these conditions is thermodynamically impossible. Instead of absorption, leakage into the GI lumen results. Thus, for the transport of many essential substances, such as sugars, amino acids, and nucleic acids, as well as some foreign molecules, the GI epithelium is equipped with a number of special transport systems. The special transport mechanisms thus are most manifest in GI absorption. These mechanisms attain much greater importance in the elimination of toxicants, however, in which special transport is important to the removal of xenobiotics and their metabolites. An important characteristic of the special transport systems, when operable, is that they allow movement of compounds with lesser lipid solubility, compounds that would ordinarily be expected to move very slowly through the highly lipophilic cell membranes. These processes are briefly described in the following. Active Transport Active transport requires energy and permits the absorption of the compound even against a concentration gradient. The following features characterize an active transport process:

Copyright 2002 by Marcel Dekker. All Rights Reserved.

1. 2.

3.

4.

Chemicals are moved against electrochemical gradients. The transport system is saturated and a transport mechanism exhibited at high substrate concentrations. The transport system is selective. Therefore, certain basic structural requirements exist for chemicals to be transported by the same mechanism with the potential for competitive inhibition. The system requires the expenditure of energy so that metabolic inhibitors block the transport process.

Compounds that are actively transported across a cell membrane are presumed to pass into the cell interior by forming a complex with a macromolecular carrier, generally proteins, on one side of the membrane. The complex subsequently traverses to the other side of the membrane, where the compound is released. The carrier molecule then returns again to the original surface to repeat the transport cycle. Carrier proteins involved in the active transport processes generally have specificities for certain kinds of chemical groups and configurations (Crane, 1979). This type of active transport system can be inhibited by a variety of compounds. For example, the transport of sugars and amino acids can be inhibited by cyanide, dinitrophenol, malonate, fluoroacetate, arsenate, and copper (Ther and Winne, 1971). The carrier system can also be saturated. This mechanism imposes a limit on the rate of absorption, even though the amount absorbed generally is a function of the dose. Many exogenous compounds can also compete with the carrier systems of essential nutrients, especially substances that structurally resemble the nutrients and other endogenous physiologically essential compounds. The active transport of organic chemicals appears to be closely associated with the sodium transport. Thus, compounds that inhibit sodium transport also inhibit the transport of the organic compounds (Ther and Winne, 1971; Concon, 1988). The energy-requiring active transport mechanism can also be inhibited by interference in the metabolic sources of energy. Thus, compounds that are inhibitors of oxidative phosphorylation also inhibit active transport of a variety of nutrients. Various sections of the GI tract seem to have specific preference for the active absorption of a compound or groups of compounds. For example, sugars as well as the neutral amino acids are largely absorbed in the middle portions of the small intestine, whereas the basic amino acids are absorbed equally in all parts of the small intestine (Concon, 1988). Bile salts and vitamin B12 are absorbed

mostly in the ileum; Ca 2+, Fe2+, and Cl– are absorbed mostly in the upper small intestine. In contrast, Na+ appears to be equally absorbed in all parts of the small intestine and colon; H + is absorbed most in the ileum and colon. The active transport process is of fundamental importance in toxicology. It is involved in the elimination of foreign compounds from the organism. To transport substances out of the cerebrospinal fluid, the central nervous system has two transport systems at the choroid plexus, one each for organic acids and organic bases (Klaassen, 1986a). Likewise, the kidney has two active transport systems that eliminate foreign compounds from the body, and the liver has at least four active transport systems, two for organic acids, one for organic bases, and one for neutral organic compounds. The active transport system itself is also potentially a target of many toxic compounds, which inhibit the process in one way or another. From the nutritional standpoint, active transport’s being subject to competitive inhibition even among the nutrients is also relevant to the toxic effects of nutrient excesses.

cess therefore explains the absorption of protein toxins and other toxic materials that otherwise would be excluded from the intestinal epithelium for reasons of molecular size alone. Since many toxic substances in foods are macromolecules, their toxicity is therefore related to the ability of the small intestine to absorb them.

2.5

FACTORS AFFECTING INTESTINAL ABSORPTION

The toxicity of compounds absorbed through the GI tract is generally much less than that of compounds that gain entry through other routes. This is because the GI tract imposes certain limitations on their rates of absorption. Most substances absorbed in the GI tract must pass through the liver, where they can be metabolized to derivatives of lesser or greater toxicity. In addition, several other factors can influence the absorption of toxicant through the GI tract. Their importance in the manifestations of toxic effects is briefly described. 2.5.1 Effect of Blood Flow

Facilitated Transport The facilitated transport mechanism is similar to active transport except that in this carrier-mediated transport system, the substrate is not moved against a concentration gradient. Also, this process does not require any energy expenditure. Hence, the term facilitated diffusion was coined by Danielli (1954). The transport of glucose from the GI tract into blood, from plasma into red blood cells, and from blood into the central nervous system is thought to occur through facilitated diffusion. There is evidence that facilitated transport may also apply to exogenous chemicals (Fiese and Perrin 1969). Similarly to active transport, facilitated diffusion is also subject to saturation phenomena, competitive inhibition of similar compounds, stimulation by sodium ions, and a temperature effect. Endocytosis Pinocytosis (for liquids) and phagocytosis (for solids) are specialized transport processes in which the membrane invaginates or flows around a toxicant, allowing more ready transfer across cell membranes. This type of transfer across cell membranes is important to removal of particulate matter from the alveoli by alveolar phagocytes and for removal of some toxic substances from the blood by the reticuloendothelial system of liver and spleen. A defect in the intestinal epithelium may enhance pinocytosis of macromolecules (Concon, 1988). This pro-

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Compounds that influence the blood flow generally also influence the rate of absorption. Thus, vasoconstrictive drugs such as serotonin, norepinephrine, and vasopressin diminish blood flow and consequently absorption of water. In contrast, ethanol, which increases the blood flow rate, is absorbed at a rapid rate in the stomach (Magnussen, 1968). Blood flow can also influence absorption by its effect on the supply of oxygen and other nutrients. This property follows from the fact that active transport requires oxygen. Indeed, there appears to be a critical blood flow rate through the splanchnic area below which active transport ceases (Robinson et al., 1964, 1966). The loss of active transport capacity of the intestinal epithelium is also observed in intestinal ischemia. Such loss of active transport capacity can be prevented by perfusion of the ischemic tissues with solution saturated with oxygen. The draining effect of blood increases the rate of absorption simply from a consideration of Fick’s law, since the removal of absorbed substances in the serosal side of the GI epithelium maintains a large concentration gradient. Normally, the rate of blood flow in the portal vein (Figure 2.2) is approximately 1.2 L/hr/kg, with a 30% increase in blood flow through the splanchnic area after a meal (Concon, 1988). Therefore, an increased absorption of toxicant may result if it is ingested during a meal, assuming that the pH is favorable and the toxicant is not bound to other components in the food. As a corollary, a decrease in blood flow rate lowers the intestinal absorption

of toxicants. Thus, a normal blood flow rate in the intestinal epithelium is essential to maintenance of a normal rate of absorption of chemical; compounds that increase the blood flow generally displaying an enhanced toxicity. 2.5.2 Effect of Lymph Flow Rate The lymphatic flow rate, only about one-six hundredth to one-thousandth of that of the blood, is important for highly toxic substances, such as the botulinum toxin, that are transported by the drag effect and bulk flow mechanism. However, very little is known about the effect of lymph flow rate on the absorption of exogenous toxicants. 2.5.3 Gut Motility and Emptying Time Gut motility and the rate of passage and elimination of food from the GI tract also influence the rate of absorption of chemicals. Higher absorption rates are observed with increased residence times of food in the GI tract. Therefore, any conditions that decrease or increase gut motility and emptying or passage time have a corresponding effect on the toxicity of compounds. If smaller amounts of compounds are presented slowly for absorption in the GI tract, there may be sufficient time and several ways to dispose of the undesirable compounds. Consequently, a toxicologically insignificant amount, if any, ultimately reaches its target organ or tissue. The emptying or passage time in the GI tract can also influence the toxicity of compounds. The intestinal microflora play an important role in this regard. For example, a compound that is made toxic by action of the intestinal microflora may show no toxic effects if it moves slowly through the intestinal tract. The reason is that only small amounts unabsorbed by the intestine reach the colon, where microflora are most abundant. In contrast, a substance may move rapidly in the GI tract for various reasons (e.g., stimulation by cathartics), so that a fraction of the toxic dose is absorbed. The same reasoning applies to stomach emptying for those compounds absorbed in the ileum or colon. The rate of appearance of substances in the colon also regulates their metabolism by the microflora, influencing both their absorption and their toxicity. The gastric emptying time is influenced by the type and volume of the meal, the acidity of the gastric content, the neutralization process in the duodenum, and drugs such as the pressor amines, norepinephrine, histamine, and tyramine (Levine and Walsh, 1975; Holz, 1968; Concon, 1988). These drugs are found in many types of food products even though their concentrations are often too low to affect normal intestinal function. They also have potent ef-

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fects on the motility of the intestines and colon (Holz, 1968). Microbial infections of the intestinal wall and other disease symptoms can also affect the intestinal motility. Such diseases produce a rapid transit time of intestinal contents, as in diarrhea, or the opposite effect, as in constipation. These conditions are extreme examples of contrasting effects of gut motility and transit time. Pathological conditions in the GI tract may also affect the integrity of intestinal mucosa and the toxicant absorption. For example, because many toxic substances are lipid soluble and are absorbed and transported in association with lipids via the lymphatic system, any malabsorption of fats may have far-reaching toxicological implications other than those involving these substances directly. 2.5.4 Chemical Factors Affecting Absorption Chemicals may affect the absorption of compounds by (a) formation of insoluble precipitates, or complexes with specific substances, or formation of chelates that facilitate or inhibit absorption and solubilization; (b) competition for binding or carrier proteins involved in absorption; and (c) modification of the motility or absorptive capacity of the GI mucosa. Compounds such as phytic acid, oxalates, and gossypol can form insoluble complexes with bivalent metal ions, amino acids, and proteins. Insoluble precipitates can also be formed by phosphates, fatty acids, and alkalis (e.g., antacids). Sometimes, chelation can also improve the absorption of toxicants. For example, citric acid can increase the absorption of lead (Graber and Wei, 1974), and magnesium improves the absorption of dicoumarol (Ambre and Fischer, 1973). Competition for carrier proteins also influences the absorption of toxicants. In this regard, similarity in general structure may be sufficient to influence the absorption of compounds. Toxicants can also modify the absorptive capacity of the intestinal mucosa by interaction with its structural constituents. Lectins, for example, bind strongly with specific receptors in cell membranes. The absorptive capacity of the intestinal mucosa may be affected by changes in the acidity of the intestinal mucosa. Thus, compounds that inhibit carbonic anhydrase cause a decrease in intestinal pH (Concon, 1988). The general metabolic integrity of the GI mucosal tissues is thus essential to their structural and functional status. Any substance or condition that destroys the metabolic integrity of these tissues has an adverse effect on

their function and structure, thereby affecting the absorption of compounds.

The specific classes of molecules and their potential sensitivity to alterations by toxicants are briefly described in the following sections:

2.6

Proteins

BIOLOGICAL TARGETS OF TOXIC COMPOUNDS

A knowledge of the interactions of a toxicant with specific molecular targets, the role that molecular target plays in the chemical dynamics of the cell, and the response of the cell to either the presence of the toxicant or the perturbations the toxicant elicits is fundamental to our understanding of the manifestations of toxic effects. In this section, the potential molecular, subcellular, and cellular targets available to a toxicant for interaction are briefly described. 2.6.1 Molecular Targets The four basic macromolecules, viz., proteins, lipids, carbohydrates, and nucleic acids, involved in the dynamic execution of living processes in the biological systems are frequently the target of toxic compounds. The small metabolites of the cell are quickly and easily replaced after modifications, since they are part of the flux of material throughout the metabolic pathways. In contrast, the macromolecules are of complex biosynthetic origin. Their replacement in the cellular system is energy intensive and requires a dietary supply of precursors, such as essential amino acids, fatty acids, and vitamins. Generally, interactions of toxicant with macromolecules, especially cellular enzymes involved in important metabolic pathways, often result in improper levels (excessive or deficient) of a cellular component. This effect, in turn, may produce a range of subtle but pervasive effects, varying from a disturbance of the osmotic strength of the cell’s cytoplasm to the interruption of energy metabolism. In the cell, one perturbation may trigger another, in a cascading series of reactions that may intensify the potential for harm and, spatially and temporally, obscure the initial triggering reaction. At some point in the series of reactions, the system is irreversibly altered, even dies. At present, our knowledge of such interactions at cellular level is confined to those of a relatively few wellcharacterized toxicants, especially those that are very specific in action and are potent, and in which the exposure produces an acute effect. The toxicity of compounds that either act chronically by a mechanism different from that of their acute action or produce latent symptoms (e.g., the mutagens, carcinogens, and teratogens) is extremely difficult to describe.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Proteins are the first product of a cell’s genetic potential. They can be broadly grouped into the following five categories: 1. 2. 3. 4. 5.

Structural proteins, e.g., collagen Catalytic proteins, e.g., various enzymes Carrier or storage proteins, e.g., hemoglobin, transferrin, ceruloplasmin Informational or regulatory proteins, e.g., peptide hormones such as insulin or repressor proteins Immunological proteins, e.g., immunoglobulins involved in defense mechanisms

Specific proteins that may fill several roles simultaneously are not unusual. Toxicants primarily interact with the side chains of amino acids that constitute the primary backbone of the proteins. Because these side chains are also involved in and primarily determine the secondary and tertiary structures of the protein, their interactions with toxicants can disturb protein structure. Usually, a disturbance at any level of protein structure, especially enzymes, can alter the protein’s catalytic or biological function. The relative order of nucleophilicity (i.e., capacity of any atom containing an unshared pair of electrons or an excess of electrons to participate in covalent bond formation) relative to the major groups in biological molecules can be summarized as follows: R-S– > R-SH R-NH2 > R-NH3+ R-COO– > R-COOH R-O– > R-OH R-OH = H-OH and finally R-S– > R-NH2 > R-COO– = R-O– Amino acids whose side chains are capable of reacting with toxic chemicals are listed in Table 2.3. From the preceding relationships it is obvious that the strongest nucleophile in protein molecules is the sulfhydryl group of cysteine, particularly in the ionized, thiolate form. Next in line are the amine groups in their uncharged, unprotonated forms, including the α-amines at the N terminals, the ε-amines of lysyl side chains, the secondary amines of histidine imidazolyl groups and tryptophan indole rings, and

Table 2.3 Amino Acids with Side Chain Functionalities and Their pKa Values Amino acid All All Lysine Histidine Arginine Tyrosine Aspartic acid Glutamic acid Cysteine

Functional group

pKa range

α-Amino, N terminus α-Carboxyl, C terminus ε-Amino Imidazolyl nitrogen Guanidinyl group Phenolic hydroxyl β-Carboxyl γ-Carboxyl Sulfhydryl

7.6–8.0 2.1–2.4 9.3–9.5 6.7–7.1 > 12 9.7–10.1 3.7–4.0 4.2–4.5 8.8–9.1

the guanidino amines of arginine residues. Finally, the least potent nucleophiles are the oxygen-containing ionizable groups, including the α-carboxylate at the C terminals, the β-carboxyl of aspartic acid, the γ-carboxyl of glutamic acid, and the phenolate of tyrosine residues. These side chains of amino acids in a protein molecule are predominantly involved in interactions with various toxicants. Coenzymes Coenzymes are a class of biomolecules that participate in several enzymatic reactions and are present in limited concentrations within the cell. Their synthesis is complex, and their replacement is in part dependent on the dietary supply of vitamins. The coenzymes themselves may be subject to direct attack by toxicants. For example, some symptoms of heavy metal poisoning are similar to those of vitamin B1 deficiency. Metalloproteins, especially the heme proteins possessing the iron-containing protoporphyrin ring, are also quite vulnerable to attack. Cyanide poisoning of cytochrome c oxidase, a heme-containing protein involved in the terminal electron transport process during respiration, is well known. Nucleic Acids Nucleic acids are the building blocks of a cell’s genetic material, viz., the deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The DNA molecule offers a target of considerable size for interaction with toxic chemicals. The introduction of an error in the DNA results in the loss of quality or quantity of biological information. The error produces a faulty protein molecule or results in a level of an RNA and/or protein species that is inappropriate to the cell’s state of differentiation. A permanent modification of DNA is a mutation, which, if expressed, may lead to a carcinogenic and teratogenic event.

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Aside from the inhibition of enzymes involved in their synthesis, toxic compounds may also affect DNA and RNA formation and function by reacting with the macromolecules themselves. For example, DNA replication, RNA translation, and consequently protein synthesis may be interfered with by alkylation of the DNA or RNA purine or pyrimidine bases by N-nitroso compounds, such as the nitrosamines. Lipids Lipids primarily serve three cellular functions: storage, structural, and informational. Triglyceride stores in the cell and the adipose tissue are mobilized in times of stress or food deprivation to yield fatty acids and glycerol for energy production. This process is not regarded as an essential function. However, lipids are an integral part of membranes. Their length, their degree of unsaturation, and the nonlipid moieties attached to them essentially govern the permeability, excitability, and fusion properties of the membrane, as well as influence the activity of membranebound enzymes complexes. Lipids, in the form of steroids, also serve as hormones. The most susceptible function to interactions with exogenous toxicants seems to be related to the lipid’s role in membrane structure and function. Free radicals generated by exogenous agents can cause oxidative changes in the unsaturated fatty acid constituents of the membranes. Such oxidative changes may in turn lead to carcinogenesis, mutagenesis, and cellular aging mechanisms. Disturbances in steroid metabolism may also lead to cancer. Carbohydrates Carbohydrate polymers serve structural, recognition, and storage functions in the cell. Carbohydrate moieties on the surface of a cell are also involved in regulation by the body’s immune system. Cells that have been transformed to tumor cells display an altered carbohydrate surface determinant. Generally, any substantial modification of carbohydrate structure or function occurs by modification of the enzymes involved in carbohydrate metabolism. The molecular weight and the redundancy of their structure in the cell simply render them too diffuse and inert to suffer attack directly by an exogenous chemical. Thus, as compared to other macromolecules, carbohydrates are not sensitive or frequent targets of exogenous chemicals. 2.6.2 Subcellular Targets Many toxicants show little discrimination in their attack on molecular targets in vitro but do elicit specific patho-

logical effects in vivo. Thus the accessibility of a particular cellular structural component, as well as the component’s influence on the integrity of one or another organelle, determine the specificity of attack. Organelles possess enzyme systems specific to their purpose. This compartmentalization and specialization of function explain in part the susceptibility of one organelle to a toxicant to which another is impervious. The availability of such targets also explains why a toxicant elicits one type of damage in one cell population but has a totally different effect in another. Some specific subcellular targets available for the exogenous toxicants are described next.

compounds makes this subcellular system a frequent target of toxicants. The activity is particularly high in the liver, although the system exists to a smaller extent in most cells. In some instances, the products formed by the enzyme system of the ER are more toxic to the cell than the parent compound. Furthermore, since the enzymes of the ER metabolize primarily endogenous substrates, unnatural proliferation of this system may result in abnormal levels of hormones, bile salts, and other normal metabolites. Thus an excess of enzyme activity can be as dangerous to the biological system as a deficiency.

Nuclei

The plasma membrane that surrounds the cell has considerable microheterogeneity in its structure and function. It is home to, among others, the cell antigen-recognition sites, membrane transport enzymes such as the Na+-K+ pump, and hormone receptor sites. Given the natural messengers and activators that arrive and operate at this dynamic interface, it is not surprising that several toxins also interact with and attack the plasma membrane. In fact, a general feature of cellular injury seems to be an increase in the permeability of the plasma membrane. This results in an influx of sodium and calcium ions and an efflux of potassium ions. A loss of soluble proteins from the cell may finally occur. Although it is not possible to decide whether to attribute the change in permeability to a primary attack of the toxicant on the membrane directly, a loss of selective permeability by the plasma membrane is unquestionably one of the characteristics of a dying cell.

Nuclei are the site of DNA and RNA synthesis. Toxicants can interfere with the synthesis of both DNA and RNA, generally by altering the activity of the individual enzymes involved. Mitochondria The mitochondria of the cell are the sites of oxidative phosphorylation and, therefore, are primarily responsible for adenosinetriphosphate (ATP) synthesis. A number of toxicants, some ancient and notorious, attack various parts of the mitochondrial oxidation system, producing low ATP levels and disturbance of the redox state of the cell. Lysosomes Lysosomes are intracellular vesicles of hydrolytic enzymes, including nucleases, phosphatases, and peptidases. The vesicle normally sequesters these potent hydrolases from the cytoplasm. A disruption of the lysosomal membranes by interaction with toxicant releases the hydrolytic enzymes to attack adjacent cell material.

Plasma Membrane

2.6.3 Cellular Targets The susceptibility of a cell to a toxin primarily depends on at least the following three factors: 1.

Endoplasmic Reticulum The endoplasmic reticulum (ER) is the fine filigree of intracellular membrane sheets that, upon cell disruption, yield the microsomes. The ER is divided into two basic units, rough and smooth, the former masked by the attachment of ribosomes. The ER also contains electron transfer enzymes responsible for oxidation of various lipophilic compounds, including the steroids, long-chain fatty acids, and exogenous compounds. Perturbation of the ER upon exposure to and interaction with a toxicant may result in a disturbance of the membrane and constituent enzyme activities, or a proliferation of the ER structure and/or specific enzymes attached to it. The ability of the ER to metabolize foreign

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2. 3.

The specialization of the cell, i.e., which susceptible organelles are preeminent in the cell’s economy The distribution of the toxin within the body The cell’s reaction to the presence of the toxin

An example of the first type, i.e., selective toxicity based on cell specialization, is the sensitivity of the cells of the myocardium to anoxia. These cells depend primarily on ATP generated by mitochondrial oxidative processes and, hence, are critically aerobic. An interruption of the blood supply (ischemia) quickly produces cell death (an infarction). Another example of selective toxicity based on specialization is the destruction of rapidly dividing intestinal crypt cells by DNA synthesis inhibitors, such as the nitrogen mustards.

The distribution of a toxin in the body and its route of entry and elimination are also important factors. Since a toxin rarely has homogeneous distribution, it is inevitable that some cell populations suffer high exposure. For example, the red blood cells, because of their thorough exposure to dissolved gases in the lungs and their high level of hemoglobin, are primary targets of carbon monoxide poisoning. The liver, in contrast, is infused with the blood directly from the stomach and small intestine. The nutrients, as well as all ingested toxins, therefore, impact initially on this organ. Finally, what the cell can or cannot do with the toxin also determines its relative sensitivity to exogenous toxicants. The liver has a large complement of enzymes in the microsomes that can metabolize a wide range of exogenous chemicals. In many cases, the liver is successful in eliminating or decreasing toxicity, but in some instances, it may create a more toxic metabolite to its own detriment. Other cells, with less active microsomal enzymes, either are less effective in dealing with a toxicant or are more resistant to it, depending on whether metabolism deactivates or activates that compound. It should also be emphasized here that the cell is remarkably resistant and can survive temporary disturbances

Figure 2.6

in its environment. Similarly, during the course of evolution, it has also developed several defensive mechanisms to counterattack the effects of exogenous chemicals in biological systems. Some of these mechanisms are described in Chapter 5. They are the emergency measures that operate with varying degrees of success and efficiency.

2.7

BIOCHEMICAL EFFECTS RESULTING IN TOXIC INJURY

Because the cell’s components are reactive chemicals, toxicants can react with its components and interfere in its operation. Exogenous substances that interfere in the cell’s activity are called intrinsic toxicants. Those native or familiar to the cell and toxic only when present in excess are called relative toxicants (Concon, 1988). Many of the compounds of the latter group are in fact essential to the normal operation of the cell; others are metabolic byproducts. As shown in Figure 2.6, a toxicant may be detoxified by metabolic processes and eliminated from the body, made more toxic (toxified) by metabolic processes and distributed to receptors, or passed on to receptors as a met-

Major steps involved in the overall process leading to toxic effects of chemicals.

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abolically unmodified toxicant. In every biological system, there are thus safeguards against the intrusion of unwanted chemicals. However, these safeguards are imperfect. When they fail, toxic effects result. As discussed in the preceding section, toxic effects eventually indicated by gross functional disturbances must necessarily initiate injury at the molecular level. Although the biochemical mechanisms of toxicity of a large number of compounds are still unknown, the biochemical bases of their toxicities may include one or more of the following general types. 2.7.1 Deficiency of Essential Compounds Nutrients that cannot be synthesized by higher living forms necessarily must be supplied from external sources. The urgency of delivering these compounds to the cell depends on their rate of usage and degradation, the extent of cell storage, and the magnitude of their involvement in energy production, the cell’s primary need. Nutrient deficiencies in cell can occur either through the absence of nutrients in foods, a primary deficiency, or failure of or interference in their delivery or metabolism. Such an internal obstruction is termed a secondary deficiency. One of the following mechanisms may be responsible for such failure. Inhibition of Digestive Enzymes or Other Digestive Factors Toxicants can inhibit a variety of enzymes involved in the digestive processes. Well-known examples include inhibitors of the proteolytic and amylolytic enzymes present in several legumes. Absence of Digestive Enzymes or Other Digestive Factors Lactose intolerance in certain segments of populations is well known because of the inherent genetic deficiency of β-galactosidase enzyme. Similarly, certain oligosaccharides present in legumes cannot be metabolized because of the absence of corresponding digestive enzymes. Interference in the Absorption of Essential Compounds Interference in the absorption of essential compounds may arise as a result of the following factors: 1.

2.

Chemical or physical combination of one compound with another, resulting in the formation of a nonabsorbable complex: chelation of dietary essential minerals by phytate falls in this category. Absence of the compound necessary for the absorption of the compound: for example, vitamin

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3.

4.

5.

6.

B12 is not effectively absorbed in the absence of the intrinsic factor. Interaction or modification of the GI mucosa: An example is the group of compounds known as lectins, which bind on the absorptive surfaces of the intestinal mucosa. Inhibition of enzymes involved in absorption: for example, the antibiotic actinomycin D inhibits RNA and protein synthesis, including presumably enzymes necessary for the absorption of amino acids (Yamada et al., 1967; Concon, 1988). Solubilization of essential compounds in solvents that are nonabsorbable. For example, mineral oil, which may dissolve lipid-soluble vitamins, may prevent their absorption. Increased motility of the GI tract: many factors can affect this motility. Certain factors in foodstuffs may cause diarrhea and similar rapid evacuation of intestinal contents, resulting in poor absorption of essential compounds.

Interference of the Transport of Essential Compounds to the Cells Nitrite ions, for example, interfere in the transport of oxygen to hemoglobin. Degradation of Essential Compounds Nutrients may be destroyed even before they are absorbed. For example, there are factors, such as thiaminases, that destroy thiamine; retinol and carotenoids may be destroyed by oxidizing agents; and ascorbic acid, by ascorbic acid oxidase. Inactivation of Essential Compounds Certain substances may react with some of the essential nutrients without causing degradation but rendering them biologically inactive. Cyanide, for example, may interact with cobalamin, vitamin B12, to form cyanocobalamin, which is biologically inactive. It is believed that chronic cyanide intoxication may be the cause of tropical amblyopia as a result of cyanide inactivation of cobalamin. Interference of the Uptake of Essential Compounds in the Cells or Tissues Thiocyanate inhibits the iodine uptake of thyroid cells, whereas glucose and amino acid uptake of muscle cells does not occur in the absence of insulin (Concon, 1988; Langer and Stolc, 1964).

Antagonism Between Essential Compounds Antagonism exists among leucine, isoleucine, and valine when one of these is present in relatively large excess over another (Harper et al., 1970); β-carotene and cholecalciferol are also antagonistic (Weits, 1964). 2.7.2 Inhibition of Metabolic and Other Nondigestive Enzymes Several toxicants inhibit the activity of metabolic and other nondigestive enzymes. Carbamates and organophosphate pesticides, for example, are potent inhibitors of acetylcholine esterase, an enzyme necessary for neurotransmission (Aldridge and Reiner, 1969; Concon, 1988, O’Brien, 1969a, 1969b). The inhibition of monoamine oxidase by antidepressant drugs has been shown to increase the biological activity of pressor amines found in several foods (Vettorazzi, 1974). 2.7.3 Interference with Neurotransmission Several aspects of the neurotransmission process are susceptible to the action of various toxicants. The propagation of the nervous impulse is rapid and is obviously an energyrequiring process. The complexity and rapidity of neurotransmission may also lay bases for its vulnerability. The interference of several toxicants with this process is usually quite serious and often fatal. For example, the deadly poisons tetrodotoxins, from the puffer fish, and saxitoxin, from shellfish and clams, derive their lethality from their capacity to block specifically the sodium gates of the axon (Kao, 1972; Narahashi, 1972). In contrast, DDT acts in an opposite way, by keeping the sodium channel open but partially blocking the potassium channel (Narahashi and Haas, 1967). The synaptic mechanism by which the nerve impulse propagates appears to be the specific target of many potent toxicants. The release of the neurotransmitter acetylcholine can be blocked by botulinum toxin (Brooks, 1956). Acetylcholine esterase, involved in the regeneration of acetylcholine after the transmittal of the nervous impulse, is also a target of many poisons, such as organophosphates (O’Brien, 1969a, 1969b) and the cholinesterase inhibitors in the potato, eggplant, tomato, and sugar beet (Orgell, 1963). Many exogenous substances can also mimic the effects of neurotransmitters. Muscarine, the toxic alkaloid from the mushroom Amanita muscaria, behaves in the same way as acetylcholine (Bradley et al., 1966). Its extreme toxicity is attributed to its resistance to degradation in the tissues.

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Toxic reactions directly involving the nervous system are generally more severe, with an almost immediate appearance of symptoms. Furthermore, other toxicity mechanisms, such as interference in the transport of nutrients, protein synthesis, energy metabolism, and respiration, indirectly affect the nervous system. 2.7.4 Phototoxic Reactions Certain exogenous and endogenous compounds in the skin cells, when sufficiently illuminated, may become highly reactive with cellular components. This process by which light damages tissues, in the presence of a photosensitive substance, is called phototoxic reaction and is also known as photosensitization or photodynamic action. Ippen (1969) classified two types of phototoxic reactions: photoautoreaction and photoheteroreaction. In the first type, the photosensitive substance merely induces the normal photochemical reaction of the cell as in sunburn formation. In other words, in the presence of the phototoxic compound, skin may become more readily susceptible to the deleterious effects of sunlight. Most phototoxic reactions, such as those caused by furocoumarins, are of this type. In the second type, a toxic product is formed from the photochemical reaction of the photoactive substance. This toxic derivative may be formed independently of the tissues. Chlorpromazine and sulfanilamide are examples of compounds that are involved in phototoxic reactions of the second type. Photoallergic reaction is a form of photoheteroreaction. In this case, the photochemical reaction produces an allergen. The classes of phototoxic compounds include both natural and synthetic compounds. The natural compounds include the hypericins (Blum, 1964), the furocoumarins or psoralens (Pathak, 1969), porphyrins (Clare, 1956; Rimington et al., 1967), steroids, essential oils (Spikes, 1968), riboflavin (Spikes and Glad, 1964), and flavin mononucleotide (FMN) (Frisell et al., 1959). The synthetic phototoxic compounds include many drugs prescribed routinely, such as anesthetics, antibiotics, antihistamines, diuretics, barbiturates, sulfonamides, phenothiazines, dyes and other coal tar and petroleum products, and perfumes and colognes (Spikes, 1968). 2.7.5 Interference with Genetic Material and Function Interactions of toxicants with DNA and RNA, the building blocks of a cell’s genetic material, not only affect cellular reactions such as protein synthesis, but may also lead to mutagenesis and carcinogenesis. These processes are described in greater detail in Chapter 4. Mutagenic effects in-

volving the germinal cells have the potential for hereditary transmission. Therefore, the damage of these effects can extend beyond one generation and across pedigrees and population groups.

2.8

DISTRIBUTION OF TOXICANTS

Subsequently to absorption, the toxicant is capable of distribution (translocation) throughout the biological system. The transport processes discussed earlier are the major factors operative in the distribution of the toxicant from cell to cell and organ to organ and for movement into total body water. Body fluids are distributed among three primary components: plasma water, interstitial water, and intracellular water. Vascular fluid has the important role in the distribution of absorbed toxicants. Human plasma accounts for approximately 4% of body weight but 53% of total blood volume, whereas the interstitial tissue fluids account for 13% of body weight and intracellular fluids for 41% (Guthrie and Hodgson, 1987b; Klaassen, 1986b). Although only a small amount of toxicant in the body may be in contact with the receptor or target site, it is the distribution of the bulk of the toxicant that governs the concentration and disposition of that critical proportion. If the toxicant is distributed only in plasma, a high concentration is achieved in the vascular tissue. On the contrary, if the same quantity of toxicant is also distributed in the interstitial and intracellular water, concentrations are much lower in the vascular system. The rate of distribution of the toxicant to the tissues of each organ is thus primarily determined by the blood flow through the organ and the ease with which the chemical crosses the capillary bed and penetrates the cells of the particular tissue. Its eventual disposition is largely dependent on the ability of the toxic chemical to pass through the cell membranes and its affinity for the various tissues. The following are some of the factors that affect the distribution of the toxicant in biological systems. 2.8.1 Binding to Plasma Proteins Binding by the plasma proteins has an important bearing upon the distribution of toxicants. Serum albumin is the most important protein in this regard. Because many toxicants are very lipophilic, the plasma lipoproteins also play an important role in toxicant binding. The binding is noncovalent, involving hydrogen, Van der Waals, and ionic bonds, and the proportion of the toxicant bound depends on various physicochemical factors.

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The nonbound (free) portion of the toxicant in the plasma is in equilibrium with the bound portion, but only the former passes through capillary membranes. Therefore, excessively protein-bound compounds (>90%) are restricted in terms of equilibrium (distribution) within the organism. Under steady-state conditions, the concentration in the extravascular water equilibrates with the free concentration in the plasma. Plasma protein binding sites may be saturated, or one bound compound may be displaced by another. Thus, a dose threshold for toxicity is often seen as a result of saturation of plasma protein binding sites, which results in a dramatic increase in the plasma concentration of the free compound. Such competitive binding for the same sites on a protein can have an important toxicological significance. This is especially true for highly toxic compounds possessing a very high affinity for protein binding sites. Although extensive plasma protein binding affects passive diffusion, which is concentration dependent, it has little effect on active transport processes such as secretion at the kidney. Plasma protein binding of toxicants therefore influences the distribution and the half-life of the toxicant in the body and is responsible for toxic dose thresholds. A number of methods are used to study toxicant(ligand)-protein interactions, including ultrafiltration, electrophoresis, equilibrium dialysis, solvent extraction, solvent partition, ultracentrifugation, spectrophotometry, and gel filtration or equilibrium. Such methods yield data that are often expressed in terms of the percentage of ligand bound. However, it must be noted that as ligand concentration is lowered, percentage of bound ligand increases. Thus, if a protein has a high affinity for a ligand, as often occurs with albumin, the percentage bound falls sharply when the total ligand concentration exceeds a critical value. 2.8.2 Plasma Level The plasma level of a toxicant is an important parameter in distribution as it relates more readily to the effect than the dose itself. In general, the plasma concentration more nearly reflects the concentration at the site of action, although the relationship may not be a simple one if the toxicant is sequestered in a particular tissue or organ. 2.8.3 Tissue Localization The passage of exogenous chemicals into cells and across membranes, as discussed earlier, is generally restricted to the nonionized, lipid-soluble form of the chemical. Thus, compounds that meet these criteria pass out of the blood,

diffuse through tissues, and are distributed through the body. Lipid-soluble compounds may dissolve in tissues with a high fat content and may remain sequestered there for some time. Some compounds may accumulate in a specific tissue because of their affinity for a particular macromolecule. Such is the case with the binding of carbon monoxide with hemoglobin. These sites may be the target sites for toxicity. Accumulation may also occur in tissues other than the site of action. Such storage depots for toxicants in the biological systems are described later in this chapter. Toxic compounds thus may be distributed throughout all the tissues of the body, or they may be restricted to certain tissues. Two areas for special consideration are the brain and the fetus. The blood-brain barrier does not completely prevent the passage of toxicants into the central nervous system (CNS) but rather represents a site that is less permeable than most other areas of the body. Therefore, many poisons do not enter the brain in appreciable quantities. The following are the major anatomical and physiological reasons why some toxicants have reduced capacity for entering the CNS (Klaassen, 1986b): 1.

2. 3.

The capillary endothelial cells of the CNS are tightly joined, leaving few or no pores between the cells. The capillaries of the CNS are largely surrounded by glial cell processes (astrocytes). The protein concentration in the interstitial fluid of the CNS is much lower than elsewhere in the body.

Thus, in contrast to other tissues, the toxicant has difficulty moving between capillaries and has to traverse not only the capillary endothelium itself, but also the membranes of the glial cells in order to gain access to the interstitial fluid. Furthermore, the low protein concentration of the interstitial fluid of the CNS also decreases the distribution of chemicals to the CNS. These features together act as a protective mechanism to decrease the distribution of toxicants to the CNS and thus the toxicity. In contrast, passage of compounds across the placenta occurs generally by passive diffusion. Lipid-soluble compounds are thus readily transported. However, if metabolism in utero converts the compound into a more polar metabolite, accumulation may occur in the fetus. Exogenous compounds generally achieve the same concentration in fetal plasma as in the maternal plasma water. In addition to chemicals, viruses (e.g., rubella, human immunodeficiency virus [HIV]), cellular pathogens (e.g., syphilis

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spirochete), antibody globulins, and even erythrocytes traverse the placenta (Goldstein et al., 1974). 2.8.4 Volume of Distribution As mentioned earlier, body fluids are distributed among plasma and interstitial and intracellular water. The distribution of the toxicant into each of these three fluids profoundly affects the plasma concentration. If a toxicant is distributed only in the plasma water (approximately 3 liters in the average human), the plasma concentration is obviously much higher than if it is distributed in all extracellular fluid (approximately 14 liters) or the total body water (approximately 40 liters). The volume of distribution (VD) may be calculated from VD = dose (mg)/plasma concentration (mg/L) and is expressed in liters. A more rigorous determination of the volume of distribution utilizes the area under the plasma concentration/time curve (area under the curve [AUC]) (Figure 2.7) for the calculation: VD = dose/(k × area) where k is the elimination rate constant or VD = dose/C0 where C0 is the plasma concentration at time zero gained by extrapolation of the log plasma concentration versus time plot (Figure 2.8). Ideally, the compound should be administered intravenously, unless the degree of absorption is known. The parameter VD yields useful information. For instance, a very high apparent VD may indicate sequestration in a particular tissue or localization in fat. Similarly, the total amount of a toxicant in the body, i.e., the total body

Figure 2.7 Plasma level profile for a foreign chemical. AUC, area under the curve. (From Timbrell [1982]).

Figure 2.8 A semilog plot of the plasma level of a foreign chemical against time. C0, plasma concentration at time zero. (From Timbrell [1982].)

burden, may be estimated from a knowledge of the plasma concentration and VD:

A simple linear relationship is seen if the distribution of the compound fits a single compartment model, i.e., the toxicant being distributed in plasma water alone. If the toxicant first undergoes distribution and the plasma concentration declines more slowly, governed by the process of elimination and metabolism, then a two-phase decay is seen. For detailed mathematical treatment of the topic, the readers are referred to several excellent reviews (Goldstein et al., 1974; Tuey, 1980; Klaassen, 1986b; Gibaldi and Perrier, 1982). The half-life of a toxicant reflects the various processes taking place in vivo after the administration of a compound. Thus, following the initial absorptive phase, the toxicant is distributed, metabolized, and excreted, and these processes, acting in conjunction, determine the rate of removal of the toxicant from the plasma. Changes in the half-life of the toxicant may therefore yield valuable information about changes in these processes. For example, the half-life indicates the ability of the body to metabolize and excrete the compound. When this ability is impaired, either through saturation of enzymatic or active transport processes or if the liver or kidneys are damaged, the halflife may well be prolonged. An indication of the ability of the body to metabolize and eliminate the compound may be gained from the total body clearance. This may be calculated from the parameters described earlier.

Total body burden (mg) = plasma concentration (mg/L) × VD (L) Only the free rather than the total amount in the plasma should be used for the calculation of VD, as only the former is available for distribution. 2.8.5 Plasma Half-Life The plasma half-life is also an important parameter. It can be calculated from measurements of the plasma level at the various time points. The half-life is the time required for the plasma concentration of the toxicant to decrease by half from a given point. Measurement of the plasma level of a toxicant at various times after dosing gives a curve that decays exponentially as shown in Figure 2.7. Plotting the data semilogarithmically (Figure 2.8) gives a linear relationship from which the half-life can be readily calculated as follows: log C = log C0 – (kt/2.303) Slope = –k/2.303 Half-life (t1/2) = 0.693/k where C = plasma concentration, C0 = plasma concentration at time zero, t = time after dosing, and k = elimination rate constant.

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Total body clearance = VD × k or alternatively, Total body clearance = dose/AUC where the dose is administered intravenously. The plasma level and half-life are also important parameters when the exposure to a toxicant is chronic. Thus, if the exposure is shorter than the half-life, the toxicant accumulates in the body, whereas if the half-life is very short compared to the exposure, the toxicant does not accumulate in the body. It is therefore important to measure the plasma concentration of the toxicant for an assessment of chronic toxicity.

2.9

METABOLISM/BIOTRANSFORMATION OF TOXICANTS

Metabolism is important in a number of body processes, one of which is the detoxification of foreign or exogenous compounds. The biotransformation of a foreign, toxic compound is thus an important aspect of its disposition in vivo. A metabolically unmodified toxicant is often referred to as an active parent compound, and a substance modified by metabolic processes as an active metabolite.

Both types of species may be involved in the manifestation of toxic responses. Almost any reactive chemical that is administered to or ingested by the organism is almost immediately subjected to mechanisms that may confine its translocation within the organism or terminate its existence as a free chemical (Figure 2.9). Upon absorption, a toxicant thus begins changing location, concentration, or chemical density. It may be transported independently by several components of the circulatory system, be absorbed by various tissues or stored, effect an action, be detoxified, or be activated; the parent compound or its metabolite(s) may react with body constituents, be stored, or be eliminated. The study of kinetics, known as pharmacokinetics or toxicokinetics, involved in these processes is a highly specialized branch of toxicology. During the metabolic phase, an active parent compound can be present in blood, liver, or extrahepatic tissues (nonliver tissue), and in the latter two, it may be converted to an inactive metabolite or metabolites. An inactive parent metabolite may produce a toxic metabolite or metabolites in the liver or in extrahepatic tissue; in both these locations, a toxic metabolite may be changed to an inactive form. Therefore, metabolism of a toxicant involves a number of pathways by which the compound is converted to a toxicant or to a substance that is eliminated from the biological system. One of the main results of such metabolic transformation thus is the facilitation of the removal from the body

Figure 2.9 Schematic representation of the pathways through which a toxicant or exogenous chemical may pass during its presence in humans.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

of toxic compounds, that, unless excreted, would accumulate to toxic levels. The types of biotransformations are many and varied, and the metabolic systems involved are necessarily very flexible and nonspecific. These are described in detail in Chapter 5. The major factor determining the route(s) of biotransformation is the structure of the compound itself. The elimination of the toxicant from the body is the end point for biotransformation. Kidneys are the main excretory organ of the body for foreign chemicals, and they are most efficient at eliminating polar molecules. Thus, metabolic processes that are detoxifying in nature generally involve reactions that convert nonpolar molecules into more polar ones. In most cases, these changes are advantageous to the body, but in some instances, the process converts basically nontoxic nonpolar compounds into more toxic polar (or more polar) metabolites. The nonpolar lipid-soluble compounds are generally reabsorbed from the kidney tubules or simply equilibrate between plasma and bile by passive diffusion, to no effect. Thus, metabolism may not necessarily be a detoxification process. Its primary purpose is often to facilitate elimination of the compound and alter its biological activity. In some cases, the effect of metabolism might just be to alter elimination from the urinary to the biliary route, for example, by increasing the molecular weight. Metabolism or biotransformation is therefore an important determinant of the activity of a compound, the duration of this activity, and the half-life of the compound in the body. Some very lipid-soluble compounds, such as chlorinated hydrocarbons, polychlorinated and polybrominated biphenyls, and aflatoxins, which are poorly absorbed, may have whole-body half-lives measured in months or even years. The chemical alterations that are the basis of biotransformation of foreign compounds are catalyzed by a number of enzymes, depending on the chemical structure of the compound in question. The most important is the cytochrome P-450 monooxygenase system, but numerous other enzymes may be utilized, both those involved in the intermediary metabolism and those whose main function is the metabolism of xenobiotics. Specific enzymes that recognize particular types of molecules are normally present in small quantities, and the body produces more of them when the need arises, e.g., after a significant exposure to the appropriate foreign molecule. Unfortunately, this process is not easily reversible, and the body does not revert to its preexposure status very rapidly. Thus exposure to one chemical of a particular type may lead to the presence of a large number of specific enzymes when a subsequent exposure to the same or a similar chemical occurs. This process of increasing

the enzyme levels is called enzyme induction. In general, this is beneficial since it helps the body to respond rapidly to repeated exposures. Of course, if the metabolic processes resulting from the response lead to more toxic rather than less toxic metabolites, this induction is counterproductive. Although the major organ involved in the biotransformation of exogenous compounds is the liver, other tissues and organs may be involved to a greater or lesser extent. The importance of liver in this respect relates to its position as a portal to the tissues of the body. By metabolizing and hence removing toxic substances ingested orally and absorbed via the hepatic-portal circulation, the liver protects the organism. In some cases, this metabolic conversion during the absorption phase is almost complete, resulting in a first-pass effect. The gut wall may also carry out biotransformation and, hence, be responsible for a first-pass effect, as may the lung for compounds absorbed by inhalation.

2.10 STORAGE OF TOXICANTS Toxicants are often concentrated in specific tissues in the body. Whereas some achieve the highest concentration at their site of action (e.g., carbon monoxide in hemoglobin), others are concentrated at sites other than the site of toxic action. For example, lead is stored in bone, whereas the symptoms of lead poisoning are due to lead in the soft tissues. The compartment where the toxicant is concentrated but not involved in the toxicological response can be thought of as a storage depot. These can be considered as protective mechanisms, preventing the accumulation of high concentrations of toxicants at the site of toxic action. These toxicants in these depots are always in equilibrium with free toxicant in plasma, and as they are biotransformed or excreted from the body, more is released from the storage site (Klaassen 1986b). The biological half-life of stored toxicants thus can be very long. The following are the major sites of storage for toxicants. 2.10.1

Plasma Proteins

Several proteins in the plasma have the ability to bind to exogenous chemicals as well as some normal physiological constituents (Table 2.4). The majority of foreign chemicals that are bound to plasma proteins are bound to serum albumin. It is the most abundant protein in plasma and serves as a depot protein and transport protein for several endogenous and exogenous compounds. Transferrin, a β1-globulin, is important for transport of iron in the body.

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Table 2.4 Examples of Plasma Proteins That Serve as Storage Depots for Physiological Constituents and Toxicants Protein

Physiological constituent/toxicant

Albumin

Transferrin Ceruloplasmin α- and β-Lipoproteins

Immunoglobulins (γ-globulins) α1-Acid glycoprotein

Calcium, copper and zinc ions, bilirubin, uric acid, vitamin C, adenosine, tetracyclines, chloramphenicol, digitonin, fatty acids, suramin, quinocrine, penicillin, salicylate, sulfonamides, streptomycin, acid dyes, phenol red, histamine, triiodothyronine, thyroxine, barbiturates Iron Copper Lipid-soluble vitamins, cholesterol, steroid hormones, vitamin B12, sialic acid, thyroxine Specific for individual antigens Basic compounds

Ceruloplasmin, which carries most of the copper in the serum, is the other metal-binding protein in plasma. The α- and β-lipoproteins are very important in the transport of lipid-soluble compounds, such as vitamins, cholesterol, and steroid hormones. The γ-globulins are antibodies that interact specifically with antigens. Compounds possessing basic characteristics often bind to α1-acid glycoprotein (Klaassen, 1986b; Wilkinson, 1983). Several relevant aspects of binding of toxicants to plasma proteins were described in conjunction with the distribution of toxicants in a preceding section in this chapter. 2.10.2

Liver and Kidney

Liver and kidney have a high capacity to bind chemicals. These two organs also concentrate more toxicants than other organs, primarily because both are important to the elimination of toxicants from the body. 2.10.3

Intracellular Binding Proteins

Within the liver and kidney, several intracellular proteins are important in concentrating the toxicants. Examples include Y protein or ligandin, which has a high affinity for many organic acids; azo dye carcinogens and corticosteroids, in the cytoplasm of the liver; and the cadmiumbinding protein metallothionein, found in the kidney and liver (Levi et al., 1969; Litwalk et al., 1971; Klaassen and Shoeman, 1974).

2.10.4

Fat

Since most toxicants are lipophilic in nature, they readily distribute and concentrate in body fat. Toxicants appear to accumulate in fat by simple physical dissolution in the neutral fats. Thus, a toxicant with a high lipid/water partition coefficient may be stored in the body fat to a large extent. Although such storage lowers the concentration of the toxicant in the target organ and thus may serve as a protective mechanism, intoxication can result from short-term starvation (Klaassen, 1986b). Examples of well-known toxicants stored in this way include pesticides such as chlordane and DDT and the polyhalogenated aromatics, such as polychlorinated and polybrominated biphenyls. 2.10.5

Bone

Bone can also serve as a reservoir for such compounds as fluoride, lead, and strontium. It is also a major storage site for some toxicants. Toxicants deposited in bone are not irreversibly sequestered and thus can be released by ionic exchange at the crystal surface and by dissolution of bone crystals through osteoclastic activity (Klaassen, 1986b). Compounds that accumulate in the body as a result of repeated frequent ingestion as contaminants of food have occasionally resulted in insidious harm to sizable populations of humans. Methylmercury and lead poisoning instances due to either misuse as food or industrial discharge of the compound are well documented in the literature.

2.11 EXCRETION OF TOXICANTS One important factor in the toxicity of foreign compounds is excretability. The more rapidly they are eliminated, the less likely they are to exert an adverse effect. If, on the other hand, their retention in the body is prolonged, the potential for toxic effects is greater. Although there are a wide variety of synthetic organic chemicals of recent origin, relative to the evolutionary time scale, they all can be eliminated from the body without special physiological systems. Most exogenous chemicals, however, are not readily eliminated until they are in a form similar to that utilized for the elimination of endogenous substances (Guthrie and Hodgson, 1987b). In general, a similar trend of metabolic reactions that yield metabolites of lower toxicity as a result of improved excretability (Figure 2.10) is seen. This trend is not without exceptions. For instance, the completely nonpolar molecules, such as methane and ethane, often are inert biologi-

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Figure 2.10 A generalized trend of biotransformation reactions that yield metabolites of lesser toxicity as a result of improved excretability.

cally and require no detoxification. However, if a nonpolar molecule is biologically active (e.g., benzene, carbon tetrachloride), it is not likely to be readily detoxified and is therefore very toxic. There are a number of routes of excretion, the major route is the kidneys for most nongaseous or nonvolatile compounds. In fact, more chemicals are eliminated from the body by this route than any other. The liver and the biliary system are also important. In contrast, routes of elimination such as milk, lungs, alimentary excretion, sweat, tears, hair, and semen are generally of minor importance. The major routes of excretion of foreign compounds are now briefly described. 2.11.1

Renal/Urinary Excretion

The kidneys are primarily excretory organs. In addition to removing most by-products of normal metabolism, kidneys are also the primary organ for excretion of polar xenobiotics and hydrophilic metabolites of any lipophilic toxicants that the body has encountered. The toxicants are transported to the kidneys either by solubilization in blood or by binding to plasma proteins. Toxic substances and other foreign compounds are removed from the blood as it passes through the kidneys; the blood flow represents about 25% of the cardiac output. The physicochemical principles governing the excretory processes are essentially as previously described for ab-

sorption. Three processes are primarily responsible for the elimination of the toxic substances from the body: passive glomerular filtration, passive tubular diffusion, and active tubular secretion. Glomerular Filtration The blood (plasma) is first passively filtered at the glomerulus and the filtrate passed into the tubules, where reabsorption may take place for lipid-soluble, nonionized compounds. Filtration at the glomerulus normally occurs for most compounds of molecular weight less than 70,000, as the pores in the membrane are relatively large (70–100 Å). Only the non-protein–bound forms of compounds are filtered. The concentration of the compound in the glomerular filtrate therefore approximates that in the plasma in the unbound form. Compounds too large to pass, those bound to proteins, and nonionized and very lipid-soluble compounds, are reabsorbed into the bloodstream by passive diffusion. These are subsequently eliminated by other avenues. Passive Tubular Diffusion Toxicants can also be excreted from the plasma into urine by passive diffusion through the tubule. The pH of the urine is an important determinant factor in this regard. Therefore, bases are more readily excreted if the urine is acid, and vice versa for the excretion of acids. Generally, weak acids are frequently biotransformed to stronger acids, thereby increasing the percentage in the ionic form. Active Tubular Secretion Active tubular secretion is an important mechanism of elimination for ionized compounds. It is not affected by plasma protein binding of the compound. This is because the mechanism is very rapid and not concentration dependent. Therefore, the dissociation of the protein-bound compound continuously provides more compound for active transport. Organic acids and bases appear to be transported by different secretory processes, which are located in the proximal convoluted tubules. Because it is energy dependent, this process can be inhibited by metabolic inhibitors or competitively by other organic acids or bases. Factors affecting kidney function, such as age and disease, may have a marked effect on the toxicity of compounds excreted into the urine. 2.11.2

generally involves active secretion rather than passive diffusion, and there appear to be specific transport systems for organic acids, organic bases, and neutral compounds. Quaternary ammonium compounds may be actively secreted into the bile by a separate process. This route of excretion seems to apply particularly to comparatively large, ionized polar molecules (Timbrell, 1982; Klaassen, 1986b). The liver is indeed in a very advantageous position for removing toxicants from blood after they are absorbed from the GI tract. The blood from the GI tract must first pass through the liver before reaching the general systemic circulation. Thus, liver can remove compounds from blood and prevent their distribution to other parts of the body. The liver is also the main site of biotransformation of toxicants. Thus, compounds can be metabolized and conjugated rapidly in the first pass through the liver and then directly excreted into the bile. Such biliary excretion obviates the need for metabolites to enter the bloodstream for excretion by the kidneys. A toxicant may be excreted by liver cells into bile and thus pass into small intestine and remain there. The excretion of compounds via the bile into the intestine may lead to the reabsorption of the compound if intestinal conditions permit. The most common mechanism for reabsorption is hydrolysis or metabolism of conjugates of the compound by intestinal microflora. The hydrolyzed conjugate, which is less polar, can be absorbed by the intestine and returned to the liver through the portal or enterohepatic circulation (Figure 2.11). This process may have toxicological consequences, especially if the metabolite is more toxic than the excreted conjugate, or if it prolongs the half-life of the compound. Enterohepatic

Hepatic/Biliary Excretion

Hepatic/biliary excretion is the second most significant route of elimination of toxicants from the body. This route

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Figure 2.11 Enterohepatic circulation. Circulation of the compound is indicated by arrows. (From Timbrell [1982].)

circulation can be recognized by examination of the profile of the plasma concentration of the toxicant. The plasma concentration does not show a smooth decline but rather increases after various intervals when reabsorption has taken place. High-molecular-weight conjugates are examples of compounds that undergo biliary excretion. The molecular weight of the compound, its charge, and the animal species influence biliary excretion of toxicants. A molecular weight >300 is generally a prerequisite for biliary excretion. Some compounds are almost exclusively eliminated from the body by biliary excretion, and consequently toxicity may be markedly increased if bile secretion is impaired. Inhibition of the detoxifying monooxygenase enzyme system, sex, and age also influence biliary excretion. The hepatic excretory system is also not fully developed in the newborn. This is why some compounds are more toxic in newborns than in adults (Klaassen, 1972, 1973). Alternatively, the development of the hepatic excretory mechanism can be promoted by administering microsomal enzyme inducers (Klaassen, 1974). 2.11.3

Lungs/Pulmonary Excretion

Gases and volatile compounds are usually eliminated from the body by the pulmonary route, as are the volatile metabolites of nonvolatile compounds. The rate of elimination depends on solubility in blood, rate of respiration, and blood flow to the lungs (Guthrie and Hodgson, 1987b). Pulmonary excretion takes place by simple diffusion, but generally it is very rapid. The best-known compounds subject to respiratory elimination are ethanol, anesthetic gases, pesticide fumigants, and many volatile organic solvents, such as ether. 2.11.4

Gastrointestinal Tract

Many toxic compounds are excreted in feces. Appearance in feces is generally due to the following factors (Klaassen, 1986b): 1. 2. 3. 4.

The chemical was not completely absorbed after oral ingestion. It was excreted into the bile. It was secreted in saliva, in gastric or intestinal secretory fluid, or in pancreatic secretion. It was secreted by the respiratory tract and then swallowed.

Toxicants can enter the lumen of the GI tract via passive diffusion. Intestinal excretion may be a major route of elimination of highly lipophilic compounds, such as organochlorine pesticides, 2,3,7,8-tetrachlorodibenzodioxin

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(TCDD), and the polychlorinated biphenyls (Rozman et al., 1982). The GI elimination of these compounds, each of which has a very long biological half-life, can be enhanced by increasing the lipid composition of the diet. 2.11.5

Excretion via Milk, Sweat, and Saliva

These are minor routes of excretion for some compounds. Of these, the secretion of toxic compounds into the milk is extremely important because (a) a toxic material may be passed in milk from mothers to the nursing child, and (b) compounds can be passed from cows to humans by this route (Klaassen, 1986b). Because milk is more acidic (pH ~ 6.5) than plasma, basic compounds may be concentrated in milk. Furthermore, because milk has high fat content (3%–5%), lipophilic compounds, such as DDT and polychlorinated and polybrominated biphenyls, are concentrated in it, thus forming a major route of their excretion. Toxic compounds excreted into sweat may produce dermatitis; those excreted in saliva are usually swallowed and are then available for GI absorption. Excretion via these three minor routes is dependent on diffusion of the nonionized lipid-soluble form of the toxicant.

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Ippen, H. 1969. Mechanisms of photopathological reactions. In Biological Effects of U.V. Radiation, ed. F. Urbach, pp. 136–169, Pergamon Press, Oxford, England. Kamrin, M.A. 1988. Toxicology. Lewis Publishers, Chelsea, MI. Kao, C.Y. 1972. Pharmacology of tetrodotoxin and saxitoxin. Fed. Proc. Fed. Am. Soc. Exp. Biol. 31:1117–1123. Klaassen, C.D. 1972. Immaturity of the newborn rat’s hepatic excretory function for ouabain. J. Pharmacol. Exp. Ther. 184:721–728. Klaassen, C.D. 1973. Comparison of the toxicity of chemicals in newborn rats to bile duct-ligated and sham-operated rats and mice. Toxicol. Appl. Pharmacol. 24:37–44. Klaassen, C.D. 1974. Stimulation of the development of the hepatic excretory mechanism for ouabain in newborn rats with microsomal enzyme inducers. J. Pharmacol. Exp. Ther. 191:212–218. Klaassen, C.D. 1986a. Principles of toxicology. In Toxicology: The Basic Science of Poisons, eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 11–32, Macmillan, New York. Klaassen, C.D. 1986b. Distribution, excretion and absorption of toxicants. In Toxicology: The Basic Science of Poisons, eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 33–63, Macmillan, New York. Klaassen, C.D. and Shoeman, D.W. 1974. Biliary excretion of lead in rats, rabbits and dogs. Toxicol. Appl. Pharmacol. 29:434–446. Klaassen, C.D., Amdur, M.O., and Doull, J. 1986. Toxicology. The Basic Science of Poisons, 3rd ed. Macmillan, New York. Langer, P. and Stolc, V. 1964. Relations between thiocyanate formation and goitrogenic effect of foods. V. Comparison of the effect of white cabbage and thiocyanate on the rat thyroid gland. Physiol. Chem. 335:216–220. Levi, A.J., Gatmaitan, Z., and Arias, I.M. 1969. Two hepatic cytoplasmic protein fractions, Y and Z, and their possible role in the hepatic uptake of bilirubin, sulfobromophthalein, and other anions. J. Clin. Invest. 48: 2156–2167. Levine, R.R. 1970. Factors affecting gastrointestinal absorption of drugs. Am. J. Dig. Dis. 15:171–188. Levine, R.R. and Walsh, C.T. 1975. Drug interactions in the gastrointestinal tract. In Functions of the Stomach and Intestines, ed. M.H.F. Friedman, pp. 139–151, University Park Press, Baltimore, MD. Litwack, G., Ketterer, B., and Arias, I.M. 1971. A hepatic protein which binds steroids, bilirubin, carcinogens and a number of exogenous organic anions. Nature (London) 234:466–467. Loomis, T.A. 1978. Essentials of Toxicology, 3rd ed. Lea & Febiger, Philadelphia. Magnussen, M.P. 1968. The effect of ethanol on the gastrointestinal absorption of drugs in the rat. Acta Pharmacol. Toxicol. 26:130–144. Manahan, S.E. 1992. Toxicological Chemistry, 2nd ed. Lewis Publishers, Boca Raton, FL.

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Schanker, L.S., Tocco, D.J., Brodie, B.B., and Hogben, C.A.M. 1958. Absorption of drugs from the rat small intestines. J. Pharmacol. Exp. Ther. 123:81–88. Shore, P.A., Brodie, B.B., and Hogben, C.A.M. 1957. The gastric secretion of drugs: A pH partition hypothesis. J. Pharmacol. Exp. Ther. 119:361–369. Spikes, J.D. 1968. Photodynamic action. In Photophysiology. Current Topics, Vol. III, ed. A.C. Giese, pp. 139–164, Academic Press, New York. Spikes, J.D. and Glad, B.W. 1964. Photodynamic action. Photochem. Photobiol. 3:471–487. Stine, K.E. and Brown, T.M. 1996. Principles of Toxicology. CRC Press, Boca Raton, FL. Tardiff, R.G. and Rodricks, J.V. 1987. Toxic Substances and Human Risk: Principles of Data Interpretation. Plenum Press, New York. Ther, L. and Winne, D. 1971. Drug absorption. Annu. Rev. Pharmacol. 11:57–70. Timbrell, J.A. 1982. Principles of Biochemical Toxicology. Taylor and Francis, London. Tuey, D.B. 1980. Toxicokinetics. In Introduction to Biochemical Toxicology, eds. E. Hodgson and F.E. Guthrie, , pp. 40–66, Elsevier, New York. Ulmer, D.D. 1977. Trace elements. N. Engl. J. Med. 297:318–319. Vettorozzi, G. 1974. 5-Hydroxytryptamine content of bananas and banana products. Food Cosmet. Toxicol. 12:107–113. Weits, J. 1964. The antagonism between vitamin A and vitamin D. Voeding 25:486–493. Wilkinson, G.R. 1983. Plasma and tissue binding considerations in drug disposition. Drug Metab. Rev. 14:427–465. Yamada, C., Clark, A.J., and Swendseid, M. 1967. Actinomycin D effect on amino acid absorption from rat jejunal loops. Science 158:129–130. Zawoiski, E.J., Baer, J.E., Braunschweig, L.W., Paulson, S.F., Shermer, A., and Beyer, K.H. 1958. Gastrointestinal secretion and absorption of 3-methyl-aminoisocamphane hydrochloride (mecamylamine). J. Pharmacol. Exp. Ther. 122:442–448.

3 Manifestations of Organ Toxicity

3.1

INTRODUCTION

The biochemical events following exposure to a toxic agent generally result in clinical manifestations or symptoms. These symptoms may be characteristic of the particular physiological process or organ system thus affected. Often multiple symptoms associated with the toxicological events indicate that more than one system is affected. Virtually all physiological processes and organ systems in the body are subject to toxic effects. Thus one must understand that all these symptoms are interrelated, so that toxic injury to the primary target may have repercussions in other systems. This is particularly true of damage to the central nervous and hematopoietic systems (Concon, 1988). Although, for the sake of convenience, the toxic effects are divided into acute, subacute, and chronic on the basis of time course, it is often not possible to categorize each type of toxicity or the effects of individual chemicals according to one of these three classifications. For example, lung toxicity may be an acute, subacute, or chronic effect. Similarly, a particular chemical may produce an acute effect at one exposure level and a subacute or chronic effect at another. In spite of these classification difficulties, it has been common practice to identify the different types of toxicity with one of the three time course categories. Thus organ damage has usually been classified as an acute or subacute effect, and most other types of toxicity have been assigned to the chronic classification. The former category includes neurotoxicity, hepatotoxicity, nephrotoxicity, hematotoxic-

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ity, skeletal toxicity, reproductive toxicity, and immunotoxicity. The toxic effects that are most relevant to food toxicants and are generally included in the chronic category are allergenicity, mutagenicity, teratogenicity, and carcinogenicity. This is because these effects, in many cases, may be induced by toxicants at the very low levels that are found in food. However, organ toxicity effects may be induced by small quantities of toxicants. Organ toxicity has generally been thought of as an acute or subacute effect for a number of reasons (Kamrin, 1988). One is that the effect often occurs soon after the initiation of exposure, especially at high doses. In addition, the degree of toxicity reflects the frequency and intensity of exposure in the traditional dose-response fashion. Another characteristic of organ toxicity is that the organ that is affected is directly linked to the type of chemical to which exposure has occurred. In this chapter, several important manifestations of organ toxicity are briefly described. The chronic manifestations of carcinogenesis, mutagenesis, and teratogenesis are described in Chapter 4.

3.2

NEUROTOXICITY

The brain and the central nervous system (CNS) are protected from toxicants by the blood-brain barrier. This barrier, which normally protects the brain from toxicants that can damage other soft tissues, however, does not exclude all toxic substances. Furthermore, several areas of the nervous system lack the barrier. These include the median

eminence with arcuate nucleus (Reese and Brightman, 1968), median preoptic region (Brightman and Reese, 1969), choroid plexus, and area postrema (Olsson and Hossman, 1970), all these belonging to the CNS, and the dorsal root ganglia (Brierly, 1955) and autonomic ganglia (Jacobs, 1977) of the peripheral nervous system (PNS). These areas are therefore more susceptible to some toxic compounds than other areas of the CNS and PNS, which have greater resistance. In addition to such differences in the morphological characteristics of the nervous system, even those toxic substances that can penetrate brain tissue do not affect equally all of the cell types in the brain. Different brain areas usually have different sensitivities to toxicants, reflecting unique biochemical properties of the cells as well as differences in degree of vascularization of brain areas (Norton, 1986). Toxic responses of the CNS may be manifested in two ways: structural toxicity and functional toxicity. 3.2.1 Structural Toxicity When CNS cells are damaged by exposure to toxic chemicals either by direct contact or by secondary effects such as anoxia subsequent to diminished oxygen supply, some similar effects are observed. These effects are the swelling of the cell and cytoplasmic organelles, dispersion of the rough endoplasmic reticulum, and swelling of the nucleolus (Norton, 1986, Stine and Brown, 1996). The changes are accompanied by decreases in the cytoplasmic pH, in activity of the oxidative enzyme systems, and in the synthesis of protein and other cell components. Certain cells are more sensitive to anoxia than others. The sequence of vulnerability to neurotoxicants can be described as neurons > oligodendrocytes > astrocytes > microglia > cells of the capillary endothelium. The lack of oxygen to brain and the CNS may result from one of the three types of anoxia: anoxic, ischemic, and cytotoxic. These conditions result from a failure in blood oxygenation, a decreased blood supply, or interference in oxygen transport, cellular metabolism, or respiration. Anoxic Anoxia Anoxic anoxia results from a primary lack of oxygen even in the presence of adequate blood flow. Such a primary condition can result from either direct interference with respiration by toxic substances or interference with the oxygen-carrying capacity of the blood. If respiration is restored before cardiovascular failure occurs, neurons in the CNS that are sensitive to anoxia may be destroyed without death of the organism. Examples include the production of

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carboxyhemoglobin by carbon monoxide and of methemoglobin by nitrites. Failure in blood oxygenation may also follow from respiratory paralysis by paralytic agents such as botulinum exotoxin and saxitoxin. Ischemic Anoxia Ischemic anoxia results from a decrease in arterial blood pressure to a level below that which supports the brain adequately with oxygen (Norton, 1986). The stagnation of the blood in the brain leads to an inadequate supply of needed substances and an accumulation of metabolic byproducts such as lactic acid, ammonia, and inorganic phosphate. Cardiac arrest caused by toxic substances is one obvious cause of inadequate blood flow. Severe hypotension that results from vasodilatation can also diminish the brain’s blood supply considerably, as in the effect of cyanide poisoning on the heart. The rupture of critical blood vessels in the brain, which results in hemorrhage or thrombosis, may also cause local ischemic anoxia of the brain. Cytotoxic Anoxia Cytotoxic anoxia is a consequence of interference with cell metabolism in the presence of an adequate supply of both blood and oxygen. In contrast to the great susceptibility of the neurons of the adult brain to ischemic and anoxic anoxia, it is the oligodendroglia that are more susceptible to this type of injury caused by metabolic inhibitors such as cyanide, methionine sulfoximine, azide, dinitrophenol, and malononitrile. Cytotoxic anoxia may also result from hypoglycemia, produced by an excess of insulin. Hypoglycin A from the akee fruit is an example of such a metabolic inhibitor. 3.2.2 Functional Toxicity An exposure to neurotoxicants may also result in functional toxicity of the CNS. Five types of central and peripheral nervous system function and the associated toxic manifestations are shown in Figure 3.1. For example, sensory loss is exemplified by blindness; deafness; and loss of the sensation to touch, temperature, pressure, and pain; and paresthesia. The loss of motor functions is associated with general paralysis (paresis) and muscle incoordination (ataxia). Functional neurotoxicity is also associated with signs suggesting loss of integrative functions, such as loss of memory and learning ability, seizures, convulsions, excitation, hyperkinetic behavior, depression, and coma. Many types of behavioral and emotional alterations and mental retardation are also manifestations that are definitely associated with neurotoxic effects (Wiener, 1970; Norton, 1986; Levi, 1987; Kamrin, 1988; Stine and

Figure 3.1

Central and peripheral nervous system functions and associated toxic manifestations.

Brown, 1996; Niesink et al., 1996). Examples of these effects are those associated with chronic lead poisoning. 3.2.3 Neurotoxicants Several classifications have been suggested for neurotoxicants (Scholz, 1953; Malamud, 1963; Windle, 1963; Brucher, 1967; Brierley et al., 1971). However, no one classification is ideal since a chemical may have more than one effect and the susceptibilities of the nervous system of different animal species can hardly be considered identical. Furthermore, any classification is subject to some error since it depends on data that are at best fragmentary. The following classification is based on the suggestions of Norton (1986), who classified the neurotoxicants according to their primary toxic action: 1.

2.

Agents causing anoxia: These chemicals cause anoxic damage to gray matter (neurons and astrocytes) with variation in pattern of damage depending on which of the three types of anoxia is produced. Examples include barbiturates, carbon monoxide, cyanide, azide, and nitrogen trichloride poisoning. Agents damaging myelin: These chemicals cause damage to myelin, affecting oligodendro-

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3.

4.

5.

cytes or Schwann cells, resulting in encephalopathy if central white matter is involved or polyneuritis if peripheral cells are damaged, e.g., isonicotinic acid hydrazide (INH, isoniazid), triethyltin, hexachlorophene, lead, thallium, and tellurium. Agents causing peripheral axonopathies: These are substances with a predilection for causing damage to axons of peripheral neurons. Examples of this type of neurotoxicants include alcohol, acrylamide, carbon disulfide, hexanedione, bromophenylacetylurea, and organophosphorus compounds. Agents causing primary damage to perikarya of peripheral neurons: Some neurotoxicants, such as organomercurials, vinca alkaloids (vincristine and vinblastine), and iminodipropionitrile, cause primary damage to the perikaryon, the main site of synthesis of protein of peripheral neurons. Neurotoxicants causing damage to synaptic junctions of the neuromuscular system: The synaptic clefts and the terminals of myelinated axons are uniquely vulnerable to toxic chemicals that cause severe poisoning in humans. Several well-known toxins, including botulinum toxin, tetrodotoxin, saxitoxin, batrachotoxin, organo-

6.

3.3

chlorine pesticides, pyrethrins, and lead, belong to this category of neurotoxins. Agents causing localized CNS lesions: The compounds in this group cause lesions restricted in distribution, which primarily affect localized anatomical areas in the CNS. These include mercury, lead, manganese, excessive quantities of glutamate and aspartate, methionine sulfoximine, gold, thioglucose, organotin and organochlorine pesticides, and certain nutrient antagonists. Such antinutritives are antimetabolites, antiniacinamides, and antihistamine factors.

LUNG TOXICITY

The principal function of the lungs is gas exchange, providing oxygen to the tissues and removing carbon dioxide. Lung as an organ is in a particularly vulnerable position as regards toxic substances. It may be involved in both absorption and excretion of volatile toxins. It may also suffer damage from nonvolatile toxic compounds administered by other routes. Although many different toxicants may damage the lung, the patterns of cellular injury and repair are relatively constant. One of the most obvious and familiar is irritation caused by volatile compounds such as ammonia and chlorine. A severe or persistent irritation may lead to constriction of airways and edema of lung tissue. Another wellknown symptom of lung toxicity is fibrosis, a formation of collagenous tissue caused by substances such as silica (silicosis) and asbestos (asbestosis). In addition, numerous agents, including microorganisms, spores, dust, and chemicals, are known to elicit allergic responses. Perhaps the most severe response of the lung to injury is cancer, the primary causative agent of which is inhaled cigarette smoke. Lung toxicity, however, is not of much relevance to the field of food toxicology, except perhaps in relation to workers involved in pesticide spraying of food crops.

3.4

cytochrome P-450-dependent monooxygenase system. Although most biotransformations are detoxification reactions, many oxidative reactions produce reactive metabolites that can produce lesions within the liver. Such damage is often seen in the centrilobular region, which also has higher concentrations of cytochrome P-450. 3.4.1 Types of Liver Injury On the basis of guidelines promulgated by the U.S. Public Health Service for the detection of hepatotoxicity due to drugs and chemicals, the hepatic lesions can be divided into two categories as follows (Davidson et al., 1979): Type I lesions: These are predictable, are dose- and time-dependent, and occur in most, if not all, subjects exposed to appropriate doses of the causative agent. These lesions are usually readily reproducible in animals. Type II lesions: These are nonpredictable, are doseand time-independent, occur sporadically, and often become apparent only after monitoring of a large number of exposed individuals. These lesions are usually not reproducible in animals. This classification, however, does not take into consideration the actual morphological characteristics of the liver injury. For a morphological classification, the system proposed by Popper and Schaffner (1959) is still widely used. It describes five groups of reactions: 1.

2.

HEPATOTOXICITY

The liver, the largest organ in the body, is often the target organ for chemically induced injuries. Several important factors are known to contribute to the liver’s susceptibility. First, compounds absorbed in the GI tract are transported by the hepatic portal vein to the liver (see Chapter 2). Thus the liver is the first organ perfused by chemicals absorbed in the gut. A second factor is a high concentration in the liver of xenobiotic-metabolizing enzymes, primarily the

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3.

Zonal hepatocellular alterations without inflammatory reaction: Chemicals in this category all produce zonal changes, either necrosis or fat accumulation. This type of lesion is probably the best understood type of hepatic injury (a type I lesion). It is dose dependable and predictable and can be easily reproduced in several animal species. Intrahepatic cholestasis: This category contains drugs of unrelated chemical structures that produce a type of jaundice resembling that produced by extrahepatic biliary obstruction in a very small percentage of the population. Inflammation or blocking of the bile ducts results in retention of bile salts as well as accumulation of bilirubin, which causes jaundice. There is no relationship between dose and response, and production of lesions in animals is not possible (a type II lesion). Hepatic necrosis with inflammatory reaction: This type of liver injury is characterized by pro-

4.

5.

gression to a massive necrosis characteristic of viral hepatitis. This is also a type II lesion. Unclassified group: This category contains a variety of hepatic injuries that do not fit into any type of scheme. For some, the lesions are associated with manifestations of abnormality in several other organs. Hepatic cancer: A number of chemicals are now being recognized as hepatocarcinogens in animals. The most common type of primary liver tumor is hepatocellular carcinoma; other types include cholangiocarcinoma, angiosarcoma, glandular carcinoma, and undifferentiated liver cell carcinoma. However, only a few hepatocarcinogens, such as vinyl chloride, which causes angiosarcoma, are human carcinogens.

Unless they occur on a massive scale, necrotic lesions due to cell death are not necessarily critical because of the regenerating capability of the liver. Cell necrosis is preceded by a number of morphological changes such as cytoplasmic edema, dilatation of endoplasmic reticulum, disaggregation of polysomes, accumulation of triglycerides, swelling of mitochondria with disruption of cristae, and dissolution of organelles and nucleus (Levi, 1987; Plaa, 1986; Niesink et al., 1996). Biochemical events that may lead to these changes include binding of reactive metabolites to proteins and unsaturated lipids (including lipid peroxidation and subsequent membrane destruction), disturbance of cellular Ca2+ homeostasis, interference with metabolic pathways, shifts in Na+ and K+ balance, and inhibition of protein synthesis. Fatty liver results from an abnormal accumulation of fats, mainly triglycerides, in the parenchymal cells. Excess lipid can result from oversupply of free fatty acids from adipose tissues or, more commonly, from impaired release of triglycerides from the liver into plasma (Plaa, 1986; Levi, 1987; Stine and Brown, 1996). The importance of fatty liver in liver injury is not clearly understood, and fatty liver in itself does not necessarily indicate liver dysfunction. Another type of liver injury that has received great attention is cirrhosis. This progressive disease is characterized pathologically by the presence of collagen throughout most of the liver. Cirrhosis is often associated with liver dysfunction and frequently results in jaundice. In humans, chronic use of alcohol is the single most important cause of cirrhosis. Well-known examples of hepatotoxic agents and associated liver injury are presented in Table 3.1.

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Table 3.1 Examples of Hepatotoxic Agents and Associated Liver Injury Liver necrosis Acetaminophen, allyl alcohol, beryllium, bromobenzene, furosemide, thioacetamide Fatty liver Allyl formate, cerium, cycloheximide, emetine, ethanol, thionine, mitomycin C, puromycin, tetracycline Both liver necrosis and fatty liver Aflatoxins, azaserine, carbon tetrachloride, chloroform, dimethylnitrosamine, galactosamine, phosphorus, pyrrolizidine alkaloids, tannic acid, tetrachloroethane, trichloroethylene Cholestasis (drug-induced) Promazine, amitriptyline, diazepam, estradiol, sulfanilamide, mepazine acetate, phenindione, methimazole Hepatitis (drug-induced) Iproniazid, isoniazid, imipramine, colchicine, halothane, methyldopa Carcinogenesis (experimental animals) Aflatoxins, pyrrolizidine alkaloids, safrole, cycasin, polychlorinated biphenyls, vinyl chloride, urethane Source: Compiled from Levi (1987), Plaa (1986), and Zimmerman (1978).

3.5

NEPHROTOXICITY

The kidneys have a greater likelihood of toxic injury from ingested substances than any other organ in the body. Several factors may be involved in this sensitivity, but perhaps the most important is high renal blood flow (Levi, 1987; Timbrell, 1982; Hook and Hewitt, 1986; Stine and Brown, 1996). Although the two kidneys constitute <1% of the total body mass, they receive approximately 20%–25% of the resting cardiac output. This large volume of blood— more than 400 ml/100 g/min of kidney cortex—indicates that a large amount of circulating toxic substances contacts kidney tissues. A second factor affecting the kidney’s sensitivity to chemicals is its ability to concentrate substances. Substances are concentrated after glomerular filtration of the plasma water in the tubular lumen by reabsorption of 98%–99% of the sodium and water. The concentration of the toxic substances in the tubular lumen and surrounding renal parenchymal cells may therefore be quite high. Since the ratio of tubular fluid concentration to plasma concentration may reach values of 500:1, a nontoxic concentration in the plasma may reach toxic concentrations in the tubular fluid.

The transport characteristics of the kidney also contribute to concentration of toxicants within the cells. If the chemical is actively secreted from the blood into the tubular urine, it is accumulated initially within the cells of the proximal tubules, or, if the substance is readsorbed from the urine, it passes through the epithelial cells in a relatively high concentration. The biotransformation of the parent compound to a toxic metabolite is yet another important factor in nephrotoxicity (Levi, 1987; Stine and Brown, 1996). Although the kidney does not possess the high levels of xenobioticmetabolizing enzymes (such as the cytochrome P-450-dependent monooxygenase system) that are found in the liver, many of the same enzymatic reactions have been shown to occur in the kidney. The levels of cytochrome P450 are highest in the cells of the pars recta of the proximal tubule, an area that is particularly susceptible to toxic damage. Considering the unstable nature of many reactive metabolites, it seems most likely that covalent binding to tissue macromolecules occurs in close proximity to the site of activation. Thus, chemicals that exert their toxicity by a reactive intermediate are probably activated directly in the kidney rather than being activated in the liver and then transported to the kidney. The kidney thus is in a particularly vulnerable position. 3.5.1 Clinical Manifestations Nephrotoxic compounds may lead to chronic or acute renal failure, which is manifested by the uremic syndrome. Uremia is characterized by oliguria and increases in blood nitrogen, mostly in the form of urea, and other substances normally excreted in the urine (Concon, 1988; Hook and Hewitt, 1986). Renal failure may also be complete, resulting in anuria. This condition is fatal unless prompt medical treatment is received. Renal damage by toxicants may be assessed by an evaluation of kidney function. Basically, the concentration of normal urine constituents is measured. For example, the concentration of NaCl in the urine may indicate a failure to concentrate urine or reabsorb water and sodium. Abnormal excretion of glucose in the absence of hyperglycemia, high urinary amino acid–creatine ratio (amino aciduria), proteins and sediments, and urinary enzymes, such as glutamate oxalacetate transaminase (Prescott and Ansari, 1969) and alkaline and acid phosphatases (Nomiyama et al., 1973), are all also indicative of renal failure. Clearance of specific substances, such as para-aminohippurate (PAH), may also indicate renal damage (McCurdy et al., 1968). Although the signs described are associated with renal damage, symptoms may not be perceptible in mild

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Table 3.2

Examples of Nephrotoxicants

Heavy metals Uranium, mercury, cadmium, lead, platinum, chromium, arsenic, gold, antimony, thallium Antibiotics Aminoglycosides (streptomycin, neomycin, kanamycin, gentamycin, tobramycin, amikacin), cephalosporins (cephaloridine), probenecid, tetracyclines, penicillins, sulfonamides Halogenated hydrocarbons Carbon tetrachloride, chloroform, hexachlorobutadiene, bromobenzene Mycotoxins Aflatoxins, rubratoxin B, sterigmatocystin, ochratoxin A, citrinin Therapeutic agents (other than antibiotics) Analgesics, anesthetics Environmental contaminants Pesticides, herbicides, polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs)

cases or early in the development of kidney damage (Sharratt and Frazer, 1963). 3.5.2 Nephrotoxicants Not all compounds that influence renal function affect the kidney directly. Renal function may be altered secondarily to change in blood pressure, blood volume, neural or hormonal influences, and a variety of destructive systemic effects. Many substances that can damage the liver may also affect the kidneys. Thus, both kidney and liver damage can occur in carbon tetrachloride and mushroom poisoning. Renal damage may also be induced by hypertension, which may be related to excessive sodium intake. Some common nephrotoxicants are summarized in Table 3.2.

3.6

HEMATOTOXICITY

Toxic injury to the blood cells and blood-forming tissues is known as hematotoxicity. In humans, the bone marrow constitutes the principal blood-forming tissue. The bone marrow produces stem cells, which are precursors of the red blood cells (RBCs, erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). The RBCs primarily deliver oxygen to all cells in the body and remove carbon dioxide from these cells. They contain hemoglobin, the iron-containing hemeprotein that is responsible for their respiratory function. Injury to the RBCs or hemoglo-

bin can result in impairment of oxygen transport and consequent peripheral hypoxia. The symptoms associated with this condition reflect the damage to the CNS and/or heart, which are most sensitive to oxygen deprivation. Hematotoxicity is manifested by anemia, agranulocytosis, methemoglobinemia, polycythemia, leukemia, thrombocytopenia and other blood-clotting disorders, and defects in the immune system (Concon, 1988; Smith, 1986; Stine and Brown, 1996; Niesink et al., 1996). 3.6.1 Anemias The anemias are characterized by a reduction in the number of circulating RBCs, in the volume of packed red cells per unit volume of blood (hematocrit), in hemoglobin concentration per 100 ml of blood, or in a combination of two or all these factors. Anemias can result from damage to the bone marrow and other erythropoietin-producing tissues, damage to the kidneys, increased rate of destruction over rate of production of RBCs, decreased nucleic acid synthesis, deficiency of iron and other minerals, vitamin deficiencies, and excessive hemorrhage. The characteristics of some common types of anemias are summarized in Table 3.3. The clinical signs of anemia include weakness, vertigo, headache, sore tongue, drowsiness, general malaise, dyspnea, tachycardia, palpitation, angina pectoris, GI dis-

Table 3.3 Common Types of Anemias Resulting from Hematotoxicity Hypochromic anemia (associated with iron deficiency) Factors interfering in the absorption or availability of iron, including high-fat diets, vitamin C and copper deficiency, and lead poisoning Aplastic anemia (damage to bone marrow) Benzene, pesticides Megaloblastic anemia (failure of RBC maturation) Deficiency of folic acid and vitamin B12, antimetabolites and antagonists such as antifolacin and anticobalamin Macrocystic anemia (presence of large number of macrocytes [large RBCs]) Deficiency of folic acid and vitamin B12, antimetabolites and antagonists such as antifolacin and anticobalamin Pernicious anemia (characterized by the presence of both macrocytes and megaloblasts) Vitamin B12 deficiency, damage to stomach glandular mucosa by toxicants Hemolytic anemia (rupture or hemolysis of RBCs) Favism, a hereditary condition caused by the ingestion of fava beans Methemoglobinemia (oxidation of hemoglobin to methemoglobin) Nitrites, free radicals, aromatic amines, arylnitro compounds

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turbances, amenorrhea, lack of libido, slight fever, and pallor of the skin, fingernail beds, and mucous membranes (Thomas, 1977; Smith, 1986). The basal metabolic rate may be increased in severe cases. 3.6.2 Other Blood Abnormalities Toxic substances can cause several other blood abnormalities. One is polycythemia, a condition marked by abnormal proliferation of RBCs. This condition may be induced primarily by the ingestion of large amounts of cobalt. Other causes include low-protein diets (Alexander, 1969) and thiamine deficiency (Grinvalsky and Fitch, 1969). Pancytopenia, a condition marked by decreased formation of all three major types of blood cells, is another blood abnormality. Many types of toxicants, such as benzene and arsenic, can promote this blood abnormality (Harris and Kellermeyer, 1970). Toxic chemicals having cytotoxic effects on bone marrow can also induce agranulocytosis (Pisciotta, 1971).

3.7

SKELETAL TOXICITY

Toxic injuries to the bones may arise from interference in bone metabolism and from deposition of radioactive and other toxic metals. 3.7.1 Interference with Bone Metabolism Bone metabolism is under the influence of several hormones, including growth, thyroid, and sex hormone corticotropin (ACTH); adrenocorticoids; and parathormone (Budy, 1975). Thus, any chemical adversely affecting the function of these hormones may indirectly have deleterious effects on bones. For example, if the secretion of a growth hormone is diminished, growth and development of bones are retarded. Overproduction, in contrast, causes an enlargement of bones, resulting in a grotesque condition known as acromegaly. Similarly, although thyroid hormones are essential for the normal development of the bones, excessive production or administration of them, in fact, leads to retardation of bone growth. Excessive production or administration of adrenocorticoids causes osteoporosis (loss of bone mineral). Parathormone regulates the mobilization of calcium in bone, so that excessive secretion of this hormone leads to excessive calcium loss (Budy, 1975; Concon, 1988). Several vitamins are also involved in the formation of bones. Cholecalciferol (vitamin D3), in its active form, regulates the mineralization of bone. Excessive doses of this vitamin cause the resorption of bones, poor calcifica-

tion, and formation of a matrix that is uncalcifiable. The toxic symptoms associated with cholecalciferol poisoning also include headache, nausea, anorexia, weakness, digestive disturbances, irreversible kidney damage, excessive urination (polyurea), and calcification of the soft tissues (Orten and Neuhaus, 1975). A deficiency of cholecalciferol produces rickets in children and osteomalacia in adults. Rickets is associated with defective mineralization, so that the bones become soft and pliable. Both excesses and deficiencies of retinoids (vitamin A) can also lead to toxic injury to bones. Excessive ingestion of this vitamin causes bone fragility and fractures of the long bones as well as other symptoms such as anorexia, irritability, fissures at the corner of the mouth, cracking and bleeding of lips, loss of hair, liver enlargement, and pain in the joints and bones (Concon, 1988). A deficiency of vitamin A leads to retardation of bone growth, especially the formation of endochondral bone. Any factor that retards the absorption of cholecalciferol also retards the absorption of retinoids. Vitamin C (ascorbic acid) is also involved in the formation and maintenance of collagen. Its deficiency causes defective collagen that cannot be calcified (Budy, 1975). 3.7.2 Deposition of Toxicants Many metals tend to concentrate in the bone, some replacing calcium in the crystal lattice. Others are bound to specific bone proteins such as sialoproteins. Those metals that tend to replace calcium include strontium, barium, radium, lead, vanadium, and cadmium (Concon, 1988). Others, such as beryllium, deposit on the crystal surfaces (Mclean and Budy, 1964). Metals that bind to bone proteins include lanthanum, thorium, and plutonium. Toxic injury also occurs when radioactive metals are deposited in the bones. From the food toxicology perspective, the main toxic effects that can be related to chronic ingestion of radioactive “bone-seeking” elements are bone cancers (Finkel et al., 1969; Goldman and Bustad, 1972). 3.8

REPRODUCTIVE TOXICITY

The reproductive system is sensitive to many toxic substances. Toxicants can have direct and indirect effects on the reproductive system: indirectly, by affecting hormones originating outside the reproductive organs; directly, by affecting the egg, sperm, and supporting structures or tissues. The main clinical manifestation of reproductive toxicity is infertility or sterility. Examples of toxicants that affect the reproductive system are summarized in Table 3.4. Of these, estrogens

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Table 3.4 Toxicants That Affect Male and Female Reproductive Capacity Steroids Natural and synthetic androgens (antiandrogens), estrogens (antiestrogens), and progestins Antineoplastic agents Alkaloids, alkylating agents, antimetabolites, antitumor antibiotics Therapeutic agents Appetite suppressants, narcotic and nonnarcotic analgesics, tranquilizers, diuretics Metals and trace elements Arsenic, lead, lithium, mercury, nickel, selenium Insecticides Organochlorines, organophosphates, carbamate Herbicides Diquat, paraquat, chlorinated phenoxyacetic acids Fungicides Captan, thiocarbamates Food additives and contaminants Aflatoxins, cyclamate, gossypol, diethylstilbestrol, monosodium glutamate Personal habits Alcohol consumption, tobacco smoking

are of particular interest to food toxicologists. Estrogenic compounds are found in many foods in the form of naturally occurring compounds, additives, or food contaminants. Beer contains estrogenic bitter acids derived from hops (Humulus lupulus) used during brewing to impart bitter taste. Other naturally occurring estrogens found in forage plants have been implicated in causing sterility in livestock. Diethylstilbestrol (DES), a contraceptive that was formerly used as a growth-promoting hormone in beef cattle, is now prohibited for use in the United States. It has been shown to cause cancer in the female offspring of women administered therapeutic doses of DES.

3.9

IMMUNOTOXICITY

The immune system functions in resistance to infectious agents, homeostasis of leukocyte maturation, immunoglobulin (antibody) production, and immune surveillance against arising neoplastic cells. The interaction of environmental chemicals or drugs with lymphoid tissue may alter the delicate balance of the immune system and result in four types of undesirable effects (Dean et al., 1986). 1. 2.

Immunosuppression Uncontrolled proliferation, i.e., leukemia and lymphoma

Table 3.5

Examples of Immunotoxic Chemicals

Immunosuppressants Alkylating agents, antiinflammatory agents, organochlorine and organophosphate insecticides, airborne pollutants, heavy metals, vinca alkaloids, antibiotics, antifungal agents, estrogens, cyclosporine, ethanol, cannabinoids, cocaine, opiates Lympholytic or lymphomodulatory agents Corticosteroids, cyclophosphamide Autoimmunity induction Heavy metals, penicillins, methyldopa, salicylates

3. 4.

Alterations of host defense mechanisms against pathogens and neoplasia Allergy and autoimmunity

Several toxicants and drugs can inhibit immune function (Table 3.5). This eventually impairs the ability of the host to fight infections. Immunosuppression is of particular clinical importance in the prolongation of allograft survival and in the treatment of autoimmune disorders. The second type of immunotoxicologic response is one in which the immune response is enhanced, frequently because a chemical agent is able to combine with and alter host proteins so that they become antigenic. Thus, the immune system perceives the altered protein as foreign and responds accordingly. This phenomenon is also commonly known as autoimmunity. The lympholytic or lymphomodulatory agents generally act by directly destroying the lymphocytes or lethally damaging their ability to undergo mitosis. Common examples include the corticosteroids, which cause massive lympholysis in some species and act primarily through modulation of lymphocyte trafficking and effector functions in other species, including humans.

Hypersensitivity and allergic manifestations of immunotoxicity are described in the following section.

3.10 HYPERSENSITIVITY AND ALLERGY Chemical exposures are endemic to our modern industrial society. There is growing evidence that there are chemically sensitive individuals in our society. Many, it is believed, may have acquired the sensitivity through chronic exposure (Spengler, 1988). But even without frank illness, the syndrome of irritation, fatigue, shortness of breath, and nausea associated with exposure to certain chemicals and incidents results in lost productivity and wasteful investigations and litigation. Individuals differ in their responses to increasing doses of a toxic substance. The underlying causes of interindividual variability include age, sex, and genetic makeup; life-style and behavioral factors, including nutritional and dietary factors; alcohol, tobacco, and drug use; environmental factors; and preexisting disease. In the classic, toxicological use of the word sensitivity, those individuals who require relatively lower doses to induce a particular response are said to be more sensitive than those who require relatively higher doses to experience the same response (Ashford and Miller, 1991). A hypothetical distribution of sensitivities, i.e., the minimal doses necessary to cause individuals in a population to exhibit a harmful effect, is shown in Figure 3.2. This distribution describes the traditional toxicological concept of sensitivity. Curve A illustrates that health effects of classic diseases and toxicants are seen in a significant portion of the normal population at a certain dose; the sensitive and resilient populations are found in the tails of the distribution.

Figure 3.2 Hypothetical distribution of different types of sensitivities as a function of dose. A, sensitivity distribution for classic toxicity; sensitive individuals are found in the left-handed tail of the distribution. B, sensitivity distribution of atopic or allergic individuals in the population who are sensitive to an allergen. C, sensitivity distribution for individuals with multiple chemical sensitivities who, because they are already sensitized, subsequently respond to particular incitants.

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A second meaning of the word sensitivity appears in the context of classic immunoglobulin E– (IgE)-mediated allergy (atopy); IgE is one of five classes of antibodies made by the body, and is, from the perspective of classically allergic individuals, the most important antibody (Deshpande, 1996). Atopic individuals have IgE directed against specific environmental or food incitants. Positive skin test results in these individuals correlate with a rapid onset of symptoms when they are actually exposed to those allergens. Atopic individuals exhibit a reaction, whereas nonallergic persons do not, even at the highest doses of allergens normally encountered. A hypothetical sensitivity distribution for an atopic effect is shown in curve B of Figure 3.2. In contrast, in individuals with multiple chemical sensitivities, multiple organ systems may be affected and multiple substances may trigger the effects. Over time, sensitivities seem to spread, in terms of both the types of triggering substances and the systems affected. Avoidance of the offending substances is usually effective but much more difficult to achieve for these individuals than for classically sensitive patients because symptoms may occur at extremely low doses and the exposures are ubiquitous. A hypothetical sensitivity distribution for a single symptom for the already chemically sensitive individual in response to a single substance trigger is shown in curve C of Figure 3.2. The term allergy is used to identify true immunologically based adverse reactions that result from exposure to certain chemicals. For nonimmunological responses to chemicals, the term chemical intolerance is generally preferred. Allergic reactions are mediated by activated lymphocytes or by antibody specific for the antigen, or for another antigen with similar determinants (antigenic chemical groups). Pseudoallergy, in contrast, mimics allergy in clinical manifestations but is not due to antigenspecific immunological reactions. The same endogenous pharmacological substances, e.g., histamine or prostaglandins that are released or synthesized after an antibody- or lymphocyte-mediated immune response, induce the clinical signs of pseudoallergies. However, in pseudoallergy, the inducing substance has a direct action to release the pharmacological substances without participation by immunological mediators. On the basis of mechanism of immunological involvement, allergic responses can be divided into four general categories (Coombs and Gell, 1975; Wells, 1982; Dean et al., 1986), which are summarized in Table 3.6. The type I or anaphylactic reactions occur when mast cells or basophils, sensitized passively with antibody (IgE in humans), are exposed to specific antigens. This reaction may entail release of histamine, serotonin, kinins, heparin, and slow-reacting substances of anaphylaxis

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Table 3.6

Classification of Allergic Reactions

Classification Type I anaphylaxis (immediate hypersensitivity) Type II Cytotoxic Type III Arthus

Type IV delayed-type hypersensitivity

Mechanisms

Clinical examples

Tissue passive sensitizing antibody Antibody or cell mediated Precipitating antibody

Allergic rhinitis, asthma, urticaria

Cell mediated; controlled by T lymphocytes

Hemolytic anemia, eczema, purpura Systemic lupus erythematosus, glomerular nephritis, rheumatoid arthritis, serum sickness Contact dermatitis, eczema, Crohn’s disease

(SRSA). The main targets of this type of reaction are the GI tract (food allergies), the skin (urticaria and atopic dermatitis), the respiratory system (rhinitis and asthma), and the vasculature (anaphylactic shock). These reactions tend to occur quickly after rechallenge with an antigen to which the individual has been sensitized and are termed immediate hypersensitivity. Both IgG and IgM antibodies mediate the type II or cytotoxic reactions. These reactions are usually attributed to the antibody’s ability to fix complement, opsonize particles, or function in an antibody-dependent cellular cytotoxicity reaction (Dean et al., 1986). The major target is often tissues of the circulatory system, including red and white blood cells and platelets. The interaction of cytotoxic antibody with these cells or their progenitors results in depletion and the production of hemolytic anemia, leukopenia, or thrombocytopenia. Additional target organs include the lungs and kidneys. In these type II reactions, antibodies may develop to respiratory and glomerular basement membranes, resulting in glomerulonephritis and pulmonary hemorrhaging. The type III or Arthus reactions result from formation of complexes of antigen with antibody (IgG or IgM) that activate complement. The complexes then become deposited in the vascular endothelium, where a destructive inflammatory response occurs. The main target tissues are the skin (lupus), the joints (rheumatoid arthritis), the kidneys (glomerulonephritis), the lungs (hypersensitivity pneumonitis), and the circulatory system (serum sickness). The antigens responsible for these types of reactions may be self-antigens, which are thought to cause lupus and rheumatoid arthritis, or foreign antigens, as in serum sickness. The type IV or delayed-hypersensitivity responses are not mediated by antibodies, but rather by macrophages

and sensitized T lymphocytes. When sensitized T lymphocytes are in contact with the sensitizing antigen, an inflammatory reaction is generated: lymphokines are produced, and an influx of granulocytes and macrophages follows. The target for this type of reaction can be almost any organ; the classic example is skin (Dean et al., 1986). The symptoms and diseases caused by food and chemical exposures involve any and every system of the body. Some commonly observed acute reactions are summarized in Table 3.7.

Table 3.7 Possible Acute Reactions to Allergens During Provocation Nasal

Throat, mouth

Ears

Lungs

Heart

Blood vessels Joints Muscles Skin

Eyes Vision

Cerebral, head

Genitourinary

Gastrointestinal

Urge to sneeze, itching, rubbing, obstruction, discharge, postnasal drip, sinus discomfort, stuffy feeling Itching, sore, tight, swollen, dysphagia, difficulty in swallowing, choking, weak voice, hoarse, salivation, mucus, bad or metallic taste Itching, full, blocked, erythema of pinna (reddening), tinnitus (ringing in ears), earache, hearing loss, hyperacusis (abnormal sensitivity to sound) Coughing, sneezing, reduced air flow, retracting, shortness of breath, heavy, tight chest, hyperventilation Chest pain, tachycardia (rapid pulse), palpitations (rapid violent or throbbing pulses, extra or skipped beats) Spontaneous bruising and petechiae, cold sensitivity, swelling, acneform lesions Ache, pain, stiff, erythema, warmth, redness Tight, stiff, aches, soreness Itching (local or general), scratching, moist, sweating, flushing, hives, pallor (white or ghostly) Itching, burning pain, lacrimation (tearing), light sensitive, feel heavy Blurring, acuity decreased, spots, flashes, darker, vision loss, photophobia, diplopia, dyslexia Headache (throbbing, stabbing), fainting, depression, mood swings, hallucinations, hyperactivity, irritability, fatigue, apathy, confusion, lethargy, blackouts, insomnia, somnolence Voided, mild urge, frequency in voiding, dysuria, genital itch, vaginal discharge, yeast infection Nausea, belching, full, bloated, vomiting, cramps, flatus, diarrhea, gallbladder symptoms, hunger, thirst, hyperacidity

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REFERENCES Alexander, C.S. 1969. Cobalt and the heart. Ann. Intern. Med. 79:411. Ashford, N.A. and Miller, C.S. 1991. Chemical Exposures: Low Levels and High Stakes. Van Nostrand Reinhold, New York. Brierley, J.R., Brown, A.W., and Meldrum, B.S. 1971. The nature and time course of the neuronal alterations resulting from oligaemia and hypoglycaemia in the brain of Macaca mulatta. Brain Res. 25:483–499. Brierly, J.B. 1955. The sensory ganglia—recent anatomical, physiological and pathological contributions. Acta Psychiatr. Neurol. Scand. 30:553–576. Brightman, M.W. and Reese, T.S. 1969. Junctions between intimately opposed cell membranes in the vertebrate brain. J. Cell Biol. 40:648–677. Brucher, J.M. 1967. Neuropathological problems posed by carbon monoxide poisoning and anoxia. Prog. Brain Res. 24:75–100. Budy, A. 1975. Toxicology of the skeletal system. In Toxicology: The Basic Science of Poisons, eds. L.J. Casarett and J. Doull. Macmillan, New York. Concon, J.M. 1988. Food Toxicology. Parts A and B. Marcel Dekker, New York. Coombs, R.R.A. and Gell, P.G.H. 1975. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In Clinical Aspects of Immunology, 3rd ed., eds. P.G.H. Gell, R.R.A. Coombs, and P.J. Lachman, pp. 761–781, Blackwell Scientific, Oxford. Davidson, C.S., Leevy, C.M., and Chamberlayne, E.C. 1979. Guidelines for Detection of Hepatotoxicity Due to Drugs and Chemicals. NIH Publ. No. 79-313. U.S. Department of Health, Education, and Welfare, Washington, D.C. Dean, J.H., Murray, M.J., and Ward, E.C. 1986. Toxic responses of the immune system. In Toxicology: The Basic Science of Poisons, 3rd ed. ed. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 245–285, Macmillan, New York. Deshpande, S.S. 1996. Enzyme Immunoassays: From Concept to Product Development. Chapman & Hall, New York. Finkel, A.J., Miller, C.E., and Hasterlik, R.J. 1969. Radium-induced malignant tumors in man. In Delayed Effects of Bone-Seeking Radionuclides, ed. C.W. Mays. University of Utah Press, Salt Lake City, UT. Goldman, M. and Bustad, L.K. 1972. Biomedical Implications and Radiostrontium Exposure. AEC Symp. Ser. 25. Atomic Energy Commission, Office of Information Services, Springfield, VA. Grinvalsky, H.T. and Fitch, D.M. 1969. A distinctive myocardiopathy occurring in Omaha, Nebraska: Pathological aspects. Ann. N.Y. Acad. Sci. 156:544–565. Harris, J.W. and Kellermeyer, R.W. 1970. The Red Cell Production, Metabolism, Destruction: Normal and Abnormal. Harvard University Press, Cambridge, MA. Hook, J.B. and Hewitt, W.R. 1986. Toxic responses of the kidney. In Toxicology: The Basic Science of Poisons. 3rd ed., eds.

C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 310–329, Macmillan, New York. Jacobs, J.M. 1977. Penetration of systemically injected horseradish peroxidase into ganglia and nerves of the autonomic nervous system. J. Neurocytol. 6:607–618. Kamrin, M.A. 1988. Toxicology. Lewis Publishers, Chelsea, MI. Levi, P.E. 1987. Toxic action. In Modern Toxicology, eds. E. Hodgson and P.E. Levi, pp. 133–184, Elsevier, New York. Malamud, N. 1963. Patterns of CNS vulnerability in neonatal hyperemia. In Selective Vulnerability of the Central Nervous System in Hypoxaemia, eds. J.F. Schade and W.H. McMenemey, pp. 208–238, F.A. Davis, Philadelphia. McCurdy, D.K., Frederick, M., and Elkinton, J.R. 1968. Renal tubular acidosis due to amphotericin. N. Engl. J. Med. 278:124–131. McLean, F.C. and Budy, A.M. 1964. Radiation, Isotopes, and Bone. Academic Press, New York. Niesink, R.J.M., de Vries, J., and Hollinger M.A. 1996. Toxicology: Principles and Applications. CRC Press, Boca Raton, FL. Nomiyama, K., Sato, C., and Yamamoto, A. 1973. Early signs of cadmium intoxication in rabbits. Toxicol. Appl. Pharmacol. 24:625–635. Norton, S. 1986. Toxic responses of the central nervous system. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 359–386, Macmillan, New York. Olsson, Y. and Hossman, K.A. 1970. Fine structural localization of exudated protein tracers in the brain. Acta Neuropathol. (Berl.) 10:26–33. Orten, J.M. and Neuhaus, O.W. 1975. Human Biochemistry. Mosby, St. Louis. Pisciotta, A.V. 1971. Drug-induced leukopenia and aplastic anemia. Clin. Pharmacol. Ther. 12:13–43. Plaa, G.L. 1986. Toxic responses of the liver. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 286–309, Macmillan, New York. Popper, H. and Schaffner, F. 1959. Drug-induced hepatic injury. Ann. Intern. Med. 51:1230–1252.

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Prescott, L.F. and Ansari, S. 1969. The effects of repeated administration of mercuric chloride on exfoliation of renal tubular cells and urinary glutamic-oxaloacetic transaminase activity in the rat. Toxicol. Appl. Pharmacol. 14:97–107. Reese, T.S. and Brightman, M.W. 1968. Similarity in structure and permeability to peroxidase of epithelia overlying fenestrated cerebral capillaries. Anat. Rec. 160:414. Scholz, W. 1953. Selective neuronal necrosis and its topistic patterns in hypoxemia and oligenia. J. Neuropathol. Exp. Neurol. 12:249–261. Sharratt, M. and Frazer, A.C. 1963. The sensitivity of function tests in detecting renal damage in the rat. Toxicol. Appl. Pharmacol. 5:36–48. Smith, R.P. 1986. Toxic responses of the blood. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 223–244, Macmillan, New York. Spengler, J. 1988. Testimony Before the Ways and Means Committee, California State Legislature, Sacramento, CA, February 22. Stine, K.E. and Brown, T.M. 1996. Principles of Toxicology. CRC Press, Boca Raton, FL. Thomas, C.L. 1977. Taber’s Cyclopedic Medical Dictionary, 13th ed., F.A. Davis, Philadelphia. Timbrell, J.A. 1982. Principles of Biochemical Toxicology. Taylor and Francis, London. Wells, J.V. 1982. Immune mechanisms in tissue damage. In Basic and Clinical Immunology, eds. D.P. Stites, J.D. Stobo, H.H. Fudenberg, and J.V. Wells, pp. 136–150, Lange Medical Publications, Los Altos, CA. Wiener, G. 1970. Varying psychological sequelae of lead ingestion in children. Public Health Rep. 85:19–24. Windle, W.F. 1963. Selective vulnerability of central nervous system of rhesus monkeys to asphyxia during birth. In Selective Vulnerability of the Central Nervous System in Hypoxaemia, eds. J.F. Schade and W.H. McMenemey, pp. 167–194, F.A. Davis, Philadelphia. Zimmerman, H.L. 1978. Hepatotoxicity. Appleton-CenturyCrofts, New York.

4 Carcinogenesis, Mutagenesis, and Teratogenesis

4.1

CARCINOGENESIS

Cancer is a major public health problem. It is also the chronic toxic effect that is of most concern to the general population. Apparently, about 80%–90% of all incident cancers are determined by potentially controllable external factors. Thus food as well as life-style may supply many carcinogenic substances. Although, in many cases, there is still a lack of definitive evidence about which dietary characteristics most influence cancer risk, substances such as coffee, alcohol, pyrolysis products, nitrites, amines, fat, smoke, pesticides, and most kinds of mutagens have been implicated as etiological factors in cancer (Deshpande et al., 1995). Thus, both synthetic as well as naturally occurring chemicals may cause cancer. The role of xenobiotic chemicals in causing cancer is called chemical carcinogenesis. Cancer occurs in humans in all age groups and in all races as well as in animal species. The incidence, geographical distribution, and behavior of specific types of cancer are related to multiple factors, including sex, age, race, genetic predisposition, and exposure to environmental carcinogens. Cancer also shows dramatically differing frequencies in different parts of the globe (Table 4.1). For example, colon cancer is 10 times less common in Nigeria than in Connecticut, in the United States. Liver cancer, in contrast, appears 70 times more frequently in Mozambique than in Norway. Stomach cancer rates in Japan are as much as 25 times higher than in Uganda. In addition to the factors listed, such differing frequencies in the occurrence of cancer could be directly attributed to people’s

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life-styles and habits as well as exposure to environmental carcinogens. Some of the environmental agents implicated in human cancer causation are listed in Table 4.2. Certain herpes group DNA viruses and type C RNA virus particles implicated as etiological agents in a wide variety of animal cancers might possibly cause human malignancies as well. These viruses carry a number of oncogenes. Oncogenic RNA viruses listed in Table 4.3 contain a reverse transcriptase enzyme that may permit reading of the oncogenic message of viral RNA into the base sequence of host cell DNA. It appears that all DNA or RNA viruses show an oncogenic potential if they can, after infection, become an integral part of the infected cell’s DNA. Even if a viral genome is proved to be essential to the initiation of certain human cancers, it is likely that additional hereditary and environmental factors modulate the neoplastic expression of latent virus infections. Indeed, epidemiological studies suggest that chemical carcinogens such as those present in tobacco smoke, azo dyes, aflatoxins, and estrogens may be far more important environmental carcinogens than viruses. 4.1.1 Characteristics Cancer is a condition characterized by the uncontrolled replication and growth of the body’s own cells (somatic cells). A cancerous tumor may be defined as an abnormal lump or mass of tissue. Its growth exceeds, and is not coordinated with, that of the normal tissue, continuing after the stimuli that initiated it have ceased. Cancerous tumors may be benign or malignant. Unlike the malignant

Table 4.1

Geographic Variation in the Incidence of Common Cancers

Cancer type Males Skin Esophagus Lung Stomach Liver Prostate Colon Mouth Rectum Bladder Nasopharynx Females Cervix Breast Uterus Ovary a

Highest incidence

Risk up to age 75 (%)

Range of variationa

Lowest incidence

Queensland, Australia Northeast Iran United Kingdom Japan Mozambique U.S.A. (blacks) Connecticut, U.S.A. India Denmark Connecticut, U.S.A. Singapore (Chinese)

Over 20 20 11 11 8 7 3 Over 2 2 2 2

Over 200 300 35 25 70 30 10 Over 25 20 4 2

Mumbai, India Nigeria Nigeria Uganda Norway Japan Nigeria Denmark Nigeria Japan United Kingdom

10 7 3 2

15 15 30 6

Colombia Connecticut, U.S.A. California, U.S.A. Denmark

Israel Uganda Japan Japan

The highest incidence observed divided by the lowest incidence observed.

tumors, the benign tumors are encapsulated and do not invade surrounding tissues; nor do they form metastases, i.e., dispersed satellite foci of tumor tissue in distant organs. Benign tumors can usually be removed surgically and do not recur. In contrast, malignant tumors are often fatal. Thus cancer is basically a disease of cells characterized by impairment or ineffectiveness of the normal cellular control and maturation mechanisms that regulate multiplication and other functions required for homeosta-

sis in complex multicellular organisms. The six distinguishing features of cancer are the following: 1. 2. 3. 4. 5.

Table 4.2 Some Environmental Agents Implicated in Human Cancer Causation Agent Aflatoxins Alcohol Asbestos X-rays Sunlight (UV) Aniline dyes Chewing tobacco Tobacco smoke Hepatitis B virus (HBV) Human T-cell leukemia virus Epstein-Barr virus Human papillomaviruses

Target organ for tumors Liver Pharynx, larynx, esophagus, liver, (breast?) Pleura of lung Bone marrow (leukemia) Skin Bladder Mouth Mouth, lung, bladder, esophagus, pancreas Liver Thymus-spleen (leukemias) Bone marrow (lymphoma), nasopharynx Uterine, cervix

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6.

Excessive cell growth, usually in the form of a tumor Undifferentiated cells and tissues, similar to embryonic tissues Invasiveness, the ability to grow into adjacent tissue (a basic distinction from normal tissue) Ability to metastasize, spread to new sites and establish new growths Type of “acquired heredity” in which the progeny of the cancer cells retain the same cancerous properties Shift of metabolism toward increased building of macromolecules from nucleosides and amino acids, and an increased catabolism of carbohydrates for cellular energy

The abnormal behavior of the cancer cell leads to illness in the host as a result of the pressure effects due to local tumor growth, of destruction of organs involved with the primary tumor or its distant metastases, and of deleterious systemic effects secondary to the growths. Each of the many cell types within the body provides a potential site for the formation of a tumor, which is then categorized by the tissue from which it arises. Of the four major types of cells listed in Table 4.4, epithelial cells generate more than 90% of human cancers. This is probably because they may be inherently more susceptible to growth deregulation and are certainly exposed to and in di-

Table 4.3

Retroviral Oncogenes Implicated in Human Cancers

Viral oncogene (v-onc) src yes myc erbA erbB jun rel mos abl raf fos Ha-ras Ki-ras fms kit sis

Prototype virus Rous sarcoma virus Yamaguchi 73 sarcoma virus Myelocytomatosis-29 virus Avian erythroblastosis virus Avian erythroblastosis virus Avian sarcoma virus 17 Reticuloendotheliosis virus, strain T Moloney murine sarcoma virus Abelson murine leukemia virus Murine sarcoma virus 3611 Mouse (osteo) sarcoma virus Harvey murine sarcoma virus Kirsten murine sarcoma virus Susan McDonough feline sarcoma virus Hardy-Zuckerman 4 feline sarcoma virus Simian sarcoma virus

rect contact with many carcinogenic agents. Cancer cases derived from epithelium are known as carcinoma, whereas those derived from mesothelium are called sarcomas. The eight most common cancers account for over 70% of cancer incidence, and about 70% of these cases have a fatal outcome. The most common cancers are lung carcinoma, colon and rectal cancer, female breast cancer, uterine carcinoma, prostatic carcinoma, bladder and kidney cancer, lymphoma, and leukemia. In many cases, early diagnosis reduces cancer mortality rate.

Table 4.4

Chicken Chicken Chicken Chicken Chicken Chicken Turkey Mouse Mouse Mouse Mouse Rat Rat Cat Cat Woolly monkey

4.1.2 The Carcinogenic Process The overall process for the induction of cancer may be quite complex, involving numerous steps and interactions between environmental and endogenous factors. DNA is a critical target in carcinogenesis (Williams and Weisburger, 1986). This view is supported by several observations: 1.

Many carcinogens are or can be biotransformed to electrophiles that react covalently with DNA. Consequently, such carcinogens are mutagens.

Cell Types and the Type of Cancer Formed

Cell type Epithelial

Connective tissue

Blood-forming Nerve

Species of origin

Tissue Breast, lung, stomach Liver, uterus, colon Skin Mouth Urinary bladder Uterine cervix Cartilage Bone Muscle Blood vessel Bone marrow, spleen Peripheral nervous system (spinal cord ganglia, adrenal cortex) Central nervous system (notably brain)

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Derived tumors Adenocarcinomas (from secretory tissue) Adenocarcinomas (from secretory tissue) Squamous carcinomas (from protective linings) Squamous carcinomas (from protective linings) Squamous carcinomas (from protective linings) Squamous carcinomas (from protective linings) Chondrosarcomas Osteosarcomas Rhabdomyosarcomas Angiosarcomas Lymphomas, myelomas, erythroleukemias, lymphocytic and myelogenous leukemias Neuroblastomas Gliomas, astrocytomas, medulloblastomas, neuromas, schwannomas

2. 3. 4. 5. 6. 7. 8.

Defects in DNA repair, such as in xeroderma pigmentosum, predispose to cancer development. Several heritable or chromosomal abnormalities predispose to cancer development. Initiated dormant tumor cells are persistent, as is consistent with a change in DNA. Cancer is heritable at cellular level and, therefore, may result from an alteration of DNA. Most, if not all, cancers display chromosomal abnormalities. Many cancers display aberrant gene expression. Cells from many cancers contain activated oncogenes.

The transformation of a normal cell into a neoplastic cell is considered to proceed through at least three phases: initiation, promotion, and progression (Figure 4.1). This multistage process is called carcinogenesis, and agents that induce it are called carcinogens. Human and experimentally induced animal tumors share a similarity in the natural history of their development and with respect to their gross and microscopic pathological features. A latency period occurs between exposure to a carcinogen and development of cancer. This latency period is analogous to the time and/or age dependency for the development of several common human cancers. The latency

Figure 4.1

A multistep carcinogenesis model.

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period comprises a series of stages that can be characterized in both experimental protocols and human studies (Table 4.5). Initiation has been described as the irreversible genetic damage that results from exposure to a carcinogen. Therefore, when coupled with a proliferative stimulus, carcinogen exposure could result in the formation of an initiated cell population with an increased susceptibility to further neoplastic development. The clonal proliferative growth of such initiated cells constitutes the succeeding stage of promotion and accounts for part of the time necessary for the development of human and experimental neoplasms. Thus, in the stage of promotion, an acceleration of the growth of previously initiated cells occurs. During progression, additional genetic damage is accompanied by the development of the characteristics of aggressive malignant growth. Thus, in experimental models, carcinogenesis has been divided into the stages of initiation, promotion, and progression. Agents with a potential carcinogenic risk can be classified as acting at any one or combination of these stages (Pitot et al., 1989). Initiation Initiation of a normal cell into a neoplastic or cancerous cell involves permanent alteration of the genetic informa-

Table 4.5 Characteristics of Multistage Nature of Carcinogenesis Initiation Irreversible Additive No threshold Environmentally modifiable Promotion Reversible Maximal response Threshold Environmentally modifiable Progression Irreversible Aneuploidy Karyotypic instability Initially responds to environmental stimuli; later, relative autonomy

tion (mutation) in the cell. Although mutation per se does not necessarily lead to cancer, it is indicative of increased risk of neoplasia. Initiation may occur through reaction of a DNA-reactive species with DNA or through the action of epigenetic carcinogen that does not react with DNA and is carcinogenic by some other mechanism. Most DNA-reactive chemicals are genotoxic carcinogens because they are also mutagens. These chemicals are either electrophilic or, more commonly, metabolically activated to form electrophilic species and react irreversibly with DNA. Once initiation has begun, the cell has the potential to develop into a tumor, provided that the other essential steps of carcinogenesis take place. If these other steps do not take place, the initiated cell may remain dormant indefinitely with no apparent effect on the organism. Because initiated cells cannot be distinguished readily from normal cells morphologically, the growth of clonally derived preneoplastic lesions after promotion typically is used as an end point for the detection of initiation (Pitot, 1990).

Many carcinogens, when given in large doses, are both initiators and promoters. These are called complete carcinogens. However, others are not in themselves carcinogens but, when given after a low dose of an initiating agent increase cancer incidence. These are called promoters. Promoters may either increase or decrease the latency period. They are usually not electrophiles and thus do not bind to DNA. The primary characteristic of promotion that distinguishes it from initiation and progression (discussed later) is the phenotypic instability of the altered genetic expression. The irreversibility of initiation by a carcinogenic insult has been demonstrated in several model systems by the administration of the promoting stimulus after a lengthy delay after exposure to the initiating carcinogen. In addition, suspension of administration of the promoting stimulus from previously initiated cells, followed by readministration, results in the rapid reinstatement of the clonal cell population, further suggesting the irreversible nature of initiation. The reversible nature of promotion is based on several types of studies, including the doseresponse characteristics of promoting agents (Pitot et al., 1987). The dependence of preneoplastic lesions, including transplanted focal hepatocytes, on the continued presence of the promoting stimulus is compatible with the reversible nature of the promotion process. Further, the dependence of the growth of preneoplastic lesions on the frequency of administration of the promoting agent indicates the presence of a threshold for the dose response of tumor promoter action (Xu et al., 1991). The following features characterize the two initial phases of the carcinogenic process (Williams and Weisburger, 1986). 1. 2.

Promotion Initiation is followed by promotion, which can be a very slow process in humans. For many common cancers, the latency period may be as long as 10–30 years. Because the time frame involved is long and because promotion is generally reversible (Figure 4.1), this step is often a very attractive target for intervention with cancer chemoprotective agents. During this stage, the initiated cell clones itself and expands within a given tissue. As the number of these target cells increases, there are many sites where changes can produce fully malignant cells.

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3.

4.

5.

The initiator must be given first; no tumors or very few tumors result if the promoter is given first. The initiator, if given once at a subchronic dose, does not produce tumors during the life of the animal; however, repeated doses of the initiator may elicit tumors even in the absence of the promoter. The action of the initiator is irreversible; tumors result in nearly the same yield if the interval between initiation and promotion is extended from 1 week to 1 year. The initiator is an electrophile or is metabolically activated to an electrophile that binds covalently to DNA to bring about a mutagenic change. The essential function of the tumor promoter is to complete the carcinogenic process started by the initiator.

6.

7.

Promoters have not been found to be electrophilic, and there is no evidence of covalent binding to macromolecules. The action of the promoter is reversible at an early stage and usually requires repeated exposures; thus, there is probably a threshold level of exposure.

Progression Some neoplastic cells undergo qualitative changes in their phenotypic properties, possibly including transition from benign to malignant (Foulds, 1969). This process probably reflects the selection during growth of a population with a genotypic coding for advantageous phenotypic properties. New genotypes could arise in neoplasms through errors in DNA replication, alterations in chromosome constitution, or hybridization of different cell types (Williams and Weisburger, 1986). Neoplasms also display abnormalities in expression of numerous gene products. Foulds (1954) suggested that the entire process in the development of cancer after initiation be called progression, however, since the irreversible changes that occur late in the carcinogenic process are distinct from the reversible characteristics of the period of promotion, the stage of progression is a distinct third stage of carcinogenesis (Pitot, 1991). The process of progression involves additional irreversible genetic damage that is coupled with increasing karyotypic instability associated with increased growth rate, metastasis, invasiveness, and autonomy. Since epidemiological studies have suggested that cancer is the result of a minimum of two genetic insults (Moolgavkar and Knudson, 1981), the initiation-promotion-progression multistage model of carcinogenesis can mimic the development of many human cancers (Dragan et al., 1992). 4.1.3 Types of Chemical Carcinogens Chemical carcinogens can be broadly classified into two groups: DNA-reactive (genotoxic) and epigenetic (Table 4.6). The DNA-reactive, genotoxic category comprises carcinogens that chemically interact with DNA and includes most of the “classic” organic carcinogens. This category consists mainly of carcinogens that function as electrophilic reactants. They can be further subdivided according to whether they are active in their parent form (primary carcinogens) or require metabolic activation (procarcinogens or secondary carcinogens). The latter usually do not produce cancer at the site of application but rather are carcinogenic to distant tissues where metabolic activation occurs. Unlike the primary carcinogens, which are synthetic products and not found in nature, the large

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class of procarcinogens includes both naturally occurring substances and synthetic chemicals. Procarcinogens that have been detected in foods are summarized in Table 4.7. Metabolic activation of procarcinogens implies the presence of enzyme systems whose main function is to promote their excretion by increasing their water solubility. This process results in decreased chemical reactivity and, for certain compounds, carcinogenicity as well. Thus, metabolic transformation may result in either activation or deactivation (detoxification). Metabolic activation reactions involving carcinogens include epoxidation (e.g., benz(a)pyrene, aflatoxins), hydroxylation and esterification (e.g., safrole), oxidation and carbonium ion formation (e.g., nitrosamines), and hydrolysis by intestinal microflora (e.g., cycasin from cycad seeds). The second broad category, designated as epigenetic carcinogens, comprises chemicals that have been reliably demonstrated not to react with DNA and that exert another kind of biological effect that appears to be the basis for their carcinogenicity. This category contains chemicals with diverse modes of action (Table 4.6). Promoters are defined as agents that facilitate the growth of dormant neoplastic cells into tumors (Berenblum 1974). Many agents that have promoting activity are also carcinogenic. Cytotoxic agents that produce cell death leading to compensatory proliferation can also give rise to cancer (Stott and Watanabe, 1982). Although the exact mechanisms are not clearly understood, they may involve enhanced susceptibility to environmental carcinogens, mutations during DNA replication, aberrant methylation, and chromosome effects (Williams and Weisburger, 1986; Barrows, 1986). Hormones are also known modifiers of chemical carcinogenesis (Levi, 1987). Estrogens, which are well known to cause cancer in laboratory animals, as well as agents that perturb the physiological processes of endocrine organs, leading to an increase in neoplasia in those organs, are in this category. Immune processes can influence carcinogenesis in a variety of ways (Becker, 1982; Williams and Weisburger, 1986; Concon, 1988). The carcinogenicity of some immunosuppressant drugs and chemicals may stem from an epigenetic phenomenon by which immunosuppression allows development of tumors initiated by a distinct genetic event (Tarr and Olsen, 1985). Solid-state or foreign body carcinogenesis is associated with the presence of films or disks of plastic or metals lodged in tissues. In general, the neoplastic process is independent of the chemical nature of the film or disk, since both plastic and chemically inert metallic foils or films can induce sarcomas (Concon, 1988). Asbestos, the best known example of this type, has been associated with

Table 4.6

Classification of Carcinogens

DNA-reactive (genotoxic) carcinogens A. Primary carcinogens (direct-acting) Biological alkylating agents including S-mustards, N-mustards, epoxides, aziridines, alkyl alkane sulfonates, strained ring lactones, and nitrosamides B. Procarcinogens (require metabolic activation) Polycyclic or heterocyclic hydrocarbons, aromatic and heterocyclic amines, quinolines and azarenes, halogenated hydrocarbons, nitrosamines C. Inorganica Certain metals and metalloids Epigenetic carcinogens A. Promoters Organochlorine pesticides, saccharin, drugs, bile acid, phorbol ester B. Cytotoxic Nitrilotriacetic acid C. Hormone-modifying Estrogens, androgens, amitrole D. Immunosuppressor Purine analogs E. Solid state Asbestos, plastics F. Cocarcinogens Phorbol ester, catechol Unclassified A. Peroxisome proliferators Clofibrate, phthalate esters B. Miscellaneous Dioxane a

Some are tentatively categorized as genotoxic because of evidence for damage of DNA; others may operate through epigenetic mechanisms such as alterations in fidelity of DNA polymerases. Source: Compiled from Levi (1987), Williams (1984), and Williams and Weisburger (1986).

bronchogenic cancer and mesothelioma, a rare form of cancer of the pleura and peritoneum. The fact that it may be a contaminant in water and food makes it a potential food-borne or waterborne carcinogen. Cocarcinogens are agents that enhance the overall carcinogenic process initiated by a genotoxic carcinogen when administered before or together with the carcinogen or at a time when carcinogen damage to DNA is still persistent (Levi, 1987; Williams and Weisburger, 1986). Accordingly, cocarcinogenesis applies to events that occur during neoplastic conversion (Figure 4.1). Sometimes promoters can also act as cocarcinogens. Cocarcinogenesis can result from such factors as hormones, viruses, immunological factors, nutritional factors, physical trauma, and skin abrasion. Examples include croton oil, citrus oils, surface-active agents such as polysorbate (Tween) or sorbitan (Span), phenol and a number of its derivatives, n-dodecane and other alkanes, and 1-alkyl alcohols. Cocarcinogens influence the initiating process by a number of mechanisms (Williams, 1984; Levi, 1987), including the following: 1. 2. 3. 4.

Increased cellular uptake of carcinogen Increased proportion of carcinogen activated Depletion of competing nucleophiles Inhibition of DNA repair mechanisms

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5.

Enhanced conversion of DNA lesions to permanent alterations

In addition, there are a number of carcinogens that have not been demonstrated to react covalently with DNA but have a mode of action that is still not sufficiently well understood to permit assignment to a specific class of epigenetic agents. Dioxane, benzene, thioamides, halogenated hydrocarbons, and methapyrilene are examples of this type. Some chemicals have the ability to increase the numbers of peroxisomes in rodent livers, producing liver tumors. Such peroxisome proliferators are sometimes considered as a class of carcinogenic chemicals (Reddy and Lalwani, 1983). Examples of this class of chemicals include hypolipidemic drugs, clofibrate, fenofibrate, gemfibrozil, and tibric acid. 4.1.4 Factors Affecting Carcinogenesis Several extrinsic and intrinsic factors affect the carcinogenic response to a particular chemical. These include dose (including frequency of exposure), age, sex, hormonal status, genetic background, immunological factors, specific chemical modifiers other than promoters and cocarcinogens, nutritional factors, and route of entry. The importance of these factors in carcinogenic process is briefly described in the following sections.

Table 4.7 in Foods

Procarcinogens (Secondary Carcinogens) Found

Carcinogen Polycyclic aromatic hydrocarbon Nitrosamines

Cycasin Pyrrolizidine alkaloids Aflatoxins Sterigmatocystin Luteoskyrin Pesticide residues Saccharin Safrole Estragole Tannins Psoralens

Food Smoked and broiled products, vegetable oils, margarine, mayonnaise, tea, coffee, cereals, shellfish Fried bacon; fried, salted, and fresh fish; cheese; mushrooms; luncheon meat; Danish pork; Hungarian salami Cycad products Cereal flour contaminants Peanuts and other agricultural products; milk and dairy products Cereals, flour, legumes Rice All types of foodstuffs Diet beverages and other products Sassafras tea and various spices Certain spices and flavorings Tea, wine Parsnip, citrus fruits, spices, flavors

Dose Similarly to toxicants, carcinogens possess dual toxic responses depending on the dose: the immediate or acute response and the chronic or delayed, carcinogenic response. Two observed properties of the carcinogenic response that are dependent on the dose are latency and tumor incidence. Latency, also known as specific tumor induction time, is defined as that period from the initial exposure to the carcinogen to the appearance of the first tumor in the exposed population (Concon, 1988). Within certain limits, latency is an inverse logarithmic function of the dose. It is sometimes expressed as the median latent period, which is the time after exposure when tumors develop in 50% of a given population. In contrast, mean latency is the arithmetic average of the individual latencies. The latency may vary from a few weeks to several years. Tumor incidence is defined as that part or percentage of the surviving exposed population in whom tumors develop at a predetermined time or age. In experimental oncology, the effective group is defined as the number of surviving animals at the time the first tumor appears, or the number of animals surviving up to the time of the mean latent period (Williams, 1984). Although the dose-latency and dose-tumor incidence phenomena have been demonstrated mostly with experimental animals, similar observations have been reported

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from epidemiological studies involving human beings. Unlike in animals with short life spans, in humans the reported latency induced by occupational carcinogens ranges from a short 10 months for epithelioma formation from pitch to as long as 50 to 60 years for the less carcinogenic shale and mineral oils (Henry, 1947). The latency of tobacco-induced lung cancer has been estimated at 20 or more years (Cairns, 1975). The dose-tumor incidence phenomenon in humans has been more thoroughly studied for tobacco-induced cancers. This effect has been clearly demonstrated in both prospective and retrospective studies: i.e., the greater the number of cigarettes smoked, the greater the risk for lung cancer (USPHS, 1964, 1971, 1972, 1973). Age There are three aspects of the effect of age on the carcinogenic process: 1. 2. 3.

Specific sensitivity of the young, especially the fetus and neonate, to a carcinogen Decreased sensitivity of older persons Age-related prevalence or increase in tumor incidence among older persons

Sensitivity of the very young, especially the fetus and neonate, to a carcinogen has been well documented in experimental animals (Della Porta and Terracini, 1969; Toth, 1968; Williams and Weisburger, 1986; Concon, 1988). The fetal brain of rats is about 60 times more sensitive to carcinogens than that of the adult. There is also evidence for placental transfer of carcinogens. Fetal induction of cancer in humans was also evident in the form of a rare vaginal cancer in prepubertal girls born to mothers treated with the synthetic estrogen, diethylstilbestrol (Miller, 1971, 1973). The increased sensitivity of the fetus and the neonate to carcinogens has firm molecular and cellular bases. The developing young tissues have higher levels of cellular replication. As shown in adults, this could, in part, increase the sensitivity of the very young to carcinogens. Similarly, enzymes involved in drug metabolism are usually at very low levels in the young (Brodie and Gillette, 1971). Thus, detoxification of chemical carcinogens is slow. The other age-related aspect of carcinogenesis is the observed high incidence of cancer with increasing age. This, of course, is related to the latency period. The events leading to cancer begin very much earlier in life and have been postulated to be specific mutations (Cairns, 1975). These mutational events may be initiated in the cell or its progeny at any time during the life of the individual. Therefore, the probability of occurrence of a mutation that includes neoplastic transformation increases with age. The cancer ob-

served later in life may in fact have been initiated at a very early age. It is therefore important that exposure of children, especially of newborns, to carcinogens be minimized. Sex Epidemiological data show that some types of cancer occur more frequently in one sex than the other (ACS, 1986; Schottenfeld and Fraumeni, 1982). The sensitivity of different sexes toward the development of neoplasia may be related to their different personal habits, occupations, and other sex-related factors. Besides the obvious hormonal differences in males and females, other sex-linked biochemical differences may account for the greater cancer susceptibility in one sex than in the other. Hormonal Influences Hormones are secreted in response to external or internal stimuli, or according to predetermined rhythms or cycles. Specialized hormone-producing glands include the hypothalamus, pituitary, thyroid, pineal, pancreas, adrenals, parathyroids, ovaries, and testes. Hormone-producing cells are also located in the GI mucosa and other tissues. Hormones regulate certain critical or rate-limiting reactions in metabolic and physiological processes. Thus, they have far reaching influences in metabolic activities of cells. Their influence in carcinogenesis is therefore to be expected. This is particularly true in tissues, such as mammary gland, that are highly dependent on hormonal regulation (ACS, 1983). Pituitary, adrenal, thyroid, and insulin hormones also play an important role in causing cancer (Concon, 1988). Genetic Influences Whether all types of cancer or their susceptibilities are heritable is still questionable. Most studies on the metabolism of carcinogens show that the metabolic activation of a procarcinogen is a prerequisite for carcinogenicity. This observation immediately points to a possible variation in this capacity among individuals, ethnic groups, or races. Cancer susceptibility presumes, in part, a heritable condition in which the individual responds to an exposure to a chemical carcinogen by development of a neoplastic growth. In contrast, in those who do not possess the heritable susceptibility cancer does not develop, in spite of exposure to the carcinogen. The hereditary susceptibility or resistance to cancer may take the form of immunological (Gatti and Good, 1970, 1971), hormonal (ACS, 1983), and other biochemical characteristics, including inducibility of metabolic enzymes (Kellerman et al., 1973).

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Immunological Factors The immune mechanism appears to have a significant effect on carcinogenesis. The aberrant, cancer-prone cells are held in check by the natural immunity of the body. When this natural immunity fails, for one reason or another, neoplastic growth is manifested. The increased incidence of malignancy in persons with immunodeficiency is well documented in the literature. Gatti and Good (1971) estimate that in persons who have primary immunodeficiency diseases, the frequency of malignant neoplasms is 10,000 times that in the general age-matched population. Chemical Modifiers A number of exogenous chemicals can alter the course of the carcinogenic process. Some of these substances enhance the induction, promotion, and growth of tumors, whereas others either inhibit or retard the carcinogenic process or reverse the growth of tumors. Three general types of substances that enhance the carcinogenic process are known: promoters, cocarcinogens, and syncarcinogens. Promoters and cocarcinogens have been described earlier. A chemical syncarcinogen acts additively or synergistically with another carcinogen in inducing a tumor in the same target organ(s). In contrast, promoters and cocarcinogens are noncarcinogenic or, at most, weakly so. Two types of inhibitory substances are known: anticarcinogens and tumor inhibitors. These two terms, often used interchangeably by oncologists, refer to substances that reduce the incidence, increase the latency, or suppress the growth of tumors induced by chemical carcinogens (Homburger, 1974). However, a distinction should be made. A chemical anticarcinogen interferes with the initiation or induction of neoplasm by a carcinogen. In other words, it prevents the neoplastic transformation of a cell. Thus, an anticarcinogen may be expected to act at the enzyme or hormonal level during the initial inductive stages of carcinogenesis or compete with the binding of carcinogens in specific cell receptors. In contrast to the anticarcinogen, the tumor inhibitor interferes with the growth of established tumors or acts only after the cell’s transformation to the neoplastic stage has taken place. A special type of tumor inhibitor is the chemotherapeutic agent that acts on clinically established tumors. These substances may also interfere with the enzyme systems of the tumor, or with the hormones involved in its promotion. Additionally, tumor inhibitors may also enhance the immunological mechanisms. Thus, one may consider the anticarcinogens as preventive or prophylactic agents, whereas the tumor inhibitors are therapeutic agents.

These chemicals may inhibit tumorigenesis by one or more of the following mechanisms: 1.

2.

3.

4.

5.

6.

Promotion of increased rates of biosynthesis of microsomal enzymes and consequent detoxification of carcinogen, e.g., 3-methylcholanthrene (3-MC), phenobarbital, and 9,10-dimethyl-1,2benzanthracene (DMBA) Competition with specific procarcinogenactivating chemical groups or metabolites, e.g., acetanilide (Weisburger et al., 1972) and 8hydroxyquinoline (Yamamoto et al., 1971) Interference with hormones involved in the initiation of the tumor induced by the carcinogen, e.g., interference of analog I with gonadotropinreleasing factor (Johnson et al., 1976), of goitrogens with thyroid hormones (Concon, 1988), and of 4-hydroxypropiophenone with pituitary hormones (Baba, 1957) Enhancement of the rate of excretion of a specific metabolite, e.g., phenobarbital (Wyatt and Cramer, 1970) Interference with the binding of the carcinogen with a specific cell component, and thus with the reaction with critical cell receptors, e.g., 7,8benzoflavone inhibition of DNA and RNA and protein binding of several carcinogens (Kinoshita and Gelboin, 1972) Inhibition of the enzymes involved in the metabolic activation of the carcinogen, e.g., inhibition of the early stages of tumor formation induced by DMBA and β-propiolactone by actinomycin D at doses that do not cause skin damage (Hemings et al. 1968).

Nutritional Factors Nutritional factors can be expected to affect tumor initiation and promotion in several ways: 1.

2.

3.

Adequate supply of nutrients is needed for the energy and metabolic requirements of the rapidly proliferating tumor mass, particularly for the biosynthesis of tissue components such as proteins. Certain vitamins and trace metals that are components of coenzymes and cofactors, respectively, are required by the enzymes that either activate or inactivate carcinogens and toxicants. The presence of certain nutrients, especially antioxidants, may inhibit the formation of carcinogens in vivo from innocuous precursors. Other nutrients, especially the transition metals, may

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4. 5.

6. 7.

enhance the induction or facilitate the promotion of tumors. Nutrients by themselves may be precursors of carcinogens formed in vivo. Fats may act as solvents of carcinogens, facilitating their absorption, transport, and storage in tissues. Nutritional status may also affect immunological competence and thus tumor development. The nature of the diet may also influence the type and quantity of intestinal microflora, which may produce carcinogens from bile acids and sterols.

Several dietary components, especially lipids, are capable of generating active oxygen radicals or superoxides (Deshpande et al., 1995). Additional support for this possibility includes studies of the effect on cancer induction by dietary fat, antioxidants, and increasing age. Because of the strong involvement of free-radical reactions in carcinogenesis, there is increasing interest in the protective effect of food antioxidants. In this regard, vitamins E and C and carotenoids appear to have a beneficial effect in the prevention of cancer. 4.1.5 Therapy Current strategies for cancer therapy are based upon a large body of empirical clinical observations that are unrelated to the genetic abnormalities that produce the cancers. The strategies do not address the underlying causes of disease but instead use methods that have worked in the past to remove or kill as many cancer cells as possible while minimizing damage to normal tissue. This approach achieves its greatest success when surgery can be employed to eradicate a tumor that has not metastasized from a primary site, particularly a site that can be sacrificed without major disability, such as a segment of skin, colon, or breast. Substantial rates of cure, with only modest degrees of disfigurement, are also possible with localized tumors in more critical organs, especially when the tumors are known to be particularly sensitive to the cell-killing effects of irradiation. Chemotherapy is often used in combination with surgery or irradiation to eliminate surviving cancer cells, especially potential metastases. Even when widespread and unsuitable for local surgery or irradiation, some tumors, particularly certain leukemias, lymphomas, and sarcomas, may respond extremely well to chemotherapy. The drugs currently in use are chosen, often in combination, because clinical experience has shown each drug or combination to be the most effective in killing certain types of cancer cells. Most of

these drugs are the so-called metabolic inhibitors, which interfere with the ability of any growing cell to make more DNA or to divide its chromosomes into daughter cells. Methotrexate, for example, inhibits the enzyme dihydrofolate reductase, required in all cells for the production of the deoxynucleotides used to synthesize DNA. Other agents, such as vincristine, bind to the cytoskeletal components of the mitotic spindle and thereby prevent chromosome separation during cell division. Yet other types of drugs, such as adriamycin, wedge themselves between the nucleotide bases in DNA or attack the bases chemically. As a result, they may either break the strands or modify the information content. Many of these drugs enter the body in an inactive form and must be acted upon by some of the same systems required to activate the chemical carcinogens. This is not surprising since carcinogens and chemotherapeutic agents often have common properties and mechanisms. Indeed, many drugs used in chemotherapy are mutagenic and inherently carcinogenic. Irrespective of their mode of action, the drugs used for chemotherapy do not attack the cancer cells with great precision and hence are also toxic to normal tissues, particularly bone marrow, hair follicles, and mucosal surfaces of the mouth, throat, and intestine. These are the places where the tissue integrity depends upon constant cell growth and division. Thus hair loss, sores, diarrhea, and depletion of the normal cellular components of the blood routinely accompany chemotherapy. Resistance to chemotherapy often appears during the course of treatment because cancer cells develop additional mutations that make them impervious to drug molecules. These mutations may change the amino acid sequence of enzymes the drugs are intended to inhibit or amplify a gene whose protein product, in excessive amounts, pumps the drugs out of the tumor cell. Regardless of the mechanism, the resistant cell has achieved a growth advantage over its siblings in the presence of anticancer drugs. Ultimately, such cells and their progeny proliferate and become, by natural selection, the predominant components of the cancer. Several new technologies are currently being investigated to exploit our new understanding of cancer cells. One such approach is targeted drug delivery, which uses distinctive properties of tumor cells to ensure that cytotoxic agents are delivered preferentially to the cancerous target cells rather than to normal cells. Often such an approach takes advantage of the appearance of novel or unusually abundant proteins on the cancer cell surface. Lethal molecules such as bacterial toxins linked to antibodies or ligands that bind to specific cell surface proteins

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can then be concentrated on cancer cells, damaging or killing them but affecting other cells minimally. Another approach involves interfering with a signaling pathway that displays exaggerated activity in a cancer cell. The growth of the cancer cells thus can be slowed, for example, by blocking an overproduced autocrine growth factor, inhibiting an overactive protein tyrosine kinase, switching off a mutant ras protein that is frozen in the active state, or reversing the effects of an overactive transcription factor. One drawback of such an approach is that the pathways to be inhibited may also be crucial to the well-being of normal cells, so any drugs effective against tumors are likely to be just as toxic to normal cells. Yet another novel approach to cancer therapy is to replace the functions normally supplied by a tumor suppressor gene that has been inactivated during malignant development. In principle, the simplest strategy is to substitute for the inactive tumor suppressor gene by infecting the cancer cells with a virus carrying a normal copy of the gene. The cancer cells should respond by returning to a more normal pattern of growth. Finally, there is renewed interest in the topic of immune response to cancers. Arguments about whether the immune system functions as a surveillance device that defends an organism against the development of many cancers over a lifetime have raged for years without satisfactory resolution. There is clear evidence, however, that immune cells are commonly present in tumors and that they are often directed against antigens displayed on the tumor cells. Some of these immune cells can damage or even kill the antigen-displaying tumor cells. Already several biotechnology companies are developing and/or marketing antibody-based drugs for cancer treatment. Some commercial successes are mentioned in the following discussion. Rituximab (Rituxan), developed by Idec Pharmaceuticals, San Diego, in collaboration with Genentech, Inc., is the first monoclonal antibody found to be effective and safe for the treatment of cancer in the United States. Rituxan is indicated for the treatment of patients with relapsed or refractory, low-grade or follicular, CD20positive, B-cell non-Hodgkin’s lymphoma (NHL), which is cancer of the lymphatic system. This disease is fatal in the large majority of patients. Rituxan is genetically engineered from portions of mouse and human antibodies and is produced through recombinant DNA technology. Monoclonal antibodies such as Rituxan are derived from a single clone of antibody-producing cells and bind only to one antigen. Rituxan binds specifically to the CD20 antigen, a molecule present on the surface of the normal and abnormal pre-B and mature B cells. More than 90% of B-cell NHL express CD20.

Once bound to B cells, Rituxan induces lysis (dissolution or destruction of the cell) through several proposed mechanisms based on in vitro data. Rituxan is believed to work with elements of the human immune system to kill CD20+ B cells through antibody-dependent cytotoxicity (ADC) and complement-dependent cytotoxicity (CDC). Through in vitro experiments, Rituxan’s binding has also been shown to induce apoptosis (programmed cell death). B-cell recovery begins at approximately 6 months after completion of treatment, and median B-cell levels return to normal by 12 months. Trastuzumab (Herceptin), developed and marketed by Genentech, Inc., South San Francisco, is the first humanized monoclonal antibody for the treatment of breast cancer. As a single agent, it is indicated for the treatment of patients with metastatic breast cancer whose tumors overexpress the human epidermal growth factor receptor2 (HER2) protein and who have received one or more chemotherapy regimens for metastatic disease. Herceptin in combination with paclitaxel is indicated for treatment of patients with metastatic breast cancer whose tumors overexpress the HER2 protein and who have not received chemotherapy for metastatic disease. Herceptin should only be used in patients whose tumors have HER2 protein overexpression. In 25% to 30% of women with metastatic breast cancer, there is a genetic alteration in the HER2 gene that produces an increased amount of the growth factor receptor protein on the tumor cell surface. This HER2 protein overexpression is associated with more aggressive disease. Recently, ImClone Systems, Inc., New York, has filed for FDA approval of IMC-C225 antibody for the treatment of colon cancer. This antibody will likely also be effective against a variety of other cancers, since it works on a very important cell-signaling pathway. One of the most important cellular signals produced by the oncogene is epidermal growth factor (EGF). Normally in short supply, EGF is found in excess amounts in up to half of all types of malignant tumors. Whereas the surface of a normal cell may have about 10,000 EGF receptors, cancer cells can have 1 million or more. EGF binds to its receptors on the surface of a cell and then triggers a cascade of enzymes inside that play an important role in keeping the tumor alive, well nourished, and spreading. The IMC-C225 antibody identifies and locks onto the receptors of a cancer cell before EGF can reach it. The growth enzymes thus are not activated, and the cell eventually stops dividing. The recent elucidation of the human genome will definitely open new vistas in cancer research at the basic genetic level. It will deepen our understanding of the multitude of genes involved, the various ways in which their

Copyright 2002 by Marcel Dekker. All Rights Reserved.

products affect growth, and the several pathways that lead to cancers in different tissues. New ideas about prevention, risk assessment, detection, diagnosis, and therapy based upon these glimmerings of comprehension will begin to emerge. The mechanisms that go awry in the development of a cancer cell are central to many biological processes, and insights gained into the lives of cancer cells will enhance our knowledge of many different aspects of human biology, including our normal development, evolutionary origins, immune defenses, even aging and death. Yet a realistic view of the causes of cancer and their links to fundamental aspects of life, the mutagens in our environment and the genes required for normal growth and development, suggests that cancer is intrinsic to multicellular life and that the total eradication of cancer from our species is implausible.

4.2

MUTAGENESIS

Mutagenesis is the phenomenon in which inheritable traits result from alterations of DNA. Although it is a naturally occurring process giving rise to diversity in species, most mutations are harmful. The toxicants that cause mutations are known as mutagens. Physical and chemical agents known to produce such alterations in the DNA include ionizing radiation, sulfur and nitrogen mustards, epoxides, ethyleneimine, and methylsulfonate. 4.2.1 Types of Alterations in the Genetic Material In general, the alterations in the genetic material can be divided into four categories. Base-Pair Transformations (Point Mutations) In base-pair transformation, a base, either a purine or a pyrimidine, may be replaced by another base. Base-pair transitions involve the replacement of one base with another of the same type, e.g., replacement of a purine by another purine. If a purine replaces a pyrimidine, it is termed a basepair transversion. These point mutations can occur in at least three ways: by chemical modification, by incorporation of abnormal analogs into the DNA, and by alkylating agents. Base-Pair Addition or Deletion The complete removal or incorporation of a base-pair is more serious than a point mutation. Here the order of the genetic code is altered and, hence, the reading from that base code is shifted. These are known as frameshift muta-

tions. If the addition of another base follows in close proximity to a deletion, the production of functional or partially functional proteins may occur. Some chemicals, such as acridine, are known to induce frameshifts. In addition, errors occurring during chromatid cross-over may also lead to frameshift mutations. Large Deletions and Rearrangements (Chromosomal Mutations) Breakage of the complete DNA molecule may occur at one or more sites. This may then be followed by reconstitution of the molecule. One result of this may be erroneous reconstitution, leading to a large change in the genetic code. This type of mutation may also occur at the level of the chromosome when segments become inverted. Both mono- and bifunctional alkylating agents cause chromosome breakage, the latter type probably by causing cross-linking across the DNA molecule. Inhibition of DNA repair may be another cause of this type of mutation. Unequal Partition or Nondisjunction (Genome Mutations) An unequal division of chromosomes during mitosis may sometimes occur. This may be the result of exposure of the cell to agents that damage or disturb the spindle fibers or interfere in some way with the process of cell division. The result is a cell with more or fewer chromosomes than normal, which may or may not be viable. Thus, in polyploidy, more than one set of chromosomes is present, whereas in aneuploidy, there are either missing or extra chromosomes in a set. However, the extra chromosomes are not sufficient to make an entire additional set of chromosomes. An example of an aneuploid condition with an extra chromosome is trisomy in mongolism. Thus there are two types of mutagens: those acting directly on DNA and those acting on the replication or the partition of chromosomes. Mutagens of the latter type may therefore only be effective at certain times in the cell cycle. Also, some mutagens may not be able to cross the nuclear membrane and are therefore only active at mitosis, when the nuclear material is in the cytoplasm. Mutations can occur in both somatic and germinal cells. However, because of the transmissibility of the mutated traits to several generations, deleterious mutations in the germinal cells have far-reaching implications to the future generations. In these cases, a cure for an inherited disease may be almost impossible. Germinal mutations, however, can be lethal to the cell, in which case, hereditary transmission of a deleterious mutation is averted.

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4.2.2 Mechanisms of Mutagenicity Mutagenesis underlies the process of carcinogenesis in that oncogenes are derived from their normal cellular counterparts, protooncogenes, via mutation (Bishop, 1991; Holstein et al., 1991; Vogelstein and Kinzler, 1992). Spontaneous and mutagen-induced processes are likely to contribute to carcinogenesis. Different mutagens appear to induce different kinds of mutations preferentially: i.e., they show a different mutagenic specificity. For a chemical (or radiation) to cause a cell to become tumorigenic, it should react readily with DNA to give DNA adducts by binding to it covalently, and these adducts or their breakdown products must be efficient in causing mutations. DNA adducts are generally regarded as an important determinant in mutagenesis. Mechanisms of mutagenicity involving DNA adducts can be placed in one of three categories (Table 4.8): 1.

2.

3.

Table 4.8

A misinformational mechanism implies that a DNA polymerase attempts to “read” the base moiety of a DNA adduct but misinterprets it. A noninformational mechanism implies that a DNA polymerase encounters an adduct that is uninterpretable and chooses to incorporate a particular deoxynucleoside triphosphate (dNTP) for reasons other than an attempt to “read” the adduct. Reasons for choosing a particular dNTP could range from its being dictated by an inherent property of the DNA polymerase to the formation of a DNA structure that best can be accommodated by the active site of a DNA polymerase. Other mechanisms constitute a general category that includes all mechanisms that do not involve DNA polymerase bypass of primary adducts and include (a) the possibility that many lesions are processed to a common intermediate that is then the mutagenic species, and (b) mechanisms in which DNA polymerase is not involved in the

Potential mutagenic mechanisms

Misinformational 1. Chemical perturbations a. Adduct-induced base tautomerization b. Adduct-induced base ionization 2. Structural perturbations a. Adduct-induced base rotation b. Adduct-induced base wobble Noninformational Other

fixation of the mutation (e.g., mutagenic mechanisms involving topoisomerases). Mutations involving misinformational lesion can, in principle, be divided into several categories (Table 4.8). First the mutagenic moiety of an adduct may induce a chemical perturbation in the base moiety of the adduct, which may improve the probability of misreplication. Second, the mutagenic moiety of the adduct also may affect the position of the base moiety of the adduct, resulting in a structural perturbation that might improve the probability of misreplication. These possibilities are briefly discussed later. Adduct-Induced Base Tautomerization The alkyl group in O6AlkGua adduct chemically locks the guanine moiety of the adduct in its enol (i.e., imidate ester) tautomeric form. This is the classic example of adduct-induced base tautomerization (Topal and Fresco, 1976). Because O6AlkGua, at least when the methyl group is anti with respect to N1 position, can base pair with thymine, O6AlkGua adducts might induce G→A transition mutations (Leonard et al., 1990; Loechler 1991). Adduct-Induced Base Ionization Adduction at certain atoms can perturb the pKa of the base moiety of the adduct inductively (Topal and Fresco, 1976). For example, N7-guanine adducts are formed positively charged; therefore, they lower the pKa of the N1 proton. This effect in turn increases the probability that N1 is deprotonated, and deprotonation may increase the probability that base pairing will occur between the zwitterionic adduct and lead to base pairing with thymine during replication. This mechanism has been proposed for aflatoxin B1 mutagenesis (Sambamurti et al., 1988; Sahasrabudhe et al., 1989). Adduct-Induced Base Rotation Adduct-induced base rotation involves anti to syn base rotation. The major 2-AF adduct (AF-C8-guanine) of 2-aminofluorine (2-AF) was shown to form a base pair with adenine after an anti to syn base rotation (Norman et al., 1989) and may be related to the mechanism by which 2-AF induces GC→TA transversion mutations (Reid et al., 1990). Adduct-Induced Base Wobble A second example of an adduct-induced structural perturbation is adduct-induced base wobble. By this mechanism, the mutagenic residue perturbs the position of the base

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moiety of the adduct with respect to the helix axis. No firmly established example of this mechanism exists, although it has been offered as one possible mechanism by which cis-thymine glycol might induce T→C mutations (Basu et al., 1989). Noninformational Lesions Although difficult to define precisely, the term noninformational lesions implies that such lesions are uninterpretable when encountered by DNA polymerase and that rules other than simple base-pairing schemes govern which base is incorporated opposite the lesion. Noninformational lesions share several characteristics: 1.

2.

3.

They block the progress of DNA polymerase in most primer extension studies in vitro (Strauss, 1989). They are not mutagenic in bacteria in the absence of the induction of the SOS response (Loeb and Preston, 1986). Adenine (i.e., dATP) appears to be incorporated preferentially opposite noninformational lesions (Strauss, 1989).

No satisfying direct mechanism(s) has been proposed to account for these apparent characteristics of noninformational lesions. 4.2.3 Mutagenic Agents Most of the chemical mutagens can be grouped into seven general classes of compounds (Freese, 1971): (a) alkylating agents, (b) free radical–forming agents, (c) purine and pyrimidine analogs, (d) oxidizing agents, (e) DNA synthesis inhibitors, (f) intercalating agents, and (g) metals. Alkylating agents, free radical generators, oxidizing agents, and metals are widespread in foods. The others have more or less limited distribution. Alkylating Agents Alkylating agents are mutagens that react readily with nucleophilic groups: amino, sulfhydryl, thio ester, and ionized acids. Their alkylation of phosphate groups and ring nitrogens in the purine and pyrimidine bases, however, is the basis for their mutagenic effects. As mentioned earlier, the N7 position of the guanine in double-stranded DNA, or the N1 of adenine in single-stranded or denatured DNA, appears to be the principal target. After alkylation, guanine or adenine is hydrolyzed off, thus destabilizing the DNA backbone and breaking the macromolecule. Alkylation of the phosphate groups also causes the DNA

backbone to break. Therefore, alkylating agents cause point mutations (mainly transitions), chromosome breaks, and chromosome mutations. These chemicals, therefore, are extremely toxic and have been implicated as teratogens and carcinogens. Examples of alkylating agents commonly found in the human food chain include cycasin, nitrosamines, and epoxides, which are the metabolic intermediates of the polycyclic aromatic hydrocarbons as well as aflatoxins; reactive acetones (aflatoxin and other mycotoxins); and organic residues such as the pesticides endrin, dieldrin, ethylene bromide, and Aramite.

Deoxyribonucleic Acid Synthesis Inhibitors Deoxyribonucleic acid synthesis inhibitors are compounds that prevent the incorporation of specific groups into DNA. Most noteworthy are the folic and glutamic acid antagonists used in cancer chemotherapy. In foods, such compounds are rarely found. Deoxyribonucleic Acid Intercalating Agents Intercalators may cause frameshift point mutations, which are deletions or insertions of one or more bases. Metals

Free Radical Formers Free radical formation is the basis of DNA alkylation reactions. Free radicals can damage DNA directly and indirectly. Direct damage appears to be a remote possibility because it can occur only when the free radicals are formed adjacent to the DNA and protective mechanisms (e.g., catalases and peroxidases, tocopherols, and other free radical scavengers) cannot interfere. Indirect damage is more likely because less interference occurs. It can follow formation of reactive compounds. Free radicals can react with each other and produce reactive compounds that can then diffuse to DNA and inflict damage. Ionizing radiation (x-, α-, β-, and γ-rays) generally affects chromosomes by formation of free radicals, but seldom by a direct hit. This is because the chromosomes are in an aqueous medium that shields against ionizing radiation. Purine and Pyrimidine Analogs Certain types of bases that are not normal constituents of the DNA and RNA can be incorporated into nucleic acids. Their incorporation results in a transition error or point mutation, for example, a substitution of an A-T pair for a G-C pair happens in the next replication. Such base analogs can also cause chromatid and chromosome breakage. Thus the purines, theophylline and theobromine, xanthines, and especially caffeine have all been shown to induce mutagenic effects in both bacterial and human cells in vitro (Concon, 1988). Oxidizing Agents The most notable oxidizing agent to cause mutation is nitrous acid, which can deaminate cytosine to uracil, adenine to hypoxanthine, and guanine to xanthine. These deaminations are mutagenic, resulting in base-pair transitions. Xanthine cannot pair with either thymine or cytosine, and this inability to pair bases inactivates the DNA (Freese, 1971).

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Some heavy metals, such as Co, Ni, Cr, Zn, Mn, and mercurials, can produce mutations in the DNA. Their mechanisms are not fully understood but may involve free radical formation from peroxides. 4.2.4 Deoxyribonucleic Acid Repair in Mutagenesis At least two major cellular processes, DNA replication and DNA repair, have been identified as important determinants in the fixation of spontaneous and chemically induced mutation. One mechanism hypothesized to result in fixation of mutation is misreplication. As described earlier, this may be caused by spontaneous or chemically induced template alterations, such as modification of the base structure, formation of rare base tautomeric forms, depurination, deaminations, or looping out of bases from helix. Several modes of DNA repair have been identified and studied in both prokaryotic and eukaryotic organisms. Some damaged bases, such as thymidine dimers, can be fixed by a group of excision repair enzymes. These enzymes produce a nick near the lesion, excise an area of DNA containing the lesion, resynthesize the DNA, and then ligate (seal) the gap between the newly repaired region and the preexisting DNA on the same strand. Another form of excision repair is performed by glycosylases that catalyze the excision of a single base from the sugar backbone (Lindahl, 1979). Other modes of repair, which may be either inducible or constitutive, have been postulated to excise mismatched bases, correct errors that follow replication, or involve genetic recombination. Uncharacterized repair systems also exist to correct single-strand breaks, doublestrand breaks, cross-linked DNA strands, and other damage. These examples illustrate the complexity and variety of DNA repair systems. Mutagenesis shows a number of characteristics in common with carcinogenicity and some with teratogenesis

and reproductive toxicity. For example, the frequency of the toxic effect is related to the extent of exposure but not necessarily the severity: i.e., the extent of the toxic effect may be related to the type of alteration that has occurred rather than the degree of exposure. Another similarity to carcinogenicity is that mutagenicity may cause its effects through the reproduction or proliferation of abnormal cells rather than loss of function, as is common in organ toxicity. A third similarity is the possible absence of a threshold. It is conceivable that a single mutational event may lead to overt toxicity. The carcinogenic potency of a compound is correlated with its mutagenic ability, suggesting that DNA is the ultimate target of carcinogenic initiation. Indeed, many chemicals known to be carcinogenic have been found to be mutagenic as well; likewise, many known mutagens have been found to be carcinogenic. Because of the high correlation of mutagenicity of chemicals to carcinogenic potential, the testing of chemicals as mutagens has now become an important screening tool in assessing potential carcinogenic risk of compounds. Teratogenesis can also result from a mutagenic event. In this situation, the genetic material of a cell is altered so that when it divides and multiplies, it leads to formation of an organ or tissue that cannot function properly. It is also possible that a mutagenic event can affect molecules that are necessary for normal differentiation and so may interfere with the correct development of the embryo or fetus. Mutagenesis, however, is not required for teratogenesis. Furthermore, mutagenic changes are distinguished from teratogenic responses in that the former are transmissible to future generations, whereas the latter are confined to a single generation. Whereas teratogens inflict damage on separate individuals of a single generation, mutagens may adversely affect several generations of unborn humans. Mutagens can damage the human gene pool, and the magnitude of this effect, though broad and potentially long lasting, is unknown.

Table 4.9

TERATOGENESIS

Teratogenesis is the formation of birth defects in offspring, often as a result of maternal or paternal exposure to a toxicant. These abnormalities can arise in a number of ways but most commonly result from alteration of the developing cells, which leads to improper functioning of these cells or interference with differentiation so that the proper cell types do not form or do not form in the proper number or location. Teratogenic effects are thus initiated in two ways: through gene or chromosome mutation in germinal cells and through alterations or interference with the normal processes of the developing embryo. It is possible that the types of events that result in teratogenesis also may result in death of the embryo or fetus. The time of greatest susceptibility to teratogens, as far as the induction of gross anatomical defects, occurs during the period of germ-layer formation and organogenesis (Wilson and Fraser, 1977; Levi, 1987). In humans, this corresponds to approximately 20–56 days after conception. The type of teratogenic response is determined by the developmental stage of the fetus at the time of exposure: i.e., there are “critical periods” for different malformations of organ systems. Because the early events in organ formation are the most sensitive, during testing the teratogen is administered during or just before the development of that organ. Considerable similarity exists in the timing of preimplantation development across several mammalian species, regardless of the total length of gestation (Table 4.9). During this period, biochemical changes in the uterine endometrium controlled by progesterone and estrogen result in the development of endometrial sensitivity to the blastocyst. The preimplantation embryo appears to be susceptible to lethality but rarely to teratogenicity with chemical insult. After implantation, organogenesis takes place. The division, migration, and association of cells into primitive organ rudiments characterize this period. The basic structural templates for organization of tissues and organs are

Timing of Early Development in Some Mammalian Speciesa

Species Mouse Rat Rabbit Sheep Monkey (rhesus) Human a

4.3

Blastocyte formation

Implantation

Organogenesis period

Length of gestation

3–4 3–4 3–4 6–7 5–7 5–8

4–5 5–6 7–8 17–18 9–11 8–13

6–15 6–15 6–18 14–36 20–45 21–56

19 22 33 150 164 267

Developmental ages are days from the time of ovulation.

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established on the molecular, cellular, and morphological levels. This period is the most susceptible to chemical insult in inducing structural birth defects. Within the organogenesis period, individual organ systems possess highly specific periods of vulnerability to teratogenic insult. Most human teratogens influence the development of several organ systems and induce syndromes of malformations rather than just single anomalies. According to Becker (1975), no teratogenic effects can occur after the end of organogenesis. Chemical teratogens can initiate these changes only during the formation of tissues, cells, and physiological and biochemical systems. This conclusion is based on the observation that no major congenital malformation is evident after the completion of organogenesis, especially the last trimester of intrauterine life. Teratogens, however, can cause damage during this period even though gross malformations are not observed. These effects may only be recognized after the appearance of functional abnormalities after birth (Langman et al., 1975). 4.3.1 Mechanisms of Action Because the initiating events of abnormal development usually occur at the subcellular or molecular level, the

Figure 4.2

damage is not readily detected until cell death, morphological damage, or functional disability is observed. Probably 8–10 mechanisms are responsible for the initial molecular damage (Figure 4.2). Despite the influence of time of exposure and the complex interaction between maternal toxicity and embryolethality on pregnancy outcome, teratogens display specificity of action in a dose-dependent manner. The most common modes of action are now briefly described. Cytotoxic Teratogens The majority of well-known developmental toxicants produce both malformations and embryolethality. Such a pattern or response is typical of agents that are cytotoxic to replicating cells via alterations in replication, transcription, translation, or cell division (Manson, 1986). Examples of such chemicals include alkylating agents, antineoplastic agents, and many mutagens. The rationale for the susceptibility of the embryo to these agents is that the rate of cell division is extremely high during the organogenesis period. Low doses of cytotoxic agents administered relatively early in the critical period may produce levels of cell death that can be replaced through compensatory hyperplasia of surviving cells. This process in turn results in the formation of growth-retarded but morph-

Possible mechanisms in the formation of a developmental defect.

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ologically normal fetuses at term. Higher doses adminstered later during the critical period may cause substantial depletion of cell number, leaving insufficient time for replacement prior to the occurrence of critical morphogenetic events. Damage at this stage results in the induction of severe malformations. With cytotoxic teratogens, the embryo is usually far more susceptible than the mother. These agents can induce a full spectrum of malformations. The site specificity is primarily determined by the time of exposure. Those organ rudiments undergoing rapid proliferation at the time of exposure are likely to be the sites of future malformation. Many of the resulting malformations involve reduction deformities, or missing elements, presumably because insufficient cells were available to form the organ rudiment. Teratogens Affecting Differentiation Events Some teratogens disrupt development by highly specific mechanisms of action not involving excessive necrosis or embryolethality. These specific teratogens usually induce a subset of all possible malformations at a given time of exposure, and usually at narrow time points within the organogenesis period. A well-defined structural anomaly or a distinct malformation syndrome results from prenatal exposure to these agents. Generalizations, however, are difficult to make since all agents appear to operate according to their own unique mechanism of action. Some well-known examples of teratogens of this class are thalidomide, glucocorticoids, and nitrofen.

deficiency during pregnancy lead to severe growth retardation, thyroid deficiencies, and delays in CNS maturation that are not reversible with augmentation of the food supply to the neonate (Shrader et al., 1977). Other examples that cause such perturbations include teratogens that reduce the transport of nutrients from the maternal to the embryonic compartment through specific interference with placental function and those causing alteration of uteroplacental blood flow. 4.3.2 Factors Affecting Teratogenicity of Compounds Several factors influence chemical teratogenesis. These include dose and duration of exposure, maternal and fetal regulatory factors, transport and access to the developing embryo, time of exposure during embryonal development, and individual susceptibility. Dose and Duration of Treatment The existence of a teratogenic threshold or no effect level is almost universally accepted. Generally, a single dose of a teratogen is more effective in inducing malformations than multiple exposures (King et al., 1965; Robens, 1969; Concon, 1988). This is because the effective dose is probably decreased since the repeated exposures may induce specific metabolizing enzymes. However, repeated exposures may cause liver and kidney damage, so that metabolic detoxification and excretion of the compound or its metabolites may be inhibited.

Nonspecific Developmental Teratogens Toxins of the nonspecific development teratogen class cause growth retardation and embryolethality without teratogenicity. Examples of this type include the mitochondrial protein synthesis inhibitors chloramphenicol and thiamphenicol (Neubert et al., 1980). Inhibition of cellular processes as fundamental as mitochondrial function is believed to result in nonspecific effects such as overall growth retardation and lethality. There is no basis for target organ susceptibility in the early embryo for perturbation of such a fundamental cellular process, and consequently all tissues appear to be affected to an equal extent. Perturbations in Maternal and Placental Homeostasis Perturbations in homeostatis are embryotoxic through indirect effects on the conceptus that result from alterations in the maternal system. The best examples of these are perturbations that lead to maternal nutritional deficiencies. Generalized malnutrition, caloric restriction, and protein

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Maternal and Fetal Regulatory Factors The ultimate concentration of a teratogen reaching the target embryonic tissue or cells obviously is a function of the physiological mechanisms that tend to reduce this concentration. Thus, the rates of absorption in the GI tract, excretion in the urine and bile, protein binding, and tissue storage are factors that tend to dissipate the concentration of a teratogen before it reaches the embryo (Wilson, 1973). The maternal and fetal metabolic processes, including those of the placenta, also diminish the dose of a teratogen reaching its target tissue. Generally, the initial dose entering the maternal bloodstream and the effectiveness of the metabolic processes of the mother to detoxify these chemicals largely determine the extent and occurrence of teratogenic injury. Transport and Access to Developing Embryo The placenta regulates the concentration of a substance reaching the embryo. Thus, the concentrations of many

harmful chemicals in the fetal fluids and tissues may be lower than those in the maternal plasma. However, the placenta does not exclude many types of toxicants. Time of Exposure During Fetal Development As described earlier, the timing of exposure to a teratogen has a critical influence on the development of malformations. When proliferation and differentiation of tissues and organs are virtually complete, exposure to teratogens may have little effect on the occurrence of gross malformations or embryonic death. However, the toxic compounds may produce growth and functional retardation. Individual Susceptibility A fetus may inherit anatomical, physiological, and biochemical characters that predispose it to teratogenic effects. For example, the absence of critical detoxifying enzymes, such as conjugases, may prevent effective elimination of a teratogen. The so-called polygenic or multifactorial inheritance may provide the necessary characteristics that increase the predisposition toward a specific toxin-induced teratogenic effect (Carter, 1969; Edwards, 1969). 4.3.3 Teratogenic Compounds Many teratogenic compounds are also known carcinogens. These include aflatoxins, aminoazobenzenes, benzpyrenes, cycasin, carbon tetrachloride, pyrrolizidine alkaloids, podophyllotoxin, caffeine, methylmercury, and ethylene thiourea (Concon, 1988). Many mutagens and/or carcinogens generally can be considered as potential teratogens (Connors, 1975). In terms of human developmental abnormality, about 3%–7% of human babies are born with malformations serious enough to require treatment. Among possible causes are genetic causes (mutant gene); chromosomal abnormalities; environmental agents, including ionizing radiation; viral infections (rubella, cytomegalovirus, herpes); maternal metabolic imbalances (endemic cretinism i.e., phenylketonuria, diabetes); and drugs and environmental chemicals (androgenic hormone, alcohol, folate antagonists, thalidomide, oral anticoagulants, methylmercury, etc.); and multifactorial causes (Wilson, 1973). Teratogenesis is usually classified as a chronic effect, although the toxicity appears within a relatively short time as compared to the lifetime of the individual. Indeed, it might more properly be labeled a subacute effect on the basis of time course. However, some of the birth defects that result from toxicant exposure are heritable and may appear in future generations as well as the present one.

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REFERENCES ACS. 1983. Cancer Facts and Figures. American Chemical Society, New York. Baba, T. 1957. Inhibitory effect of p-hydroxypropiophenone (PHP) upon experimental induction of hepatoma in rats fed butter-yellow (DAB). Gann 48:148–158. Barrows, L.R. 1986. Methylation of DNA guanine via the 1-carbon pool in dimethylnitrosamine-treated rats. Mutat. Res. 173:73–79. Basu, A.K., Loechler, E.L., Leadon, S.A., and Essigmann, J.M. 1989. Genetic effects of cis-thymine glycol: Site-specific mutagenesis and molecular modeling studies. Proc. Natl. Acad. Sci. U.S.A. 86:7677–7681. Becker, B.A. 1975. Teratogens. In Toxicology: The Basic Science of Poisons, eds. L.J. Casarett and J. Doull, pp. 221–240, Macmillan, New York. Becker, F.F. 1982. Cancer: A Comprehensive Treatise. 2nd ed., Plenum Press, New York. Berenblum, I. 1954. Carcinogenesis and tumor pathogenesis. Adv. Cancer Res. 2:129–175. Bishop, J.M. 1991. Molecular themes in oncogenesis. Cell 64:235–248. Brodie, B.B. and Gillette, J.R. 1971. Concepts in Chemical Pharmacology. Springer, New York. Cairns, J. 1975. The cancer problem. Sci. Am. 233(5):64–79. Carter, C.O. 1969. Genetics of common disorders. Br. Med. Bull. 25:52–57. Concon, J.M. 1988. Food Toxicology, Parts A and B. Marcel Dekker, New York. Connors, T.A. 1975. Cytotoxic agents in teratogenic research. In Teratology, Trends and Applications, eds. C.L. Berry and D.E. Poswillo, pp. 137–155, Springer Verlag, New York. Della Porta, G. and Terracini, B. 1969. Chemical carcinogenesis in infant animals. Prog. Exp. Tumor Res. 11:334–363. Deshpande, S.S., Deshpande, U.S., and Salunkhe, D.K. 1995. Nutritional and health aspects of food antioxidants. In Food Antioxidants, eds. D.L. Madhavi, S.S. Deshpande, and D.K. Salunkhe, pp. 361–469, Marcel Dekker, New York. Dragan, Y.P., Xu, Y.H., Xu, Y.I., Sargent, L.M., and Pitot, H.C. 1992. Multistage hepatocarcinogenesis in the rat: Insights into risk estimation. In Relevance of Animal Studies to the Evaluation of Human Cancer Risk, eds. R. D’Amato, T.J. Slaga, W.H. Farland, and C. Henry, pp. 261–279, WileyLiss, New York. Edwards, J.H. 1969. Familial predisposition in man. Br. Med. Bull. 25:58–64. Foulds, L. 1954. The experimental study of tumor progression: A review. Cancer Res. 14:327–339. Foulds, L. 1969. Neoplastic Development. Academic Press, New York. Freese, E. 1971. Molecular mechanisms of mutations. In Chemical Mutagens, Principles and Methods for Their Detection, Vol. 1, ed. A. Hollaender, pp. 126–159, Plenum Press, New York.

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Miller, R.W. 1973. New hypotheses on the etiology of cancer. Proc. 7th National Cancer Conference, J.B. Lippincott Co., Philadelphia, PA. Moolgavkar, S.H. and Kundson, A.G. 1981. Mutation and cancer: A model for carcinogenesis. J. Natl. Cancer Inst. 66:1037–1052. Neubert, D., Barrach, H.J., and Merker, H.J. 1980. Drug induced damage to the embryo or fetus. Curr. Top. Pathol. 69:242–324. Norman, D., Abuaf, P., Hingerty, B.E., Live, D., Grunberger, D., Broyde, S., and Patel, D. 1989. NMR and computation characterization of the N-(deoxyguanosine-8-yl) aminofluorine adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn]:A[anti] pair formation and its pH dependence. Biochemistry 28:7462–7476. Pitot, H.C. 1990. Altered hepatic foci: Their role in murine hepatocarcinogenesis. Annu. Rev. Pharmacol. Toxicol. 30:465–500. Pitot, H.C. 1991. Characterization of the stage of progression in hepatocarcinogenesis in the rat. In Boundaries Between Promotion and Progression, eds. O. Sudilovsky, L. Liotta, and H.C. Pitot, pp. 3–18. Plenum Press, New York. Pitot, H.C., Campbell, H.A., Maronpot, R.R., Bawa, N., Rizvi, T.A., Xu, Y.H., Sargent, L., Dragan, Y., and Pyron, M. 1989. Critical parameters in the quantitation of the stages of initiation, promotion, and progression in one model of hepatocarcinogenesis in the rat. Toxicol. Pathol. 17:594–612. Pitot, H.C., Goldsworthy, T.L., Moran, S., Keenan, W., Glauert, H.P., Maronpot, R.R., and Campbell, H.A. 1987. A method to quantitated the relative initiating and promoting potencies of hepatocarcinogenic agents in their dose-response relationships to altered hepatic foci. Carcinogenesis 8:1491–1499. Reddy, J.K. and Lalwani, N.D. 1983. Carcinogenesis by hepatic peroxisome proliferators: Evaluation of the risk of hypolipidemic drugs and industrial plasticizers to humans. CRC Crit. Rev. Toxicol. 12:1–58. Reid, T.M., Lee, M.S., and King, C.M. 1990. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency. Biochemistry 29:6153–6161. Robens, J.F. 1969. Teratologic studies of carbaryl, diazinon, norea, disulfiram and thiram in small laboratory animals. Toxicol. Appl. Pharmacol. 15:152–163. Sahasrabudhe, S.R., Sambamurti, K., and Humayun, M.Z. 1989. Base-substitution mechanisms and the origin of strand bias. Mol. Gen. Genet. 217:20–25. Sambamurti, K., Callahan, J., Perkins, C.P., Jacobson, J.S., and Humayun, M.Z. 1988. Mechanisms of mutagenesis by bulky DNA lesions at the guanine N7 position. Genetics 120:863–873. Schottenfeld, D. and Fraumeni, J.F. 1982. Cancer Epidemiology and Prevention. W.B. Saunders, Philadelphia. Shrader, R.E., Ferlatte, M.I., Hastings-Roberts, M.H., Schoenborne, B.M., Hoernicke, C.A., and Zeman, F.J. 1977. Thy-

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Vogelstein, B. and Kinzler, K.W. 1992. Carcinogens leave fingerprints. Nature (London) 355:209–210. Weisburger, J.H. 1972. On the sulfate ester of N-hydroxy-N-2fluorenylacetamide as a key ultimate hepatocarcinogen in the rat. Cancer Res. 32:491–500. Williams, G.M. 1984. Modulation of chemical carcinogenesis by xenobiotics. Fundam. Appl. Toxicol. 4:325–344. Williams, G.M. and Weisburger, J.H. 1986. Chemical carcinogens. In Toxicology: The Basic Science of Poisons, eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 99–173, Macmillan, New York. Williams, G.M., Dunkel, V.C., and Ray, V.A. 1983. Cellular systems for toxicity testing. Ann. N.Y. Acad. Sci. 407:328–333. Wilson, J.G. 1973. Environment and Birth Defects. Academic Press, New York. Wilson, J.G. and Fraser, F.C. 1977. Handbook of Teratology, Vol. 1 and 2. Plenum Press, New York. Wyatt, P.L. and Cramer, J.W. 1970. Urinary excretion of N-hydroxy-2-acetyl-aminofluorene (N-OH-AAF) by rats given phenobarbital (PB). Proc. Am. Assoc. Cancer Res. 11:83–91. Xu, Y.D., Dragan, Y.P., Young, T., and Pitot, H.C. 1991. The effect of the format of administration and the total dose of phenobarbital on altered hepatic foci following initiation in female rats with diethylnitrosamine. Carcinogenesis 12:1009–1016. Yamamoto, R.S., Williams, G.M., Frankel, H.H., and Weisburger, J.H. 1971. 8-Hydroxyquinoline: Chronic toxicity and the inhibitory effect on the carcinogenicity of N-2-fluorenylacetamide. Toxicol. Appl. Pharmacol. 19:687–698.

5 Biotransformation of Xenobiotics

5.1

INTRODUCTION

The term metabolism is commonly used to describe the biochemical changes that substances undergo in living organisms. In a more restricted sense, it refers to the processes by which chemical compounds are broken down in an organism, largely by enzymatic action, to produce energy and components for the synthesis of biomolecules required for life-sustaining processes. Metabolism thus provides two essentials for life, energy and raw materials. Aside from normal food intake, both humans and animals are also constantly exposed in their environment to a number of chemicals that are foreign to their bodies. Irrespective of whether they have nutritive value, these compounds, often referred to as xenobiotics, are also subjected to metabolic transformations. Such compounds can be of natural or synthetic origin. They may interfere with the normal metabolic pathways, resulting in toxic action. Alternatively, they themselves may be toxic and need to be metabolized to other materials that are usually, though not invariably, less toxic and more readily eliminated from the organism. The term detoxification is often used to describe the metabolism of xenobiotics. However, this expression is misleading because it implies that metabolic transformations invariably involve reduction of toxicity. For example, the parent compound may or may not have toxicological activity, which may reside in its metabolite(s). Metabolism may therefore not necessarily be a detoxification process, in that the metabolites may be more toxic or active than the parent compound. This is particularly true for some chemical carcinogens, organophosphates, and a number of compounds

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that cause cell necrosis in the lung, liver, and kidney. These reactive intermediates are often implicated in the events that ultimately result in cell death, chemically induced cancer, teratogenesis, and a number of other toxic effects. Examples listed in Table 5.1 show the variable success of detoxification during xenobiotic metabolism. Detoxification is achieved very successfully in some cases (e.g., cyanide to thiocyanide, reducing toxicity by a factor of about 200) and less successfully in other cases (e.g., benzoate to hippurate, reducing the toxicity by a factor of only about 2). There are also instances in which the metabolism achieves no detoxification whatsoever or even involves a reverse effect (e.g., conversion of pyridine to methylpyridinium ion, which increases toxicity by a factor of about 6). Several other instances in which metabolic conversion of a xenobiotic chemical results in the formation of products that are more toxic than the original compound are listed in Table 5.2. For these reactions detoxification is indeed a misnomer, and the alternate expression intoxication has occasionally been suggested (Parke, 1968). However, the same metabolic pathways may lead to a decrease or increase in the toxicity of different xenobiotic compounds, and it is often recommended to refer to all such reactions as biotransformations. It is now evident that the determination of the toxicity of any compound that is metabolically transformed is in essence the determination of the toxicity of the parent compound and its metabolites. Among the first biotransformation reactions discovered were hippurate biosynthesis, ethereal sulfate biosynthesis, and glucuronate biosynthesis. Research conducted during the 20th century on xenobiotic metabolism substan-

Table 5.1

Xenobiotic Metabolism with Varying Degrees of Detoxification

Compound –

CN Pyramidon Benzoate p-Aminobenzoate Sulfadiazine Pyridine

LD50, g/kg 0.002 0.25 2 3 1.5 1.2

Metabolite

LD50, g/kg

SCN 4-Aminoantipyrine Hippurate p-Aminohippurate N4-Acetylsulfadiazine N-Methylpyridinium ion

0.4 1.25 4 3 0.5 0.2

–

tially improved our understanding of species-specific differences in the action of pesticides, mutagens, and carcinogens. All biotransformation reactions occurring in animal species extant today are the product of age-long biological evolution. Naturally, reactions that were successful in terms of detoxification conferred a competitive advantage on their bearers, and that natural selection has produced a continuously improving pattern of detoxifying capabilities up the evolutionary ladder. However, this could only be true with xenobiotic compounds that were part of the natural environment. The industrial revolution and accompanying ecological changes produced a multitude of new compounds to which no substantial accommodation could have developed during the relatively short

Table 5.2 Increased Toxicity of Some Xenobiotics Resulting from Metabolic Conversion Compound Sulfanilamide Ethylene glycol Methanol Fluoroacetate Parathion Tremorine Tri-o-cresyl phosphate Dimethylnitrosamine Schradan Heptachlor Pyridine Chloral hydrate Nitrobenzene Acetanilid Pentavalent arsenicals Selenate 2-Naphthylamine Codeine Phenylthiourea

Product Acetylsulfanilamide Oxalic acid Formate Fluorocitrate Paraxon Oxytremorine Cyclic phosphate Diazomethane Schradan-N-oxide Heptachlor epoxide n-Methyl pyridinium chloride Trichloroethanol chloride Nitrosobenzene, phenylhydroxylamine Aniline Trivalent arsenicals Selenite 2-Amino-1-naphthol Morphine Hydrogen sulfide

Source: Compiled from Loomis (1978), Sipes and Gandolfi (1986), and Concon (1988).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

timespan since their introduction. Coping with new environmental chemicals may involve the induction of new enzymes or the modification of preexistent enzymes to deal with new substrates and the gradual elimination of genetic lines incapable of making this adjustment. This may require several generations over a very long timespan. It is thus not possible to predict at present the ultimate effect of industrialization on human biochemical characteristics. However, it is quite likely that sufficient change of the environment necessarily involves alteration in the survival value of certain genetic traits and/or adaptive capabilities. In considering metabolic and biotransformation processes, it is important to keep in mind the pathways of nutrients and xenobiotics in living organisms (Figure 5.1). The gastrointestinal (GI) tract is the region where essentially all ingestion of food, water, and associated contaminants occurs. Environmental and occupational exposure to toxicants, however, may also occur through the respiratory tract and through skin. In general, the more lipophilic compounds are readily absorbed through the skin, across the lungs, or through the GI tract. Constant or even intermittent exposure to these lipophilic chemicals can result in their accumulation within the organism to toxic levels, unless effective means of elimination are present. Biotransformation is the sum of the processes by which a xenobiotic compound is subjected to chemical change by living organisms (Figure 5.2). This definition implies that a particular chemical may undergo a number of chemical changes. It may also mean that the parent molecule is chemically modified at a number of positions or that a particular metabolite of the parent compound may undergo additional modification. The result of the biotransformation reaction(s) is that the metabolites are chemically distinct from the parent compound. Metabolites are usually more hydrophilic than the parent compound. This enhanced water solubility reduces the ability of the metabolite to partition into biological membranes and thus restricts the distribution of the metabolites to the various tissues, decreases the renal tubular and intestinal reabsorption of the metabolite(s), and ultimately promotes

Ingestion

Inhaled air (O2 uptake)

Gastrointestinal tract

Exhaled air (CO2 elimination)

Bile Liver

Pulmonary system (lung and alveoli)

Fecal excretion

Blood and lymph system distribution

Kidney Cell, cellular metabolism

Fat, storage Bladder

Urinary excretion

Figure 5.1

Major routes and sites of absorption, metabolism, binding, and excretion of xenobiotics in the body.

the excretion of the chemical by the urinary and biliary fecal routes (Sipes and Gandolfi, 1986). The likelihood that a xeonobiotic compound will undergo biotransformation in the body depends upon the chemical nature of the species. Compounds with a high degree of polarity, such as relatively ionizable carboxylic acids, are less likely to enter the body system and, when they do, tend to be quickly excreted. Therefore, such compounds are unavailable, or only available for a short time, for enzymatic metabolism. In contrast, volatile compounds such as

dichloromethane or diethylether are expelled so quickly from the lungs that enzymatic metabolism is less likely. This leaves as the most likely candidates for biotransformation reaction nonpolar lipophilic compounds, those that are relatively less soluble in aqueous biological fluids and more attracted to lipid species. Of these, the ones that are resistant to enzymatic attack (e.g., polychlorinated biphenyls [PCBs]) tend to bioaccumulate in lipid tissue. Therefore, it is apparent why lipophilic xenobiotics can accumulate within the body; they are readily absorbed, but poorly excreted.

PHASE I

PHASE II

Expose or add functional groups

XENOBIOTIC Foreign compounds

Primary product Oxidation Reduction Hydrolysis

Biosynthetic

Secondary product

conjugation

EXCRETION

LIPOPHILIC

Figure 5.2

HYDROPHILIC (Ionizable)

Integration of phase I and phase II biotransformation reactions in the metabolism of xenobiotics.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

XENOBIOTICS Increase molecular size

Increase polarity

Increase ionization

Increase excretability Decrease toxicity

Figure 5.3 General trends of biotransformations yielding metabolites of lesser toxicity.

One important factor in the toxicity of xenobiotic compounds is excretability. The vertebrate kidney is built in such a way that it can handle electrolytes better than nonelectrolytes, and, for example, among organic acids, the more completely they are ionized at physiological pH, the more readily the kidney excretes them. Ionization in turn depends on the dipole moment (degree of polarity) of the compound, i.e., the distance between the geometric centers of all positive charges versus all negative charges in the molecule. It is apparent that low dipole moments require near-symmetry of the molecule according to all spatial axes, and the larger the molecule becomes, the lower is the probability of such symmetry. On this basis it is possible to generalize the trend of biotransformations that yield metabolites of lesser toxicity as a result of improved excretability as shown in Figure 5.3. This rule is not without exceptions, the completely nonpolar molecules (e.g., methane, ethane) particularly are often inert biologically and require no detoxification. However, if a nonpolar molecule is biologically active (e.g., benzene, carbon tetrachloride), it is not likely to be detoxified readily and therefore is very toxic.

5.2

SITES OF BIOTRANSFORMATIONS

Xenobiotic compounds may be metabolized in a wide variety of body tissues and organs. As part of the body’s defense against the entry of xenobiotic species, the most prominent sites of xenobiotic metabolism are those associated with entry into the body (Figure 5.1). Although the skin, lungs, and gut wall through which xenobiotic species enter the body from the GI tract are also sites of significant

Copyright 2002 by Marcel Dekker. All Rights Reserved.

xenobiotic compound metabolism, the liver is of particular significance in this regard. The enzymes or enzyme systems that catalyze the biotransformation of xenobiotics are primarily localized in the liver. Furthermore, materials entering systemic circulation from the GI tract must first traverse the liver. The liver receives all the blood, containing nutrients and other foreign substances, that has perfused the splanchnic area. Because of this the liver has developed the capacity to extract these substances readily from the blood and to modify chemically many of these substances before they are stored, secreted into bile, or released into the general circulation. The metabolism of xenobiotic compounds usually occurs in the microsomal fraction (smooth endoplasmic reticulum) of liver cells. Some biotransformations are nonmicrosomal, e.g., redox reactions involving alcohols, aldehydes, and ketones. Although practically every tissue in the body shows some activity toward xenobiotic metabolism, these activities are limited with respect to the diversity of chemicals they can handle, and, thus, their contribution to the overall biotransformation of xenobiotics is limited. However, biotransformation of a chemical within an extrahepatic tissue may have an important toxicological implication for that particular tissue. In this regard, the microflora inhabiting the GI tract play an important role in modifying the toxicological properties of xenobiotic compounds. The dominant varieties in the intestinal microflora are of the genera Bacteroides and Bifidobacterium. Both are non-spore-forming anaerobes. The species commonly encountered include Bacteroides fragilis, Bacteroides melaninogenicus, and Bifidobacterium adolescentis (Williams and Drasar, 1972; Concon, 1988). The aerobic microorganisms found in the GI tract belong to the genera Enterobacteria (e.g., Klebsiella aerogenes, Proteus mirabilis, and mostly Escherichia coli), Streptococcus (S. viridans [mitior] and S. faecalis), and Lactobacillus (L. acidophilus and L. casei). Clostridium (e.g., C. perfringens and C. sporogenes) and Veillonella (e.g., V. parvula and V. alcalescens) species are also common anaerobic inhabitants of the GI tract. The density of microbial populations increases toward the distal segments of the GI tract. The stomach is generally sterile when no food is present or when the acidity falls below pH 3 (Drasar et al., 1969). The bacterial count in this organ may increase to 105 bacteria/ml of gastric content. After eating, most of these are derived from the mouth and the food and include species of Streptococci, Enterobacteria, Bacteroides, and Bifidobacteria. This bacterial population drops drastically toward the latter part of gastric digestion, when the pH of the stomach content drops significantly.

Bacterial density begins to increase in the small intestines, but in the proximal part of the organ, including the proximal ileum, each particular species rarely exceeds 105 microorganisms present (Drasar et al., 1969). The distal ileum is more heavily laden with microorganisms; both aerobes and anaerobes are represented in the bacterial populations. Like those of the stomach, the bacterial populations of the small intestines, particularly the proximal sections, also increase after a meal and may be as high as 105 microorganisms/ml of intestinal content (Gorbach et al., 1967; Concon, 1988). As expected, the bacterial count in the large intestines and rectum is much greater than that of the small intestines. The colonal and rectal bacterial count can be as high as 109 bacteria/g of sample. In the feces, the total viable bacterial count can be as high as 1011 bacteria/g of sample (Moore et al., 1969). Several types of biotransformation reactions have been demonstrated with these intestinal microorganisms. These include hydrolysis, decarboxylation, deamination, O-demethylation, ring opening of heterocyclic compounds, reduction, dehydroxylation, aromatization, and dehalogenation. Very often reaction types are degradative. Given these facts, the intestinal microflora must be included in the assessment of the toxicological properties of xenobiotic compounds. It is also likely that certain diseases, particularly cancer of the GI tract, must involve the activity of intestinal bacteria in their causes (Hill et al., 1971; Hill, 1977; Concon, 1988). Evaluating the role of intestinal bacteria in causing a disease is difficult, considering the vast number of exogenous and endogenous compounds modified by them. Even more staggering is the number of possible metabolites that can be produced by the bacterial reactions in the GI tract.

5.3

BIOTRANSFORMATION ENZYME SYSTEMS

Biotransformation processes are enzymatically induced and result in either alteration of the parent molecule or formation of products involving combinations of normally occurring substances and the parent molecule. These reactions are generally accomplished in the liver by several remarkable enzyme systems. These can be broadly classified as phase I and phase II enzymes. The phase I enzymes introduce reactive, polar functional groups onto lipophilic toxicant molecules; the products are usually more watersoluble than the parent xenobiotic species. More importantly, the phase I reactions introduce a “chemical handle” to which a substrate material in the body may become attached so that the toxicant can be eliminated from the body. The phase II enzymatic reactions thus produce a

Copyright 2002 by Marcel Dekker. All Rights Reserved.

conjugation product that is amenable to excretion from the body. The changes in structure and properties of a xenobiotic compound that result from a phase I reaction are relatively mild. Phase II reactions, in contrast, usually produce metabolites that are much different from the parent compounds. It should be noted that not all xenobiotic compounds undergo both phase I and phase II reactions. Some may undergo only a phase I reaction and be excreted directly from the body. As a corollary, a compound that already possesses an appropriate function group capable of conjugation may undergo a phase II reaction without a preceding phase I reaction. Two distinct categories of biotransformation enzyme systems are known to exist in mammals. One consists of enzymes that normally occur in the tissues and are responsible for transformation of normal endogenous chemicals in the tissues. The second consists of enzymes that alter the structure of many foreign xenobiotic chemicals but have no established normal endogenous substrates. Several enzyme systems that induce the transformation of normal chemical substrates in the body are also active in catalyzing alterations of xenobiotics that structurally are sufficient similar to the normal substrate. For example, the nonspecific esterase hydrolyzing enzyme cholinesterase not only hydrolyzes acetylcholine (a normally occurring neurotransmitter), but also hydrolyzes the local anesthetic agent procaine as well as the muscle-paralyzing drug succinylcholine. Similarly, monoamine oxidase, important in the metabolism of normally occurring biological amines, such as epinephrine and tyramine, also oxidizes xenobiotic short-chain amines such as benzylamine. The phase I enzymes are located primarily in the endoplasmic reticulum, a network of interconnected channels present in the cytoplasm of most cells. It can be visualized with the electron microscope as filament-like structures of two types, smooth and rough surface filaments. When the liver cells are ruptured by homogenization, the tubular endoplasmic reticulum breaks up and fragments of the membrane are sealed off to form microvesicles, referred to as microsomes, which can be isolated by differential ultracentrifugation of the liver homogenate. The phase I enzymes are primarily located in the precipitated microsomal fraction obtained from the smooth reticular endothelium. These enzymes are membrane-bound, since the endoplasmic reticulum is basically a contiguous membrane composed of lipids and proteins. The presence of enzymes within a lipoprotein matrix is critical, since the lipophilic substrates preferentially partition into the lipid membranes, the site of biotransformation. The supernatant or cytosol obtained from centrifugation contains a number of soluble enzymes. Many of

these enzymes are involved in phase II biotransformation reactions. Many of the important biotransformation enzymes are often referred to as cytosolic or microsomal to indicate their subcellular location. 5.3.1 Phase I Enzymes Monooxygenations of xenobiotics are catalyzed either by the cytochrome P-450–dependent monooxygenase system (also referred to as the polysubstrate monooxygenase system, or the mixed-function oxygenase [MFO] system) or by the flavin adenine dinucleotide (FAD)-containing monooxygenase. The latter is sometimes referred to as the mixed-function amine oxidase. Both enzyme systems add a hydroxyl group to the xenobiotic molecule and are primarily located in the endoplasmic reticulum of the cell. Monooxygenations, also known as mixed-function oxidations, are those oxidations in which one atom of a molecule of oxygen is incorporated into the substrate while the other is reduced to water. The electrons involved in the reduction of cytochrome P-450 or FAD are derived from reduced nicotinamide-adenine dinucleotide phosphate (NADPH), which reduces a component of the microsomal enzymes that react with molecular oxygen to form an active oxygen intermediate, which oxidizes the xenobiotic compound. In addition to the two monooxygenase systems, the phase I enzymes include a family of hydrolytic enzymes, esterases and amidases. The cleavage of the ester or amide bond, regardless of the remaining chemical structure, produces two functional groups for further biotransformations: a carboxylic acid plus either an amine (from an amide) or an alcohol (from an ester). Finally, a variety of oxidation-reduction systems can be considered part of the phase I enzymes since these are redox enzymes and often alter the oxidation state of a carbon to allow it to be more readily excreted or biotransformed by the phase II enzymes. Cytochrome P-450–Dependent Monooxygenase System The most important enzyme systems involved in phase I reactions are the cytochrome P-450–containing monooxygenases. Cytochrome P-450s, the carbon monoxide–binding pigments of microsomes, are hemoproteins of the b cytochrome type. Unlike most cytochromes, they are named not from the absorption maximum of the reduced form in the visible region but from the unique wavelength of the absorption maximum of the carbon monoxide derivative of the reduced form, namely, 450 nm. The cytochrome P-450 system is actually a coupled enzyme system composed of two enzymes: NADPH-cytochrome P-450 reductase, and a heme-containing enzyme,

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cytochrome P-450. These enzymes are embedded in the phospholipid matrix of the endoplasmic reticulum. The phospholipid phophatidylcholine is not involved directly in the electron transfer but plays a crucial role in facilitating the interaction between these two enzymes. The NADPH-cytochrome P-450 reductase has a preference for NADPH as its cofactor. It is a flavoprotein capable of transferring one or two electrons to cytochrome P-450. In vertebrates, the liver is the richest source of cytochrome P-450 and is most active in the monooxygenations of xenobiotics. Cytochrome P-450 and other components of the monooxygenase system dependent on it are also found in skin, nasal mucosa, lung, and GI tract. In addition to these organs, its presence has been demonstrated in kidney, adrenal cortex and medulla, placenta, testes, ovaries, fetal and embryonic liver, corpus luteum, aorta, blood platelets, and the nervous system. In humans, cytochrome P-450 has been demonstrated in fetal and adult liver, placenta, kidney, testes, fetal and adult adrenal gland, skin, blood platelets, and lymphocytes. The mammalian liver cells contain more than one cytochrome P-450. To date, at least 10 forms of cytochrome P-450 have been isolated from rat liver microsomes. These differ in both the structure of the polypeptide chain and the specificity of the reactions they catalyze (Sipes and Gandolfi, 1986). In addition, the types and amounts of cytochrome P-450 vary with species, organ, age, health, sex, stress, and chemical exposure. In contrast to cytochrome P-450, only one NADPHcytochrome P-450 reductase has been isolated from a single source. Its concentration is usually one-tenth to onethirtieth that of cytochrome P-450. Therefore, this enzyme must mediate the reduction of the many different forms of cytochrome P-450. The mechanism of cytochrome P-450 function is not clearly understood; the generally recognized steps are shown in Figure 5.4. The initial step consists of the binding of the substrate to oxidized cytochrome P-450, followed by a one-electron reduction catalyzed by NADPHcytochrome P-450 reductase to form a reduced cytochrome-substrate complex. This complex can interact with CO to form the CO-complex, which gives rise to the wellknown difference spectrum with a peak at 450 nm and also inhibits monooxygenase activity. The next several steps are less well understood. They involve an initial interaction with molecular oxygen to form a ternary oxygenated complex. This ternary complex accepts a second electron, resulting in the further formation of one or more poorly understood complexes. One of these, however, is probably the equivalent of the peroxide anion derivative of the substrate-bound hemoprotein. Under some conditions, this complex may break down to

Lipid OOH

ROH

NADPH

Lipid

R

Cyt-Fe3+R

Cyt-Fe3+

O

NADPH Cytochrome reductase

e-

XOOH

H2O

e-

O2

Cyt-Fe3+R

Cyt-Fe2+R

O2

CO hv

H2O2 Cyt-Fe2+R

Cyt-Fe2+R CO

O2

O2 Cyt-Fe3+R O2

NADPH Cytochrome P-450 reductase

or

Cytochrome b5 e-

e-

Cyt-Fe2+R O2

NADH-Cyt b5 reductase

NADPH

eNADH

Figure 5.4

Generalized scheme showing the sequence of events for cytochrome P-450 monooxygenations in phase I biotransformations.

yield hydrogen peroxide and the oxidized cytochromesubstrate complex. Normally, however, one atom of molecular oxygen is transferred to the substrate and the other is reduced to water, followed by dismutation reactions leading to the formation of the oxygenated product, water, and the oxidized cytochrome. Examples of the reactions catalyzed by the microsomal cytochrome P-450 system are shown in Table 5.3. Sipes and Gandolfi (1986) have suggested the following criteria for the participation of cytochrome P-450 in a biotransformation reaction: 1. 2. 3. 4.

Enzymatic activity increased by induction Enzymatic activity decreased by inhibitors Substrates that produce characteristic difference spectra Enzymatic activity reconstituted with individual purified components

Microsomal Flavin Adenine Dinucleotide (FAD)-Containing Monooxygenase Tertiary amines such as trimethylamine and dimethylaniline are metabolized to N-oxides by an amine oxidase,

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which is microsomal but not dependent on cytochrome P450. This enzyme, now known as the microsomal FADcontaining monooxygenase, is also dependent on NADPH and O2 and has been purified to homogeneity from pig and mouse liver microsomes. This enzyme is historically referred to as mixed-function amine oxidase.

Table 5.3 Oxidation Reactions Catalyzed by the Cytochrome P-450–Containing Monooxygenases Epoxidation and aromatic hydroxylation Aliphatic hydroxylation Aliphatic epoxidation Dealkylation: O-, N-, and S-dealkylation N-Oxidation Oxidative deamination S-Oxidation P-Oxidation Desulfuration and ester cleavage Methylenedioxy (benzodioxole) ring cleavage Oxidative dehalogenation

The FAD-containing monooxygenase is now known to have much wider substrate specificity than formerly believed. It includes tertiary and secondary amines as well as a number of different types of sulfur compounds such as sulfides, thioethers, thiols, and thiocarbamates. This enzyme competes with the cytochrome P-450 system in the oxidation of amines. It converts tertiary amines to amine oxides, secondary amines to hydroxylamines and nitrones, and primary amines to hydroxylamines and oximes. It is believed that the endogenous substrate for the flavin-containing monooxygenase is cysteamine, which is oxidized to cystamine. Epoxide Hydrolase Formerly known as epoxide hydrase or epoxide hydratase, the enzyme epoxide hydrolase is thought to be located in close proximity to the microsomal cytochrome P-450 monooxygenases. It catalyzes the hydration of arene oxides and aliphatic epoxides to their corresponding trans-1,2-dihydrodiols. The latter are less electrophilic and, therefore, chemically less reactive than the epoxides. Arene oxides (epoxides of aromatic compounds) are generally unstable and rearrange to the corresponding phenol. The phenol is then available to participate in various phase II conjugation reactions. Epoxide hydrolase has been found in a wide variety of tissues, including liver, testis, ovary, lung, kidney, skin, intestine, colon, spleen, thymus, brain, and heart (Sipes and Gandolfi, 1986). Its close proximity to the site of formation of its substrates suggests that epoxide hydrolase may have evolved as an important means of detoxifying arene oxides and aliphatic epoxides. Epoxide hydrolase–catalyzed hydration of oxides occurs through activation of water to a nucleophilic species. The resulting nucleophilic species attacks the least hindered carbon atom from the side opposite the oxide ring. Consequently, ring opening is directed away from the hydroxylation and the resulting diols have a trans configuration. This enzyme is considered a detoxication enzyme, since it inactivates a number of highly reactive oxides that have been implicated in tissue injury and mutagenicity. Its activity is regulated by a number of factors. Alcohol Dehydrogenases Alcohol dehydrogenases are enzymes that catalyze the conversion of alcohols to aldehydes or ketones. These reactions are reversible; the carbonyl compounds are reduced to alcohols. This enzyme is found in the soluble fraction of the liver, kidney, and lung and is probably the most important enzyme involved in the metabolism of foreign alcohols. It can use either NAD or NADP as a co-

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enzyme, but the reaction proceeds at a much slower rate with NADP. The enzyme readily converts primary alcohols to aldehydes and secondary alcohols at slower rates to ketones; tertiary alcohols are not readily oxidized. Alcohol dehydrogenase is inhibited by a number of heterocyclic compounds such as pyrazole, imidazole, and their derivatives. Aldehyde Dehydrogenase The enzyme aldehyde dehydrogenase catalyzes the formation of acids from aliphatic and aromatic aldehydes with oxidized nicotinamide-adenine dinulceotide (NAD+) as the cofactor; the acids are then available as substrates for phase II conjugating enzymes. Other enzymes in the soluble fraction of liver that oxidize aldehydes are aldehyde oxidase and xanthine oxidase, both flavoproteins that contain molybdenum; however, their primary role seems to be the oxidation of endogenous aldehydes formed as a result of deamination reactions. The inhibition of aldehyde dehydrogenase usually results in toxicity of xenobiotics requiring this route of biotransformation. Esterases and Amidases Mammalian tissues contain a large number of nonspecific esterases and amidases that can hydrolyze ester and amide linkages in xenobiotic compounds. This hydrolytic cleavage of ester and amide linkages liberates carboxyl groups and an alcohol function in the case of esters and an amine or NH3 in the case of amides. These carboxyl, alcohol, and amine groups then undergo a variety of phase II conjugation reactions. In some instances, this group of enzymes can hydrolyze even thioesters. These enzymes are both cytosolic and microsomal enzymes. The cytosolic esterases are usually associated with a specific reaction (e.g., acetyl cholinesterase and pseudocholinesterase), whereas the microsomal-associated esterases handle a diverse array of xenobiotic esters (Sipes and Gandolfi, 1986; Manahan, 1992). 5.3.2 Phase II Enzymes The biotransformation reactions in phase II processes are biosynthetic in nature and thus require energy to drive them. This is usually accomplished by activating the cofactors or by producing high-energy intermediates. Since the cofactors are activated either directly or indirectly with adenosine triphosphate (ATP), the energy status of the organ is important in determining the cofactor availability. Some of the important enzyme systems involved in phase II biotransformation processes are briefly described. For a

detailed review of this subject, the readers are referred to an excellent review by Sipes and Gandolfi (1986). Glucuronosyltransferases Glucuronidation is one of the major phase II biotransformation reactions in the conversion of both exogenous and endogenous compounds to polar, water-soluble compounds. The resulting glucuronides are then eliminated from the body in the urine or bile. The enzyme that carries out the reaction is uridine glucuronosyltransferase or UDP-glucuronosyltransferase. It catalyzes the interaction between the high-energy nucleotides, either UDP glucose (UDPG) or UDP-glururonic acid (UDPGA), and the functional group on the acceptor molecule. The enzymatic activity is localized in the endoplasmic reticulum of numerous tissues, whereas most phase II enzymes are cytosolic enzymes. Quantitatively, the liver is the most important tissue. However, the activity is also present in the kidney, intestine, skin, brain, and spleen. The location of this enzyme in the microsomal membrane is important physiologically, since it may have direct access to the products formed by the action of microsomal cytochrome P-450. This highly integrated system within the microsomal membrane results in the sequestration of highly lipophilic compounds, the addition or unmasking of a functional group, and the conjugation of this functional group with the highly polar glucuronic acid moiety. Numerous functional groups present in both xenobiotic and endogenous compounds that undergo conjugation with glucuronic acid are listed in Table 5.4. These include aliphatic and aromatic alcohols and carboxylic acids, primary and secondary aromatic and aliphatic amines, and free sulfhydryl groups. These form O-, N-, and S-glucuronides, respectively. Certain nucleophilic carbon atoms have also been shown to form C-glucuronides (Sipes and Gandolfi, 1986). During the conjugation reaction, the UDP-glucuronic acid cofactor, which is in the α configuration, undergoes inversion that leads to glucuronides that have a β configuration. Glucuronidation reactions contribute a carboxyl group, which exists primarily in the ionized form at physiological pH. This group promotes excretion not only because it confers water solubility, but also because it can participate in biliary and renal organic anion transport systems that recognize this group. Glucuronides are excreted from the body in either bile or urine, depending on the size of the aglycone. If this moiety has a molecular weight below about 250, the glucuronide is cleared by renal tubular organic acid secretion into urine. Those above 350 are often secreted into bile; either pathway may excrete aglycones in the molecular weight range 250 to 350.

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Table 5.4 Examples of Different Classes of Glucuronide Conjugates Functional group O-Glucuronide Alcohols Aliphatic Alicyclic Benzylic Phenolic Carboxylic acids Aliphatic Aromatic α,β-Unsaturated ketone N-Hydroxy N-Glucuronide Carbamate Arylamine Aliphatic tertiary amine Sulfonamide S-Glucuronide Aryl thiol Dithiocarbamic acid C-Glucuronide 1,3-Dicarbonyl systems

Example

Trichloroethanol Hexobarbital Methylphenylcarbinol Estrone α-Ethylhexanoic acid o-Aminobenzoic acid Progesterone N-Acetyl-N-phenylhydroxylamine Meprobamate 2-Naphthylamine Tripelennamine Sulfadimethoxine Thiophenol N,N-Diethyldithiocarbamic acid Phenylbutazone

Sulfotransferases Sulfation of hydroxyl groups is an important conjugation reaction during phase II biotransformation. Sulfation is an effective means of decreasing the pharmacological and toxicological activity of compounds. Sulfotransferases, a group of soluble enzymes found primarily in liver, kidney, intestinal tract, and lungs, transfer inorganic sulfate to the hydroxyl group present on phenols and aliphatic alcohols, yielding sulfate esters or ethereal sulfates. In addition, sulfation of aromatic amines and hydroxylamines to form the corresponding sulfamate and N-O-sulfates can be seen. The products of sulfation reaction are ionized organic sulfates that are more readily excreted than the parent compound or hydroxylated metabolites. There are four classes of sulfotransferases involved in detoxification processes (Sipes and Gandolfi, 1986). Aryl sulfotransferase conjugates phenols, catecholamines, and organic hydroxylamines. Hydroxysteroid sulfotransferase conjugates hydroxysteroids and certain primary and secondary alcohols. Estrone sulfotransferase is active with phenolic groups on the aromatic ring of steroids; bile salt sulfotransferases catalyze the sulfation of both conjugated and unconjugated bile acids. The activity of these enzymes varies considerably with the sex and age of animals.

The sulfate donor for these reactions is 3′-phosphoadenosine-5′-phosphosulfate (PAPS), which is synthesized from inorganic sulfate and ATP. The major source of sulfate required for the synthesis of PAPS is derived from cysteine through a complex oxidation sequence. Since the concentration of free cysteine is limited, an important determinant in the extent of sulfation of xenobiotics is the availability of PAPS. In reactions catalyzed by the sulfotransferases, the SO3– group of PAPS is readily transferred in a reaction involving nucleophilic attack of the phenolic oxygen or the amine nitrogen on the sulfur atom and the subsequent displacement of adenosine-3′,5′diphosphate (Sipes and Gandolfi, 1986). Although sulfate conjugation usually results in detoxification, in some cases conjugation results in toxication. Certain sulfate conjugates, e.g., N-O-sulfate esters of N-hydroxy-2-acetylaminofluorene, are chemically unstable and degrade to form potent electrophilic species. Sulfate conjugates of xenobiotics are excreted mainly in urine, and some can be degraded enzymatically. Methyl Transferases Various specific (histamine and indole) and nonspecific Nmethyl transferases are believed to have evolved in the liver to handle the evolution and uptake of hydrogen sulfide produced by anaerobic bacteria in the intestinal tract. The hydrogen sulfide is methylated to methane thiol, which is further methylated to dimethylsulfide. The nonspecific N-methyl transferases are of most concern since these enzymes are capable of methylating a variety of primary, secondary, and tertiary exogenous and endogenous amines, such as serotonin, benzylamine, amphetamine, and pyridine. Methylation is not usually a quantitatively important pathway for xenobiotic metabolism. It actually masks functional groups, thereby reducing the water solubility of the chemical, and/or impairing its ability to participate in other conjugation reactions. The functional groups involved in methylation are aliphatic and aromatic amines, N-heterocyclics, mono- and polyhydric phenols, and sulfhydryl-containing compounds. The methyl group is transferred to the xenobiotic from a high-energy cofactor, Sadenosyl methionine (SAM). The methyl group bound to the sulfonium ion in SAM has the characteristics of a carbonium ion and is transferred by nucleophilic attack of the alcohol oxygen, the amine nitrogen, or the thiol sulfur on the methyl group, giving S-adenosylhomocysteine and the methylated substrate as products. The largest source of substrates for these S-methyl transferase reactions appear to be the thio ethers of glutathione conjugates. Certain glutathione thio ethers are hydrolyzed to cysteine conjugates in the kidney prior to acetylation and excretion.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

N-Acetyl Transferases Arylamines are often biotransformed by the acetylation of the amine function. The enzymes that catalyze these reactions are designated acetyl coenzyme A (CoA):amine Nacetyl transferases. The cofactor for these reactions is acetyl CoA. These are cytosolic enzymes found in many tissues. The major substrates for these reactions include aromatic primary amines, hydrazines, hydrazides, sulfonamides, and certain primary aliphatic amines. Acetylation of arylamines occurs in two sequential steps. Initially the acetyl group from acetyl CoA is transferred to the N-acetyl transferase to form acetyl-N-acetyl transferase as an intermediate. The second step is acetylation of the amino group of the arylamine substrate with regeneration of the enzyme. The interaction between the amine and the acetyl group results in the formation of an amide bond. This reaction also masks a functional group, and the N-acetyl derivatives are less water-soluble than the parent compound. N-Acyl Transferases The conjugation of xenobiotics containing a carboxylic acid group with one of a variety of amino acids is an important reaction. These reactions result in the formation of an amide (peptide) bond between the carboxylic acid group of the xenobiotic and the amino group of the amino acid. Substrates for these reactions include aromatic carboxylic acids, arylacetic acids, and aryl-substituted acrylic acids. Although the most common reaction involves glycine, conjugation with glutamine is more prevalent in humans. The formation of the peptide bond is a two-step coupled reaction catalyzed by different enzymes. The first reaction involves ATP-dependent activation of the acid to a thioester derivative of coenzyme A. The enzymes that catalyze this activation are called ATP-dependent acid:CoA ligases. The coenzyme A thioester then transfers its acyl moiety to the amino group of the acceptor amino acid. The ligase and the N-acyltransferase are soluble enzymes. Amino acid conjugates are eliminated primarily in urine. Glutathione S-Transferases The glutathione S-transferases are a family of enzymes that catalyze the initial step in the formation of N-acetylcysteine (mercapturic acid) derivatives of a diverse group of foreign compounds. At least seven different enzymes have been isolated from the cytosol of rat livers with a broad display of overlapping substrate specificity. These enzymes have molecular weights in the range of 45,000–50,000 daltons and consist of two subunits. Glutathione S-transferases are localized in both the cytoplasm and the endoplasmic reticulum; the cytoplasmic activities are gen-

erally 5 to 40 times greater than the microsomal activity. These enzymes are ubiquitous; the greatest activity occurs in the testis, liver, intestine, kidney, and adrenal gland. The cofactor for reactions catalyzed by these enzymes is the tripeptide glutathione (GSH), which is composed of glycine, glutamic acid, and cysteine. The enzymes catalyze the reaction of the nucleophilic sulfhydryl of glutathione with compounds containing electrophilic carbon atoms. The reaction of the glutathione thiolate anion (GS–) results in formation of a thioether bond between the carbon atom and the sulfhydryl group of glutathione. Compounds that are substrates for the glutathione Stransferases share the following three basic features: 1. 2. 3.

They must be hydrophobic to some degree. They must contain an electrophilic carbon atom. They must react nonenzymatically with glutathione at some measurable rate.

The glutathione conjugates are subsequently cleaved to cysteine derivatives, primarily by enzymes located in the kidney. These derivatives are then acetylated to give the N-acetylcysteine (mercapturic acid) conjugates that are readily excreted in urine. The importance of the nucleophilic reactions catalyzed by glutathione S-transferases has become increasingly apparent over the last few years. These enzymes provide a means of reacting the diverse array of electrophilic xenobiotics with the endogenous nucleophile glutathione, thus preventing, to a degree, the reaction of these compounds with essential constituents of the cell (Sipes and Gandolfi, 1986; Manahan, 1992). Furthermore, these enzymes also detoxify reactive intermediates produced by the cytochrome P-450 system. Rhodanese The mitochondrial enzyme rhodanese is involved in the detoxication of cyanide. Thiosulfate can act as the donor of sulfur; however, it is not known with certainty which of the components of the sulfite pool is the true cofactor. Since the product of the reaction, thiocyanate, is far less toxic than cyanide, rhodanese catalyzes an unmistakable detoxification reaction.

5.4

INDUCTION OF BIOTRANSFORMATION ENZYMES

The activities of the enzymes involved in biotransformation processes may be enhanced after treatment of animals or humans with chemicals. A multitude of compounds with diverse chemical structures have been shown to in-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

duce monooxygenase and other enzymes involved in detoxification processes. These compounds include drugs, insecticides, polycyclic hydrocarbons, industrial chemicals, and natural products. Generally, this enhanced activity results from an increase in the rate of synthesis of the biotransformation enzymes. The induction of microsomal enzymes has been demonstrated in many different species, including humans, and in several different tissues from these species. Induction may require the repeated administration of the compound or chronic exposure to it. Although the most well-known and studied microsomal enzyme system stimulated by other compounds is the cytochrome P-450 monooxygenase system, other enzymes also may be induced. Microsomal enzyme inducers can induce microsomal enzyme-catalyzed reduction, glucuronyl transferase, UDP-glucose dehydrogenase, glutahione transferases and esterases, as well as some of the enzymes involved in steroid metabolism. The major types of microsomal enzyme inducer are the barbiturates, typified by phenobarbital, and the polycyclic hydrocarbons, of which 3-methylcholanthrene (3MC) is the best known. However, these two inducers produce different effects (Table 5.5). Although the two compounds induce the microsomal monooxygenase system, each induces a different form of the cytochrome P450 with different spectral properties. Phenobarbital causes a proliferation of the smooth endoplasmic reticulum, whereas 3-methylcholanthrene does not. It seems that the induction caused by phenobarbital is fairly general, whereas that of 3-methylcholanthrene is more specific. As well as increasing the amount of cytochrome P-450, phenobarbital increases NADPH-cytochrome P-450 reductase and ribonucleic acid (RNA) synthesis. Conney (1967) has summarized the possible interactions. Inducers may interact (a) with deoxyribonucleic acid (DNA) to stimulate DNA-directed synthesis of messenger RNA (mRNA); (b) with repressors or other regulators of gene function; (c) with the endoplasmic reticulum to enhance translation of mRNA on the ribosomes; and (d) with monooxygenase to reduce activity and, hence, allow an endogenous inducing agent to accumulate. In terms of toxicity, enzyme induction may either increase or decrease the toxic effects of a particular compound, depending on the basis for that toxicity. Different inducers also influence toxicity differently. For instance, of the inducers dichlorodiphenyl trichloroethane (DDT), 3MC, and chlordane, only treatment with chlordane protects against parathion toxicity. Phenobarbital induction increases the toxicity of phosphorothionate insecticides but reduces that of other organophosphate insecticides. The pharmacological activity of codeine is increased by induction, which increases demethylation to morphine. The tox-

Table 5.5 Characteristics of the Hepatic Effects of Phenobarbital and Polycyclic Aromatic Hydrocarbons Characteristics Onset of effects Time of maximal effect Persistence of induction Liver enlargement Protein synthesis Phospholipid synthesis Liver blood flow Biliary flow Enzyme components Cytochrome P-450 Cytochrome P-448 NADPH-cytochrome c reductase Substrate specificity N-Demethylation Aliphatic hydroxylation Polycyclic hydrocarbon hydroxylation Reductive dehalogenation Glucuronidation Glutathione conjugation Epoxide hydrolase Cytosolic receptor

Phenobarbital

PAHs

8–12 Hours 3–5 Days 5–7 Days Marked Large increase Marked increase Increase Increase

3–6 Hours 24–48 Hours 5–12 Days Slight Small increase No effect No effect No effect

Increase No effect Increase

No effect Increase No effect

Increase Increase Small increase

No effect No effect Increase

Increase

No effect

Increase Small increase Increase None identified

Small increase Small increase Small increase Identified

NADPH, reduced nicotinamide-adenine dinucleotide phosphate; PAH, polycyclic aromatic hydrocarbon. Source: Compiled from Sipes and Gandolfi (1986), Concon (1988), and Manahan (1992).

icity of cyclophosphamide is increased by induction with the inducer chlordane (Snyder and Remmer, 1979).

5.5

INHIBITION OF BIOTRANSFORMATION ENZYMES

In contrast to enzyme induction, inhibition generally requires only a single dose of inhibitor rather than repeated doses. Although environmentally it may be of less consequence than induction, in the case of drug interactions it is probably of greater importance. The inhibitory mechanisms include competition for active sites or cofactors of the enzymes, inhibition of transport components in multienzyme systems, decreased biosynthesis or increased breakdown of enzymes or their cofactors, as well as allosteric changes in enzyme conformation and even loss of functional tissue (e.g., hepatic necrosis). Inhibition of xenobiotic biotransformation in vivo is a complex process. Many chemicals produce multiple ef-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

fects, such as inhibition of both phase I and phase II reactions. Inhibitors may show pronounced effects on only certain cytochrome P-450 isozymes, and, therefore, the degree of inhibition varies in untreated animals versus those treated with inducing agents. The degree of inhibition is also time-dependent. Many inhibitors of mammalian monooxygenase activity can also act as inducers. Inhibition of microsomal monooxygenase activity is fairly rapid and involves a direct interaction with the cytochrome, whereas induction is a slower process. Therefore, after a single injection of a suitable compound, an initial decrease due to inhibition would be followed by an inductive phase. As the compound and its metabolites are eliminated, the levels would be expected to return to control values. Some of the best examples of such compounds are the methylenedioxyphenyl synergists, such as piperonyl butoxide. Because cytochrome P-450 combined with methylenedioxyphenyl compounds in the type III inhibitory complex cannot interact with CO, the cytochrome P-450 titer would appear to follow the same curve.

5.6

BIOTRANSFORMATION REACTIONS

Biotransformation of most xenobiotics can be conveniently divided into two categories, phase I reactions and phase II reactions. A phase I reaction introduces reactive, polar functional groups (e.g., hydroxyl, amino, carboxyl, epoxide, hydroxylamine, sulfhydryl) onto lipophilic toxicant molecules. In their unmodified forms, such toxicants tend to pass through lipid-containing cell membranes and may be bound to lipoproteins, in which form they are transported through the body. Because of the functional group attached, the product of phase I reaction is usually more water-soluble than the parent xenobiotic molecule and, more importantly, possesses a “chemical handle” to which a substrate material in the body may become attached so that the toxicant can be eliminated from the body. Phase I reactions are generally oxidative, reductive, or hydrolytic processes that provide the necessary chemical structure for phase II reactions, which are generally conjugations. In general, the changes in structure and properties of a compound that result from a phase I reaction are relatively mild. Phase II processes, however, usually produce species that are much different from the parent compounds. Not all xenobiotic compounds undergo both phase I and phase II reactions. Some may undergo only a phase I reaction and be excreted directly from the body. Alternatively, a compound may already possess an appropriate functional group capable of conjugation and may undergo a phase II reaction without a preceding phase I reaction.

5.6.1 Phase I Reactions

Table 5.6 Phase I Oxidation Reactions Involved in the Biotransformation of Xenobiotics

The overall processes involved in a phase I reaction are shown in Figure 5.5. Normally, a phase I reaction adds a functional group to a hydrocarbon chain or ring or modifies one that is already present. The product is a chemical species that readily undergoes conjugation with some other species naturally present in the body to form a substance that can be readily excreted. Phase I reactions are of several types, of which oxidation of C, N, S, and P is most important. Reduction may occur on reducible functionalities such as alkenyl, carbonyl, and nitro groups. Phase I reactions may also consist of hydrolysis processes, which require that the xenobiotic compound have a hydrolysable group. Some of the important phase I reactions are described in the following discussion. Oxidation Reactions The biochemical essence of animal life is oxidation, and the enzymatic makeup of living organisms is geared toward oxidizing foodstuff. Any xenobiotic compound that can be handled by identical or analogous routes is therefore metabolized with relative ease, although this does not mean that it is always successfully detoxified. The majority of oxidation reactions that xenobiotics undergo are catalyzed by the microsomal monooxygenases based on cytochrome P-450, although mitochondrial and soluble fraction enzymes may also be involved (Table 5.6).

Microsomal oxidations Epoxidation Aromatic hydroxylation Aliphatic hydroxylation Alicyclic hydroxylation Heterocyclic hydroxylation N-, S-, and O-Dealkylation N-Oxidation N-Hydroxylation S- and P-Oxidation Desulfuration Deamination Nonmicrosomal oxidations Amine oxidation Alcohol and aldehyde oxidation Purine oxidation Aromatization

Epoxidation Epoxidation consists of adding an oxygen atom between two carbon atoms in an unsaturated system or into aromatic hydrocarbons such as benzene (Figure 5.6). Epoxidation is a particularly important means of metabolic attack upon aromatic rings that abound in many xenobiotics. Cytochrome P-450 is involved in epoxidation reactions. Stable epoxides occur as major metabolites of the

Xenobiotic compound usually lipophilic with affinity for biological membranes

Enzyme system with multiple, nonspecific enzymes, primarily cytochrome P-450

Metabolites with reactive, polar functional group Epoxide, Hydroxide, Sulfhydryl, Hydroxylamine, Amino, Carboxyl, Others

To Phase II Biotransformation Reactions Figure 5.5

Overall process of phase I reactions in biotransformation metabolic processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Aromatic hydroxylation generally proceeds via the formation of an epoxide intermediate (see the conversion of benzene to phenol in Figure 5.6). Unsaturated aliphatic compounds and heterocyclic compounds may also be metabolized via epoxide intermediates. In the case of the furan ipomeanol and vinyl chloride, the epoxide intermediate is thought to be responsible for the toxicity. Other examples of unsaturated aliphatic compounds that may be toxic and are metabolized via epoxides are diethylstilbestrol; allyisopropyl acetamide, which destroys cytochrome P-450; sedormid, and secobarbital. Aromatic hydroxylation may also occur through a mechanism other than epoxidation. Thus, m-hydroxylation of chlorobenzene is thought to proceed via a direct insertion mechanism (Figure 5.9). Hydroxylation can consist of the addition of more than one epoxide group. Hydroxylation and epoxidation are responsible for making several xenobiotic compounds toxic through metabolic processes. A prominent example of this phenomenon is the metabolic production of the carcinogenic 7,8-diol-9,10-epoxide of benz(a)pyrene (Figure 5.10). These metabolites arise by prior formation

insecticides heptachlor and aldrin. Epoxides are invariably very toxic and often mutagenic or carcinogenic. Unstable epoxides may be formed as short-lived intermediates in the hydroxylation of aromatic ring systems into phenolic compounds. The leukemogenic activity of benzene is perhaps due to such an intermediate. Epoxide formation in the case of aflatoxin is believed to be the final step in the expression of carcinogenicity (Figure 5.6). Aromatic Hydroxylation Aromatic hydroxylation (Figure 5.7) is an extremely important phase I biotransformation reaction. In these reactions, a hydroxyl is introduced into the aromatic ring. When the aromatic compounds contain substituents, the hydroxyl may be introduced o- or p- to the substituents. The major products of aromatic hydroxylation are phenols, but catechols and quinols may also be formed, arising by further metabolism. Other metabolites such as diols and glutathione conjugates may also be produced. Consequently, a number of hydroxylated metabolites may be produced from the aromatic hydroxylation of a single xenobiotic compound (Figure 5.8).

Cl

O

H C

Cl

C

Cl Cl Trichloroethylene

C

C

Cl

H

Cl

Cl

CHCl

H2C

O

C

C

CHCl

Cl

O

H 2C

C

H

H

Trichloroaldehyde

Cl

O H 2C

Cl

Chloroacetaldehyde

Vinyl chloride OH O O O Benzene

Benzene epoxide

O

O Furan-2,3-epoxide

Furan

Phenol

O

O

O

O

O

O O

O

Aflatoxin B1

Figure 5.6

OCH3

O

Aflatoxin B1 epoxide

Examples of epoxide reactions in phase I biotransformation processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O

OCH3

Benzene

OH

OH

OH

OH

OH

OH

OH OH

OH Quinol

Figure 5.7

Resorcinol

OH

Phenol (Major product)

1,2,4-Trihyhdroxybenzene

Catechol

Aromatic products of the microsomal oxidation of benzene. Phenol is the major metabolite.

of the 7,8-epoxide, which gives rise to 7,8-dihydrodiol through the action of epoxide hydrolase. This is further metabolized by the cytochrome P-450–dependent monooxygenase system to the 7,8-diol-9,10-epoxides, which are both potent mutagens and unsuitable substrates for the further action of epoxide hydrolase. Benz(a)pyrene is classified as a procarcinogen, or precarcinogen, in that metabolic

action is required to convert it to a species that is carcinogenic as such. The nature of the substituent in a substituted aromatic compound influences the position of hydroxylation. Thus, o-p-directing substituents, such as amino groups, result in o- and p-hydroxylated metabolites such as the o- and p-aminophenols from aniline (Figure

Naphthalene

OH

OH OH

OH

H

OH H OH

1-Naphthol

Figure 5.8

2-Naphthol

1,2-Dihydroxynaphthalene

Hydroxylated products of naphthalene metabolism.

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1,2-Dihydro-1,2dihydroxynaphthalene

Cl

Cl

OH

m-Chlorophenol

Chlorobenzene

Figure 5.9 m-Hydroxylation of chlorobenzene via direct insertion mechanism.

5.11a). Metadirecting substituents such as nitro groups lead to m- and p-hydroxylated products; for example, nitrobenzene is hydroxylated to m- and p-nitrophenols (Figure 5.11b). Aliphatic Hydroxylation Aliphatic hydroxylation of alkane chains can occur on the terminal carbon atom (methyl group or ω-carbon) or on the carbon atom next to the last one (ω-1 carbon) by the insertion of an oxygen atom between carbon and hydrogen (Figure 5.12a). The initial oxidation product is an alcohol, which is oxidized further to a carboxylic acid.

The carboxylic acid (a fatty acid) is oxidized further by βoxidation. Although simple aliphatic molecules such as n-butane, n-pentane, and n-hexane, as well as alicyclic compounds such as cyclohexane, are oxidized to alcohols, aliphatic hydrocarbon chains are not readily metabolized. The alkyl side chains of aromatic compounds are more readily oxidized, often at more than one position. The initial products of microsomal enzyme-mediated hydroxylation are primary and secondary alcohols. Thus, the npropyl side chain of n-propyl benzene can be hydroxylated in three positions, giving the primary alcohol 3-phenylpropan-1-ol by ω-oxidation, and the two secondary alcohols, 3-phenylpropan-2-ol (benzylmethylcarbinol) by ω-1 oxidation, and 3-phenylpropan-3-ol (ethylphenylcarbinol) by α-oxidation (Figure 5.12b). Further oxidation of the primary alcohol may take place to give the corresponding acid, phenylpropionic acid, which may be further metabolized to benzoic acid, probably by oxidation of the β carbon to the carboxylic acid. Alicyclic Hydroxylation Hydroxylation of saturated rings yields monohydric and dihydric alcohols. For instance, cyclohexane is me-

7,8-Epoxide of benz(a)pyrene

Benz(a)pyrene

O

O

HO OH 7,8-Diol-9,10 epoxides of benz(a)pyrene

HO OH O 7,8-Dihydrodiol of benz(a)pyrene

HO OH

Figure 5.10 Examples of epoxidation and aromatic hydroxylation.

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(a) NH2

NH2

NH2 OH

OH Aniline

o-Aminophenol

p-Aminophenol

(b) NO2

NO2

NO2

OH OH Nitrobenzene

m-Nitrophenol

p-Nitrophenol

Figure 5.11 The nature of the substituent in aromatic compounds influences the position of hydroxylation in (a) aniline and (b) nitrobenzene.

tabolized to cyclohexanol, which is further hydroxylated to cyclohexane-1,2-diol (Figure 5.13). With mixed alicyclic/aromatic, saturated, and unsaturated systems, alicyclic hydroxylation appears to predominate, as shown for the compound tetralin (Figure 5.14). Heterocyclic Hydroxylation Nitrogen heterocycles, such as pyridine and quinoline, undergo microsomal hydroxylation at the 3 position. In quinoline, the aromatic ring is also hydroxylated in positions o- and p- to the nitrogen atom. Aldehyde oxidase, a soluble enzyme, may also be involved in the oxidation of quinoline to give 2-hydroxyquinoline (Figure 5.15). Another example of heterocyclic oxidation is the microsomal oxidation of coumarin to 7-hydroxycoumarin (Figure 5.16). N-, S-, and O-Dealkylation Dealkylation is the removal of alkyl groups from nitrogen, sulfur, and oxygen atoms (Figure 5.17). The microsomal enzymes catalyze these reactions. These reactions proceed, presumably, by initial introduction of a hydroxyl to the alkyl group, forming an unstable hemiacetal, which decomposes to an alcohol and an aldehyde. N-Dealkylation is a common reaction in the metabolism of drugs, insecticides, and other xenobiotics. The reaction may proceed via an unstable oxidized intermediate,

Copyright 2002 by Marcel Dekker. All Rights Reserved.

which spontaneously rearranges with loss of the corresponding aldehyde. Both the N-alkyl and the N,N-dialkyl carbamates are readily dealkylated; in some cases, the methylol intermediates are stable enough to be isolated. N,N-Dimethyl-p-nitrophenyl carbamate is a useful model compound for this reaction (Figure 5.18). The insecticide carbaryl undergoes several monooxygenations, including attack on the N-methyl group. In this case, the methylol derivative is stable enough to be isolated or to be conjugated in vivo. S-Dealkylation is believed to occur with a number of thioethers, including methylmercaptan and 6-methylthiopurine. A microsomal enzyme system catalyzes Sdealkylation with oxidative removal of the alkyl group to yield the corresponding aldehyde, as with N-dealkylation (Figure 5.17). However, certain differences from the Ndealkylation reaction suggest that different enzymes may be involved. The S-demethylation of 6-methylthiopurine to 6-mercaptopurine is shown in Figure 5.19. Aromatic methyl and ethyl ethers may be metabolized to give the phenol and the corresponding aldehyde (Figure 5.17). Ethers with longer alkyl chains are less readily O-dealkylated; the preferred route is ω-1-hydroxylation. Probably the best-known example of O-dealkylation is the demethylation of p-nitroanisole. Because of the ease with which the product, p-nitrophenol, can be measured, it is frequently used as a substrate for the demonstration of cytochrome P-450–dependent monooxygenase

(a)

H3C

(CH2)4

CH3

O

H3C

Alkane

(CH2)5

O

OH

H3C

Alcohol

(b)

(CH2)4

COOH

Carboxylic acid

n-Propylbenzene CH2CH2CH3

ω-oxidation

α-oxidation

(ω-1)-oxidation

OH

OH CH2CH2CH2OH

3-Phenylpropan-1-ol

CHCH2CH3

CH2CHCH3

3-Phenylpropan-2-ol (benzylmethylcarbinol)

3-Phenylpropan-3-ol (ethylphenylcarbinol)

Figure 5.12 Aliphatic oxidation of (a) alkanes and (b) alkyl side chains of aromatic compounds.

activity. The reaction is believed to proceed by an unstable methylol intermediate (Figure 5.20a). The O-dealkylation of organophosphorus triesters differs from that of p-nitroanisole in that it involves the dealkylation of an ester rather than ether. The reaction was first described for the insecticide chlorfenvinphos and is known to occur with a wide variety of vinyl, phenyl, phe-

nylvinyl, and naphthyl phosphate and thionophosphate triesters (Figure 5.20b). N-Oxidation The oxidation of secondary and tertiary amines is catalyzed by a liver microsomal amine oxidase that re-

OH

OH OH

Cyclohexane

Cyclohexanol

Figure 5.13 Hydroxylation of cyclohexane to mono- and dihydric alcohols.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

trans-Cyclohexane1,2-diol

Tetralin Major route

Minor route

OH HO

OH

1-Tetrol

2-Tetrol

5,6,7,8-Tetrahydro-2-naphthol

Figure 5.14 Alicyclic hydroxylation of tetralin.

quires NADPH and molecular oxygen but is not dependent on cytochrome P-450. N-Oxidation can occur in a number of ways, including hydroxylamine formation, oxime formation, and N-oxide formation, although the latter is primarily dependent on the FAD-containing monooxygenase. Hydroxylamine formation occurs with a number of amines such as aniline and many of its substi-

tuted derivatives. In the case of 2-acetylaminofluorene, the product is a potent carcinogen, and thus the reaction is an activation reaction (Grantham et al., 1968) (Figure 5.21a). Oximes can be formed by the N-hydroxylation of imines and primary amines. Imines have been suggested as intermediates in the formation of oximes from primary amines (Figure 5.21b).

Quinoline

N Aldehyde oxidase

Cytochrome P-450

Cytochrome P-450

OH

N

OH

2-Hydroxyquinoline Figure 5.15 Hydroxylation of quinoline.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

N

3-Hydroxyquinoline

HO

N

6-Hydroxyquinoline

O

O

HO

Coumarin

O

O

7-Hydroxycoumarin

Figure 5.16 Microsomal enzyme-mediated heterocyclic hydroxylation of coumarin.

N-Hydroxylation N-Hydroxylation of primary arylamines, arylamides, and hydrazines is also catalyzed by the microsomal cytochrome P-450–dependent mixed-function oxidase system. It requires NADPH and molecular oxygen. The N-hydroxylation of aniline is shown in Figure 5.22. The N-hydroxylated product phenylhydroxylamine is responsible for the production of methemoglobinemia after aniline administration to experimental animals. This may occur by further oxidation of phenylhydroxylamine to nitrosobenzene, which may then be reduced back to phenylhydroxylamine. This reaction lowers the reduced glutathione concentration in the red blood cells, removing the protection of hemoglobin against oxidative damage. N-Hydroxylated prod-

CH3 R

CH3

N

R CH3

N H

HCHO

ucts are chemically unstable and dehydrate, thereby producing a reactive electrophile such as an imine or imino-quinone. N-Hydroxylation is one of the reactions responsible for converting some compounds (e.g., 2-acetylaminofluorene, Figure 5.21a) into potent carcinogens. S-Oxidation Aromatic and aliphatic sulfides or thioethers are oxidized by microsomal monooxygenases to form sulfoxides, some of which are further oxidized to sulfones (Figure 5.23). This reaction is very common among insecticides of several different chemical classes, including carbamates, organophosphates, and chlorinated hydrocarbons. The organophosphates include phorate and demeton, whereas among the chlorinated hydrocarbons, endosulfan is oxidized to endosulfan sulfate and methiochlor to a series of sulfoxides and sulfones, eventually yielding the bis-sulfone. Among carbamates, methiocarb yields the sulfoxide and sulfone, and drugs such as chloropromazine and solvents such as dimethyl sulfoxide are also subject to S-oxidation. The metabolism of the pesticide temik (aldicarb) by this route is shown in Figure 5.24. P-Oxidation

R

R

CH3CHO

OC2H5

OH

P-Oxidation, a little known reaction, involves the conversion of trisubstituted phosphines to phosphine oxides, e.g., diphenylmethylphosphine to diphenylmethylphosphine oxide. Although described as a typical cytochrome P-450–dependent monooxygenation, it too is known to be catalyzed by the FAD-containing monooxygenase as well. Desulfuration

R

S

CH3

RSH HCHO

Figure 5.17 Microsomal enzyme-mediated N-, O-, and Sdealkylation.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Replacement of sulfur by oxygen is known to occur in a number of cases, and the oxygenation of the insecticide parathion to give the more toxic paraoxon is a good example (Figure 5.25). This reaction is also important for other phosphorothionate and phosphorodithioate pesticides. The replacement of sulfur by oxygen converts compounds from being relatively inactive toward cholinesterases into being potent cholinesterase inhibitors.

O

O

O CH2OH

OCN(CH3)2

OCNHCH3

OCN CH3

NO2

HCHO

NO2

NO2

N,N-Dimethyl-p-nitrophenyl carbamate

N-Methyl-p-nitrophenyl carbamate

Figure 5.18 Dealkylation of N,N-dialkyl carbamates by microsomal monooxygenases.

Much of the splitting of the phosphorus ester bonds in organophosphate pesticides, formerly believed to be due to hydrolysis, is now known to be due to oxidative dearylation. This is a typical cytochrome P-450–dependent monooxygenation, requiring NADPH and molecular oxygen and being inhibited by CO. Oxidative desulfuration at the C-S bond may also occur, such as in thiobarbital, which is metabolized to barbital, or in the metabolism of phenylthiourea to phenylurea (Figure 5.26) Deamination Deamination of compounds such as amphetamine may be catalyzed by a microsomal amine oxidase that requires NADPH and molecular oxygen. The product of deamination of a primary amine is the corresponding ketone (Figure 5.27). This overall reaction may in fact represent several steps: initial carbon oxidation followed by rearrangement to give the ketone with the loss of ammonia. The metabolic reaction is carbon oxidation rather than oxidation at the nitrogen atom. Monoamine oxidase, which may also be involved in the deamination of amines, is a mitochondrial enzyme.

SH

SCH3 N

N N

N H

6-Methylthiopurine

N

N N

N H

6-Mercaptopurine

Figure 5.19 Oxidative S-demethylation of 6-methylthiopurine.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Methylenedioxy (Benzodioxole) Ring Cleavage Methylenedioxyphenyl compounds, such as safrole or the insecticide synergist piperonyl butoxide, many of which are effective inhibitors of cytochrome P-450–dependent monooxygenations, are themselves metabolized to catechols. The most probable mechanism appears to be an attack on the methylene carbon, followed by elimination of water to yield a carbene. The highly reactive carbene either reacts with the heme iron to form a cytochrome P-450–inhibitory complex or breaks down to yield the catechol (Figure 5.28). In addition to the microsomal monooxygenases, other enzymes are involved in the oxidation of xenobiotics. These enzymes are located in the mitochondria or in the soluble cytoplasm of the cell. The major nonmicrosomal oxidation reactions are described later. Amine Oxidation The most important function of amine oxidases appears to be the oxidation of amines formed during normal processes. Two types of amine oxidases are involved with the oxidative deamination of both endogenous and exogenous amines. The monoamine oxidases are a family of flavoproteins found in the mitochondria of a wide variety of tissues, including liver, kidney, brain, intestine, and blood platelets. They are a group of similar enzymes with overlapping substrate specificities and inhibition. The diamine oxidases are soluble enzymes also found in a number of tissues that oxidize amines to aldehydes. These enzymes prefer primary amines; secondary and tertiary amines are less readily deaminated. The oxidation of endogenous compounds such as 5-hydroxytryptamine and diamines such as putrescine is shown in Figure 5.29. The products of oxidation of both monoamines and diamines are aldehydes.

(a)

OCH3

OH

OCH2OH

H2O

NO2

NO2

NO2

p-Nitroanisole

(b)

CH3CH2O

p-Nitrophenol

O P

CH3CH2O

CHCl

OC

Cl

OH CH2CHO

CH3CHO

O

HO

P CH3CH2O

OR

CH3CH2O

O P OR

Cl Chlorfenvinphos

Figure 5.20 Examples of O-dealkylation during phase I biotransformations. (a) p-Nitroanisole, an aromatic methyl ether; (b) organophosphorus triester, chlorfenvinphos.

(a) Hydroxylamine Formation H

OH

N

N COCH3

COCH3 N-Hydroxy-2-acetylaminofluorene

2-Acetylaminofluorene

(b) Oxime Formation CH3

O CCH

H3C

CH3 NH

CH3

Trimethylacetophenone imine

O CCH

H 3C

NOH

CH3

Trimethylacetophenone oxime

Figure 5.21 Examples of N-oxidation reaction during phase I biotransformations: (a) hydroxylamine formation, (b) oxime formation.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

NH2

NHOH

Phenylhydroxylamine

Aniline

N

O

Nitrosobenzene

Figure 5.22 N-Hydroxylation of aniline catalyzed by the microsomal cytochrome P-450–dependent mixed-function oxidase.

(Figure 5.31a). Xanthine oxidase also catalyzes the oxidation of foreign compounds, such as the nitrogen heterocycle phthalazine (Figure 5.31b). This compound is also a substrate for aldehyde oxidase, giving the same product.

Alcohol and Aldehyde Oxidation Although in vitro a microsomal enzyme system that oxidizes ethanol has been demonstrated, probably the more important enzyme in vivo is alcohol dehydrogenase. It is found in the soluble fraction of the liver, kidney, and lung and is probably the most important enzyme involved in the metabolism of foreign alcohols. The coenzyme for this fairly nonspecific enzyme is usually NAD, although NADP may also be utilized. Primary alcohols are oxidized to aldehydes; n-butanol is the substrate oxidized at the highest rate (Figure 5.30a). Although secondary alcohols are oxidized to corresponding ketones, the rate is less than that for primary alcohols, and tertiary alcohols are not readily oxidized. The aldehydes produced by these reactions may be further oxidized by aldehyde dehydrogenase to the corresponding acid (Figure 5.30b). The acids are then available as substrates for conjugating enzymes. This enzyme also requires NAD and is found in the soluble fraction. Other enzymes may also be involved in the oxidation of aldehydes, particularly aldehyde oxidase and xanthine oxidase. Both of these enzymes are found in the soluble fraction of the cell, contain molybdenum, and utilize flavoproteins. Xanthine oxidase is also involved in the oxidation of purines.

Aromatization of Alicyclic Compounds Cyclohexane carboxylic acids of the general formula C6H11(CH2)nCOOH, if n is an even number (including 0), are oxidized to benzoic acid by a liver and kidney mitochondrial enzyme system (Figure 5.32a). This enzyme system requires CoA, ATP, and oxygen and is involved in three sequential dehydrogenation steps after the initial formation of the cyclohexanoyl CoA. If n is an odd number, aromatization does not occur and the cleavage products of the alicyclic ring are excreted (Figure 5.32b). Reduction Reactions Reductions in xenobiotic metabolism are much less common than oxidations because they go counter to the general trend of biochemical reactions in living tissue, yet it must be realized that all enzymatic reactions are fundamentally reversible. The prevailing direction depends on the chemical equilibrium. If the reduced form of a redox equilibrium is more readily excretable than the oxidized form, then the laws of mass action push the reaction in the direction of reduction. The enzymes responsible for reduction may be located in both the microsomal fraction and the soluble cell fraction. The location of the reductase depends on the particular type of reduction being catalyzed. The reductions

Purine Oxidation The oxidation of purines and purine derivatives is catalyzed by xanthine oxidase. For example, the enzyme oxidizes hypoxanthine to xanthine and then to uric acid

O R

S

R'

R

S

O R'

R

S

R'

O Sulfoxide

Sulfone

Figure 5.23 S-Oxidation to form a sulfoxide and sulfone by microsomal monooxygenases. These reactions are very common among insecticides of different classes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Temik CH3

Sulfoxide CH3

O

CH3SCCH

NOCNHCH3

CH3SOCCH

CH3

Sulfone O

NOCNHCH3

CH3SCCH

CH3

CH3 NOH

CH3SOCCH

CH3

NOCNHCH3

CH3SO2CCH

CH3

CH3

O

CH3

CH3 NOH

CH3SO2CCH

CH3

NOH

CH3

Oxime derivative

Figure 5.24 S-Oxidation of the pesticide temik (aldicarb).

catalyzed by these enzymes take place under anaerobic conditions and are dependent upon NADH or NADPH. The involvement of FAD may simply be as a nonezymatic electron donor. Also, bacteria in the intestines mediate most reductions of xenobiotic compounds. The contents of the lower bowel may contain as many as 1010 anaerobic bacteria per gram (Renwick and George, 1989). The compounds reduced by this gut flora may enter the lower bowel either by oral ingestion (without having been absorbed through the intestinal wall) or by secretion with bile. In the latter case, the compounds may be parent materials or metabolic products of substances absorbed in upper regions of the gastrointestinal (GI) tract. Intestinal flora are also known to mediate the reduction of organic xenobiotic sulfones and sulfoxides to sulfides. A number of functional groups, such as nitro, diazo, carbonyl, disulfide sulfoxide, alkene, and pentavalent arsenic, are susceptible to reduction (Figure 5.33), although in many cases it is difficult to tell whether the reaction proceeds enzymatically or nonenzymatically by the action of such biological reducing agents as reduced flavins or re-

EtO

duced pyridine nucleotides. Examples of such reactions are given later. Nitro and Azo Reduction Aromatic amines are susceptible to reduction by both bacterial and mammalian nitroreductase systems. This reaction sequence is catalyzed by cytochrome P-450. It is inhibited by oxygen, although NADPH is still consumed. Also, high concentrations of FAD or flavin mononucleotide (FMN) catalyze the nonenzymatic reduction of the nitro group. The reduction of nitro compounds proceeds through several stages to yield the fully reduced primary amine, as illustrated with nitrobenzene (Figure 5.33a). The intermediates are nitrosobenzene and phenylhydroxylamine. Aryl hydroxylamines, whether derived from nitro compounds by reduction or by N-hydroxylation of amines, have been shown to play a very important role in the toxicity of a number of compounds. Requirements for azo reduction are similar to those for nitro reduction, viz., anaerobic conditions and NADPH. They are also inhibited by CO and presumably involve cytochrome P-450. The ability of mammalian cells

EtO P

O

EtO

NO2

P

O

EtO S

O Parathion

Paraoxon

Figure 5.25 Oxidative desulfuration of parathion to the more toxic metabolite paraoxon.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

NO2

C2H5

C2H5

NH

NH C2H5

C2H5 O

O

S

N H

O

N H

Thiobarbital

Barbital

Sulfoxide Reduction

S

O

NHCNH2

NHCNH2

Phenylthiourea

Phenylurea

Figure 5.26 Oxidative desulfuration of thiobarbital and phenylthiourea.

to reduce azo bonds is rather poor, and the gut microflora play an important role in the reduction of many azo compounds (Figure 5.33b). Disulfide Reduction Disulfide reactions are split into free thiols, analogous to the cystine-cysteine reaction. Many of these reactions are three-step sequences, the last reaction of which is catalyzed by glutathione reductase. The reduction of the drug disulfiram (Antabuse) is shown in Figure 5.33c. Ketone and Aldehyde Reduction In addition to the reduction of aldehyde and ketones through the reversible reaction of alcohol dehydrogenase,

CH3 CH2CHNH2

CH3 CH2C

O

NH3

Amphetamine

Phenylacetone

Figure 5.27 Oxidative deamination of amphetamine to the corresponding ketone. The reaction catalyzed by microsomal amine oxidase requires NADPH and molecular oxygen. NADPH, reduced nicotinamide-adenine dinucleotide phosphate.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

a family of aldehyde reductases also reduces these compounds (Figure 5.33d). These reductases are NADPH-dependent, cytoplasmic enzymes of low molecular weight, which have been found in liver, brain, kidney, and other tissues.

The reduction of sulfoxides has been reported to occur in mammalian tissues (Figure 5.33e). Soluble thioredoxin-dependent enzymes in the liver are known to be responsible in some cases. It is believed that the oxidation in the endoplasmic reticulum followed by reduction in the cytoplasm may be a form of recycling that could extend the in vivo half-life of certain toxicants. Dehalogenation An important step in the metabolism of the many xenobiotic compounds that contain covalently bound halogens (F, Cl, Br, I) is the removal of halogen atoms, a process called dehalogenation. The microsomal enzymemediated removal of a halogen atom from a xenobiotic compound may be either oxidative or reductive. For example, the volatile anaesthetic halothane undergoes both oxidative (Figure 5.34) and reductive (Figure 5.35) dehalogenation. The latter metabolic pathway is possibly responsible for the occasional hepatotoxicity of the drug. It seems that there are several metabolic pathways; some occur under anaerobic conditions, others under aerobic conditions. Carbon tetrachloride also undergoes reductive dechlorination to give chloroform. This is catalyzed by the cytochrome P-450 system in vitro under anaerobic conditions and probably involves a free-radical reaction. Dehalogenation may also involve glutathione, as in the dehydrohalogenation of the insecticide DDT (Figure 5.36). DDT-dehydrochlorinase enzyme occurs in both mammals and insects in the soluble fraction of cell homogenates. The enzyme also catalyzes the dehydrochlorination of a number of other DDT analogs. In all cases, the p,p configuration is required; o,p and other analogs are not utilized as substrates. Hydrolysis Certain xenobiotic compounds require degradation or cleavage before they can be further metabolized. The most common of such reactions is the hydrolysis of esters, but amides, hydrazides, and nitriles may also be hydrolyzed. Alkyl groups in nonester linkages are sometimes eliminated in these reactions. Alicyclic or heterocyclic compounds may undergo ring scission.

Catechol R

OH

R1

OH HCHO

R

R

O

O

CH2

O

C

O

R1

R

H

O

R1

C OH

O

R1 Carbene

Complexes with Fe+2 of cytochrome P-450 Figure 5.28 Monooxygenation of methylenedioxyphenyl compounds during phase I biotransformation.

(a) O2, H2O

HO CH2CH2NH2

HO

oxidase

N H

CH2CHO

Monoamine

NH3, H2O2

N H

5-Hydroxytryptamine

(b)

O2, H2O Diamine

H2N(CH2)4NH2

oxidase

Putrescine

NH3, H2O2

Figure 5.29 (a) Mono- and (b) diamine oxidation during phase I biotransformations.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

H2N(CH2)3CHO

(a) Alcohol dehydrogenase

RCH2OH + NAD+

RCHO + NADH + H+

(b) Aldehyde dehydrogenase

RCHO + NAD+

RCOOH + NADH + H+

Figure 5.30 Oxidation of (a) primary alcohols and (b) aldehydes by dehydrogenases.

Hydrolysis of esters is catalyzed by a number of enzymes of generally low specificity and wide distribution. These can be classified as aryl esterases (aromatic esters), alkyl esterases (aliphatic esters), choline esterases, and acetyl esterases. Enzymes such as trypsin and chymotrypsin may also hydrolyze certain carboxyl esters. The hydrolysis of esters yields the corresponding acid and alcohol or phenol (Figure 5.37a). The hydrolysis of amides, hydrazides, and nitriles proceeds in an analogous manner. Examples of each reaction type are shown in Figure 5.37b–5.37d. The amidasecatalyzed hydrolysis of amides is rather slower than that of esters. The hydrolysis of some amides may be catalyzed by a liver microsomal carboxyl esterase, as in the case of phenacetin. Hydrazides are hydrolyzed to the correspond-

ing acid and hydrazine. These hydrolysis reactions are probably catalyzed by amidases and are inhibited by organophosphate inhibitors such as bis-p-nitrophenyl phosphate. Epoxide Hydration Epoxides, three-membered rings containing an oxygen atom, of alkene and arene compounds are hydrated by enzymes known as epoxide hydrolases (or hydratases). These enzymes add water to the epoxide to yield a transdihydrodiol (Figure 5.38). The enzyme is found in the microsomal fraction of the cell in close proximity to the cytochrome P-450 group of monooxygenases. Epoxide hydrolase is therefore well placed to carry out its important role in detoxifying the chemically unstable and often

(a) N

HN

O

O

O

N

HN

H N

HN

O N

N H

O

N H

Hypoxanthine

N H

N H

O

Uric acid

Xanthine

O

(b) Phthalazine

N

NH

N

N Phthalazinone

Figure 5.31 Oxidation of (a) hypoxanthine and (b) phthalazine by xanthine oxidase.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

N H

(a) (CH2)n

COOH

COOH mitochondrial enzyme

Even-numbered (n = 0, 2, 4, 6, . . . ) cyclohexane carboxylic acids

Benzoic acid

(b) (CH2)n

COOH mitochondrial enzyme

Ring cleavage products

Odd-numbered (n = 1, 3, 5, 7, . . . ) cyclohexane carboxylic acids Figure 5.32 Aromatization of cyclohexane carboxylic acids by a mitochondrial enzyme system requiring CoA, ATP, and oxygen during phase I transformations. (a) Even-numbered and (b) odd-numbered cyclohexane carboxylic acids. CoA, coenzyme A; ATP, adenosine triphosphate.

toxic epoxide intermediates produced by cytochrome P450–mediated hydroxylation. Ring Scission or Opening Microsomal oxidation may also open rings in cyclic compounds. The result of ring opening is the formation of polar groups (Parke, 1968). Thus, opening the pyrrole ring in an indole after oxidation and hydrolysis leads to the formation of anthranilate containing both carboxyl and amino groups (Figure 5.39a). The oxidation and hydrolysis of the lactone ring in coumarin produce both hydroxyl and carboxyl groups in the product 2-hydroxyphenylacetic acid (Figure 5.39b). The oxidative cleavage of benzene produces a dicarboxylic, trans-muconic acid (Figure 5.39c). The opposite of ring opening, cyclization, has also been observed in rare instances. Thus, after exposure to 2hydroxyethyl aniline, increased excretion of indican is noted (Figure 5.40) (Parke, 1968). 5.6.2 Phase II Reactions The biotransformation reactions described for phase I are adaptations of biochemical reactions originally developed for the metabolism of nutrients. Phase II reactions or conjugations, in contrast, seem to have developed primarily for the metabolism of xenobiotic compounds. These types

Copyright 2002 by Marcel Dekker. All Rights Reserved.

of reactions are, therefore, most often successful in terms of detoxification. The overall process for the conjugation of a xenobiotic compound is shown in Figure 5.41. Metabolism of phase I products and other xenobiotics containing functional groups such as hydroxyl, amino, carboxyl, epoxide, or halogen can undergo conjugation reaction with endogenous metabolites. The latter include sugars, amino acids, glutathione, and sulfate. The groups donated in conjugation reactions are often involved in intermediary metabolism. Conjugation products, with only rare exceptions, are more polar, less toxic, and more readily excreted than are their parent compounds. Conjugation reactions usually involve activation by some high-energy intermediate and have been classified into two general types: type I, in which an activated conjugating agent combines with the substrate to yield the conjugated product, and type II, in which the substrate is activated and then combines with an amino acid to yield a conjugated product. The formation of glycosides and sulfates exemplifies type I, whereas type II consists primarily of amino acid conjugations. The coenzymes involved in these reactions include CoA with acetate and other short-chain fatty acids; adenosine or phosphoadenosine phosphate (PAP) with sulfate, methionine, and ethionine; and uridine diphosphate (UDP) with glucose and glucuronic acid. The important phase II biotransformation reactions are described later.

(a) Nitro Reduction NO2

NO

Nitrobenzene

NHOH

NH2

Phenylhydroxylamine

Nitrosobenzene

Aniline

(b) Azo Reduction H3C

H 3C

H 3C

H2N

N

H3C H N

H N

H2N

N

Hydrazo derivative

O-Aminoazotoluene

H3C

H3C H2N

H2N

NH2

(c) Disulfide Reduction (C2H5)2NCSS

SSCN(C2H5)2

2 (C2H5)2NCSSH Dimethyldithiocarbamic acid

Disulfiram

(d) Aldehyde Reduction

(e) Sulfoxide Reduction S

CHO

CHO

O

(C2H5O)2PSCH2S

Cl

Carbophenothion sulfoxide

Cl

p-Chlorobenzaldehyde

Cl

p-Chlorobenzyl alcohol

S (C2H5O)2PSCH2S Carbophenothion

Figure 5.33 Examples of metabolic reduction reactions in phase I biotransformation processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Cl

F F

Cl

C

C

F

Br

H

O2

F

F

Cl

C

C

F

Br

OH

F

F

Cl

C

C

O

F

HBr

F

F

O

C

C

OH

F

HCl

Halothane

Trifluoroacetic acid

Figure 5.34 Oxidative dehalogenation of halothane.

phate (Figure 5.42); UDPGA is able to donate glucuronic acid to a wide variety of substrates, including endogenous substances. Conjugation with glucuronic acid involves nucleophilic attack by the oxygen, sulfur, or nitrogen atom at the C-1 carbon atom of the glucuronic acid moiety. Glucuronides are therefore generally β in configuration. Conjugation with hydroxyl groups gives ether glucuronides and with carboxylic acids, ester glucuronides. Amino groups may be conjugated directly or through an oxygen atom. Certain thiols may also be conjugated directly through the sulfur atom. A great variety of xenobiotic compounds are subject to glucuronide conjugation. Susceptible functional groups include alcohols, phenols, enols, carboxylic acids, amines, hydroxylamines, carbamides, sulfonamides, and thiols (Figure 5.44). The reaction qualifies as the most frequent method of xenobiotic compound excretion in mammals. A few endogenous compounds (e.g., thyroxine, bilirubin) are also excreted as glucuronides. Although conjugation generally decreases biological activity, including toxicity, occasionally the latter is increased, as in the case of acetylaminofluorene. The N-hydroxyglucuronide is a more potent carcinogen than N-hydroxyacetylaminofluorene (Figure 5.45). Glucuronic acid has substantial advantages over glucose as a detoxifying agent because glucuronides are ionizable, whereas glucosides are not. This difference is

Glycoside Conjugation The formation of an activated intermediate, either uridine diphosphate glucose (UDPG) or uridine diphosphate glucuronic acid (UDPGA), is required for glycoside formation. The enzymes involved occur in the soluble fraction of the liver and other organs. The activation sequence and examples of the various types of glycosides described in the following section are shown in Figures 5.42 and 5.43, respectively. Glucuronides In most vertebrates and all mammals, the 6-carboxyl derivative of glucose, viz., glucuronic acid, has evolved for xenobiotic conjugation. The conjugation reaction involves the transfer of glucuronic acid from UDPGA. The atoms to which glucuronic acid may be attached are oxygen in hydroxyl and carboxyl groups, and in some cases, sulfur and nitrogen atoms. The enzymes catalyzing the conjugation reactions, the glucuronyl transferases, are found in the microsomal fraction where hydroxylated phase I metabolites of lipophilic xenobiotic compounds are produced. As a result, the lifetime of the phase I metabolites is often quite brief. The donor UDP-glucuronic acid (UDPGA) is formed in the soluble fraction of hepatic cells from glucose-1-phos-

F Cl F

C

C H

F

Br

F Cl e-

F

F

C

C H

F

Br

e-

F

C

C H

F Cl

Br-

Halothane

H+

H

F C

F

C

F Cl

1,1-Difluoro-2-chloroethylene

Figure 5.35 Reductive dehalogenation of halothane.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

F H

F-

C

C H

F Cl 1,1,1-Trifluoro-2-chloroethane

H Cl

C

GSH

Cl

Cl

C

Cl

Dehydrochlorinase

CCl3

CCl2

DDT

DDE

Figure 5.36 Dehydrohalogenation of the insecticide DDT. The reaction requires glutathione. DDT, dichlorodiphenyltrichloroethane; GSH, glutathione; DDE, 1,1-dichloro-2,2-bis(chlorophenyl) ethylene.

sometimes cited as the reason why higher animals have better resistance to environmental chemicals than the lower animals, and as the basis for the selective action of pesticides. Glucosides Although rare in vertebrates, glucosides formed from xenobiotics are common in insects, mollusks, and

(a)

plants. Formed from UDP-glucose, they appear to fall into the same classes as the glucuronides. Generally, glucoside formation is not a very efficient method of detoxification because the sugar contributes no ionizable group to the conjugate. The reaction therefore appears to have been abandoned during evolution and does not occur in mammals.

RCOOR' + H2O

RCOOH + R'OH

Alkyl ester

NH4+

(b) CONH2

COOH

Benzamide

Benzoate

CONHNH2

COOH

(c)

NH2 N Isonicotinic acid hydrazide (Isoniazid)

NH3+

N Isonicotinic acid

NH4+

(d) CH2CN Benzylcyanide

CH2COOH Phenylacetic acid

Figure 5.37 Hydrolysis of (a) esters, (b) amides, (c) hydrazides, and (d) nitriles during phase I biotransformations.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

OH Epoxide hydratase

H H

O

OH Benzene-trans-1,2-dihydrodiol

Benzene epoxide Figure 5.38 Hydration of benzene epoxide by epoxide hydratase.

Sulfate (Ethereal) Conjugation In general, sulfate conjugation probably ranks with glucuronic acid and mercapturic acid conjugations as the most commonly occurring excretory biotransformation process for xenobiotic compounds. The sulfated compounds become salts of strong acids and are therefore highly ionized at the pH of the body; consequently, the sulfate conjugates are very soluble and promptly eliminated in the urine. The sulfate ion is derived from methionine via cysteine. Sulfate esters are formed with xenobiotics such as aliphatic alcohols, phenols, and arylamines, and with

endogenous compounds such as steroids and carbohydrates. This process requires the prior activation of sulfate ions to 3′-phosphoadenosine-5′-phosphosulfate (PAPS) (Figure 5.46), a reaction sequence requiring the consumption of ATP, and hence, using a considerable amount of energy. In addition to inorganic sulfate and ATP, the formation of PAPS requires the sequential action of ATP sulfurylase and adenosine-5′-phosphosulfate kinase. ATP sulfurylase from rat liver is a large molecule of about 500,000 daltons. Several group VI anions other than sulfate can also serve as substrates, although the resultant an-

(a) O H2O N H

N H

O

COOH H2O

COOH

NHCHO

NH2

Indole

Anthranilic acid

(b)

O O

O

O

O

CH2CCOOH

CH2COOH

OH

OH

H2O OH Coumarin

2-Hydroxy-2-phenyl acetic acid

(c) OH HOOC OH Benzene

Pyrocatechol

C H

H C

C H

H C

COOH

trans-Muconic acid

Figure 5.39 Ring scission of cyclic xenobiotics in the phase I biotransformation processes: (a) indole, (b) coumarin, and (c) benzene.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

CH2CH2OH

CH2CHO

O

- H2O

NH2

N H

NH2

2-Hydroxyethyl aniline O

OSO3H

OH SO4-2 PAPS, K+

N

N

Indican

Indoxyl

Figure 5.40 Cyclization of 2-hydroxyethyl aniline during phase I biotransformation.

hydrides are unstable. Because this instability would lead to the overall consumption of ATP, these other anions can exert a toxic effect by depleting the cell of ATP. The second enzyme, the kinase, is not well known from mammalian tissues, but that from yeast shows a high affinity for adenosine-5′-phophosulfate (APS). The reaction is essentially irreversible.

A family of related sulfotransferases that have been classified as follows catalyzes the final step: aryl sulfotransferases, hydroxysteroid sulfotransferases, estrone sulfotransferases, and bile salt sulfotransferases. These enzymes exist in several different forms. Selected examples of sulfate conjugates of some xenobiotic compounds are shown in Figure 5.47.

Xenobiotic compound, often phase I reaction product Functional groups Carboxyl

Hydroxyl

Halogen

Epoxide

Endogenous conjugating agent

Conjugation product Higher polarity Greater water solubility More easily excreted

Figure 5.41 Overall process of conjugation in the phase II biotransformation processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Amino

Glucose-1-phosphate

CH2OH O COOH

OH O

P

O

OH 2 NADH2

O

O

UTP

UDP

UDP-α-D-glucuronic acid (UDPGA) PPi UDPG dehydrogenase

CH2OH

2 NAD+ H2O

O OH O

P

O

OH O

O

P

OCH2

NH O

O

N

O

UDP-α-D-glucose (UDPG)

Figure 5.42 Formation of uridine diphosphate glucuronic acid (UDPGA).

Methylation (Alkylation) Amino, hydroxyl, and thiol groups in xenobiotic compounds may undergo methylation in vivo, as do certain endogenous compounds such as catecholamines. These reactions are catalyzed by several N-, O-, and S-methyl transferases. The sulfur-containing amino acid methionine is the chief methyl donor. The methyl group is released only from its activated form, S-adenosyl methionine (SAM) (Figure 5.48), formed from methionine and ATP. S-Adenosylethionine, formed from the amino acid ethionine, acts analogously to an ethyl donor in transethylation reactions. Even though these reactions may involve a decrease in water solubility, they are generally detoxification reactions. Examples of biological methylation reactions are shown in Figure 5.49. Several enzymes are known that catalyze N-methylation reactions. They include histamine N-methyltransferase, a highly specific enzyme that occurs in the soluble fraction of the cell; phenylethanolamine N-methyltransferase, which catalyzes the methylation of noradrenaline to adrenaline as well as the methylation of other phenylethanolamine derivatives; and indoethylamine N-methyltransferase or nonspecific N-methyl transferase. The latter methylates endogenous compounds such as serotonin and

Copyright 2002 by Marcel Dekker. All Rights Reserved.

tryptamine, and exogenous compounds such as nornicotine and norcodeine. O-Methyltransferases can be found in both the soluble and the microsomal fractions of several tissues. The soluble enzyme preferentially methylates epinephrine, norepinephrine, and other catechol derivatives; the microsomal enzyme methylates a number of alkyl-, methoxy-, and halophenols. The S-methyltransferase is also a microsomal enzyme and can methylate a wide variety of substrates, including thioacetanilide, mercaptoethanol, and phenylsulfide. This enzyme may also be important in the detoxification of hydrogen sulfide, which is methylated in two steps, first to the highly toxic methanethiol, then to dimethylsulfide. Acetylation Acetylation is an important route of metabolism for aromatic amines, sulfonamides, and hydrazines. The enzyme that catalyzes this reaction is N-acetyl transferase. It is found in the cytosol of hepatic reticuloendothelial cells, in the GI mucosal cells, and also in white blood cells. The coenzyme utilized in the acetyl transferase–catalyzed reactions is acetyl CoA, which transfers the acetyl group

O-Glucuronide Formation

COOH

OH

O O

HO

UDP glucuronosyl

UDPGA

OH

transferase

OH

UDP

COOH

N-Glucuronide Formation O

H N

NH2

HO

UDP glucuronosyl

UDPGA

OH

transferase

OH

S-Glucuronide Formation

UDP

COOH O UDP glucuronosyl

UDPGA

SH

S

HO

transferase

OH OH

UDP

C-Glucuronide Formation

UDP glucuronosyl

UDPGA N

N

N COOH

O

N

transferase

O (CH2)3CH3

O

O (CH2)3CH3

O OH

UDP

HO OH

O-Glucoside Formation

CH2OH

OH

O

HO

UDP glucosyl

UDPG

O

OH NO2

NO2

Figure 5.43 Examples of glycoside formation in phase II biotransformation processes.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

UDP OH

transferase

CH2O

C6H9O6

COO

Benzyl glucuronide (alcohol type)

O

C6H9O6

O

Benzoyl glucuronide (carboxylic acid type)

C6H9O6

S

Phenyl glucuronide (phenol type)

Thiophenol glucuronide (thiol type)

O

SO2 O

C6H9O6

C6H9O6

HN

C6H9O6

N

C6H9O6

Sulfathiazole-N1-glucuronide (sulfonamide type)

Aniline glucuronide (amine type)

4-Hydroxycoumarin glucuronide (enol type)

N

COCH3 C3H7 N

CH2OCOCH3 C

O

C6H9O6

N-Hydroxy-2-aceteminofluorene glucuronide (hydroxylamine type)

H3C

CH2OCONH

C6H9O6

Meprobamate glucuronide (carbamide type)

Figure 5.44 Examples of xenobiotic compounds subject to glucuronide conjugation in phase II reactions.

to the enzyme, which in turn acetylates the substrate. As does transmethylation, acetylation results primarily in detoxification by masking a reactive amino group, rather than increased water solubility of the metabolite. The resulting amide is less water-soluble than the parent compound. Typical acetylation reactions are shown in Figure 5.50. Acetylation of certain xenobiotic compounds shows wide interindividual variation in humans and in the rabbit.

OH N

COCH3

This variation has both a developmental and a genetic basis and shows a bimodal distribution; the two phenotypes are termed rapid and slow acetylators. This polymorphism probably reflects different forms of acetyltransferase enzyme. The acetylation polymorphism has a number of toxicological consequences. Only certain substrates are polymorphically acetylated; others, notably sulfanilamide, p-aminosalicylic acid, and p-aminobenzoic acid, are monomorphically acetylated.

O

Glucuronic acid

N

COCH3

Glucuronyl transferase

N-Hydroxyacetylaminofluorene

N-Hydroxyacetylaminofluorene glucuronide

Figure 5.45 N-Hydroxyacetylaminofluorene glucuronide, a more potent carcinogen than its parent compound, N-hydroxyacetylaminofluorene.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O N CH

CH2

CH

O

CHOH

CH2

N

O

N N

HO O

P S

P

O O

HO

O NH2

OH

OH

Figure 5.46 Chemical structure of 3′-phospho-adenosine-5′phosphosulfate (PAPS).

Amino Acid Conjugation In the second type of acylation reaction, amino acid conjugation, xenobiotic compounds containing carboxylic acids are excreted as peptide conjugates. The amino acids most commonly utilized in mammals are glycine and glutamine, but conjugates with ornithine (reptiles and birds) and taurine (fish) are also known. The reaction involves acylation

NH2

of the amino group of the amino acid by the xenobiotic carboxylic acid group. It is therefore the converse of acetylation as described earlier. The activating enzyme occurs in the mitochondria and belongs to a class of enzymes known as the ATP-dependent acid:CoA ligases (AMP) but has been also known as acyl CoA synthetase and acid-activating enzyme. It appears to be identical to the intermediate-chain-length fatty acyl-CoA synthetase. Exogenous carboxylic acids are activated to form SCoA derivatives in a reaction involving ATP and CoA. These CoA derivatives then acylate the amino groups of a variety of amino acids. These reactions are schematically shown for conjugation of benzoic acid and phenylacetic acid with glycine and glutamine, respectively, in Figure 5.51. The conjugates with substituted benzoic acids or heterocyclic acids and glycine are sometimes broadly designated as hippuric acids. Phosphate Conjugation Phosphorylation of xenobiotics is not a widely distributed conjugation reaction; insects are the only major group of

NHOSO3H OH

Aniline

OSO3H

2-Naphthol NH2

NHSO3H

2-Naphthylamine O CH3

HO

O CH3

HO3SO Estrone

Figure 5.47 Examples of conjugation by sulfate in phase II biotransformation processes. 3′-Phospho-adenosine-5′-phosphosulfate (PAPS) is required for the prior activation of sulfate ions in these reactions.

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O N CH2

CH

CHOH

CHOH

N

CH

S+

H3C

N N

CH2

CH2

CH

COOH NH2

NH2

Figure 5.48 Chemical structure of S-adenosylmethionine synthesized from methionine and ATP.

animals in which it occurs. The enzyme from the gut of cockroaches utilizes ATP, requires Mg2+, and is active in the phosphorylation of 1-naphthol and p-nitrophenol. Glutathione Conjugation Several types of xenobiotic compounds are excreted as conjugates with N-acetylcysteine (mercapturic acid conjugates). These conjugates generally result from an initial

conjugation with glutathione (a tripeptide containing GluCys-Gly) followed by metabolic cleavage of the glutamyl and glycinyl residues and then acetylation of the cysteine moiety. The initial conjugation reactions are catalyzed by glutathione-S-transferases. These enzymes are widely distributed and are found in essentially all groups of living organisms. They are primarily found in the soluble fraction of the cell but are also present in the microsomes.

N-Methylase

N H

N CH3

SAM

SAH

N

N Nornicotine

Nicotine

O-Methylase O

O

CH3CHN

OH

SAM

CH3CHN

Hydroxyacetanilide

OCH3

SAH

p-Methoxyacetanilide

S-Methylase

Cl

SH

p-Chlorothiophenol

SAM

Cl

SCH3

SAH

p-Chloro-S-methylthiophenol

Figure 5.49 Examples of methyl transferase reactions in phase II biotransformation processes: SAM, S-adenosylmethionine, SAH, S-adenosylhomocysteine.

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CONHNHCOCH3

CONHNH2 Acetyl CoA

N

N Isoniazid

Acetylisoniazide

NHCOCH3

NH2

Acetyl CoA

SO2NHCOCH3

SO2NH2

N1,N4-Diacetylsulfanilamide

Sulfanilamide

Figure 5.50 Typical acetylation reactions in phase II biotransformation processes.

COOH

COSCoA

CONHCH2COOH Glycine

ATP CoASH

Benzoyl CoA

Benzoic acid

ATP

Benzoyl glycine (Hippuric acid)

Glutamine

CH2CONH2

CoASH

CH2 CH2COOH Phenylacetic acid

CH2COSCoA Phenylacetyl CoA

CH2CONHCHCOOH Phenylacetylglutamine

Figure 5.51 Conjugation of xenobiotics with amino acids: (top) glycine, (bottom) glutamine.

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RX + HSCH2CHC(O)NHCH2COOH NHC(O)CH2CH2C(NH2)COOH Glutathione-S-transferase

RSCH2CHC(O)NHCH2COOH NHC(O)CH2CH2(NH2)COOH γ-Glutamyl transpeptidase

RSCH2CHC(O)NHCH2COOH NH2 Cysteinyl glycinase

RSCH2CH(NH2)COOH Acetylase

RSCH2CHCOOH HNC(O)CH3

MERCAPTURIC ACID

The overall pathway and examples of reactions mentioned later are shown in Figure 5.52. In many cases, the sulfhydryl group of glutathione acts as a nucleophile, attacking the reactive electrophilic center in the xenobiotic molecule. This reaction is catalyzed by one of the various forms of glutathione transferase. This is followed by the transfer of glutamate by γ-glutamyltranspeptidase (a membrane-bound glycoprotein found in kidney and liver cells), by loss of glycine through cysteinyl glycinase, and finally by acetylation of the cysteine amino group. The overall sequence, but particularly the initial reaction, is extremely important toxicologically. By removing reactive electrophiles, it protects vital nucleophilic groups in macromolecules such as proteins and nucleic acids. The mercapturic acids formed can be excreted either in the bile or in the urine. There are a variety of substrates for this reaction, the mechanism of which may vary. Thus, aromatic hydrocarbons, alkyl halides, aryl halides, aryl epoxides, alkyl epoxides, aromatic nitro compounds, and alkenes may all be conjugated with glutathione and excreted as mercapturic acids (Table 5.7). In some cases, enzymatic catalysis may not be necessary; a chemical reaction between the activated substrate and glutathione is sufficient. Selected examples of glutathione conjugation reactions are shown in Figure 5.53.

Figure 5.52 Glutathione transferase reaction and formation of mercapturic acids during phase II biotransformation of xenobiotic compounds.

Table 5.7

Classes of Compounds Metabolized to Mercapturates

Compound class Aromatic hydrocarbon Arylamine Arylhalide Halogenonitrobenzene Aralkyl ester Aralkyl halide Alkyl halide Alkyl phenol Nitroalkane Halogenocycloalkane Carboxylic acid Ester Sulfonamide Sulfur mustard α,b-Unsaturated compound

Example

Thiol-reacting group

Benzene, naphthalene, anthracene, benz(a)anthracene Aniline, 2-naphthylamine Chlorobenzene, 1,2-dichlorobenzene, 1-chloronaphthalene 1,2-Dichloro-4-nitrobenzene, pentachloronitrobenzene Benzyl acetate Benzyl chloride Allyl chloride, bromomethane 3,5-di-tert-butyl-4-hydroxytoluene 1-Nitropropane Bromocyclohexane Maleic acid Ethyl methanesulfonate, urethane Benzothiazolesulfonamide Bis-β-chloroethyl sulfride Arecoline, ethacrynic acid

Epoxide intermediates Hydroxylamine intermediates Epoxide intermediates Cl, NO2 OCOCH3 Cl Cl, Br H of tolueyl CH3 NO2 Br α,β=double bond CH3SO3, OCONH2 SO2NH2 Cl α,β=double bond

Source: Concon (1988).

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Aryl transferase

Aralkyl transferase

Cl

SG NO2

CH2SG

CH2Cl NO2

GSH

GSH

HCl

HCl NO2

NO2

Epoxide transferase SG

O

Alkene transferase

OCH2CHCH2 CHCOOC2H5

OCH2CHCH2

CHCOOC2H5

GSH

CHCOOC2H5

OH GSH

GSCHCOOC2H5

Sulfate ester transferase CH2OSO3H

NO2

CH2SG

NO2

GSH H2SO4

Figure 5.53 Examples of glutathione transferase phase II biotransformation reactions.

5.7

FACTORS AFFECTING XENOBIOTIC METABOLISM

It is quite obvious from the foregoing discussion that biotransformation reactions present tremendous variability. Seldom does a unique and exclusive route, even in the same species, metabolize a compound. Genetic and environmental factors, sex, age, nutrition, health status, and size of the dose all influence the relative utilization of different pathways. The same individual may excrete a simple substance such as phenol in three to four different forms (Figure 5.54). Sometimes metabolism is distinctly species-specific, as in the case of phenylacetic acid (Figure 5.55). Among more complex compounds, metabolic differences between closely related species may be subtle, but even such subtle differences may have long-reaching consequences. For instance, different rodents oxidize 1,2,5,6dibenzanthracene in different molecular positions (Figure 5.56). On the position of the oxidation depends the carcinogenicity of the resulting molecule. Occasionally compounds in a homologous series may be metabolized according to the size of the molecule. A good example of this is the extent of glucuronide conjugation of secondary alcohols in rabbits. Up to a certain

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molecular size (2-propanol to 2-heptanol), the percentage excreted as glucuronide increases from 10% to 55%. However, only about 15% is excreted as glucuronide conjugate with 2-octanol. This sudden reversal is attributed to the metabolic cleavage of the molecule into two halves prior to glucuronide conjugation. In view of these circumstances, no firm general rules should be formulated on biotransformation routes. Only relatively few of the millions of organic compounds known today have been studied exhaustively from a toxicological viewpoint. Predictions based on chemical structure or extrapolations from one species to another can therefore frequently lead to error. Nevertheless, an understanding of the role of both physiological as well as extraneous factors affecting xenobiotic biotransformations can provide essential guidelines in the assessment of toxic hazards of specific compounds. Despite the complexity of biotransformation reactions, the following general conclusions can be drawn (Sipes and Gandolfi, 1986; Concon, 1988; Hodgson, 1987): 1.

Phase I metabolism generally introduces into a xenobiotic a functional group that enables conjugation to an endogenous metabolite to occur during the phase II metabolism.

Phenol

OH

50%

40%

<1%

10%

OH

OH O C6H9O6 Phenylglucuronide

O SO3K Potassium phenyl sulfate

OH

OH

Quinol

Catechol

Figure 5.54 Different forms of excretion of phenol by the same individual.

2.

3.

The conjugates produced by phase II metabolism are considerably more water-soluble than either the parent compound or the phase I metabolite(s) and hence are more excretable. During the course of metabolism, and particularly during phase I biotransformations, reactive intermediates that are much more toxic than the parent compound may be produced. Thus, xeno-

4.

biotic metabolism may be either a detoxification or an intoxication (activation) process. Because the number of enzymes involved in phase I and phase II reactions is large and many different sites on organic molecules are susceptible to metabolic attack, the number of potential metabolites and intermediates that can be derived from a single substrate is frequently very large.

Phenylacetic acid

CH2COOH Primates

Birds and reptiles

Lower primates

COOH

COOH

CH2CONHCHCH2CH2CONH2 Phenylacetylglutamine

CH2CONHCHCH2CH2CH2NH2 Phenylacetylornithurate

CH2CONHCH2COOH Phenylacetylglycine (hippurate)

Figure 5.55 Species-specific differences in the biotransformation of phenylacetic acid.

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1,2,5,6-Dibenzanthracene

OH

O O

Mouse

Rabbit

Rat and mouse

OH

Mouse

Mouse

O

HO

*

OH

Mouse

Mouse

O O O HO

*

Mouse

HO

O

OH O O

Figure 5.56

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Differences in the metabolism of 1,2,5,6-dibenzanthracene in closely related species. *, carcinogenic metabolites.

*

5.

6.

7.

8.

Because both qualitative and quantitative differences exist among species, strains, individual organs, and cell types, a particular toxicant may have different effects in different circumstances. Because exogenous chemicals can be inducers and/or inhibitors of the xenobiotic-metabolizing enzymes of which they are substrates, such chemicals may interact to bring about toxic sequelae different from those that might be expected from any of them administered alone. Because endogenous factors also affect the enzymes of xenobiotic metabolism, the toxic sequelae to be expected from a particular toxicant vary with developmental stage, nutrition status, health or physiological status, sex, stress, or environment. It has become increasingly clear that most enzymes involved in xenobiotic metabolism occur as several isozymes and that these coexist within the same individual and, frequently, within the same subcellular organelle. An understanding of the biochemical and molecular genetics processes of these isozymes may lead to an understanding of the variation among species, individuals, organs, sexes, and developmental stages.

REFERENCES Concon, J. M. 1988. Food Toxicology. Parts A and B. Marcel Dekker, New York. Conney, A. H. 1967. Pharmacological implications of microsomal enzyme induction. Pharmacol. Rev. 19:317–366. Drasar, B. S., Shiner, M., and McLeod, G. M. 1969. Studies on the intestinal flora. 1. The bacterial flora of the gastrointestinal tract of healthy and achlorhydric persons. Gastroenterology 56:71–79.

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Grantham, P. H., Mohan, L., Yamamoto, R. S., Weisburger, E. K., and Weisburger, J. H. 1968. Alteration of the metabolism of the carcinogen N-fluoren-2-ylacetamide by acetanilide. Toxicol. Appl. Pharmacol. 13:118–130. Gorbach, S. L., Nahas, L., Lerner, P. I., and Weinstein, L. 1967. Studies of intestinal microflora. I. Effect of diet, age, and periodic sampling on numbers of fecal microorganisms in man. Gastroenterology 53:845–855. Hill, M. J. 1977. The role of unsaturated bile acids in the etiology of large bowel cancer. In Origins of Human Cancer, eds. C. H. H. Hiatt, J. D. Watson, and J. A. Winsten, pp. 79–98, Cold Spring Harbor Laboratory, New York. Hill, M. J., Crowther, J. S., and Drasar, B. S. 1971. Bacteria and the etiology of cancer of large bowel. Lancet 1:95–100. Hodgson, E. 1987. Metabolism of toxicants. In Modern Toxicology, eds. E. Hodgson and P. E. Levi, pp. 51–84, Elsevier, New York. Loomis, T. A. 1978. Essentials of Toxicology, 3rd ed., Lea and Febiger, Philadelphia, PA. Manahan, S. E. 1992. Toxicological Chemistry. 2nd ed., Lewis Publishers, Chelsea, MI. Moore, W. E. C., Cato, E. P., and Haldeman, L. V. 1969. Anaerobic bacteria of the gastrointestinal flora and their occurrence in clinical infections. J. Infect. Dis. 119:641–649. Parke, D. V. 1968. The Biochemistry of Foreign Compounds. Pergamon Press, London. Renwick, A. G. and George, C. F. 1989. Metabolism of xenobiotics in the gastrointestinal tract. In Intermediary Xenobiotic Metabolism in Animals: Methodology, Mechanisms, and Significance, eds. D. H. Hutson, J. Caldwell, and G. D. Paulson, pp. 3–12, Taylor and Francis, London. Sipes, I. G. and Gandolfi, A. J. 1986. Biotransformation of toxicants. In Casarett and Doull’s Toxicology: The Basic Science of Poisons, 3rd ed., eds. C. D. Klaassen, M. O. Amdur, and J. Doull, MacMillan, New York. Snyder, R. and Remmer, H. 1979. Classes of hepatic microsomal mixed function oxidase inducers. Pharmacol. Ther. 7:203–209. Williams, R. E. O. and Drasar, B. S. 1972. Alterations in gut bacterial flora in disease. In Recent Advances in Gastroenterology. 2nd ed., eds. J. Badenoch and B. N. Brooke, pp. 102–127, Churchill-Livingston, London.

6 Measurement of Toxicants and Toxicity

6.1

MEASUREMENT OF TOXICANTS

Qualitative and quantitative analytical techniques are of tremendous importance in all fields and disciplines of life sciences for the detection, identification, and measurement of concentration of a wide variety of biologically important molecules. These analytical techniques can be classified into three general categories: biological techniques or bioassays, those based on physical and/or chemical methods, and those that depend on noncovalent binding of one reactant to another. Techniques that fall under the third category are also commonly referred to as binding assays. Since the 1980s tremendous advances have been made in the sensitivity or the level of detection of chemicals by using sophisticated analytical instruments. In most cases, the values derived are accurate to levels that were unattainable before. Instrumentation, however, is only one part of the analytical process, which consists of a series of six operations culminating in the determination of the extent and quantity of the chemical present (Leidy and Hodgson, 1987). A generalized protocol for the quantitative measurement of toxicants is shown in Figure 6.1. A prerequisite for use of any qualitative and/or quantitative analytical technique is a clear definition of the goal, which varies with circumstances. On the basis of the nature of the toxicant, a strategy for sampling and subsequent analysis first must be formulated. Such strategy would obviously differ for the analysis of toxicant metabolites versus the determination of an accidental overdose of a chemical.

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The actual sampling step depends on the type of matrix involved. The sample must be a representative of the object of the study, since the resulting data are only as good as the samples from which they are derived. A special attention to sampling procedures is often necessary. Isolation and extraction are the next important steps in the overall process of identification and quantitation of toxicants (Figure 6.1), which involve the physical extraction of the toxicant and the removal of other interfering substances from the sample medium. The methods used for extraction are generally dictated by the chemical and physical characteristics of the toxicant and the sample matrix. Simple blending, washing (for surface contaminants), or continuous extraction of the solid samples using one or more often, a combination, of solvents is commonly used for this purpose. Toxicants from the liquid samples are generally extracted by shaking with an appropriate solvent or solvent combination. Other extraction methods, such as boiling, grinding, or distilling the sample with appropriate solvents, are used less frequently. After extraction, an isolation or separation step may precede the actual analysis of the toxicant. During extraction processes, many undesirable components are also released from the sample matrix. These must be removed to obtain accurate quantitative results from certain instruments. These compounds include plant and animal pigments, lipids, organic material from soil and water, and inorganic compounds. Unless removed, these impurities can decrease the sensitivity of the detectors and columns used in many analytical instruments, mask peaks, or produce extraneous peaks on chromatograms (Leidy and

quantitation can be done by an analytical instrument. If the reaction used for detection is highly specific (e.g., immunoassay), the analysis can be carried out on relatively impure samples. The final step in the measurement of toxicants is an evaluation of the data to determine whether additional sampling is required. Automatic data handling systems are used widely with the modern sophisticated instruments. Many are connected directly to computers to facilitate rapid identification and quantitation. It should be noted that although the data are based on either external or internal standards, the calculated values are only as good as the efficiency of the analytical method used. As mentioned earlier, an indication of the latter can be easily obtained by including an internal reference standard during the extraction and quantitation procedure. The salient features of commonly used analytical techniques for the identification and quantitation of toxicants are now described. Figure 6.1 A generalized protocol for the quantitative measurement of toxicants in biological samples.

6.1.1 Biological Techniques or Bioassays

Hodgson, 1987; Thoma et al., 1977; Pickering, 1977). Some modern instruments now have the capacity to remove these substances and concentrate the samples to small volumes for quantitative analysis. However, these instruments are often expensive. Most laboratories, therefore, rely on methods such as adsorption, chromatography, thin-layer chromatography (TLC), and solvent partitioning to isolate the compound of interest. The procedures used for the extraction and isolation of compounds must be able to give a true concentration. The efficiency of extraction procedures can be determined to a great extent by adding known amounts of the compound being analyzed to a sample matrix known to be free of the toxicant. Analysis of such fortified or “spiked” samples along with the unknown allows the analyst to determine the efficiency of the analytical method. Adding the same amount of material to solvent and analyzing it without performing the extraction and cleanup allow a further check on efficiency. Generally, at least an 80% recovery rate is considered necessary for an analytical method to be adequate, although some methods effect only 50%–60% recovery (Leidy and Hodgson, 1987). Recovery samples, in addition to indicating efficiency, provide information on how the instruments are functioning. Thus, one can correct data from a particular set of samples to reflect daily variation. Once the sample is isolated, extracted, or separated, and sometimes concentrated, the actual identification and

Biological techniques or bioassays measure the response that follows the application of a stimulus to a biological system. The applied stimulus is represented by standard or test samples that contain the biologically active substance or analyte. The biological system that receives the stimulus may be a whole, multicellular organism such as an animal or plant; isolated organs or tissues from multicellular organisms; or whole cells or microorganisms (Hawcroft et al., 1987; Odell, 1983; Clausen, 1988; Deshpande, 1996). In the field of analytical toxicology, the organisms most commonly used for bioassays are aquatic and include plants as well as vertebrate and invertebrate animals (Table 6.1). Among these, the water flea, Daphnia species (spp.), is commonly used for screening tests to obtain an index of the toxicity of pure or mixed compounds in water or waste treatment processes or to determine which of several processes may contribute to the eventual toxicity of plant effluents (Leidy and Hodgson, 1987). The end point of the tests is acute lethality over a 24- to 48-hr period. A variety of chronic and multigeneration tests can also be carried out by using Daphnia spp. Grass shrimp (Palaemonetes spp.) are also used widely in a variety of bioassays to identify toxic effluents, to estimate pure compounds, and to measure the toxic potential of dissolved pesticides, chlorine, heavy metals, and industrial wastes. Mortality is usually the end point used with groups of 10–20 shrimp exposed to each of five or six concentrations of the test compound. A positive control using a compound of known toxicity is run simultaneously

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Table 6.1 Organisms and Animals Commonly Used in Bioassay and Toxicity Measurement protocols Bioassay procedures Vertebrates Fathead minnow Pimephales promelas Brook trout Solvelinus fontinalis Sheepshead minnow Cyprinodon variegatus Invertebrates Water fleas Daphnia spp. Grass shrimp Palaemonetes spp. Shrimp Penaeus spp. Algae Green algae (Chlorophytes) Selenastrum capricomutum, Chlorella spp. Blue-green algae (Cyanophytes) Anabaena flos-aquae Red algae (Rhodophytes) Porphyridium cruentum Animal toxicity tests (in approximate order of use) Mice, rats, chickens, amphibians, guinea pigs, hamsters, rabbits, fish, dogs, reptiles, cats, quail, turkey, nonhuman primates, gerbils, pigeons

for comparison (Buikema and Cairns, 1980; Glass, 1973; Rand and Petrocelli, 1985). Although vertebrate animal bioassays require more time and laboratory space, their use has the advantage that they are closer to mammals in activation and detoxification pathways as well as in mode of toxic action. Among vertebrates, fish are commonly used as bioassay organisms. The most useful species among freshwater fish is the fat-head minnow (Pimephales promelas); among saltwater species, the sheepshead minnow (Cyprinodon variegatus) is widely used (Table 6.1). Higher vertebrates are most commonly used in toxicity testing using bioassays. Their use is described later in this chapter. Although the use of algae in bioassays is not uncommon, such techniques are beset with several problems. Because their growth rate is quite variable, not only a number of concentrations are used, but also enough replicates to permit statistical analyses. Similarly, physical conditions and composition of the medium used also must be clearly controlled. Algal growth can be monitored in several ways, by counting the cells manually under the microscope or by using a cell counter, measuring chlorophyll spectrophotometrically, or measuring ATP or the assimilation of a suitable 14C-labeled precursor. The response that follows the application of a toxicant, measured as a change in some aspect of the biological system used, may be a positive response associated with an increased activity or a negative response that is inhibitory or even lethal to the biological system. It relates to a biological activity that is normally attributed to the ana-

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lyte and is often expressed in concentration units, generally relative to an arbitrary although internationally recognized standard. All bioassays are comparative and require a standard preparation, ideally from the same source, with which each test sample can be compared. The use of “in-house” standards often results in a lack of interlaboratory correlations and, therefore, is not advisable. Better correlations are achieved by the use of International Standards for the Calibration of Bioassays. These standards are determined by the World Health Organization and are based on the recommendations of the Expert Committee on Biological Standardization (Bangham, 1983; Kirkwood, 1977; Campbell, 1974; Deshpande, 1996). Once the assays are completed, the data can be analyzed in several different ways to establish the relationship between the dose and the intensity of the response. Representative examples of dose-response curves are shown in Figure 6.2 (A–D). Although only positive relationships are shown here, it is not uncommon to observe inverse relationships in these types of studies. The intensity of the bio-

Figure 6.2 Typical dose-response relationships observed in bioassays: (A) ideal response with adequate sensitivity and wide detection limits; (B) although sensitivity is adequate, detection limits are restricted to higher doses of chemicals; (C) bioassays with wide detection limits but lacking sensitivity; (D) high sensitivity that restricts the detection limits of the bioassays to lower doses of chemicals.

logical response is dependent on both the slope of the curve as well as its position relative to the abscissa; these determine the sensitivity and detection limits (or range) of the bioassay, respectively. Ideally, the response to an applied stimulus should be sufficiently sensitive to allow differentiation of small changes in doses but not so great as to restrict the detection limits of the assay. Representative examples of this class of analytical techniques include monitoring the potency of pharmaceutical drugs at target sites, tissues, or the organism as a whole and measuring of LD50 values to determine the toxicity of chemicals that are potentially hazardous to human health. Bioassays can only be used for substances that produce a biological response in living organisms and tissues. This restriction precludes their widespread use as a general analytical technique in several fields. Similarly, the general public is increasingly viewing the use of animals or their tissues in scientific research as unethical. Advances in cell culture techniques, however, have minimized the necessity for the use of animals in bioassays. At present, most initial screening tests for measuring the biological potency of various drugs involve the use of cells cultured in an artificial medium. By monitoring the obvious morphological or biochemical changes in the cell culture populations, these techniques greatly facilitate the elimination of potentially hazardous drugs in the early stages and thereby restrict the number of tests to be carried out with actual animal systems to a minimum. Clinical drug trials with selected human populations, although they fall under this category, are generally not viewed as bioassays. Another major disadvantage of bioassays that use animal model systems is the inherent biological variability often found in test animals and their tissues, which results in poor precision and reproducibility and necessitates the extent of replication. Therefore, the use of animal model systems is cost-prohibitive and time-consuming. Similarly, in some types of bioassays, the animals may die or have to be slaughtered for further tissue analysis. In this respect, microbiological assays using broth cultures (tube assays) and assays on semisolid culture media (plate assays) offer several advantages. Although yeasts, protozoa, and algae can be used for microbiological assays, most methods use bacteria because of their relative ease of use. Microbiological assays do not require highly specialized and expensive equipment. They also provide highly homogeneous cell populations for test and thus result in better assay precision. These methods, therefore, are ideal in situations in which the number of tests is likely to be small and, hence, large capital expenditure cannot be justified. Microbiological assays, however, are limited only to those analytes that either promote or inhibit

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microbiological growth. This characteristic mandates that no other substance be present in the test sample that either promotes or inhibits growth or modifies the response to the analyte. In spite of these limitations, their general specificity for biologically active forms of the analyte is a good reason for actually using bioassays. Bioassays are the preferred analytical methods when the test material contains a mixture of active and inactive forms of the analyte that cannot be separated effectively. Alternatively, the analyte may occur in a variety of active forms that affect the same target site but with different biological activities and are present in unknown relative quantities. In addition to having high specificity, bioassays are also often very sensitive. Bioassays, therefore, are used when no suitable alternative methods are available. 6.1.2 Physical and Chemical Methods The classical biochemical techniques involving the use of spectrophotometry, gas chromatography (GC), mass spectrometry (MS), high-performance liquid chromatography (HPLC), paper and thin-layer chromatography (PC and TLC), and electrophoretic techniques coupled with ultraviolet (UV) and fluorescence detection methods are widely used for the identification and quantitation of biologically important compounds. These basic analytical tools exploit the physical and/or chemical characteristics of the compounds. The major limitations of these techniques include the cost of equipment and consumables, sample throughput, and the level of experience and skill required for analysis. Moreover, such methods are often tedious and laborious and may require elaborate sample cleanup and concentration procedures. The basic principles of these techniques are briefly described. Chromatography Chromatography encompasses a variety of techniques that resolve solutes by differential migration during passage through a porous medium. In these processes, various solute components are separated as a result of differential affinity of the components for a stationary phase (a solid or liquid) or for a mobile phase (a gas or liquid). The chief phenomena responsible for this affinity for the stationary phase are adsorption, ion exchange, affinity, and solution in a stationary solvent or matrix. The various forms of chromatography all include at least one of these phenomena. In fact, during the course of application of any chromatographic procedure, two or more of these phenomena are usually working at once.

Paper Chromatography Paper chromatography, an example of partition chromatography involving differential migration of solutes resulting from differences in distribution between two immiscible solvents, is one of the earliest chromatographic techniques developed. The aqueous constituent of the solvent system, which is adsorbed onto the paper, represents the stationary phase; the organic constituents are the moving or the mobile phase. Separation is effected by partition between the two phases as the solvent system moves over the paper. Although many variations exist, including reverse-phase paper chromatography, in which the paper is treated with a hydrophobic material, and use of ion exchange cellulose paper, these techniques have been superseded by equivalent systems involving TLC, primarily because of the speed and the greater resolving power of the latter technique. Thin-Layer Chromatography TLC techniques are widely used to separate many toxicants from interfering compounds. The adsorbent (various forms of alumina or silica gel) is spread as a thin layer (250–2000 µm) on a glass plate, resistant plastic, or fiberglass backing. When the sample is applied in solution along a base line near the bottom of the plate, the solvent migrates up the plate and the compounds move up with the solvent. Differential rates of migration result in separation. The compounds can be scraped from the plate and eluted from the adsorbent with suitable solvents. Recent developments in TLC adsorbents allow toxicants and other materials to be quantitated at the nanogram (10 –9 g) and picogram (10–12 g) levels. Adsorption Chromatographic Techniques In its simplest form, adsorption chromatography consists of separation of substances by filtration of a solution through a column of finely powdered adsorbent. The substances are adsorbed at the top of the column and then slowly move down from the column as a suitable solvent or solvent mixture flows through the column. The relative affinity of the solutes for the adsorbent is a function of the chemical constitution of the substances being separated, the nature of the solvent, and the nature of the adsorbent. The substances that have more affinity for the solvent (i.e., less tendency to be adsorbed) move more quickly down the column. The development proceeds until the separated compounds emerge from the column and appear in the filtrate. This continuous filtrate is then fractionated into a series of fractions or cuts. The separated solutes in these fractions are finally analyzed by suitable means.

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Several variations of adsorption column chromatographic techniques are available. The most commonly used ones are described next. Ion Exchange Chromatography. Ion-exchange chromatography utilizes the differential affinity of charged ions or molecules in solution for inert, immobile (insoluble) charged substances. When the charged ion or molecule contains one or more positive charges that exchange with another positively charged component ionically bound to a negatively charged immobile phase, this process is called cation exchange, the inverse process is anion exchange. Compounds so bound are eluted by changes in pH (or ionic strength) and, since the net charge depends on the relationship between the pH of the solution and the isoelectric point of the compounds, compounds of different isoelectric points can be sequentially eluted. Gel Permeation/Filtration Chromatography. Gel permeation techniques are based on molecular sieve effects of the adsorbent particles that separate molecules on the basis of their size and thus molecular weight. Molecules large enough to be excluded from the pores of the porous material move through the column faster than those not excluded and thus can be separated from them. Cross-linked dextrans (such as Sephadex) or agarose (Sepharose), are commonly used materials. Affinity Chromatography. Affinity chromatography is a potent tool for separating biologically active macromolecules. It is, however, seldom used for purifying small molecules, such as most toxicants. This technique utilizes biospecific separation, group-specific interactions, or chemisorption of the target molecules on appropriate solid phases. Affinity chromatography occupies a unique place in separation technology. It is the only technique that allows purification of almost any biomolecule on the basis of its biological function or its unique chemical structure. Compared with other liquid chromatographic methods, such as ion-exchange and gel permeation chromatography, affinity chromatographic techniques allow high specific separation, isolation, and purification of native, biologically active substances, as well as their synthetic counterparts, from more or less large amounts of contaminants. Furthermore, their concentrating effect allows the isolation of a desired substance from larger sample volumes. Purification of the target molecule is often of the order of several thousandfold, and recoveries of the active material are generally very high. Although affinity chromatography is a method based on specific and reversible molecular interactions of biologically active substances, it involves several variations in

which the matrix-bound ligand does not originate exclusively from biological matter. Some such examples of affinity-based separation techniques include metal chelate chromatography, charge transfer adsorption chromatography, hydrophobic interaction chromatography, dye-ligand chromatography, and covalent chromatography. In all these cases, affinity chromatography is due to chosen groups anchored as ligands on the matrix material. Furthermore, there is no reason to avoid using the term affinity chromatography when the substance to be separated by means of one of these techniques is not of biological origin. Deshpande (1995) has reviewed the basic principles, theoretical considerations involved, and variations available. Gas Chromatography or Gas-Liquid Partition Chromatography GC, or as it is sometimes called, vapor-phase chromatography (VPC), consists of separations of vaporized components by a combination of partition chromatography, varied degrees of adsorption chromatography, and the varied relative volatility (boiling points) of the vaporized components. GC has become the method most commonly used for the separation and quantitation of organic toxicants. A few microliters of solvent containing one or more dissolved components is initially injected through a septum into a heated injection chamber. The heat of this chamber should be high enough to vaporize all of the components rapidly, thereby allowing them to be swept in a stream of hot carrier gas (nitrogen or helium) into a long GC column coiled in a constant high-temperature compartment. The GC column is packed with any commercially available, usually inert, usually inorganic, solid supports coated with one or more nonvolatile organic coatings. The support with its organic (liquid phase) coating constitutes the stationary phase of gas liquid partition chromatography systems. As the volatilized components pass through the column they partition between the stationary phase material in the column and the mobile phase, that is, the stream of inert carrier gas. This partitioning also reflects the relative volatility of the vaporized components at the temperature in question; compounds of lower boiling points usually have less affinity for the stationary phase. The vaporized components then exit from the column in the inverse order of their affinity for the stationary phase, and the component with the highest affinity for the stationary phase exits last. The exiting components are then detected, and their passage is recorded on a linear chart recorder. Most modern instruments use a computerized data collection and analysis package. Five types of detectors are used widely in toxicant detection (Leidy and Hodgson, 1987). These are the flame ionization (FID), flame photometric (FPD), electron cap-

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ture (ECD), alkali flame, and conductivity detectors. The FID operates on the principle of ion formation from compounds being burned in a hydrogen flame as they elute from a column. The ions cause a current to flow between two electrodes held at a constant potential, thus sending a signal to the electrometer. The FPD is a specific detector in that it detects either phosphorus- or sulfur-containing compounds. When atoms of a given element are burned in a hydrogen-rich flame, the excitation energy supplied to these atoms produces a unique emission spectrum. The intensity of the wavelengths of light emitted by these atoms is directly proportional to the number of atoms excited. Larger concentrations cause a great number of atoms to reach the excitation energy level, thus increasing the intensity of the emission spectrum. The change in intensity is detected by a photomultiplier, amplified by the electrometer, and recorded. Filters that allow only the emission wavelength of phosphorus (526 nm) or sulfur (394 nm) are inserted between the flame and the photomultiplier to give this detector its specificity (Leidy and Hodgson, 1987). The ECD is used to detect halogen-containing compounds, although it produces a response to any electronegative compound. When a negative direct current (DC) voltage is applied to a radioactive source (e.g., 63Ni or 3H), low-energy β particles are emitted, producing secondary electrons by ionizing the carrier gas as it passes through the detector. The secondary electron stream flows from the source (cathode) to a collector (anode), where the amount of current generated is amplified and recorded. As electronegative compounds pass from the column into the detector, electrons are removed or “captured,” and the current is reduced. The reduction, which is related to both the concentration and the electronegativity of the compound passing through, produces a response that is recorded. The sensitivity of the ECD is greater than that of any of the other detectors currently available. Early electrolytic conductivity detectors operated on the principle of component combustion, which produced simple molecular species that readily ionized, thus altering the conductivity of deionized water. The changes were monitored by a DC bridge circuit and recorded. By varying the conditions, the detector can be made selective for different types of compounds, e.g., chlorine-containing or nitrogen-containing compounds (Leidy and Hodgson, 1987). The alkali flame detector can also be made selective. Enhanced response to compounds containing arsenic, boron, halogen, nitrogen, and phosphorus results when the collector (cathode) of an FID is coated with different alkali metal salts such as KBr, KCl, Na2SO4, and Rb2SO4. As with conductivity detectors, by varying gas flow rates,

type of salt, and electrode configuration, enhanced responses are obtained. High-Performance Liquid Chromatography Although relatively new to the field of analytical chemistry, HPLC has become very popular for the following reasons: 1. 2. 3. 4.

It can be run at ambient temperatures. It is nondestructive to the compounds of interest, which can be collected intact. In many instances, derivatization is not necessary for response. The columns can be loaded with large quantities of the material for detection of low levels.

The basic HPLC instrument consists of a solvent reservoir, a gradient-forming device, a high-pressure pumping device, an injector column, and a detector. The principle of operation is very similar to that of GC, except that the mobile phase is a liquid instead of a gas. Separation is effected by the composition of the mobile phase and its flow rate. Most separation columns use finely divided packing (3–10 µM in diameter), some have bonded phases, and others use alumina or silica. The columns normally are 15–20 cm in length, with small diameters (approximately 4.6-mm inner diameter). A high-pressure pump is required to force the solvent through this type of column. The major detectors presently used for HPLC are UV fluorescence spectrophotometers, photodiode array, and differential refractometers. Spectroscopic Techniques Spectroscopic techniques are concerned with the changes in atoms and molecules when electromagnetic radiation is absorbed or emitted. A number of such techniques are routinely used in the field of toxicant analysis. These are described next. Ultraviolet/Visible Spectrophotometry Photometry involves the qualitative and quantitative use of absorption and emission data obtained from compounds that absorb light in the ultraviolet (200–400 nm) and visible (VIS) (400–800 nm) regions of the electromagnetic spectrum. Many inorganic and organic molecules show maximal absorption at specific wavelengths in the UV/VIS range, and these can be used to identify and quantitate compounds. The basic UV/VIS instrument includes a source (usually a tungsten lamp for VIS measurements or a hydrogen discharge lamp for UV), sample chamber, monochromator (device used to isolate spectral regions), and detector. Most modern instruments now include computerized data analysis packages.

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Infrared Spectrophotometry Within molecules, atoms are in constant motion, and associated with these motions are molecular energy levels that correspond to the energies of quanta of infrared (IR) radiation. These motions can be resolved into rotation of the whole molecule in space and into motions corresponding to the vibration of atoms with respect to one another by bending or stretching of covalent bonds. The vibrational motions are very useful in identifying complex molecules, because functional groups (e.g., OH, C, O, and S-H) within the molecule have characteristic absorption bands (Leidy and Hodgson, 1987). The principal functional groups can be determined and used to identify compounds in cases in which chemical evidence permits relatively few possible structures. Standard IR spectrophotometers cover the spectral range from 2.5 to 15.4 µm (wave number equivalent to 4000–650 cm–1) and use a source of radiation that passes through the sample and reference cells into a monochromator. The radiation is then collected, amplified, and recorded. Most modern instruments use microprocessors, allowing a number of refinements that have increased the versatility of IR instruments so that more precise qualitative and quantitative data can be obtained. Atomic Absorption Spectroscopy Atomic absorption (AA) spectroscopy is routinely used to detect metal-containing toxicants. Samples are vaporized either by aspiration into an acetylene flame or by carbon rod atomization in a graphic cup or tube (flameless AA). The atomic vapor formed contains free atoms of an element in their ground state, and, when illuminated by a light source that radiates light of a frequency characteristic of that element, the atom absorbs a photon of wavelength corresponding to its AA spectrum, thus exciting it. The amount of absorption is a function of concentration. The flameless instruments are much more sensitive than conventional flame AA, with sensitivity three orders of magnitude greater than that of conventional flame AA. Nuclear Magnetic Resonance Spectroscopy Nuclear magnetic resonance (NMR) detects atoms that have nuclei that possess a magnetic moment. These are usually atoms containing nuclei with an odd number of protons (charges). Such nuclei can exist in two states, a low-energy state with the nuclear spin aligned parallel to the magnetic field, and a high-energy state with the spin perpendicular to the field. Basically, the instrument measures the absorption or radiowave necessary to change the nuclei from a low- to a high-energy state as the magnetic field is varied. It is most commonly used for hydrogen at-

oms, although 13C and 31P are also suitable. Because the field seen by a proton varies with its molecular environment, such molecular arrangements as CH3, CH2, and CH all give different signs, providing much information about the structure of the molecule in question. Mass Spectroscopy The mass spectrometer is easily the most outstanding instrument for the identification of compounds. In toxicant analysis, mass spectroscopy (MS) is used widely as a highly sensitive detection method for GLC and is increasingly being used with the HPLC. Both these instruments can be interfaced to the mass spectrometer. A portion of the column effluent from the GC or the HPLC is passed into the mass spectrometer, where an electron beam bombards it. This process removes electrons or negative groups, and the ions produced are accelerated. After acceleration, they pass through a magnetic field, where the ion species are separated by the different curvatures of their paths under gravity. Normally, only single positive ions are detected. The resulting pattern is characteristic of the molecule under study. By interfacing the detector with a com-

Table 6.2

puter system, data reduction, analysis, and quantitation are performed automatically. Large libraries of mass spectra have been developed for computing systems and with technological advances substances in femtogram (10–15 g) quantities are detected and quantitated (Leidy and Hodgson, 1987). One disadvantage of MS is its high cost ($250,000–$750,000 per instrument). A summary of various spectroscopic techniques is presented in Table 6.2. 6.1.3 Binding Assays Binding assays comprise a variety of methods that utilize the specific reaction between a ligand and a binding protein. The basic principle of a binding assay is a reversible reaction between a ligand (L) and its binding protein (BP) that obeys the law of mass action as follows: L + BP → L–BP The ligand combines with the binding protein to form the L–BP complex at rate constant k1. At equilibrium, the L–BP complex dissociates with a rate constant k2

Characteristics of Spectroscopic Techniques

Type

Principle

Uses

Visible and ultraviolet (UV) spectrophotometry

Energy transitions of bonding and nonbonding outer electrons of molecules, usually delocalized electrons

Spectrofluorimetry

Absorbed radiation emitted at longer wavelengths

Infrared and Raman spectroscopy

Flame spectrophotometry (emission and absorption)

Atomic vibrations involving a change in dipole moment and a change in polarizability, respectively Energy transitions of outer electrons of atoms after volatilization in a flame

Routine qualitative and quantitative biochemical analysis, including many colorimetric assays; enzyme assays, kinetic studies, and difference spectra Routine quantitative analysis, enzyme analysis and kinetics; more sensitive at lower concentrations than visible and UV absorption Qualitative analysis and fingerprinting of purified molecules of intermediate size

Electron spin resonance (ESR) spectrometry

Detection of magnetic moment associated with unpaired electrons

Nuclear magnetic resonance (NMR) spectrometry

Detection of magnetic moment associated with an odd number of protons in an atomic nucleus Determination of the abundance of positively ionized molecules and fragments

Mass spectrometry

Source: Compiled from Williams and Wilson (1975) and Leidy and Hodgson (1987).

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Qualitative and quantitative analysis of metals; emission techniques; routine determination of alkali metals; range of metals that may be determined and sensitivity extended by absorption technique Research on metalloproteins, particularly enzymes and changes in the environment of free radicals introduced into biological assemblies, e.g., membranes Determination of structure of organic molecules of molecular weight < 20,000 Qualitative analysis of small quantities of material (10–6 to 10–9 g), particularly in conjunction with gas liquid chromatography

to form free ligand and binding protein. The ligand is invariably referred to as an antigen, hapten, or analyte. Binding assays can be further classified into three general categories: immunoassays in which the ligand is an antigen and the binding protein its specific antibody, receptor binding assays in which the receptor protein is usually extracted from the target organ of a hormone or drug, and circulating binding protein assays that employ a naturally occurring plasma protein such as thyroxine-binding globulin (TBG) for the assay of thyroxine (T 4 ) and transcortin for the assay of cortisol or progesterone. Immunoassays are by far the most common form of binding assays. In fact, of all the methods described, immunoassays have developed into an extremely versatile analytical technique with a diverse range of assay protocols. In comparison to other analytical methods, an immunoassay provides a rapid, economical, highly sensitive, and specific analysis that is relatively simple to perform and interpret. Three factors have been primarily responsible for the rapid development of immunoassays as a powerful analytical tool in many fields of life sciences, including the following: 1.

2.

3.

The generation of antibodies that display marked specificity against an immense range of compounds The high affinity with which they bind their respective antigen, thus enabling great sensitivity to be achieved and indeed defining the ultimate sensitivity that can be attained The ease of their use in detecting the concentration of antigen, or in detecting the presence of antibodies

As compared to bioassays, which provide estimates of concentration of an analyte based on its biological activity or function, immunoassays are structural assays, which assess only a part of the antigen’s structure or its antigenic determinant. Therefore, the values obtained by a bioassay may be considerably lower than those determined by an immunoassay method. However, even when an analyte can feasibly be determined by chromatographic, colorimetric, or other standard analytic procedures, quantitative immunological methods are often used because of their speed, simplicity, and relatively low cost. Other reasons for the growing popularity of immunoassays include the potential of rapidly measuring a minute quantity of specific analyte from within a complex sample matrix, often with little or no sample cleanup; the development of more sensitive detection systems; the relative ease of performing the assays; as well as the lower cost of the assays relative to that of most conven-

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tional analytical techniques (Deshpande and Sharma, 1993; Deshpande, 1996). Immunoassays can be performed with a wide variety of designs and formats. The three most commonly used assay formats are the antibody capture assays, the antigen capture assays, and the two-antibody immunometric sandwich assays (Figure 6.3). Detailed descriptions of immunoassay technology are available in several books and monographs (Deshpande, 1996; Wild, 1994; Tijssen, 1985; Nakamura et al., 1992; Price and Newman, 1996).

6.2

MEASUREMENT OF TOXICITY

An understanding of food safety is improved by defining two basic concepts, toxicity and hazard. Toxicity is the capacity of a substance to produce harm or injury of any kind (chronic or acute) under any conditions. This might include the capacity to damage the developing fetus (teratogenicity), to alter the genetic code (mutagenicity), or to induce cancer (carcinogenicity). Furthermore, any deviation from normal is viewed as a possible negative effect, even though the change may seem to be positive, such as increased growth rate or enhanced nutrient absorption. The change is assumed to be negative until proved beneficial. In contrast, hazard is the relative probability that harm or injury will result when the substance is used in a proposed manner and quantity. Assessments of whether a food or ingredient is safe should not be based on whether it has inherent toxicity but on whether or not a hazard is created. Toxicity testing is performed in a number of different ways, many of which grew historically in response to particular concerns and regulations. For example, tests for acute, or short-term, toxicity were developed largely in response to concerns about the most highly exposed groups, mainly workers. In contrast, testing for long-term, chronic effects has evolved in response to concerns about the general population as well as subgroups. The most well developed chronic test, the carcinogenesis bioassay, has gradually evolved from a test to determine whether a chemical is a carcinogen to one aimed at quantitatively establishing the dose at which it is expected to produce a specific cancer incidence in an exposed human population. Most of the biological methods developed for the measurement of toxicity are thus the result of the practical need to obtain as much information as possible about the effects of chemicals insofar as they may be pertinent to our continued well-being. In fact, for those chemicals that are to be administered intentionally to humans, such as food additives, food substitutes, or drugs, it is necessary to obtain the most toxicity data economically possible. In this

Figure 6.3 Assay formats commonly used for enzyme immunoassay products: (A, B, C) antibody capture formats. (A, B) Antigen is bound to the solid phase to capture the desired antibody. The bound antibody is then quantitated by using (A) an enzyme-labeled second antibody specific for the primary antibody or (B) a “ladder” of secondary antibodies to increase assay specificity. (C) Assay format is used to capture class-specific antibodies by using a solid-phase coated with either polyclonal or monoclonal antibody specific for the class of immunoglobulins to be captured and detected by using an enzyme-labeled antigen. (D) Example of an immunometrical, reagent-excess sandwich immunoassay commonly used for polyvalent antigens; (E, F) examples of reagent-limited, competitive immunoassays using enzyme-labeled antigen and enzymelabeled antibody, respectively, on antibody-coated or antigen-coated solid phase. IgG, immunoglobulin G.

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section, the parameters used for toxicity testing and assessment, the dose-response relationships, and the commonly used testing protocols are briefly described. 6.2.1 Toxicological Units Several different toxicological units are used to express the toxicity potential of chemicals (Table 6.3). These can be broadly grouped under three categories: 1. 2. 3.

Units used in animal studies Units used to obtain acceptable human exposure levels Risk indices used in epidemiological studies

The simplest measurement of toxicity is lethality. Trevan (1927) first reported the use of the LD50, or median lethal dose (Table 6.3), as a measure of acute toxicity. Since then, different methods have evolved for determining LD50, which is defined as the calculated dose of a substance that is expected to cause the death of 50% of an entire defined experimental animal population (NIOSH, 1983). The LD50 is determined by administering graduated doses of test material to groups of experimental animals (OECD, 1981). Generally, three or more dose levels are selected to produce mortality rates bracketing 50%. When information to estimate proper dose levels is not available, a limit test or LD50 screen using a single dose level (usually 5 g/kg body weight for oral and 2 g/kg body weight for dermal administration) may be conducted. If mortality is not observed in the limit test, a study to determine an LD 50 would generally be unnecessary since mortality above these dose levels would have limited toxicological significance (Yermakoff, 1987). If mortality is observed, this information can be used to set dose levels for a complete LD50 study. Alternatively, an LD50 can be estimated by the up-and-down method (Brownlee et al., 1953; Murray and Gibson, 1972). With this procedure, test groups are dosed sequentially, and the results from each group are used to predict for succeeding groups the doses that will most efficiently bracket the LD50. Selection of a given route of administration for an LD50 study is generally based on anticipated human exposure. For example, drugs and food additives are commonly tested by oral administration, whereas chemicals associated with occupational exposures are most appropriately tested dermally or by inhalation (Yermakoff, 1987). Other parenteral routes might include subcutaneous, intraperitoneal, intravenous, and intratracheal administration. LD50 values for components of a chemical mixture can be used to generate an estimate of the acute toxicity of the mixture. For example, the European Economic Com-

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mission (EEC) (1983) has proposed a mathematical weighting of component LD50 values to classify acute toxicity for warning labels on hazardous mixtures. In addition to LD50, other measures of lethality can also be derived from dose versus percentage lethality curves generated from the LD50 studies. The LD99 and LD01 values are used to estimate the minimal and maximal dose lethal to an entire animal population, respectively (Table 6.3). However, as they are at the extreme ends of the dose/lethality curves, they suffer from the lack of precision. The lowest lethal dose (LDL0, Table 6.3) values for human exposure, which are commonly obtained from accidental exposures or drug overdoses, can provide valuable information by eliminating the need to extrapolate toxicity information from animals to humans. The effective and toxic dose parameters do not use lethality as an end point, but rather graded responses by identifying a value above which the response is defined as positive (Table 6.3). The median effective dose (ED50) and median toxic dose (TD50) are often used together to estimate the margin of safety, or therapeutic index, which is a measure of the selectivity of a drug or other chemical for a given response. These parameters are further described in the discussion of dose-response relationships later in this chapter. Similar to LD01, ED01 and TD01 parameters, effective or toxic in only 1% of treated animals, are associated with poor precision because of the large confidence limits at the extreme ends of the dose-response curves. The median lethal concentration parameters, LC50 and LCt50, unlike the LD50, are based on the concentration and duration of exposure to the test material rather than the dose administered (Table 6.3). These parameters are used to assess toxicity of chemicals via the inhalation route. The maximal tolerated dose (MTD) is often used for carcinogenicity bioassays. The no-effect level (NEL) and other similar parameters (Table 6.3) were first proposed by the Food and Agriculture Organization/World Health Organization (FAO/WHO) Expert Committee on Food Additives (1974) to assess the toxicity and safety of chemicals for use as additives in food processing and preservation. Several parameters are used to set the acceptable human exposure levels for chemicals (Table 6.3). Threshold limit values (TLVs) are recommended atmospheric concentrations of workplace substances to which workers may be exposed without adverse health effects. The acceptable daily intake (ADI), defined by the FAO/WHO Expert Committee on Food Additives (1974), was designed to indicate safe levels of food additives. The concept is also applicable to contaminants in food, air, and water. The SNARLs (Table 6.3) are levels of drinking water contaminants below which no adverse effects are expected to occur after a specified exposure period. These are not legal

Table 6.3

Toxicological Units Commonly Used to Define and Monitor Exposure to Chemicals

Units used in animal studies LD50 (lethal dose/50% response): calculated dose of a substance that is expected to cause the death of 50% of an entire defined experimental animal population LD01 (lethal dose/1% response): maximal dose of a substance that is likely to be sublethal to an entire experimental animal population LD99 (lethal dose/99% response): minimal dose of a substance that is likely to be lethal to an entire experimental animal population LDL0 (lowest lethal dose): dose of a substance administered over any given period in one or more divided portions and reported to have caused death in humans or animals ED50 (effective dose/50% response): median effective dose obtained from a dose versus percentage response curve that uses not lethality as end point but rather a graded response by identifying a value above which the response is defined as positive; may use any clearly defined measure of efficiency ED01 (effective dose/1% response): dose effective in only 1% of treated animals ED99 (effective dose/99% response): dose effective in 99% of the experimental population of animals TD01, TD50, and TD99 (toxic dose/1%, 50%, and 99% response): similar to effective dose (ED) but uses a clearly defined measure of clinical toxicity LC50 (lethal concentration/50% response, inhalation exposure): median lethal concentration of an inhaled chemical defined as a “statistically derived concentration of a substance that can be expected to cause death during exposure” LCt50 (lethal concentration x time/50% response, inhalation exposure): statistically derived concentration of a substance that can be expected to cause death within a fixed time after exposure in 50% of the animals exposed for the specific time LCL0 (lowest lethal concentration, inhalation exposure): dose of a substance inhaled over any given period in one or more divided portions reported to have caused death in humans or animals LC50 (lethal concentration/50% response, exposures other than inhalation): for aquatic toxicity: concentration of a chemical in water killing 50% of a test batch of fish within a particular period of exposure; for in vitro toxicity testing using cultured hamster cells: concentration of a chemical that causes transformation of 50% cells in response to carcinogens without toxicity-induced selection of subpopulations MTD (maximal tolerated dose): highest dose of the test agent given during the chronic study that can be predicted not to alter the animal’s longevity from effects other than carcinogenicity; should not produce greater than 10% inhibition of weight gain, produce clinical evidence of toxicity or pathological lesions, or alter longevity except as a result of carcinogenesis NEL (no-effect level): level of a substance that can be included in the diet of a group of animals without toxic effects; used interchangeably with no-adverse-effect level (NAEL), no-observed-effect level (NOEL), and no-observed-adverse-effect level (NOAEL) MED (minimal effective dose): minimal effective dose used as an alternative to the NEL: minimal dose that produces an observed effect; used interchangeably with lowest-effect level (LEL), lowest-observed-effect level (LOEL), and lowest-observed-adverse-effect level (LOAEL) Units used to estimate acceptable human exposure levels TLV (threshold limit value): recommended atmospheric concentrations of workplace substances (ppm or mg/m3) to which workers may be exposed without adverse health effects TLV-TWA (threshold limit value–time-weighted average): same as TLV except that it represents a time-weighted average concentration for an 8-hr workday and a 40-hr workweek; can be exceeded for short periods during the workday without producing adverse health effects as long as the average concentration is at or below TLV. TLV-STEL (threshold limit value–short-term exposure limit): used for chemicals that may produce adverse effects when TLV is exceeded for only a brief period; time-weighted concentration limit for 15 min; no more than four such exposure periods permitted per day, and maximum of 60 min must elapse between exposures TLV-C (threshold limit value-ceiling): stringent maximal permissible exposure that may not be exceeded even for short periods; similar to maximal acceptable (formerly allowable) concentrations (MACs); applied to fast-acting highly toxic or extremely irritating substances (e.g., acetic anhydride) for which even brief exposure periods may cause serious toxicity ADI (acceptable daily intake): amount of food additive that can be taken daily in the diet, even over a lifetime, without risk; concept also applicable to contaminants in food, air, and water SNARL (suggested no-adverse-response level): level of drinking water contaminants below which no adverse effects are expected after specified exposure period, usually defined for 24-hr, 7-day, or chronic (lifetime) exposure

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Table 6.3

(continued)

Risk indices used in epidemiology RR (relative risk): ratio of risk in a group of subjects exposed to a chemical of interest to that in an unexposed group, widely used in epidemiological studies PAR (population attributable risk): proportion of a disease attributable to a given etiological agent; used for studying long-term disease and mortality SMR (standardized mortality ratio): ratio of observed to expected total number of deaths, where expected number is computed by applying the schedule of age-specific mortality rates in the comparison population to the distribution of person-years in the study population; widely used as a measure of chemical-related mortality

standards for drinking water, but are intended to assist efforts to protect the public health from contaminated drinking water supplies until pollutants can be reduced to acceptable levels. The parameter relative risk (RR) is commonly used in epidemiological studies as a measure of association (not causation) between a chemical and a disease (Table 6.3). It can be determined directly or indirectly, depending on the method of sampling used in the study. The population attributable risk (PAR) is used as a measure of the contribution of chemical exposure to a population’s disease burden. It is particularly suitable for the study of long-term disease and mortality but has some limitations when applied to short-term recurrent disease (Park, 1981). The standardized mortality ratio (SMR) is widely used as a measure of chemical-related mortality.

3.

The measurable end point of toxicity in such studies may be a pharmacological, biochemical, or pathological change that shows percentage or proportional change, or an “all-or-none” type of effect such as death or loss of consciousness. Thus the observed responses can be classified as all-or-none or quantal responses (Trevan’s original assumptions) and as graded responses. However, for most practical and sound conceptual reasons, they can be considered to be identical. In either case, the dose-response relationship is graded between a dose at which no effect is measurable and one at which the maximal effect is demonstrated. The dose-response relationship is predicted on the following assumptions: 1.

6.2.2 Dose-Response Relationships 2. The relationship between the dose of a compound and its toxicity is the most fundamental concept in toxicology. It was almost 400 years after Paracelsus’ famous dictum relating dose to effect before Trevan (1927) developed the concept of the quantal median effective dose in a large group or population, the ED50, and its derivative, the LD50, the median lethal dose. Trevan’s method and the modifications and derivatives that followed are based on the following assumptions: 1.

2.

The quantal effect, the all-or-none predetermined level of response that demarcates a positive effect from no-effect: All individuals responding at or above a predetermined level are equally positive; all below are nonresponsive, not negative and not zero, thus the demarcation of sleep or no sleep, convulsion or no convulsion, death or no death. Drowsiness, agitation, and moribundity are no-effects. The percentage of the numbers responding positively, in any group large or small, is a function of the dose: larger doses, greater numbers.

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Trevan’s method and its variants followed normal or Gaussian distribution statistics.

3.

The toxic response is a function of the concentration of the chemical at the site of action. The concentration at the site of action is related to the dose. The response is causally related to the chemical given.

The site of action may be an enzyme, a pharmacological receptor, other macromolecules, or a cell organelle or structure. The interaction of the toxicant at the site of action may be reversible or irreversible, and each gives rise to different types of response. In irreversible interactions, a single interaction is theoretically sufficient. Furthermore, continuous or repeated exposure allows a cumulative effect dependent on the turnover of the toxinreceptor complex. In contrast, in reversible interactions, there may be a threshold below which no response is observed. The response may also be very short as it depends on the concentration of the toxicant at the site of action, which may only be transient. In some instances, as with chemical carcinogens, time is also an important factor, both for the appearance of the effect and for the length of exposure. The second assumption implicit in the doseresponse curve is that the concentration in tissues is gen-

erally related to the dose of the chemical or toxicant. However, as described in Chapter 2, various factors affect the absorption of a chemical from the site of exposure, its distribution in the tissues, and its metabolism and excretion. Thus the concentration of the chemical may not be directly proportional to the dose, so the dose-response relationship may not be straightforward or marked thresholds may occur. Similarly, in some cases, the response may be only indirectly related to the chemical. For many chemicals, a toxicological spectrum showing the transition of the biological effects as a function of dose can be constructed. Chemicals can be categorized into two groups based on the transition of the types of biological effects on the host with increasing dose (Figure 6.4). Type I chemicals primarily comprise most toxicants with no known beneficial effects. On the basis of therapeutic effects, the type II chemicals may be further subdivided into three subtypes: only nutritional, only therapeutic, and nutritional and therapeutic, for which there may be a transition from the nutritional to the therapeutic effects with increasing dose. The first and third subtypes are clearly limited to that group of compounds that comprises the nutrients. The second subtype consists primarily of the various drugs, medicines, and antibiotics as well as many nonnutritive compounds present in foodstuffs. A typical dose-response curve is shown in Figure 6.5. A wide variety of doses are administered to experimental animals to determine which dose of a particular chemical causes which kinds of toxic effects. Generally, very small doses cause no observable effects, higher doses some toxicity, still higher doses greater toxicity, and at a high enough dose, the animal dies. In toxicology, the quantal dose-response is used extensively. Determination of the median lethal dose (LD50) is usually the first experi-

Figure 6.4 Classification of chemicals based on their biological effects as a function of dose.

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Figure 6.5 or toxicant.

Dose-response relationship for a typical chemical

ment performed with a new chemical. In practice, this is experimentally determined usually by using mice or rats. At least 10 animals are used per dose, and a range of doses is administered so that at least 3 and preferably more of the doses result in producing some deaths and some survivals, i.e., kill less than 100% and more than 0%. If a large number of doses is used with a large number of animals per dose, a sigmoid dose-response curve, as is shown in Figure 6.5, is observed. A normally distributed sigmoid curve approaches a response of 0% as the dose is decreased and approaches 100% as the dose is increased but theoretically never passes through 0% and 100%. However, the minimally effective dose of any chemical that evokes a stated all-ornone response is called the threshold dose, even though it cannot be determined experimentally (Klaassen, 1986). The shape of the dose-response curve depends on a number of factors, but it is basically derived from the familiar bell-shaped Gaussian curve (Figure 6.6A), which describes a normal distribution in biological system. Each point on such a bell-shaped curve represents the percentage of animals responding at each dose minus the percentage responding at the immediately lower dose. Thus, only a few animals respond at low doses and others at high doses, but the majority respond at around the median dose. Those animals responding at the lower end of the curve are referred to as hypersusceptible and those at the higher end of the curve as resistant. The more animals are used, the closer the curve is to a true sigmoid shape. Such a curve has a relatively linear portion between 16% and 84%, representing the limits of one standard deviation (SD) of the mean. Although the LD50 parameter can be determined from such a curve, because of the limited sample size often used in such studies, which does not often define adequately the sigmoid

Table 6.4 Probit Transformation of Quantal Dose-Response Obtained from a Normally Derived Population % Response

Standard deviation

Probit

0.14 2.30 6.70 15.90 30.90 50.00 69.10 84.10 93.30 97.70 99.38 99.86

–3.0 –2.0 –1.5 –1.0 –0.5 0 0.5 1.0 1.5 2.0 2.5 3.0

2.0 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0

The therapeutic index (TI) is defined as the ratio of the dose required to produce a toxic effect and the dose needed to elicit desired therapeutic response. Therapeutic index (TI) = TD50/ED50 or LD50/ED50 Figure 6.6 Dose-response relationship expressed as a frequency distribution (A) derived from a normally distributed Gaussian population and (B) as expressed in probit units.

curve, probit (derived from probability units) analysis is used for such determinations (Figure 6.6B). The probit analysis uses SD units. The sigmoid dose-response curve is divided into multiples of the SD from the median dose, which is the point at which 50% of the animals being used respond. To avoid negative numbers, probit units define the median as probit 5 and then each SD unit is added or subtracted from the median probit as shown in Table 6.4. The sigmoid dose-response curve thus can be converted into a straight line when transformed into probit units (Figure 6.7). The LD50 is then obtained by drawing a horizontal line from the probit unit 5, which is the 50% mortality point, to the dose-effect line. At the point of intersection, a vertical line, which intersects the abscissa at the LD50 point, is drawn. The quantal or all-or-none response is not limited to lethality alone. Similar dose-response curves can be constructed for other types of pharmacological (ED50) or toxic (TD50) effects, as well as beneficial therapeutic responses (Figure 6.8).

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The TI represents the relative safety of a chemical or a drug. The larger the ratio, the greater is the relative safety. However, the use of median doses for the calculation of TI does not take into consideration the slopes of the dose-response curves. The slope of the dose-response curve is related to the degree of individual variability of the exposed animals in their response to the chemical. Dose-response curves with a shallow slope indicate a high degree of variability of the

Figure 6.7 Comparison of the toxicity of two compounds, A and B. Although the LD50 is the same (5 mg/kg), toxicity occurs with A at a much lower dose than B, but the minimal to maximal effect is achieved with B over a very much narrower dose range. See also Figure 6.2.

2.

3. 4. 5. Figure 6.8 Dose-response curves for pharmacological effect, toxic effect, and lethal effect, illustrating ED50, TD50, and LD50. The proximity of the curves for efficacy and toxicity indicates the margin of safety for the compound and the likelihood of toxicity in certain individuals after doses necessary for the desired effect.

population with respect to the response, whereas a steep slope implies low variability and therefore a relatively uniform response. Thus, even though two chemicals may have similar LD50 indices, they may exhibit strikingly different toxicity behavior (Figure 6.7). Hence, to overcome the inherent limitation in the calculation of the TI, the margin of safety using the ED99 for the desired effect and the LD01 (or TD01) for the undesired effect is established as follows: Margin of safety = TD01/ED99

If properly conducted, acute toxicity tests yield not only the LD50, but also information on other acute effects such as cause of death, time of

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It should also be noted that the dose-response curves under special conditions might take on shapes that are considerably different from the normally expected relationship. Although the traditional dose-response curve represents the range of sensitivities (i.e., biological variation) in a population, encompassing the most sensitive and the most resistant individuals, the individual sensitivities are assumed to be unimodally distributed. If a significant proportion of the population is more sensitive to a chemical, then the resultant bimodal distribution of the doseresponse curve would appear as shown in Figure 6.9 (A and B). These curves never take on a negative slope, since all the sensitive individuals that have reacted at a lower dose will also respond at the higher dose. These curves thus represent the summation of the sensitivities of the two groups. As the proportion of an unusually sensitive group

or LD01/ED99

The LD50 value is not an absolute biological constant. It depends on a large number of factors, including the absorption of chemical from the site of exposure, its distribution in the tissues, and its subsequent metabolism and excretion. Moreover, the results of LD50 tests are known to vary with factors such as animal species and strain used, age, weight, sex, health, nutrition, gut contents, route of toxicant administration, housing, temperature, time of day, season, and human error. Similarly, because the most important information needed for regulatory purposes concerns chronic toxicity, little useful information is derived from the LD50 test. Therefore, despite standardization of test species and conditions for measurement, the LD50 value for a given chemical may vary considerably in different determinations in different laboratories. Comparison of LD50 values must therefore be undertaken with caution and regard for these limitations. In spite of its obvious limitations, continued use of the LD50 tests is advocated for the following reasons: 1.

death, symptoms, nonlethal acute effects, organs affected, and reversibility of nonlethal effects. Information concerning mode of action and metabolic detoxification can be inferred from the slope of the mortality curve. The results can form the basis for the design of subsequent subchronic studies. The test is useful as a first approximation of hazards to workers. The test is rapidly completed.

Figure 6.9 Dose-response curves seen in unusually sensitive populations: (A) bimodal distribution of sensitivities; (B) minor segment of population responds at much lower doses than majority of population.

decreases, the differences in sensitivities must be correspondingly greater before they can be differentiated from the normal spectrum of individual variations within the main group. Similarly, in some toxicological studies, the competing effects observed do not occur in isolation from one another. For example, when studying liver damage due to exposure to carbon tetrachloride, some of the animals exposed to the high dose levels may die of the more immediate effects on the central nervous system without ever exhibiting measurable liver damage. Similarly, in many carcinogenicity assays, the selected dose levels may be so high that animals exposed to the highest doses may succumb before sufficient time elapses for tumors to develop. In both instances, the resulting dose-response curve, which summarizes the events through time t, shows a segment at high dose rates with negative slope (Figure 6.10). Substances that perform metabolically essential functions also show unusual dose-response curves. Such curves exhibit a negative slope at low dose rates (reversed dose-response curves) that are below the region of minimumal requirements (Figure 6.11). At higher dose rates, the essential compounds can reach levels that produce toxic responses, resulting in the traditional dose-response curve. For some compounds, such as vitamin C and some amino acids, the range of doses between essentiality and toxicity may be great; for others, such as selenium and vitamins A and D, the range may be small, so that only a small window is available for optimal exposure. The complex dose-response relationships seen with unusually sensitive subgroups in population when competing effects occur, or when the question is one of essentiality versus toxicity, present additional difficulties in establishing the margins of safety for chemicals. The commonly used mathematical extrapolation models cannot accommodate data involving these phenomena without significant modifications.

Figure 6.11 Dose-response curves for substances that perform essential biological functions: (A) negative slope (reverse dose-response curve) seen at low dose rates that are below the region of minimal requirements for biological functional integrity; (B) at higher dose rates, essential compounds can produce toxic responses that generate a more traditional dose-response curve.

6.2.3 Animal Toxicity Tests The extent to which a chemical is tested for toxicity with animal models depends on the intended use of the compound. Those that are intended for introduction into humans, such as drugs or food additives, obviously require extensive toxicological testing. Extensive toxicological testing means that the compound is subjected to a series of individual short-term tests that are designed to detect specific types of toxicity. In addition, at least two different species of animals must be exposed to the compound for at least a major portion of the lifetime of the animals. If the compound is eventually to become an environmental agent, these tests may even involve investigations utilizing insects, fish, wildfowl, or any animal. A general outline of the principal categories of tests commonly conducted for toxicological purposes is presented in Table 6.5. Two main principles underlie all descriptive animal toxicity testing: 1.

2.

Figure 6.10 Dose-response curve in situations in which competing effects can result in a negative slope of the curve a high dose rates.

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The effects produced by the compound in laboratory animals, when properly qualified, are applicable to humans. The exposure of experimental animals to toxic agents in high doses is a necessary and valid method of discovering possible hazards in humans.

On the basis of dose per unit of body surface, toxic effects in humans are usually in the same range as those in experimental animals (Klaassen, 1986). On a body weight basis, however, humans are generally more vulnerable, probably by a factor of about 10. An understanding of such quantitative differences as well as species variation is essential in formulating appropriate safety factors and relatively safe dosages for humans.

Table 6.5 1.

2.

3.

4.

5.

Summary of Types of Tests for Toxicity

Chemical and physical properties For the compound in question, probable contaminants from the industrial synthesis as well as intermediates (and waste products) in the synthetic process Exposure and environmental fate A. Degradation studies, e.g., hydrolysis, photodegradation B. Degradation in soil, water, etc., under various conditions C. Mobility and dissipation in soil, water, and air D. Accumulation in plants, aquatic animals, wild terrestrial animals, food plants and animals, etc. In vivo tests A. Acute tests (single dose) I. LD50 and/or LC50 (24-hr test and survivors followed for 14 days) a. Two species (usually rats and mice) b. Two routes of administration (one by intended route of use) c. Topical effects on rabbit skin (if intended route of use is topical; evaluated at 24 hr and at 7 days) B. Prolonged (subchronic) tests (daily doses) I. Duration 90-day feeding, 30- to 90-day dermal or inhalation exposure II. Two species (usually rats and dogs) III. Route of administration according to intended route of use IV. Evaluation of state of health a. All animals weighed weekly b. Complete physical examination weekly c. Blood chemical, urinary, hematological, and function tests performed on all ill animals V. All animals subjected to complete autopsy including histological evaluation of all organ systems C. Chronic tests (daily doses, including oncogenicity, teratogenicity, and reproductive toxicity tests) I. Duration 2–7 yr depending on species; multigenerational studies for assessing reproductive toxicity II. Species selected from results of prior prolonged tests, pharmacodynamic studies on several species of animals, possible single-dose human trial studies; otherwise two species used III. Minimum of two dose levels IV. Route of administration according to intended route of use V. Animal care and examination a. All animals weighed weekly b. Complete physical examination weekly c. Blood chemical, urinary, hematological examination and function tests on all animals at 3- to 6mo intervals and on all ill or abnormal animals VI. All animals subjected to complete autopsy including histological examination of all organ systems D. Special tests I. Neurotoxicity (delayed neuropathy) II. Potentiation III. Metabolism IV. Pharmacodynamics V. Behavioral In vitro tests A. Mutagenicity prokaryote (Ames test) B. Mutagenicity eukaryote (Drosophila sp., mouse, etc.) C. Chromosome aberration (Drosophila sp., sister chromatid exchange, etc.) Effects on wildlife Selected species of wild mammals, birds, fish, and invertebrates: acute toxicity, accumulation, and reproduction in laboratory-simulated field conditions or actual field conditions

Source: Compiled from Loomis (1978) and Hodgson (1987).

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The second principle is dictated by the fact that the number of animals used in toxicology experiments will always be small compared to the size of human populations similarly at risk. It is based on the quantal dose-response concept that the incidence of an effect in a population is greater as the dose or exposure increases. Hence, relatively large doses are tested in relatively small groups of animals; then, using the toxicological principles, the results are extrapolated to estimate risk at low doses. Toxicity tests are not designed to demonstrate that a chemical is safe, but rather to characterize the toxic effects it can produce. Not all tests are required for all potentially toxic chemicals; any of the tests shown in Table 6.5 may be required by the regulations imposed under a particular law. The Food and Drug Administration (FDA), the Department of Labor (Occupational Safety and Health Act), and the Environmental Protection Agency (EPA) are the principal regulatory bodies involved in the regulation of toxic chemicals in the United States. These agencies have also formulated guidelines for conducting animal toxicity tests. The principles of the most commonly used animal toxicity tests are briefly described in the following sections. Chemical and Physical Properties The determination of chemical and physical properties of known or potential toxicants does not constitute a test for toxicity. However, it is an essential preliminary for such tests (Hodgson, 1987). The information obtained can be used as follows: 1.

2. 3.

4.

5.

For structure-activity comparisons with other known toxicants, which may indicate the most probable hazards As an aid in identification in subsequent poisoning episodes In determining the stability to light, oxidizing or reducing agents, heat, etc., which may allow preliminary estimates of persistence in the environment as well as indicate the most likely breakdown products that themselves may require testing for toxicity In establishing such properties as lipid solubility or lipid/water partition coefficient, which may allow preliminary estimates of rate of uptake and persistence in living organisms (e.g., use of vapor pressure to indicate whether the respiratory system is a probable route of entry) In acquiring knowledge of the chemical and physical properties needed to develop analytical

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methods for the measurement of the compound and its degradation products Exposure and Environmental Fate Data on exposure and environmental fate are needed not to determine toxicity but to provide information that may be useful in the prediction of possible exposure in the event that the chemical is toxic (Hodgson, 1987). Primarily useful for chemicals released into the environment such as pesticides, these tests include the rate of breakdown under aerobic and anaerobic conditions in soils of various types and the rate of movement toward groundwater. The effect of physical factors on degradation through photolysis and hydrolysis studies and the identification of the products formed can indicate the rate of loss of the hazardous chemical or the possible formation of hazardous degradation products. In Vivo Tests In vivo tests using animal models are the principal tool of the toxicologist for the determination of toxicity of chemicals. The most commonly used laboratory mammals are mice, rats, hamsters, gerbils, guinea pigs, rabbits, cats, dogs, and certain nonhuman primates. Frequently used birds include chickens, quail, pigeons, and turkeys. Certain amphibians, reptiles, and fish can also provide information related to their unique physiological adaptations to their living environments. The commonly used in vivo tests for toxicity assessment are now described. Acute Toxicity (LD50) Testing Perhaps the simplest and most commonly applied toxicity test is the single-exposure study, with death as the criterion for toxicity. However, other acute effects, such as eye or skin irritation, are also subject to such tests. The species most often used are the mouse and rat, but sometimes the rabbit and dog are also employed. Administering a single dose to a limited number of animals and determining the number of deaths after 14 days constitute the study. In addition to mortality and weight, periodic examination of test animals is conducted for signs of intoxication, lethargy, behavioral modifications, morbidity, etc. The acute toxicity test is designed to determine the LD50 parameter. It also provides information on clinical manifestations of acute toxicity (Table 6.6) and gives dose-ranging guidance for subsequent studies. Subacute Toxicity Testing The LD50 is a very crude measure of toxicity because it does not recognize any toxic effects short of death.

Table 6.6

Clinical Manifestations of Acute Toxicity

Structure/function affected Respiratory Motor activities Reflex Ocular Cardiovascular Gastrointestinal Dermal

Possible effects Changes in rate or depth of breathing Changes in frequency and nature of movement Response to external stimuli Tearing Changes in heart rate Vomiting, cramps Swelling or redness

Therefore, other tests are employed to examine toxic effects that are less extreme. The subacute toxicity tests are performed to obtain information on the toxicity of the chemical after repeated administration and to help establish the doses for the subchronic studies. A typical protocol is to give four different dosages of the chemical to the animals by mixing it in the feed. For rats, 10 animals per sex per dose are often used, whereas for dogs three dosages and 3 animals per sex are used (Klaassen, 1986).

Table 6.7

Clinical chemical and histopathological evaluations are made after 14 days’ exposure (Table 6.7). Subchronic Toxicity Testing Subchronic tests examine toxicity caused by repeated dosing over an extended period but not one that is so long as to constitute a significant portion of the expected lifespan of the species tested. Typically, 90 days is the most common test duration. The subchronic study is usually conducted in two species (rats and dogs) by the route of intended exposure (usually oral). At least three doses are employed: a high dose that produces toxicity but does not cause more than 10% fatalities, a low dose that produces no apparent toxic effects, and an intermediate dose. Twenty rats or six dogs of each sex are used at each dose. Animals are then examined for parameters summarized in Table 6.7. Subchronic toxicity testing provides information on essentially all types of chronic toxicity other than carcinogenicity. It also provides data for a more reasonable prediction of appropriate doses for the chronic exposure studies. These tests are also frequently used as the basis for the determination of the no-observable-effect level

Clinical Indications of Subacute, Subchronic, and Chronic Toxicity Tests

Tests performed Hematological and clinical chemical evaluation Urinalysis Organ weights: liver, kidneys, heart, gonads, brain, adrenals Histopathological analysis (all tissues) Gross lesions Skin Mandibular and mesenteric lymph node (all studies) Bronchial and mediastinal lymph nodes for inhalation studies Mammary glands with adjacent skin Salivary glands Thigh muscle Sciatic nerve Sternebrae, femur, or vertebrae including marrow Costochondral junction, rib Thymus Oral cavity, larynx, and pharynx Trachea Lungs and bronchi Heart and aorta Thyroid Parathyroids Esophagus Stomach Tongue

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Small intestine (duodenum, jejunum, ileum) Large intestine (cecum, colon, rectum) Tissue masses or suspect tumors and regional lymph nodes Liver Gallbladder (mice) Pancreas Spleen Kidneys Adrenals Urinary bladder Seminal vesicles Prostate Testes, epididymis, vaginal tunics of testes and scrotal sac Ovaries Uterus Pituitary Spinal cord Eyes Preputial or clitoral glands Zymbal glands (auditory sebaceous glands)

(NOEL) or no-observable-adverse-effect level (NOAEL). This value is often defined as the highest dose level at which no deleterious or abnormal effect can be measured and is used in risk assessment calculations. Subchronic tests are also useful in providing information on target organs and on the potential of the test chemical to accumulate in the organism. Chronic Toxicity Testing Long-term or chronic exposure studies are performed similarly to the subchronic studies except that the period of exposure is longer. The length of exposure is somewhat dependent on the intended period of exposure in humans. The most important tests of this type are chronic toxicity, carcinogenicity, teratogenicity, and reproductive toxicity. Chronic exposure studies are often used to determine the carcinogenic potential of chemicals. These studies are usually performed in two species, one of which is either a rat or a mouse strain. Thus, for a rat, exposure is 2 years. To assure that 30 rats per dose survive the 2-year study, 60 rats per group per sex are often started in the study. Both gross and microscopic pathological examinations are made, not only on those animals that survive the chronic exposure but also on those that die early. The principal end point is tumor incidence as determined by histological examination. The teratogenic effect and the effect of chemicals on reproduction and development also need to be determined. Teratology is the study of defects induced during development between conception and birth. Several different experimental protocols are used, including both single and multigeneration studies (Wilson and Fraser, 1977; Hodgson, 1987; Klaassen, 1986). The end points observed in these studies are summarized in Table 6.8. In addition, epidemiological studies are used to establish links between exposures to particular toxic substances and specific chronic effects. There are two types of epidemiological studies. The first type is the case-control, or retrospective type, in which two well-matched populations are studied. The second type of epidemiological study, the prospective or cohort study, follows a population from a set time into the future. The basic problem with epidemiological studies is that they are uncontrolled and are done on a very variable population. Nonetheless, they provide valuable information on toxicity potential of a given chemical. A summary of the characteristics of the four main types of toxicological assays is presented in Table 6.9, which clearly shows the differences in the types of testing done to assess different types of toxicity. It also suggests why it is so difficult to make comparisons among the dif-

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Table 6.8 End Points Used in Single-Generation and Multigeneration Studies for Assessing Reproductive Toxicity and Teratogenic Potential of Toxicants Single-generation tests Preimplantation death: number of corpora lutea in the ovaries relative to number of implantation sites Postimplantation death: number of resorption sites in uterus relative to number of implantation sites Gross effects on male or female reproductive system Duration of gestation Litter size and condition, number of dead and live pups, weight of pups, gender of pups, gross morphological variation in pups Subsequent survival and performance of dam and pups, weight gain, mortality, etc. Gross and visceral abnormalities in weanlings Multigeneration tests Fertility index, number of pregnancies relative to the number of matings Number of live births, relative to the number of total births Gender and initial weight of pups Growth rate of pups Survival of pups relative to number born (or relative to number to which litters are culled) Gross deformities at birth Internal abnormalities at weaning Histological changes at weaning (third generation only)

ferent types of tests. This difficulty is reflected in problems of regulation and of communication of risk to the public. Special Tests Special tests are usually not required but may be carried out as useful adjuncts to current testing protocols. This is one of the rapidly evolving areas in toxicology. These tests include the following: 1. 2. 3. 4. 5. 6.

Neurotoxicity, including delayed neuropathy Potentiation and synergism between combinations of chemicals Toxicokinetics and metabolism Behavioral testing Covalent binding of toxicants, particularly to genetic material Immunotoxicity

In Vitro and Other Short-Term Tests Several in vitro tests are also used for assessing the toxic and/or carcinogenic potential of chemicals. The Ames test for mutagenicity (Ames et al., 1975) is probably widely used. It depends on the ability of mutagenic chemicals to

Table 6.9

Characteristics of Toxicological Assays Used in Human Risk Assessment

Type of assay Acute toxicity Subacute toxicity Chronic (carcinogenesis bioassay) Chronic (epidemiology)

Usual subjects

End point

Dose criterion

Extrapolation method used

Rodents Rodents Rodents

Death Multiple Cancer

50% Lethality Safe level Acceptable risk level

Not generally done Safety factor Mathematical modeling

Humans

Multiple

Variable

Not applicable

bring about reverse mutations in mutant Salmonella typhimurium strains that have defects in the histidine biosynthesis pathway. These strains do not grow in the absence of histidine but can be caused to mutate back to the wild type, which can synthesize histidine and hence can grow in its absence. The number of reverse mutations is then quantitated by number of bacterial colonies that grow in a histidine-deficient medium. The test can be performed in the presence of the S-9 fraction from rat liver to allow metabolic activation of promutagens. There is a high correlation between bacterial mutagenicity and carcinogenicity of chemicals. Other related tests include tests based on reverse mutations, as in the Ames test, as well as those based on forward mutations. Some reverse mutation tests utilize tryptophan, nicotinic acid, and arginine mutants of Escherichia coli. Others follow forward mutations in E. coli from galactose-nonfermenting to galactose-fermenting mutants and in S. typhimurium conferring resistance to 8-azaguanine. Polymerase-deficient and thus DNA repair-deficient E. coli and Bacilus subtilis strains are also used to study DNA-damaging potential of chemicals via the DNA repair method. Cell culture techniques using Chinese hamster ovary cells and mouse lymphoma cells are widely used for studying mammalian cell mutations. Other commonly used gene mutation test systems include Drosophila sp. (fruit fly) sex-linked recessive lethal test, reciprocal mitotic recombination (mitotic crossing-over), and nonreciprocal mitotic recombination in yeast (Saccharomyces cerevisiae), and the spot test for mutations in mice. A number of tests are also available for studying DNA damage and repair. These use events at the DNA level as end points. Given the number of tests, long- and short-term and in vivo and in vitro, available for monitoring acute and chronic toxicity, it is practically impossible to apply a complete series of tests to all commercial chemicals and all their derivatives in food, water, and the environment. Thus, the challenge of toxicity testing is to identify the most effective set or sequence of tests necessary to describe the apparent and potential toxicities of a particular chemical or a mixture of chemicals. The enormous empha-

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sis on in vitro or short-term tests in the last decade or so had its roots in the need to find substitutes for lifetime feeding studies in experimental animals or, at the very least, to suggest a sequence of tests that would enable setting of priorities for which chemicals should be subjected to chronic tests. Such tests are also used to eliminate the need for chronic testing for chemicals that either clearly possess the potential for toxicity or clearly do not. However, in spite of the sound scientific bases these tests are based upon, there still is no consensus on test sequences or on the circumstances in which short-term tests may substitute for chronic ones. Thus, short-term tests not only are often required, but also are required in addition to all the other tests required before their development (Hodgson, 1987). It is important, however, that test sequences have been suggested and considered by regulatory agencies in the United States and in the EEC. One such sequence is shown in Table 6.10.

Table 6.10 Three-Tier Decision Point Protocol for Evaluating Chemicals for Mutagenesis and Carcinogenesis A. Structure of chemical B. In vitro tests 1. Bacterial mutagenesis (Ames test) 2. Mammalian mutagenesis 3. Deoxyribonucleic acid (DNA) repair 4. Chromosome tests (sister chromatid exchange, micronucleus, etc.) 5. Cell transformation Decision point 1: evaluation of tests from A and B and selection of appropriate tests for C C. In vivo testing—limited bioassays 1. Skin tumor induction in mice 2. Pulmonary tumor induction in mice 3. Breast cancer induction in female rats 4. Altered foci induction in rodent liver 5. Assays for promoters Decision point 2: evaluation of data from tests A–C D. Long-term bioassay Decision point 3: final evaluation of all results Source: Adapted from Weisburger and Williams (1981).

Figure 6.12 The safety decision tree.

The National Academy of Sciences, after a 2-year study, suggested that focus should be directed toward compounds of high toxicity and high rates of use. Their report suggested that raw commodities, ingredients, and additives (both intentional and unintentional) should receive the same scrutiny (Clausi, 1979; Hutt and Sloan, 1979). Toxicity would be assessed by using the food safety decision tree method (Figure 6.12) developed by the Scientific Committee of the Food Safety Council (FSC, 1978, 1982).

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Overall, toxicity testing is a rather inexact science. The primary aim is not to establish with certainty the doses at which effects will occur in humans, but rather to establish levels at which there is reasonable confidence that effects will not occur. The overall goal should be to identify those compounds that present an unacceptable potential for risk to humans or the environment and thus ought to be banned but, at the same time, provide an accurate assessment of the risk to humans and the environ-

ment for the less toxic compounds so that their use can be regulated.

6.3

Table 6.11 Information Used in Health Risk Assessment 1.

RISK ASSESSMENT

The word risk implies uncertainty along with possible danger, injury, or loss. Risk can be physical, psychological, or monetary. It is not the same as hazard, but rather a way of judging the degree of hazard. Two concepts are embodied in the term: both the magnitude of loss due to the occurrence of an undesirable event and the probability of its occurrence (Munro and Krewski, 1983; Jones, 1992). As described in Chapter 1, we tolerate varying degrees of risk, depending on the situation. However, neither risk nor benefit can be easily quantified. In the area of food science and nutrition, the U.S. FDA and the food industry use a procedure called risk assessment. This approach uses a base of scientific research to establish possible harm that may result to an individual or population from exposure to a substance or process. It is based on exposure level and frequency together with inherent toxicity. Further, it must consider all available data, including historical and epidemiological data. For example, comparing the structure in question with that of known carcinogens can predict the carcinogenic potential of chemicals. Those with similar structures are more likely to be carcinogenic. Thus highly probable risks are separated from improbable ones, to enable scientists and regulatory agencies to concentrate their limited resources on those chemicals most likely to present a problem for humans. Similarly, correlations derived from epidemiological data also can identify risks. However, extreme care should be taken in evaluating correlations, and one must be constantly aware that the relationships found through correlations may not be causal. Risk assessment can be defined as the process of assessing the possible adverse health effects in humans resulting from exposure to substances or other potential hazards. This definition allows ordering the data, identifying the data gaps and uncertainties, assigning priorities, and determining research needs. On the basis of information in the risk assessment, a regulatory agency can then develop regulatory options; evaluate public health, economic, social, and political consequences of the regulatory options; and implement agency decisions and actions. These decisions and options are the core of the risk management process. The various types of information used in health risk assessment are summarized in Table 6.11. The elements involved in hazard identification and dose-

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2.

3.

4.

Hazard identification A. Human data a. Monitoring and surveillance b. Epidemiological studies c. Clinical studies B. Animal data C. In vitro data D. Structure-activity relationships Hazard characterization (dose-response assessment) A. Human studies B. Epidemiological studies a. Clinical studies C. Animal studies a. Minimal effects determination b. Dose-response modeling c. Special issues, including interspecies conversion and high- to low-dose extrapolation D. Pharmacokinetic studies based on physiological features Exposure characterization A. Demographic information B. Ecological analyses C. Monitoring and surveillance systems a. Animals b. Humans D. Biological monitoring of high-risk individuals E. Disposition and transport modeling (mathematical) F. Integrated exposure assessments a. Over time b. Over hazard (synergism) Risk determination A. Mathematical a. Unit and population risk estimates b. Threshold determination (e.g., safety factor approach, no-observable-adverse-effect level [NOAEL]) B. Formal decision analysis C. Interrisk comparisons D. Qualitative panel reviews E. Quantitative informal scientific advice F. Risk-benefit analysis

Source: DHHS (1986).

response assessments are summarized in Tables 6.12 and 6.13. There are two approaches adopted for establishing safe or virtually safe exposures for chemical hazards identified in animal or human studies (Figure 6.13). The approach should depend on the nature of the toxic effect or hazard that is the basis for risk assessment (Renwick, 1999). For nongenotoxic effects, it is considered that there is a threshold of exposure below which no effect will be

Table 6.12 Elements to Consider in Hazard Identification Animal bioassay data What are the most common data available? Assume that results from animal experiments are applicable to humans. Epidemiological data What is the association between exposure to a substance and disease? Risk is often low, number of people exposed is small, latency period is long, and exposures are mixed and multiple. Structure-activity relationships What chemicals are known to cause adverse health effects? What substances are structurally related and/or have similar mechanisms of toxicity? Source: From DHHS (1986).

produced; therefore, it is possible to calculate exposures for humans that would be without significant adverse health effects (safety assurance). The threshold dose for the most critical effect in a test is the highest exposure level without adverse, i.e., toxicologically relevant, effects. The basic differences between the nongenotoxic and genotoxic compounds are listed in Table 6.14. In contrast, for genotoxic chemicals it is considered that there may be no threshold for the effect, and therefore estimates are made of the possible magnitude of the risk at human exposures (dose-response extrapolation). Each of these approaches usually involves the uncertainties of extrapolating from high-dose animal studies to low-dose human exposure and from small groups of inbred animals to the larger and more diverse human population. Safety assurance is based on using the doseresponse relationship to define an approximation of the threshold in the animal study. This is called the no-observed-adverse-effect-level or NOAEL. It is a level of exposure in which the treated animals do not differ from untreated control animals in relation to the hazard or effect recognized at higher doses. For the determination of the

Table 6.13 Dose-Response Assessment • •

• •

Define the relationship between dose and response. In general, as the dose of many toxicants increases, toxicity increases; however, the manner in which toxicity increases varies. It is customary to extrapolate from the high doses administered to animals to low doses experienced by humans. The validity of these extrapolations must be considered, and the statistical and biological uncertainties defined.

Source: DHHS (1986).

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NOAEL, a series of doses is used. In order to establish the dose-effect relationship, the dose levels are chosen in such a way that the highest dose causes an adverse effect that is not observed after the lowest dose. As described earlier, ideally in a long-term toxicity study the highest dose should evoke symptoms of toxicity without causing excessive mortality, and the lowest dose should not interfere with development, normal growth, and longevity. In between, doses sufficiently high to induce minimal toxic effects should be selected. The determination of an adverse effect in a particular study depends not only on the doses tested, but also on the types of parameters measured and the ability to distinguish between a real adverse effect and a false positive finding. In long-term toxicity tests, the average value of a specific parameter at a particular dose level is compared with the average value of the parameter in control animals. An effect can then be defined in purely statistical terms as a significant deviation of a control value. However, in determining an adverse effect, the biological relevance of this deviation should be taken into consideration. If, for example, a slight but significant alteration is only observed at the highest dose level, it is difficult to define it as a real adverse effect. More weight should be given to a particular change in a parameter, if a dose-response relationship can be established, or if the observed change is related to changes in other functional or morphological parameters. If an effect is irreversible, its relevance is unquestionable. In some cases, however, the biological relevance of an effect must be interpreted in relation to historical control values. This is often the case when the value of the particular parameter is highly variable among the control animals used in a number of different toxicological studies. The historical control data should originate from the same species, strain, age, sex, supplier, and laboratory to allow proper comparison. The NOAEL (or any other benchmark parameter used) expressed on a body weight basis (e.g., mg/kg/day) is divided by a large uncertainty or safety factor to derive the “safe” level of human exposure. Most regulatory bodies adopt a similar approach despite the differences in nomenclature (Dourson et al., 1996). The safe human exposure is usually termed acceptable for an additive or tolerable for a contaminant together with a time base that is related to the exposure profile linked to toxicity, e.g., acceptable daily intake (ADI), tolerable daily intake (TDI), or provisional tolerable weekly intake (PTWI) for chemicals that accumulate. The latter is based on the observation that at a certain level of weekly intake, the intake is balanced by elimination, and therefore, no accumulation in the body takes place. Provisional means that the available safety data do not warrant a final conclusion. These assess-

Figure 6.13 Two approaches to risk assessment for establishing safety (nongenotoxic) or virtually safe (genotoxic) exposures to chemical hazards in the human food chain.

ments may be dated to indicate the time at which the database was assessed (Rubery et al., 1990). Safety factors provide an adequate safety margin for the consumer in the extrapolation of animal data to humans (Figure 6.14). Usually, most national as well as international regulatory bodies traditionally apply a safety factor of 10 for interspecies variation between animals and humans. A further factor of 10 that assumes that the variation in sensitivity within the human population is in a 10fold range is included, yielding an overall safety factor of 100. Each of these safety factors has to allow for differences in two areas: the delivery of the chemical to the site of toxicity (toxicokinetics) and its activity at the target site (toxicodynamics). A scheme that subdivides each of the 10-fold factors to allow relevant data to replace part of the uncertainty inherent in converting a NOAEL into an ADI or TDI has been developed (Renwick, 1993). For contami-

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nants for which a risk assessment may have to be undertaken on a nonideal database, additional uncertainty factors may be used to allow for extrapolating from subchronic to chronic or when the lowest dose studied produces an effect; therefore, this dose (the lowest-observableadverse-effect-level [LOAEL]) must be used in place of a NOAEL (WHO, 1994). Although the standard safety or uncertainty factor is 100, implying in practice that this additional factor often equals 1, extrapolation factors up to 2000 may be applied. If toxicity data in human beings are available, such data take precedence over animal data, and, generally, in such cases a safety factor of 10 is appropriate. A lower safety factor may suffice if the substance under investigation is identical to traditional food components, e.g., nutrients such as vitamins and amino acids, if the substance is metabolized into endogenous compounds, or if it lacks

Table 6.14 Characterization of Risks for Noncarcinogens and Genotoxic Carcinogenic Compounds Noncarcinogens • For food additives, the no-observed-adverse-effect-level (NOAEL) is divided by a safety or uncertainty factor to estimate an acceptable daily intake (ADI). • For systemic toxicants, the U.S. EPA developed the reference dose (RfD) approach: the NOAEL is divided by an uncertainty factor and a modifying factor. Generally, the RfD is an estimate of a daily exposure to the human population (including sensitive subgroups) that is likely to have no appreciable risk of harmful effects during a lifetime. Genotoxic carcinogens • Risk is estimated from the cumulative dose and/or the dose-response curve extrapolation. • Mathematical models are used to extrapolate to low-dose response. • A range of risks might be produced by using different models and assumptions about dose-response curves and relative sensitivities of humans and animals and for different estimates of human doses.

overt signs of toxicity. For substances serving as essential sources of energy in the human diet, the safety factor 100 is not applied. A food additive, for example, is considered safe for its intended use if the human intake figure is less than or equivalent to ADI. As mentioned earlier, the ADI is usually derived from the results of lifetime studies in animals and therefore related to lifetime use in humans. This practice provides a sufficient safety margin so that no particu-

lar concern is felt if humans are exposed to levels higher than the ADI in the short term, provided that the average intake over longer periods does not exceed it. Using an ADI derived from a NOAEL found in an appropriate animal study and a suitable safety factor implies an intrinsic conservatism reflecting the uncertainty of the extrapolation of experimental animal data to the diverse human population. In the case of contaminants, extrapolation is difficult from high-dose animal experiments

Figure 6.14 Use of safety factors for setting acceptable daily intake (ADI). Small safety margins (2–10) are acceptable for essential nutrients, e.g., selenium and vitamin A. Conversely, large safety margins (>100) should be set for contaminants and known toxins. Additives fall in between (usually ~100).

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to human conditions, in which lower doses are consumed. The ADI also makes some allowance for the possible synergistic effects humans experience when additives are consumed together in foodstuffs. The effect of the interacting additives may be different from the responses to the individual additives. As compared to compounds exhibiting nongenotoxic effects, genotoxic carcinogenic substances are assumed to exhibit no threshold in their dose-response relationship. Therefore, no absolute safe human exposure level can be defined. An important problem a toxicologist confronts with this group of substances is that the dose levels needed to establish the dose-response relationship in experimental animals are many orders of magnitude higher than those likely to be encountered in human exposure situations (Derks et al., 1997). Simple linear extrapolation from these high doses to find the dose associated with negligible risk is considered to be safe, but rather conservative. The negligible risk is often called the virtually safe dose or risk-specific dose and is assumed to cause one extra tumor in 106 human subjects after lifetime exposure. More often, mathematical models based on certain assumptions about the mechanism of carcinogenesis are used to fit the high-dose data obtained in animals and to predict effects at low dose levels. An often-used mathematical model is the multistage model, which assumes carcinogenesis to be a multistage process, and tumor incidence to depend on the probabilities of transition of each stage into the next. The number of stages and the transition probabilities are estimated by curve fitting of the experimental data. Other mathematical models that have been introduced may differ in behavior in the low-dose region and show no differences at high dose levels. Usually, no adjustments are made to correct for interspecies differences in sensitivity to carcinogenic substances. However, in some cases, dose levels have been normalized to body surface area rather than to body weight. Such normalization may correct for interspecies differences in the pharmacokinetic behavior of xenobiotics. The subdivision of carcinogens into genotoxic and nongenotoxic raises the interesting question of whether chronic carcinogenicity studies over 2 years are necessary when a compound is nongenotoxic (Purchase, 1994; Renwick, 1999). A nongenotoxic compound would normally be assessed by the NOAEL and uncertainty factor approach described, because 6- or 12-month studies may be more sensitive for revealing target organ toxicity as a result of the absence of aging changes. Therefore, it would probably be logical to reduce the duration of chronic bioassays for nongenotoxic chemicals to optimize the detection of noncancer end points. Linearized low-dose risk assessment for genotoxic and nongenotoxic chemicals in-

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volves the linear extrapolation of cancer risk from an incidence of more than 1 in 100 (animals) to 10–5 to 10–6 (humans). Because the cancer dose response in animals is usually closely related to the maximal tolerated dose (MTD), it has been suggested that the human “acceptable” risk or “virtually safe dose” could be estimated simply by dividing the MTD by 740,000 without the need for a cancer bioassay (Gaylor and Gold, 1995). The outcome of either safety assessment or quantitative risk assessment (low-dose extrapolation) is the definition of a daily or weekly exposure that is acceptable, tolerable, or virtually safe. The concentrations of the chemical in different foods are then established for a food additive or contaminant such that the intake by a high consumer would not exceed the safe intake. In the case of food additives, a simple method to assess the adequacy of the safety margins currently available that adopts a combination of hazard-identification data and per capita exposure estimates has been proposed. The socalled R-value is the ratio of the estimated exposure (derived from total poundage estimates assuming that only 10% of the population consume 100% of the product, i.e., a simple per capita estimate) to the LOAEL from the critical study. A review of toxicity and intake data for 159 substances showed an average safety margin (1/R) of about 10,000 (Rulis, 1987). Although this analysis is not equivalent to a normal uncertainty factor because it is based on a LOAEL, it clearly reveals the effectiveness of current risk assessment procedures for food additives.

6.4

STANDARD SETTING

Within the framework of public health legislation, national regulatory authorities are responsible for standard setting with regard to food safety. The authorities can carry out the process of standard setting as a separate national affair or adopt standards set by international bodies such as the World Health Organization (WHO), the U.S. FDA, or the European Union (EU). To achieve harmonization in food standards many countries adopt standards set by the WHO. However, since 1992 the member countries of the EU are required to accept the decisions taken by the European Commission (EC) and enforce Union Standards into their own national legislation. 6.4.1 World Health Organization The WHO is an international advisory body with the overall aim of protecting human health. As far as toxicological risk assessment is concerned, it is not a legislative body. It

backs national authorities in setting standards for the protection of human health. The International Program on Chemical Safety (IPCS) plays a guiding role in the international procedure of evaluating risks from chemicals and setting tolerances for residues of chemicals in food. Through the IPCS, the WHO participates in two joint committees of the WHO and the FAO. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the Joint Meeting on Pesticide Residues (JMPR) serve as scientific advisory bodies of the Codex Alimentarius Commission, a joint FAO/WHO commission that sets standards for chemicals in foods. The Codex Alimentarius Commission is responsible for the implementation of the Joint FAO/WHO Food Standards Program. It has the following objectives: 1. 2.

3.

4.

5.

Protect the health of the consumer and ensure fair practice in food trade. Promote coordination of all food regulatory activities carried out by international governmental and nongovernmental organizations. Establish priorities and initiate and give guidance to the preparation of provisional standards by and with the aid of appropriate organizations. Finalize provisional standards and, after acceptance by governments, publish them in a Codex Alimentarius. Amend published standards, after appropriate survey, in view of certain developments.

as grains and meat tend to favor relatively high MRLs, whereas those that are importers tend to favor low MRL standards. In tackling these differences, the Codex Commission follows a thorough stepwise procedure, leading to the acceptance of a formal Codex Standard (Figure 6.15). This procedure gives members an opportunity to participate in the decision process and to use the final result for their own national standard setting. However, national or regional policy sometimes disturbs this ideal in standard setting, for example, when the EU uses other MRLs based on the recommendations of one of its own Scientific Committees. Joint FAO/WHO Expert Committee on Food Additives The Joint FAO/WHO Expert Committee on Food Additives evaluates food additives, food contaminants, and residues of veterinary drugs in the human food chain. The JECFA was first convened in 1956 with the following mandate: 1.

2. Although the Codex Alimentarius and FAO/WHO do not have any legal authority and the standards they propose are not standards as defined, the Codex standards have been shown to be of great value in the harmonization of world food standards. Codex also offers proposals for maximal residue limits (MRLs) to national governments for acceptance into the prevailing national registration or standardization system. There are Codex Committees on food additives and contaminants, on pesticide residues, and on veterinary drug residues. The membership of the Codex Committees is open to all nations, and their meetings are attended by formal national delegations. Whereas the considerations of JECFA and JMPR are purely scientific, the proposals of the Codex Committees are partly based on national politics. The regional differences in the use of additives, pesticides, or veterinary drugs are a problem in the harmonization of worldwide MRLs. Officially recommended use rates for pesticides are usually higher in those countries where extreme climatic conditions favor the development of pests or diseases than in more temperate climates. Further, countries that are important exporters of foods such

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Formulate general principles governing the use of food additives, with special reference to their legal authorization, on the basis of considerations such as innocuousness, purity, limits of tolerance, and the social, economic, physiological, and technical reasons for their use, taking into account work already done on the subject by national and other international bodies. Recommend, as far as possible, suitable uniform methods for the physical, chemical, biochemical, pharmacological, toxicological, and biological examination of food additives and of any degradation products formed during the processing, for the pathological examination of experimental animals and for the assessment and interpretation of the results.

This mandate was later expanded to include food contaminants and veterinary drugs in 1987. For food additives, the JECFA calculates the ADIs or provisional ADIs when the available information does not warrant a final conclusion. This parameter, as described earlier, indicates the safe daily dietary intake of a substance. The actual daily dietary intake should not exceed the ADI; therefore, information on the dietary intake is necessary. It can be obtained from market-basket or total diet studies. In the case of major food components and some novel foods (e.g., modified starches, polyols, modified celluloses), it is often not necessary to calculate the ADI, since the effects observed in toxicity experiments concern the nutritional value. In such cases, no numerical

Figure 6.15 Procedures leading to the acceptance of a formal Codex Standard.

value for the ADI is given: i.e., the ADI is not specified. These products, therefore, are believed to be acceptable. For residues of veterinary drugs, the WHO panel of the Joint Expert Committee evaluates the toxicological information and establishes, if possible, the ADIs (or provisional ADIs). The FAO panel proposes limits (MRLs or provisional MRLs) for residues of veterinary drugs in products of animal origin, based on the WHO ADIs and on information about the distribution of the residues in tissues of the target animals. In setting the MRLs, the maximal theoretical intake should not exceed the ADI. This maximal theoretical intake is estimated by using the exaggerated consumption package for products of animal origin (Table 6.15).

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Veterinary drug residues include parent drugs as well as their metabolites. The metabolites are taken into account if they are toxicologically relevant, i.e., present in a considerable quantity or having a toxicological or pharmacological potential. The MRL is expressed in terms of parent drug levels or in terms of levels of a marker metabolite, if the percentage of the marker metabolite formed from the parent drug is known. However, JECFA concluded that application of these recommended MRLs does not pose a risk to the consumer, since the NOAEL used for the calculation of the ADI was very conservative, and the consumption data used in Table 6.15 are at the upper limits of the range for the individual intake of animal products. Thus, in practice, the safety

Table 6.15 Exaggerated Average Daily Consumption of Animal Products Used for Calculating the Maximal Theoretical Intake and Maximal Residue Limits for Residues of Veterinary Drugs in Products of Animal Origin Animal product Cattle/swine Muscle Liver Kidney Fat Milk Poultry Muscle Liver Kidney Skin Eggs Fish Muscle Liver

Average daily consumption, g 300 100 50 50 1.5 L 300 100 40 60 100 300 100

rules are interpreted with a certain flexibility though strict rules are applied for the derivation of health-based recommendations. Joint Meeting on Pesticide Residues The Joint Meeting on Pesticide Resides (JMPR) was convened for the first time in 1963 to evaluate pesticide residues on the basis of toxicological and biochemical data. If the data are inadequate, the JMPR allocates an ADI for each individual pesticide under investigation. The FAO panel of the JMPR evaluates disposition of residues and resulting residue levels under conditions of Good Agricultural Practice, on the basis of data on patterns of use. In order to evaluate the acceptability of a proposed MRL, it is necessary to compare the dietary intake of pesticide residues calculated on the basis of the MRL with the ADI. The dietary intake is calculated by multiplying each MRL with the quantity of the corresponding diet component, followed by summation of the residue quantities obtained. It should be noted that the use of the MRL in the calculation of total intake may lead to a higher value than the actual intake, since the actual residue levels are often lower than the recommended MRLs. Food consumption patterns vary considerably from one country to another, and from one culture to another. At the international level, the total intake is calculated on the basis of a hypothetical average global food consumption package, composed according to the recommendations in the FAO Food Balance Sheets, i.e., consisting of compo-

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nents of each cultural diet. At the national level, the total intake is calculated on the basis of actual consumption data. In practice, these are cultural diet data. There are three different ways of calculating the daily intake of pesticide residues: theoretical maximal daily intake (TMDI), estimated maximal daily intake (EMDI), and estimated daily intake (EDI). The EDI is a refinement of the EMDI at national level, based on adequate actual data. The procedure in which the dietary intake of pesticide residues is compared with the ADI starts with the intake parameter that can be the highest, i.e., the TMDI. If TMDI does not exceed ADI, it is highly unlikely that the ADI will be exceeded in practice, and therefore, the MRL proposals can be considered to be acceptable. If the TMDI is higher than the ADI, a parameter concerning the actual situation (EMDI) should be used in order to eliminate the pesticide from further consideration. For veterinary drugs, a different procedure is used. MRLs for veterinary drugs are based on theoretical maximal consumption data. Furthermore, veterinary drug residue limits are set for the fresh animal products, and the effects of industrial or in-house processing on the residue content are not taken into account. International Program on Chemical Safety Within the framework of the International Program on Chemical Safety (IPCS), WHO has drawn guidelines for the protection of drinking water quality. According to WHO, these guidelines should be applied in setting national standards, not only for community piped-water supplies but also for all sorts of drinking water except bottled mineral waters. Adoption of these worldwide guidelines is dependent on national priorities and socioeconomic factors. Since water is one of the primary needs for life maintenance, it must be available even if the quality is not entirely satisfactory; this assumption implies that setting standards that are too stringent could limit the availability of water. Such limited availability is considered unacceptable, in particular in regions with water shortage. The information used for drawing guidelines for drinking water include both toxicological data as well as data on the occurrence of contaminants in drinking water, physical properties like solubility, and aesthetic and organoleptic aspects. In cases in which threshold doses were exceeded, ADIs were calculated or adopted if they were available from other international bodies. For genotoxic carcinogens that may be present as contaminants in drinking water, the risks were assessed on the basis of an acceptable risk of 1 additional case of tumorigenesis per population of 1 million lifelong exposed persons.

Since exposure to substances whose guidelines are under revision not only occurs via drinking water but also via other routes (food, air), the ADI may be partly ingested. In general, intake via drinking water amounts to 10% of the ADI. Since for most pesticides exposure via other routes is extensive, an intake value of 1% of the ADI is employed. For disinfectants used for the purification of the drinking water, exposure via other routes is negligible. Therefore, higher intake values (up to 50%) are applied. The toxicological guide values calculated according to the procedure were compared with taste and odor thresholds. If the latter values were lower, the standards were based on organoleptic quality. 6.4.2 United States Food Safety System The food safety system in the United States is quite complex and multileveled. It is also essentially uncoordinated. As a consequence, the government’s role is also complex, fragmented, and in many ways, uncoordinated. The U.S. food supply is abundant and affordable and is judged by many to present an acceptable level of risk to health. The system has evolved from one that provided consumers with minimally processed basic commodities that were predominantly for home preparation to the present system of highly processed products designed either to be ready to eat or to require minimal preparation in the home. As a result of many technological advances, the food system has progressed dramatically from traditional food preservation processes such as salting and curing to today’s marketplace with frozen ready-to-eat meals and take-out foods. At least a dozen federal agencies implementing more than 35 statutes make up the federal part of the food safety system in the United States. They are involved in the key functions of safety: monitoring, surveillance, inspection, enforcement, outbreak management, research, and education. The major federal agencies involved include the Agricultural Marketing Service, the Animal and Plant Health Inspection Service, the Agricultural Research Service, the Cooperative State Research, Education and Extension Service, the Economic Research Service, the Food Safety and Inspection Service, and the Grain Inspection, Packers and Stockyards Administration of the United States Department of Agriculture; the Centers for Disease Control and Prevention, the Food and Drug Administration, and the National Institute of Health of the Department of Health and Human Services; the National Marine Fisheries Service of the Department of Commerce; and the Environmental Protection Agency. In addition, 28 House and Senate committees provide oversight of the 35 statutes. The primary congressional committees responsible

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for food safety are the Agriculture Committee and Commerce Committee in the House; the Agriculture, Nutrition, and Forestry Committee and the Labor and Human Resources Committee in the Senate; and the House and Senate Agriculture, Rural Development, and Related Agencies Appropriating Subcommittees. Four agencies play major roles in carrying out food safety regulatory activities in the United States: the Food and Drug Administration (FDA), which is part of the Department of Health and Human Services (DHHS); the Food Safety and Inspection Service (FSIS) of the U.S. Department of Agriculture (USDA); the Environmental Protection Agency (EPA); and the National Marine Fisheries Service (NMFS) of the Department of Commerce. More than 50 interagency agreements have been developed to tie together the activities of the various agencies. The recent proposal to create a Joint Food Safety Research Institute of the USDA and the FDA is an obvious outgrowth of such efforts. The U.S. FDA has jurisdiction over domestic and imported foods that are marketed in interstate commerce, except meat and poultry products. FDA’s Center for Food Safety and Applied Nutrition (CFSAN) seeks to ensure that these foods are safe, sanitary, nutritious, wholesome, and honestly and adequately labeled. CFSAN exercises jurisdiction over food processing plants and has responsibility for approval and surveillance of food-animal drugs, feed additives, and all food additives (including coloring agents, preservatives, food packaging, sanitizers, and boiler water additives) that can become part of food. CFSAN enforces tolerances for pesticide residues that are set by the EPA and shares with FSIS responsibilities for egg products. The FDA’s statutes give CFSAN jurisdiction over restaurants, but it has always ceded this responsibility to states and localities. The agency provides leadership for state regulation of retail and institutional food service through the development of a model Food Code, which it recommends be adopted by states and localities (DHHS, 1995, 1997a; NRC, 1998). The U.S. FDA has oversight responsibility for an estimated 53,000 domestic food establishments (Rawson and Vogt, 1998). In fiscal year 1997, the agency devoted 2728 staff-years to food safety activities. Food safety consumes about 23.5% of FDA’s budget each year (OMB, 1998). In 1997, that amounted to approximately $203 million for food safety surveillance, risk assessment, research, inspection, and education out of the total FDA budget of $997 million (NRC, 1998). The largest share of FDA’s budget is devoted to its nonfood responsibilities, including drugs, cosmetics, and medical devices. Its drug approval

mission, in fact, dominates the agency’s culture and its public image. The FSIS seeks to ensure that meat and poultry products for human consumption are safe, wholesome, and correctly marked, labeled, and packaged if they move into interstate or international commerce. By the mid-1990s, roughly 7400 FSIS inspectors were responsible for inspecting 6200 meat and poultry slaughtering and processing plants by continuous carcass-by-carcass inspection during slaughter as well as by full daily inspection during processing (FSIS, 1996a; NRC, 1998). The FSIS shares responsibility with FDA for the safety of intact-shell eggs and processed egg products. Because of the statutorily mandated continuous inspection requirements, FSIS’s inspection budget is about four times that of the FDA. Food scientists believe that inspection of each animal carcass is no longer the best or most cost-effective means of preventing food-borne diseases; however, this effort is required by the statute and so is fully funded. The sensory evaluation inspection methods used in FSIS inspections were appropriate when adopted, when major concerns included gross contamination, evidence of animal disease, and other problems that are no longer acute concerns. Those methods are not appropriate or adequate to detect the major microbial and chemical hazards of current concern. Because of the FDA-USDA jurisdiction split along commodity lines, some food products that might be perceived by consumers as similar are regulated differently, depending on the content. The most cited example is pizza, which is regulated by the FDA unless topped with 2% or more of cooked meat or poultry, in which case it is USDA-regulated (FSIS, 1996b; 9 CFR 319.600). This means that inspection at pizza production facilities must be conducted simultaneously under two sets of guidelines by two different inspectors from separate agencies. The EPA licenses all pesticide products distributed in the United States and establishes tolerances for pesticide residues in or on food commodities and animal feed. The agency is responsible for the safe use of pesticides, as well as food plant detergents and sanitizers, to protect people who work with and around them and to protect the general public from exposure through air, water, and home and garden applications, as well as food uses. The EPA is also responsible for protecting against other environmental chemical and microbial contaminants in air and water that might threaten the safety of the U.S. food supply. In both these programs, it works with state and local officials. The NMFS conducts a voluntary seafood inspection and grading program, which is primarily a food quality activity. Seafood is the only major food source that is both “caught in the wild” and raised domestically. It is also an

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international commodity for which quality and safety standards vary widely from country to country. Inspection of processing is a challenge because much of it takes place at sea. Mandatory regulation of seafood processing is under the FDA and applies to all seafood-related entities in the FDA’s establishment inventory, including exporters, all foreign processors that export to the United States, and importers. However, fishing vessels, common carriers, and retail establishments are excluded. The Agricultural Marketing Service (AMS); Grain Inspection, Packers, and Stockyards Administration (GIPSA); and Animal and Plant Health Inspection Service (APHIS) of the USDA oversee the USDA’s marketing and regulatory programs. Together they play indirect roles in food safety and more direct roles in marketing, surveillance, data collection, and quality assurance (NRC, 1998). The Centers for Disease Control and Prevention (CDC) of DHHS engages in surveillance and investigation of illnesses associated with food consumption in support of the USDA and FDA regulatory missions. The Federal Trade Commission, through regulation of food advertising, plays an indirect role in food safety regulation. Several other federal agencies have smaller but important regulatory responsibilities in food safety. For example, the Department of the Treasury’s Bureau of Alcohol, Tobacco, and Firearms (ATF) is responsible for overseeing the production, distribution, and labeling of alcoholic beverages, except wines containing less than 7% alcohol, which are the responsibility of the FDA. The department’s Customs Service assists other agencies in ensuring the safety and quality of imported foods through such services as collecting samples (NRC, 1998). In addition to the various federal agencies involved in food safety monitoring, state and local health departments are responsible for surveillance at the state and local levels, and the extent to which these activities are carried out varies widely by jurisdiction. States and territories have separate departments of health and of agriculture. In addition, many counties and many cities have parallel agencies. In total, more than 3000 state and local agencies have food safety responsibilities (DHHS, 1997b). States are responsible for the inspection of meat and poultry sold in the state where they are produced, but FSIS monitors the process. The 1967 Wholesome Meat Act and the 1968 Wholesome Poultry Products Act require state inspection programs to be “at least equal to” the federal inspection programs. If a state chooses to end its inspection programs or cannot maintain the “at least equal to” standard, the FSIS must assume responsibility for inspection. In some states, state employees carry out inspections in some

federal plants under federal-state cooperative inspection agreements. FDA’s Food Code provides scientific standards and guidelines that states and localities may adopt for food safety in restaurants and institutional food settings (DHHS, 1995; 1997b). The code includes temperature standards for cooking, cooling, refrigerating, reheating, and holding food. It also recommends that inspectors visit restaurants every 6 months. Each state or locality may choose to adopt any or all of the code in its laws or regulations. Although there appears to be some recent progress toward more widespread adoption of this model code, according to one survey, there is much variation among jurisdictions in standards currently being applied to restaurants and other food establishments (DeWaal and Dahl, 1997). In contrast with the FDA Food Code, which has had varied acceptance, the Public Health Service 1924 Standard Milk Ordinance has been adopted by all 50 states, the District of Columbia, and U.S. trust territories. This model was created collaboratively by public and private entities to assist states and municipalities in initiating and maintaining effective programs for the prevention of milkborne disease. Now known as the Grade A Pasteurized Milk Ordinance, it is the standard used in the voluntary cooperative state–Public Health Service (PHS) program for certification of interstate milk shippers. Revisions are considered every 2 years on the basis of recommendations of the National Conference on Interstate Milk Shipments. The ordinance is incorporated by reference into federal specifications for procurement of milk and milk products, and it is used as the sanitary regulation for milk and milk products served on interstate carriers. Public health agencies, the milk industry, and many others recognize the ordinance as a national standard for milk sanitation, although exemptions allow for the sale of raw milk in some states (NRC, 1998). In recent years, many parts of the current food safety assurance system in the United States are in various stages of transition to Hazard Analysis Critical Control Point (HACCP) programs. The use of HACCP systems in food production, processing, distribution, and preparation is now widely recognized as the best current approach to enhancing the safety of foods. HACCP programs use a systematic approach to identify microbiological, chemical, and physical hazards in the food supply and establish critical control points that eliminate or control such hazards (NRC, 1985). The control must effectively address the identified hazard, and the effectiveness of the control point must be validated. The principles of the HACCP method and its role in product quality are described later in this chapter.

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Surveillance is also an essential part of the U.S. food safety monitoring program. Surveillance for human foodborne diseases is primarily the responsibility of state and local health departments, which are required or authorized to collect and investigate reports of communicable diseases. Although specific reporting requirements vary by state, such common and important bacterial food-borne pathogens as Salmonella, Shigella, and Campylobacter spp. and E. coli O157:H7 are reportable in most states. In addition, recognized outbreaks of food-borne diseases are reportable in most states regardless of the cause. On a national level, the CDC collects data from the states on the occurrence of specific pathogens and collects summary data on food-borne disease outbreaks investigated by local and state health departments. CDC conducts field investigations of food-borne diseases only at the request of the state health departments. The agency also plays a role in coordinating investigations of multistate or international outbreaks. 6.4.3 The European Union The European Community was founded as a free-trade association for its member countries. One of the objectives was to achieve harmonization in setting food standards. Since January 1992, all member countries have to accept the products produced in other member countries without any restriction and have to apply identical criteria for quality and safety. In practice, this means that member countries cannot approve a marketing authorization for substances used in the production of foods without the agreement of the European Community. The Commission of the European Communities formally carries out the safety evaluation of food additives or substances present in a food as a result of their use in its production process. The food safety evaluation process within the European Union (EU) is shown in Figure 6.16. Several scientific groups are involved in this process. Proposals made by these working groups for the safe use of food additives and for MRLs are, if adopted by Regulatory Committees, enforced by the Council of Ministers. Enforced proposals are published in the Official Journal of the European Union and are, from that time on, imperative for the regulatory authorities in the member countries. The Scientific Committee for Food (SCF) advises the commission with regard to directives for food additives, flavoring substances, solvents, materials in contact with food, contaminants, novel foods, and foods for particular nutritional use. Consultation of the SCF is obligatory in all cases concerning public health. The committee evaluates the available toxicological and ana-

Figure 6.16 Scientific working groups involved in food safety evaluation in the European Union.

lytical information in order to estimate the maximal limits for the safe ingestion of the substances under investigation and designates these guide values by the following classification: 1.

2.

3. 4.

5.

Acceptable daily intake (ADI, or provisional ADI if more data are required) for lifetime exposure, to be used to set standards for the use of particular food components ADI not specified, if the technological limits are believed to provide a sufficiently large safety margin Acceptable, limited, and well-defined use Not acceptable, intentional use considered unsafe; particularly for carcinogenic substances, no acceptable values Tolerable daily intake, for lifetime unintentional exposure (e.g., environmental pollutants and contaminants originating from packaging materials).

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According to the present EU regulation, any new request for the admission of a new substance that is covered by the Food Directive should no longer be addressed to the member state concerned, but directly to the commission. The evaluation of the food safety aspects of veterinary drugs used in animal production falls under the mandate of the Committee for Veterinary Medicinal Products (CVMP). Since January 1992, the decisions made by this Working Group and authorized by the CVMP overrule the national safety evaluation of veterinary drug residues. At present, no admission of a new veterinary drug in a member country is possible if a Union Standard has not been set. In contrast to the members of the other scientific committees, the members of the CVMP and of the Working Group are national representatives. This means that not only scientific judgments but national policy arguments contribute to decisions. In order to establish ADIs and MRLs, the Working Group follows a procedure as used by

the JECFA of the WHO. If possible, the it adopts ADIs and MRLs already established by the Codex Commission on Veterinary Drugs, but sometimes the scientific judgment of the Working Group differs from those of JECFA and Codex, yielding a different conclusion. In JECFA, the uncertainty with respect to the toxicological evaluation and the lack of sufficient data often lead to a number of questions to be answered by industry, and no ADI or MRL is established in such a case. The EU, however, is entitled to set residue levels for all veterinary drugs. Before 1997, about 400 biologically active substances present in veterinary drugs had to be evaluated and MRLs had to be established (van Leeuwen, 1997). This means that if there are not sufficient data available for an appropriate safety evaluation, a pragmatic approach has to be chosen that allows the establishment of provisional ADIs by applying larger uncertainty factors, resulting in the establishment of provisional MRLs. If the use of a particular component is a serious reason for concern, the MRL is also based on the detection limit. In the EU, the Scientific Committee for Pesticides (SCP) follows a procedure similar to that of JMPR. In general, this means that carcinogens are not acceptable as pesticides, and for other substances ADIs have to be established. The ADIs are compared with the estimated intakes of the residues through the consumption of various agricultural products. On the basis of this comparison, residue standards are set. The Scientific Committee on Animal Nutrition (SCAN) has already evaluated the additives used in cattle, swine, and poultry feed to prevent the outbreak of diseases as an accepted Union procedure. After the evaluation of all available toxicological data, conditions of use that were safe for the consumer were described, and these conditions were included as an annex to the veterinary drug acts in several countries. However, SCAN is now in the process of developing procedures for standard setting of feed additives, a process that, in the light of the ongoing harmonization, needs to be comparable to the procedures used by the CVMP and by JECFA.

ensuring food safety, HACCP has the advantage of being a structured system combining several proven techniques. The concept has been adopted by Codex Alimentarius (1995) and is mandatory in many countries. Although it was developed to deal with microbiological hazards, it also covers chemical and physical hazards. The National Aeronautics and Space Administration (NASA) can trace the origins of HACCP approach back to the initial period of space exploration. Absolutely safe food was required for the astronauts. However, to achieve this through the conventional end-product testing would require that all the food would have to be tested, leaving none for eating. Thus a procedure to assure safe food production was sought. Collaboration by the Pillsbury Company, NASA, and the U.S. Army laboratories proposed HACCP, which was based on the Failure, Mode and Effect Analysis (FMEA) used by NASA engineers in construction designs. The HACCP concept was introduced in the United States in 1971 at the Conference of Food Protection, where it was “recommended for widespread use” (FDA, 1972; Bauman, 1974). In the beginning the food industry showed little interest in HACCP, but microbiological problems with lowacid canned foods, particularly mushrooms, led to FDA promulgation of specific regulations for control embodying HACCP principles. Their successful introduction in the canning industry inevitably led to pressure for their wider acceptance by the food industry (Silliker, 1987). Subsequently, as a means of safe food production, it has been adopted worldwide as stated in Codex Alimentarius Commission (CAC, 1993) and the National Advisory Committee on Microbiological Criteria for Foods (NACMCF, 1992). 6.5.1 Hazard Analysis Critical Control Point (HACCP) Principles In order to produce a safe food product with negligible levels of foodborne food pathogens and toxins, three controlled stages must be established: 1.

6.5

THE HAZARD ANALYSIS CRITICAL CONTROL POINT (HACCP) SYSTEM

The food processing industry worldwide is currently implementing new management systems. One such program is Hazard Analysis Critical Control Point (HACCP) to eliminate the risks of food consumption and subsequently reduce the current increasing number of reported food poisoning outbreaks. Although it is not the only method for

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2.

Prevent microorganisms from contaminating food through hygienic production measures. This must include an examination of ingredients, premises, equipment, cleaning, and disinfection protocols, and personnel. Prevent microorganisms from growing or forming toxins in food. This can be achieved through chilling, freezing, or other processes such as reduction of water activity or pH. These processes, however, do not destroy microorganisms.

3.

Eliminate any foodborne microorganisms, for example, by using a time and temperature processing procedure, or by the addition of suitable preservatives.

These controls are central to the HACCP concept and are achieved through seven steps of principles (Figure 6.17). These principles are stated by the Codex Alimentarius Commission (CAC, 1993) and the National Advisory Committee on Microbiological Criteria for Foods (NACMCF, 1992). Although HACCP is an internationally recognized procedure, differences arise in the interpretation and implementation of these seven principles. The Codex principles are described in the following sections.

Principle 4 “Establish CCP monitoring requirements. Establish procedures from the results of monitoring to adjust the process and maintain control.” The frequency of monitoring the CCP and the identifiable person responsible for the monitoring are established. Principle 5 “Establish corrective actions to be taken when monitoring indicates a deviation from an established critical limit.” The HACCP team must establish the remedial action that must be taken, and by whom, if the critical limit is not attained.

Principle 1 “Conduct a hazard analysis. Prepare a list of steps in the process where significant hazards occur and describe the preventive measures.” A HACCP team is required to achieve this first principle (Scott, 1993). Ideally, the team is composed of production manager, engineer, microbiologist, and quality assurance staff. The multidisciplinary team is a prerequisite for identifying all the hazards. Additionally, such a team should have first hand information concerning the production process at the shop floor level. The HACCP team constructs a process flow diagram that identifies all the hazards. Principle 2 “Identify the critical control points (CCPs) in the process.” The HACCP team must identify the steps in the production process that are essential for the elimination or significant reduction of the identified hazards from Principle 1. These critical control points (CCPs) are identified through the use of decision trees such as shown in Figure 6.17. A CCP must be a quantifiable procedure in order for measurable limits and monitoring to be achievable in Principles 3 and 4. Principle 3 “Establish critical limits for preventive measures associated with each identified CCP.” These critical limits describe the difference between safe and unsafe products at the CCP. Factors constituting a critical limit can be temperature, time, pH, moisture or water activity, salt concentration, and titratable acidity. An example of a proposed approach to set criteria at CCP is shown in Figure 6.18.

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Principle 6 “Establish effective record-keeping procedures that document the HACCP system.” Records must be kept to demonstrate safe product manufacture and to indicate that appropriate action has been taken for any deviations from the critical limits. Principle 7 “Establish procedures for verification that the HACCP system is working correctly.” Verification procedures must be developed to ensure the HACCP plan is effective for the current processing procedure. In the Codex document, principles 6 and 7 are actually reversed (Mortimore and Wallace, 1994). The HACCP implementation has mainly been product-specific, with consideration of each ingredient, and so on. An alternative approach, and one more appropriate for food processors with large numbers of products, is generic HACCP (Generic HACCP for raw beef, 1993; HACCP for raw beef, 1993). Canada has been proactive in promoting HACCP for all food commodities and has developed 38 generic HACCP models. In the United States, the FSIS initiated generic HACCP models for refrigerated foods, cooked sausage, poultry slaughter, ground beef, and swine slaughter. Examples of identified CCPs in such a generic HACCP are listed in Table 6.16. Such generic models serve as suggested guidelines and can set minimal standards. Sometimes acceptable levels cannot be reached at one CCP, but the combined effect of many actions to reduce contamination, survival, and growth may lower the amount of a contaminant in the final product to an acceptable level. For example, contamination of poultry is prac-

Figure 6.17 Hazard analysis critical control point (HACCP) system and its seven steps as set out by the Codex Alimentarius Commission.

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Figure 6.18 Approach to set criteria at critical control points (CCPs) in the HACCP system. The approach is based on the use of quantitative risk analysis.

tically impossible to prevent, although good agricultural and hygienic practices can prevent accumulation of foodborne pathogens. It is usually in the kitchen where Salmonella and Campylobacter spp. in poultry can be controlled. Adequate cooking kills the pathogens, and further handling and use should prevent recontamination and growth. In this example, the most effective CCPs are at the end of the food chain. The HACCP approach appears to be much more effective in ensuring the safety of foods than traditional visual inspection practices. It institutes methods to control food safety hazards, whereas traditional inspection and

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testing procedures are not designed to detect and control contaminants that are sporadically distributed throughout foods and are not visible. Implementation of HACCP is the responsibility of food producers, processors, distributors, and consumers. The role of government is to ensure that HACCP programs are properly implemented throughout the food supply continuum by evaluation of HACCP plans and inspection of records indicating monitoring of CCPs. Implementation of this innovative approach requires a major educational effort and cultural change among federal inspectors.

Table 6.16 Identified Critical Control Points in Generic Hazard Analysis Critical Control Point Modelsa Critical control point Refrigerated foods 1. Preparation 2. Cooking 3. Chilling 4. Assembly of components 5. Gas flush 6. Package inspection 7. Labeling/code dating 8. Chilling 9. Storage Whole young chickens 1. Receiving 2. Scalding 3. Venting/opening/evisceratingMicrobiological 4. Offline procedure 5. Neck/giblet chiller 6. Final washer 7. Carcass chilling 8. Packaging/labeling 9. Storage/distribution a

Specification criteria Physical Microbiological Microbiological Microbiological Microbiological Microbiological Microbiological, chemical Microbiological Microbiological Microbiological, chemical, physical Microbiological Microbiological Microbiological, physical Microbiological Microbiological, physical Microbiological, physical Microbiological

CCP, critical control point; HACCP, hazard analysis critical control point.

6.6

TOTAL QUALITY MANAGEMENT AND LONGITUDINALLY INTEGRATED SAFETY ASSURANCE

Total quality management (TQM) regimens and longitudinally integrated safety assurance (LISA) extend the management of food safety beyond food processing to include the distribution stage and all the subsequent steps until consumption (Mossel, 1983, 1989). Control can be applied at the primary stage of harvesting, as can veterinary control of livestock, for example, the control of Salmonella spp. in poultry (Seniell, 1995); This latter has been called the longitudinally integrated safety assurance (LISA) concept. The preventive HACCP approach to food safety can also improve animal health care since there is an opportunity for preventive health action and risk management. This is at a relatively low cost in terms of labor, finance, and documentation expenditure, at both the farm and sector levels (Noordhuizen and Welpelo, 1996). However, epidemiological studies are required to identify the critical control points and to design HACCP procedures for livestock producers. The “Seven P” approach for implementing LISA is shown in Table 6.17. Public education is essential to the complete LISA strategy. The need for adequate personal hygiene at home,

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avoidance of cross-contamination in the kitchen, and control of pests must be frequently reinforced. Total quality management (TQM) is similar in emphasis to quality assurance and has been defined as “a continual activity, led by management, in which everybody recognizes personal responsibility for safety and quality” (Shapton and Shapton, 1991). This requires the company as a whole to achieve uniformity and quality of a product and thus maintain safety. TQM is broader in scope than HACCP since it includes quality and customer satisfaction in its objectives. Snyder (1995) and Webb and Marsden (1995) have extensively reviewed this topic. In 1987, the International Organization for Standardization (ISO) in Geneva, Switzerland, published the ISO 9000 standards. The ISO 9000 series is composed of five standards as follows: 1.

2.

3.

ISO 9000: quality management and quality assurance standards—guidelines for selection and use ISO 9001: quality systems—model for quality assurance in design/development, production, installation, and servicing ISO 9002: quality systems—model for quality assurance in production and installation

Table 6.17 Longitudinally Integrated Safety Assurance Implementation: The “Seven P” Approach P1 P2

P3

P4 P5

P6

P7

a

Premises: ensuring the construction and the equipment for compliance with GMDPs, with special emphasis on ease of cleaning and disinfection and screening against vermina Procurement: providing raw materials and ingredients of the best microbiological quality; if necessary, reaching an agreement with suppliers that unavoidably contaminated raw materials be decontaminated by procedures not adversely affecting the wholesomeness, quality, or acceptability of products so treated Processing: introducing, where required, microbicidal treatments as a third, essential line of defense against potentially dangerous contamination, relying on lethality levels derived from risk analysis Preservation: preventing loss of initial microbial integrity during postprocess storage, transportation, distribution or culinary preparation due to either contamination or colonization Personnel: educating and particularly motivating line staff to follow prescribed procedures, with special emphasis on providing those responsible for safety assurance with simple tools that allow them to assess the effectiveness of recommended practices Postmanufacture surveillance: (a) validating adherence to GMDPs by monitoring fresh product samples and specimens approaching the storage limit and (b) promptly identifying and rectifying incidental failures Public concern about certain processing procedures: taking seriously consumers’ anxiety about perceived adverse health impacts of certain modes of processing foods for safety; communicating the views of impartial academic expert panels on such matters

GMDP, good manufacturing and distribution practice.

4. 5.

ISO 9003: quality systems—model for quality assurance in final inspection and test ISO 9004: quality management and quality system elements—guidelines

These standards can be used as a starting point for designing TQM programs (Webb and Marsden, 1995; Deshpande, 1996). Irrespective of which approach is used, risk assessment and risk management techniques help a great deal in deciding what to do, when, and where. Even if these techniques were primarily designed for microbial hazards, it is clear that along the food chain, people have to decide what is a hazard, what is still acceptable, and what is not. The advantage of using HACCP, LISA, TQM, or the ISO standards is that such decisions have to be made and that nothing is left to chance.

6.7

THE ROLE OF INDUSTRY AND ACADEMIA

Although industry in general has no formal responsibility in the process of standard setting, it still plays an important role. The food producing and processing industries conduct substantial chemical surveillance as part of their own safety, quality, and regulatory compliance programs. Their focus is typically on pesticide residues, residues of veteri-

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nary drugs, mycotoxins, heavy metals, chemical and environmental pollutants, and bacterial pathogens that might be associated with foods. The extent and consistency of these programs are difficult to assess because the data are largely unavailable. However, some trade associations maintain a few databases, which can be used for this purpose. Although these programs play an important role in ensuring food safety, the best opportunity to establish an integrated monitoring system lies in the public sector, with oversight provided under regulatory authority. To that end, there might be opportunities for industry and agencies to share information more effectively. The food industry also provides the necessary information about the identity and purity of the substance, conditions of use, analytical methods for detection of residues, efficacy, and toxicological data that are essential for the safety evaluation. In almost all countries, the regulatory committees and agencies do allow the industry the opportunity to clarify existing problems or to comment on decisions made by these bodies. In this regard, the Codex system is unique in its potential to allow industries to participate in pre-Codex meetings and to be members of the national delegations. In these delegations the industry representatives, however, have no voting status. Further, the International Group of National Associations of Manufacturers of Agrochemical Products and the International Animal Health Industry participate as observers in the Codex

meetings without voting rights but with a limited opportunity to join in the debate. Trade associations formed in part to give members a unified voice on various issues of common interest, e.g., marketing, technical issues, and regulation, have model policies and regulatory support programs to help members enhance food safety and meet regulatory requirements. Such associations exist in many countries. For example, in the United States, the National Food Processors Association (NFPA) has developed model manuals on management of food product recalls, threats of tampering, and other crises, which can be adapted to an individual company’s needs. Videos and individual training programs are also available to members. The NFPA laboratories historically have helped members and the FDA work out questions on the safety of canned and other processed foods. In fact, an industry initiative in the early 1970s led to the lowacid canned foods regulations in the United States. In addition, consumer organizations play important roles in the promotion of food safety, including its regulatory aspects. Some organizations are also formed for general consumer protection purposes; others work primarily around issues of food safety and quality. As opinion leaders, these groups focus public attention on issues of concern, often seeking improved regulatory efforts and outcomes. Some of them, most notably Consumers Union and the Center for Science in the Public Interest (CSPI) in the United States, even have the scientific, financial, and public information resources to engage in product testing and surveillance and to disseminate their test results. Professional organizations and universities are also actively involved in the food safety system in many ways. The professional organizations offer expertise to assist both research and regulatory processes, and universities are involved in training and continuing education for professionals. Universities also train state and local regulatory professionals and provide periodic programs on food safety to update producers, processors, retailers, nutritionists, and health professionals.

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Brownlee, K. A., Hodges, J. L., and Rosenblatt, M. 1953. The up-and-down method with small samples. J. Am. Stat. Assoc. 48:262–277. Buikema, A. L. and Cairns, J. 1980. Aquatic Invertebrate Bioassays. ASTM, Special Technical Publication No. 715. American Society of Testing and Materials, Philadelphia. CAC. 1993. Codex Guidelines for the Application of the Hazard Analysis Critical Control Point (HACCP) System. Joint FAO/WHO Codex Committee on Food Hygiene. WHO/FNU/FOS/93.3, Annex II. Campbell, P. J. 1974. International biological standards and reference preparations. J. Biol. Standardization 2:249–258. Clausen, J. 1988. Immunochemical Techniques for the Identification and Estimation of Macromolecules. Elsevier, Amsterdam. Clausi, A. S. 1979. Revising the U.S. food safety policy. Food Technol. 33(11):65–67. Codex Alimentarius. 1995. Guidelines for the Application of the Hazard Analysis Critical Control Point (HACCP) System. Alinorm 95/13, Annex to Appendix III, Codex Alimentarius Commission. Food and Agriculture Organization, Rome. Derks, H.J.G.M., Groen, C., Olling, M., and Zeilmaker, M. J. 1997. Extrapolation of toxicity data in risk assessment. In Food Safety and Toxicity, ed. J. de Vries, pp. 241–254, CRC Press, Boca Raton, FL. Deshpande, S. S. 1995. Affinity chromatography. In Bioseparation Processes in Foods, eds. R. K. Singh and S.S.H. Rizvi, pp. 297–332, Marcel Dekker, New York. Deshpande, S. S. 1996. Enzyme Immunoassays: From Concept to Product Development. Chapman & Hall, New York. Deshpande, S. S. and Sharma, B. P. 1993. Immunoassays, nucleic acid probes, and biosensors: Two decades of development, current status and future projections in clinical, environmental and agricultural applications. In Diagnostics in the Year 2000, ed. P. Singh, B. P. Sharma, and P. Tyle, pp. 459–525, Van Nostrand Reinhold, New York. DeWaal, C. S. and Dahl, E. 1997. Adoption of the 1995 food code: A survey of 45 state and local health departments. J. Assoc. Food Drug Officials 61(4):15–29. DHHS. 1986. Determining Risks to Health: Federal Policy and Practice. Task Force on Health Risk Assessment, U.S. Department of Health and Human Services, Auburn House, Dover, MA. DHHS. 1995. Food Code. Public Health Service, Food and Drug Administration, Department of Health and Human Services, Department of Commerce, Springfield, VA. DHHS. 1997a. FDA approves irradiation of meat and pathogen control. HHS News, Dec. 2, 1997, Department of Health and Human Services, Washington, D.C. DHHS. 1997b. Food Code. Public Health Service, Food and Drug Administration, Department of Health and Human Services, Department of Commerce, Springfield, VA. Dourson, M.L., Felter, S.P., and Robinson, D. 1996. Evolution of science-based uncertainty factors in noncancer risk assessment. Regul. Toxicol. Pharmacol. 24:108–120.

EEC. 1983. Commission Directive of July 1983 adapting to technical progress for the fifth time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging, and labeling of dangerous substances, 83/467/EEC. European Economic Commission, Off. J. Eur. Commun. 26(L257):1–33. FAO/WHO. 1974. Toxicological Evaluation of Certain Food Additives in a Review of General Principles and Specifications. Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives, Technical Report Series No. 539. World Health Organization, Geneva. FDA. 1972. Proceedings of the 1971 National Conference on Food Protection. Food and Drug Administration, U.S. Department of Health, Education, and Welfare, Washington, D.C. FSC. 1978. Proposed system for food safety assessment. Food Cosmet. Toxicol. 16(suppl. 2):1–139. FSC. 1982. A proposed food safety evaluation process. Food Technol. 36:163–170. FSIS. 1996a. Pathogen reduction: Hazard analysis and critical control point systems, final rule. Food Safety and Inspection Service, U.S. Department of Agriculture. Fed. Reg. 61(144):38806–38989. FSIS. 1996b. Food Standards and Labeling Policy Book. Food Safety and Inspection Service, U.S. Department of Agriculture, Washington, D.C. Gaylor, D. W. and Gold, L. S. 1995. Quick estimate of the regulatory virtually safe dose based on the maximum tolerated dose for rodent bioassays. Regul. Toxicol. Pharmacol. 22:57–63. Generic HACCP for raw beef. 1993a. Food Microbiol. 10:449–488. Glass, G. E. 1973. Bioassay Techniques and Environmental Chemistry. Ann Arbor Science, Ann Arbor, MI. HACCP implementation: A generic model for chilled foods. 1993b. J. Food Protect. 56:1077–1084. Hawcroft, D., Hector, T., and Rowell, F. 1987. Quantitative Bioassay. John Wiley, New York. Hodgson, E. 1987. Measurement of toxicity. In Modern Toxicology, ed. E. Hodgson and P. E. Levi, pp. 233–285, Elsevier, New York. Hutt, P. B. and Sloan, A. E. 1979. Food Safety Report. Nutrition Policy Issues, 6. National Academy of Sciences, Washington, D.C. Jones, J. M. 1992. Food Safety. Eagan Press, St. Paul, MN. Kirkwood, T.B.L. 1977. Predicting the stability of biological standards and products. Biometrics 33:736–742. Klaassen, C. D. 1986. Principles of toxicology. In Toxicology: The Basic Science of Poisons, eds. C. D. Klaassen, M. O. Amdur, and J. Doull, pp. 11–32, Macmillan, New York. Leidy, R. B. and Hodgson, E. 1987. The measurement of toxicants. In Modern Toxicology, eds. E. Hodgson and P. E. Levi, pp. 287–307, Elsevier, New York.

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Loomis, T. A. 1978. Essentials of Toxicology, 3rd ed., Lea and Febiger, Philadelphia, PA. Mortimore, S. and Wallace, C. 1994. HACCP–a Practical Approach. Chapman and Hall, London. Mossel, D.A.A. 1983. Seventy-five years of longitudinally integrated microbiological safety assurance in the dairy industry in The Netherlands. Neth. Milk Dairy J. 37:240–245. Mossel, D.A.A. 1989. Adequate protection of the public against food-transmitted diseases of microbial etiology. Achievements and challenges, half a century after the introduction of the Prescott-Meyer-Wilson strategy. Int. J. Food Microbiol. 9:271–294. Munro, I. C. and Krewski, D. R. 1983. Regulatory considerations in risk management. In Carcinogens and Mutagens in the Environment. Vol. II. Naturally Occurring Compounds, ed. H. F. Stich, pp. 156–166, CRC Press, Boca Raton, FL. Murray, R. E. and Gibson, J. E. 1972. A comparative study of paraquat intoxication in rats, guinea pigs, and monkeys. Exp. Mol. Pathol. 17:317–325. NACMCF. 1992. Hazard analysis and critical control point system. Int. J. Food Microbiol. 16:1–23. Nakamura, R. M., Kasahara, Y., and Rechnitz, G. A. 1992. Immunochemical Assays and Biosensor Technology for the 1990s. American Society of Microbiology, Washington, D.C. NIOSH. 1983. Registry of Toxic Effects of Chemical Substances, Vols. 1–3, National Institute for Occupational Safety and Health, U.S. Government Printing Office, Washington, D.C. Noordhuizen, J.P.T.M. and Welpelo, H. J. 1996. Sustainable improvement of animal health care by systematic quality risk management according to the HACCP concept. Vet. Q. 18:121–126. NRC. 1985. Meat and Poultry Inspection: The Scientific Basis of the Nation’s Program. National Research Council, National Academy Press, Washington, D.C. NRC. 1998. Ensuring Safe Food: From Production to Consumption. Institute of Medicine, National Research Council, National Academy Press, Washington, D.C. Odell, W. D. 1983. Principles of in vitro bioassays. In Principles of Competitive Protein Binding Assays, ed. W. D. Odell and P. Franchimont, pp. 267–279, John Wiley, New York. OECD. 1981. Guidelines for Testing of Chemicals. Organization for Economic Cooperation and Development, Paris. OMB. 1998. Budget of the United States, Fiscal Year 1999. Office of Management and Budget, Washington, D.C. Park, C. B. 1981. Attributable risk for recurrent events. Am. J. Epidemiol. 113:491–493. Pickering, W. F. 1977. Pollution Evaluation: The Quantitative Aspects. Marcel Dekker, New York. Price, C. P. and Newman, D. J. 1996. Principles and Practice of Immunoassay, 2nd ed. Stockton Press, New York. Purchase, I.F.H. 1994. Current knowledge of mechanisms of carcinogenicity: genotoxins versus nongenotoxins. Hum. Exp. Toxicol. 13:17–28.

Rand, G. M. and Petrocelli, S. N. 1985. Fundamentals of Aquatic Toxicology. Hemisphere, Washington, D.C. Rawson, J. M. and Vogt, D. U. 1998. Food Safety Agencies and Authorities: A Primer. Library of Congress, Congressional Research Service, Washington, D.C. Renwick, A. G. 1993. Data-derived safety factors for the evaluation of food additives and environmental contaminants. Food Addit. Contam. 10:275–305. Renwick, A. G. 1999. Exposure estimation, toxicological requirements and risk assessment. In International Food Safety Handbook, ed. K. van der Heijden, M. Younes, L. Fishbein, and S. Miller, pp. 59–94, Marcel Dekker, New York. Rubery, E. D., Barlow, S. M., and Steadman, J. H. 1990. Criteria for setting quantitative estimates of acceptable intakes of chemicals in food in the UK. Food Addit. Contam. 7:287–302. Rulis, A. M. 1987. Safety assurance margins for food additives currently in use. Regul. Toxicol. Pharmacol. 7:160–168. Scott, V. N. 1993. Implementation of HACCP in a food processing plant. J. Food Protect. 56:548–554. Seniell, H. J. 1995. Control of foodborne infections and intoxications. Int. J. Food Microbiol. 25:209–217. Shapton, D. A. and Shapton, N. E. 1991. Principles and Practices for the Safe Processing of Foods. Butterworth-Heinemann, Oxford. Silliker, J. H. 1987. Principles and applications of the HACCP approach for the food processing industry. In Food Protection Technology, ed. C. W. Felix, pp. 137–153, Lewis Publishers, Chelsea. Snyder, O. P. 1995. HACCP-TQM for retail and food service operations. HACCP. In Meat, Poultry and Fish Processing, Vol. 10. Advances in Meat Research, ed. A. M. Pearson

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and T. R. Dutson, pp. 156–189, Blackie Academic and Professional, London. Thoma, J. J., Bond, P. B., and Sunshine, I. 1977. Guideline for Analytical Toxicology Programs, Vols. 1 and 2, CRC Press, Cleveland, OH. Tijssen, P. 1985. Practice and Theory of Enzyme Immunoassays. Elsevier, Amsterdam. Trevan, J. W. 1927. The error of determination of toxicity. Proc. R. Soc. Lond. B 101:483–514. Van Leeuwen, F.X.R. 1997. Setting toxicological standards for food safety. In Food Safety and Toxicity, ed. J. de Vries, pp. 255–266, CRC Press, Boca Raton, FL. Webb, N. B. and Marsden, J. L. 1995. Relationship of the HACCP system to Total Quality Management. In Meat, Poultry and Fish Processing, eds. A. M. Pearson and T. R. Dutson, pp. 122–139, Blackie Academic and Professional, London. Weisburger, J. H. and Williams, G. M. 1981. Carcinogen testing: Current Problems and new approaches. Science 214: 401–407. WHO. 1994. Assessing Human Health Risks of Chemicals: Derivation of Guidance Values for Health Based Exposure Limits. Environmental Health Criteria 170. World Health Organization, Geneva. Wild, D. 1994. The Immunoassay Handbook. Stockton Press, New York. Williams, B. W. and Wilson, K. 1975. Principles and Techniques of Practical Biochemistry. Edward Arnold, London. Wilson, J. G. and Fraser, F. C. 1977. Handbook of Teratology, Vols. 1 and 2, Plenum Press, New York. Yermakoff, J. K. 1987. Toxicologic units. In Toxic Substances and Human Risk. Principles of Data Interpretation, eds. R. G. Tardiff and J. V. Rodricks, pp. 13–27, Plenum Press, New York.

7 Dietary Constituents

7.1

INTRODUCTION

Essential nutrients are organic and inorganic substances required to sustain life. They are used for growth, maintenance, tissue repair, and reproduction, and foods are the vehicles for them. Different organisms require different nutrients for their existence. To sustain life and maintain health, dietary intakes of nutrients must meet certain minimal levels. An individual food may contain only a few nutrients, or it may supply many. However, no single food provides all the nutrients in amounts and proportions necessary for adequate health. If the total diet supplies all the essential nutrients, the cells and body organs can synthesize many thousands of additional metabolically important substances. The cell cannot use foods in their native state. They must first undergo digestion within the intestinal tract. The digestion process results in release of nutrients, which are then transported across the mucosal wall of the intestine. Ultimately, the nutrients enter the bloodstream and are transported to tissues, where they are utilized for various physiological and metabolic functions. To prevent the accumulation of nutrients and/or their metabolites to toxic levels in organs and body fluids, the phases of absorption, utilization, and catabolism are integrated at the wholebody level. Intakes in excess of cellular needs are excreted or enter catabolic pathways to be removed as breakdown products via routes such as urine, feces, bile, and sweat (Figure 7.1). The science of nutrition, however, is not just the science of food and its relation to life and health. We must not only be concerned with what is required in a diet, but

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with what is actually consumed. We should also be equally concerned with the problems caused by an excess of a food component as we are with the problems caused by the deficiency of an essential nutrient. In considering the physiological effects of food components, it should be noted that these effects are always related to the level of their intake. A useful concept is that for every food component, there are three ranges of intake: one associated with physiological inertness, a second with physiological function or benefit, and the third with potential hazard. Although it is arguable at what level a nutrient is physiologically inert, there is no doubt that certain levels of intake are insufficient to maintain normal body functions. The level of nutrient requirement associated with normal health, i.e., with physiological function and benefit, is well understood for most nutrients. We also know with certainty that the concept of “zero risk” cannot be considered valid anymore. One would probably not consider our food sources of energy as ever constituting a potential hazard, but there is a general consensus among nutritionists that currently the most important problem of malnutrition in the United States is obesity (Deshpande and Sathe, 1991; Bray, 1996). This example clearly shows that the margin between the level of caloric intake consistent with normal physiological function and benefit and that creating a potential hazard is narrow. Thus, for every nutrient, there is also a level of intake that contributes a potential hazard. The margin between the level of function and the level of hazard varies considerably with each and must be determined in each case. That the basic components (i.e., carbohydrates, protein, fat, minerals, and vitamins) of human diet under nor-

Figure 7.1

Schematic representation of the major stages in the utilization of nutrients.

mal conditions do not exert any adverse effects is taken for granted. However, nutrient intakes that sufficiently exceed requirements can lead to a variety of toxic effects, including death. This potential for adverse effects from both deficiencies and excesses makes nutrients very different from other chemicals in foods and underlines the importance of nutritional hazards in food safety and toxicology. Very high nutrient intakes may exceed the metabolic capacity of the organism, causing pathological effects and a deterioration of health. This state occurs relatively frequently in certain diseases, such as maple syrup urine disease (branched-chain ketoacidemia) and phenylketonuria (PKU), in which infants cannot adequately dispose of the branched-chain amino acids (leucine, isoleucine, and valine) and phenylalanine, respectively. The free amino acids rise to high levels in blood and tissues, including the brain, and if these elevated levels are maintained, mental retardation may occur. The treatment of these conditions includes diets supplying low levels of the “overabundant” nutrients (Scriver et al., 1995; Reeds and Beckett, 1996). Another example of the accumulation to toxic levels of an essential nutrient is hypervitaminosis A, due to excessive dietary intake of vitamin A. This condition is characterized by a drying and desquamation of the skin, headaches, loss of hair, and bone and joint pain (Olson, 1996). In contrast, if the intake of a nutrient is inadequate to meet the normal needs of the body, metabolic responses occur within the cells and organs to conserve their limited supply. These changes include a more effective absorption of nutrients from the intestine and/or the activation of bio-

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Table 7.1 Factors Affecting Nutrient Requirements Dietary factors Chemical form of nutrient Presence or level of other dietary constituents Food processing Bioavailability Host factors Age Sex Genetic Physiological states Growth Pregnancy Lactation Aging Pathological states Metabolic disease(s) Trauma Neoplasia Other stress and drugs Environmental factors Physical Temperature Altitude Biological Infectious agents Social Dietary habits Environmental sanitation Personal hygiene Physical activity

chemical mechanisms to improve retention and utilization of the nutrient by body tissues. Such is usually the case in iron deficiency. However, when utilization and metabolic control of nutrients are altered by genetic disease, infection, or administration of drugs, the requirements for nutrients are also affected. Unless the dietary supply is adequate to compensate for these effects, health deteriorates. Globally, several factors affect the nutrient requirements of individuals and populations (Table 7.1). A fundamental component of variation in requirements is that introduced by genetic differences. The inborn errors of metabolism, for example, are extreme examples of the nutritional implications of genetic variation in humans. Factors affecting nutrient requirements listed in Table 7.1 are too numerous to discuss extensively here; a detailed review of some 40–50 essential dietary constituents is also beyond the scope of this discussion. Therefore, in this chapter, only those potential toxic effects of normal dietary constituents that occur under unusual dietary conditions are described.

7.2

WATER

Water is a macronutrient that is more critical to the maintenance of life than food. Although humans can live without food for weeks and even months, without water death occurs within days. No other substance is as widely involved in as many diverse functions of the human body as water, which serves as a mechanism to transport nutrients and waste products between the body tissues and organs. Moist surfaces in lungs permit diffusion of oxygen and carbon dioxide between the inspired air and the capillaries in alveoli. Water dissolves waste products in urine and feces and serves as a vehicle for their excretion. It also lubricates and gives structural support to tissues and joints. The most important function of water, however, is in thermoregulation. The body generates a considerable heat burden through metabolism and muscular contraction. Water helps dissipate this load by absorbing large quantities of heat while undergoing only small changes in the temperature. By weight, water constitutes ~60% of the human male body and 50%–55% of the female body, which has a higher proportion of fat. The water content of various organs ranges from 83% in blood to only about 10% in the adipose tissue (Table 7.2). Water is distributed throughout the body but is found primarily in two compartments, one within the cells and one between cells (Table 7.3). The largest part of water in the body (~62%) is inside the cells. The extracellular water accounts for ~30% of the total body water and includes

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Table 7.2 Water Composition of Tissues and Organs by Weight Tissue

% Water

Blood Kidneys Heart Lungs Spleen Muscle Brain Intestine Skin Liver Skeleton (bone) Adipose tissue

83.0 82.7 79.2 79.0 75.8 75.6 74.8 74.5 72.0 68.3 22.0 10.0

Source: Compiled from Pivarnik and Palmer (1994) and Askew (1996).

fluid located between tissues (interstitial) and in blood plasma (~7% of the total). The primary source of daily water intake in humans is fluid consumption. The fluid content of food also contributes greatly to daily water balance. The combined water intake through fluids and foods consumed at meals is the normal route for maintaining fluid balance (Rothstein et al., 1947). The most variable and quantitatively most important routes of water loss in humans are the sweat glands and the kidneys. The volume of water lost through sweat depends upon several factors, including exercise, workload, temperature, relative humidity, hydration status, and the degree of prior heat acclimation (Sawka and Pandolf, 1990; Sawka, 1992; Askew, 1996). The total sweat loss is usually 500–700 mL/day but can be as much as 8–12 L/day (Katch and McArdle, 1993). The kidney has the ability to regulate water loss in the urine by increasing the tubular

Table 7.3 Distribution of Body Water Among Compartments Compartment Body weight (kg) Total body water (L) Intracellular Extracellular Interstitial Plasma Transcellular

Male 70 42 26 13 10 3 3

Female 55 28 17 9 6.5 2.5 2

Source: Compiled from Pivarnik and Palmer (1994) and Askew (1996).

resorption of water. Water conservation by the kidney is an important homeostatic mechanism. Although feces contain ~70% water, fecal excretion of water in the absence of diarrhea is relatively small because of the efficient resorption of water from the digested matter in the jejunum and colon. Diarrhea or vomiting can increase the normal daily water loss from 100 mL/day to 10–50 times that amount (McArdle et al., 1991). Body heat is continually transferred to the environment as water vaporizes from lung and skin surfaces. For each liter of sweat or respiratory water that the human body vaporizes, it dissipates ~2427 kJ (580 kcal) of heat. An adequate water intake is critical to sustained exercise performance, in which large quantities of heat are generated and large quantities of oxygen must be delivered to the working muscle. Hypohydration increases plasma tonicity by decreasing the plasma volume and raises the body core temperature. It also makes the cardiovascular system work harder, impairs thermoregulation, and decreases physical performance (Askew, 1996). Thermoregulation is compromised in cold as well as in heat. Fluid intake thus becomes even more important during work in extreme heat, cold, or high altitude, in which heat strain, sweating, and respiratory water losses are increased. Environmental factors acting in concert with voluntary or involuntary dehydration can result in severe hypohydration.

7.3

CARBOHYDRATES

Carbohydrates are the most widely distributed and abundant organic compounds on Earth. They have a central role in the metabolisms of animals and plants. Carbohydrate biosynthesis by green plants from carbon dioxide and water in the presence of light energy, i.e., photosynthesis, supports the existence of all other organisms. Carbohydrates are a basic food, accounting for about 50%–70% of the total caloric intake. Depending upon cultural and dietary choices, the composition of indigenous dietary carbohydrate can vary, but it generally includes starch, simple sugars, complex polymers such as dietary fiber, and minor components. In human nutrition, the first two are the major sources of energy. The dietary fiber, although not used for energy because it is not hydrolyzed by digestive enzymes, nonetheless is of importance to balanced daily nutrition. A number of other carbohydrates are also added to processed foods in quantity. These include hydrolyzed cornstarch, fructose syrups made from cornstarch, modified starches, gums, mucilages, and sugar alcohols, which are added to change texture, mouth feel, shelf life, color, viscosity, and taste.

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It is generally believed that human beings have no short-term requirement for carbohydrate in food. One cannot demonstrate deficiency symptoms by feeding a carbohydrate-free diet. However, disturbances in metabolism and intestinal function can appear. And in the long run, the lack of dietary fiber and its beneficial effects can lead to serious health risks such as diverticulitis in the aged and increased risks of certain types of cancer. 7.3.1 Inborn Errors of Carbohydrate Metabolism Normally, carbohydrates do not contain any substances that can be considered toxic in healthy individuals. Nonetheless, in some inborn errors of carbohydrate metabolism, the consumption of various mono- and disaccharides leads to some serious and at times fatal consequences (Stanbury et al., 1978). Carbohydrate malabsorption is a failure to absorb a carbohydrate in the appropriate manner at the appropriate site. This can result from an enzyme or carrier (transporter) deficiency (primary deficiency) or a deficiency induced by a disease (secondary deficiency) (Szepesi, 1996). Galactosemia In galactosemia, which results from the deficiency of galactose-1-phosphate uridyl transferase, ingestion of galactose or any galactose-containing disaccharide leads to the production of galactitol and other toxic metabolites (Segal and Berry, 1995). Toxic manifestations include rapidly progressive hepatic dysfunction that leads to hepatic necrosis if galactose ingestion is not stopped, as well as development of cataracts and renal tubular dysfunction. The mainstay of treatment is lifetime avoidance of galactose ingestion, which prevents the development of the potentially fatal hepatic complications. However, galactosemic individuals detected by neonatal screening programs and treated with galactose restriction from the first week of life still exhibit largely unexplained late sequelae. These include specific expressive speech delays, subtle intellectual dysfunction, and premature ovarian failure in females (Rhead, 1996). Fructose Intolerance In hereditary fructose intolerance due to a deficiency of fructose aldolase B, the clinical presentation is generally more chronic than that in galactosemia and consists of failure to grow, mild hepatotoxicity, and proximal renal tubular dysfunction (Gitzelmann et al., 1995). As in galactosemia, in hereditary fructose intolerance, lifetime avoidance of fructose largely prevents the development of symptoms.

Congenital Disaccharide Intolerance Although they are rare, congenital disaccharide intolerances are the inherited disorders related to the metabolism of disaccharides. Adult-onset hypolactasia is probably the most common form of a disaccharidase deficiency and is most common among populations of non-European origin (Goda et al., 1985; Szepesi, 1996). In such people, lactase level declines after weaning, so that although children can consume milk, consumption of milk or even some partially fermented milk products by adults causes abdominal distress. In such instances, the presence of undigested lactose in the intestinal lumen disturbs the osmolality because of its ability to attract water from the tissue as well as its conversion to lactic acid by the intestinal microflora. The fermentation of lactose also results in gas formation and causes abdominal discomfort due to flatulence and diarrhea. Primary low lactase activity is one of the most commonly encountered inherited enzymatic dysfunctions, but it differs widely in healthy subjects of various racial groups who live under similar conditions. Secondary low lactase activity is associated with certain diseases of the small intestine. These include infectious disorders, celiac disease, and tropical malabsorption, including sprue and protein-calorie malnutrition. In the United States and Europe, the food industry has solved this problem by marketing lactase-treated milk. Glycogen Storage and Metabolism Other inborn errors of carbohydrate metabolism are related to glycogen storage and metabolism diseases. The type I, or Gierke’s disease, which results from a deficiency of glucose-6-phosphatase, is the carbohydrate disorder with the most potent effects on hepatic and total body glycogen and glucose metabolism (Chen and Burchell, 1995). The primary biochemical consequence of this enzymatic deficiency is severe, chronic and unremitting hypoglycemia. This results in activation of pathways of lipolysis and, futilely, gluconeogenesis and glycogenolysis, either directly by biochemical or indirectly by endocrinological mechanisms. The functional result is an influx of carbon moieties to the liver, leading to the overproduction of acetyl-CoA and other compounds, such as lactic acid, cholesterol, triacylglycerol, and uric acid. In glycogen storage disease type III, which is due to a deficiency of the amylo-1,6-glucosidase debrancher enzyme, hepatic glucose production from glycogen is largely interrupted, although gluconeogenesis from other carbon sources remains intact. In early infancy, the clinical presentation of these individuals—failure to thrive, hepatomegaly, and hypoglycemia—is similar to but often less severe than that of individuals with glycogen storage disease type

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I. In later childhood and adolescence, these symptoms generally spontaneously resolve and clinical status improves (Rhead, 1996). However, later in adult life, in affected individuals a debilitating myopathy, presumably due, at least in part, to increased muscle protein turnover during fasting and sleep, may develop. The resultant amino acid flux is utilized to maintain blood glucose levels via gluconeogenesis. 7.3.2 Diabetes Mellitus Diabetes mellitus is yet another disease that involves an elevated blood glucose concentration. However, it is not a single disease but rather a group of disorders of varying causes and pathogenesis. Because of its acute and longterm complications, diabetes is a major cause of morbidity and mortality. It is increasing in prevalence in many populations around the world. In the United States alone, about 8%–10% of the total population are estimated to be diabetic (Horton and Napoli, 1996). Diabetes mellitus is currently classified into four clinically different types: insulin-dependent diabetes mellitus (IDDM or type 1), non-insulin-dependent (NIDDM or type 2), gestational diabetes mellitus (GDM), and diabetes secondary to or associated with other diseases that damage the pancreas or produce severe insulin resistance. Diabetes can be diagnosed when random plasma glucose concentration is >11.1 mmol/L (>200 mg/dL) and the patient has the classic signs and symptoms. The overall goals of therapy for diabetes mellitus are to achieve normal or near-normal carbohydrate, lipid, and protein metabolism; to prevent acute complications such as severe hypoglycemia, hyperglycemia, or ketoacidosis; and to prevent long-term complications of diabetes including microvascular disease, which damages the eyes and kidneys; neuropathy; and macrovascular disease, which leads to cardiac, cerebral, and peripheral vascular insufficiency. There is now overwhelming evidence that good glycemic control plays a major role in reducing complications due to diabetes. The major goal of nutritional therapy is to achieve and maintain blood glucose concentrations as close to normal as possible. However, there is no one “diabetic diet” that is suitable for every diabetic person. Dietary recommendations should take into account requirements for normal growth and development and general health, as well as treatment of the metabolic abnormalities associated with diabetes and its complications. The readers are referred to several excellent reviews and books for a comprehensive treatment of this subject (Davidson, 1999; Leroith, 2000; Pickup and Williams, 1994; Taylor, 1999; Gulledge and Beard,

1999; Davidson and Davidson, 1998; Hitman, 1999; Ekoe et al., 2000). 7.3.3 Dental Caries Dental caries is a chronic infectious disease of the teeth involving demineralization of the tooth surface over time by organic acids produced from bacterial fermentation of carbohydrate deposits. The development of dental caries requires the interaction of four principal factors: host (susceptible teeth and oral environment conducive to caries formation), agent (presence of viable cariogenic microorganisms), environment (diet conducive to caries, deficient fluoride exposure, and oral hygiene), and time (sufficient duration of conducive environment). Dental plaque represents an organized large mass of oral bacteria capable of hydrolyzing starches and metabolizing sugars to lactic acid. The latter is responsible for intermittent demineralization of tooth enamel and dentine. The greater the fall in pH and the more prolonged that fall is, the more severe is the process of demineralization. It is, therefore, not surprising that frequent intakes of readily fermentable carbohydrates, particularly sucrose in confectionery, which has a long mouth retention time, are associated with a markedly increased risk of dental caries (Gustaffson et al., 1954; Rugg-Gunn et al., 1984; Burt et al., 1988; Lachapelle et al., 1990; Konig and Novia, 1995; Gibney, 1999). The polyols or sugar alcohols of simple carbohydrates, produced by the hydrogenation of the corresponding reducing sugar, produce fewer dental caries than caloric sweeteners such as glucose and sucrose (Birkhed et al., 1985; Finley and Leveille, 1996). Xylitol and sorbose are more resistant to fermentation by oral microflora and produce less plaque than does glucose. Maltitol, platinate, platinose, sorbitol, and lycosin are fermented by Actinomyces, Lactobacillus, and Streptococcus spp. Although more acid is produced, these sugars seem to have no cariogenic effect with S. mutans. Fermentation-related changes in standard dental tests with sugar alcohols, glucose, and sucrose are summarized in Table 7.4. These data show that glucose and sucrose have the greatest potential for producing adverse effects leading to dental caries, whereas lactitol and xylitol exhibited the lowest potential for adverse effects. Oral hygiene, which removes the microbial mass of plaque, and fluoride exposure, through fluoridated water or fluoridated dentrifices, can significantly lower the incidence of dental caries in the presence of usual sugar intakes. Fluoride dramatically increases the natural re-

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Table 7.4 Order of Fermentation-Related Changes in Standard Dental Tests After 24 Hours with Various Carbohydrates Decrease in pH Glucose > sucrose > mannitol > sorbitol > xylitol > lactitol Tritratable acidity Glucose > sucrose > mannitol > sorbitol > lactitol > xylitol Polysaccharide formation Glucose > sucrose > mannitol > sorbitol > lactitol > xylitol Microbial growth Glucose > sucrose > sorbitol > mannitol > lactitol > xylitol Calcium dissolving Sucrose > glucose > mannitol > sorbitol > lactitol > xylitol Phosphorus dissolving Sucrose > glucose > mannitol > sorbitol > lactitol > xylitol Source: Compiled from Grenby and Phillips (1989) and Finley and Leveille (1996).

mineralization process that takes place on the normalization of oral pH. The sugar–dental caries relationship continues to be a problem in the developed world, where social, physical, or mental handicap reduces good oral hygiene. It will also be a continuing problem in developing countries where oral hygiene is poor and water is often not fluoridated (Gibney, 1999). 7.3.4 Raffinose-Family Oligosaccharides Among the various dietary sugars, oligosaccharides of the raffinose family have a more definite antinutritive effect. These sugars (raffinose, stachyose, and verbascose) occur in appreciable amounts in mature legume seeds and comprise 30% to 80% of the total soluble sugars (Deshpande and Damodaran, 1990; Deshpande and Sathe, 1991). They contain α-galactosidoglucose and α-galactosidogalactose bonds (Figure 7.2). The human gastrointestinal (GI) tract does not possess α-galactosidase activity; the mammalian invertase is a an α-glucosidase. Also, the raffinose family sugars are unable to pass through the intestinal mucosal wall. Consequently, the microflora in the lower intestinal tract metabolize these sugars to produce flatus. The accumulation of flatus in the intestinal tract results in discomfort, abdominal rumblings, cramps, pain, and diarrhea and is characterized by the production of hydrogen, carbon dioxide, and small amounts of methane gas. Sugars of the raffinose family are believed to be largely responsible for the often-reported problem of flatulence after consumption of diets containing beans and other legumes.

Figure 7.2

7.4

Structural relationships of the raffinose family oligosaccharides.

PROTEINS AND AMINO ACIDS

Amino acids, peptides, and proteins are important constituents of food. They supply the required building blocks for protein biosynthesis. In addition, they directly contribute to the flavor of food and are precursors for aroma compounds and colors formed during thermal or enzymatic reactions in production, processing, and storage of food. Proteins also contribute significantly to the physical properties of food through their ability to build or stabilize gels, foams, emulsions, and fibrillar structures. Proteins form the major cellular structural elements, are biochemical catalysts, and are important regulators of gene expression. Therefore, any discussion of protein and amino acid nutrition necessarily involves virtually every element of mammalian biochemical and physiological processes.

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Proteins and amino acids do impose some adverse and potentially harmful effects on the organism. In addition to certain proteins that are toxic or function as enzyme inhibitors, amino acid toxicity, amino acid imbalance, and amino acid antagonism are examples of some such problems. 7.4.1 Amino Acid Toxicity Since amino acids are integral components of all the dietary proteins, it is questionable whether they should be classified as naturally occurring toxicants. However, a few proteins do contain excessive amounts of some amino acids. In addition, studies have shown that increased intake of some amino acids may lead to some disturbances (Harper et al., 1970; Harper, 1994; Scriver et al., 1995; Rhead, 1996). Both the age of the subject and the ade-

quacy of the diet with respect to protein, caloric intake, vitamins, as well as the relative proportion of amino acids in the diet influence the individual’s susceptibility to the amino acid load. Interestingly, the essential amino acids seem to be less well tolerated in excessive amounts than the nonessential ones; methionine is the least well tolerated of the nutritionally important amino acids (Harper, 1973). Certain amino acids have deleterious effects on the organism when they are present in overabundance. Such effects can only be counteracted by the reduction of dietary intake. The inborn errors of amino acid metabolism present classic examples of the successful treatment and prevention of medical complications through the use of nutritional therapies. Amino acid toxicity is analogous to the toxic effects on the cells of an inherited amino acidopathy with an accumulation of the amino acids. A well-known example of this kind is the inborn error of metabolism PKU, which is due to either a deficiency of phenylalanine hydroxylase or, more rarely, a deficiency of the enzymes synthesizing or recycling the reduced tetrahydrobiopterin cofactor essential to the function of this enzyme (Scriver et al., 1995). Since the 1950s, PKU has been successfully treated with diets containing all the essential macro- and micronutrients but low in or free of phenylalanine. Phenylalanine levels are regulated by adding normal protein foods to the diet in amounts that permit normal growth and development but prevent its accumulation in cells and tissues (Rhead, 1996). Dietary restriction of phenylalanine has been highly successful in preventing the severe progressive mental retardation found in individuals with PKU. Another example of amino acid toxicity is the maple syrup urine disease or branched-chain ketoacidemia. which results from a deficiency of the branched-chain ketoacid dehydrogenase involved in the catabolism of all three branched-chain amino acids, viz., leucine, isoleucine, and valine (Chuang and Shih, 1995). However, because metabolic imbalance and accumulation of branched ketoacid intermediates produce generalized metabolic acidosis and life-threatening episodic crises, in contrast with PKU, in which toxicity is largely confined to the central nervous system, therapeutic approaches in branched-chain ketoacidemia have been less successful in preventing longterm medical and neurological sequelae than in PKU (Rhead, 1996). Providing adequate energy intake as carbohydrate and fat helps to prevent tissue protein catabolism, which occurs during fasting and illness. Such dietary intervention ameliorates chronic acidosis and reverses metabolic crises in these disorders involving amino acid catabolic pathways. Similarly, propionic and methylma-

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lonic acidemias, when not responsive to vitamins and cofactors, are treated with diets low in valine, isoleucine, threonine, and methionine—precursors to both methylmalonyl CoA and propionyl CoA (Fenton and Rosenberg, 1995a, 1995b). Leucine restriction in isovaleric acidemia due to isovaleryl CoA dehydrogenase deficiency decreases the frequency and severity of metabolic decompensations and clinical crises (Sweetman and Williams, 1995). Although humans can tolerate large intakes of protein and amino acids in a well-balanced pattern, excessive consumption sometimes leads to liver and kidney hypertrophy. This finding suggests that an increase in the metabolic load of these nutrients increases the demand for metabolic capacity and regulatory mechanisms. Glutamate, whose sodium salt is commonly used as a flavor enhancer in meat products, is associated with the socalled Chinese restaurant syndrome (Kwok, 1968). Higher than normal blood levels of glutamate can result in a feeling of weakness, palpitation, and numbness of the neck and back, and its symptoms may last up to 2 hr. 7.4.2 Amino Acid Imbalance Amino acid imbalance is a concept first discussed by Harper in 1964 to describe the change in an amino acid requirement as a consequence of an excess of another amino acid. In such situations, an excess of an amino acid may lead to a relative deficiency of another amino acid, especially when it is the limiting amino acid. Studies in experimental animals have shown that after only a few hours on an imbalanced diet, they reduce their food intake and the plasma concentration of the limiting amino acid decreases (Harper et al., 1970). A metabolic imbalance appears with a subsequent loss of muscular mass and a rise in the liver content of protein, glycogen, and fat. Gopalan and Rao (1975) even postulated the imbalance between isoleucine and leucine as an etiological factor in the pathogenesis of endemic pellagra in populations who consume corn and sorghum staple diets. This seems to be the first report of an important nutritional defect in humans caused by an amino acid imbalance. 7.4.3 Amino Acid Antagonism The concept of amino acid antagonism is closely linked to amino acid imbalance. It is related to a competition between structurally similar amino acids that share a common step in metabolism or transport. The problem is easily corrected by the supplementation of the amino acids. Amino acid antagonism has been described for branched-

chain amino acids and between lysine and arginine (Harper et al., 1970). Amino acid antagonism has been linked to several disorders involving transport defects. Cystinuria and Hartnup disease as well as several inborn errors of amino acid metabolism have been linked to this phenomenon. In Hartnup disease, decreased gastrointestinal absorption and increased urinary excretion of α-monoamino-monocarboxylic amino acids lead to systemic deficiency of several essential amino acids, notably tryptophan, a precursor for nicotinamide synthesis (Levy, 1995). In this disorder, therefore, intakes of essential amino acids must be raised to permit positive nitrogen balance, and nicotinamide supplements must be provided. However, with the exception of high-leucine–low-isoleucine corn diets in certain endemic populations, there are few indications that individual amino acids are available in foodstuffs in amounts large enough to cause any adverse effects due to amino acid imbalance and antagonism. 7.4.4 Gluten Enteropathy Gluten enteropathy, also called celiac sprue, is a syndrome of small bowel mucosal injury initiated by gluten, a protein found in certain cereal grains, including wheat, barley, rye, and oats. This disorder classically has its onset in early childhood, soon after wheat gluten is introduced into the diet, but the presentation of symptoms may be delayed even until old age (Langman et al., 1985). Available evidence suggests that the reaction to gluten is a cellmediated immune response. Direct contact between the mucosa and the gluten is required. This interaction results in an inflammatory response in the mucosa that may evolve quickly over hours or, more commonly, over many months (Kumar et al., 1979). Clinical manifestations include diarrhea, weight loss, vomiting, flatulence, abdominal distention, and weakness. The only treatment of gluten enteropathy is permanent withdrawal from the diet of all foods and drugs containing even minute quantities of gluten.

7.5

DIETARY FAT AND FATTY ACIDS

Dietary fat consists mainly of a heterogeneous mixture of triacylglycerols (triglycerides) and makes up a substantial but variable portion of total energy intake. In many European countries, fat accounts for 40%–50% of the total energy in the diet (Grundy, 1996). In the United States, fat intakes are somewhat lower, ranging between 30% and

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40% of total energy; in other populations, particularly in Asia and Africa, fat provides only 15%–25% of energy. A widely held belief is that excess dietary fat intake contributes importantly to several chronic diseases, such as coronary heart disease (CHD), stroke, diabetes mellitus, cancer, and obesity. This belief has fostered the recommendation that dietary fat intake should be kept relatively low, that is, 30% of total energy (National Cholesterol Education Program, 1991). The overall issue of dietary fat, however, is not simple. It is a major nutrient and an important source of body fuel. Furthermore, fat consists of a complex mixture of triacylglycerol molecules that can differ greatly from one another in their chemical and physical properties. Consequently, data related to the different kinds of triacylglycerols and their constituent fatty acids must be taken into account before general dietary recommendations are made. Dietary fatty acids differ considerably in their carbon chain length and the number of double bonds between carbon atoms. Triacylglycerols are composed of three molecules of fatty acids, which may be saturated, unsaturated, or polyunsaturated, esterified with one molecule of glycerol. Dietary triacylglycerols are derived either unmodified from natural sources (animal and plant fats) or modified industrially for special purposes in foods. Through various combinations of different kinds of fatty acids, myriad different species of triacylglycerols occur in nature and are consumed in the diet. Unsaturated fats (e.g., those in many vegetable oils) are normally liquid at room temperature and chemically differ from saturated fats (e.g., most animal fats) by the presence of double bonds in their structure. This makes them more susceptible to oxidative breakdown both in foods and in the body. Dietary fats also include sterols and steroids, in particular cholesterol, found in animal fats but not in vegetable fats, and phospholipids. The fat in the diet is usually derived from foods that have long been used as energy sources. Consequently, most of them have been selected over time and have very few deleterious effects. Since the 1970s, however, several unconventional and new plant food sources have been identified and introduced in human diets as a source of fat energy, generating an increased potential risk for adverse effects. The antinutritional effects of dietary fats can be broadly classified into the following four categories: (a) inborn errors of fatty acid metabolism, (b) occurrence of certain toxic fatty acids, (c) potentially hazardous effect of increased fat intake, and (d) effect of an increased intake of polyunsaturated fatty acid (PUFA) per se or of the vitamin E requirement.

7.5.1 Inborn Errors of Lipid Metabolism Most inborn errors of metabolism are related to the fatty acid β-oxidation pathway. The enzymatic deficiencies producing defective mitochondrial β-oxidation have become more prominent since 1982, when medium-chain acylCoA dehydrogenase deficiency was first identified (Divry et al., 1983). β-Oxidation disorders have a broad clinical spectrum, with marked heterogeneity even within a single enzymatic deficiency (Rhead, 1991, 1996). For example, short-chain acyl-CoA dehydrogenase deficiency has been associated with either clinical normalcy or death in the neonatal period or with early onset of significant neuromuscular retardation. Similarly, palmityl carnitine transferase I deficiency produces a complete inability of the mitochondria to transport and β-oxidize long-chain (C16) fatty acids and leads to markedly impaired ketogenesis and hypoglycemia with prolonged fasting (Rhead, 1996). However, individuals who have this deficiency are clinically normal if they have not suffered sequelae due to severe hypoglycemia during an episode of fasting. In contrast, very-long-chain acyl-CoA dehydrogenase deficiency can result in significant neuromuscular and cardiac complications (Aoyama et al., 1995). The symptoms of most of these disorders related to fatty acid β-oxidation are hypoglycemia and reduced ketogenesis during fasting. Dietary therapy includes providing diets that are low in fat, preventing hypoglycemia by frequent feeding, and, more rarely, providing supplementation with carbohydrate or cornstarch. Smith-Lemli-Opitz syndrome is yet another example of a genetic disorder; it is characterized by microcephaly, characteristic facial features, severe psychomotor retardation, genital anomalies in males, and cutaneous syndactyly (Smith et al., 1964). This syndrome results from defective cholesterol synthesis due to a deficiency of the enzyme 7dehydrocholesterol reductase (Irons et al., 1994). This enzymatic block leads to the accumulation of 7-dehydrocholesterol and a systemic cholesterol deficiency. SmithLemli-Opitz syndrome is therefore an enzymatic deficiency that blocks an important anabolic, rather than catabolic, pathway. Adrenoleukodystrophy is a rare inborn error of fatty acid metabolism. It is a peroxisomal disorder inherited in an X-linked recessive fashion (Moser et al., 1995). The enzymatic deficiency involves activation and transport of very-long-chain fatty acids across the peroxisomal membrane, after which the fatty acid chain would normally be shortened by the peroxisomal β-oxidation system. Systemic accumulation of very-long-chain fatty acids affects both the central nervous system function and the adrenal

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function to varying degrees in different affected males, even in brothers in the same family. The early-onset (before the age of 10), rapidly progressive, and fatal disease is termed adrenoleukodystrophy, whereas the later-onset (adult), slowly progressive form is called adrenomyeloneuropathy. The provision of diets high in triacylglycerols containing erucic and oleic acids and low in long-chain fat prevents chain elongation of dietary long-chain (C16) fatty acids to very-long-chain (C22–C32) fatty acids. This therapy incorporates the interesting feature of using a normal nutrient in unusually high amounts to inhibit an anabolic synthetic pathway, the end products of which, if accumulated, are deleterious. 7.5.2 Toxic Fatty Acids Among the toxic fatty acids, erucic acid (C22:1, ω9) has received the greatest attention. It accounts for as much as 50% of the fatty acid content of the oil derived from some Brassica species, such as rapeseed and mustard. As early as 1960, Roine and his collaborators reported myocardial effects in rats fed 50% to 70% of their caloric intake as rapeseed oil. Subsequent studies have shown that even an intake of 4 energy percent in the form of erucic acid can lead to certain morphological defects (Engfeldt, 1975; Ziemlanski, 1977). The worldwide production and increasing use of rapeseed and mustard seed oils in margarine and the important role they play in the farming systems of certain countries, such as Canada and India and in northern Europe have called for intensive studies in these areas. Plant breeding efforts since the 1980s have resulted in the identification and development of low-erucic-acid-content rapeseed varieties. The mechanism for the development of the abnormalities shown in experimental animals on erucic acid diets is thought to be a blockage of the β-oxidation of the fatty acids in mitochondria (Engfeldt, 1975). In addition to erucic acid, several other fatty acids have been shown to be potentially harmful. Trans-isomers of fatty acids that occur in the depot fats of animals have been much discussed with regard to their role in the organism (Vergroesen, 1975). Trans unsaturated fatty acids are also produced through hydrogenation of polyunsaturated oils. Small amounts are also present in butter fat and are produced by hydrogenation of plant oils in the rumen of cattle (Craig-Schmidt, 1992; Sommerfield, 1983). Trans fatty acids are consumed from a variety of sources. About one-third of the American intake of trans fatty acids is from household shortenings, margarines, spreads, and dressings; another third is derived from fats and oils used in food products (snacks, cookies, crackers, bread, cake, potato chips, and french fries); the rest is from

food service fats and oils and meat and dairy products (Hunter and Applewhite, 1991). The actual intake of trans fatty acids varies considerably among individuals but on average is about 3% of total energy (Hunter and Applewhite, 1986, 1991). Trans fatty acids are implicated in the elevation of serum cholesterol levels and impaired functions of cell membranes in humans (Grundy, 1996; Gibney, 1999). Therefore, they play an important role in influencing the biological risk factors for coronary heart disease (CHD). Indeed, high intakes of trans fatty acids have been associated with an increased risk of CHD independent of all known diet, life-style, and biological risk factors in large epidemiological studies using both the prospective cohort and case-control approaches (Willett et al., 1992; Ascherio et al., 1994). In addition to erucic acid and trans fatty acids, there are a number of uncommon fatty acids with marked physiological effects even at very low concentrations. These include the cyclopropene fatty acids in cottonseed oil (Bailey et al., 1966), epoxy oils in soybean and sunflower seed oils (Sen Gupta, 1972), and furanoid fatty acids found in some species of edible fish (Glass et al., 1975). 7.5.3 Increased Fat Intake High intakes of dietary fats have been implicated as an important causative factor in cardiovascular disease. There is also a common belief that high-fat diets promote the development of obesity and its complications and that they raise serum cholesterol levels. Indeed, 1996 epidemiological data indicate that overweight is more likely when the diet is high in fat (Bray, 1996). However, the role of percentage fat intake in the causation of obesity remains uncertain. In contrast, certain fatty acids in the dietary fat mix definitely raise serum cholesterol levels. The cholesterol-raising fatty acids include three saturated fatty acids (lauric, myristic, and palmitic) and the trans fatty acids (Keys et al., 1965; Hegsted et al., 1965; Zock et al., 1994; Grundy, 1996). These saturated fatty acids are relatively high in two tropical oils, coconut oil and palm kernel oil. Butterfat also is high in myristic acid. The recommendation to reduce intakes of cholesterol-raising fatty acids seems prudent. Serum cholesterol levels will generally fall, as will risk of cardiovascular disease. Unsaturated fatty acids, whether monounsaturated or polyunsaturated, in contrast, do not raise cholesterol levels. However, it should be noted that several other risk factors are causally associated with heart disease, including genetic predisposition, obesity, sex, inactivity, smoking, hy-

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pertension, diabetes, stress, and diet (Ziegler and Filer, 1996; Gibney, 1999). An extensive review of the effect of altered dietary fat composition involving 33 studies in the 1980s (Gibney, 1992) reveals the following basic principles: 1.

2.

3.

Low-fat diets inevitably lower low-density lipoprotein (LDL) cholesterol but also appear to raise plasma triglyceride level and lower plasma high-density lipoprotein (HDL) cholesterol level. Increased consumption of polyunsaturated fatty acid (PUFA) at the expense of saturated fatty acid (SFA) lowers LDL cholesterol level but may also lower HDL cholesterol level if the PUFA/SFA ratio exceeds 2.0. Increased intakes of monounsaturated fatty acids at the expense of SFA lower LDL cholesterol level with no effect on HDL cholesterol level.

These and several other studies indicate not only that saturated fatty acids remain a major determinant of total and LDL cholesterol level, but also that the modifications to diet required to achieve a given reduction in these lipids depend on age and on the starting value of serum cholesterol level. 7.5.4 Increased Intake of Polyunsaturated Fatty Acid The fat content and composition in the human diet have been much discussed during the last four decades, as have the potential hazardous effects of saturated fatty acids in causing cardiovascular diseases. This focus has led to the recommendation of an increased intake of PUFA. Nevertheless, an increased intake of PUFA also leads to some potentially toxic problems (Truswell, 1978; Grundy, 1996). It requires an increased intake of vitamin E; fortunately, the vegetable oils that are usually recommended as sources of PUFA are also rich sources of this vitamin. However, it should be noted that no human population has customarily consumed diets high in PUFA (Keys, 1970). Higher intakes of PUFA may also promote obesity (Grundy, 1996). Furthermore, prolonged storage of oils rich in PUFA leads to the formation of hydroperoxides in the presence of oxygen. Of greater interest are the autooxidation and formation of cyclic monomers during the heat treatment of polyunsaturated fat in deep fat frying operations. When consumed alone, these compounds have been shown to be quite toxic (Billek, 1973). The possible role of dietary fat as an etiological factor in colon carcinogenesis has also been discussed (Reddy et al., 1976).

7.6

MINERALS

Minerals are the constituents that remain as ash after the incineration of plant and animal tissues. They appear in foods in a variety of forms. Their chemical nature may have considerable nutritional importance, since it determines whether the nutrient is made available to the body during the process of digestion and absorption. Minerals may be divided into two categories: the main or macroinorganic elements (Na, K, Cl, Ca, Mg, and P) are present in the body in significant quantities; their combined mass is about 3 kg in adult human. The trace or microelements (Fe, Cu, Zn, Mn, I, Mo, etc.), in contrast, are required only in small amounts, usually less than 30 mg/day. Their body content is about 30 g. According to their biological roles, minerals may also be divided into essential elements, for which the biological roles are known; nonessential elements, with unknown functions, if any; and toxic elements, which may be ingested through food or water or absorbed from air. The major mineral elements, such as Na, K, Ca, Mg, and Cl, fulfill electrochemical functions. Ca, K, Mg, Cu, and Zn participate as catalysts in enzyme systems, and Ca, P, and F play a role in maintaining the structure of hard tissues, bones, and teeth. Other essential elements include iodine for the formation of thyroid hormones (tri- and tetraiodothyronine) and iron as a constituent of heme as well as enzymes involved in oxidation-reduction reactions. The importance of minerals as food ingredients depends not only on their nutritional and physiological roles; they also contribute to food flavor, activate or inhibit enzyme-catalyzed and other reactions, and affect the texture of food. The toxicological aspects of several important mineral elements are briefly described in the following sections. 7.6.1 Sodium (Chloride) Sodium is present mostly as an extracellular constituent and maintains the osmotic pressure of the extracellular fluid. The kidney is solely responsible for salt excretion and extracellular volume regulation. The intake of too little or too much sodium can result in serious disorders. From a nutritional standpoint, only excessive intake is of concern since it results in hypertension, i.e., abnormally high blood pressure. A relationship between salt intake and excretion and blood pressure (renal function curve) has been shown for humans (Guyton, 1987). With all regulatory systems intact, this relationship is extremely steep, and sodium (chloride) intakes >600 mmol/day are necessary to cause an increase in blood pressure in normal individuals (Luft et

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al., 1979). In primary and secondary hypertension, these relationships are altered (Kimura, 1987; Luft, 1996a). In most primary hypertension, the relationship generally remains extremely steep, namely, a state of salt resistance. In some forms of secondary hypertension, such as primary aldosteronism, the relationship becomes flatter (salt sensitivity). Some patients who have primary hypertension, low-renin hypertension, type 2 diabetes, and hypertension; elderly people; and obese people are also relatively saltsensitive. In other forms of secondary hypertension, such as renal vascular hypertension, the relationship remains steep. The dietary sodium and chloride intake should be limited to <100 mmol/day to alleviate the development of hypertension, increase in blood pressure with age, and cardiovascular disease morbidity and mortality. 7.6.2 Potassium Potassium is the major intracellular cation in the body and serves a variety of crucial functions in energy metabolism, membrane transport, and maintenance of the potential difference across the cell membranes. The latter function is particularly important to all neuromuscular and endocrine cells, whose function is determined by their ability to depolarize and to repolarize. Potassium enters the body through the diet and is eliminated almost exclusively by the kidneys under normal circumstances. An increase in the extracellular potassium concentration (hyperkalemia) may result from increased potassium intake, decreased renal excretion of potassium, or a shift in potassium balance across cell membranes from the inside to the outside of cells. Similarly, a decrease in extracellular potassium concentration (hypokalemia) can result only from a decrease in potassium intake, an increase in potassium excretion, or a shift of potassium from outside to inside cells. The signs and symptoms of both hyperkalemia and hypokalemia are similar: weakness, lethargy, gastric hypomotility, cardiac arrhythmias, and conduction disturbances (Luft, 1996b). Both hyperkalemia and hypokalemia can be lethal. Cardiac arrhythmias and conduction disturbances can be deadly as a result of failure of the heart to perfuse blood through the vascular bed. 7.6.3 Calcium Calcium is responsible for structural functions involving the skeleton and soft tissues and regulatory functions such as neuromuscular transmission of chemical and electrical stimuli, cellular secretion, and blood clotting. More than 99% of the body calcium is in the skeleton. The physiolog-

ical functions of calcium are so vital to survival that in the face of severe dietary deficiency or abnormal losses, the same mechanisms can demineralize bone to prevent even minor hypocalcemia. Bone provides a vital and readily available source of calcium for the maintenance of normal extracellular calcium concentrations, ~50% of which is ionized (Ca2+) and physiologically active. Because of strict homeostasis under physiological conditions, calcium toxicity is a rare occurrence. The serum calcium concentration varies little despite large changes in dietary calcium because of the adaptive alterations by which the endocrine system (primarily the parathyroid hormone [PTH] calcitonin, and a sterol hormone, calcitriol) regulates this mineral. Nonetheless, in some diseases such as hyperproteinemia (e.g., hyperglobulinemia in myelomatosis) and hypoproteinemia (e.g., hypoalbuminemia in cirrhosis of the liver and nephrosis), total plasma calcium level can, respectively, increase or decrease. A loss of body calcium, especially in older people, is often linked to increased incidence of osteoporosis. Dietary fiber and oxalates (e.g., from leafy vegetables like spinach) can chelate calcium and other minerals in the GI tract. These observations led to the concern that high-fiber diets may increase the risk of bone loss and osteoporotic fractures (McCance and Widdowsen, 1942; Ismail-Beigi et al., 1977). Although there is evidence in some but not all studies that the consumption of high amounts of dietary fiber (in particular wheat bran) may interfere with the absorption of calcium, there is little evidence that high-fiber diets alone induce calcium deficiency in individuals who otherwise consume a balanced diet. Since the 1990s, considerable new evidence from human studies has also suggested a role for dietary calcium in blood pressure regulation. Hypertensive patients have been shown to have mild hypercalciuria and lower levels of serum ionized and ultrafilterable calcium than do normotensive patients, even in the absence of differences in total serum calcium level (Folsom et al., 1986; Strazzulo et al., 1986). Although calcium metabolism is probably perturbed in primary hypertension, it is not clear whether the changes are the cause or the result of the hypertension. 7.6.4 Phosphorus Phosphorus is a key inorganic constituent of bone. In cells, it is an important part of many life-sustaining compounds, such as phospholipids, phosphoproteins, and nucleic acids; the hormonal second messengers, cyclic AMP (cAMP), cyclic guanosine monophosphate (cGMP), and inositol polyphosphates; and 2,3-diphosphoglycerate, which is the

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regulator of oxygen release by hemoglobin. Phosphorus is also the repository of metabolic energy in the form of high-energy phosphate bonds, an allosteric regulator of many enzymes and an active participant in many physiological buffer systems. Phosphorus is closely linked to calcium metabolism. The Ca/P ratio in food should be about 1. Changes in phosphorus intake by normal adult humans have important effects on calcium metabolism (decreased intestinal calcium absorption and decreased renal excretion of calcium). The ability to adapt to decreases or increases of dietary phosphorus depends on the ability of the kidney to respond by increasing or decreasing its production of calcitriol, respectively (Arnaud and Sanchez, 1996). Severe hypophosphatemia (<0.5 mmol/L) can cause both skeletal myopathy and cardiomyopathy (Bringhurst, 1989). These conditions may lead to rhabdomyolysis. Chronic moderate hypophosphatemia frequently results in osteomalacia or rickets. Generally, restoration of serum phosphate concentration to normal corrects abnormal organ function in hypophosphatemic conditions. Acute, severe hyperphosphatemia can cause hypocalcemia severe enough to cause tetany and even death (Bringhurst, 1989). The less severe hyperphosphatemia induced by phosphate ingestion rarely causes symptoms; if patients have associated disorders with a tendency toward hypocalcemia (e.g., mild hypoparathyroidism or chronic renal failure), however, frank hypocalcemia can be precipitated. 7.6.5 Magnesium Magnesium is essential for a wide range of fundamental cellular reactions. Its deficiency, therefore, can lead to serious biochemical and symptom changes. Magnesium is involved in at least 300 enzymatic steps in intermediary metabolism, including those associated with the conversion of energy-rich phosphate compounds and β-oxidation of fatty acids. It also stabilizes plasma membranes and nucleic acids. Hypomagnesemia is the hallmark of experimental depletion in all species studied thus far. Symptomatic human deficiency usually develops in a setting of predisposing and complicating disease states (Table 7.5). These disease states cause intakes of magnesium to be impaired, reduce intestinal or renal absorption, or do both, leading to increased losses. Symptomatic deficiency is best treated by intravenous or intramuscular magnesium administration in conjunction with appropriate therapy for the underlying condition and with correction of other electrolyte and acid-base abnormalities.

Table 7.5

Clinical Conditions Contributing to Hypomagnesemia

Malabsorption syndromes Inflammatory bowel disease Gluten enteropathy, sprue Intestinal fistula bypass, or restriction Bile insufficiency states Immune diseases with villous atrophy Radiation enteritis Lymphangiectasia, other fat absorption defects Primary idiopathic hypomagnesemia Gastrointestinal infections Renal dysfunction with excessive losses Tubular diseases Metabolic disorders Hormonal effects Nephrotoxic drugs and diuretics Endocrine disorders Diabetes mellitus Hyperaldosteronism Hyperparathyroidism with hypercalcemia Post parathyroidectomy (“hungry bone” syndrome) Hyperthyroidism Pediatric genetic and familial disorders Primary idiopathic hypomagnesemia Renal wasting syndrome Bartter’s syndrome Infant of diabetic or hyperparathyroid mothers Transient neonatal hypomagnesemic hypocalcemia Inadequate intake, provision, and retention of magnesium Alcoholism Protein-calorie malnutrition (usually with infection) Source: Compiled from Shils (1994, 1996).

Pharmacological effects of increased levels of magnesium have also been observed in both in vitro and in vivo studies (Shils, 1994, 1996). Acute doubling of serum magnesium concentrations causes hypotension and increased renal blood flow in patients in association with prostacyclin release from endothelium. Increased prostacyclin level can in turn inhibit platelet adhesion and aggregation. Elevated serum magnesium levels can occur when individuals ingest magnesium-containing drugs, usually antacids or cathartics, long term with serious renal insufficiency. This occurs because >20% of Mg2+ from various salts may be absorbed. The toxic effects of magnesium excess progress to lethality in their severity with increasing serum concentration (Mordes and Wacker, 1979). Nausea, vomiting, and hypotension may occur at 1.5–4.5 mmol/L (3–9 mEq/L); bradycardia and urinary retention also may occur in this range. Electrocardiographic changes, hyporeflexia, and secondary central nervous system depression may appear at 2.5–5 mmol/L (5–10 mEq/L), followed at

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higher concentrations by life-threatening respiratory depression, coma, and asystolic cardiac arrest. Calcium infusion can counteract magnesium toxicity. Avoidance of magnesium-containing medications by patients who have significant renal disease is recommended unless there are good reason for and close monitoring of their use. 7.6.6 Iron Iron is one of the most investigated and best understood of nutrients. Research on iron nutrition has been facilitated by the relative ease of sampling blood and red cells, which represent the major functional pool of iron in the body. To a large extent, iron metabolism and factors leading to iron deficiency are well defined. Iron deficiency is the most common nutritional defect worldwide, and yet it can be successfully prevented on a population basis (DeMaeyer et al., 1985; Yip and Dallman, 1996). In recent years, concern about iron overload in developed countries has spurred research in this area. The iron content of the body is about 4–5 g. The iron-containing compounds in the body can be grouped into two categories: functional (known to serve a metabolic or enzymatic function) and storage (used for storage and transport of iron). Approximately two-thirds of the total body iron is functional iron; of this about 85% is in the form of hemoglobin within the circulating erythrocytes, and the rest in myoglobin and iron-containing enzymes, such as peroxidase, catalase, hydroxylases, and flavin enzymes. Iron deficiency is the most common nutritional deficiency in the United States and worldwide, affecting mainly older infants, young children, and women of childbearing age. In developing countries, it is estimated that 30%–40% of young children and premenopausal women are affected by iron deficiency (DeMaeyer et al., 1985). Mackay (1928) was among the first to demonstrate that iron deficiency was the reason for anemia prevalent among infants in East London, and she showed that providing iron-fortified powdered milk could alleviate it. One should, however, distinguish between anemia and irondeficiency anemia. There are many causes of anemia other than iron deficiency, notably infection and even mild inflammatory disease (Reeves et al., 1984; Yip and Dallman 1988). A diagnosis of iron-deficiency anemia can easily be made when the anemia is accompanied by laboratory evidence of iron deficiency, such as low serum ferritin level (iron storage and transport protein), or when there is a rise in the hemoglobin levels in response to iron treatment. Depending on its severity, anemia causes a substantial reduction in work capacity, impaired psychomotor development

and intellectual performance, impaired capacity to maintain body temperature in a cold environment, decreased resistance to infection, increased absorption of lead and thus lead poisoning, and preterm delivery, low birth weight, and fetal death. Very severe anemia is associated with increased childhood and maternal mortality. Iron deficiency can be corrected by increasing the content and bioavailability of iron in the diet. Iron absorption is improved by including meat, fish, poultry, and ascorbic acid–rich foods in meals and by decreasing the consumption of tea and milk. Iron-fortified cereal products augment the iron content of the diet; those with added ascorbic acid also enhance iron absorption. Iron can also be provided in the form of tablets (e.g., ferrous sulfate, ferrous gluconate) or liquid supplements. In general, iron absorption from supplements is greatest in iron-deficient individuals, because absorption is inversely proportional to iron stores. The toxic potential of iron derives from its principal biological property, the ability to exist in two oxidation states: Fe2+ (ferrous form) and Fe3+ (ferric form). Iron serves as a catalyst in redox reactions by donating or accepting electrons. Some redox reactions, when not properly modulated by antioxidants or iron-binding proteins, can damage cellular components such as fatty acids, proteins, and nucleic acids. A number of health disorders are related to short-or long-term exposure to iron in amounts exceeding the physiological capacity to protect against its reactivity. These pathological conditions range from acute iron poisoning to organ damage due to chronic iron overload (Yip, 1995). In recent years, there has also been concern that “iron overnutrition” among otherwise healthy individuals may lead to increased risk of chronic diseases. Excess ingestion of iron can also interfere with the absorption and utilization of copper, zinc, and manganese, and deficient dietary phosphate enhances the toxicity of excess iron. Iron toxicity is the most dramatic form of iron excess and can lead to severe organ damage and death within hours or days. The problem occurs mainly among young children who swallow iron tablets intended for women in the household (Centers for Disease Control, 1993). The most pronounced local effect of iron poisoning is hemorrhagic necrosis of the GI tract, manifested by vomiting and bloody diarrhea that result from strong acids produced by the interaction of iron with hydrochloric acid in the stomach. Systemic effects include coagulation defects, metabolic acidosis, and shock. Over the long term, excessive accumulation of body iron not only can result in excessive iron stores, but also can damage various organs when iron cannot be adequately contained in stores. The best known and perhaps

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the most common form of chronic iron overload is hereditary hemochromatosis, a genetic defect in the regulation of iron absorption. Clinical signs of organ damage from iron overload usually become evident during the third to fifth decade of life in men and after menopause in women (Yip and Dallman, 1996). Iron is absorbed and widely dispersed in the body, particularly in the liver and pancreas, with associated liver enlargement and functional insufficiency, portal cirrhosis, bronze pigmentation of the skin, diabetes mellitus, and cardiac failure (Yip and Dallman, 1996). There is uncertainty about the relationship between dietary intakes and iron depositions as well as disagreement as to whether the effects seen are due to iron per se or are the combined results of the iron with some other nutritional, toxic, metabolic, or infectious abnormality. A less common form of iron overload is related to excessive oral iron intake without a defect in iron absorption. The best-known example is Bantu-type hemosiderosis, a syndrome seen among Bantu tribesmen in southern Africa who consume large amount of maize beer high in iron (40–80 mg/L). The high iron content results from use of iron containers for brewing the acid beer, which increases iron solubility (Bothwell et al., 1964). Evidence from 1992 suggests that genetic factors may also contribute to iron overload among Africans who have very high iron intakes (Gordeuk et al., 1992). The second major route of iron overload is blood transfusion, which typically occurs among persons who have severe and refractory anemia and depend on repeated transfusions—e.g., β-thalassemia, a major hereditary defect in hemoglobin production. Sideroblastic anemias are due to inherited or acquired disorders of hemoglobin synthesis that result in ineffective erythropoiesis (Bottomley, 1982). Other transfusion-dependent conditions include anemia from bone marrow failure and various types of severe chronic hemolytic anemia. In hemolytic disorders, the severe anemia also increases gastrointestinal iron absorption; the increase can contribute significantly to the iron burden beyond what is provided in transfusions. Experimental studies have also shown that high iron levels promote carcinogenesis or faster rates of tumor growth (Steves et al., 1988; Beard, 1993). The only strong evidence linking iron overload with cancer is the increased risk of hepatic carcinoma among individuals with hemochromatosis. This association is attributed to chronic injury to hepatic tissues due to extremely high levels of iron in the liver. 7.6.7 Copper Copper is an essential nutrient needed for the function of many important enzymes (Table 7.6), electron transport-

Table 7.6

Copper-Containing Enzymes in Mammals and Other Species

Enzyme/protein Cytochrome c oxidase

Distribution and function a

Cu/Zn superoxide dismutase (Cu/Zn SOD) Tyrosinase (catecholoxidase/phenolase)

Lysyl oxidasea,b Dopamine-4-monooxygenasea,b α-Amidating enzymeb Amine and diamine oxidasesa,b Ceruloplasmina,c Ferroxidase IIa Extracellular SOD Ascorbate oxidasea

Laccasea

Phenylalanine-4-monooxygenasea

Metallothioneinc

Ubiquitous in mitochondria, last component in the electron transport chain of oxidative phosphorylation, reduction of oxygen Ubiquitous in cytosol, protection against free radicals, dismutation of superoxide to peroxide and oxygen Widely distributed, melanin production in mammals in melanosomes, diverse functions in plants and fungi, oxidative polymerization of tyrosines, oxidation of monophenols and o-diphenols to quinines Extracellular in connective tissue, cross-linking of collagen and elastin, oxidative crosslinking of lysyl residues Central nervous system and adrenal medullary cells, formation of epinephrine and norepinephrine, hydroxylation of dopamine Granules of neurohypophysis, modification of neuropeptides, oxidative removal of carbons of C-terminal glycine residue, leaving α-amino Intracellular and extracellular; oxidative inactivation of histamine, tyramine, and polyamines; oxidative demination Blood plasma and other extracellular fluids, free radical scavenger and role in promotion of flux of iron out of storage sites, oxidation of Fe2+ to Fe3+ Blood plasma, function unknown, but (like ceruloplasmin) can oxidize Fe2+ to Fe3+ Blood plasma and other extracellular fluids, part of antioxidant defense system, dismutation of O2– to H2O2 and H2O Plants and fungi, mainly intracellular; function unclear but in plants may play a role in fruit maturation and protection of wounds; oxidizes ascorbate, catechols, flavonoids, and hydroxycinnamic acid; in terms of reactivity, belongs to the family of blue copper oxygenases that include laccase and ceruloplasmin Plants and fungi, mostly extracellular; oxidative polymerization of phenolic compounds to seal wounds (trees); decomposition of lignin, in fungi oxidation of benzene diols, belongs to the family of blue copper oxygenases Ubiquitous intracellular enzyme, has either iron or copper as a cofactor (mammalian enzyme is iron-dependent), synthesis of tyrosine and degradation of phenylalanine, hydroxylation of phenylalanine Ubiquitous intracellular with traces that are extracellular in body fluids, high cysteine–containing divalent metal ion storage protein that has some SOD activity when bound to copper

a

Reaction requires molecular mono- or dioxygen. 6-Hydroxy dopa or pyrroloquinone is a cofactor for the enzyme. c Also plays nonenzymatic roles. Source: Compiled from Linder and Hazegh-Azam (1995) and Linder (1996). b

ers, and other factors. One of these (cytochrome c oxidase) is fundamental to generation of usable adenosine triphosphate (ATP) energy by almost all living cells; others play major roles in the protection of cells and cell membranes against oxidative damage, the integrity of connective tissue and blood vessels, the formation of skin and hair pigments, the production of neurotransmitters and other hormones, and perhaps also the aspects of iron metabolism involving heme biosynthesis and flux of iron out of critical sites in liver and intestine. In blood plasma, it is bound to ceruloplasmin, which catalyzes the oxidation of Fe2+ to Fe3+. This reaction is of great physiological significance,

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since it is only the Fe3+ form in blood that is transported by the transferring protein to the iron pool in the liver. Copper’s ubiquitous and basic biochemical roles in metabolism potentially place all cells in jeopardy when a dietary deficiency arises. The body copper requirement, 1–2 mg per day, is supplied in a normal diet. The amount of copper in the body is about 100–150 mg. Because of their weight and volume, muscle and skeleton probably contain approximately 25% and 42% of the total body copper, respectively (Linder, 1996). Liver and brain are next (~9%), and blood has about 5%, 60% of which is in the plasma.

In humans and animals, absorption of copper occurs primarily in the duodenum. Almost 55% to 75% of the copper intake from normal foodstuffs is absorbed in the GI tract. The efficiency of uptake does not vary appreciably with age and sex. After being absorbed by the intestine, copper rapidly enters the blood circulation and quickly is deposited mainly in the liver. In general, copper cannot be considered a metal that is stored in the body. It usually enters the body from the intestine with ease and is also readily excreted. Copper homeostasis is maintained mainly through excretion. Copper is relatively nontoxic to most mammals and birds, including rodents, poultry, pigs, and humans. Excess intakes of copper that cause acute or even chronic toxic effects are rare. Nevertheless, there have been instances when children accidentally ingested copper sulfate used as a pesticide on certain crops (Davis and Mertz, 1987) and when there has been more chronic excess intake, as in the case of “Indian childhood cirrhosis” (Barrow and Tanner, 1988). Acute symptoms of copper poisoning include nausea, vomiting, diarrhea, headache, dizziness, and weakness. In more severe cases, tachycardia, hypertension, and coma may occur and may be followed by jaundice, hemolytic anemia, hemoglobinuria, uremia, and death. The Indian childhood cirrhosis problem arose when some of the women in India heated milk formula in brass pots that leached a great deal of the metal into the liquid. This process resulted in the development of liver cirrhosis. A special form of copper toxicosis occurs in Wilson’s disease, a rare genetic disease associated with excessive accumulation of copper in the liver, kidney, brain, and cornea (Linder, 1996). Toxic manifestations of this disease can derive from normal dietary intake levels of copper (2–5 mg daily) but are accelerated by larger intake levels. Copper accumulation leads to cirrhosis of the liver, kidney damage, and brain damage, with the characteristic brown and green corneal rings called Kayser-Fleischer rings. Wilson’s disease is progressive and ultimately fatal unless treated with copper-chelating agents to remove the copper from tissues and promote urinary excretion. Most toxic effects of copper probably result from the production of oxygen radicals by copper chelates, such as when ascorbate reduces Cu2+ (Kadiiska et al., 1993; Lind et al., 1993; Shah et al., 1992). In liver, the first recipient of most of the incoming dietary copper, damage from the oxygen radicals causes scar tissues to form, leading to changes in tissue architecture and a reduction in liver function. Scarring of other tissues and damage to cell membranes (e.g., in kidney tubules and erythrocytes) lead to cell lysis and connective tissue deposition.

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7.6.8 Zinc Zinc has been recognized as an essential nutrient for nearly a century. It is a component of a number of enzymes (e.g., alcohol dehydrogenase, lactate dehydrogenase, carboxypeptidases A and B, and carbonic anhydrase). Catalytic roles for zinc are found in enzymes from all the six representative classes of enzymes (Vallee and Galdes, 1984), and zinc has important roles in both protein and carbohydrate metabolism. The total zinc content of adult human tissue is about 2–4 g. Body deposits of zinc are apparently not readily mobilized; therefore, regular dietary intake, especially in periods of rapid growth, is required. Fortunately, zinc is widely distributed in many different foods. It is absorbed from the small intestine (Solomons and Cousins, 1984; Cousins, 1989). Homeostatic control of zinc metabolism is maintained primarily by fecal excretion of endogenous zinc (King and Turnlund, 1989). Historically, food safety problems from zinc were considered much more likely to derive from deficiencies than from excesses. Indeed, phytate in unleavened bread was believed to be a contributing factor to the development of human zinc deficiency, as was first described in the Middle East (Prasad, 1979). The dietary aspects of zinc toxicity have been well reviewed (Fox, 1989; Cousins, 1996; Mills, 1989). Acute zinc toxicity results in gastric distress, dizziness, and nausea. Gastric problems are also observed in chronic toxicity. Among other chronic effects are reductions in immune function and in HDL cholesterol level with very high supplements (300 mg zinc/day). On balance, zinc should be considered a relatively nontoxic micronutrient in moderate supplementation levels. The concern is that nutrient imbalances and interactions caused by selective supplementation may cause toxicity not encountered with normal dietary practices. 7.6.9 Manganese Manganese functions in part as a constituent of metalloenzymes and as an enzyme activator. Manganese-containing enzymes include arginase, pyruvate carboxylase, and MnSOD. Arginase, the cytosolic enzyme responsible for urea formation, contains 4 mol Mn2+/mol enzyme. For manganese-activated reactions, the metal can act by binding either to the substrate (e.g., ATP) or directly to the protein, resulting in conformational changes. In contrast to the relatively few manganese metalloenzymes, there are a large number of manganese-activated enzymes, including hydrolases, kinases, decarboxylases, and transferases (Keen et al., 1984; Wedler, 1994).

The body contains a total of 10–40 mg of manganese. The daily requirements for this mineral are easily met by the normal daily food intake. At present, our understanding of the mechanisms regulating the uptake and retention of manganese in humans is limited. Manganese absorption is though to occur throughout the length of the small intestine, and its homeostasis is primarily achieved by excretion (Keen and Zidenberg-Cherr, 1996). In marked contrast to most other essential mineral elements, mangase does not appear to have any appreciable “stores” in the body. This lack of manganese storage proteins may contribute to the considerable toxicity of this element when there are high cellular concentrations of this metal. In humans, manganese toxicity represents a serious health hazard, which results in severe abnormalities of the central nervous system (Keen et al., 1994). In its more severe forms, Mn2+ toxicity can result in a syndrome characterized by severe psychiatric symptoms, including hyperirritability, violent acts, hallucinations, disturbances of libido, and incoordination. The toxicity results in a permanent crippling of the extrapyramidal system, the morphological lesions of which are similar to those of Parkinson’s disease. There have been a number of cases of manganese toxicity in individuals who consume water containing high manganese concentrations (Keen et al., 1994; Velazuez and Du, 1994). The mechanism or mechanisms underlying the cellular toxicity of manganese have not been absolutely identified, although there is evidence that they involve manganese-initiated catechol autooxidation and excessive tissue oxidative damage (Keen et al., 1994; Lloyd, 1995). Abnormal carbohydrate metabolism may also underlie some of the effects of manganese toxicosis, given the observation that insulin production can be impaired in ani-

mals subjected to high amounts of this element (Keen et al., 1994). Manganese may also interfere with the absorption of vitamin B12. 7.6.10

Selenium

Selenium for many years was primarily noted for its toxicity in grazing animals. Animals feeding on plants that accumulate selenium from soils high in selenium content can experience toxic effects such as blindness, muscle paralysis, and death of respiratory failure. Chronic effects in such animals also include hair loss, hoof soreness, anemia, liver cirrhosis, and cardiac atrophy (Stults, 1981). Selenosis of livestock has been widely reported from several parts of the world, including China, the United States, Australia, Mexico, Canada, Colombia, Israel, and Ireland (Levander and Burk, 1996). The signs of selenium deficiency have not been observed in free-living animals whose diets are adequate in vitamin E. Despite the difficulties encountered in producing a pure selenium deficiency in animals, selenium is considered an essential element for humans. Most selenium in animal tissues is present in two forms: selenomethionine, which is incorporated in place of methionine in a variety of proteins, and selenocysteine in selenoproteins such as glutathione peroxidase, iodothyronine deiodinase, and selenoprotein P (Figure 7.3). Selenomethionine in tissues is derived from the diet because it cannot be synthesized in the body. This form of selenium is not regulated by the selenium status of the animal and can be regarded as an unregulated storage compartment (Levander and Burk, 1996). When the dietary selenium supply is interrupted, the turnover of the selenomethionine pool provides selenium to the organism.

Figure 7.3 Relationships of dietary forms to tissue forms of selenium. Excretory metabolites and the transport form are also present in tissues but only in relatively small quantities.

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Selenocysteine is the form of selenium known to account for its biological activities. Selenocysteine is tightly regulated in the body since its reactivity would likely interfere with its biochemical function if it were free in the cell. The selenium content in humans is 10–15 mg, whereas the daily dietary intake is about 0.05–0.1 mg. Depending on the region, it can vary greatly because of the varying content of selenium in the soil. About 80% of the organic selenium ingested from foods appears to be absorbed; absorption is generally greater from plant foods than from meat or other animal products (Levander and Burk, 1996). Metabolically, selenium occurs as a prosthetic group of glutathione peroxidase enzymes (Combs and Combs, 1986). The enzyme plays an important role as a free radical scavenger in human metabolism. Through its interactions, selenium also appears to counteract the toxicity of several heavy metals, including cadmium, mercury, and silver. The metabolic effects of selenium deficiency include susceptibility to certain types of oxidative injury, alterations in thyroid hormone metabolism, increased susceptibility to injury by heavy metals, alterations in activities of biotransformation enzymes, and increase in plasma glutathione concentration (Hill et al., 1987; Burk et al., 1995; Berry and Larsen, 1992; Arthur and Beckett, 1994; Reiter and Wendel, 1983). Selenium deficiency has been associated with Keshan disease, an endemic cardiomyopathy that primarily affects children and women of childbearing age in some areas of China that have selenium-poor soils (Yang et al., 1988). Large-scale intervention trials have demonstrated conclusively the efficacy of selenium supplementation in controlling Keshan disease (Yang et al., 1984). Kaschin-Beck disease, an endemic osteoarthritis that occurs during the preadolescent or adolescent years, is another disease that has been linked to low-selenium status in China (Yang et al., 1988; Allander, 1994). However, the selenium deficiency hypothesis does not seem to be as widely accepted for the Kaschin-Beck disease as for Keshan disease, and other etiological theories (mycotoxins in grain, mineral imbalance, organic contaminants in drinking water, etc.) have been proposed (WHO, 1990). Cases of human selenium poisoning by consumption of toxic foods containing high levels of selenium have been reported in China and the United States (Yang et al., 1988; Jensen et al., 1984; Anonymous, 1984; Helzlsouer et al., 1985). Excessive intake of selenium in the human diet results in dermatitis, dizziness, brittle nails, gastric disturbances, hair loss, and a garlic odor on the breath. The margin of safety between essential trace levels of selenium in the human diet and for the manifestation of its toxic symptoms appears to be quite small.

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The biochemical basis of selenium toxicity is not fully understood, but several possible reaction mechanisms have been suggested, such as interference with sulfur metabolism, catalytic oxidation of sulfhydryl groups, and inhibition of protein synthesis (Levander, 1983; Levander and Burk, 1996). Selenium has also been indicated as an animal carcinogen on the basis of rat studies (Stults, 1981). However, its carcinogenic effects have not yet been confirmed in humans under normal dietary conditions. Furthermore, there is some limited epidemiological evidence interpreted to indicate that selenium may, in fact, inhibit cancer in humans (Levander and Burk, 1996). 7.6.11

Fluoride

Fluoride was once considered an essential nutrient (NRC, 1974), but the Food and Nutrition Board has backed away from that position because an essential role for fluoride could not be confirmed. However, fluoride is the only nutrient that has been demonstrated to reduce the prevalence and severity of dental caries in both children and adults. Because of fluoride’s positive impact on dental health, the National Research Council’s Food and Nutrition Board now considers it to be a “beneficial element for humans” (NRC, 1989). Although fluoride is no longer considered an essential factor for human growth and development, many believe that there is an optimal dose of systemic fluoride for maximal benefit against dental caries. On the basis of empirical evidence, it appears that 0.05–0.07 mg/kg body weight per day is a fair estimate of that optimal dose (Burt, 1992). Fluoride is ubiquitous, occurring in minute amounts in all foodstuffs and water supplies. The major influence on total fluoride intake, however, is the fluoride content of the drinking water (0.5–1.5 ppm) (USDHHS, 1993). About 75%–90% of the ingested fluoride is absorbed rapidly and readily from the GI tract; the remaining is excreted in urine and feces (USDHHS, 1991). Once absorbed, it passes into the blood for distribution throughout the body and for partial excretion. Most of the ionic fluoride retained in the body enters into the calcified tissues (bone and developing teeth) either by substitution for the hydroxyl ion or bicarbonate ion in hydroxyapatite in bone or enamel to form fluoroapatite or by ionic exchange within the hydration shell of the crystalline surface. Except increased susceptibility to dental caries, adverse effects of deficiencies in fluoride have generally not been noted. However, some relationship between the lowfluoride content of the drinking water and adverse effects on the bone structure has been reported in the literature (Stults, 1981). In addition, alleviation of osteoporosis has

been accomplished by treatment with high levels of fluoride and calcium (Phipps, 1996; Rich and Ensinck, 1961; Pak, 1989; Gruber and Baylink, 1991; Kleerekoper and Mendlovic, 1993). The body protects against potentially toxic levels of fluoride by increased urinary excretion and deposition of retained fluoride in the bones. Gradual accumulation with age in humans can lead, with excessive exposure, to calcification of joints and in advanced cases to emaciation and death. The signs and symptoms of acute fluoride toxicity are nausea, vomiting, diarrhea, abdominal pain, excessive salivation and lacrimation, pulmonary disturbances, cardiac insufficiency and weakness, convulsions, sensory disturbances, paralysis, and coma (Duxbury et al., 1982). The only known adverse effect associated with the chronic ingestion of relatively low levels of fluoride (1–2 ppm in the drinking water) is dental fluorosis, which is hypomineralization of enamel that results from contact of excessive fluoride with teeth during developmental stages (Fejerskov et al., 1990). Chronic ingestion of high doses of fluoride can cause skeletal fluorosis, which may occur in people who ingest 10–25 mg fluoride/day for 7–20 years (NRC, 1993; Felsenfeld and Roberts, 1991). The symptoms range from occasional stiffness or pain in the joints to osteoporosis of long bones, and, in severe cases, muscle wasting and neurological defects (Smith and Hodge, 1979). 7.6.12

Chromium

Chromium was designated an essential element on the basis of its role in restoring glucose tolerance in rats (Schwarz and Mertz, 1959). The essentiality of trivalent chromium (Cr3+) in humans, its most stable form in biological systems, is based in part on animal evidence that indicates an important role in carbohydrate and lipid metabolism and glucose utilization. It activates several enzymes and stimulates fatty acid and cholesterol synthesis. Chromium also potentiates insulin action by activating the enzyme phosphoglucomutase. The chromium content of the body varies considerably, depending on the region; the range is 6–12 mg. The daily intake also varies greatly, from 5 to 200 µg. Intestinal absorption of Cr3+ is low (Stoecker, 1996). In humans, it is accumulated in the liver, spleen, soft tissue, and bone (Lim et al., 1983). Its homeostasis is maintained via urinary excretion. Because chromium is ubiquitous in the environment, it is difficult to produce a clear chromium deficiency in laboratory animals. However, it plays an important role in restoring glucose tolerance in rats. Metabolic disorders of carbohydrate metabolism responsive to chromium treatment have also been reported in the literature (Stoecker,

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1996). Chromium concentrations in serum or plasma are near the detection limits of the currently available instruments and do not appear to be good indicators of actual chromium status (Anderson et al., 1985). Intakes of Cr3+ that are adequate to produce a physiological effect are considered safe (Mertz, 1969, 1993). Furthermore, the safety of 200-µg chromium supplements given as chromium chloride has been established in a number of studies (Mertz, 1993). Because Cr3+ is poorly absorbed, high oral intakes may be necessary to attain toxic levels. The toxic effects of the hexavalent form (Cr6+) have been attributed primarily to occupational exposure to chromate dust. The symptoms include allergic dermatitis, skin and nasal septum lesions, and increased incidence of lung cancer (Losi et al., 1994; Von Burg and Liu, 1993; O’Flaherty, 1994). 7.6.13

Iodine

The only role that iodine plays in the economy of the body is that of an essential component of the thyroid hormones. Thyroid hormone is essential to development. The principal secretion product of the thyroid gland is thyroxine, which contains four iodine atoms. It is largely converted in the periphery to the principal metabolically active species, triiodothyronine. The thyroid hormones circulate noncovalently bonded to carrier proteins and exert their biological effects through specific receptors. The content of iodine in the human body is about 10 mg, and the daily requirement is about 100–200 µg. Iodine absorption from food occurs exclusively and rapidly as iodide in the stomach and upper small bowel. The absorbed iodide enters the blood in free and dialyzable form. Its homeostasis is maintained via fecal excretion. A deficiency of iodine in animals and humans has important consequences to both embryonic and postnatal development (Delange, 1994). These disorders are among the most important and prevalent of the diseases of humankind. In fact, iodine deficiency is the most prevalent cause of preventable mental retardation in the world (Stanbury, 1994, 1996). The reason for this prevalence is that iodine is deficient in soils in most parts of the world, especially in mountain regions, the Gangetic plain of northeastern India, and the Tacloman Desert of central western China. Disorders arising from iodine deficiency include goiter, cognitive and neuromuscular impairment, increased embryonal and postnatal mortality rate, deafmutism, and impaired fertility. Iodine deficiency of long standing induces thyroid enlargement (goiter) and nodule formation. Some elements of the thyroid under this circumstance become autonomous of normal control, and this loss of con-

trol may be permanent. The most severe form of the iodine deficiency disorders is cretinism, characterized by markedly impaired mentation and neural disorders. All these disorders vanish among newborns when iodine is introduced prophylactically before the third trimester of pregnancy. Except unusual and rare instances of hypersensitivity to iodide, humans are remarkably tolerant of high levels of iodine intake. Large intakes of iodide result in a prompt but transient inhibition of thyroxine synthesis in humans without hyperthyroidism, the so-called WolffChaikoff effect. If excessive intake (200 mg/day) is continued for weeks or months, individuals with normal thyroid function usually adapt, but those with thyroid abnormalities do not and become functionally hypothyroid, often with severe thyroid enlargement (Stults, 1981). The existence of endemic goiter in certain parts of the world has also been traced to very high intakes of iodine (≤ 200 mg/day) The action of excess iodine in producing thyrotoxicosis is not clearly understood. Increases in frequency of hospital admissions for thyrotoxicosis have been observed in the United States, several European countries, Zimbabwe, and Zaire and are particularly well documented in Tasmania and Australia (Connolly et al., 1970). Generally, this endemic has died down after a few years but has risen again in the iodine-deficient regions when the level of iodine in salt has been raised. This observation is consistent with the contention that once autonomy develops in the thyroid, in either nodular or paranodular locations, it never resumes normal control. 7.6.14

Molybdenum

The evidence for the essentiality of molybdenum first appeared in 1953 when xanthine oxidase was identified as a molybdenum metalloenzyme (Mills and Davis, 1987; Rajagopalan, 1988). It is also a constituent of various flavindependent enzymes. Molybdoenzymes catalyze the hydroxylation of various substrates. Molybdenum may also be involved in stabilizing the steroid-binding ability of the glucocorticoid receptor (Nielsen, 1996). The body contains 8–10 mg of molybdenum. The daily intake in food is approximately 0.3 mg. It is rapidly absorbed from the intestinal tract and readily excreted in urine. Thus, excretion, rather than regulated absorption, is the major homeostatic mechanism for molybdenum (Turnlund et al., 1993). Molybdenum absorbed into the blood is loosely attached to erythrocytes and tends to bind specifically to A2-macroglobulin. Large oral doses are necessary to overcome the homeostatic control of molybdenum. Thus, it is a relatively nontoxic element. There is little information relating mo-

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lybdenum to toxicity in humans. In humans, both occupational and high dietary exposure to molybdenum have been linked through epidemiological methods to elevated uric acid level in blood and increased incidence of gout and multiple sclerosis (Stults, 1981; Nielsen, 1996). 7.6.15

Cobalt

Cobalt serves its primary known nutritional function in humans as a component of vitamin B12 and to be of nutritional value must be ingested as vitamin B12. The total cobalt content of the body is 1–2 mg. The only known deficiency problem is pernicious anemia, which results from poor diet or physiological disorders associated with vitamin B12 itself. Cobalt toxicity is classically identified with several instances of severe cardiac failure in heavy beer drinkers (Stults, 1981). In addition to congestive heart failure, in individuals who drink large amounts of beer, containing 1–2 ppm of cobalt salts added as a foam stabilizer, in many cases polycythemia, pericardial effusion, thyroid epithelial hyperplasia, and neurological abnormalities also developed. In general, cobalt toxicity is virtually impossible through normal diets because of the small amounts found in foods. 7.6.16

Silicon

Ample circumstantial evidence indicates that silicon is an essential nutrient for higher animals, including humans. Findings from animals indicate that silicon nutriture affects macromolecules such as glycosaminoglycans, collagen, and elastin (Nielsen, 1996; Carlisle, 1981, 1984, 1988). Although more should be known about the physiological or biochemical function and requirement for silicon, speculation has materialized on the possible involvement of silicon deprivation in the occurrence of several human disorders, including atherosclerosis, osteoarthritis, osteoporosis, hypertension, and Alzheimer’s disease (Nielsen, 1996). This speculation indicates the need for more work to clarify the consequences of silicon deficiency in humans. The silicon content of the body is approximately 1 g. The intake in food amounts to 21–46 mg/day. Little is known about its metabolism. Connective tissue (including aorta, trachea, tendon, bone, and skin) and its appendages contain much of the silicon that is retained in the body (Adler et al., 1986). The high silicon content of connective tissues may be the result of its presence as an integral component of the glycosaminoglycans and their protein complexes that contribute to structural framework.

Silicon is not protein-bound in plasma; it is believed to exist in plasma almost entirely in the undissociated monomeric silicic acid form, Si(OH)4 (Carlisle, 1984; Berlyne et al., 1986). The elimination of absorbed silicon occurs mainly via the urine, where it probably exists as magnesium orthosilicate. Most silicon compounds are essentially nontoxic when taken orally. Humans have used magnesium trisilicate as an antacid for over 50 years without any obvious deleterious effects. Other silicates are food additives used as anticaking or antifoaming agents (Villota and Hawkes, 1986). The toxicity of silicic acid is apparent only at concentrations >100 mg/kg body weight.

sociate some side effects with excess intakes of the watersoluble vitamins. On the basis of safety and toxicological considerations, vitamins can be divided into two broad categories (Table 7.7). 1.

2. 7.7

VITAMINS

Vitamins are minor but essential constituents of food. They are required in relatively small amounts for the normal growth, maintenance, and functioning of the human body. The vitamin requirement of the body is usually adequately supported by a balanced diet. A deficiency can result in hypovitaminosis and, if more severe, in avitaminosis. Both can occur as a consequence not only of insufficient supply of vitamins by food intake, but of disturbances in resorption, stress, and disease. An assessment of the extent of vitamin supply can be made by determining the vitamin content in blood plasma or by measuring a biological activity that is dependent on the presence of a vitamin, as are many enzyme activities. Vitamins are usually divided into two general classes: the fat-soluble vitamins, such as A, D, E, and K, and the water-soluble vitamins, B1, B2, B6, nicotinamide (niacin), pantothenic acid, biotin, folic acid, B12, and C. Under normal dietary conditions, vitamins do not pose any serious health hazard. However, in the recent past, the prescription of vitamin megadoses as a cure for certain ailments has made a potential risk of vitamin intoxication a reality. There is also a common tendency to fortify and enrich various food products with vitamin and mineral supplements. This may lead to a risk of overconsumption of some vitamins and calls for some guidelines. Most reports of vitamin toxicity are related to the fat-soluble vitamins. Unlike the water-soluble vitamins, they are not excreted but rather are stored within the body tissues. Excessive consumption of these vitamins from specific foods or from supplements may further aggravate the condition. In contrast, excess intakes of the water-soluble vitamins result only from the use of supplements and merely lead to the body’s excreting what it does not need in urine and sweat. However, research is beginning to as-

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Those with a safety level at least 50–100 times the recommended daily allowance (RDA) and no clear indication of serious adverse reactions above that level. This level should be adequate to match any pharmacological dose, and these vitamins should be recognized as safe for elevated dose use, not necessarily controlled by a physician. Those with a safety ratio of about 10 times, often influenced by the health status of the individual, or those with serious irreversible adverse reactions. These vitamins can be used safely at an RDA level but should only be administered at higher dosage under medical supervision to prevent dose escalation.

Several other factors also influence the safety of vitamins. These include the number of doses to be given and the interval between the doses, the mode of administration (e.g., oral or parenteral), the health of the individual (e.g., pregnancy, youth and old age, any pathological conditions), and interference by food or drugs. Generally the latter interactions reduce the toxicity of vitamins rather than increase it. The safety and toxicological aspects of vitamins in human nutrition are described in the sections that follow.

Table 7.7

Categorization of the Safety of Vitamins

Type Water-soluble

Fat-soluble

a

Safe for nonmedical use Thiamine Niacin Riboflavin Pantothenic acid Biotin Folic acid Cobalamins Ascorbic acid Tocopherols

RDA, recommended daily allowance.

Safe at RDA, therapeutic use under medical supervisiona Pyridoxine

Retinol Calciferols Phylloquinone

7.7.1 Vitamin A (Retinol) Vitamin A was the first in the long series of vitamin discoveries that began early in the 20th century. It was identified as a necessary fat-soluble factor for rat growth in 1914 and was structurally elucidated in 1930. The biological conversion of β-carotene to vitamin A was shown in the same year. These early studies on vitamin A are well reviewed in Moore’s fine treatise (Moore, 1957). In a nutritional sense, the vitamin A family includes all naturally occurring derivatives of β-ionone (other than carotenoids) that possess the biological activity of alltrans-retinol or are closely related to it structurally. Major compounds belonging to the vitamin A group are shown in Figure 7.4. Because provitamin A carotenoids are nutritionally active, they are also included in the vitamin A family. Only 50 of approximately 600 carotenoids found in nature are converted to vitamin A, but most carotenoids, including those with provitamin A activity, also can serve as singlet oxygen quenchers and as antioxidants under certain conditions. These characteristics are not possessed by retinol. The daily requirement for vitamin A is 1.5–1.8 mg. Approximately 75% is provided by retinol intake (as fatty acid esters, primarily retinyl palmitate), and the remaining 25% is through β-carotene and other provitamin A active carotenoids. Only about 60% of the β-carotene is converted into active retinol. The absorption efficiency of dietary vitamin A in healthy adults who ingest significant amounts of fat (>10 g/day) is greater than 80%. The intestinal absorption of carotenoids is much more critically dependent on the presence of bile salts than is that of vitamin A (Olson, 1994). Vitamin A is very well stored in the body; >90% of the total is found in the liver, essentially in the form of fatty acid esters. In a vitamin A–depleted state, the relative amounts of vitamin A in the kidney and in most epithelial tissues in relation to that in the liver are increased. Its content in the liver is about 250 µg/g tissue: i.e., a total of about 240–250 mg is stored. The liver supplies the blood with free retinol, which then binds to proteins in blood. Vitamin A concentration is 45–84 µg/100 mL plasma in adults; values below 15–24 µg/100 mL indicate a deficiency. Vitamin A occurs only in animal tissue. Plants are devoid of vitamin A but do contain carotenoids, which yield vitamin A by cleavage of the centrally located double bond. The best-defined function of vitamin A is in vision. Retinol, in the form of 11-cis-retinal (II), is the chromophore component of the visual cycle chromoproteins in three types of cone cells, viz., blue, green, and red (λmax 435, 540, and 565, respectively), and of rods of retina. Its deficiency results in night blindness and some eye disor-

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ders. The other major biological functions of this vitamin include cell differentiation, embryonic development, spermatogenesis, immune response, taste, hearing, appetite, and growth (Olson, 1996). Most of these processes depend directly or indirectly on cellular differentiation. Vitamin A deficiency is a major public health problem in many areas of the less industrialized world. It was estimated that 500,000 preschool-age children become blind each year because of vitamin A deficiency (Underwood, 1994). Most blind children do not survive. The common clinical signs of vitamin A deficiency are night blindness and xerophthalmia. The most diagnostic clinical signs in young children are Bitot’s spots, which are foamy white accumulations of sloughed cells that usually appear on the temporal quadrant of the conjunctiva. In addition to clinical deficiency, more than 100 million children suffer from vitamin A inadequacy in the absence of clinical signs of acute deficiency (Underwood, 1994). These children generally have a higher mortality rate and a higher incidence of severe infections than do vitamin A–sufficient children. An inadequate vitamin A status is commonly associated with protein-calorie malnutrition, a low intake of fat, lipid malabsorption syndromes, and febrile diseases. Among adults, lactating women are at most risk. Retinol is one of the vitamins for which the safety margin between the RDA and the dose that produces adverse reactions is relatively small. Its adverse reactions are very much influenced by the health of the individual and particularly by the functional integrity of the liver. In the presence of liver damage, there are an increased level of unbound circulating retinol and a higher incidence of adverse reactions. Vitamin A toxicity is seldom reported to result from ingesting natural foodstuffs. Exceptions are examples of acute toxicity following the consumption of such uncommon items as polar bear liver or shark, halibut, or cod liver, which contain 13,000–100,000 IU/g tissue (Hayes and Hegsted, 1973), 1 IU equals 0.3 µg of retinol or 0.6 µg of β-carotene. The early signs of acute toxicity include nausea, vomiting, headache, vertigo, blurred vision, muscular incoordination, and, in infants, bulging of the fontanelle. When the dose is very large, a second phase, characterized by drowsiness, malaise, inappetence, physical inactivity, itching, skin exfoliation, and recurrent vomiting, follows by the next week (Macapinlac and Olson, 1981). Lethal doses induce coma, convulsions, respiratory abnormalities, and then death by respiratory failure or convulsions, within 1–6 days. Nonetheless, after acute dosing, with smaller but still toxic amounts, recovery is usually complete within a few weeks. Chronic toxicity, which is much more common than acute toxicity, is induced by the recurrent ingestion over a

Figure 7.4 Major compounds of the vitamin-A group. β-Carotene, a provitamin A compound, is the most abundant of carotenoids found in human foods and also contains the highest vitamin A activity.

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period of weeks to years of excessive doses of vitamin A, those usually ≤ 10 times the RDA. Toxic signs commonly include headache, alopecia, cracking of the lips, dry and itchy skin, hepatomegaly, and bone and joint pain (Bauernfeind, 1980; Hathcock et al., 1990; Armstrong et al., 1994). Most cases of chronic hypervitaminosis A were reported in children with daily intakes of 12,000–600,000 IU and in adults of 50,000–1,000,000 IU. Hypervitaminosis A during pregnancy has also been implicated in teratogenic effects in humans (Pick et al., 1981). Fetal resorption, absorption, birth defects, and permanent learning disabilities in the progeny are the most serious teratogenic effects of vitamin A. Therefore, caution should be exercised in the use of retinol (as indeed with all therapy) during pregnancy. Carotenoids in foods are not known to be toxic even when ingested in large amounts. Hypercarotenosis, a benign condition characterized by a jaundice-like yellowing of the skin and high plasma carotenoid concentrations, however, can result when large amounts of carotene-rich foods (e.g., tomato and carrot juice, or daily β-carotene supplements >30 mg) are consumed (Micozzi et al., 1988). The only known toxic manifestations of carotenoids intake is canthaxanthin retinopathy, which may occur in patients treated therapeutically with large daily doses (50–100 mg) of this 4,4′-diketo derivative of β-carotene for long periods (Weber et al., 1992). After cessation of intake, however, the crystalline canthaxanthin inclusion bodies in the retina slowly disappear. 7.7.2 Vitamin D (Calciferol) Vitamin D is one of the most important biological regulators of calcium metabolism. These effects are achieved by one of its metabolites, 1α,25(OH)2D3, which is considered a steroid hormone. Humans are reported to have been aware since early antiquity of the substance we now know as vitamin D (Soleki, 1971). The first scientific description of a vitamin D deficiency, namely, rickets, was provided in the 17th century by both Dr. Daniel Whistler (1645) and Professor Francis Glisson (1650) (Norman, 1979). The major breakthroughs in understanding the causative factors of rickets resulted from the development of nutrition as an experimental science and the appreciation of the existence of vitamins. Vitamin D is popularly known as the “sunshine vitamin.” Its common form, vitamin D3 (cholecalciferol), can be produced photochemically by the action of sunlight or UV light from the precursor sterol, 7-dehydrocholesterol (Figure 7.5). The latter is present in the epidermis or skin of most higher animals. Vitamin D3 can be endogenously produced as long as animals and humans have access on a

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regular basis to sunlight. Under such conditions, there is no dietary requirement for this vitamin (Norman, 1996). The other common form, vitamin D2 (ergocalciferol), is a completely synthetic form of vitamin D that is produced by irradiation of the plant steroid ergosterol (Figure 7.6). The daily requirement for this vitamin is about 10 µg. Artificial fortification of foods, such as milk, margarine and butter, and cereals, helps in meeting the RDA for this vitamin. The major circulating form of vitamin D is the active metabolite 25-hydroxycholecalciferol, which is further hydroxylated to related metabolites, the most potent of which for intestinal calcium transport is 1α,25(OH)2D 3. The most important point of regulation of the vitamin D endocrine system occurs through the stringent control of the activity of the renal 1-hydroxylase. In this way, the production of the hormone 1α,25(OH)2D3 can be modulated according to the calcium and other endocrine needs of the organism. Probably the most important determinant of activity of the 1-hydroxylase is the vitamin D status of the animal (Henry et al., 1974). When circulating concentrations of 1α,25(OH)2D3 are low, its production by the kidney is high, and when the circulating concentrations are high, its output by the kidney is sharply reduced. Apart from mediating the calcium homeostasis, the vitamin D endocrine system embraces many more target tissues than simply the intestine, bone, and the kidney (Figure 7.7). Notable additions to this list include the pancreas, pituitary gland, breast tissue, placenta, hematopoietic cells, skin, and cancer cells of various organs (Norman, 1996). The biological responses of 1α,25(OH)2D3 are initiated via regulation of gene transcription as well as a separate signal transduction pathway(s), which generates biological responses very rapidly. Excessive amounts of vitamin D are not normally available from usual dietary sources; thus reports of vitamin D toxicity are rare. However, there is always the possibility that vitamin D toxicity may occur in individuals who are taking excessive amounts of supplemental vitamins. In fact, among the fat-soluble vitamins, vitamin D is one of the most potent toxic vitamins, in which the range among the therapeutic, pharmacological, and toxicological levels is the least. The biological basis for toxicity resulting from the excess intake of the parent vitamin D3 is believed to be the unrestrained metabolism by the liver of the vitamin D3 to 25(OH) D3; this is a largely unregulated metabolic step (Bhattacharya and DeLuca, 1973). The vitamin D toxicity is thought to occur as a result of high plasma levels of 25(OH) D3 rather than those of 1α,25(OH)2D3. Large concentrations of the former can mimic the actions of the latter, resulting in a massive stimulation of Ca2+ absorption

Figure 7.5 Chemical characteristics and irradiation pathway for production of vitamin D3 (a natural process by exposure to sunlight). The provitamin 7-dehydrocholesterol, characterized by the presence in the B ring of ∆5, 7 conjugated double-bond system, is converted to the seco-B previtamin steroid, where the 9, 10 carbon-carbon bond has been broken. Then the previtamin D, in a process independent of ultraviolet light, thermally isomerizes to the active vitamin form, which is characterized by a ∆6, 7; ∆8, 9; and ∆10, 19 conjugated double-bond system. In biological systems, vitamin D is capable of assuming a large number of conformational shapes because of rotation about 6,7 carbon-carbon single bond of the B ring. Here both the 6-s-cis conformer (the steroidlike shape) and the 6-s-trans conformer (the extended shape) are shown.

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Figure 7.6 Chemical characteristics and irradiation pathway for commercial production of vitamin D2 from ergosterol. For details, see the legend for Figure 7.5.

and bone Ca2+ resorption, and ultimately soft-tissue calcification and kidney stones (Hartenbower et al., 1977). Among the sites susceptible to dangerous calcification are the blood vessels, heart, and lung; renal calcification can lead to renal failure and death (Taylor, 1972). It is now a well-established fact that the safety factor for orally administered vitamin D is small. On a chronic administration basis, current evidence suggests that the levels of intake of vitamin D should not exceed 10 times the RDA.

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7.7.3

Vitamin E (α-Tocopherol)

The term vitamin E applies to a family of eight related compounds, the tocopherols and the tocotrienols, which consist of substituted hydroxylated ring systems (chromanol ring) lined to a phytyl side chain (Figure 7.8). The four major forms of vitamin E are designated α, β, δ, and γ, on the basis of the number and position of methyl groups on the chromanol ring. The tocotrienols have three double bonds in the phytyl side chain but otherwise resem-

Figure 7.7

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Human disease states related to vitamin D.

Figure 7.8

Chemical structures of tocopherol and tocotrienol series of compounds having vitamin E activity.

Substitution

Tocopherol

Tocotrienol

R1, R2, R3 = CH3 R1, R3 = CH3; R2 = H R1, R2 = CH3, R3 = H R1 = CH3, R2, R3 = H

α-Tocopherol (α-T) β-Tocopherol (β-T) γ-Tocopherol (γ-T) δ-Tocopherol (δ-T)

α -Tocotrienol (α-T-3) β-Tocotrienol (β-T-3) γ-Tocotrienol (γ-T-3) δ-Tocotrienol (δ-T-3)

ble the tocopherols. Less widely distributed in nature, the tocotrienols may have biological activity comparable with that of the tocopherol, but are generally considered of less nutritional importance. The potential physiological effects of vitamin E are related to its antioxidant property. It is a scavenger of free radicals, thus preventing free radical or oxidant damage to PUFA in cell membranes, thiol-rich protein constituents of membranes and the cytoskeleton, and nucleic acids (Deshpande and Deshpande, 1997). It is also involved in the conversion of arachidonic acid to prostaglandins and slows down the aggregation of blood platelets. The daily requirement for this vitamin is about 15 mg of α-tocopherol. It increases when the diet contains a high content of PUFA. A normal supply results in a concentration of 0.7–1.6 mg/100 mL in blood plasma. A level less than 0.4 mg/100 mL is considered a deficiency. Vitamin E is absorbed similarly to dietary fat. It must be solubilized by the bile acids secreted from the liver so that it can traverse the aqueous environment in the intestinal lumen to reach the surface of absorptive intestinal cells. It is absorbed by a nonsaturable, non-carrier-mediated, passive diffusion process. Vitamin E is transported by the lipopro-

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teins in the circulatory system. The liver functions as a rapid turnover store of vitamin E, never accumulating large amounts because of the functions of the tocopherol transfer protein (Sokol, 1996). Vitamin E is found almost exclusively in the adipose cell fat droplet, all cell membranes, and circulating lipoproteins. The deficiency of vitamin E at the cellular level is generally accompanied by an increase of lipid peroxidation of cellular membranes. This may lead to decreased energy production by the mitochondria, oxidation and mutation of the DNA, and alterations of normal transport processes of the plasma membrane. Symptomatic deficiency of vitamin E in humans rarely, if ever, occurs because of inadequate oral intake of this vitamin, presumably because of its ubiquitous distribution in nature. The most common disorders associated with low plasma levels of vitamin E are cystic fibrosis (Sokol et al., 1989), abetalipoproteinemia (Rader and Brewer, 1993), chronic cholestatic liver diseases (Sokol, 1994), celiac disease, short bowel syndrome, and other forms of chronic diarrhea (Satyamurti et al., 1986). Compared with the other fat-soluble vitamins, vitamin E is relatively nontoxic when taken orally. The clini-

cal experience indicates that the safety margin with αtocopherol is substantial even with prolonged daily administration (Briggs, 1978). Large intakes of vitamin E, however, may interfere with the absorption of vitamin A and vitamin K. More importantly, intakes of vitamin E in excess of 1200 mg/day can interfere with the metabolism of vitamin K1, thus potentiating the anticoagulation effect of drugs such as coumadin (Corrigan, 1979). With this exception, the safety factor appears to be substantially in excess of 100 times the RDA. 7.7.4 Vitamin K (Phylloquinone, Phytomenadione) The K-group vitamins are naphthoquinone derivatives, which differ in their side chains (Figure 7.9). It occurs naturally as phylloquinone (vitamin K1) of green plants and menaquinone (vitamin K2) of animal tissues, intestinal bacteria, and other microorganisms. A synthetic, watersoluble product, menadione, or vitamin K3, is also available. It is biologically active since the body readily converts it to a menaquinone (Olson, 1980). The 10th edition of the National Research Council’s Recommended Dietary Allowances was the first to include a recommendation for vitamin K (NRC, 1989), which is 1 µg phylloquinone/kg body weight for adults. The major

source of this vitamin in the diet is phylloquinone of plant origin. Normal diets in the United States provide on the order of 300–500 µg/day, well above the safe and adequate adult level of 70 to 140 µg. It is absorbed from the gut via the lymphatic system, then transported in chylomicrons to target tissues. Excretion of phylloquinone occurs predominantly in feces via the bile, but significant amounts are also excreted in the urine (Suttie, 1996). In contrast to the other fat-soluble vitamins, vitamin K has a very rapid turnover in the liver. The hemorrhagic condition that results from the dietary lack of vitamin K is related to a lower concentration of plasma prothrombin (factor II) and a depressed synthesis of other clotting factors VII, IX, and X. Primary vitamin K deficiency is uncommon in healthy humans. The hemorrhagic disease of newborns is, however, a long-recognized syndrome that is at least partly responsive to vitamin K (Lane and Hathaway, 1985). Vitamin K stores are low at birth because of poor placental transfer, and the sterile gut precludes any possible use of menaquinones during early life. The low vitamin K content of breast milk and low milk intake are contributing factors to vitamin K deficiency in newborns (Canfield and Hopkinson, 1989; Kries et al., 1987). Commercial formulas are now routinely supplemented with vitamin K. The information relating to the safety of vitamin K by the oral route is small. There is no known toxicity associated with the administration of high doses of phylloquinone (NRC, 1987). The administration of menadione to infants is associated with hemolytic anemia and liver toxicity, and phylloquinone is now prescribed to prevent hemorrhagic disease of newborns. The toxicity of dietary menadione is, however, relatively low, and animals have been fed as much as 1000 times the daily requirement with no adverse effects. 7.7.5 Interactions of Vitamins A, D, E, and K

Figure 7.9

Biologically active forms of vitamin K.

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The toxicity of the fat-soluble vitamins is difficult to elucidate completely in certain cases because of their interactions (Stults, 1981). High levels of one of these vitamins may well lessen the toxicity symptoms of another. Thus, an otherwise toxic level of a fat-soluble vitamin may result in reduced adverse effects if high, or at least adequate, levels of another fat-soluble vitamin are present. For example, the varying time of onset for the toxic effects associated with excess consumption of cod liver oil may be due to the relative proportions of vitamins A and D present rather than solely to the vitamin A. Adequate or excess levels of vitamin E also appear to protect against high levels of vitamins A and D, and vitamin K protects against high levels of vitamin A.

The converse of the protective interactions should also be noted. Deficiency of one or more of these vitamins may extend the toxic effects of others. For example, the toxic effects of vitamins A and D can be exacerbated in the presence of vitamin E and/or K deficiencies, and vitamins A and E are mutually antagonistic. Whatever the exact nature of the interactions among the fat-soluble vitamins may be, prudence still dictates avoidance of excess levels of consumption except for specific therapeutic uses under the direction of a physician. 7.7.6 Thiamine (Vitamin B1) The first of the B vitamins to be discovered, thiamine or vitamin B1 is essential for the proper functioning of several important metabolic reactions. The typical disease resulting from its deficiency is beriberi. Thiamine, in the form of its pyrophosphate (TPP) (Figure 7.10), is the coenzyme of several important enzymes, such as pyruvate dehydrogenase, transketolase, phosphoketolase, and α-ketoglutarate dehydrogenase, in reactions involving the transfer of an activated aldehyde unit. The daily adult requirement for thiamine is 1–2 mg. Since it is a key substance in the carbohydrate metabolism, the requirement increases in a carbohydrate-enriched diet. Thus, thiamine requirements are related to energy consumption. Body stores of thiamine are relatively small and, hence, regular intake is necessary, especially because large single doses of this vitamin are absorbed poorly. The process of thiamine absorption involves two mechanisms (Hoyumpa et al., 1982; Rindi, 1984, 1992). At concentrations <1 mmol/L, thiamine is absorbed mainly by an active carrier-mediated system that involves phosphorylation of the vitamin and is age-related (Rindi and Ferrari, 1977; Gastaldi et al., 1992). At higher concentrations, passive diffusion prevails. Absorption takes place primarily in the jejunum. In blood, thiamine is transported in erythrocytes, which contain free thiamine and its phosphorylated forms, and in plasma, which contains only the

Figure 7.10 Pyrophosphate ester of vitamin B1.

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free thiamine and its monophosphorylated analog (Rindi et al., 1968). In adult humans, the total thiamine content is estimated to be ~30 mg; its biological half-life is 9.5–18.5 days (Ariaey-Nejad et al., 1970). Inadequate dietary intake is the main cause of human thiamine deficiency in the underdeveloped countries, whereas alcoholism is the primary cause in the industrialized countries. Beriberi is the ultimate consequence of an inadequate intake of thiamine in humans. The main manifestations of the disease affect the cardiovascular (heart hypertrophy and dilatation of the right ventricle, tachycardia, and respiratory distress) and the nervous system (exaggeration of tendon reflexes, polyneuritis, muscle weakness, pain, and convulsions). In subclinical deficiency, the symptoms, which may include tiredness, headache, and reduced productivity, are less prominent and can be induced by chronic alcoholism. Some congenital defects in thiamine metabolism include the maple syrup urine disease (branched-chain ketoacidemia), lactate acidosis, Leigh disease, and thiamine-responsive megaloblastic anemia (Rindi, 1996; Rhead, 1996). Apart from the hypersensitivity reaction, which has only been reported extremely rarely after oral administration and then nearly always in the range of 5–10 mg single dose, toxic reactions to thiamine are virtually unknown (Itokawa, 1978; Rindi, 1996). With parenteral administration, in contrast, reactions occurred at a more usual therapeutic level (5–10 mg) (Kolz et al., 1980). For this reason, it is advised that the parenteral administration be preceded by a skin test dose of this vitamin. For chronic oral administration, the safe dose can be placed at least at 50–100 times the RDA (i.e., about 100 mg daily). 7.7.7 Riboflavin (Vitamin B2) Riboflavin is the prosthetic group of flavin enzymes important in a wide variety of reactions in the intermediary metabolism. The two major coenzyme derivatives are riboflavin-5′-phosphate (flavin mononucleotide [FMN] and flavin adenine dinucleotide (FAD) (Figure 7.11).

Figure 7.11 Riboflavin and the two coenzymes derived from it, riboflavin-5′-phosphate (flavin mononucleotide [FMN]) and flavin adenine dinucleotide (FAD). FMN is formed from riboflavin by the addition in the 5′ position of a phosphate group derived from ATP. FAD is formed from FMN after combination with a molecule of ATP. ATP, adenosine triphosphate.

The daily requirement for this vitamin is 1.6–2.6 mg. Deficiency symptoms are rarely observed with a normal diet and, since the riboflavin pool in the body is very stable, even in a deficient diet it is not depleted by more 30%–50%. The riboflavin content of urine is an indicator of riboflavin supply levels. Values above 800 µg/g creatinine are normal; 27–79 µg/g is low; and less than 27 µg/g strongly suggests a vitamin-deficient diet. Glutathione reductase activity assay can also provide similar information. Because the dietary sources of riboflavin are largely in the form of coenzyme derivatives, these molecules must be hydrolyzed before absorption. The absorptive process occurs in the upper GI tract by specialized transport involving a phosphorylation-dephosphorylation mechanism, rather than passive diffusion (Jusko and Levy, 1967). This process is sodium-dependent and involves an adenosine

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triphosphatase (ATPase) active transport system that can be saturated. In the human blood, the transport of flavins involves loose binding to albumin and tight binding to a number of globulins, especially the immunoglobulins immunoglobulin A (IgA), IgG, and IgM (Innis et al., 1985). The urinary excretion of flavins occurs predominantly in the form of riboflavin; FMN and FAD are not found in urine. As mentioned earlier, the major function of riboflavin is to serve as the precursor of the coenzymes FMN and FAD and of covalently bound flavins. Riboflavin catalyzes numerous oxidation-reduction reactions. Because FAD is part of the respiratory chain, riboflavin is central to energy production. Other major functions include drug metabolism in conjunction with the cytochrome P-450 enzymes and lipid metabolism. The redox functions of flavoco-

enzymes include one-electron transfers, two-electron transfers from the substrate to flavin are also accomplished. Flavoproteins catalyze dehydroxylation reactions, as well as hydroxylations, oxidative decarboxylations, dioxygenations, and reduction of oxygen to hydrogen peroxide. Thus, many different kinds of reactions are catalyzed by the flavoproteins (McCormick, 1990). Riboflavin coenzymes are also involved in the metabolism of four other vitamins: folic acid, pyridoxine, vitamin K, and niacin (Rivlin, 1991). Riboflavin also has powerful antioxidant activity that derives from its role as a precursor to FMN and FAD (Dutta, 1993). Among the FAD-requiring enzymes is glutathione reductase. Riboflavin deficiency is, therefore, associated with increased lipid peroxidation. The clinical features of human riboflavin deficiency do not have the specificity that may characterize deficits of some other vitamins. Isolated deficiency is rarely encountered. The early symptoms may include weakness, fatigue, mouth pain and tenderness, burning and itchy eyes, and possibly personality changes (Rivlin, 1996). More advanced deficiency may give rise to cheilosis, angular stomatitis, dermatitis, corneal vascularization, anemia, and brain dysfunction. There is general agreement that dietary riboflavin intake at many times the RDA produces no demonstrable toxicity (McCormick, 1994; Cooperman and Lopez, 1984; NRC, 1989, Rivlin, 1979). Because riboflavin absorption is limited to ~25 mg as a maximum at any one time, consuming megadoses of this vitamin would not be expected to increase the amount absorbed. Several protective mechanisms prevent tissue accumulation of this vitamin. Because riboflavin also has very low solubility, even intravenous administration of this vitamin would not introduce large amounts into the body (Rivlin, 1996). Certainly the safe dose for riboflavin can be placed at substantially above 100 times the RDA. 7.7.8 Niacin The term niacin is the generic descriptor for nicotinic acid (pyridine-3-carboxylic acid) and derivatives that qualitatively exhibit the biological activity of nicotinamide (nicotinic acid amide) (Figure 7.12). Nicotinic acid was isolated as a pure chemical substance in 1867, but not until 1937 was it demonstrated to be the anti–black tongue factor in dogs and the antipellagra vitamin for humans (Spies et al., 1938). Niacin is essential in the form of the coenzymes NAD and NADP in which the nicotinamide moiety acts as electron acceptor or hydrogen donor in many biological redox reactions. Thus, NAD functions as an electron car-

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rier for intracellular respiration as well as a codehydrogenase with enzymes involved in the oxidation of fuel molecules, such as glyceraldehyde-3-phosphate, lactate, alcohol, 3-hydroxybutyrate, pyruvate, and α-ketoglutarate dehydrogenases. NADP functions as a hydrogen donor in reductive biosyntheses, as in fatty acid and steroid syntheses, and, like NAD, as a cohydrogenase, as in the oxidation of glucose-6-phosphate to ribose-5-phosphate in the pentose phosphate pathway. The niacin cofactor NAD is also required for important nonredox reactions. It is the substrate for three classes of enzymes that cleave the β-N-glycosylic bond of NAD to free nicotinamide and catalyze the transfer of ADP-ribose (Lautier et al., 1993). Two classes of enzymes catalyze the ADP-ribose transfer to proteins: mono-ADP-ribosyltransferases and poly-ADP-ribose polymerase (PARP). A third class of enzymes promotes the formation of cyclic ADPribose, which mobilizes calcium from intracellular stores in many types of cells (Kim et al., 1994). The vitamin occurs in food as nicotinic acid, either as its amide or as a coenzyme. Animal organs, such as liver and lean meat; cereals; yeast; and mushrooms are abundant sources of niacin. The RDA expressed as niacin equivalent (NE) ranges from 13 to 19 NE/day for adults or 1.6 NE/MJ (6.6 NE/1000 kcal; 1 NE = 1 mg of niacin) (NRC, 1989). Additional amounts of 5 NE/day are recommended for lactating women. The RDA provides for differences in various diets consumed in terms of the bioavailability of niacin and the contribution of tryptophan. Recognizing that variations occur in the amount of tryptophan converted to niacin, the Food and Nutrition Board recommended that an average value of 60 mg tryptophan be considered to be equivalent to 1 mg niacin. Nicotinic acid and nicotinamide are rapidly absorbed from the stomach and the intestine (Bechgaard and Jespersen, 1977). At low concentrations, absorption occurs as sodium-dependent facilitated diffusion, but at higher concentrations, passive diffusion predominates. Niacin, 3 to 4 g given orally, can be almost completely absorbed. Nicotinamide is the major form in the bloodstream and arises from enzymatic hydrolysis of NAD in the intestinal mucosa and liver (Henderson and Gross, 1979). In liver, excess plasma nicotinamide is converted to storage NAD (i.e., NAD not bound to enzymes) and to metabolites of niacin that are excreted. Tryptophan and nicotinic acid also contribute to storage NAD. Excess niacin is methylated in the liver to N1-methylnicotinamide (NMN) which is excreted in the urine along with the 2- and 4-pyridone oxidation products of NMN (Figure 7.12). The two major excretion products are NMN and 2-pyridone; minor amounts of niacin or niacin oxide and hydroxyl forms are also excreted (Mrocheck et al.,

Figure 7.12 Niacin-related structures. ADP, adenosine diphosphate; NAD+, oxidized nicotinamide-adenine dinucleotide; NADP+, oxidized nicotinamide-adenine dinucleotide phosphate.

1976). The pattern of niacin products excreted after niacin ingestion depends somewhat on the amount and form of niacin ingested and the niacin status of the individual. The classic dietary deficiency disease of niacin is pellagra. The most common signs of a niacin deficiency are changes in the skin; mucosa of the mouth, tongue, stomach, and intestinal tract; and nervous system. The symptoms associated with the skin are the most characteristic. A pigmented rash develops symmetrically in areas exposed to sunlight and is similar to sunburn, although in chronic cases, a darker color may develop. Changes in the digestive tract are associated with vomiting, constipation, or diarrhea, and the tongue becomes bright red. Neurological symptoms include depression, apathy, headache, fatigue, and loss of memory. Pellagra was common in the United States and parts of Europe in the early 20th century, but it has now virtually disappeared from industrialized countries except for its occurrence in some alcoholics. It still appears in India and parts of China and Africa (Jacob and Swendseid, 1996; Malfait et al., 1993). The adverse effects of niacin are primarily the side effects associated with large therapeutic doses prescribed for nutritional deficiency. Nicotinic acid, but not nicotinamide, has marked vasodilative effects on administration of large doses (100–300 mg orally or 20 mg intravenously). Flushing reactions, headache, cramps, and nausea have been recorded (Mosher, 1970; Hayes and Hegsted, 1973; Estep et al., 1977). Because of the side effects of nicotinic acid, the physiologically active amide form (i.e., nicotinamide) is now the favored therapeutic form. Doses of 200 mg to 10 g of the amide daily have been given for periods of at least 10 years under medical supervision. Occasional side effects at the higher doses

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have included rashes, excessive pigmentation, reduced glucose tolerance in diabetics, and rarely, liver abnormalities as demonstrated by liver function test results (Watermann, 1978; Marks, 1985, 1989). A direct relationship must be in doubt for at least some of these adverse effects for they have often cleared despite continuation of therapy. On the basis of the current evidence, the safe long-term dose for niacin can be placed at at least 50–100 times the RDA with negligible danger substantially above this level. 7.7.9 Pyridoxine (Vitamin B6) Vitamin B6, a group of nitrogen-containing compounds, occurs naturally in three primary forms: pyridoxine (PN), pyridoxal (PL), and pyridoxamine (PM) (Figure 7.13). The metabolically active form, pyridoxal phosphate (PLP), functions as a coenzyme in more than 100 enzymatic reactions in the metabolism of amino acids and other nitrogencontaining compounds. The six primary types of enzymatic reactions are summarized in Table 7.8. Details of the complex biochemical characteristics of these enzymes have been reviewed (Dolphin et al., 1986; Korpela and Christen, 1987). Other functions of PLP include a role in gluconeogenesis, niacin formation, lipid metabolism, nervous system function and biochemical reactions, immune system, and hormone modulation (Leklem, 1996). The intake of the vitamin occurs usually in the form of PL or PM. The RDA for vitamin B6 is 1.6–2.0 mg/day for adults. Food processing and the amount of pyridoxine glucoside in foods affect the bioavailability of this vitamin. The requirement for vitamin B 6 is affected by pregnancy, lactation, possibly age, protein intake, bioavailability, and exercise. All three forms of this vitamin are

Figure 7.13 Structures of vitamin B6 vitamers.

readily and efficiently absorbed in the jejunum by a nonsaturable process (Leklem, 1991). After absorption, each of the forms can be phosphorylated in the liver and thus retained by a process called metabolic trapping. The metabolism of vitamin B6 is highly regulated in the liver and probably also in other tissues. There are numerous body pools of vitamin B 6 (Coburn et al., 1988; Coburn, 1990). Of the total body pool, estimated to be 1000 µmol, 80%–90% is present in muscle, where a majority of the vitamin B6 is present as PLP bound to glycogen phosphorylase. In comparison, the total amount in circulation is <1 µmol. Pyridoxine deficiency in the diet causes disorders in protein metabolism, e.g., in hemoglobin synthesis. Hydroxykynurenic and xanthurenic acids accumulate, since

Table 7.8 Six Primary Types of Enzymatic Reactions Involving Pyridoxal-5′-Phosphate Type of reaction

Examples

Aminotransferase

Alanine aminotransferase, aspartate aminotransferase Tryptophan decarboxylase, tyrosine decarboxylase δ-Amino levulinate synthetase, serine palmitoyltransferase Serine hydroxymethyltransferase, cystathionase L-Serine dehydratase Interconversion of D and L amino acids (only in bacteria)

Decarboxylation Decarboxylation with C-C bond formation Side chain cleavage Dehydratase Racemization

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the conversion of tryptophan to nicotinic acid, a step regulated by the kynureninase enzyme, is interrupted. Convulsion disorders in infants caused by a deficiency of vitamin B6 have been reported (Leklem, 1996). Pharmacological doses of pyridoxine have been used in the prophylaxis and treatment of deficiency disease, as well as in the treatment of various dermatological, neuromuscular, and neurological conditions. These include certain anemias, hyperoxaluria, levodopa-induced dystonia (Sauberlich and Canham, 1980); isoniazid toxicity (Dipalma and Ritchie, 1977); nausea and vomiting of pregnancy, undesirable lactation (Marcus, 1975); and chorea, depression, and schizophrenia (Khera, 1975). Doses in the 100- to 400-mg/day range are generally used, although as much as 3 g/day has been administered for prolonged periods in the treatment of schizophrenia (Phillips et al., 1978). The therapeutic use of pyridoxine has not been proved effective except in association with inborn errors of vitamin B6 metabolism, presence of a vitamin B6 antagonist, or true dietary deficiency of the vitamin (Miller and Hayes, 1982). Although its toxicity is low, vitamin B6 is one of the vitamins about which there is still a dispute on the question of the safety margin. It is abundantly clear that doses in the range of 500 mg–6 g daily on chronic administration can provoke a reversible neuropathy (Berger and Schaumberg, 1984). In contrast, doses up to 500 mg have been given for months or years without any adverse effects (Mitwalli et al., 1984) if the rare, mild, and transient dependency state is excluded. The main point of contention is whether adverse reactions occur at 200–500 mg in women being treated longterm for premenstrual tension. Most of the evidence favors safety of doses of at least 500 mg used chronically, though caution should be exercised at present above 200 mg daily. Thus, the safe level for vitamin B6 intake can be defined as being at least 100 times the RDA (200 mg daily). However, the safety margin of pyridoxine is less good than that of most other water-soluble vitamins. 7.7.10

Folic Acid (Folacin)

Folic acid (or pterolglutamic acid and related glutamateconjugated compounds) is the term most commonly used to refer to a family of vitamers with related biological activity (Figure 7.14). Other terms, folate, folates, and folacin, are generally interchangeable. Folic acid functions in its reduced form, tetrahydrofolic acid, and is an essential cofactor in methyl transfer reactions such as those of purine and pyrimidine synthesis, so critical to normal nucleic acid replication and cell mitosis.

Figure 7.14 Folic acid and the tetrahydrofolate derivative of folic acid.

The daily adult requirement is estimated to be 0.4–0.8 mg. The level of free folic acid in blood serum or erythrocytes can monitor a sufficient supply. Serum values of 5–20 ng/ml are normal, whereas less than 5 ng/ml is a deficiency level. When folic acid is lacking, there is a increased excretion of formiminoglutamic acid, which is formed from histidine. The conversion of the latter to glutamic acid as the final step in histidine degradation is dependent on folic acid. Intestinal folate absorption occurs at the monoglutamate level; hence, hydrolysis of folyl polyglutamates to monoglutamyl derivatives is a necessary step for this process. In foods, folic acid is mainly bound to oligo-γ-Lglutamates made up of 1–8 glutamic acid residues. Unlike that of free folic acid, the resorption of this conjugated form is limited and occurs only after the glutamic acid moiety is cleaved by the enzyme folic acid conjugase, a process that occurs in the intestinal mucosa. The intestinal transport of monoglutamyl folates occurs by a carriermediated process that is equally shared by reduced and oxidized monoglutamyl derivatives as well as by the antifolate methotrexate (Selhub and Rosenberg, 1996). This transport is highly pH-dependent, with an optimumal pH of 5.0–6.0, and declines sharply with increasing pH. A second transport mechanism is simple diffusion. This transport predominates at higher luminal pH or at pharmacological folate concentrations. The crucial role of folic acid in cell growth is evident in that deficiency is not uncommon in poorly nourished infants and pregnant women and is expressed by abnormality in the most rapidly growing tissues, viz., pancytopenia, megaloblastic anemia, hyperplastic bone marrow, and GI inflammation and atrophy (Herbert et al.,

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1980; Miller and Hayes, 1982; Selhub and Rosenberg, 1996). A folic acid deficiency is also linked to neural tube defects, cancer, and heart disease. To prevent deficiency, particularly in pregnant women, daily folate supplements of 100 µg are recommended, whereas therapeutic doses of 1–15 mg are administered in cases of acute deficiency disease or as treatment for folate antagonists such as methotrexate and diphenylhydantoin. For a long time, folic acid was considered virtually nontoxic. However, one potential danger of excessive folate intake is that it can interfere with the diagnosis of vitamin B12 deficiency (Hayes and Hegsted, 1973; Miller and Hayes, 1982). In doses greater than 1 mg/day, folic acid may correct the pernicious anemia of vitamin B12 deficiency but has no effect on the progressive nerve degeneration of B12 deficiency. Therefore, in the case of multiple deficiencies, excessive folate supplementation without added vitamin B12 may result in irreversible spinal nerve degeneration, optic neuropathy, and other neurological damage before the underlying vitamin B12 deficiency is detected. For this reason, excessive intake of folic acid should be prevented and prophylactic vitamin supplementation should always include vitamin B12 along with folic acid. The safety level for folic acid must currently be estimated at between 50 and 100 times the RDA (Marks, 1989). 7.7.11

Pantothenic Acid

Pantothenic acid, a relatively late addition to the B-vitamin family, is the building unit of coenzyme A (CoA), the main carrier of acetyl and other acyl groups in cell metabolism (Figure 7.15). Acyl groups are linked to CoA by a

Figure 7.15 Chemical structure of pantothenic acid.

thioester bond. Pantothenic acid is essential to the synthesis of fatty acids and membrane phospholipids, including sphingolipids, as well as to the oxidative degradation of fatty acids and amino acids. The synthesis of amino acids such as leucine, arginine, and methionine includes a pantothenate-dependent step. Pantothenic acid, in CoA, is also required for the synthesis of isoprenoid-derived compounds such as cholesterol, steroid hormones, dolichol, vitamin A, vitamin D, and heme A. Further, CoA is essential to the synthesis of δ-aminolevulinic acid, a precursor of the corrin ring in vitamin B12 and the porphyrin rings in hemoglobin and cytochromes. It contributes an essential acetyl group to the neurotransmitter acetylcholine and to the sugars N-acetyl-glucosamine, N-acetyl-galactosamine, and N-acetyl-neuraminic acid, components of glycoproteins and glycolipids. Its central role in many metabolic processes has been comprehensively reviewed (PlesofskyVig, 1996; Metzler, 1977). Although a formal RDA has not been established for pantothenic acid, the recommended daily intake is about 6–8 mg for adults. It is extremely widespread in foods, and a deficiency of this vitamin is unlikely. CoA from dietary sources is hydrolyzed in the intestinal lumen to pantothenic acid, which is absorbed into the bloodstream by a sodium-dependent transport mechanism (Fenstermacher and Rose, 1986). From plasma, it is taken up into most cells by cotransport with sodium ions. Tissue CoA levels appear to be independent of pantothenate availability. Mitochondria may be the final site of CoA synthesis, since 95% of CoA is located in mitochondrial membrane. Nevertheless, all required synthetic enzymes have been found in the cytosol (Robishaw et al., 1982). Pantothenate is released from CoA by multiple steps of hydrolysis; the final, unique step is the hydrolysis of pantetheine to pantothenate and cysteamine (Wittwer et al., 1983). Free pantothenic acid is excreted in urine. Because of the widespread availability of pantothenic acid, human diets rarely are deficient in it. When pantothenic acid deficiency was induced in humans by the combined administration of the antagonist ω-methylpantothenate and a pantothenate-deficient diet, the most common symptoms were headache, fatigue, insomnia, intestinal disturbances, and paresthesia of hands and feet (Hodges et al., 1958).

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The toxicity of pantothenic acid is minimal. No clearly defined adverse reactions were seen even at high doses (10–12 g daily) (Hayes and Hegsted, 1973; Sauberlich, 1980; Marks, 1989). Similarly, no pharmacodynamic effects from pantothenic acid have been reported. On the basis of the current evidence, the safe dose for chronic administration of pantothenic acid is at least 100 times the suggested range for the daily requirement. 7.7.12

Biotin

Biotin (Figure 7.16) is the prosthetic group of carboxylating enzymes such as acetyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase and, therefore, plays an important role in fatty acid biosynthesis and in gluconeogenesis. The carboxyl group of biotin forms an amide bond with the ε-amino group of a lysine residue of the particular enzyme protein. Only the compound D-(+)biotin is biologically active. The daily adult requirement for biotin is 150–300 µg. It is widely distributed in natural foodstuffs. The majority of biotin in foods appears to be protein-bound. Neither the mechanism of the intestinal hydrolysis of proteinbound biotin nor the relationship of digestion of proteinbound biotin to its bioavailability has been clearly defined. A biotin transporter is present in the intestinal brush border membrane (Mock, 1996). Biotin transport also occurs by simple diffusion, which predominates at higher, pharmacological concentrations. Biotin deficiency is extremely rare; it occurs only with prolonged consumption of raw eggs, which contain the biotin-binding protein avidin. Biotin toxicity in humans has never been reported even in individuals who have received daily doses of as much as 200 mg orally and 20 mg intravenously to treat biotin-responsive inborn errors of metabolism and acquired biotin deficiency (Mock, 1996). For practical pur-

Figure 7.16 The structure of biotin.

poses, a figure of at least 100 times the probable RDA may be regarded as safe. 7.7.13

Cyanocobalamin (Vitamin B12)

Vitamin B12, like folic acid, is involved in methylation reactions and is thus essential for cell replication. In addition, it is required for the normal development and function of the nervous system. The daily adult requirement of vitamin B12 is 3–4 µg. It is present in all animal, but rarely in plant, foods. The resorption of vitamin B12 is achieved with the aid of a glycoprotein, the intrinsic factor formed by the stomach mucosa. The deficiency of vitamin B12 is usually caused by impaired resorption due to inadequate formation of the intrinsic factor. Vitamin B12 deficiency is most likely to occur in strict vegetarians and individuals with malabsorption conditions, gradually giving rise to a megaloblastic anemia and neurological problems, which may become quite severe with continued lack of the vitamin (Herbert et al., 1980). Cyanocobalamin, the common supplemental form of vitamin B12, is given intramuscularly at a dose of 100 µg for acute deficiency disease, and doses of 1–10 µg are frequently given for maintenance. Vitamin B12 has been found to have extremely low toxicity. There is a limiting mechanism for cyanocobalamin intestinal absorption, which depends on the vitamin status of the body (Raccuglia et al., 1969). Doses as high as 30 mg daily (i.e., 10,000 times the RDA) have been given without any adverse effects (Reisner, 1967; Marks, 1989; Raccuglia et al., 1969), so that the safety margin is quite large. 7.7.14

Choline, Inositol, and p-Aminobenzoic Acid

The substances choline, inositol, and p-aminobenzoic acid are not actually essential vitamins but may be considered “conditional” B vitamins (Moran and Greene, 1979; Miller and Hayes, 1982). Humans under special circumstances require them. Choline, a basic constituent of lecithin and other phospholipids, is the precursor of the neurotransmitter acetylcholine and acts as a source of labile methyl groups for many reactions. It has been used in the treatment of alcohol- or protein deficiency–induced fatty liver without demonstrable benefits (Appel and Briggs, 1980a), whereas pharmacological doses seem to alleviate symptoms of tardive dyskinesia, Huntington’s disease, and other neurological disorders (Davis et al., 1976; Growden et al., 1977). Doses of up to 20 g/day for several weeks have been used; some patients have experienced depression, dizziness,

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nausea, diarrhea, abnormal electrocardiogram results, and a fishy odor of breath. myo-Inositol, the metabolic active isomer of inositol, is present in tissues as part of phospholipids and certain glycoprotein complexes. Although the metabolism of this substance is altered in diabetes and renal disease, the therapeutic use of myo-inositol for these conditions has had no observable benefits (Appel and Briggs, 1980b). It has been used experimentally to lower cholesterol level in hyperlipidemic patients, with some reports of significant reductions in β-lipoprotein cholesterol level (Agusti et al., 1978). Its toxicity appears to be relatively low, and humans fed 3 g myo-inositol per day or injected with 1 g of the substance showed no ill effects (Appel and Briggs, 1980b). p-Aminobenzoic acid (PABA) is sometimes included in multivitamin preparations; adverse effects from oral doses have not been reported. It has also gained acceptance as a sunscreen, and topical preparations containing PABA are widely used. There have been several reports of allergic contact dermatitis to PABA (Thompson et al., 1977; Kaidbey and Kligman, 1978; Mathias et al., 1978), which, although transient, may be quite severe, compounding the phototoxicity for which it is applied. PABA or other related substances are also capable of actually inducing photoallergic reactions (Kaidbey and Kligman, 1978) or systemic lupus erythematosus (PereyoTorrellas, 1978). 7.7.15

Ascorbic Acid (Vitamin C)

Vitamin C is also known as ascorbic acid, ascorbate, or ascorbate monoanion. Ascorbate is the biochemically active form of the vitamin (Figure 7.17). It is a cofactor or cosubstrate for eight isolated enzymes (Table 7.9). Three enzymes require ascorbate for proline or lysine hydroxylation in collagen biosynthesis, depending on whether proline or lysine is hydroxylated and where hydroxylation occurs on the amino acid. Two require ascorbate in the biosynthetic pathway for carnitine, which in turn is used for transmembrane electron transfers in ATP synthesis. Two enzymes, dopamine β-monooxygenase and peptidylglycine α-monooxygenase, contain an active site copper moiety and require ascorbate for hormone biosynthesis. The former enzyme is necessary for norepinephrine synthesis from dopamine. The latter mediates the amidation at the carboxy terminus of peptide hormones, thereby conferring stability to hormones such as thyrotropin-releasing hormone, corticotropin-releasing hormone (ACTH), vasopressin, oxytocin, and cholecystokinin. Ascorbate is also required for the enzymatic metabolism of tyrosine. It pri-

Figure 7.17 Ascorbic acid (vitamin C) and its oxidation products. Dehydroascorbic acid can be reduced back to the intermediate free radical and then to vitamin C. However, hydrolysis of dehydroascorbic acid (not shown here) is irreversible with a loss of vitamin activity.

marily acts as an electron donor (or reducing agent) for intra- and extracellular chemical reactions. Semidehydroascorbic acid (Figure 7.17) may be biochemically active, but it is more likely that its activity is based on its reduction to ascorbate. Likewise, dehydroascorbic acid action is indirect in that its activity is based on the reduction to ascorbate (Welch et al., 1995). The daily adult requirement for vitamin C, 45–80 mg, is based on preventing signs and symptoms of scurvy for at least 4 weeks if vitamin C ingestion ceased, esti-

Table 7.9

mates of catabolic rates of vitamin C metabolism, and the dose at which urinary excretion occurs (NAS, 1989). Ascorbate appears to be absorbed by active transport in the human gut by an intestinal transporter (Levine et al., 1996). Intestinal absorption of dehydroascorbic acid has not been well characterized. About 3% of the body’s vitamin C pool, which is 20–50 mg/kg body weight, is excreted in the urine as ascorbic acid, dehydroascorbic acid (a combined total of 25%), and their metabolites, 2,3diketo-L-gulonic acid (20%) and oxalic acid (55%). An in-

Ascorbic Acid and Enzyme Function

Enzyme Proline hydroxylase (EC 1.14.11.2) Procollagen-proline 2 oxoglutarate-3-dioxygenase (EC 1.14.11.7) Lysine hydroxylase (EC 1.14.11.4) γ-Butyrobetaine 2-oxoglutarate 4-dioxygenase (EC 1.14.11.1) Trimethyllysine 2-oxoglutarate dioxygenase (EC 1.14.11.8) Dopamine β-monooxygenase (EC 1.14.17.3) Peptidyl glycine α-amidating monooxygenase (EC 1.14.17.3) 4-Hydroxylphenylpyruvate dioxygenase (EC 1.13.11.27)

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Function

Reference

Collagen synthesis Collagen synthesis Collagen synthesis Carnitine synthesis Carnitine synthesis Catecholamine synthesis Peptide amidation Tyrosine metabolism

Peterkokshy and Udenfriend (1965) Kivirikko et al. (1989) Puistola et al. (1980) Lindblad et al. (1977) Dunn et al. (1984) Friedman and Kaufman (1965) Levine (1986) La Du and Zannoni (1961)

crease in excreted oxalic acid occurs only with a very high intake of ascorbic acid. Scurvy is caused by a dietary deficiency of vitamin C. The symptoms include femoral neuropathy, hair loss, edema, anemia, and psychological abnormalities including hypochondriasis, depression, and hysteria. Fatigue and lethargy are reported as relatively late symptoms (Levine et al., 1996). Vitamin C is perhaps the most controversial vitamin in terms of polarization of views on the benefits and risks of high doses. With the very extensive use made of ascorbic acid in therapy and the food trade, it is scarcely surprising that the level of safety has been the subject of extensive review over the past few decades. Vitamin C has little frank toxicity. Adverse effects, however, do occur and are dose-dependent (Levine et al., 1995, 1996). Diarrhea, abdominal bloating, or both can occur when several grams are taken at once, although there should be no need for ingestion of these large doses. Because ascorbate maintains iron in its reduced form, iron absorption is facilitated by ascorbate. In patients who are iron-overloaded, ascorbate may further enhance the iron overload or be otherwise harmful (Cohen et al., 1981; Nienhuis, 1981; Young et al., 1994). Data are conflicting concerning the effect of ascorbate on urate and oxalate excretion, cyanocobalamin depletion, mutagenicity, rebound scurvy, hypoglycemia, infertility, decreased immunological tolerance, and elevation of iron absorption to dangerous levels in normal individuals. These harmful effects have been mistakenly attributed to ascorbate ingestion (Levine et al., 1996; Marks, 1989). On the basis of the available evidence, the safe daily level for this vitamin is at least 100 times the RDA. 7.7.16

Interactions Involving Water-Soluble Vitamins

There is some evidence that the intake of some members of the B-group vitamins may affect the requirements of others (Marks, 1989). It is not clear whether this applies only at a low intake or whether there may be interactions involving high dosage. For practical purposes, vitamin deficiency rarely involves individual vitamins, and hence, at low dosage, the use of a mixed B group preparation is desirable. However, there is currently no evidence that a similar relationship applies in high-dose therapy.

7.8

MISCELLANEOUS FACTORS

In addition to the dietary deficiencies and excess consumption of various nutrients described, several factors in-

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terfere with or affect the metabolism and bioavailability of nutrients. Detailed discussion of such factors is beyond the scope of this chapter. However, for the benefit of the readers, the information provided in this chapter is summarized in several tables. The important functions and deficiency aspects of essential nutrients are summarized in Table 7.10. The physiological, pharmacological, and toxic doses of some vitamins are presented in Table 7.11. Among the fatsoluble vitamins, vitamin D is one of the most potent toxic vitamins, in which the range of therapeutic, pharmacological, and toxicological levels is the least. With respect to the water-soluble vitamins, the greatest attention has been devoted to vitamin C and niacin. At this point, it is also useful to consider the adverse effects of those compounds that inhibit the activity of a vitamin in a metabolic reaction. The collection of compounds involved in these phenomena are quite diverse and do not lend themselves to simple characterization. In the beginning, such compounds were observed to have similar structure, to produce symptoms similar to those produced by deficiencies of specific vitamins, and to compete with specific vitamins. They were, therefore, termed antivitamins. Subsequently, it was noted that antivitamins were of two basic types: structurally similar antimetabolites and structure-modifying compounds that inhibit the effect of a vitamin by forming inactive complexes with, or otherwise modifying, the molecule. Although the precise mechanism of action is not known in many cases, the antivitamins or vitamin antagonists are now recognized as a varied class of compounds that cancel or reduce the effect of a vitamin. Some examples of the antivitamins and their characteristics are shown in Table 7.12. Some of the more commonly occurring inborn errors of metabolism and principles of dietary therapy are summarized in Table 7.13. Some affect anabolic pathways that are involved in the synthesis of compounds important for cell metabolism and health or in the interconversion of substrates. However, most inborn errors of metabolism involve catabolic pathways, in which the accumulation of toxic intermediates negatively interferes with or destroys essential functions of specific tissues and organs. Principles of treatment include complete avoidance of nonessential nutrients that generate toxic metabolites (e.g., hereditary fructose intolerance and galactosemia). In other disorders, essential nutrients become toxic in and of themselves at high intracellular concentrations or generate toxic metabolites; thus, their intake must be restricted to the minimum adequate for normal growth and development. Examples of such disorders include the hyperphenylalaninemias (PKUs) and the hyperammonemias of the urea cycle disorders (Table 7.13). In other diseases, essential or

Table 7.10 Functions and Deficiency Aspects of Essential Nutrients Nutrient

Functions

Major nutrients Water

Transport of nutrients and waste products, thermoregulation, participation in metabolic reactions, lubrication of tissues and joints Protein Provision of essential amino acids required for metabolism, growth, and repair of all body tissues; precursors of structural protein, enzymes, antibodies, hormones, and metabolically active compounds Carbohydrate Storage and provision of energy, detoxification and elimination of unwanted substances, regulation of protein and fat metabolism Fat Storage and provision of energy, provision of essential fatty acids; cell membrane structure and function; precursors of prostaglandins; hormone synthesis; transport of fat-soluble vitamins and lipid precursors; insulation Water-soluble vitamins Coenzyme (TPP) in energy yielding and energy transfer Thiamine reactions (vitamin B1)

Riboflavin (vitamin B2)

Constituent of two flavin nucleotide coenzymes (FAD and FMN) involved in energy metabolism

Niacin

Constituent of two coenzymes (NAD and NADP) involved in oxidation-reduction reactions Coenzyme (PLP) involved in amino acid and fat metabolism

Pyridoxine (vitamin B6) Pantothenic acid Folic acid

Biotin

Choline Cyanocobalamin (vitamin B12) Ascorbic acid (vitamin C)

Constituent of coenzyme A, acyl transfer in lipid, carbohydrate, and protein metabolism Coenzyme involved in the transfer of one-carbon units (methyl donor), nucleic acid synthesis and formation, maturation of red blood cells Coenzyme in carboxylation and deamination reactions required for fat synthesis, amino acid metabolism, and glycogen formation Constituent of phospholipids, precursor of putative neurotransmitter acetylcholine Coenzyme involved in transfer of single-carbon units in nucleic acid metabolism, synthesis of red blood cells and nerve myelin sheath Coenzyme, synthesis of collagen and connective tissue, hormone synthesis, maintenance of intercellular matrix of cartilage, bone and dentine

Fat-soluble vitamins Vitamin A Constituent of rhodopsin (visual pigment); regulation of pro(retinol) tein synthesis, cellular differentiation, and growth; role in mucopolysaccharide synthesis

Deficiency diseases or symptoms Thirst, dehydration

Impaired growth and repair of tissue, lowered resistance to disease, kwashiorkor and, coupled with low energy intake, marasmus None known, energy deficits and ketosis with minimal intakes Poor growth, dry scaly skin, impaired absorption of fat-soluble vitamins

Beriberi (peripheral neuropathy, edema, heart failure), irritability, fatigue, emotional instability, depression, loss of appetite, growth retardation Cheilosis, angular stomatitis, glossitis, seborrheic dermatitis, itching and burning of eyes, increased sensitivity to light Pellagra, dermatitis, dementia, diarrhea, death Cheilosis, nervous system disorders, convulsions, muscular twitching, depression, vomiting, mucous membrane lesions, seborrhea Fatigue, sleep disturbances, anorexia, nausea, impaired coordination (rare in humans) Megaloblastic anemia, glossitis, gastrointestinal disturbances, diarrhea Fatigue, depression, nausea, dermatitis, muscular pain, anemia Not reported in humans Pernicious anemia, demyelination, neurological disorders Scurvy (degeneration of skin, teeth, blood vessels, epithelial hemorrhages)

Night blindness, xerophthalmia (keratinization of ocular tissue), permanent blindness, impaired growth, lowered resistance to infection (table continues)

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Table 7.10 (continued) Nutrient

Functions

Fat-soluble vitamins (continued) Vitamin D Regulation of protein synthesis in GI tract, calcium and (calciferol) phosphorus levels in the blood; promotion of growth and mineralization of bones and teeth; increase of absorption of calcium Vitamin E Function as an antioxidant to prevent cell membrane damage (tocopherols) Vitamin K Important role in blood clotting, involvement in the forma(phylloquinone, tion of active prothrombin menaquinone) Macroinorganic elements Sodium Major extracellular cation, acid-base balance, body water balance, nerve function, cell membrane transport, and osmotic equilibrium Potassium Major intracellular cation, acid-base balance, body water balance, nerve function, cofactor for protein synthesis and energy metabolism Chlorine Extracellular anion, formation of gastric juice, acid-base balance Calcium Bone and tooth formation, blood clotting, protein synthesis, nerve transmission, cell membrane transport, cofactor in enzyme systems Phosphorus Bone and teeth formation, acid-base balance, essential component of cellular energy transfer Magnesium Intracellular cation, cofactor for enzymes in energy metabolism and protein synthesis Trace elements Iron Zinc Copper Manganese Iodine

Constituent of hemoglobin and enzymes involved in energy metabolism, cellular respiration Cofactor for enzymes in energy, protein, and nucleic acid metabolism Constituent of enzymes associated with iron metabolism Cofactor for enzymes in protein synthesis and energy metabolism Constituent of thyroid hormones

Fluorine Molybdenum Chromium Cobalt Selenium

Improvement of crystallization of bones and teeth Cofactor for enzyme xanthine oxidase Cofactor in glucose transport with insulin Constituent of cyanocobalamin (vitamin B12) Constituent of selenoaminoacids (selenomethionine and selenocysteine) and glutathione peroxidase enzyme; function in close association with vitamin E

Silicon

Effect on metabolism of macromolecules such as collagen, elastin, and glycosaminoglycans

Deficiency diseases or symptoms Rickets (bone deformities) in children, osteomalacia in adults

Hemolytic anemia, creatinuria, ceroid deposits in smooth muscles Hemorrhage, impaired blood clotting mechanism

Hyponatremia, muscle cramps, mental apathy, reduced appetite, decreased extracellular fluid volume Hypokalemia, muscular weakness, paralysis

Muscle cramps, mental apathy, reduced appetite Stunted growth, rickets, osteoporosis, convulsions, tetany Bone pain and demineralization, anorexia, negative calcium balance Anorexia, growth failure, impaired nerve and muscle functions, spasms, behavioral disturbances, weakness Iron-deficiency hypochromic, microcytic anemia Impaired growth, anorexia, small sex glands, skin lesions, loss of sense of taste and smell Anemia, bone demineralization, low white cell count Poor growth, disturbances of nervous system, reproductive abnormalities Goiter (enlarged thyroid), impaired energy metabolism, listlessness Higher frequency of tooth decay Not known Impaired ability to metabolize glucose Not known or same as vitamin B12 deficiency Increased heavy metal toxicity and oxidative injury, alterations in thyroid metabolism, Keshan (cardiomyopathy) and Kaschin-Beck (osteoarthritis) disease Atherosclerosis, osteoarthritis, osteoporosis, hypertension, Alzheimer’s disease

TPP, thiamine pyrophosphate; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; NAD, nicotinamide-adenine dinucleotide; NADP, nicotinamide-adenine dinucleotide phosphate; PLP, pyridoxal phosphate.

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Table 7.11 Physiological, Proposed Pharmacological, and Toxic Doses of Some Vitamins

Vitamin

Physiological dose

Vitamin A

5000 IU

50,000–100,000 IU (Acne)

100,000–500,000 IU

Vitamin B6

2.0 mg

10,000 mg

Niacin

20 mg

50 mg (Against symptoms of oral contraceptives) 2000–5000 mg (Hypercholesterolemia)

β-Tocopherol

15 mg

300–1200 mg (Cardiovascular disorders)

Vitamin D

400 IU

Vitamin C

45 mg

50,000–100,000 IU (Hypophosphatemic rickets) 100–2500 mg (Colds)

Pharmacological dosea

Toxic dose

Manifestations of toxicity Headache, nausea, vomiting, pseudotumor cerebri Abnormal hepatic enzymes

100 mg 1000 mg 2000–6000 mg 1000 mg/kg (Animals)

1000–3000 IU (Children) 150,000 IU (adults) 2000–4000 mg

Cutaneous flush Carbohydrate intolerance Gastritis Increased deposition of cholesterol in aorta, decreased tolerance to ethanol Hypercalcemia, renal failure Induction of nephrolithiasis, induction of vitamin C–dependent syndrome, inactivation of vitamin B12, reversal of effects of anticoagulants, reproductive failure

a Not always accepted as proper treatment. Source: Compiled from Hodges (1976), Hambraeus (1982), and Deshpande and Sathe (1991).

nonessential nutrients become necessary and therapeutic when ingested in far greater than normal amounts; examples are carbohydrate therapy in glycogen storage disease type I and nicotinamide therapy in Hartnup disease. In certain disorders, nutrients, cofactors, or vitamins become therapeutic either in doses that are pharmacological rather than physiological or when administered parenterally rather than enterally. Examples include the entire family of vitamin- and cofactor-responsive inborn errors of metabolism, such as vitamin B12–responsive methylmalonic acidemia; vitamin B6–responsive homocystinuria; the disorders of biotin metabolism and usage, including biotinidase and holocarboxylase synthetase deficiencies; and

certain disorders of folate metabolism (Table 7.13). In many diseases, effective dietary treatment requires that natural foods be replaced with artificial nutrient mixtures in diets that lower intakes of potentially toxic nutrients while meeting essential nutritional requirements. Examples of such diseases include PKU, the urea cycle disorders, branched-chain ketoacidemia, methylmalonic acidemia, and propionic acidemia. In certain disorders, the enzymatic deficiency blocks anabolic pathways, thus rendering nutrients essential that are not usually so or raising the dietary requirements necessary to prevent nutritional deficiency and clinical disease. Examples include cholesterol in Smith-Lemli-Optiz syndrome, arginine in the urea

Table 7.12 Examples of Compounds Possessing Antivitamin Activity Antagonist (vitamin) Thiaminase (thiamine) Hypoglycin A (riboflavin) Niacytin and leucine (niacin) Avidin (biotin) Linatine (pyridoxine) Excess β-carotene (vitamin D) Polyenic acids (vitamin E) Dicumarol and related synthetic products (vitamin K)

Type of inhibition

Source

Modifies structure, deactivated by heat Mechanism unknown, counteracted by riboflavin Mechanism unknown, reversed by alkaline medium Forms complex, action prevented by heat Alters structure of all three pyridoxine forms Mechanism unknown Mechanism unknown, counteracted by vitamin E Mechanism unknown, may be competition in prothrombin synthesis

Varied, including plants, fish, and shellfish Ackee plum Wheat bran, millets Egg white Flaxseed Varied Varied Anticoagulants

Source: Compiled from Somogyi (1973, 1978).

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Table 7.13 Inborn Errors of Metabolism and Dietary Therapy Principle

Disorder

Complete avoidance of nonessential nutrients rendered toxic by the enzymatic deficiency Reduction of essential nutrient intake to a minimum to prevent toxicity Increased requirement for essential or nonessential nutrients

Vitamin- and cofactor-responsive disorders

Provision of specific nutrients to activate alternative pathways for toxic metabolite disposal Nutrients that inhibit normal metabolic pathways to prevent accumulation of toxic metabolites Provision of modified biochemicals that have become essential nutrients as a result of enzymatic deficiency

Nutrient

Galactosemia, hereditary fructose intolerance, urea cycle disorders

Galactose, fructose, nonessential amino acids

Hyperphenylalaninemias, urea cycle disorders, branched-chain ketoacidemia Hartnup disease, urea cycle disorders, Smith-Lemli-Opitz syndrome, biotinidase, prolidase deficiency, hereditary orotic acidemia Methylmalonic acidemia, homocystinuria, holocarboxylase synthetase, fatty acid oxidation disorders, hyperphenylalaninemias Isovaleric academia, fatty acid oxidation disorders

Phenylalanine protein, essential amino acids, leucine, isoleucine, valine Nicotinamide, essential amino acids, arginine, biotin, cholesterol, essential fatty acids, glycine, proline, uridine, hydroxylproline Vitamin B12, vitamin B6, biotin, riboflavin, biopterin, tetrahydrobiopterin

Urea cycle disorders, adrenoleukodystrophy, hereditary orotic aciduria

Arginine, citrulline, erucic and oleic acids, uridine

Functional methionine synthetase deficiency, glutathione synthetase deficiency

Hydroxycobalamin, glutathione alcohol esters

Glycine, carnitine

Source: Compiled from Scriver et al (1995) and Rhead (1996).

cycle disorders, nicotinamide in Hartnup disease, and biotin in biotinidase deficiency. In many disorders the provision of pharmacological amounts of specific nutrients activates alternate metabolic pathways, thus disposing of the accumulated toxic intermediates in the blocked primary metabolic pathway (e.g., isovaleric acidemia and carnitine therapy in disorders of fatty acid β-oxidation). Some new classes of nutritional therapies involve altering normal pathways of metabolism to prevent endogenous synthesis and accumulation of toxic compounds, such as using erucic and oleic acids in adrenoleukodystrophy to prevent synthesis of toxic very-

long-chain fatty acids from shorter-chain precursors (Table 7.13). Ethanol, a dietary substance with an energy value of 7.1 kcal/g (29.7 kJ/g), constitutes ~5% of the daily energy intake of U.S. adults when consumed in moderation (Mitchell and Herlong, 1986). When consumed in excess, it is the most widely abused drug worldwide, is unique in causing liver and other organ damage, and is responsible for 1 in 20 deaths in the United States (McGinnis and Foege, 1993). Nutrient deficiencies associated with excessive alcohol consumption are summarized in Table 7.14. Abundant evidence from studies of binge-drinking alco-

Table 7.14 Nutrient Deficiencies Associated with Alcohol Abuse Micronutrient Folate Thiamine Vitamin A Zinc Protein Fat

Cause

Consequences

Diet, malabsorption, oxidation Diet, malabsorption Diet, malabsorption, enhanced biliary excretion Diet, malabsorption, enhanced urine and fecal excretion Diet, malabsorption, catabolism Diet, malabsorption

Anemia, diarrhea, cancer Neurological (Wernicke), cardiomyopathy Night blindness, immunity, cancer risk Anorexia, immunity, skin rash, poor wound healing Muscle wasting, immunity Energy depletion

Source: Compiled from Halsted (1996) and Leevy et al. (1965).

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holics and animal models indicates that excessive alcohol consumption is probably the major cause of deficiencies of folate, thiamine, pyridoxine, vitamin A, and zinc in adult Americans (Halsted, 1996). These deficiencies are multiple in most instances and are more prevalent in the presence of alcoholic cirrhosis (Leevy et al., 1965). Similarly, the distinction between chemicals ingested as micronutrients and those ingested as drugs is often not clearly drawn. Recent examples of toxic syndromes resulting from the overly enthusiastic use of vitamins and amino acids as health supplements remind us that the traditional emphasis on the importance of balance in nutrition continues to be valid (Schaumberg et al., 1983; The clinical spectrum, 1990). In contrast, some drugs (e.g., neomycin) are prescribed for ingestion in multigram daily dosages that can influence nutrient disposition on a mass basis. Chemicals entering the GI tract together may combine in a way that markedly alters their absorption (Table 7.15). Although the literature has emphasized the reduced bioavailability of the more important or the limited member of a pair of complexing molecules, there may be other effects. The failure of tetracyclines to be absorbed after complexing with di- and trivalent cations is well documented in the literature. Similarly, the excessive use of antacids has been found occasionally to cause clinically significant phosphate depletion by the formation of insoluble aluminum or magnesium phosphates (Baker et al., 1974). A similar loss of dietary heavy metals occurs with the chronic ingestion of large quantities of plant-derived phytates, which are polyphosphate inositol derivatives that readily bind metals. Gastrointestinal diseases are yet another major cause of nutrient malabsorption. The nutritional health of humans is very much dependent on normal gut function, and many intestinal diseases result in disturbance of normal nutrition. The most striking disturbance of intestinal function is arguably the short bowel syndrome, in which insufficient intestinal length is present to allow normal

Table 7.16 Nutritional Consequence of Short Bowel Syndrome Area of restriction Duodenum Jejunum, ileum Distal ileum

Colon

Nutritional consequence Iron, folate, calcium Protein-energy, iron, water-soluble vitamins, trace elements, electrolytes Vitamin B12 steatorrhea and deficiency of fat-soluble vitamins caused by loss of bile acids Water, electrolytes

absorption. The degree and spectrum of the resulting malabsorption depend not only on the extent of restriction, but also on the site restricted (Table 7.16). In contrast, inflammatory bowel diseases, such as Crohn’s disease and ulcerative colitis, are commonly associated with significant malnutrition. In childhood, growth failure is a major feature and must be dealt with quickly and effectively before the potential for normal growth is lost. The combination of inadequate intake due to symptoms, increased enteric losses, malabsorption, and side effects of medications or inadequacy of prescribed special diets may lead to deficiencies of minerals and vitamins (Table 7.17). Finally, as a handy reference, the RDAs, minimal observed adverse effect levels, and safety margins for various macro- and micronutrients are summarized in Table 7.18. The preceding discussion of individual macro- and micronutrients indicates that consumption of a varied and balanced diet does not present significant food safety problems for normal, healthy individuals. Safety problems are invariably associated with particular deficits or excesses of individual nutrients or combinations thereof. To put the nutritional aspects of food safety in perspective, it

Table 7.17 Nutritional Deficiencies in Inflammatory Bowel Disease Table 7.15 Examples of Physical Interactions Between Nutrients and Drugs Agents Tetracyclines, phytates Neomycin, cholestyramine

Mineral oil Antacids

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Effects Bind heavy metals into nonabsorbed complexes Bind bile acids and lower absorption of lipid-soluble vitamins Dissolves and removes lipophilic drugs, vitamins Form insoluble phosphates

Protein-energy Trace elements Zinc Magnesium Selenium Vitamins Vitamin A Vitamin E Thiamine Riboflavin Pyridoxine Niacin

Table 7.18 Recommended Dietary Allowances, Minimal Observed Adverse Effect Levels, and Safety Margins for Various Macro- and Micronutrientsa

Nutrient Macronutrients Fat Carbohydrate Protein Micronutrients Vitamins Vitamin A Vitamin D Vitamin E Vitamin C Niacin Thiamine Pyridoxine Cyanocobalamin Biotin Minerals Iron Sodium Potassium Calcium Iodine Zinc Selenium Fluorine Copper Magnesium a

Unit Energy % Energy % Energy %

Recommended dietary allowance

Minimal observed adverse effect level

35 60 7

µg µg mg mg mg mg mg µg µg

1000 10 10 60 20 1.5 2 2 100

mg mg mg mg µg mg µg mg mg mg

10 500 2000 800 150 15 150 1 3 5

50 90 30

Safety margin 1.4 1.5 4.3

15,000 50 >900 >12,000 1000 >500 2,000 >100 >10,000

15 5 >80 >200 50 >300 1000 >50 >100

180 2,500 18,000 >2,500 >2,000 150 5,000 10 >35 10

18 5 9 >3 >130 10 33 10 >13 2

The recommended dietary allowances (RDAs) are expressed in terms of average daily intake over time for adults.

is important to examine overall dietary trends, diet modifications, and fad diets as well as the recommended national dietary goals and dietary guidelines in different populations. For example, with respect to the nutrient content of the diet in the United States, one of the most noteworthy changes since the 1980s has been the increase in fat intake. Increased fat intakes are directly correlated with increased risks of obesity and complications associated with it. One must also consider diet modifications. In the affluent Western societies, the trend has been toward increased consumption of pre-prepared or restaurant foods compared to traditional home-cooked meals. Similarly, the safety of fad diets, particularly the weight-reduction diets, has not yet been thoroughly addressed. The potential hazards associated with radical departures from normal diets (such as the ingestion of large excesses of protein, vitamins, and/or minerals) must be evaluated from a long-term nutritional viewpoint. In this regard, the best advice is still to try to

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achieve a balanced intake from a variety of foods, to use caution in undertaking radical changes in diet, and to avoid excessive dietary supplements of vitamins and minerals except when there is evidence of actual need. The relatively well-known classic diseases and related adverse effects associated with deficient intakes of the essential nutrients illustrate the basic importance of nutritional hazards in food safety. That importance is amplified by the less well-known, but nonetheless significant toxicity produced by excessive intakes of individual nutrients. Furthermore, complex interactions among nutrients can exacerbate the effects of both deficient as well as excessive nutrient intakes. Interactions between various drugs and excessive alcohol and nutrient intakes present still another facet of nutritional hazards. Fortunately, adequate diets present no significant hazards to normal healthy individuals. However, misuse of individual foods, drastic departures for weight loss or other purposes, and

excessive ingestion of nutrient supplements in the absence of demonstrated need, especially from unproven and unconventional sources, all can pose substantial hazards. Finally, the nature of nutritional hazards is such that the best available advice is moderation; eat a variety of foods, avoid excessive intake of calories from any one source, and avoid overconsumption of any food or nutrient. For those at high risk of major diseases, dietary advice from a qualified source is a prudent course of action. Drastic changes in diet including excessive ingestion of nutrient supplements should be undertaken only when the need to do so has been determined and guidance from a qualified source has been obtained.

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ed. D. Klimis-Tavantzis, pp. 4–37, CRC Press, Boca Raton, FL. Welch, R. W., Wang, Y., and Crossman, A. 1995. Accumulation of vitamin C (ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J. Biol. Chem. 270:12584–12592. WHO. 1990. Kashin-Beck Disease and Noncommunicable Diseases. World Health Organization, Geneva. Willett, W. C., Stampfer, M. J., Manson, J. E., Colditz, G. A., Speiger, F. E., Rosner, B. A., Sampson, L. A., an Hennekens, C. H. 1992. Intake of trans fatty acids and risk of coronary heart disease among women. Lancet 341: 581–585. Wittwer, C. T., Burkhard, D., and Ririe, K. 1983. Purification and properties of a panthetheine hydrolysing enzyme from pig kidney. J. Biol. Chem. 258:9733–9738. Yang, G., Chen, J., and Wen, Z. 1984. The role of selenium in Keshan disease. Adv. Nutr. Res. 6:203–231. Yang, G., Ge, K., Chen, J., and Chen, X. 1988. Selenium-related endemic diseases and the daily selenium requirement of humans. World Rev. Nutr. Diet. 55:98–152. Yip, R. 1995. Toxicity of essential and beneficial metal irons: Iron. In Handbook on Metal Ligands Interactions of Biological Fluid, ed. G. Berthon, pp. 179–221, Marcel Dekker, NY. Yip, R. and Dallman, P. R. 1988. The role of inflammation and iron deficiency as causes of anemia. Am. J. Clin. Nutr. 48:1295–1300. Yip, R. and Dallman, P. R. 1996. Iron. In Present Knowledge in Nutrition, 7th ed., eds. E. E. Ziegler and L. J. Filer, pp. 277–292. ILSI Press, Washington, D.C. Young, I. S., Trouton, T. G., and Torney, J. J. 1994. Antioxidant status and lipid peroxidation in hereditary haemochromatosis. Free Radic. Biol. Med. 16:393–397. Ziegler, E. E. and Filer, L. J. 1996. Present Knowledge in Nutrition, 7th ed. ILSI Press, Washington, D.C. Ziemlanski, S. 1977. Pathophysiological effects of long-chain fatty acids. Bibl. Nutr. Dieta. 25:134–139. Zock, P. L., de Vries, J.H.M., and Katan, M. B. 1994. Impact of myristic acid versus palmitic acid on serum lipid and lipoprotein levels in healthy women and men. Arterioscler. Thromb. Vasc. Biol., 14:567–575.

8 Food Additives

8.1

INTRODUCTION

Food additives are substances that are intentionally added to food to maintain or improve its appearance, texture, flavor, and nutritional value, as well as to prevent microbial spoilage. The definition of food additives has evolved considerably between the time of the first deliberations of a joint FAO/WHO committee of nutrition experts in 1955 and the present. Initially, the FAO/WHO Joint Expert Committee for Food Additives (JECFA) defined a food additive as “non-nutritive substances added intentionally to food, generally in small quantities, to improve its appearance, flavor, texture, or storage properties” (Davis, 1967). This definition was rather narrow and did not include flavorings and added nutrients. Since these early beginnings, the Joint FAO/WHO Codex Alimentarius Commission has broadened the scope of the definition: A food additive is any substance not normally consumed as a food by itself and not normally used as a typical ingredient of the food, whether or not it has nutritive value, the intentional addition of which to food for a technological (including organoleptic) purpose in the manufacture, processing, preparation, treatment, packing, packaging, transport or holding of such food results, or may reasonably be expected to result (directly or indirectly) in it or its byproducts becoming a component of or otherwise affecting the characteristics of such foods. The term does not include “contaminants” or substances added to food for maintaining or improving nutritional qualities.

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The official definition of food additives in the EEC is given in the Guide Directive 89/107/EEC (EEC, 1989) as follows: For the purposes of this Directive “food additive” means any substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value, the intentional addition of which to food for a technological purpose in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food results, or may be reasonably expected to result, in it or its byproducts becoming directly or indirectly a component of such foods. In contrast, the Food Protection Committee of the U.S. National Research Council (NRC)/National Academy of Sciences (NAS) defines food additives as “substances added to foods either directly or intentionally for a functional purpose, or indirectly during some phase of production, processing, storage, or packaging without intending that they remain in, or serve a purpose in the final product” (NAS, 1970). This definition thus also includes many substances that happen to be a component of the food, and these encompass just about everything. The legal definition of food additives in the United States is as follows: includes all substances not exempted by section 201(s) of the act [Federal Food, Drug and Cosmetic Act of 1938; U.S. Congress 1938], the intended use of which results, directly, either in their becoming a component of food or otherwise affecting the characteristics of food, including any substances in-

tended for use in producing, manufacturing, packing, processing, preparing, treating, packaging, transporting, or holding food (and including any source of radiation intended for such use), if such substance is not generally recognized, among experts qualified by scientific training and experience to evaluate its safety, as having been adequately shown through scientific procedures (or, in the case of a substance used in food prior to January 1, 1958, through either scientific procedures or experience based on common use in foods) to be safe under the conditions of its intended use. (U.S. Congress, 1959) The U.S. definition, therefore, excludes many substances that are technically considered as food additives. These include substances that are generally recognized as safe (GRAS), food colors, and food ingredients that have sanction for use in foods from the U.S. Food and Drug Administration (U.S. FDA) and the U.S. Department of Agriculture (USDA) before January 1, 1958. The U.S. definition also includes the so-called indirect or incidental food additives (see Chapters 16–18), such as components that migrate from food packaging materials that are in direct contact with the food products, residues of fumigants, solvents, and propellants, which have no desirable technical effect on the food products. According to this definition, pesticide residues are not food additives before a food is processed; they become additives after processing. Similarly, a contaminant of the ingredients added to a food product or an unsafe component of a container for foods is also considered a food additive. Another strange feature of the U.S. definition of food additives is the inclusion of sources of radiation as additives. The use of radiation presumably could contaminate food with radionuclides and, therefore, must be regulated. It must, however, be noted that the legal definition was so designed by the U.S. Congress solely for regulatory purposes. Furthermore, the status of a substance may change. For example, a substance that is GRAS and, therefore, not considered a food additive becomes one if it can be shown to be unsafe by contemporary testing methods; such a substance, therefore, is subject to legal tolerance specifications. Within the framework of public health legislation, national regulatory authorities are responsible for standard setting with regard to food safety. The authorities can carry out the process of standard setting as a separate national process or adopt standards set by international bodies such as the World Health Organization (WHO) and the European Union (EU). To achieve harmonization in food standards, many countries adopt standards set by the WHO. However, since 1992 the member countries of the EU are

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required to accept the decisions taken by the European Commission and enforce Union Standards into their own national legislation. Unlike the U.S. FDA and the EU, the Codex Alimentarius and FAO/WHO do not have any legal authority in enforcing the standards.

8.2

GENERAL PRINCIPLES FOR USE

Justification can be made for the use of each food additive. Whether such justification is valid or not must depend on the technical necessity of using the additive, the benefits derived by the consumer, and a demonstration of safety of the food additive. These conditions are regulated or specified by law, and no additive can be sold in the marketplace without satisfying these three conditions. The criterion of safety, of course, takes precedence over all else. In compliance with safety requirements, and if no other economic or technically practical means are available, the following six general principles issued by the Joint FAO/WHO Codex Alimentarius Commission (1973) may serve as justification for the use of additives in food: 1.

2. 3. 4.

5.

All actual or proposed food additives must be tested and evaluated toxicologically in all pertinent aspects, including cumulative, synergistic, and potentiating effects. Only food additives that are judged safe at the level of intended use will be endorsed. All food additives will be reevaluated in the light of new information on their use and safety. Food additives should always confirm to approved specifications, e.g., specifications approved by the Codex Alimentarius Commission. Justification for use of food additives may be one of the following, consistent with safety requirements, and if no other economical or technically practical means are available: a. Preservation of nutritional quality of food. Reduction in nutritive value of foods is justified for consumers with special dietary needs, and when the food is not a significant item in the diet. b. Usage for specific foods required by consumers with special dietary needs. c. Enhancement of the keeping quality, stability, and organoleptic properties of foods provided it does not drastically change the nature and quality of the food so as to deceive the consumer. d. Use as aids in food production, transport, and storage provided no deception as to

6.

8.3

faulty raw materials or undesirable practices or techniques is intended. Temporary or permanent approval of the use of a food additive must consider: a. Limitation to specific foods, purpose, and conditions of use b. Lowest level necessary to achieve desired effects c. Acceptable daily intake for humans or equivalent assessments, and probable daily intake from all sources, taking into consideration the probable intakes of special consumer groups.

SAFETY ASSESSMENT

Toxicological standard setting is a process carried out by legally qualified national authorities to protect the public health or the quality of the environment. A toxicological standard for a substance can be defined as a limit value for its content in food, drinking water, soil, or air. These standards are based not only on toxicological knowledge, but also on a thorough risk-benefit analysis. In the process of standard setting, toxicological guide values or healthbased recommendations are weighed against technical feasibility and check possibilities, and socioeconomical and political interests. Thus, standards are based on scientific as well as practical considerations. Such standards are only of value if they can be implemented and enforced. Usually, health-based recommendations or guide values are based on data obtained from toxicological studies in experimental animals, and only sometimes on observations in humans. Such safety evaluation is essential to identify the type(s) of adverse effect and to establish and quantify the dose-response relationships over certain periods. Adequate toxicological data thus are essential to determine the level at which human exposure to a substance can be considered safe. During the 1950s, the major principles of toxicological assessment concerning substances present in foods, either by accident or by design, as in the case of food additives, were elaborated. Protection of the health of consumers was the aim of these deliberations initiated by several international organizations such as the WHO, FAO, the International Union against Cancer, the Council of Europe, and the European Committee for the Protection against the Hazards of Chronic Toxicity, more commonly known as Eurotox (Pascal, 1999). In 1955, a joint FAO/WHO conference recommended that the general managers of the agencies organize regular meetings of a joint FAO/WHO committee of ex-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

perts to study the problems inherent in use of the food additives, thus resulting in the creation of JECFA. During its inaugural meeting in 1956, JECFA outlined the general principles that were to govern the use of the food additives, as described in the preceding section. They also insisted in particular on the principle of positive lists to protect consumers’ health. The main effect of this decision was to prohibit the use of any substance not authorized on sufficient toxicological grounds. During their second meeting in 1957, JECFA experts, in their final report, “Evaluation of Concentrations Probably Harmless to Man,” concluded: “From these various investigations a dosage level can be established that causes no demonstrable effect in the animals used. In the extrapolation of this figure to man some margin of safety is desirable.” The concept of acceptable daily intake (ADI) was beginning to take shape and was later elaborated by Truhaut during the sixth JECFA meeting in 1961. Since 1961, the definition of ADI has been discussed and clarified. The definition retained in the WHO report (WHO, 1987) is expressed as follows: “Acceptable daily intake: an estimate by JECFA of the amount of a food additive expressed on a body weight basis that can be ingested daily over a lifetime without appreciable health risk.” ADI is expressed as a range, from 0 to an upper limit in milligrams per kilogram (mg/kg) body weight, and is considered to be the limit of acceptability of the substance. As a rule, ADIs are given only for substances excreted by the organism within 24 hours, which consequently do not accumulate (Pascal, 1999). Sometimes, provisional ADIs are calculated for substances or additives when the available information does not warrant a final conclusion. For noncarcinogenic components, the ADI is derived from the no-observed-adverse-effect level (NOAEL). The highest dose with NOAEL found in animal studies is divided by a safety factor. In the case of food additives, the safety factor is generally set at 100. The 100-fold factor can be regarded as comprising two main factors of 10 each: 10 for taking into account the extrapolation from animals to humans, and 10 for taking into account a susceptible human subpopulation, such as infants. According to this principle, if a NOAEL may be defined from observations made in humans, a safety factor of 10 may be chosen (Lu, 1988). For the determination of the NOAEL, a series of doses is used. In order to establish the dose-effect relationship, the dose levels are chosen in such a way that the highest dose causes an adverse effect that is not observed after the lowest dose. Ideally, in a long-term toxicity study, the highest dose should evoke symptoms of toxicity without causing an excessive mortality rate, and the lowest

dose should not interfere with development, normal growth, and longevity. In between, sufficiently high doses should be selected to induce minimal toxic effects. The ADI does not imply an affirmation of total safety of the specific toxic effects and for the specific toxic effects for the entire human population. It simply indicates an extremely high probability that the products are safe at the permitted level of intake even with prolonged consumption by normal individuals. It does not address itself to the safety of the pertinent food additive in abnormal or disease states, in spite of the 100-fold safety factor. The ADI also is based on specific effects and, therefore, may not apply to certain toxic manifestations. Again, the ADI is also subject to uncertainties associated with applying results of animal tests to humans. The guide value is not called ADI for all food components. For environmental pollutants and contaminants that can accumulate in the organism, it is often expressed as tolerable daily intake (TDI), since such compounds are not added to food intentionally and are therefore tolerable rather than “acceptable.” For cumulative chemicals, JECFA included in 1972 a provisionally tolerable weekly intake (PTWI) (JECFA, 1972). PTWI is based on the observation that at a certain level of weekly intake, intake is balanced by elimination, and therefore no accumulation in the body takes place. Provisional means that the available safety data do not warrant a final conclusion. For carcinogenic compounds, ADI is not derived from an NOAEL. In the United States, for example, for substances indicated as carcinogens, and especially for components initiating cancer, a zero-risk approach is followed. The so-called Delaney clause of the Food Additives Amendment shows that some concern existed in 1958. This clause, sponsored by Rep. James J. Delaney of New York and others, banned from U.S. food and beverages any food additive found to be carcinogenic at any level in humans or animals. The clause states, “No additive shall be deemed to be safe if it is found to induce cancer when ingested by man or animals, or if it is found, after tests which are appropriate for the evaluation of the safety of food additives, to induce cancer in man or animal.” Much controversy has been generated over this clause since its passage. It is considered by some to be scientifically untenable because it is a zero tolerance law: i.e., if cancer is shown in any species when the chemical is given in any amount by any means, not even necessarily fed, then the substance must be banned. The way the law is written means that a substance that causes cancer in animals but is known not to be harmful to humans must nevertheless be banned. Several attempts have been made to amend the Delaney clause. At present, risk assessment of genotoxic

Copyright 2002 by Marcel Dekker. All Rights Reserved.

carcinogens presupposes that mathematical models are used to define minuscule risks, i.e., risks so low that they have no practical effect. To this purpose, a so-called calculated mortality procedure is used, involving linear extrapolation to a virtual low risk level (e.g., 106 over a lifetime). It is assumed that carcinogenesis starts with a cell mutation and that the risk of cancer development is related to the daily dose of the component concerned to the power m, which corresponds to the number of hits of the carcinogenic component that is necessary for the initiation of cancer. Generally, for m the value 1 is used: this is a conservative model in which exposure to a component and cancer incidence is linearly related. By using information on the carcinogenic dose of a component in animal experiments, the daily dose that would induce extra cancer incidence in humans can be estimated. Currently, the maximal tolerable extra cancer risk is estimated at 10–6 per lifetime. In 1985, the FDA commissioner, Frank Young, interpreted the Delaney clause in a new way, using the concept of de minimis. In this instance, two cosmetic color additives were added to the permanent listings. The decision was based on the fact that although these color additives did cause cancer in laboratory animals, the risk to humans was so trivial that it need not be considered under the law, hence, the de minimis status (Smith, 1987). The de minimis concept is currently being used by several regulatory agencies as an exception to the Delaney clause. It is used when a substance is known to cause cancer in laboratory animals but has a dietary risk that is deemed negligible. The substance is then allowed to be used. For regulatory purposes, negligible means that the lifetime risk of a cancer is increased by no more than 1 in a million (Federal Register, October 19, 1988, 53FR 41104 and 41125). This interpretation became necessary because analytical capabilities have increased so dramatically.

8.4

TYPES OF FOOD ADDITIVES

Approximately 2800 compounds are approved for food additive use in the United States. Approximately 1300 of these are food flavoring compounds that are used at quite low levels because of their low flavor thresholds. Also, most food flavors are composed of a multitude of compounds, and very seldom is only one compound utilized as a food flavorant. In contrast, the E list of additives permitted in Europe approaches only 400 compounds (Maga, 1995). According to the Federal Food, Drug and Cosmetic Act, there are five broad categories of compounds associated with human food. These include GRAS, which represents approximately 1600 substances; pesticide residues

and unavoidable contaminants; color additives; prohibited substances; and intentional food additives. The U.S. FDA introduced the GRAS list because many of these substances had a history of prolonged use with apparent safety, and there was a need to minimize the cost of safety testing. Initially, this list comprised around 687 items (Hall, 1973). The criteria that are used to judge whether or not a substance will be included in this list are as follows (U.S. FDA, 1983): 1.

2. 3.

4.

5. 6.

Common use in foods prior to January 1, 1958, without known detrimental effects or health hazards. Determination of safety by appropriate scientific tests. Determination of safety under criterion 1 or 2 by scientists “qualified by training and experience to evaluate the safety of substances added directly and indirectly to foods.” Substances included in the GRAS list can maintain their GRAS status only if the following additional criteria are met: Compliance with any applicable food-grade specifications of the Food Chemical Codex (NRC, 1972). Performance of appropriate function in the food product in which they are found. Usage at a level no higher than necessary to achieve the intended purpose in that food.

Substances that are naturally found in food that is widely consumed for a long time with apparent safety or are human or animal metabolites may be regarded as GRAS, depending on the level of their use in food. However, this view must be accepted with caution because it is not valid in several instances. Affirmation of GRAS status is subject to specific limitations as to the manner and condition of use, types of food in which the additives are to be used, functional use of additives, and level(s) of use in the food. Any significant deviation from the conditions or limitations on which the GRAS affirmation was based may automatically invalidate the original GRAS affirmation. In such cases, the manufacturer must establish that the new application or modification is GRAS; otherwise a food additive regulation specifying a prohibition of use, a tolerance level, or a condition of use will be imposed. In 1969, President Nixon directed the FDA to update the assessment of safety of all GRAS substances on the basis of current information. In 1972, the Select Committee on GRAS Substances (SCOGS) (1977) was formed under the auspices of the Federation of American Societies

Copyright 2002 by Marcel Dekker. All Rights Reserved.

for Experimental Biology to review the safety of all GRAS substances on the basis of published and other available information. The committee placed all reviewed substances in five categories: 1.

2.

3.

4.

5.

Category I are those substances whose GRAS status was reaffirmed. This means that the available information presented no evidence of toxic hazard of the substances in question. Category II are those substances whose GRAS status was reaffirmed at current level of use. This means that the available information presented no evidence of toxic hazard at the level of current use and practice. However, additional information is needed to determine whether increased consumption would constitute a hazard. Category III are substances whose safety is reaffirmed at the level of current use and practice. However, uncertainties exist so that additional studies are required to affirm their safety. Category IV are those substances for which information is incomplete to “reaffirm safety.” This means that evidence of toxicity has been reported; however, at the level and manner of current use the information is insufficient to determine whether the reported adverse effects have public health significance. Category V are those substances of which no biological studies are available with which to judge their safety.

On the basis of these classifications, the FDA may continue the GRAS status of those in category I with only good manufacturing practice (GMP) as the limitation. GRAS status may also be retained on those in category II, limited only by the amounts used. Substances in category III retain GRAS status in the interim until further tests on their safety are completed. Those in category IV require establishment of safe conditions of use or the GRAS status may be rescinded altogether. Those in category V may be placed on a doubtful classification until additional data are available for evaluation. Otherwise, the GRAS status may be rescinded as well. Since one of the prerequisites for food additive approval is effectiveness of function in a food system, one practical way to attempt to classify nearly 2800 compounds would be to use their technical effect or functionality in foods. Several types of food additives are listed in Table 8.1. In the following section, the potential toxic effects of selected food additives of several different classes are described.

Table 8.1

Types of Additives Classified by Their Functionality in Foodsa

Additive

Function

Anticaking agents and free-flow agents

Prevent caking and lumping

Antimicrobial agents (preservatives)

Prevent microbial growth

Antioxidants

Prevent rancidity and enzymatic browning

Clouding and crystallizing agents and inhibitors

Aid in crystal formation of sucrose and glucose; prevent crystallization of vegetable oils Impart desired colors

Colorants Condiments and seasonings Curing and pickling agents

Impart characteristic taste and/or flavor to foods Impart desirable, unique texture, flavor, and color of meat, vegetables

Dough conditioners and strengtheners

Modify starch and gluten of flour to produce more stable dough

Drying agents Emulsifiers

Absorb moisture Disperse fats and oils in aqueous medium to establish uniform dispersion or emulsion

Enzymes

Promote specific reaction in food processing

Firming agents

Precipite pectins to strengthen supporting tissues of pickles and other vegetables Impart characteristic flavor to foods, adjuvants; increase usefulness of flavors

Flavoring agents and adjuvants

Flavor enhancers

Flour-treating agents (e.g., bleaching and maturing agents) Formulation aids (carriers, binders, fillers, plasticizers, film formers, etc.) Humectants

Supplement, enhance, or modify original flavor without enhancing its own characteristic flavor Improve color and/or baking qualities of flour Promote or produce desired texture in foods

Malting aid

Retain moisture, prevent dusting, prevent drying out Produce CO2 in baked goods to impart light, fluffy texture Prevent foods from sticking on contact surfaces Provide bulk, plasticity, and mouth feel of gums Assist fermentation of barley

Nutritive sweeteners

Impart sweet taste

Leavening agents Lubricants and release agents Chewing gum base

Examples Calcium stearate, calcium silicate, magnesium carbonate, cornstarch, tricalcium phosphate, SiO2 Sodium benzoate, calcium propionate, potassium sorbate, sodium nitrite Ascorbic acid, BHT, BHA, EDTA ethoxyquin Methyl glucoside–coconut oil ester, oxystearin Carotenes and other natural pigments, synthetic food colors NaCl, vinegar, lactic acid, citric acid, lemon juice, and other acids Sodium nitrate, sodium nitrite, sodium chloride, sodium metaphosphate, ascorbic acid Potassium bromate, acetone peroxide, calcium phosphate, calcium sulfate, glyceryl monostearate Cornstarch, anhydrous dextrose Cornstarch, lecithin, mono- and diglycerides, sorbitan monostearate, polysorbates, propylene glycol, sodium lauryl sulfate Rennet, papain, pectinase, peroxidase, glucose oxidase Calcium salts, aluminum sulfate Spices and herbs, salt, soy sauce, and about 1500 natural and synthetic flavoring compounds MSG, disodium inosinate, disodium guanylate Acetone peroxide, benzoyl peroxide, potassium bromate Solvents for flavoring agents, starch, modified starch, gum acacia, magnesium stearate, sodium caseinate Sorbitol, propylene glycol, sodium tripolyphosphate Yeast, baking powder ingredients such as Na2CO3, CaCO3, NaH2PO4 Oleic acid, hydrogenated sperm oil and vegetable oils, mineral oil Chicle, rubber, paraffin, glycerol esters of rosin Gibberellic acid and its potassium salt, potassium bromate Sucrose, lactose, glucose, fructose, corn syrup, maple syrup, molasses, honey (table continues)

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Table 8.1

(continued)

Additive

Function

Nonnutritive sweeteners Nutrient supplements Oxidizing and reducing agents pH Control agents

Sequestrants Solvent vehicles Stabilizers and thickeners (includes suspending agents, bodying agents, setting agents) Surface active agents

Surface finishing agents Synergists

Examples

Impart sweet taste with less than 2% of caloric value of sucrose Increase nutritive value of foods Produce stable products by oxidation or reduction of food ingredients Act as buffers; reduce or increase acidity of foods Binds metals to prevent catalyzing oxidation of some food components Dissolve other additives such as colors and flavors Produce viscous solutions of dispersions; impart body, improve consistency, or stabilize emulsions Permit rapid wetting, solubilization, dispersion, rehydration, whipping, foaming, or defoaming Coat fruits and candies, increase palatability Increase effects of other specific food additives Preserve desirable appearance or feel of foods

Texturizers

Saccharin, cyclamate, aspartame, xylitol Most vitamins, minerals, amino acids, fatty acids, sugars Hydrogen peroxide, SO2, sodium sulfite, stannous chloride Acidifying: vinegar (acetic acid), HCl, H2SO4, citric acid; alkalizing: NaOH, NaHCO3 EDTA, citric acid, H3PO4, sodium metaphosphate Ethanol, glycerol, triacetin, acetone, propylene glycol, triethyl citrate Starch, modified starch, natural and synthetic gums, carrageenans, carob bean, hydroxypropyl cellulose, hyhdroxypropyl methylcellulose Dioctyl sodium sulfosuccinate, sodium lauryl sulfate, lactylic esters of fatty acids, dimethyl polysiloxane Various natural waxes, oxidized polyethylene, rosin, polyvinylpyrrolidone Citric acid, tricalcium phosphate, other phosphates NaHCO3, glycerine, corn syrup, modified starch

a

Indirect food additives are not included in this table. BHT, butylated hydroxytoluene; BHA, butylated hydroxyanisole; EDTA, ethylenediamine tetraacetic acid; MSG, monosodium glutamate. Source: From Concon (1988).

8.5

FOOD COLORS

The natural pigments associated with most fresh foods, especially fruits and vegetables, are vivid and brilliant. However, these pigments are subjected to adverse physical and chemical conditions during processing that cause their partial degradation. Processing therefore often results in undesirable changes in color and thus diminishes the visual perception of foods. In the developed countries, as much as 75% of the food is processed in some form before it reaches the consumer. Food manufacturers and processors therefore must replace the lost color if the acceptable attractive appearance is to be restored. Colors are primarily used in foods for the following reasons (NAS, 1971): 1.

2.

To restore the original appearance of the food when heat processing and subsequent storage have destroyed the natural colors To ensure uniformity of color as a result of natural variations in color intensity

Copyright 2002 by Marcel Dekker. All Rights Reserved.

3.

4. 5.

6.

7.

To intensify colors naturally occurring in foods to meet consumer expectations (e.g., fruit yogurts, sauces, and soft drinks) To help protect flavor and light-sensitive vitamins during shelf storage To give an attractive appearance to foods, such as colorless gelatin-based desserts, that would otherwise look unappetizing To help preserve the identity or character by which foods are recognized and thus aid in product identification To serve as visual indication of food quality

Both synthetic and natural food colors, therefore, play a significant role in enhancement of the aesthetic appeal of processed foods as well as in food manufacture, storage, and quality control. They are also important ingredients in many convenience foods such as confectionery, instant desserts, ice creams, snacks, and beverages, since many of these are naturally colorless. Colors are also used

to supplement the natural appearance of a given food system and to ensure batch-to-batch uniformity when raw materials have varying color intensities. Prior to 1900, there was no regulation in the United States on the usage of food colors in processed foods. Many of the colors were toxic, and their overuse often resulted in food poisoning. They were often used to make foods of inferior quality acceptable. The National Academy of Sciences (NAS, 1971) cites the following cases in which colors were deliberately used in foods to defraud the consumer or to disguise adulteration: 1.

2.

3.

4.

5.

6.

7.

In 1920, Frederick Accum mentioned the fate of a woman who habitually ate pickles colored green with copper sulfate while at her hairdresser’s and later became ill and died. Cheese colored with vermilion (HgS) and red lead (Pb3O4) also caused several cases of food poisoning. In a Manchester tea shop, stocks of copper arsenite, lead chromate, and indigo were found on hand to color used tea leaves for resale. Because of the lack of synthetic dyes, candies were generally colored with mineral pigments. An analysis of 100 samples of candy revealed that 59 contained lead chromate, 12 red lead, 6 vermilion, and 4 white lead (basic lead carbonate). In Boston, 46% of the candy sampled in the year 1880 contained one or more mineral pigments. The primary pigment found was lead chromate. In 1860, a caterer who wished to have a green pudding at a public dinner asked a druggist to provide a color. The copper arsenite he received and used caused two deaths. In London around 1900, the addition of yellow coloring to milk was so common that housewives refused to buy uncolored milk, thinking it had been adulterated. The yellow tint was commonly added to prevent detection of skimmed or watered milk. It was not until 1925 that British law prohibited the coloring of milk.

During the course of the 20th century, food colors have been evaluated rigorously for their technical suitability and toxicological properties. The stricter government guidelines have greatly reduced the range of colors available for food use. However, these guidelines and a stricter government policy have greatly helped to eliminate dishonest practices and protect consumer health and safety. In the following section, the toxicology of natural and synthetic colorants is described.

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8.5.1 History The discovery of the first synthetic dye, mauve, in 1856 is credited to Sir William Henry Perkins. This discovery was followed soon by the synthesis of a host of new and different synthetic dyes. These colors, as compared with natural extracts from animals, vegetables, and minerals, were superior in tinctorial strengths, hues, and stability. They were also readily available in many different shades and hues. Some of these synthetic dyes were used almost immediately in Europe. The United States first legalized the use of synthetic organic dyes in foods by an act of Congress that authorized the addition of coloring matter to butter in 1886. The second such act was passed in 1896 to add synthetic colors to cheese. However, by the year 1900, some 80 different food colors were being used in a wide variety of products including ketchup, jellies, cordials, butter, cheese, ice cream, candy, sausage, noodles, and wine. Colors were also increasingly being used in several drug and cosmetic products. The U.S. Congress soon recognized the proliferation in the use of color additives as a threat to public health. Of particular concern were that substances known to be poisonous were often incorporated into foods and that dyes were frequently used to hide poor quality and to add weight or bulk to certain items. Moreover, the synthetic colors, also known as aniline dyes, were manufactured from coal tar derivatives. Although they were sold in highly purified forms, the negative connotation of their association with coal tar resulted in much unfavorable publicity. This association also prompted the development of synthetic dyes from petrochemicals. The increasing public concern over the misuse of colors in foods prompted the U.S. government to initiate a comprehensive testing program to investigate the relationship of coloring matters to public health and to establish principles that should be followed to govern their use. By 1904, the U.S. Department of Agriculture issued a series of Food Inspection Decisions. Under the guidance of Dr. Bernard C. Hesse, a German dye expert, the department also undertook the arduous task of studying the chemical and physiological properties of the then nearly 700 extant coal tar dyes as well as the laws of various countries and states regarding their use in food products. Until then, very few colorants were ever tested for their effects on health. Of the 80 colorants used widely in foods in the United States by the turn of the century, 30 were never tested, and their safety was simply unknown. Of the remaining colorants, 26 had been tested but the results were contradictory: 8 were considered by most experts to be unsafe, and the remaining 18 were deemed more or less harmless. These 18 colors were then tested physiologically by deter-

mining their acute short-range effects in dogs, rabbits, and human beings. On the basis of these studies and Hesse’s recommendations, the first comprehensive legislation, the Food and Drug Act of 1906, listed seven dyes for use in foods: amaranth, Ponceau 3R, erythrosine, indigotine, Light Green SF, Napththol Yellow S, and Orange 1. These new regulations also established a system for voluntary certification of synthetic organic food colors by the Department of Agriculture. The original list of seven dyes, however, could not fulfill the industry’s need for additional colors. Hence, during the following three decades, several new colorants were added to the system after rigorous physiological testing. A chronological history of synthetic, certifiable food colors is presented in Table 8.2. In 1938, the Federal Food, Drug and Cosmetic Act, which superseded the act of 1906, established mandatory certification, requiring submission of samples from each batch of colorants for evaluation of purity. The act also created three new categories of synthetic coal tar dyes for various applications: 1.

Table 8.2

FD&C colors: those certifiable for use in coloring foods, drugs, and cosmetics

2.

3.

D&C colors: dyes and pigments considered safe in drugs and cosmetics when in contact with mucous membranes or when ingested External D&C colors: colorants that, because of their oral toxicity, were not certifiable for use in products intended for ingestion but were considered safe for use in products externally applied

Under this new law, all common names of dyes and colors were also changed to color prefixes and numbers. Thus certified amaranth became FD&C Red No. 2. During the following two decades, the situation with regard to color additives appeared finally to be under control. In the early 1950s, several cases of sickness of children who had eaten candy and popcorn colored with excessive amounts of dyes were reported. The Food and Drug Administration (FDA) also initiated a new round of pharmacological testing of food colors. These studies were conducted at higher levels and for longer test periods than any of the earlier test studies and hence refuted the assumption of safety of such widely used colors as FD&C Orange No. 1, FD&C Orange No. 2, and FD&C Red No. 32. After a series of

A Chronological History of Synthetic Food Colors in the United States

Year listed for food use

Common name

FDA namea

Color Index Number

Year delisted

Current status for food use

1907 1907 1907 1907 1907 1907 1907 1916 1918 1918 1918 1922 1927 1929 1929 1929 1939 1939 1939 1950 1959 1966 1971

Ponceau 3R Amaranth Erythrosine Orange 1 Naphthol Yellow S Light Green SD Yellowish Indigotine Tartrazine Sudan 1 Butter Yellow Yellow AB Guinea Green B Fast Green FCF Ponceau SX Sunset Yellow FCF Brilliant Blue FCF Naphthol Yellow S Orange SS Oil Red XO Benzyl Violet 4B Citrus Red No. 2 Orange B Allura Red AC

FD&C Red No. 1 FD&C Red No. 2 FD&C Red No. 3 FD&C Orange No. 1 FD&C Yellow No. 1 FD&C Green No. 2 FD&C Blue No. 2 FD&C Yellow No. 5 — — FD&C Yellow No. 3 FD&C Green No. 1 FD&C Green No. 3 FD&C Red No. 4 FD&C Yellow No. 6 FD&C Blue No. 1 FD&C Yellow No. 2 FD&C Orange No. 2 FD&C Red No. 32 FD&C Violet No. 1 Citrus Red No. 2 Orange B FD&C Red No. 40

16155 16185 45430 14600 10316 42095 73015 19140 12055 — 11380 42085 42053 14700 15985 42090 10316 12100 12140 42640 12156 19235 16035

1961 1976 — 1956 1959 1966 — — 1918 1918 1959 1966 — 1976 — — 1959 1956 1956 1973 — — —

Not allowed Not allowed Allowed Not allowed Not allowed Not allowed Allowed Allowed Not allowed Not allowed Not allowed Not allowed Allowed Not allowed Allowed Allowed Not allowed Not allowed Not allowed Not allowed Allowed Allowed Allowed

a FDA, U.S. Food and Drug Administration. Source: From Marmion (1984) and Ghorpade et al. (1995).

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legal battles in which the validity of the 1938 act to set quantity limitation was questioned, and through the efforts of the Certified Color Industry and the FDA, a new law was passed. The Color Additives Amendments of 1960 defines a color additive as “any dye, pigment or other substance made or obtained from a vegetable, animal or mineral or other source capable of coloring a food, drug or cosmetic or any part of the human body.” The law consists of two parts or titles. Title I of the Delaney-type clause prohibited addition to food of any colorant found to induce cancer in humans or animals. Title II of the clause allowed the use of existing color additives under a provisional listing pending the completion of scientific investigations needed to ascertain their safety for permanent listing. Synthetic colorants were thus required to undergo premarketing safety clearance, and previously authorized colorants were reevaluated. Natural colorants were also required to undergo similar testing but were not required to undergo certification for purity specification. The law also eliminated any distinction between “coal tar” colors and other color additives and empowered the secretary of health, education and welfare to decide which colors must be certified and which could be exempted from certification on the basis of their relationship to public health. The law also allowed the secretary to list color additives for specific uses and to set conditions and tolerances on the use of color additives. Under the provisions of this law, both producers and consumers of the color additives were required to provide the necessary scientific data to obtain “permanent” listing of a color additive. The economic considerations dictated testing of only those colors of commercial importance, and consequently many previously certifiable colors were eventually delisted by default. Most colorants are now “permanently” listed; those that are not continue to be listed provisionally. With the passage of the Medical Device Amendment of 1976, the Congress created a new category of color additive by mandating the separate listing of colorants for use in medical devices if the color additive in them had direct contact with the body for a significant period. Food colors that are currently allowed for use in foods in the United States and their current status are summarized in Table 8.3. To add any new colors to this list, the FDA (U.S. FDA, 1982) now requires results from the following toxicological studies: 1. 2. 3.

One subchronic feeding study, of 90-day duration, in a nonrodent species, usually dog Acute toxicity studies in rats Chronic feeding studies in at least two animal species, e.g., rats and mice (one with in utero exposure), lasting at least 24–30 months

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4. 5. 6.

One teratology study One multigeneration reproduction study using mice One mutagenicity test

The development and use of food colors in European countries have been somewhat different from those in the United States. For example, the United Kingdom did not establish any legislation for food colorants until the mid1950s (Haveland-Smith and Combes, 1980). Some countries still permit the use of any colorants, and others prohibit the use of all synthetic colorants for food applications. A major difficulty involved in the lack of a universal regulatory policy appears to be controversy regarding the physiological and pharmacological testing of synthetic colorants. Nevertheless, several synthetic colorants approved for use in foods by the FDA are also commonly used in Europe. The United Kingdom at present monitors food colors on the basis of a Color Index System. Hence, for the benefit of the readers, wherever applicable, the Color Index Numbers are also provided for the synthetic colorants approved for use in foods in both Europe and the United States. 8.5.2 Classification The color additives permitted for use in foods can be broadly classified into two categories: colorants subject to certification and those exempt from certification. A schematic diagram for the classification of various food colors is shown in Figure 8.1. Colorants Subject to Certification The synthetic food colors subject to certification are normally very pure chemicals with standardized color strengths. Because of the starting materials used in their manufacture in the past, certified colors have also been known as “coal tar” dyes. However, at present they are manufactured as a by-product of the petroleum industry. The chemical structures of several of these dyes are shown in Figure 8.2. Certified colors are available as water-soluble dyes or as insoluble pigments or “lakes.” The FDA considers lakes toxicologically equivalent to their corresponding dyes. The agency, however, has not yet established regulations for their use in foods. Except for the lake of FD&C Red No. 40, all lakes are provisionally listed by the FDA (Table 8.4). The soluble dyes readily dissolve in water and certain polyhydric solvents such as propylene glycol. They are available as is (“primary colors”) or as admixtures with other certified colors (“secondary mixes”). A nearly infi-

Table 8.3

A Summary of Colorants Permitted and Their Current Status for Use in Foods

FDA official name

Color index no.

Limitationsa

Current status

Subject to Certification FD&C Blue No. 1 FD&C Blue No. 2 FD&C Green No. 3 FD&C Red No. 3 FD&C Red No. 40 FD&C Yellow No. 5 FD&C Yellow No. 6 Citrus Red No. 2 Orange B

42090 73015 42053 45430 16035 19140 15985 12156 19235

Annatto extract β-Apo-8′-carotenolb Canthaxanthinb Caramel

75120 40820 40850 75130 (natural) 40800 (synthetic)

Orange skins only, 2.0 ppm max., based on weight of whole fruit Sausage and frankfurter casings or surfaces only, 150 ppm max., based on weight of finished products

Listed Provisional Listed Listed Listed Listed Provisional Listed Listed

Exempt from Certification

Carrot oil Cochineal extract and carmine Corn endosperm oil Dehydrated beets and beet powder Dried algae meal Ferrous gluconateb Fruit juice Grape color extract Grape skin extract Paprika Paprika oleoresin Riboflavinb Saffron Synthetic iron oxideb

Tagetes meal and extract Titanium dioxide Toasted, partially defatted, and cooked cottonseed flour Turmeric Turmeric oleoresin Ultramarine blueb Vegetable juice a

Maximum 15 mg/lb solid or semisolid food, or pint of liquid Maximum 30 mg/lb solid or semisolid food, or pint of liquid

75470 Feed only for enhanced color of chickens and eggs Not to exceed 15 mg/lb of food Feed only for enhanced color of chickens and eggs Ripe olives only Nonbeverage food only Beverages only

75100 77491 77492 77499 75125 77891

75300 75300 77007

Dog and cat food only, 0.25% (w/w) max.

Feed only for enhanced color of chickens and eggs 1% (w/w) Maximum in finished food

Salt for animal feed only, 0.5% (w/w) max.

Listed Listed Listed Listed

Listed Listed Listed Listed Listed Listed Listed Listed Listed Listed Listed Listed Listed Listed

Listed Listed Listed Listed Listed Listed Listed

No color additive or product containing one can be used in the area of the eye, in surgical sutures, or in injections, unless so stated. Also, no colorant can be used to color foods for which Standards of Identity have been promulgated under Section 401 of the Food, Drug and Cosmetic Act, unless the use of added color is authorized by the standard. b Synthetic but nature-identical.

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Food Colorants

Subject to Certification

Azo FD&C Red No. 40 FD&C Yellow No. 5 FD&C Yellow No. 6 Orange B Citrus Red No. 2

Exempt from Certification

Triphenylmethane

Xanthene

Sulfonated indigo

FD&C Blue No. 1 FD&C Green No. 3

FD&C Red No. 3

FD&C Blue No. 2

Nonsynthetic (Natural) Annatto Extract Anthocyanins Betalains Chlorophylls Caramel Paprika Turmeric Saffron Cochineal extract (carmine)

Figure 8.1

Carrot oil Carthamus yellow Corn endosperm oil Cottonseed flour Dried algae meal Grape color extract Grape skin extract Lutein Fruit and vegetable juices

Synthetic (Nature Identical) Beta-carotene Beta-apo-8′-carotenal Canthaxanthin Riboflavin Ferrous gluconate

Inorganic Titanium dioxide Ultramarines Iron oxides & hydroxides Calcium carbonate Silver Gold Aluminum

Classification of food colorants.

nite number of shades can be prepared by properly blending the available primary colors. FD&C dyes must contain a minimum of 85% pure dye; however, some that are commercially available have 90%–93% pure dye content. Law must certify each batch of colorant certified for purity prior to approval for use in foods. Certification requires that the color manufacturer submit a representative sample from each color batch to the FDA for chemical analysis. If the sample complies with the specifications, a certificate is issued and that batch of color is released for use (Kassner, 1987). The certification process ensures that every new batch is chemically identical to the pigment used in the animal feeding studies on which the approval of the color was based. The dyes are available as powders, pastes, granules, and solutions. In addition, the blends are available in jelly or fatbased sticks for convenience of use. Colorants Exempt from Certification The class of color additives exempt from certification comprises the so-called natural colors. Although according to the Color Additives Amendment Act of 1960 the exempt colorants need not be certified prior to their sale, they are still subject to surveillance by FDA to ensure that they

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meet the government regulations and are used in accordance with the law. All exempt colorants in use before the 1960 amendment continue to be provisionally listed, pending the completion of testing to obtain their permanent listing. Colorants exempt from certification could be broadly classified as nonsynthetic (natural), nature-identical, and inorganic colorants (Figure 8.1). The nonsynthetic colorants, which comprise a wide variety of organic and inorganic compounds, are extracted from animal, plant, and mineral sources. The nature-identical colorants are the synthetic counterparts of the naturally occurring pigments. The chemical structures of active ingredients of several of these natural colorants are shown in Figure 8.3. Several inorganic pigments as well as extracts from natural foods are also used as colorants in many parts of the world. In the United States, several of these are exempt from certification. However, they have been defined in the Code of Federal Regulations. 8.5.3 Toxicological Characteristics of Colorants Subject to Certification Colorants subject to certification in the United States include the FD&C dyes and the FD&C lakes. These colors

and their toxicological properties are briefly described in the following sections. Although only nine FD&C colors are permitted for use in food, several others that have been delisted in the United States are still used in many parts of the world. Hence, information is also provided on some of these delisted colorants. FD&C Red No. 1 FD&C Red No. 1 (Ponceau 3R, Color Index No. 16155) was one of the seven synthetic colorants approved for food use by the Food and Drug Act of 1906. This disodium salt of 1-pseudocumylazo-2-naphthol-3,6-disulfonic acid belongs to the monoazo group of synthetic dyes (Figure 8.2). FD&C Red No. 1 is a dark red powder that dissolves readily in water to yield a poppy red solution. FD&C Red No. 1 has proved to be a liver carcinogen when fed to both sexes of Osborne-Mendel rats at 0.5%, 1.0%, 2.0%, and 5.0% levels (Hansen et al., 1963; Mannell, 1964). Grice and associates (1961) have also reported a dose-related incidence of trabecular cell carcinoma of the liver. Its hepatocarcinogenicity has also been observed in mice and dogs. The toxicity of FD&C Red No. 1 appears to be due to its two trimethylaniline derivatives, mesidine and pseudocumidine, since xylidines were also shown to be hepatotoxic to rats and dogs (Magnusson et al., 1971). The hepatocarcinogenic nature of FD&C Red No. 1 led to its delisting in 1961. FD&C Red No. 2 FD&C Red No. 2 (amaranth, Color Index No. 16185) belongs to the monoazo group of synthetic dyes. It was also one of the seven original dyes certified for use by the Food and Drug Act of 1906. It is synthesized by coupling one mole of diazotized naphthionic acid with one mole of 2naphthol-3,6-disulfonic acid (Figure 8.2). Amaranth is a reddish brown powder that dissolves rapidly in water to yield a magenta red or bluish red solution. Early physiological studies of amaranth as up to 5% of the total diet found no pathological damage, mutagenicity, or increase in tumor incidence in rats (Cook et al., 1940; Willheim and Ivy, 1953; Mannell et al., 1958). Long-term, 7-year-duration studies conducted by the FDA also showed no evidence of pathological damage in dogs when the dye was fed at 2% level (U.S. FDA, 1974). Similarly, several studies in mice, rats, rabbits, hamsters, cats, and dogs have also shown no significant teratogenic, reproductive, or other harmful effects (WHO, 1975). Amaranth, therefore, was being considered for permanent listing. However, two studies from the former Soviet Union reported carcinogenic and embryotoxic responses

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in rats fed 0.8%–1.6% amaranth in the diet (Baigusheva, 1968; Andrianova, 1970). On the basis of the overwhelming data supporting the nontoxic nature of amaranth, FDA discredited these two studies. In early 1971, FDA initiated its own in-house study to confirm the embryotoxigenicity of amaranth in rats, which found a “statistically significant increase in a variety of malignant neoplasms among aged Osborne-Mendel female rats.” In 1975, FDA appointed a Toxicological Advisory Committee to consider all aspects of controversy surrounding FD&C Red No. 2. On the basis of all the available data, the committee concluded that the earlier pharmacological studies of FD&C Red No. 2 did not meet the then newly established rigorous standards of testing with respect to the number of animals and the extent of histopathological examination. On the basis of the committee’s recommendation, FDA terminated the provisional listing of amaranth in 1976. A subsequent petition for permanent listing of amaranth was denied in 1980, again on the basis of insufficient evidence (U.S. FDA, 1980). However, amaranth is still used to color foods in Canada, Japan, and several countries of the European Economic Community. FD&C Red No. 3 FD&C Red No. 3 (erythrosine, Color Index No. 45430), synthesized by the iodination of fluorescein, belongs to the xanthine group of dyes (Figure 8.2). Erythrosine is a brown-colored powder that yields a red solution with a slight fluorescence in 95% alcohol. In the United States, FD&C Red No. 3 has been approved for use in foods since 1907. Its use in foods, drugs, and cosmetics is widespread. With the exception of an International Research and Development Corporation (IRDC) study, most chronic and subchronic studies in rats, mice, gerbils, hamsters, dogs, and swine have shown it to be noncarcinogenic (Borzelleca and Hallagan, 1987; Borzelleca et al., 1987; Lin and Brusick, 1986). Its acute oral toxicity is low; the LD50 level in the rat is 7400 mg/kg body weight (Butterworth et al., 1976). Its property of being poorly absorbed is one reason for its low toxicity (Daniel, 1962; Parkinson and Brown, 1981; Radomaski, 1974; Webb et al., 1962). The IRDC study (1982), requested by the FDA prior to permanent listing of this dye, found adverse in utero effects and development of thyroid tumors in rats fed diets containing 4% erythrosine. Further investigations showed that the tumors were due to a secondary metabolism. No adverse effects, however, were observed at lower levels of 0.1%, 0.5%, and 1% dye in the diet. The FDA has determined that the Delaney clause does not apply to substances that act secondarily or indirectly, nor to those for

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Figure 8.2

(Facing page and above) Chemical structures of selected synthetic food colorants subject to certification.

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Table 8.4 Lakes Listed for Use in Food and Their Current Status Lake

Current status

FD&C Red No. 40 FD&C Yellow No. 6 FD&C Red No. 3 FD&C Blue No. 1 FD&C No. 2 FD&C Green No. 3 FD&C Yellow No. 5

Permanent Provisional Provisional Provisional Provisional Provisional Provisional

which no effect levels can reasonably be established (The latest status, 1985; Malaspina, 1987). Subchronic feeding studies indicated that erythrosine inhibits conversion of thyroxine to triiodothyronine, thereby causing increased secretion of thyrotropin by the pituitary gland. This effect in turn results in an increased stimulation of the thyroid and, hence, the tumor formation. No effect levels for the tumor formation process in male rats have been established at 0.5% (302 mg/kg body weight/day). Human studies, however, have failed to identify any adverse effects after the ingestion of FD&C Red No. 3 (Anderson et al., 1964; Bora et al., 1969). FD&C Red No. 3 is partially deiodinated in the gut to lower-iodinated fluoresceins. Its high iodine content has made researchers question whether it would have any effects on the thyroid (JECFA, 1987). In vitro studies indicate that erythrosine may inhibit neurotransmitters (Augustine and Levitan, 1980; Mailman et al., 1980). Several researchers have studied the effects of this dye on neurotransmitter release and iron transfer across cell membranes (Swanson and Logan, 1980; Augustine and Levitan, 1983; Silbergeld et al., 1983; Vorhees et al., 1983; Kantor et al., 1984). However, no conclusive evidence that was found linked this color to possible adverse behavioral effects. Other adverse effects have been reported, including effects on blood and gene mutations in some strains of Escherichia coli. In contrast, no mutations were seen when using the Ames test (JECFA, 1987). FD&C Red No. 3 is permanently listed in the United States and is approved for use by the EEC. A temporary ADI was set at 0.05 mg/kg (JECFA, 1989). However, Red No. 3 lake was never so listed and was banned by the FDA in 1990. FD&C Red No. 4 FD&C Red No. 4 (Ponceau SX, Color Index No. 14700) was approved for food use in 1929. This monoazo dye (Figure 8.2) is synthesized by coupling one mole each of

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diazotized 1-amino-2,4-dimethylbenzene-5-sulfonic acid and 1-naphthol-4-sulfonic acid. A red-colored powder, it is readily soluble in water, yielding orange-red solution. FD&C Red No. 4 was originally approved to color butter and margarine. It was found to be noncarcinogenic to rats when fed at 5% level in the diet for up to 2 years (Davis, 1966). These researchers, however, found the dye to be toxic to dogs. When fed at a 1% level in the diet for a period of 7 years, FD&C Red No. 4 produced chronic follicular cystitis with hematomatous projections into the urinary bladder, hemosiderotic focal lesions in the liver, and atrophy of the zona glomerulosa of the adrenals. In one study, three of the five dogs fed a diet containing 2% dye died prematurely within 6 months. Thus, its provisional listing as a permissible food color was terminated in 1976 (U.S. FDA 1983b). FD&C Red No. 32 FD&C Red No. 32 (Oil Red XO, Color Index No. 12140) was approved for food use in 1939. This monoazo dye (Figure 8.2) is synthesized by diazotizing one mole of xylidine mixtures from which the meta components are partially removed with one mole of 2-naphthol. It is a brownish red powder soluble in oil. FD&C Red No. 32 was found to be a strong cathartic in dogs and rats (Radomaski, 1961). It is also highly toxic to rats (Fitzhugh et al., 1956). Rats fed the dye at 0.1% level in the diet for 2 years showed growth retardation, damage to liver and heart tissue, and higher mortality rates when compared to those on control diets. Dogs thus treated also showed similar toxic effects (Fitzhugh et al., 1956). The dye was banned in 1956 for food use in the United States. FD&C Red No. 40 FD&C Red No. 40 (Allura Red AC, Color Index No. 16035) is a monoazo dye (Figure 8.2). Developed in the mid-1960s, it is manufactured by coupling diazotized 5amino-4-methoxy-2-toluenesulfonic acid with 6-hydroxy2-naphthalenesulfonic acid. It is the most widely used of all FD&C colors in the United States (Jones, 1992) and in 1970 became permanently listed for food use. Lifetime studies on rats and mice have indicated that Red No. 40 is neither carcinogenic nor teratogenic (Borzelleca, 1990; Borzelleca et al., 1989; Brown and Dietrich, 1983; Hazleton Laboratories, 1978; Parkinson and Brown, 1981; Vettorazzi, 1980). No consistent adverse effects have been shown except moderate growth depression in rats receiving the highest dose, over 5% of the diet (Borzelleca et al., 1989; Parkinson and Brown, 1981).

Red No. 40 has been reported to have some psychotoxicity on the basis of a controversial rat study (Vorhees et al., 1983). The rats were exposed to dye and then mated. The females were dosed during gestation, and the offspring were also fed this colorant. The offspring were then rated on a series of performance tests such as swimming. The test is controversial because doses as high as 10% of the diet were used. Although this test clearly indicates an adverse effect, opponents argue that these dose levels are particularly inappropriate for tests on psychotoxicity. Their rationale is that no person could ever eat anything approaching that amount of food color and that at lower levels no observable effects on performance could be seen. In the United States, FD&C Red No. 40 was approved for food use in 1971. However, it was refused similar legal status in Canada after the Health and Protection Branch (HPB) of Health and Welfare Canada concluded that the data submitted by the manufacturers to support its safety were inadequate (IFT, 1986). These toxicological studies were terminated after 21 months, when pneumonia swept the rat colony, instead of after the required 24month period. Although the Canadian HPB deemed these studies inadequate, FDA accepted the test results as being adequate proof of safety. Later studies proved its safety for food use, thus allowing its use in Canada. The dye, however, is not permitted for food use in the United Kingdom, Switzerland, Sweden, the Netherlands, and other countries of the EEC. However, an ADI was established by the JECFA in 1989 at 0–7 mg/kg body weight, with a maximal anticipated daily intake calculated at 0–19 mg/kg. Thus, similarly to FD&C Red No. 2, it has contradictory regulations in the United States and many other Western countries. Citrus Red No. 2 Citrus Red No. 2 (Solvent Red 80, Color Index No. 12156) is principally 1-(2,5-dimethoxy-phenylazo)-2-naphthol (Figure 8.2). It belongs to the monoazo group of dyes. Its use is limited to coloring skins of oranges not intended for processing. Toxicological properties of Citrus Red No. 2 have been well studied. Feeding studies in rats and dogs have shown it to be noncarcinogenic (Radomaski, 1962). In the same study, its metabolite, 1-amino-2-naphthol, was also found to be noncarcinogenic when fed to rats and dogs. However, several studies later reported this dye to be a carcinogen. When the dye was injected subcutaneously in female mice, Sharratt and associates (1966) observed an increased incidence of malignant tumors, such as adenocarcinomas of the lungs and lymphosarcomas. Clayson and colleagues (1968) also observed a similar, statistically

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significant increase in bladder cancer in mice after implantation of a pellet containing the dye into the lumen of the bladder. Dacre (1965) reported hyperplasia and benign tumors of the bladder in rats and mice fed diets containing Citrus Red No. 2. Although all these studies reported the carcinogenic nature of the dye, FDA still would not remove its listing as a permitted dye for coloring orange skins (U.S. FDA, 1983a). This decision was based on the opinions of Grasso (1970) and the JECFA (1969), who suggested that only those tests involving the oral route are relevant to the question of carcinogenicity of the ingested product. Similarly, a number of 2-N-hydroxy metabolites that are known carcinogens from compounds of these types have not been detected for Citrus Red No. 2 or any other permissible azo dyes. Furthermore, even if Citrus Red No. 2 is unequivocally shown to be carcinogenic by the oral route, it still would pose negligible hazards to humans, unless, of course, the dyed skins of oranges were ingested in products such as orange marmalade. FD&C Green No. 3 FD&C Green No. 3 (Fast Green FCF, Color Index No. 42053) belongs to the triphenylmethane group of dyes (Figure 8.2). It is synthesized by a condensation reaction of p-hydroxybenzaldehyde-o-sulfonic acid with α-(N-ethylanilino)-m-toluenesulfonic acid, followed by oxidation and conversion to a disodium salt. The dye is a reddish or brownish violet powder. It is readily soluble in water, yielding bluish green solutions. Toxicological studies have shown both Indigo Carmine and FD&C Green No. 3 to induce sister chromatid exchanges in bone marrow cells, when the dyes were injected intraperitonially into mice at doses exceeding 25 and 10 mg/kg body weight, respectively (Ghorpade et al., 1995). Earlier biochemical studies, however, had shown that this dye is poorly absorbed and almost completely excreted (Hess and Fitzhugh, 1955). It is not mutagenic by the Ames test (Parkinson and Brown, 1981). A 2-year study feeding FD&C Green No. 3 to rats, dogs, and mice at concentrations as high as 5% of the diet showed no carcinogenic or toxic effects (Radomaski, 1974). No-effect levels have been reported as 1500–4000 mg/kg/day in rats and 8800–11,800 in mice (Borzelleca, 1990). In 1986, JECFA established the ADI for humans at 0–25 mg/kg, with an anticipated maximal intake at 0.0003 mg/kg/day. Repeated subcutaneous injections of FD&C Green No. 3 have been shown to produce sarcomas in rats at the site of injection. The JECFA originally considered this finding cause for concern and judged the toxicological data inadequate to meet its requirements (Vettorazzi,

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Figure 8.3

Chemical structures of selected food colorants exempt from certification.

1980). FD&C Green No. 3 was introduced in 1929 and is currently a permanently listed dye for food use in the United States. However, it has been used in neither the United Kingdom nor the EEC. FD&C Blue No. 1 FD&C Blue No. 1 (Brilliant Blue FCF, Color Index No. 42090) belongs to the triphenylmethane group of synthetic dyes (Figure 8.2). It is manufactured by a condensation reaction involving benzaldehyde-o-sulfonic acid and α-(Nethylanilino)-m-toluenesulfonic acid. FD&C Blue No. 1 is a bronze-purple powder and dissolves readily in water, giving a greenish blue solution. Like other dyes of similar chemical class, FD&C Blue No. 1 is poorly absorbed (Brown et al., 1980). After administration, over 90% is recovered unchanged in the feces. Feeding studies in rats for over 2 years with this dye at concentrations as high as 5% of the diet showed no effect. The toxicological effects of this dye are concentration-dependent. Borzelleca and associates (1990) studied lifetime feeding of the dye in Charles River CD rats and CD1 mice. Dietary concentrations of 0.1%, 1.0%, and 2.0% were used in the rat study. In addition to two independent control groups, 70 animals of each sex were used at each dosage level. The exposure was begun in utero.

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The F0 rats were fed the dye for approximately 2 months prior to mating. The maximum exposure times for the F1 animals were 116 weeks for males and 111 weeks for females. Whereas the no-observed-adverse-effect level (NOEL) in males was estimated at 2% of the dye level, it was found to be only 1% for the female rats. The latter finding was based on a 15% decrease in the body weight and a significant decrease in survival rate when the dye was fed at 2% level in the diet. In the preceding study (Borzelleca et al., 1990), groups of mice (60 of each sex) were also fed the dye at 0.5%, 1.5%, and 5% levels. The maximal exposure time was 104 weeks for both sexes, and the NOEL was estimated to be 0.5% of the dye in the diet. As with other dyes in this class, injection of FD&C Blue No. 1 leads to tumors at the site of injection. Much controversy exists over the significance of these findings, as data from feeding studies indicate no tumors or other adverse effects. Regulators in the United States decided that the feeding data were the data critical to decision making for food color safety. FD&C Blue No. 1 was introduced for food use in 1929. After being tested for chronic toxicity and having undergone teratology and reproduction studies, it was listed permanently for food use in the United States. The

FAO/WHO, however, is critical of the adequacy of the research on it. It initially was not allowed as a food color in most EEC countries or the United Kingdom because of the tumors seen at the site of injection. The United Kingdom reinstated it after noting that many surface-active colors and substances caused sarcomas at the site of injection. Analysis of intakes in the United Kingdom showed that the average intake is at least 5000 times less than the ADI (Drake, 1980). Its ADI, according to JECFA, is 0–12.5 mg/kg, with an anticipated maximal intake of 0.022 mg/kg/day. FD&C Blue No. 2 FD&C Blue No. 2 (Indigotine, Indigo Carmine, Color Index No. 73015) was one of the seven original dyes allowed for food use by the Food and Drug Act of 1906. It belongs to the indigoid family of synthetic dyes (Figure 8.2) and is manufactured by the sulfonation of indigo dye. It is a blue, brown to reddish powder and is readily soluble in water, yielding blue solutions. When tested for at a concentration of 0.5 g/100 mL in cultures of E. coli, FD&C Blue No. 2 showed no significant mutagenic effects (Lueck and Rickerl, 1960). Similar observations were made in an FDA study with rats. Chronic toxicity studies even at the highest feeding levels in mice, rats, dogs, pigs, and beagles indicate that this substance is innocuous (Borzelleca and Hogan, 1985; Borzelleca et al., 1985; Parkinson and Brown, 1981; Radomaski, 1974). It is poorly absorbed (<5%) from the gut in rats (Lethco and Webb, 1966). As do certain other food dyes, this one produces tumors at the site of injection. The metabolic studies on this color are fairly complete and do not suggest any potential toxicity problems concerning its use in foods. FD&C Blue No. 2 was permanently listed in the United States in 1987. The Scientific Committee of the EEC, which states that the toxicological data are currently adequate, also allows it. The ADI is 2.5 mg/kg. As a comparison, the ADI established by the JECFA in 1969 was 0–17 mg/kg, with a calculated maximal daily intake of 0.009 mg/kg. The average intake in the United Kingdom is 1000 times less than the ADI (Drake, 1980). FD&C Yellow No. 3 and FD&C Yellow No. 4 Both FD&C Yellow No. 3 and FD&C Yellow No. 4 belong to monoazo group of dyes (Figure 8.2). FD&C Yellow No. 3 (Yellow AB, Color Index No. 11380) is prepared by coupling diazotized aniline and 2-naphthylamine in equimolar ratios. FD&C Yellow No. 4 (Yellow OB, Color Index No. 11390), in contrast, is synthesized by reacting diazotized

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o-toluidine with 2-naphthylamine. Both are orange-colored powders and are oil-soluble dyes. Both these dyes were introduced in the United States for food use in 1918 for coloring oleomargarines. Allamark and coworkers (1955) showed both to be hepatotoxic to rats and dogs. Even at 0.05% dietary levels, both these dyes induced significant weight loss in experimental animals (Hansen et al., 1963). Right-side cardiac atrophy and hypertrophy were observed in rats fed diets containing greater than 0.05% dye for a period of up to 2 years. These two dyes are metabolized under the acid conditions of the stomach to by-products of 2-naphthylamine (Harrow and Jones, 1954; Radomaski and Harrow, 1966). 2-Naphthylamine is a known liver carcinogen in mice and a bladder carcinogen in dogs (Clayson and Gardner, 1976). In the United States, both of these dyes were delisted from the category of certified colors in 1959. FD&C Yellow No. 5 FD&C Yellow No. 5 (Tartrazine, Color Index No. 19140) is a monoazo dye having pyrazoline ring structure (Figure 8.2). It is synthesized by condensing phenylhydrazine-psulfonic acid with oxalacetic ester. The reaction product is then coupled with diazotized sulfanilic acid. The resulting ester is then hydrolyzed with sodium hydroxide. Alternatively, tartrazine can also be synthesized by condensing two moles of phenylhydrazine-p-sulfonic acid with one mole of dihydroxytartaric acid. FD&C Yellow No. 5 is an orange-yellow powder. It is readily soluble in water, yielding golden yellow solutions. Chronic toxicity and carcinogenicity studies in rodents feeding at the 5% level showed no adverse effects of this colorant (Borzelleca and Hallagan, 1988a, 1988b). Mutagenic and other reproductive and developmental studies in rats and rabbits have not incriminated it (Burnett et al., 1974; Cordas, 1978; Pierce et al., 1974; Sobotka et al, 1977; Brown and Dietrich, 1983). After an extensive review of all the scientific evidence from tests conducted prior to tartrazine’s approval for food use, FDA concluded that the colorant is neither carcinogenic nor genotoxic. Tartrazine, however, is known to cause allergic reactions in a few sensitive individuals (Kevansky and Kingsley, 1964; Chafee and Settipane, 1967; Mitchell, 1971; Lockey, 1972). It has also been implicated in the induction of asthma (Chafee and Settipane, 1967). Azo dyes, particularly tartrazine, have been implicated in adverse food reactions involving immune mechanisms such as urticaria (Chafee and Settipane, 1967; Weber et al., 1979). Gerber and associates (1979) also noted nonimmunological reactions such as bronchospasm in asthmatic aspirin-intolerant individuals and those with chronic urticaria. Symptoms in

sensitive individuals include itching hives, tissue swelling, asthma, and rhinitis. Miller (1982) also noted that such symptoms appear more often in asthmatic allergic and aspirin-intolerant individuals than in the general public. Zlotlow and Settipane (1977) reported a clinical case of tartrazine-induced chronic urticaria in a 16-year-old white male patient. Population studies have indicated that of about 1 million aspirin-sensitive individuals in the United States, about 15% are also sensitive to tartrazine (Chafee and Settipane, 1974; Settipane et al., 1974; Tse, 1982). Studies by Loblay and Swain (1985) suggested that the reactions to tartrazine represented individual idiosyncrasy and that the propensity to react to tartrazine and other natural as well as artificial food colorants is probably genetically determined. Tartrazine does not inhibit the action of cyclooxygenase and has no effect on prostaglandin formation. This characteristic led Morales and colleagues (1985) to conclude that the tartrazine sensitivity in aspirinintolerant individuals is not surprising. It is not yet well documented or established that the impurities in the colorant, rather than the dye itself, may also be involved in such adverse reaction mechanisms. Listed for food use in the United States since 1916, tartrazine is the second most commonly used FD&C color. Most persons ingest tartrazine on a daily basis with a maximal per capita daily dose of around 16 mg. It was permanently listed for use in food in 1969 (Malaspina, 1987). The dye is also used in some 60 countries around the world for coloring foods. However, its association with allergic-type reactions in sensitive individuals has resulted in regulations that either limit its use or require declaration of its name in the list of ingredients (44FR 3721226, June 1976). The food can no longer be simply labeled as “artificially colored.” This requirement was extended to all FD&C certified colors in 1991. FD&C Yellow No. 6 FD&C Yellow No. 6 (Sunset Yellow FCF, Color Index No. 15985) is a monoazo dye (Figure 8.2). It is synthesized by coupling diazotized sulfanilic acid with 2-naphthol-6-sulfonic acid. The dye is an orange-red powder and is readily soluble in water, giving an orange-yellow solution. Similarly to amaranth and tartrazine, FD&C Yellow No. 6 also causes allergic reactions and induces urticaria in sensitive individuals. The controversies surrounding the dye are primarily due to the results obtained in an FDA lifetime feeding study of rats. In this study, in a number of female rats proliferative renal lesions developed when the dye was fed at 5% level in the diet. Such a high concentration would result in an average daily consumption of 3926 mg/kg/day, whereas the normal consumption in hu-

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mans for this dye is only about 0.15 mg/kg/day (Newsome, 1990). FDA later submitted the data to the National Toxicology Program (NTP) for a peer review (50FR 51455, December 1985). An earlier NTP study in which the rats received a lower dietary concentration of the color (1.25% and 2.5%) for a period of 24 months did not show any harmful effects related to the consumption of this dye. A concurrent study in mice also yielded negative results. The NTP Expert Committee, after reviewing the data provided by FDA, concluded that FD&C Yellow No. 6 is noncarcinogenic. NOEL for this colorant has been determined to be 500–1600 mg/kg/day in rats and 2600–11,400 in mice (CCMA, 1983). An ADI was established by the JECFA in 1982 at 0–2.5 mg/kg with an anticipated maximum of 0.11 mg/kg. Similarly to tartrazine, the use of FD&C Yellow No. 6 in foods must be accompanied by specific inclusion of its name in the list of ingredients. It is also approved for use in several countries around the world. Orange B Orange B is principally the disodium salt of 1-(4-sulfophenyl)-3-ethylcarboxy-4-(4-sulfonaphthylazo)-5-hydroxypyrazole (Figure 8.2). It belongs to the pyrazolone group of azo dyes. It is manufactured by reacting phenylhydrazine p-sulfonic acid with the sodium derivative of diethyl hydroxymaleate. It is then partially hydrolyzed to remove one ethyl group, followed by coupling with diazotized naphthionic acid (Marmion, 1984). Orange B is permanently listed as a synthetic food dye. Its usage, however, is restricted to a level not to exceed 150 ppm by weight and is allowed only in casings or on the surfaces of sausages and frankfurters. Orange B has been shown to have no adverse effects when fed at a 5% level to rats and mice and at a 1% level to dogs (NAS, 1971). The safe level for Orange B for human ingestion, calculated on the basis of 0.01% of the maximal NOEL established by the long-term animal studies for the most sensitive species and assuming a daily dietary intake of 1814 g/day for humans, was tentatively established at 181 mg/person/day. On the basis of the CCIC (1968) survey, the estimated maximal ingestion of Orange B in human nutrition was found to be only 0.31 mg/person/day. 8.5.4 Toxicological Characteristics of Colorants Exempt from Certification Natural food colors are either obtained from direct use of approved pigmented animal, vegetable, or fruit products or concentrated extracts from these materials or are their syn-

thetic equivalents (Table 8.4). The FDA has approved a number of such substances that are exempt from certification. However, the amounts used are limited (Freund, 1985). Unlike the certified colors, most of the natural colors have been subject to very limited toxicity testing. The toxicological properties of some of the important natural colorants are briefly described in the following. Nonsynthetic (Natural) Colorants Annatto Extracts The annatto extract pigments are extracted from the pericarp of the seeds of the annatto tree (Bixa orellana L.). Annatto extracts are prepared by leaching the seeds with one or more approved food-grade solvents such as edible vegetable oils and fats and alkaline and alcoholic solutions. The major coloring compound of the oil-soluble extract is the carotenoid bixin (Color Index No. 75120) (Figure 8.3). The mutagenic action of annatto extracts was tested at 0.5 g/100 ml in cultures of E. coli. No adverse effects were found (Lueck and Rickerl, 1960). The administration of aqueous extracts of bixa roots depresses the spontaneity of motor activity in mice; the intraperitoneal effective dose ED50 is 21 mg/kg body weight. The extract also affects the volume of gastric secretion but not its pH when injected intraduodenally at a 400-mg/kg level. Annatto extracts are also antispasmodic (1 mg/ml, isolated guinea pig ileum) and hypotensive (intravenous at 50 mg/kg body weight in rats) (Durham and Allard, 1960). Several long-term tests in mice and rats performed on well-defined types of extracts containing 0.1%–2.6% bixin, however, have not shown any carcinogenic potential associated with their use. Anthocyanins Fruit juices and grape skin extracts provide a wide spectrum of anthocyanins, which typically color foods blue, rose red, or violet. They are the glycosides of anthocyanidins consisting of 2-phenyl benzopyrillium (flavylium) structure (Figure 8.3). Widely used in a variety of food products, anthocyanins seldom pose any toxicity concerns. Dehydrated Beets (Beet Powder, Betalains, Betacyanins) Betalains are found in the members of the Centrospermae family of plants such as red beets, chards, cactus fruits, pokeberries, bougainvillea, and amaranthus flowers. The color additive beet powder is defined as “a dark red powder prepared by dehydrating sound, mature, good

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quality, edible beets” (Marmion, 1984). Betanin (Figure 8.3) is the principal pigment in beet colorant, accounting for 75%–95% of the total betacyanins. Pink lemonade can get its color from the addition of beet extract. Even though this colorant is natural, the beverage is labeled as artificially colored because lemonade is not naturally pink. Chlorophylls The most abundant of naturally occurring plant pigments, chlorophylls are the green and olive-green pigments (Figure 8.3) in green plants. Chlorophyll extracts are not permitted for food use in the United States. However, they may be added to foods in the form of green vegetables. In such cases, they are classified as food ingredients. Chlorophylls marketed as chlorophyll-copper complexes are permitted food colorants in Canada and Europe (Newsome, 1990). Caramel Caramel belongs to the group of melanoidin pigments responsible for the attractive red-brown color of cooked foods. Approximately 75%–85% of the caramel produced in the United States is used in soft drinks, especially root beers and colas. In toxicological studies, no abnormalities were detected after observation of animals for 14 days after administration of 12 different caramel color products (Foote et al., 1958; Chacharonis, 1960, 1963). Sharratt (1971) showed that a single dose of caramel, up to 10 g/kg body weight in mice and 15 g/kg in rabbits, did not cause convulsions or other signs of distress. Several short-term toxicological studies in rats at levels of 10–15 g caramel/kg body weight or 10%–20% caramel/kg body weight did not show any abnormalities or significant differences in results of histochemical and hematological studies (Prier, 1960; Haldi and Calandra, 1962; Key and Calandra, 1962). Similarly, many long-term toxicological and reproduction studies (WHO, 1975) also showed no adverse effects in up to three generations. Cochineal Extract (Carmine, Carminic Acid) Cochineal extract (Color Index No. 75470) is the concentrated solution obtained after the removal of alcohol from an aqueous, alcoholic extract of cochineal. It is obtained from the dried bodies of the female insect Coccus cacti (Dactylopius coccus costa). Cochineal extracts consist mainly of carminic acid (Figure 8.3). Short-term toxicological studies on cochineal extracts were conducted in mice and rabbits. When 1%–2%

aqueous solutions of the lithium salt of carminic acid were injected intraperitoneally for a period of 60 days, the only abnormality observed in mice was a proliferation of spleen tissue (WHO, 1975). Similar effects were also observed in rabbits given intravenous injections of 3–10 ml of 2%–4% aqueous solution of the lithium salt of carminic acid every 5–6 days. In another study, groups of 40 rats received cochineal carmine in 0.4% aqueous gum tragacanth at 0, 2.5, 5.0, and 10 mg/kg body weight, by intubation. The process was carried out 5 days per week for a period of 13 weeks. No adverse hematological effects were noted (WHO, 1975). Turmeric and Turmeric Oleoresin Turmeric (Color Index No. 75300) is the fluorescent yellow extract of the dried, ground rhizome of Curcuma longa. It is a perennial herb of the Zingiberaceae family native to southern Asia and is cultivated widely in China, India, South America, and the East Indies. Turmeric oleoresin is the combination of flavor and color principles obtained from turmeric by solvent extraction. The major pigment in turmeric, and its oleoresin is curcumin (1,6-heptadiene-3,5-dione-1,7-bis[4-hydroxy-3methoxyphenyl]) (Figure 8.3). They are often used to replace FD&C Yellow No. 5 in a variety of foods. It is a GRAS substance in the United States. Little is known about the toxicity of curcumin. Nature-Identical Colorants

β-Carotene (Provitamin A) β-Carotene (Color Index No. 75310) (Figure 8.3) is an isomer of the naturally occurring carotenoid pigment carotene. It was one of the first “natural” colorants synthetically produced on a commercial scale. Its use in food as a permitted colorant eventually led to the creation of the colorants exempt from certification category (Marmion, 1984). Unlike for other nature-identical carotenoid colorants, the FDA permits the addition of β-carotene to color foods at any levels consistent with good manufacturing practice (GMP). In humans, about 30%–90% of the ingested β-carotene is normally excreted in the feces. A concomitant intake of fat does not improve its absorption. Excessive doses of β-carotene depress the vitamin A activity of the absorbed fraction; only a small fraction appears in the serum. When dissolved in oil, as much as 10%–41% and 50%–80% of β-carotene is absorbed in adults and children, respectively (Fraps and Meinke, 1945). Although hypercarotenemia associated with excessive intakes of βcarotene is harmless and produces no adverse symptoms,

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it usually disappears when the vitamin intake is discontinued (Abrahamson and Abrahamson, 1962; Nieman et al., 1954). Bagdon and coworkers (1960) also noted the absence of hypervitaminosis in human volunteers given βcarotene over an extended period. Similarly, Greenberg and associates (1959) saw no symptoms of hypervitaminosis in 15 individuals who received daily doses of 60 mg of β-carotene over a 3-month period. Serum carotene levels rose from 120 µg/100 ml to a maximum of 308 µg/100 ml after 1 month, while vitamin A levels remained essentially unchanged. Long-term studies of up to four generations in rats fed 0 to 100 ppm of β-carotene daily for 110 weeks also showed no adverse effects in any of the generations (Bagdon et al., 1960).

β-Apo-8′-Carotenal (Apocarotenal) β-Apo-8′-carotenal (Color Index No. 40820) (Figure 8.3) is an aldehydic carotenoid that occurs naturally in oranges, spinach, grass, tangerines, and marigolds. It is available commercially as a synthetic color. In toxicological studies, no adverse effects were observed in dogs of both sexes when apocarotenal was fed at daily levels of 0.1 or 1 g per animal during a 14-week period. In other studies, three- to fivefold higher vitamin A levels were found in test animals as compared to the respective controls. Histopathological examination also showed pigmentation of the kidney, adipose tissue, and adrenal cortex (Bagdon et al., 1962). Canthaxanthin The carotenoid pigment canthaxanthin (Color Index No. 40850) (Figure 8.3) occurs naturally in an edible mushroom (Cantharellus cinnabarinus), sea trout, algae, daphnia, salmon, brine shrimp, and several species of flamingo. It has been available commercially as a synthetic food color since 1969. Toxicological studies conducted with three dogs of each sex fed 100 and 400 mg/kg body weight canthaxanthin for 1 week showed no adverse effects on body weight or general health of the animals as compared to those of the control dogs (WHO, 1975). Similarly, when tested at 1000-ppm levels, canthaxanthin did not produce any adverse teratological effects in three generations of rats. Along with β-carotene and apocarotenal, the FDA permanently lists canthaxanthin as an uncertified color additive (Dziezak, 1987). 8.5.5 Food Uses and Consumption Patterns Typical usages of certified and exempt colorants in various food systems are summarized in Tables 8.5 and 8.6. Data

Table 8.5

Food Applications of Synthetic Colors Regulated in the United Kingdom and the United States

Color

FD&C no.

Yellow/orange colors Tartrazine

Yellow No. 5

Yellow 2G Quinoline Yellow Sunset Yellow FCF

Yellow No. 6

Orange RN Orange G Red colors Carmoisine Ponceau 4R Amaranth Red 2G Erythrosine

Red No. 3

Allura® Red Citrus Red No. 2

Red No. 40 Citrus Red No. 2

Blue colors Indigocarmine Patent Blue V Brilliant Blue FCF Green colors Green S Fast Green FCF

Blue No. 2

Applications General-purpose, powdered desserts, confectionery, ice cream, dairy products, soft drinks, pickles, sauces, fish, bakery products General-purpose General-purpose, soft drinks, desserts, ice cream, dairy products, confectionery General-purpose, soft drinks (not recommended if calcium ions present), ice cream, canned foods, confectionery, baked goods, desserts General-purpose General-purpose Confectionery, soft drinks, ice cream, desserts, canned fruit Soft drinks, confectionery, jellies, canned goods, fish, lake to color cheese rind, and coated confections Canned foods, soft drinks, jams, ice cream, powdered desserts Meat products, sugar confectionery, jams Only red color used with maraschino cherries and glacé; also used in meat products, confectionery, and canned foods General-purpose Coloring of orange skins only

Blue No. 1

Confectionery General-purpose, confectionery, drinks, icings General-purpose, confectionery, drinks, icings

Green No. 3

General-purpose, often blended with yellow to produce leafy green hues General-purpose, oftend blended to produce various shades

Brown colors Brown FK Chocolate Brown FB Chocolate Brown HT

Coloring of fish in brine without precipitation Baked cereal products, sugar confectionery, desserts General-purpose, baked products, vinegar, confectionery

Black colors Brilliand Black BN Black 7984

General-purpose color used in blends, also in fish roe products and confectionery General-purpose

Source: From Marmion (1984), Newsome (1990), Rayner (1991), and Ghorpade et al. (1995).

on the major categories of processed foods manufactured with certified colors and the levels of colorant used are presented in Table 8.7. These figures were obtained by the Certified Color Industry Committee from a survey of sales records of certified colorants used by the various segments of the color industries (CCIC, 1968). Ten percent of the food in the United States contains added color (NAS/NRC, 1989). Because of the public concern over the increasing use of food additives in processed foods, the NAS and the NRC conducted an extensive survey of more than 12,000 individuals and estimated their average daily intake of food additives, including certified

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and exempt food colorants (NAS/NRC, 1979). The results are summarized in Table 8.8. The average daily intake of the certified FD&C colorants by Americans above the age of 2 years ranged from 3.1 to 100 mg/day/person, whereas that of colorants exempt from certification ranged from 0.43 to 250 mg/day/person. Because the U.S. food supply is very complex and different food colorants can be used interchangeably in foods to achieve similar technical effects, the NAS estimated actual intakes to be approximately one fifth of the reported amount. There is some concern that the intake in children is higher than that in the general population, as they are heavier consumers of foods

Table 8.6

Food Applications of Colorants Exempt from Certification

Anthocyanins (blue-red shades) Soft drinks, alcoholic drinks, sugar confectionery, preserves, fruit toppings and sauces, pickles, dry mixes, canned and frozen foods, dairy products Annatto extracts (orange shades) Oil-soluble bixin: dairy and fat-based products, processed cheeses, butter, margarine, creams, desserts, baked and snack foods Water-soluble norbixin: sugar and flour confectionery, cheese, smoked fish, ice cream and dairy products, desserts, custard powders, cereal products, and bread crumbs β-Carotene (yellow to orange) Butter, margarine, fats, oils, processed cheeses, water-dispersible forms in soft drinks, fruit juices, sugar and flour confectionery, ice cream, yogurts, desserts, cheese, soups, and canned products β-Apo-8′-carotenal (orange to orange-red) Cheese, sauces, spreads, oils, fats, ice cream, cake mixes, cake toppings, snack foods, and soft drinks Canthaxanthin (orange-red to red) Sugar confectionery, sauces, soups, meat and fish dishes, ice cream, biscuits, bread crumbs, salad dressings Paprika (orange to red) Meat products, snack seasonings, soups, relishes, salad dressings, processed cheeses, sugar confectionery, fruit sauces and toppings Saffron (yellow) Baked goods, rice dishes, soups, meat dishes, sugar confectionery Crocin (yellow) Smoked white fish, dairy products, sugar and flour confectionery, jams and preserves, rice and pasta Lutein (yellow) Salad dressings, ice cream, dairy products, sugar and flour confectionery, soft drinks Beet powder (bluish red) Frozen and short shelf life foods, ice cream, flavored milks, yogurts, dry mix desserts, jelly crystals Cochineal (orange) Soft drinks and alcoholic drinks Cochineal carmine (bluish red) Soft drinks, sugar and flour confectionery, flavored milks, desserts, sauces, canned and frozen products, pickles and relishes, preserves and soups Sandalwood (orange to orange-red) Fish processing, alcoholic drinks, seafood dressings, bread crumbs, snack seasonings, meat products Alkannet (red) Sugar confectionery, ice cream, alcoholic drinks Chlorophyll (olive green) Sugar confectionery, soups, sauces, fruit products, dairy products, pickles and relishes, jams and preserves, pet foods, drinks Copper chlorophyll (green) Flour and sugar confectionery, soups, sauces, pickles, relishes, fruit products, ice cream, yogurts, jelly, desserts, dry mix desserts, sauces and soups, soft drinks Caramel (yellowish tan to red-brown) Alcoholic and soft drinks, sugar and flour confectionery, soups, sauces, desserts, dairy products, ice cream, dry mixes, pickles, and relishes Malt extract (light brown) Alcoholic and soft drinks, sugar and flour confectionery, soups, sauces, desserts, dairy products, ice cream, dry mixes, pickles, and relishes Turmeric (bright yellow) Ice cream, yogurt, frozen products, pickles and relishes, flour and some sugar confectionery, dry mixes, yellow fats (table continues)

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Table 8.6

(continued)

Riboflavin (yellow) Cereal products, sugar-coated confectionery, sorbet, ice cream Vegetable carbon black (black) Sugar confectionery, shading color Orchil (red) Soft drinks, alcoholic drinks, sugar confectionery Safflower (yellow) Soft drinks, alcoholic drinks Titanium dioxide Sugar-coated confectionery Ferrous gluconate Ripe olives Iron oxides Sugar-coated confectionery, pet foods, meat and fish pastes Silver, gold, and aluminum Surface coating of sugar confectionery, cake decorations a Not all colors are permitted for food use in the United States. Source: From Marmion (1984), Newsome (1990), Rayner (1991), and Ghorpade et al. (1995).

that contain more coloring. Unfortunately, data on the intake of food colors by various segments of the population are not available. On the basis of U.S. food consumption patterns and the amount of FD&C colors certified by the FDA during the 1978–1981 period, Marmion (1984) estimated the

Table 8.7 Major Categories of Processed Foods Manufactured Using Certified Colors and Levels of Color Used Level of color used (ppm) Category Candy and confections Beverages (liquid and powdered) Dessert powders Cereals Maraschino cherries Pet foods Bakery foods Ice cream and sherbets Sausage (surface) Snack foods Miscellaneousa a

Range

Average

10–400 5–200 5–600 200–500 100–400 100–400 10–500 10–200 40–250 25–500 5–400

100 75 140 350 200 200 50 30 125 200 —

Includes nuts, salad dressings, gravy, spices, jams, jellies, and food packaging. Source: From CCIC (1968).

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consumption of certified color additives as 0.024 lb/day/ person. Labeling Issues When color is added to a food, the label must state artificially colored or artificial color added. The term natural color may not be used even if the color is derived from nature. This labeling is intended to protect the consumer so that there can be no misunderstanding about whether it is the actual color of the item or one that has been enhanced by the addition of color of any kind. The alternate way to label the product is to declare on the label “colored with _________” or “__________ color,” where the blanks are filled in with specific name(s) of the colorant(s) used. The declaration of the specific colorant FD&C Yellow No. 5 (tartrazine) on the label has been required since 1986, since some individuals are sensitive to it. However, the Nutrition Labeling and Education Act passed by the U.S. Congress in November 1990 required that all certified colors be shown on the label after November 1991. 8.5.6 Food Colorants and Hyperkinesis Food additive–induced hyperkinesis is characterized by several types of aberrant behavior whereby individuals show one or more signs of the following: hyperactivity and fidgetiness, compulsive aggression, excitability, impul-

Table 8.8 Colorants

Consumption of Certified FD&C and Exempt Food

Category/colorant

Average daily intake (mg/kg/person)a

Certified FD&C colorants FD&C Red No. 3 FD&C Red No. 40 FD&C Blue No. 1 FD&C Blue No. 2 FD&C Yellow No. 5 FD&C Yellow No. 6 FD&C Green No. 3 Orange B FD&C Red No. 3 Lake FD&C Red No. 40 Lake FD&C Blue No. 1 Lake FD&C Blue No. 2 Lake FD&C Yellow No. 5 Lake FD&C Yellow No. 6 Lake

24 100.44 16 7.8 43 37 4.3 17.8 15 27 6.6 3.1 22 14

Colorants exempt from certification β-Apo-8′-carotenal Annatto extract Paprika Paprika oleoresin Turmeric Turmeric oleoresin Saffron Cochineal extract (carmine) Grape skin extract (enocianina) Beet powder (dehydrated beets) Carrot oil Canthaxanthin

2.0 3.2 61 2.7 4.7 0.44 0.43 7.1 46 23 250 250

a

Data represent the 99th percentile of persons over 2 years of age in the “eaters group” (those who consumed one or more foods containing the additive in question during the 14-day survey period). Ninety-nine percent of the population sampled was estimated to have intakes equal or below the value shown. Total sample size was 12,000 persons. Source: From NAS/NRC (1979).

siveness, impatience, short attention span, poor sleep habits, and gross and fine muscle incoordination. Such behavior is generally accompanied by learning disabilities in the form of difficulty in reasoning, lack of auditory and visual memory, and difficulty in understanding ideas and concepts. Several clinical trials have confirmed that additive-free diets can indeed improve the behavior of hyperkinetic children (Brenner, 1977; Conners, 1980). Such studies, however, should not be expected to provide definite conclusions about the hyperkinetic effects of food additives, especially the synthetic food colors.

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Among other findings, these studies are highly subject to the placebo effect (Spring and Sandoval, 1976; Harley and Matthews, 1977; Wender, 1977). Harley and colleagues (1978), while studying nine boys selected from a group of 46 hyperactive subjects, found that only one subject responded with increased undesirable behavior when challenged with cookies and candy bars containing 26 mg of a blend of eight approved food colors. In a similar study, Weiss and coworkers (1980) observed that, among 27 hyperactive children (22 males and 5 females, aged 2.5–7 years) who previously responded favorably to additivefree diets, only 2 showed statistically significant adverse responses when challenged daily with a mixture of about 35 mg of certified FD&C colors. One 3-year-old boy had a mild response on several criteria. The food colors used in this study were FD&C Yellow No. 5, FD&C Yellow No. 6, FD&C Red No. 40, FD&C Red No. 3, FD&C Blue No. 1, FD&C Blue No. 2, and FD&C Green No. 3. Yet another aspect of such double-blind studies was that the food colors elicited hyperkinetic behavior rapidly and briefly. This is contrary to Feingold’s (1975) initial claim that the effects persisted for several days. Thus if two different observers were to note behavioral changes several hours apart, they might report opposite effects. For example, Williams and associates (1978) have reported that only the teachers, who were in a better position to observe early behavioral changes than were the parents, noted the improvement or worsening of the hyperactive behavior of test children. Goytte and colleagues (1978) and Conners (1980) have observed hyperkinetic effects within 3 hours after the food additive challenge. Levy and coworkers (1978) reported that significant effects in terms of deterioration in behavior could be detected only when measured within a few hours after a tartrazine challenge; the effect could not be observed after 24 hours. The failure of some earlier studies (Harley et al., 1978; Levy et al., 1978; Swanson and Kinsbourne, 1979a, 1979b) to detect effects of food additive challenges may have been due to the low doses (1 to 26 mg of food colors) used in their experiments. Thus when much higher doses (up to 150 mg) of certified FD&C food color blends were used, Swanson and Kinsbourne (1980) were able to document impaired behavior on a laboratory learning test based on the Conners scale in all 20 confirmed hyperactive children. Once again, the measurements were made within 3.5 hours after the challenge with a blend of nine dyes. The nonhyperactive children, in contrast, did not show any adverse effects. The amount of food color used in this study, according to an FDA estimate, was at the 90th percentile for artificial food colors consumed by 5- to 12-year-old children in the United States (Sobotka, 1976). Swanson

and Kinsbourne (1980) have commented that the time course of the appearance of the effect, i.e., the initial appearance of 0.5 hour after administration, peaking at 1.5 hours and lasting up to at least 3.5 hours, suggested that the food additive effect was nonimmunological. The nonimmunological nature of the hyperkinetic effect of food colorants was also observed in animal studies. Mailman and associates (1980) showed that the administration of 50–300 mg/kg body weight FD&C Red No. 3 (erythrosine) to rats attenuates the suppressive effect of punishments monitored by the number of electric shocks received by the animals in an approach-and-avoidance test. This observed effect in rats is similar to that seen with barbiturate and benzodiazepine drugs, which also aggravate hyperkinesis in humans. In contrast, amphetamine reverses such effects (Cantwell, 1975). Levitan (1977) has reported membrane interactions with FD&C Red No. 3 dye, whereas Logan and Swanson (1979) have observed that this dye also decreases the uptake of several neurotransmitters by homogenates prepared from rat brains. However, at least with dopamine, a major portion of the observed effects may have been an artifact resulting from its nonspecific interaction with brain membranes (Mailman et al., 1980). The dye also irreversibly increases acetylcholine release when applied to isolated neuromuscular synapses in the frog (Augustine and Levitan, 1980). The neurotransmitter release, which normally depends on the presence of calcium ions in the presynaptic terminals, was also independent of its concentration. The studies cited essentially support the basic premise of Feingold that food additives do induce certain behavioral changes in humans. However, at least with the artificial food colors, the hyperkinetic syndrome in humans may be induced or exacerbated in a subset of children. The evidence also shows that the basic mechanism may involve the central nervous system. Thus one important aspect of food toxicological mechanisms, the behavioral toxicity of food components, is underscored by the experience with artificial food colors. These studies perhaps will create a greater interest in this field and the unexplored aspects of behavioral food toxicology.

8.6

SWEETENERS

Sweeteners present the consumers with one of the most important taste sensations. This is reflected by the world production of sugar, which increased from 8 million tons in 1900 to over 90 million tons in 1990s. For nutritional and health reasons, however, there is a growing need for sugar substitutes in food that are nonnutritive, i.e., noncaloric and noncarcinogenic.

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Noncaloric sweeteners lead all other food additives in dollar sales, and their use appears to be growing. Concerns about sweeteners seem to grow along with their acceptance. The banning of cyclamate, the subsequent controversy about saccharin, and spurious reports about aspartame (Roberts, 1990) have raised consumer concerns about the safety of sweeteners and other additives. The cyclamate banning sparked a systematic review of all additives. Interest in artificial sweeteners continues because of the strong interest in dieting and because saccharin, aspartame, and other sweeteners are frequently in the news (Lecos, 1985; Stamp, 1990; Jones, 1992). The toxicological properties of three of the most important sweeteners, viz., cyclamates, saccharin, and aspartame, are described in the following sections. 8.6.1 Cyclamates The sodium and calcium salts of N-cyclohexylsulfamic acid (Figure 8.4), commonly known as cyclamates, were introduced commercially in 1950. These synthetic sweeteners are about 30 times as sweet as sucrose, and about one tenth as sweet as saccharin. Cyclamate’s sweetness coupled with a less bitter aftertaste than saccharin made it very viable commercially as an artificial sweetener. Cyclamates are sold generally as a mixture of 1 part saccharin and 10 parts cyclamate. A commercial brand, Sucaryl, containing such a mixture was widely available in the United States prior to its banning in 1970. Since cyclamate was 20 times less expensive than saccharin, although less sweet, the use of cyclamate-saccharin mixtures soared. In 1955, the NAS reported cyclamate safe for human consumption. Since it was used in the food supply before 1958, cyclamate was classified as GRAS with the passage of the Food Additives Amendment Act. In the early 1960s, because of its popularity and increasing consumption, especially in carbonated beverages, the FDA again requested the NAS to assess the safety of cyclamate at the then-current levels of use. The NAS reported to the FDA that although reasonable quantities of cyclamate posed no hazard to humans, additional studies were needed to resolve some questions about its safety. In 1969, Oser (1975) revealed the results of their experiments whereby Wister-derived rats were fed a 10:1 cyclamate/saccharin mixture, and after 78 weeks, some of the animals were fed cyclohexylamine. The rats were on treatment for 104 weeks, at 500, 1120, or 2500 mg mixture/kg body weight. Papillary carcinomas of the bladder were reported in 12 (9 males, 3 females) survivors that were fed 2500 mg/kg; none was found with the lower doses or the control. When the FDA learned of the results, cyclamates were removed from the GRAS list on October

Figure 8.4

Chemical structures of nonnutritive sweeteners.

18, 1968. The FDA further ordered in 1969 the halting of the production of general-purpose products containing cyclamates. In addition, all beverages and packaged mixes containing cyclamates were ordered removed from the marketplace beginning January 1, 1970. Two other studies in mice have shown cyclamates to be tumorigenic. Bryan and Erturk (1970) implanted cholesterol pellets containing one part sodium cyclamate and four parts cholesterol into the bladder of 60- to 90-day-old mice; all surviving animals were killed at 13 months. Bladder tumors were observed in 34/69 of treated animals compared to 13/106 of control (p < 0.001). Rudali and colleagues (1969) administered sodium cyclamate in drinking water of mice throughout their lifespan at a dose of 20 to 25 mg/kg/day. In treated female XVII/G mice, 16/20 lung tumors predominantly developed as compared to 3/16 in the control group (p < 0.01). Among treated male C3H × RIII mice, 28/34 predominantly showed liver tumors compared to the 16/28 in the control (p < 0.05). A shorter latent period also was observed in the cyclamate-treated animals. However, these tumors were diagnosed on the basis of gross appearance only. More significantly, the experiments of Hicks and coworkers (1975) demonstrated the cocarcinogenicity of sodium cyclamate in Wistar rats. In this case, the animals were given a single 2-mg intramuscular subcarcinogenic dose of N-methyl-1-nitrosourea (NMU), then fed sodium cyclamate in the diet equivalent to 1 or 2 g/kg/day. Among

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those given the higher dose, bladder tumors developed in 34/69 of the treated group; in this group, 18 kidney tumors also were detected. Neither type of tumor was seen in 124 controls (p < 0.001, in both cases). No tumors were found among animals treated with NMU or cyclophosphamide, both known to cause this type of tumor. These observations pointed to an important aspect of the potential health hazards of cyclamates: i.e., the possible influence of other exogenous compounds. In contrast to these positive effects, 22 other studies by investigators around the world using sodium cyclamates, and in some, calcium cyclamate or a mixture of these sweeteners and saccharin, have yielded negative results. Almost all of these studies employed a dose of 5% sodium cyclamate in the diet. These studies employed rats and mice but also hamsters, monkeys, and beagle dogs. However, in the opinion of the National Cancer Institute committee (TCRDCC, 1976), at least 10 of these studies were deficient in various factors, so that their negative conclusions cannot be given much credence. Nevertheless, five studies with negative results were, at a minimum, properly executed. Because of the negative findings in several feeding studies and the questioned human applicability or uncertainties of those studies showing positive results, even though the experimental protocols and results were valid, the Temporary Committee for the Review of the Data on the Carcinogenicity of Cyclamate (TCRDCC, 1976) con-

cluded, “The present evidence does not establish the carcinogenicity of cyclamate nor its major metabolite, cyclohexylamine, in experimental animals.” Thus, the committee implied that the available evidence neither proved nor disproved the suspected carcinogenicity of cyclamates. Indeed the committee stated that the studies of Bryan and Erturk (1970), Hicks and associates (1975), and Oser (1975) were not invalid but that these studies underscored the uncertainties associated with bioassay techniques. The work of Oser (1975) did not address itself to the question of cyclamate carcinogenicity because a saccharin-cyclamate mixture was used. The Bryan and Erturk (1970) technique involving bladder implantation cannot be related to the mode of human exposure, since “highly artificial conditions were being used.” The cocarcinogenicity study of Hicks and colleagues (1975) involving exposure of the animals to a subcarcinogenic dose of N-methyl-Nnitrosourea prior to feeding cyclamate has not yet been shown to be valid as a means of detecting bladder carcinogens. Finally, the insensitivity of bioassay techniques used renders judgment regarding weak carcinogens impossible. For example, it has been calculated that a total of 51,968 animals would be required to detect a 1% difference in bladder tumor incidence between test and control groups; this total assumes a survival of 18 to 24 months, and a bladder tumor incidence in the control (spontaneous) of 1%. Even so, some major uncertainties still remain. In 1973 Abbott Laboratories sought FDA’s permission to remarket the sweetener in foods designed for special dietary purposes. In the intervening years, there have been several attempts to reinstate cyclamate. One new petition included nearly 500 new toxicological assessments attesting to cyclamate’s safety. However, even after a series of petitions and court challenges, cyclamate was still not permitted in the food supply. In 1985, the NAS reviewed all existing data and concluded that cyclamate and its incriminated metabolite, cyclohexylamine, were not themselves carcinogens but could be cancer promoters (NAS/NRC, 1985). The Society of Toxicology published an article stating that the FDA’s cyclamate decision was an example of how not to do and interpret animal studies (Munro, 1987). In 1984, another petition to reinstate cyclamate was submitted to the FDA. This included over two dozen studies indicating that high doses of cyclamate throughout the lives of laboratory animals did not cause cancer. Further, 15 epidemiological studies showed no significant increase in the relationship between bladder cancer risk and the use of artificial sweeteners, both saccharin and cyclamate (Calorie Control Council, 1985; Morris and Przybyla, 1985). The FDA’s Cancer Assessment Committee has now

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exonerated cyclamate as a carcinogen (Newberne and Conner, 1986). Despite these extensive studies, cyclamate is still not allowed as a food additive in the United States. It is, however, used in over 40 countries around the world. It is also considered a safe additive by the WHO and the EEC (Malaspina, 1987). The current ADI for cyclamate of 0–11 mg/kg body weight is derived by using the NOEL for cyclohexylamine-induced testicular toxicity in rats during subchronic administration, assuming approximately a level of 18% transformation of cyclamate to cyclohexylamine (which assumes 30% metabolism of the 60% cyclamate that is not absorbed and thus can be metabolized by the intestinal microflora) and applying a safety factor of 200. The NOEL of 100 mg/kg body weight for cyclohexylamine was consistent in the different studies reviewed by Bopp and associates (1986) and is one of the best validated NOEL values available for food additives. 8.6.2 Saccharin Saccharin (Figure 8.4) was discovered accidentally in 1879 (Fahlberg and Remsen, 1879) and began to be sold commercially in 1900. It was originally used as an antibacterial agent and food preservative. It is 200 to 700 times sweeter than sucrose but must be used below a concentration of 0.1% because of its bitter aftertaste. This flavor quality was overcome somewhat in the 1950s when a 1:10 mixture of saccharin and cyclamate gained popularity because the sweetness imparted by the mixture was greater than the sum of the sweetness of each component, and the bitterness of saccharin was held below its threshold of perception for most consumers. The safety of saccharin has probably been investigated more and certainly has been reviewed and debated more than that of any food additive. The suspicion regarding its safety occurred early in its commercial introduction. It was banned in 1912, but the ban was lifted during World War I because of the shortage of sugar. The first report that suggested the possible carcinogenicity of saccharin was that of Fitzhugh and colleagues (1951), who reported an increased incidence of lymphosarcoma in rats fed a 5% saccharin diet. Because the control also had a high incidence of tumors, this initial finding was inconclusive (U.S. FDA, 1977). Therefore, in 1955 and again in 1968, the Food Protection Committee of the U.S. National Academy of Sciences (1968) concluded that a saccharin intake of as much as 1 g/day by an adult could be considered safe. However, the Wisconsin Alumni Research Foundation (WARF, 1972, 1973) reported the result of a two-generation study in which the parent (male and female)

Sprague-Dawley rats (F0) and their offspring (F1) were fed 0.05%, 0.5%, and 5% saccharin in the diet throughout their lifespan. In the males of the F1 generation a statistically significant increased incidence of bladder tumors (treated: 8/14, control, 0/14, p = 0.001) developed. A similar study by the U.S. FDA (1973) also showed a statistically significantly increased incidence of bladder tumors among males of the F1 generation when fed a 7.5% saccharin diet for 2 years (treated, 7/23; control, 1/25, p = 0.018). Thus, when the results were published, the U.S. FDA (1972) issued a regulation restricting general use of saccharin. Despite these results, a committee of the National Academy of Sciences (NAS, 1974) charged by the FDA to study the evidence concluded that partly because of the possible presence of impurities such as orthotoluenesulfonamide (OTS), the evidence did not conclusively establish the carcinogenicity of saccharin; the committee recommended further tests. OTS is the most common impurity in commercial saccharin preparation. Between 1973 and 1977, 11 single-generation studies in many laboratories in the United States and in other countries did not confirm these results. However, a twogeneration study using Charles River–Sprague-Dawley rats conducted by the Canadian National Health and Welfare Ministry (Arnold, 1977) confirmed the FDA and WARF studies (treated, 12/45; control, 0/42, p = 0.002). These studies also demonstrated that under the conditions of the experiments, the F0 generation showed a significant incidence of bladder tumors among male rats (treated, 7/38; control, 1/36, p = 0.033). Whereas the saccharin samples used in the FDA and WARF studies contained OTS estimated at 20 to 368 ppm and 12.5 to 18.0 ppm, respectively (NAS, 1974), the samples used by the Canadian study contained none. OTS was shown not to produce bladder tumors when fed at a level of as high as 250 mg/kg/day in the diet or in drinking water with added 1% ammonium chloride (Arnold, 1977). The latter was added to prevent the formation of alkaline urine, which has been associated with the formation of bladder calculi; ammonium chloride was correlated with the formation of bladder tumors in mice. The U.S. National Research Council/National Academy of Sciences Committee for a Study on Saccharin and Food Safety Policy (NAS/NRC, 1978) reviewed the pertinent toxicological studies. The committee concluded the following: 1.

Saccharin is absorbed rapidly in the GI tract, is distributed throughout the body, and crosses the placental wall. It is not metabolized in humans as far as present analytical methods permit, and

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2.

3.

4.

5.

6.

7.

only to a small extent in animals; it is eliminated mainly in the urine. Saccharin is a bladder carcinogen in male rats only as shown by two-generation studies in which the rats were fed continuously with saccharin (5% or greater) in utero and throughout life. In addition, in one two-generation study in rats, the males of the parent generation also showed a significant increase in bladder cancer. Saccharin promotes bladder tumor development in the presence of some other chemical carcinogens. Thus, the carcinogenic risk from saccharin as a tumor promoter may be considerably greater than that by itself, since humans are subject to multiple exposures to environmental carcinogens. However, in this case, the state of the science at present does not permit estimation of the human risk. Factors in design of the two-generation studies do not present doubtful interpretation of results. These factors include: doses studied (maximum tolerated dose), exposure in utero, high sodium diet (owing to use of sodium cyclamate) of treated compared to control animals, and the possible presence of microcalculi in the urinary bladders of treated compared to control rats. A few studies suggested an increase in benign uterine tumors and ovarian lesions in saccharintreated rats. OTS, the main impurity in some commercial saccharin preparations, is not a carcinogen in rats. That other impurities in saccharin may be carcinogenic is a remote possibility for the following reasons: a. Even though the saccharin used in two studies (FDA and WARF) had different patterns of impurities, the same carcinogenic responses (bladder cancer) were obtained. b. The very much purer saccharin used in the Canadian study produced no other tumors in males but those in the bladder, and c. If the impurities in the latter were carcinogenic, they would have to be very potent, since they are present at very low concentrations. Sixteen short-term assays for genetic effects produced negative results, whereas results of five others were positive. These variations in results might be expected because the assays evaluated various types of genetic effects and because saccharin is a weak carcinogen. Thus, these results are compatible with the in vivo car-

cinogenic effects. Definitive interpretation of the health risk to humans has not been provided by these results. The FDA in 1977 proposed a total ban of saccharin, invoking both the Delaney clause and the general safety clause of the Food, Drug, and Cosmetic Act. Since saccharin was at that time the only available artificial sweetener, the expected ensuing public furor resulted in the passage of the Saccharin Study and Labeling Act, which was signed into law in November 1977. Briefly, the act required the posting of warning signs regarding the health hazards of saccharin in stores, established an 18-month extension before the ban could be enforced, and called upon the National Academy of Sciences to study, among other points, the question pertinent to the decision regarding the safety of saccharin. The U.S. Congress has continued to enact extensions of the moratorium on the ban. Since 1977 many further studies have been done to assess long-term hazards of using saccharin. Metabolic studies showed it to be metabolically unchanged after being slowly absorbed and rapidly excreted. This is important evidence against its carcinogenicity, as no known carcinogens are excreted unchanged. A 1983 study (IFT Expert Panel, 1986) involving 2500 second-generation male rats revealed that high doses of saccharin caused changes in rat bladder tissue if the rat was exposed to saccharin during the suckling period but not if exposure occurred during the fetal period or after the suckling period. The incidence of tumors was clearly a function of dose, as number of tumors declined sharply as dose decreased. From this experiment, the risk of consumption of two cans of diet soda daily was extrapolated, and the increased risk of human bladder cancer was calculated at less than 1 in 1 million. Variation in risk using different methods of extrapolation based on rodent experiments ranged from 0.2 cancer to 144,000 cancers in the next 70 years in the United States (Munro, 1987). Epidemiological studies in Scandinavia, Japan, England, and the United States have not revealed an overall association between saccharin ingestion and bladder cancer. Data from these studies include individuals whose exposure to artificial sweeteners began decades ago (Concon, 1988; Newberne and Conner, 1986). One study found no elevated risk for the population in general, but they did find a positive association for several subgroups (Hoover, 1980). These included white males who were heavy smokers and nonwhite females with no known exposure to bladder carcinogens. The American Medical Association’s Council on Scientific Affairs recommends a moratorium on the saccharin ban, since evidence for the carcinogenic-

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ity of saccharin in humans has not been forthcoming. It also recommends careful monitoring of any adverse effects of saccharin and warns that young children and pregnant women carefully consider the use of saccharin (AMA, 1985, 1986). Legally, saccharin is now classified as a cocarcinogen (tumor promoter) with very low potency. Extrapolations suggest that saccharin at 30–300 mg/day (0.43–4.3 mg/kg/day) does not increase human cancer risk (Byard, 1984). It is allowed in the United States under the congressional moratorium on banning its use. Because the rat is the only species that has been reported to show an increase in the incidence of bladder tumors at high dietary concentrations of sodium saccharin, the JECFA concluded in 1993: from the long-term feeding studies . . . the dose-related carcinogenic activity of sodium saccharin on the urinary bladder was specific to the male rat and . . . exposure during the neonatal period was critical for the subsequent development of these tumors in the absence of an initiator or stimulus such as freeze ulceration. The critical events during the neonatal phase that lead to an increase in the population of initiated cells have not been identified. Saccharin is approved for use in 80 countries and has been determined to be safe by both the FAO/WHO JECFA and the Scientific Committee for Foods of the European Economic Community (Arnold and Clayson, 1985; Arnold and Munro, 1983). Saccharin provides a good example of the difficulty of evaluating the actual risk for humans on the basis of animal experiments without a complete knowledge of the mechanism of action. The approach adopted by the expert committees is considered appropriate since the epidemiological studies on saccharin did not show any evidence that its ingestion increases the incidence of bladder cancer in the human population. 8.6.3 Aspartame Aspartame, a dipeptide formed from the two naturally occurring amino acids phenylalanine and aspartic acid (Figure 8.4), is about 150 to 200 times sweeter than sucrose. Its sweet taste and lack of the bitter aftertaste often associated with artificial sweeteners make aspartame advantageous from a sensory viewpoint. Since it hydrolyzes under acid conditions or at high temperatures, its use is limited to cold nonacid products that do not require prolonged storage.

G. D. Searle filed a food additive petition that included 113 studies for approval of aspartame in 1973 to the FDA and in Canada to the Health Protection Branch. In 1974, the FDA approved the use of aspartame as a sweetener in certain foods. However, the approval was stayed in December 5, 1976, after formal objections were filed. These were based on allegations that aspartame might lead to brain damage and might cause mental retardation and endocrine dysfunction. Searle agreed to postpone the marketing of aspartame until the safety issues were resolved. One objection was based on the finding of uterine polyps in rats in a long-term feeding study involving diketopiperazine (Select Committee on Small Business, 1977), a derivative of this compound formed in solution from aspartame (Figure 8.5). Both mono- and dinitroso derivatives of piperazine are known to cause several cancers in experimental animals (Concon, 1988). However, feeding studies for up to 2 years on Charles River mice, SpragueDawley rats, and for more than 106 weeks on beagle dogs of both sexes produced no evidence that aspartame causes a statistically significant incidence of neoplasms compared to that in control animals (Searle and Co., 1973a, 1973b; 1974a). Furthermore, no evidence of transplacental carcinogenicity has been demonstrated in rats, even after the animals were exposed to aspartame-containing diets (2 g/kg and 4 g/kg) throughout their lifespan, beginning with in utero exposure (Searle and Co., 1974b). Bladder implantation experiments in mice with both aspartame and its hydrolysis product, diketopiperazine, also have produced no evidence of carcinogenicity (Searle and Co., 1973c).

Figure 8.5 Cyclization of aspartame in aqueous solutions to a diketopiperazine derivative.

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Ishii and colleagues (1981) also demonstrated the noncarcinogenicity of aspartame or a mixture of aspartame and diketopiperazine in the diet in Wistar rats. Therefore, in 1981 the FDA approved aspartame for use in certain types of foods and as a tabletop sweetener (U.S. FDA, 1981). The second objection involved the possible neurotoxicity of aspartate as a result of the aspartate moiety. The latter becomes monosodium aspartate under the digestive conditions in the GI tract (Olney, 1971). It has been contended that overconsumption of aspartame-sweetened products, which are most likely to be eaten by children, may result in brain damage in that population group similar to that seen in animals (Reynolds et al., 1976). Because the neurotoxicity of aspartate depends on the occurrence of a rapid rise in blood aspartate levels, as in the ingestion of the free amino acid, Stegink (1979) fed aspartame at different levels to normal adults, 1-year-old infants, and individuals who were heterozygous for phenylketonuria (PKU) (See Chapter 7). They found that even with high doses of aspartame (100 to 200 mg/kg), aspartate was rapidly metabolized in both adults and infants such that only small increases in blood aspartate levels above control values occurred. These results precluded any rapid rise in aspartate to levels, which can cause brain damage, as stated by Olney (1971). Aspartame can also yield relatively high amounts of phenylalanine, which are generally inconsequential in normal adults, but damaging to phenylketonuric infants. Thus, the FDA regulation regarding the initial approval of aspartame specified a proviso that the food in question bear a conspicuous warning label directed to phenylketonuric individuals. However, Stegink (1979) showed that even with the ingestion of 100 to 200 mg/kg by human infants or adults, the mean peak value of 49 µmol phenylalanine/100 ml blood was obtained at 45- to 90-minute ingestion. This level is below the toxic dose for these susceptible subjects. Both the American Medical Association (AMA, 1986) and the American Academy of Pediatrics (AAP, 1985) have concluded that aspartame use is safe for humans who do not suffer from PKU and is safe for the fetus during pregnancy at levels currently being used. The American Diabetes Association (ADA) has also endorsed the safety of aspartame (ADA, 1985). Furthermore, studies have indicated that aspartame had no effect on blood glucose levels (Nehrling et al., 1985). The American Dental Association has also issued a positive statement about the safety of aspartame and has endorsed its use because it does not promote tooth decay (Goodman et al., 1987). JECFA has currently established an ADI of 40 mg/kg for aspartame (Wells, 1989). Aspartame use is currently allowed in over 50 countries.

8.6.4 Acesulfame-K

8.7

Acesulfame-K is a derivative of acetoacetic acid that is about 130 times sweeter than sucrose (Wells, 1989). A large number of pharmacological and toxicological tests have initially given acesulfame-K a clean slate. Metabolic studies show it to be excreted unchanged (O’Sullivan, 1983). The JECFA has approved its use, and it is being used in 20 countries. It received approval for use in the United States in the summer of 1988. Packets of this sweetener are available on the consumer market.

Apart from microbial spoilage and browning reactions, oxidative degradation of unsaturated fatty acids, primarily oleic, linoleic, linolenic, and arachidonic acids in food lipids, and similar other unsaturated compounds is primarily responsible for determining the shelf life of food products. The reaction, known as peroxidation, self-propagates by a chain reaction that eventually leads to rancidity. These chain reactions are catalyzed by heat, light, ionizing radiation, trace metals (particularly iron and copper), and metalloproteins such as heme, and enzymatically by lipoxygenases. Rancid food products, recognized by their undesirable odor and taste, not only are unacceptable, but also contain toxic compounds. Lipid oxidation products are also implicated in causing several diseases, including arteriosclerosis, coronary heart disease, and cancer. Strategies that inhibit oxidation in foods containing fats include the following:

8.6.5 Sugar Alcohols Sorbitol, mannitol, maltitol, and xylitol are all sugar alcohols (polyols) that occur naturally in small amounts in fruits and vegetables. Theoretically, these sugar alcohols yield the same number of calories as sucrose, but in actuality they are incompletely metabolized, so the net energy yield is lower (Wursch and Anantharaman, 1989). The lower energy yield is, however, offset by a sweetening power that is much lower than that of sucrose for all polyols except xylitol. Most are of little use to the lucrative diet market. However, their main advantage is the demonstrated ability to diminish caries formation markedly, by over 60%, in humans when substituted for sucrose (Scheinin, 1979; Voirol, 1979). These naturally occurring polyols have all yielded negative results with respect to mutagenicity tests (JECFA, 1986), but one study indicated that xylitol may be tumorigenic (ADA, 1980). The market for sweeteners is still growing, and the situation where the ADI for the known sweeteners is reached is not inconceivable. There is, however, a need for sweeteners that are stable under specific technological conditions and are less controversial than saccharin. Although since the introduction of aspartame the use of saccharin has slowly declined, aspartame cannot replace saccharin completely because of its instability when heated under acidic conditions. Therefore, the search for new sweeteners continues. One such sweetener, thaumatin (Talin) is found in the fruit of a West African plant, katemfe. It is a protein about 5000 times sweeter than sucrose. However, the sweetness develops slowly and leaves a licorice-like aftertaste. Thaumatin is approved for use in the United Kingdom, Mexico, Japan, and Australia (except in baby food). In the United States, the only allowed use is in chewing gum, but petitions for other uses have been filed. In 1985, JECFA agreed it was safe for use in food but did not specify an ADI (Wells, 1989).

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ANTIOXIDANTS

1. 2. 3. 4. 5.

Rapid production and timely consumption of food Storage of food under vacuum or modified atmosphere Hydrogenation of fat to reduce the degree of unsaturation Avoidance of prooxidants, such as metal ions Use of natural or synthetic antioxidants

In this section, the chemical properties of free radical formation and the toxicological aspects of most commonly used antioxidants in food processing and preservation are described. 8.7.1 Free Radical Chemical Characteristics A free radical is defined as “any species capable of independent existence that contains one or more unpaired electrons.” An unpaired electron is one that occupies an atomic or molecular orbital by itself. The presence of one or more unpaired electrons causes the species to be paramagnetic and sometimes makes it highly reactive. Because electrons tend to occur in pairs such that their spins are antiparallel to each other, the pair per se has no net spin. This property is energetically most favorable, and therefore almost all chemical bonds tend to have two electrons. Free radicals can be generated by the gain or loss of a single electron from a nonradical. The two-electron bonds can break in two ways: symmetrically by homolytic fission or asymmetrically by heterolytic fission. In homolytic fission, a covalent bond is broken such that one electron (indicated by an asterisk in the following equa-

tions) from each member of the shared pair remains with each atom as shown: R—H → R* + H*

(1)

In contrast, when bonds are broken heterolytically, only one atom receives both electrons: R—H → R:– + H+

(2)

Only chemical species formed by the homolytic processes are called free radicals, because they have an unpaired electron. Because electrons are more stable when paired in orbitals, radicals are in general more reactive than nonradicals. Their reactivity, however, varies greatly depending on the chemical species. Free radicals can react with other molecules in a number of ways. Thus two free radicals can share their unpaired electrons and join to form a covalent bond: R* + R* → R—R

(3)

Similarly, a radical might donate its unpaired electron to another molecule (a reducing radical), it might take an electron from another molecule to form a pair (an oxidizing radical), or it might add on to a nonradical species. However, if a radical gives one electron to, takes one electron from, or adds itself on to a nonradical, that nonradical becomes a free radical. Thus, a general feature of the reactions of free radicals is that they tend to proceed as chain reactions. The oxidation process is terminated by the reaction of two free radicals to form stable polymeric compounds as shown in equation (3). Detailed mechanisms of the oxidation of different unsaturated fatty acids are described in several reviews (Labuza, 1971; Frankel, 1984; Kappus, 1991; Madhavi and Salunkhe, 1995; Jadhav et al., 1996). The self-propagation of free radical reactions can be prevented by antioxidants. Some such as α-tocopherols and their congeners and ascorbic acid occur naturally. The most widely used and highly effective synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), gallates, and tertiary butyl hydroquinone (TBHQ). These are often used in combination with metal chelators, such as citric acid. Madhavi and Salunkhe (1995, 1996) reviewed the toxicological characteristics of both naturally occurring and synthetic antioxidants in the mid-1990s. Here, only the toxicological aspects of some of the more important commercially used synthetic antioxidants are described. 8.7.2 Butylated Hydroxyanisole Butylated hydroxyanisole (BHA; tert-butyl-4-hydroxyanisole) is the most widely used antioxidant in the food in-

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dustry. It is a mixture of two isomers: 2-tert-butyl-4hydroxyanisole (2-BHA) and 3-tert-butyl-4-hydroxyanisole (3-BHA) (Figure 8.6); the commercial compound contains over 90% of the 3-isomer (Buck, 1985; Madhavi and Salunkhe, 1996). The absorption and metabolism of BHA have been extensively studied in several species, including humans. It is rapidly absorbed from the GI tract, rapidly metabolized, and completely excreted. The major metabolites of BHA were the glucuronides, ether sulfates, and free phenols (Figure 8.7). The metabolites are generally excreted in urine, whereas the unchanged BHA is eliminated in the feces. Short-term feeding studies conducted in a number of species such as mice, rat, rabbit, and dogs at levels up to 500–600 mg/kg body weight indicate a depressed growth rate and reduced activity of several enzymes, including catalase, peroxidase, and cholinesterase (Karplyuk, 1962; Martin and Gilbert, 1968; Cha and Heine, 1982). Other deleterious effects include increased excretion of sodium and potassium in urine, liver enlargement, and proliferation of smooth endoplasmic reticulum. However, nucleolar abnormalities have not been observed. The antioxidant also induced a number of hepatic enzymes in rats and mice such as epoxide hydrolase, glutathione-S-transferase, glucose-6phosphate dehydrogenase, and biphenyl-4-hydroxylase. In some of the earlier long-term studies, BHA was found to be safe in rats after 22 months (Wilder and Kraybill, 1948; Brown et al., 1959; Karplyuk, 1962) and in dogs after 15 months (Wilder et al., 1960). In the early 1980s, Ito and coworkers (Ito et al., 1982; 1983a, 1983b; 1984) reported that BHA induced cancer of the nonglandular stomach (forestomach) in male F344 rats. In one study, 2% BHA in the rat diet produced a high incidence of papilloma in almost all the treated animals and squamous cell carcinoma in about 30% of the test animals. At lower dose levels of 0.5%, no neoplastic lesions were observed, although hyperplasia was induced. These studies have also shown adverse effects of crude BHA and the 3isomer, whereas the 2-isomer had no effect. In addition, two metabolites, viz., p-tert-butyl phenol and 2-tert-butyl4-methylphenol, also induced forestomach papillomas. Since these observations, many studies have been performed in several species to determine whether BHA acts specifically on the forestomach epithelium and whether there is a threshold for hyperplasia (Altmann et al., 1986; Iverson et al., 1986; Tobe et al., 1986; Ikeda et al., 1986; Amo et al., 1990). Depending on the duration of the experiment, BHA has been shown to produce hyperplasia and/or tumors specifically in the forestomach of rats, mice, and hamsters. The hyperplasia was reversible, but the time necessary for reversal was dependent on dura-

Figure 8.6 Commonly used synthetic antioxidants: A, 3-BHA; B, 2-BHA; C, Gallates; R = C3H7 (propyl gallate), R = C8H17 (octyl gallate), R = C12H25 (dodecyl gallate); D, BHT; E, TBHQ. BHA, butylated hydroxyanisole; BHT, butylated hydroxytoluene; TBHQ, tertiary butyl hydroxyquinone.

tion and level of dosage of BHA. The NOEL for hyperplasia in the rat forestomach was 0.125% BHA in the diet. In animals without a forestomach (e.g., guinea pig, dog, pig, monkey), BHA produced no histological effects, including hyperplasia, in the esophagus or glandular stomach. In gavage studies in the monkey, the mitotic index in the “target” squamous epithelium of the distal esophagus was raised at high dose levels, but not at 250 mg/kg body weight. On this basis, the Scientific Committee for Food of the EEC (SCF, 1989) concluded that “the action of BHA in producing forestomach hyperplasia and tumors in rodents may not be relevant for man.” In addition to the carcinogenic effect of BHA on the forestomach in rodents, several studies have reported an enhancing effect by BHA of carcinogen-initiated tumorigenesis in the forestomach. Whysner and coworkers (1994) reported an analysis of the lifetime exposure of male F344 rats to BHA after initial treatment with Nmethyl-N′-nitro-N-nitrosoguanidine (MNNG). They reported that BHA clearly acts as a tumor promoter in the forestomach of rodents only at high levels of exposure, and that these levels are well above exposures to humans resulting from the use of BHA as a food additive. The mechanisms of BHA tumor promotion appeared to in-

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volve cell damage resulting in enhanced cell proliferation. BHA has a NOEL that is consistent with its role in tumor promotion and carcinogenesis. Furthermore, this study also suggested some indication that at lower doses, closer to those that reflect its use as a food additive, BHA may have an inhibitory effect in forestomach tumorigenesis. Humans’ lack of a forestomach further decreases the likelihood that BHA would have any harmful effects in humans. BHA also does not seem to have any adverse mutagenic or teratologic effects in several animal species (Clegg, 1965; Hansen and Meyer, 1978; Allen, 1976; Stokes et al., 1972; Williams, 1977, 1986; Tong and Williams, 1980; Rogers et al., 1985). In the United States, a maximum of 0.02% of the fat content as BHA is GRAS. However, in certain rancidityprone but low-fat foods (e.g., dry breakfast cereals, instant potatoes), BHA at up to 50 ppm of the food itself is permissible as a regulated food additive. In a 1989 reevaluation, the JECFA allocated an ADI of 0–0.5 mg/kg (FAO/WHO, 1989). Accurate data on the daily intake of BHA and other similar antioxidants in humans are not available. Generally, estimates are in the range of 1 to 5 mg/day (Janssen, 1997).

Figure 8.7

Metabolites of BHA. BHA, butylated hydroxyanisole; TBHQ, tertiary butyl hydroquinone.

8.7.3 Butylated Hydroxytoluene Butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-pcresol); (Figure 8.6) is another synthetic antioxidant widely used alone or in combination with other antioxidants such as BHA, propyl gallate, and citric acid. The absorption, metabolism, and excretion of BHT have been studied extensively in several animal species. Its oxidative metabolism is mediated by the microsomal monooxygenase system. However, there is some species variation. In rats, rabbits, dogs, and monkeys, oxidation of the p-methyl group predominates, whereas in humans, the tert-butyl groups are oxidized (Madhavi and Salunkhe, 1995, 1996). Oxidation of both p-methyl and tert-butyl groups is observed in mice. The major metabolites of BHT are shown in Figure 8.8. BHT is also one of the additives in which toxicity has been most intensively studied (JECFA, 1996). Earlier investigators found no harmful effects in rats fed 0.2%, 0.5%, or 0.8% BHT in the diet for 24 months (Deichman et al., 1955), or 0.03%, 0.1%, and 0.3% in the diet that contained 20% fat and was fed for 10 weeks (Frawley et al., 1965). In other such studies, reduced weight gain and increased weights of the liver, brain, and other organs were observed (Gaunt et al., 1965; Johnson and Hewgill, 1961).

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In a 1992 study, Takahashi (1992) reported that BHT in very high doses (1.35%–5% for 30 days) caused a doserelated toxic nephrosis with tubular lesions in mice. The lesions appeared as irregular patches or wedge-shaped proximal tubules, necrosis, and cyst formation. Renal toxicity has also been reported in rats (Meyer et al., 1978; Nakagawa and Tayama, 1988). Later studies showed a variety of multiple effects of BHT on lung, liver, kidneys, thyroid metabolism, blood coagulation, electrolyte excretion, and promotion or inhibition of carcinogenesis. A hepatocarcinogenic effect of BHT was demonstrated in a long-term carcinogenicitytoxicity study utilizing an exposure in utero, during the suckling period, and during a further 40-week period after the standard 2-year exposure (Olsen et al., 1986). A newer study carried out under similar conditions examined in detail hepatic modifications due to BHT and confirmed that BHT causes an enlargement of the centrilobular hepatocytes, which was indicative of proliferation of smooth endoplasmic reticulum consistent with an induction of mixed-function oxidases (Price, 1988). BHT administration resulted in persistent, marked induction of cytochrome P-450 2B and γ-glutamyl transpeptidase activity. In view of the probable involvement of hepatic enzyme induction in the development of the hepatocyte damage asso-

Figure 8.8

Metabolites of BHT. BHT, butylated hydroxytoluene.

ciated with repeated doses of BHT, JECFA (1995) decided that, in this case, enzyme induction was the most sensitive index of effect on the liver. A well-defined threshold was demonstrated and a NOEL of 25 mg/kg body weight determined, taking into consideration effects observed in the reproduction aspect of the in utero–chronic exposure. An ADI of 0–0.3 mg/kg body weight was allocated. Several studies have been conducted on the modifying effects of BHT on chemical carcinogenesis. These effects depend on a number of factors, including target organs, type of carcinogen, species and strain differences, type of diet used, and time of administration. In general, BHT inhibited the induction of neoplasms in the lung and forestomach in mice and lung, liver, and forestomach neoplasms in rats when given before or with the carcinogen. BHT also had a promoting effect on urinary bladder, thyroid, and lung carcinogenesis (Ito et al., 1987). In the 1987, the Scientific Committee for Food of the EEC considered that since the NOEL for hepatic carcinogenesis was higher than that obtained for other toxicological effects, the ADI should be determined for the latter. An ADI of 0–0.05 mg/kg body weight based on a NOEL of 5 mg/kg body weight on thyroid, reproduction, and coagulation effects in the rat, was established.

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These different interpretations illustrate the difficulties encountered in extrapolation from animal experimentation to humans. The effects of BHT on blood coagulation and on lung, tested on animals, constitute a good example of these difficulties. Takahashi and Hiraga (1978a) demonstrated for the first time that BHT could cause a high mortality rate that was due to massive hemorrhage when BHT was included in the diet of rats at a concentration of 0.69% or more. They also showed a significant dose-dependent reduction in prothrombin index at a 40-fold lower concentration with as little as 0.017% BHT in the diet (Takahashi and Hiraga, 1978b). Vitamin K–dependent clotting factors II (prothrombin), VII, IX, and X were rapidly decreased by BHT, which exerted its hemorrhagic effect through metabolic activation. BHT quinone methide appears to be more active than the parent compound. Hemorrhagic deaths occurred among male rats of all strains tested and female rats of the Fischer strain, but not among female rats of the Donryu and Sprague-Dawley strains. Such effects were not observed in quails, rabbits, or dogs. Effects on coagulation in guinea pigs and mice were equivocal (Takahashi, 1992). In 1990, JECFA suggested that the hemorrhagic effects of BHT (Takahashi and Hiraga, 1978a) were attribut-

able to the use of animals that were vitamin K–deficient. However, they did not consider this effect to be critical with respect to the safety evaluation of BHT as a food additive in the human population (JECFA, 1991). Cottrell and associates (1994) reported that dose levels of BHT (600 mg/kg/day) that caused clotting defects in the rat were far greater than any likely human exposure. These levels were used in these investigations solely to demonstrate and study a toxicological mechanism. The dose-response data obtained indicated that the human relevance of such clotting abnormalities is small, if not negligible, since no effect was observed in the rat at a dose 1000 times the human ADI (0.125 mg/kg/day, JECFA, 1991) and the lowest observed effect level was 4800 times the ADI, which is well above the usual margin for safety. These data (Takahashi and Hiraga, 1978a, 1978b) were taken into account when JECFA allocated BHT an ADI of 0–0.3 mg/kg body weight in 1995. BHT appears to have no adverse effects on reproduction and does not appear to be teratogenic in singlegeneration and multigeneration reproduction studies in rats, mice, hamsters, rabbits, and monkeys at lower doses; the NOEL was equivalent to 50 mg/kg body weight (Clegg, 1965; Johnson, 1965; Allen, 1976; Olsen et al., 1986). BHT was also found to have no effect in several strains of Salmonella typhimurium with or without metabolic activation (Shelef and Chin, 1980; Hageman et al., 1988). Mutagenic effects were observed in rats at high doses of BHT in two dominant lethal tests (Stanford Research Institute, 1972, 1977).

tarded growth, and caused renal damage accompanied by an approximately 40% mortality rate in the first month. In the survivors, no pathological changes were observed (Orten et al., 1948). In similar studies, at high doses PG produced growth retardation, anemia, hyperplasia in the outer kidney medulla, and an increase in the activity of cytoplasmic and microsomal hepatic drug-metabolizing enzymes (Van Der Heijden et al., 1986). In long-term feeding studies, similar observations were made. Whereas lower concentrations (<1% in diet) of PG showed no adverse effects, concentrations above 1% showed reduced food intake, growth retardation, patchy hyperplasia of stomach, increased incidence of hepatic vacuolization and suppurative inflammation of the prostrate gland in male rats, and sometimes an increased mortality rate (Lehman et al., 1951; Orten et al., 1948; Dacre, 1974; Abdo et al., 1986a, 1986b). Carcinogenic effects were seldom observed. Similarly, no adverse teratological or mutagenic effects were shown for PG (Rosin and Stich, 1980; Sluis, 1951; Tanaka et al., 1979). In 1980, JECFA allocated a group ADI of 0–0.2 mg/kg body weight. However, later studies indicated that octyl gallate (OG) and dodecyl gallate (DG) may have an adverse effect on reproduction. Hence, in a reevaluation in 1987, an ADI of 0–2.5 mg/kg was allocated to PG and no ADIs were allocated to OG and DG because of lack of toxicological information (FAO/WHO, 1987).

8.7.4 Gallates

Tertiary butyl hydroquinone (TBHQ) (Figure 8.6) was introduced in the 1970s and was approved as a food-grade antioxidant in 1972. It is often regarded as the best antioxidant for the protection of frying oils and the fried product (Buck, 1984). TBHQ is currently used in the United States and some other countries. It is, however, not permitted in EEC countries or Japan because of the lack of adequate toxicological data (Sherwin, 1976; EEC Commission Documents, 1982). In a 1987 reevaluation, the JECFA assigned a temporary ADI of 0–0.2 mg/kg body weight (FAO/WHO, 1987). Absorption and metabolism studies have been conducted in rats, dogs, and humans. In all three species, over 90% of an orally administered dose of TBHQ was rapidly absorbed, and nearly 80% was excreted in the urine in the first 24 hours. The excretion was essentially complete within 48 hours (Astill et al., 1975). In short-term feeding studies in rats given 100 and 200 mg/kg body weight TBHQ for 1 month or fed 1% TBHQ for 22 days, no adverse effects on growth rate or mortality rate, or histopathological features were observed

The gallate group of antioxidants comprises the propyl, octyl, and dodecyl esters of gallic acid (3,4,5-trihydroxybenzoic acid) (Figure 8.6). Of the three, propyl gallate (PG) is more effective as an antioxidant and is widely used in several food products. Nearly 70% of the ingested PG is absorbed in the GI tract. The other two are absorbed to a lesser extent. All three esters are hydrolyzed to gallic acid and free alcohol. The latter is metabolized through the Krebs cycle. Gallic acid is methylated to yield 4-o-methyl gallic acid, which is the main metabolite found in urine either free or conjugated with glucuronic acid. Small quantities of gallic acid are also excreted as glucuronide, as well as in the free form. Unchanged esters are excreted in the feces (Madhavi and Salunkhe, 1996). In short-term feeding studies and at doses up to 0.5% PG in the diet, no adverse changes were observed in body weight, liver weight, and total liver lipid content (Johnson and Hewgill, 1961). However, at doses of about 1% in the diet, PG caused a reduction in weight gain, re-

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8.7.5 Tertiary Butyl Hydroquinone

(Fassett et al., 1968). It did have a slight inductive effect on some of the liver microsomal mixed-function oxidases (Astill et al., 1975). Similarly, in long-term feeding studies with doses up to 0.5% TBHQ in the diet, no deleterious effects on food intake, growth rate, mortality rate, and organ weight were observed (Nera et al., 1984; Astill et al., 1975). Levels >2%, however, caused mild hyperplasia of the forestomach with focally increased hyperplasia of the basal cells in rats (Altmann et al., 1986). TBHQ does not seem to have any adverse effects on reproduction and is found to be nonteratogenic and nonmutagenic.

8.8

ACIDULANTS AND SEQUESTRANTS

Acidulants serve several important functions in food processing and preservation. Important functions of food acidulants include roles as pH control agents, preservatives, chelating agents–antioxidant synergists, flavor adjuncts, and viscosity and melting modifiers. Both inorganic and organic acids are commonly used for this purpose. Among the inorganic acids, phosphoric acid and its derivatives are the most widely used food acidulants (Deshpande et al., 1995). Phosphoric acid is also the highest-value-added inorganic acid marketed in the United States and the second largest in terms of volume after sulfuric acid. Hydrochloric and sulfuric acids are seldom used as direct food acidulants. Nevertheless, they are used indirectly in the chemical synthesis of several food-grade chemicals. They are also used in the cornstarch-processing industry to manufacture corn syrups. As a group, the organic acids constitute the most widely used acidulants in the food industry. Among the carboxylic acids, citric acid alone commands over 60% of the market share of all the acidulants used in food processing. The phenolic benzoic acid and the fatty sorbic acid are primarily used as preservatives; other fatty acids such as butyric and caprylic acids find limited application as acidulants and flavoring ingredients. The lactones ascorbic acid and glucono δ-lactone are both used as acidulants. Ascorbic acid is also an excellent antioxidant synergist. Both are capable of chelating metal ions that are responsible for enhancing the rancidity of fatrich foods. Lysine, glutamic acid, and cysteine are amino acids that find limited application as acidulants. Of these, cysteine and its derivatives are primarily used as dough conditioners in the baking industry, and the sodium salt of glutamic acid is used as a flavor enhancer in numerous products. Lysine is primarily used to fortify wheat-based products to enhance their protein nutritional value. Other

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essential amino acids are primarily used as nutrients, especially in parenteral nutrition formulations. In this section, the toxicological aspects of selected acidulants are briefly reviewed. 8.8.1 Phosphoric Acid and Phosphates Phosphoric acid is the only inorganic acid extensively used as a food acidulant and accounts for about 25% of the weight of all the acids used in foods, compared to 60% for citric acid and 15% for the other food acidulants (Gardner, 1972; Deshpande et al., 1995). The U.S. FDA in its Code of Federal Regulations (CFR, 1988) lists orthophosphoric and polyphosphoric acids and their calcium, potassium, sodium, and ammonium salts as GRAS food additives. The various groups are listed in Table 8.9. Each food additive is listed under functional categories for its applications. The straight-chain polymeric phosphates are also allowed since they are hydrolyzed to orthophosphates. Although doubts were cast earlier on the use of cyclic metaphosphates as GRAS additives, subsequent studies have shown that little, if any, cyclic metaphosphate is absorbed through the intestinal wall when administered orally (Ellinger, 1972). The metaphosphates must first be hydrolyzed to tripolyphosphate and then to orthophosphate, which then can be absorbed. Sodium metaphosphate is allowed as a GRAS additive, provided it meets the Food Chemicals Codex specifications. The ammonium, calcium, and potassium salts are equally acceptable. Several researchers have extensively investigated the toxicity of various phosphate derivatives commonly used in foods. Similarly to other inorganic salts, phosphates in excess quantities can be toxic. Excessive amounts of inorganic salts not only upset mineral balance in the body, but also affect the osmotic pressure of body fluids (Deshpande et al., 1995). The Food and Agriculture Organization, in collaboration with the World Health Organization, has published two extensive reports on the toxicology of various phosphates (FAO/WHO, 1967; FAO, 1974). Data summarized in Table 8.10 on the acute toxicity of various phosphates indicate that orthophosphates and the shorter-chain polyphosphates are more toxic than sodium chloride when given orally, whereas the longer-chain and the cyclic polyphosphates are less toxic. In general, all phosphates are significantly more toxic when introduced into the body in a manner that circumvents the digestive system. Intraperitoneal or intravenous injection of the phosphates, including the higher polyphosphates, produces only small differences in the LD50 levels, probably as a result of rapid enzymatic hydrolysis of the polyphos-

Table 8.9 the FDAa

Phosphate Food Additives Recognized as GRAS by

Miscellaneous and/or general-purpose food additives Phosphoric acid Ammonium phosphate (mono-, dibasic) Sodium acid pyrophosphate Sodium aluminum phosphate Calcium phosphate (mono-, di-, tribasic) Sodium phosphate (mono-, di-, tribasic) Sodium tripolyphosphate Sequestrants Calcium hexametaphosphate Calcium phosphate (monobasic) Dipotassium phosphate Sodium acid phosphate Sodium hexametaphosphate Sodium metaphosphate Sodium phosphate (mono-, di-, tribasic) Sodium pyrophosphate Tetrasodium pyrophosphate Sodium tripolyphosphate Nutrient and/or dietary supplements Calcium glycerophosphate Calcium phosphate (mono-, di-, tribasic) Calcium pyrophosphate Ferric phosphate Ferric pyrophosphate Ferric sodium pyrophosphate Magnesium phosphate (di-, tribasic) Manganese glycerophosphate Manganese hypophosphite Potassium glycerophosphate Sodium phosphate (mono-, di-, tribasic) Emulsifying agents Monosodium phosphate derivatives of mono- and diglycerides a GRAS, generally regarded as safe; FDA, U.S. Food and Drug Administration Source: From CFR (1988).

phate chains to orthophosphate in the blood (Ellinger, 1972; Deshpande et al., 1995). The introduction of phosphate salts into the body by nonoral routes is meaningless in evaluating the toxicity of phosphates as food ingredients. These studies do not take into consideration the changes that salts undergo before or during absorption through the intestinal wall. The feeding studies, in contrast, reflect the toxicity of phosphates as food ingredients more accurately. Even then, definitive conclusions are difficult to draw, because in most studies, optimal ratios of dietary essential minerals either were not used or were not mentioned in detail. The interrelationship

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between calcium and phosphorus in human is well established (see Chapter 7). A proper ratio of these two minerals in the human diet is essential to maintain bone. Whereas short-term studies, sometimes conducted at concentrations as high as 2%–4% in the diet, generally do not indicate any adverse effects of phosphates, most long-term feeding studies indicate some retardation of growth rates, calcium deposition in various tissues and organs, and, more noticeably, heart and kidney damage in test animals (Table 8.11). Toxic symptoms that result from chelation by the phosphate anion of calcium, iron, magnesium, copper, and similar ions essential to human metabolism may also be manifested over time. The results of animal-feeding studies reported in the scientific literature indicate that levels of 0.5% of phosphates can be tolerated in the diet without any adverse physiological effects. Higher levels may be tolerated if a proper balance of other ions, particularly calcium, magnesium, and potassium, is maintained (Ellinger, 1972; FAO, 1974; Deshpande et al., 1995). Few, if any, applications for phosphates require a concentration of >0.5% to obtain the desired effect; in fact, higher levels of phosphates often produce adverse physical and chemical effects and off-flavors in food products. According to Ellinger (1972), a 0.5% phosphate level is highly unlikely ever to appear in the total human diet. While setting the dietary intake guidelines for phosphoric acid and its salt additives, the FAO (1974) has taken into consideration the important role phosphates play in human metabolism, especially in bone, teeth, and many enzyme systems. Phosphorus is also a key element in carbohydrate, fat, and protein metabolism and is the primary source of energy stores in the plant and animal kingdom. FAO (1974) estimated that adult humans require a minimum of 0.88 g of phosphorus in their daily diet. The blood serum of human adults normally carries 2.5–4.5 mg phosphorus/100 ml; that of children carries a higher level. While setting the guidelines for phosphorus intake in the diet, in addition to the available toxicological data on various phosphates, the FAO (1974) has taken into consideration the levels of calcium and other minerals in the human diet that affect the levels of phosphates that produce the earliest signs of adverse effects. The committee has recommended an unconditional acceptance level for total dietary phosphorus of <30 mg/kg body weight/day in the human nutrition. This level is considered safe under all dietary conditions. The FAO (1974) has also suggested a conditional acceptance level of 30–70 mg/kg body weight/day when the dietary calcium level is high.

Table 8.10 Acute Toxicity Levels of Phosphates in Animals

Phosphate H3PO4 NaH2PO4 NaH2PO4 NaH2PO4 Na2HPO4 NaH2PO4 + Na2HPO4 Na2H2P2O7 Na2H2P2O7 Na2H2P2O7 Na2H2P2O7 Na4P2O7 Na4P2O7 Na4P2O7 Na4P2O7 Na4P2O7 Na4P2O7 Na4P2O7 Na5P3O10 Na5P3O10 Na5P3O10 Na5P3O10 Na6P4O13 Na6P4O13 (KPO3)η + pyro (KPO3)η + pyro Hexametaphosphateb Hexametaphosphate Hexametaphosphate Hexametaphosphate Hexametaphosphate (NaPO3)η = 6c (NaPO3)η = 11 (NaPO3)η = 27 (NaPO3)η = 47 (NaPO3)η = 65 (NaPO3)3 cyclic (NaPO3)3 cyclic (NaPO3)3 cyclic NaCl (table salt) NaCl (table salt) NaCl (table salt)

Animal

Routea

Rabbit Mouse Guinea pig Rat Rabbit Rat

IV Oral Oral IP IV IV

Mouse Mouse Mouse Rat Rat Rat Mouse Mouse Mouse Mouse Rabbit Mouse Mouse Mouse Rat Rat Rat Rat Rat Rabbit Mouse Mouse Mouse Mouse Rat Rat Rat Rat Rat Mouse Mouse Mouse Mouse Mouse Mouse

Oral SC IV Oral IP IV IP Oral SC IV IV Oral SC IV IP Oral SC Oral IV IV Oral Oral SC IV IP IP IP IP IP Oral SC IV Oral SC IV

a

LD50 (mg/kg)

Approximate lethal dose (mg/kg) 1,010 >100 >2,000 >36 >985, ≤ 1,075

>500 2,650 480 59 >4,000 233 10 0–500 ca. 40 2,980 400 69 ca. 50 3,210 900 71 134 3,920 875 4,000 ca. 18 ca. 140 >100 7,250 1,300 62 192 200 326 70 40 10,300 5,940 1,165 5,890 3,000 645

IP, intraperitoneal; IV, intravenous; SC, subcutaneous; oral, by mouth (in diet, by stomach tube, etc.) Average chain lengths (η) were not given. c Polyphosphate preparations for which η was determined. Source: From Ellinger (1972), FAO (1974), and Deshpande et al. (1995). b

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Table 8.11 Short-Term and Long-Term Feeding Studies with Phosphates Length of testa

Maximal level tolerated

Effect of excess phosphate

Phosphate

Test animal

Orthophosphoric acid H3PO4 (36.4%) H3PO4 (36.4%) H3PO4 (36.4%)

Humans Rats Rats

Variable >12 Months 44 Days

17–26 g/d >0.75% <2.94%

No adverse effectsb No adverse effects Kidney damage

Na and K orthophosphates MSPc MSP MSP MSP MSP + DSP DSP DSP DKP

Humans Rats Guinea pigs Guinea pigs Rats Rats Rats Rats

Variable 42 Days 200 Days 12–32 Weeks 3 Generations 6 Months 1 Month 150 Days

5–7 g/day >3.4 g/kg/day >2.2%, <4.0% 4%–8%d >0.5%, <1.0% >1.8%, <3.0% <5.0% >5.1%

No adverse effects Kidney damage Calcium deposits, reduced growth Calcium deposits, reduced growth Kidney damage Kidney damage Kidney damage No adverse effects

Pyrophosphates TSPP TSPP SAPP + TSPP + (KPO3)η

Rats Rats Rats

6 Months 16 Weeks 3 Generations

>1.8%, <3.0% <1.0% >0.5%, <1.0%

Kidney damage Kidney damage Kidney damage

Tripolyphosphates STP STP STP STP STP

Rats Rats Rats Dog Dog

6 Months 1 Month 2 Years 1 Month 5 Months

>1.8%, <3.0% >0.2%, <2.0% >0.5%, <5.0% >0.1 g/kg/day <4.0 g/kg/day

Kidney damage Kidney damage Kidney damage No adverse effects Kidney, heart damage

Polyphosphates SHMP SHMP SHMP SHMP SHMP Graham’s salt (KPO3)η + SAPP +TSPP

Rats Rats Rats Dog Dog Rats Rats

150 Days 1 Month 3 Generations 1 Month 5 Months 6 Months 3 Generations

>0.9%, <3.5% >0.2%, <2.0% >0.5%, <5.0% >0.1 g/kg/day <2.5 g/kg/day >1.8%, <3.0% >0.5%, <1.0%

Slight growth reduction Kidney damage Kidney damage No adverse effects Kidney, heart damage Kidney damage Kidney damage

Cyclic phosphates (NaPO3)3 (NaPO3)3 (NaPO3)3 (NaPO3)3 (NaPO3)3 (NaPO3)4 (NaPO3)4 (NaPO3)4

Rats Rats Rats Dog Dog Rats Dog Dog

1 Month 2 Years 3 Generations 1 Month 5 Months 1 Month 1 Month 5 Months

>2.0%, <10.0% >0.1%, <1.0% >0.05% >0.1 g/kg/day <4.0 g/kg/day >2.0%, <10.0% >0.1 g/kg/day <4.0 g/kg/day

Kidney damage Retarded growth of males No adverse effects No adverse effects Kidney, heart damage Kidney, heart damage No adverse effects Kidney, heart damage

a

Some studies did not give definite periods. No adverse effects: no physiological damage noticeable at any level of phosphate tested. c MSP, monosodium phosphate; DSP, disodium phosphate; DKP, dipotassium phosphate; TSPP, trisodium pyrophosphate; SAPP, sodium acid pyrophosphate; STP, sodium tripolyphosphate; SHMP, sodium hexametaphosphate. d Guinea pigs tolerated higher levels of phosphate only when increased levels of Mg2+ and K+ were present in diets. Source: From Ellinger (1972), FAO (1974), and Deshpande et al. (1995). b

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8.8.2 Hydrochloric Acid

8.8.4 Acetic Acid and Its Salts

Hydrochloric acid is rarely used as an acidulant as classically defined. Nevertheless, it finds many applications in the food industry. Hydrochloric acid is permitted as a food acidulant by the FAO (1974). It is also used in processes that require hydrolysis of starting materials such as proteins and starches. It can be used in the production of corn syrups by varying degrees of controlled acid hydrolysis of corn starch. Hydrochloric acid is also used to produce the chloride salts of several important food additives. The constituent ions of hydrochloric acid are normal participants in animal and human metabolism and, per se, of no toxicological significance. Toxicological considerations are involved either in the purely local action of the corrosive acid or in the effects of the addition of large quantities of either hydrogen or chloride ions to the electrolyte pool of the body (FAO, 1974). Concentrated solutions of hydrochloric acid cause severe burns; permanent visual damage may also occur. Prolonged exposures also result in dermatitis and photosensitization. Inhalation of acid vapors results in cough, choking, and inflammation and ulceration of the respiratory tract. Ingestion of the strong solutions of the acid corrodes mucous membranes, esophagus, and stomach and causes dysphagia, nausea, vomiting, intense thirst, and diarrhea. In extreme cases, circulatory collapse and death may occur (Budavari, 1989). Because of the physiological role of hydrochloric acid, the FAO (1974) concluded that in concentrations approaching the physiological pH of the gastric juice it probably has no toxicological significance. The FAO also found no need to limit its use on toxicological grounds when used in accordance with GMP.

Vinegar, a dilute aqueous solution of acetic acid, has been used since the earliest recorded human history. Very little pure acetic acid is used in foods, although it is classified by the FDA as a GRAS substance. Consequently, it may be used in products not covered by the Definitions and Standards of Identity. Vinegar is used as an acidifier, flavor enhancer, flavoring agent, pH control agent, pickling agent, and solvent and is used for its antimicrobial properties. Vinegars have extensive food applications. Acetic acid enters naturally into the metabolism of the body. It is absorbed from the GI tract and is completely utilized in oxidative metabolism or in anabolic syntheses. Isotope experiments have shown acetates to be utilized in the formation of glycogen intermediates of carbohydrates and fatty acid synthesis, as well as in cholesterol synthesis (FAO, 1974). Acetic acid also participates in the acetylation of amines and may be converted to alanine by transamination and, therefore, incorporated into proteins of plasma, liver, kidney, gut mucosa, muscle, and brain (Documenta, 1970). Acetic acid solutions in water or organic solvents can be very strongly corrosive to the skin and can cause irreparable scarring of tissues of the eyes, nose, and mouth. It causes a variety of adverse effects in humans, which range from allergic-type symptoms such as canker sores (Tuft and Ettelson, 1956), cold sensitivities (Wiseman and Adler, 1956), and epidermal reactions (Weil and Rogers, 1951) to death (Palmer, 1932). Humans who have ingested acetic acid in high concentrations at a low pH have exhibited burned lips, stomach, and intestinal mucosa; corroded lung tissue; and subsequent pneumonia resulting from the inhalation of vapors (Gerhartz, 1949). Acidosis and renal failure, reduction of clotting efficiencies, and interference in blood coagulation have also been reported (Paar et al., 1968; Fin’ko, 1969). In short-term feeding studies, Sollman (1921) fed groups of three to six rats 0.01%, 0.1%, 0.25%, and 0.5% acetic acid in drinking water for periods of up to 9–15 weeks. Although mortality rate was unaffected, rats given 0.5% acetic acid showed a progressive reduction in body weight gain, loss of appetite, and a 27% decrease in food consumption. In other studies, Okabe and colleagues (1971) observed ulceration of gastric mucosa at 10% level in rat diets, whereas a 20% concentration was required for the same effects in cats. Since acetic acid occurs naturally in plant and animal tissues and is involved in fatty acid and carbohydrate metabolism as acetyl CoA, and because humans consume about 1 g/day acetic acid in vinegar and other foods and

8.8.3 Sulfuric Acid Similarly to hydrochloric acid, sulfuric acid is not directly used as an acidulant in foods but has several applications in the manufacture and synthesis of various food additives. It is also used in the hydrolysis of cornstarch for the production of corn syrups. The various sulfate derivatives used in food applications are also prepared by using sulfuric acid. Sulfuric acid is corrosive to all body tissues. Inhalation of concentrated vapors of the acid causes serious lung damage, and contact with eyes results in total loss of vision. Skin contact with strong sulfuric acid solutions produces severe necrosis; frequent skin contact with dilute solutions causes dermatitis (Gosselin, 1984).

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beverages, the FAO has set no limit on its ADI for humans (FAO, 1974). 8.8.5 Lactic Acid and Its Derivatives One of the most widely distributed organic acids in nature, lactic acid is also one of the earliest used as a food additive. It is present in many foods. It is a primary acid component in sour milk and also occurs naturally in sauerkraut, pickles, beer, buttermilk, and cheese. Lactic acid is used as an acidifier, antimicrobial agent, curing agent, flavor enhancer, flavoring agent, pH control agent, pickling agent, solvent, and carrier. The FDA classifies it as a GRAS food additive when used at a level not exceeding the amount reasonably required to accomplish the intended effect. In evaluating the ADI of lactic acid in the human diet, the FAO/WHO (1967) has taken into consideration its well-established metabolic pathway after normal consumption in humans. It does not allow the use of D(–)- or the DL-lactic acid in infant foods. Although no limits were set for the consumption of L(+)-lactic acid, the FAO/WHO (1967) has set a conditional daily intake for D(–) isomer at 100 mg/kg body weight/day in adult humans. 8.8.6 Succinic Acid and Succinic Anhydride Succinic acid, a dicarboxylic acid, is a relatively new product approved for food uses. The FDA has approved it as a flavor enhancer, miscellaneous and general-purpose food chemical, neutralizing agent, and pH control agent (CFR, 1988). It is a GRAS chemical when used at levels not exceeding GMP. Succinic acid, however, is not mentioned specifically in any of the Definitions and Standards of Identity; it is covered under “other edible organic acids” as an optional ingredient. Succinic acid is moderately toxic by subcutaneous route (Lewis, 1989). It is also a severe eye irritant. Dye and associates (1944) conducted short-term studies on rats that gradually received up to 2.0 mg/day at 4 weeks, and the studies continued at this level for 100 days. When compared with the control animals, the test animals did not show any abnormalities in reproduction, hair appearance, tooth eruption, or eye opening. Dye and colleagues (1944) also found no abnormalities in the development of chick embryos when comparable dosages were administered into the air sacs. Since succinic acid occurs naturally in small amounts in several fruits and vegetables and as an intermediate in the Krebs cycle, no limit has been set on the ADI of succinic acid in the human diet.

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8.8.7 Fumaric Acid and Its Salts Fumaric acid is a polyfunctional chemical of significant commercial interest worldwide. It is used extensively in fruit juice drinks, gelatin desserts, pie fillings, refrigerated biscuit doughs, maraschino cherries, and wines (Citric challenged, 1964; Deshpande et al., 1995). A poison by the intraperitoneal route, fumaric acid is mildly toxic by ingestion and skin contact, and is a skin and eye irritant (Lewis, 1989). In short-term feeding trials, Packman and coworkers (1963) observed no abnormalities in the normal blood and urine components of rabbits fed 320–2080 mg/kg body weight disodium fumarate (equivalent to 6.9% salt or 5% of the free acid). Levey and coworkers (1946) have conducted extensive long-term feeding studies in rats. Eight groups of 14 weanling rats were fed diets containing 0%, 0.1%, and 1.0% fumaric acid and 1.38% sodium fumarate for 1 year (half of the groups) or 2 years. No adverse effects were noted on the rate of weight gain, hemoglobin, normal blood constituents, calcium balance as studied by bone histological features, or histological characteristics of liver, kidney, spleen, and stomach. In the only human study conducted thus far, Levey and associates (1946) gave 75 chronically disabled subjects, ranging in age from 29 to 91 years, 500 mg fumaric acid daily for 1 year without any toxic manifestations in hemoglobin level, red and white blood cell counts, nonprotein nitrogen and creatinine levels, and bromosulfonaphthalein and phenosulfonaphthalein excretion. On the basis of their observations, Levey and colleagues (1946) recommended an LD50 of 8000 mg/kg for fumaric acid and its disodium salt. In another study, five groups of 12 male and 12 female rats were fed diets containing 0%, 0.1%, 0.5%, 0.8%, and 1.2% fumaric acid for 2 years; no toxic effects on growth or food consumption were observed (Fitzhugh and Nelson, 1947). In the same study, when a further four groups of 12 male rats were fed for 2 years diets containing 0%, 0.5%, 1.0%, and 1.5% fumaric acid, only the group with the 1.5% fumaric acid diet showed a “very slight increase” in mortality rate and testicular atrophy. Further gross and microscopic examination of other major organs revealed no abnormalities; the tumor incidence was not significantly different among the groups. Primarily on the basis of the 2-year study of Fitzhugh and Nelson (1947), the FAO/WHO (1967) established the level causing no toxicological effects in rats at 1.2% or 12,000 ppm of fumaric acid in the diet (equivalent to 600 mg/kg body weight/day) and for sodium fumarate at 1.38% or 13,800 ppm (equivalent to 690 mg/kg/day).

Similar levels for humans were established at 500 mg/day, equivalent to 10 mg/kg/day. The FAO/WHO (1967) has also approved the use of fumaric acid and its salts at 0–6 mg/kg body weight/day as an unconditional ADI for humans, and 6–10 mg/kg/day conditionally. 8.8.8 Malic Acid and Malic Anhydride L-Malic acid is widely distributed in small amounts, especially in several fruits and vegetables. It is the predominant acid in apples, apricots, bananas, cherries, grapes, orange peels, peaches, pears, plums, quinces, broccoli, carrots, peas, potatoes, and rhubarb (Gardner, 1972). It is present in the second-largest acid quantity in citrus fruits, many berries, figs, beans, and tomatoes. The FDA allows the use of malic acid in foods as an acidifier, flavor enhancer, flavoring agent, pH control agent, and synergist for antioxidants. It is one of the miscellaneous and/or general-purpose food additives in the FDA list of GRAS chemicals. The following limits have been set for its use in different food products: 3.4% in nonalcoholic beverages; 3.0% in chewing gum; 0.8% in gelatins, puddings, and fillings; 6.9% in hard candy; 2.6% in jams and jellies; 3.5% in processed fruits and fruit juices; 3.0% in soft candy; and 0.7% in all other foods when used in accordance with GMP. Long-term studies using rats fed 500 and 5000 ppm malic acid in their basal diets showed no reduction in growth rates or abnormal changes in hematological, urine analysis, and histological characteristics. Diets containing 50,000 ppm malic acid, however, significantly decreased food consumption and growth (Hazleton Laboratories, 1971a). Dogs fed the same three levels of malic acid showed no effects when the same parameters were used (Hazleton Laboratories, 1971b). Reproductive experiments using rats fed 1000 and 10,000 ppm malic acid prior to mating showed no significant differences between those rats and controls (Hazleton Laboratories, 1970). According to a FAO/WHO (1967) report, the need to impose a severe limitation on the content of malic acid in foods arises from the established nephrotoxicity of maleic acid. Male rats fed diets containing 1% or more maleic acid showed growth retardation, increased mortality rate, and changes in the renal proximal convoluted tubes (Fitzhugh and Nelson, 1947). Intraperitoneal administration of 0.1 M sodium maleate in daily doses of 1–2 mL/kg for 2–3 weeks produced glucosuria, phosphaturia, and aminoaciduria. The lethal doses by the oral route were established at 5000 mg L(+)-malic acid/kg for rabbits and at 1000 mg sodium maleate/kg body weight for dogs.

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In evaluating the acceptance of malic acid, the FAO/WHO (1967) has placed emphasis on its well-established metabolic pathway. It has not set any limits for the ADI of the L(+)-isomer of malic acid; the D(–)-isomer is conditionally recommended at 0–100 mg/kg body weight/day in the human diet. Excepting therapeutic purposes, the FAO/WHO (1967) does not allow the use of D(–) and DL-malic acids in baby food formulas. 8.8.9 Tartaric Acid and Its Salts Tartaric acid and cream of tartar are manufactured as byproducts of the wine industry. The CFR (1988) allows tartaric acid and its salts for use in foods as an acidifier, firming agent, flavor enhancer, flavoring agent, humectant, pH control agent, and sequestrant. It is listed as an optional ingredient in standards for fruit butters, fruit jellies, preserves and jams, artificially sweetened jellies and preserves, and fruit sherbets. In short-term studies, rats fed 7.7% sodium tartrate (5.0% free acid) did not show any adverse effects on food consumption, growth, mortality rate, and gross history (Packman et al., 1963). In a long-term feeding study, 21day-old weanling rats were fed diets containing, 0%, 0.1%, 0.5%, 0.8%, and 1.2% tartaric acid (Fitzhugh and Nelson, 1947). No significant differences were observed in weight gain, mortality rate, histopathological effects, and tumor incidence. In teratogenic testing, no abnormalities were observed in mice, rats, hamsters, or rabbits at levels of 274, 181, 225, and 215 mg/kg body weight, respectively (Food and Drug Research Laboratories, 1973). The FAO (1974) has set an unconditional ADI limit of 30 mg/kg body weight for tartaric acid as the L(+) form or its salt in human nutrition. 8.8.10

Adipic Acid

From a commercial viewpoint, adipic acid is the most important of all the aliphatic dicarboxylic acids; worldwide annual production exceeds 2 million metric tons (Deshpande et al., 1995). The CFR (1988) allows the use of adipic acid as a flavoring agent, leavening agent, neutralizing agent, and pH control agent. The FDA has classified adipic acid as a miscellaneous and/or general purpose food additive and has set the following limitations on its use in different food categories: 0.05% in baked goods, 0.005% in nonalcoholic beverages, 0.5% in condiments and relishes, 0.45% in dairy product analogs, 0.3% in fats and oils, 0.0004% in frozen dairy desserts, 0.55% in gelatin and puddings, 0.1% in gravies, 0.3% in meat products, 1.3% in

snack foods, and 0.02% in other food categories when used in accordance with GMP. The capacity of humans and animals to metabolize adipic acid appears to be limited to metabolism of a relatively small amount (FAO/WHO, 1967). While summarizing the acute toxicity data by various routes of administration of adipic acid in rats and mice, Doores (1983) noted that the acid caused hemorrhaging in the small intestine in conjunction with distention of the stomach by administration of the acid via the oral route. Both intravenous and intraperitoneal injections produced hemorrhaging in the lungs, in addition to acidosis by the former route and adhesions of the intestines by the latter. The FAO/WHO (1967) has established the level of adipic acid that has no toxicological effect in rats at 10,000 ppm in the diet, equivalent to 500 mg/kg body weight/day. They have also set a conditional daily intake of 0–5 mg adipic acid/kg/day for humans. 8.8.11

Citric Acid and Its Salts

Citric acid is the most versatile and widely used organic acid in foods and pharmaceuticals. It is approved for food uses as an acidifier, curing accelerator, dispersing agent, flavoring agent, sequestrant, and synergist for antioxidants. The FDA classifies citric acid and its sodium and potassium salts as GRAS food additives. In short-term feeding studies, three dogs given a daily oral dose of 1380 mg citric acid/kg body weight for 112–120 days had no symptoms or evidence of renal damage (FAO, 1974). Similar studies with rats fed 0%, 0.2%, 2.4%, and 4.8% citric acid in a commercial diet showed a depressed food intake with a concurrent lowering of body weight and slight blood chemical abnormalities at the 4.8% level as compared with characteristics of the control group (Yokotani et al., 1971). Test animals in this group also showed a slight atrophy of thymus and spleen. In contrast, Packman and coworkers (1963) did not observe any abnormal blood or urine analytical or histological changes in rabbit fed 7.7% sodium citrate (equivalent to 5% citric acid) for up to 60 days. Bonting and Jansen (1956) conducted long-term studies involving three generations of rats fed diets containing 1.2% citric acid. The test animals did not show any acidosis, abnormal serum composition, or abnormalities in the reproductive system. In a 2-year long-term feeding study using male rats fed a basal diet containing 3% or 5% citric acid, Horn and associates (1957) found no adverse survival rate or histopathological effects in comparisons with the control animals. Weight gains in the treated

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groups, however, were significantly lower than in controls, and food consumption was greatly lowered in the 5% group. Cramer and associates (1956) investigated the relationship of the intake of sodium citrate and citric acid in rats fed vitamin D–free diets containing low levels of phosphorus but adequate levels of calcium. In the absence of the vitamin in diets, the citrates completely prevented the absorption of calcium, although there was no adverse effect on weight gain. The calcium was excreted as calcium citrate in the urine. On the basis of their studies, Cramer and colleagues (1956) concluded that citrates have a rachitogenic effect on the test animals. Dalderup (1960) evaluated the effects of 1.5, 4.5, and 12 g citric acid/kg food in a noncarcinogenic diet on the formation of dental caries in rats. No differences were noted in the number of cavities formed, although animals in the highest-dosage group did show more enamel erosion when compared with the control group. In other studies evaluating the effects of citrates on calcium metabolism, dogs given subcutaneous injections of 320–1200 mg of sodium citrate/kg body weight showed decreased blood calcium levels, while showing increased excretion of urinary calcium (Gomori and Gulyas, 1944). Gruber and Halbeisen (1948) suggested that several of the symptoms produced through the use of high levels of citrates in the diets resembled calcium deficiency. They concluded further that such effects may have been due to the chelation of calcium by the acid moiety rather than the salt itself. Martindale’s Extra Pharmacopoeia (1972) summarizes the effects of citric acid in human beings. Frequent or large-dose ingestion of citric acid may cause erosion of teeth and local irritation, probably because of low pH. Such effects are also seen with lemon juice that contains 7% citric acid and has pH below 3. Citric acid is an important intermediate in the oxidative metabolic pathway of the Krebs cycle. Moreover, potassium and sodium citrates in doses of up to 4 g have been routinely used in medical practice for years without giving rise to ill effects. Both compounds are used as mild diuretics and as means of lowering urine acidity in daily doses of up to 10 g (Martindale’s Extra Pharmacopoeia, 1972). On the basis of these facts, the FAO (1974) concluded that citric acid and its sodium and potassium salts do not constitute a significant toxicological hazard to humans and, hence, set no limits on their ADI in the human diet. Available acute toxicity data, expressed as LD50 values, for various acidulants and their derivatives are summarized in Table 8.12.

Table 8.12 LD50 Values for Various Acidulants Used in the Food Industry Acidulant

Test animal

Route of administration

Phosphoric acid

Rat Rabbit Rat Rat Rabbit Rabbit Mouse Mouse Rat Rabbit Rabbit Rabbit Rat Mouse Mouse Rat Mouse Rat Rat Mouse Mouse Rabbit Rat Rat Rat Rat Rat Guinea pig Mouse Rat Mouse Rat Rat Rat Rabbit Rabbit Rat Dog Dog Mouse Mouse Mouse Mouse Rat Rabbit Rabbit Mouse Mouse Mouse Mouse Rat Rat

Oral Subcutaneous Oral Oral Intravenous Intragastric Oral Intravenous Oral Rectal Subcutaneous Oral Oral Oral Intravenous Intravenous Intravenous Oral Oral Intravenous Subcutaneous Skin Oral Intravenous Oral Intravenous Oral Oral Oral Intraperitoneal Oral Oral Oral Oral Oral Oral Oral Oral Oral Intravenous Oral Intravenous Intraperitoneal Oral Oral Intravenous Oral Intravenous Intraperitoneal Subcutaneous Oral Intraperitoneal

Sodium hydrogen phosphate Sodium dihydrogen phosphate Trisodium phosphate Hydrochloric acid Acetic acid

Sodium acetate

Calcium acetate Dehydroacetic acid Propionic acid Sodium propionate

Calcium propionate Lactic acid

Sodium lactate Ferrous lactate Succinic acid Fumaric acid Sodium fumarate Disodium fumarate L-(+)-Malic acid Sodium malate Tartaric acid Adipic acid

Citric acid

LD50 (mg/kg body weight) 1,530 2,740 17,000 8,290 1,580 300 4,960 525 3,310–3,530 1,200 1,200 1,200 3,530–4,960 3,310 380 147 52 1,000 2,600–3,500 625 2,100 1,640 5,100 1,380–3,200 3,340 580–1,020 3,730 1,810 4,875 2,000 147 2,260 10,700 8,000 3,600–4,800 5,000 1,600 1,000 5,000 485 1,900 680 275 3,600 2,430–4,860 2,430 5,040–5,790 203–960 961 2,700 11,700 725–884 (table continues)

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Table 8.12 (continued) Acidulant

Test animal

Route of administration

Citric acid (continued)

Rat Rabbit Mouse Mouse Rat Rabbit Dog Rat Rat Rat Rabbit Rabbit Dog Mouse Mouse Mouse Mouse Mouse Guinea pig Rabbit Dog Mouse Guinea pig Rabbit Dog Mouse Mouse Rat Rat Mouse Mouse Rat Mouse Rabbit

Subcutaneous Intravenous Intravenous Intraperitoneal Intraperitoneal Intravenous Intravenous Oral Oral Intravenous Oral Subcutaneous Oral Oral Intraperitoneal Oral Intraperitoneal Oral Oral Oral Oral Oral Oral Oral Oral Oral Intraperitoneal Oral Oral Intraperitoneal Intraperitoneal Oral Intravenous Intravenous

Sodium citrate

Potassium citrate Benzoic acid Sodium benzoate

Butyl p-hydroxybenzoate (free acid) Butyl p-hydroxybenzoate (sodium salt) Ethyl p-hydroxybenzoate

Methyl p-hydroxybenzoate

Propyl p-hydroxybenzoate Sorbic acid Sodium sorbate Potassium sorbate Caprylic acid δ-Gluconolactone

LD50 (mg/kg body weight) 5,500 330 44 1,460 1,210 338 167 17,000–40,000 2,700 1,714 2,000 2,000 2,000 5,000 230 950 230 8,000 2,000–2,400 5,000 5,000 8,000 3,000–3,600 6,000 6,000 8,000 400 7,360–10,500 4,000–7,160 2,500 1,300 4,920–6,170 600 7,630

Source: From FAO/WHO (1962, 1963, 1967), Ellinger (1972), FAO (1974), Chipley (1993), Doores (1983, 1989), Sofos and Busta (1983), and Lewis (1989).

8.9

FLAVORING AGENTS

Flavor is the property of a food or beverage that causes the simultaneous reaction of taste on the tongue and odor in the olfactory center of the nose. Flavoring agents are substances that when added to a food or beverage impart flavor, i.e., evoke those two simultaneous responses. Flavorings are primarily used for three purposes: (a) to impart the characteristic flavor of the flavoring; (b) to augment, complement, or modify a flavor; and (c) to mask the original flavor. Recently, the use of flavorings has been expanded to include new roles as antioxidants and as antimicrobials.

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Over 70% of the 1547 additives and 1175 GRAS substances recognized by the FDA in the U.S. food supply are food flavor ingredients. They are derived from a variety of sources: culinary herbs and spices, essential oils from various natural sources, semisynthetic flavor chemicals produced by chemical modification of compounds isolated from natural raw materials, synthetic flavor compounds, biochemically derived flavorings, enzymatic and nonezymatic browning, and processes such as smoking and fermentation. Furthermore, they belong to a number of different chemical groups or classes, including terpene hydrocarbons, sesquiterpenes, fatty acids, alcohols, esters, ethers, aldehydes, ketones, lactones, phenols, and hetero-

cyclic compounds. One common property appears to be that almost all are compounds of molecular weight less than 300. Although the sheer number (>2000) of food flavoring ingredients available today seems staggering, the quantities used are quite small. Of the natural or synthetic flavorings used by the entire food industry worldwide, 78% were used in quantities of less than 1000 pounds (0.0006 mg/day/capita), 61% in quantities less than 100 pounds, and 38% in quantities less than 10 pounds (Senti, 1983). Therefore, although flavor additives are numerous, the quantities used are usually insignificant. Only about 22% of the flavorings are used in concentrations that exceed 100 ppm (Swaine, 1995). Hall and Merwin (1981) estimated the annual per capita consumption of the majority of flavoring agents as less than 10 mg. Consumption of essential oils and higher-volume flavorings, such as vanillin, is 10–100 mg. Salt consumption, in contrast, is estimated to be 7–10 kg, and sugar more than 50 kg. Reineccius (1989) estimated that the flavor compounds already present in the food account for over 98% of ingested flavor, and added flavor compounds less than 2%. The safety of flavors, whether natural or synthetic, is a concern. However, there is a general agreement that it is neither desirable nor possible to evaluate flavor ingredients in the same way as other additives because of their sheer number and the low concentrations used, as their very nature precludes overuse. In addition, vast majorities of flavoring substances occur widely in traditional foodstuffs. The chemical structures of most of them are not related to substances of demonstrated toxicity (Grundschober and Stofberg, 1986). Furthermore, for most flavors, only very limited amounts of material are generally available for testing. Regulatory agencies the world over agree that too many flavoring compounds exist to warrant adequate test-

ing of all of them and assigning them equal importance to materials with unequal risks would be an injudicious use of resources. As a result, JECFA set up a ranking for testing based on the following (Jones, 1992): 1. 2. 3.

The total amount of flavoring likely to be ingested Structural similarity to substances with known biochemical or toxicological properties The nature-identical or totally synthetic (not found in nature) property of the substance

Flavoring ingredients that are synthetic and not nature-identical can be dealt with in the same way as any other food additive. Flavorings derived from nature or that are nature-identical are subject to an evaluation based on their consumption ratio. Stofberg and Kirschman (1985), recognizing the impracticality of testing thoroughly all components commonly used as flavorings, proposed the consumption ratio (CR) as a mechanism for establishing priority for safety evaluation. The CR relies on the facts that most flavoring agents are components of natural foods widely used in the traditional preparation of meals and that exposure or dose of such substance is paramount to its toxicity. The CR is the ratio between the quantity of flavoring agent that is consumed as an ingredient of basic and traditional foods and the quantity of that same substance consumed as a component of added flavorings by the same population over the same period (Stofberg, 1983). A flavoring agent would be said to be food-predominant if the CR were greater than 1: i.e., this substance is predominantly consumed as a component of traditionally prepared food. Conversely, a CR of less than 1 would indicate that a flavoring agent was consumed predominantly as an ingredient in added flavorings. Stofberg (1983) has proposed the use of the CR as a means of classification for safety evaluation. The consumption ratios of some common flavoring agents are shown in Table 8.13.

Table 8.13 Consumption Ratios of Selected Flavoring Agents

Flavoring agent

Annual consumption (kg) via food

Annual consumption (kg) as flavoring

Consumption ratio

Food-predominanta

39,543.7 57,945.9 37,478.0 9,655.2 1,300,995.1 61,731.4 20,674.7

597.0 156,738.0 137,259.0 5.4 68,403.0 27,768.0 475,650.0

66.24 0.37 0.24 1,788.00 19.02 2.22 0.042

++ — — ++ ++ + —

Acetaldehyde Benzaldehyde Ethyl butyrate 3-Ethyl-2,6-dimethylpyrazine Limonene Methyl anthranilate Vanillin a

++, predominant consumption as component of traditional foods; +, moderate consumption via traditional foods; —, consumed predominantly as added flavoring. Source: From Stofberg (1983) and Swaine (1995).

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The following discussion is primarily confined to flavoring agents or constituents of flavoring extracts that are regarded as toxic, have chemical structures analogous to those of toxic compounds, and are used in high volumes.

Ingestion may also induce sweating and salivation. It is a vesicant when applied in concentrated form to the skin.

8.9.1 Allyl Isothiocyanate

d-Limonene is the major component of all citrus oils and is also present in several spice and herb oils. The National Toxicology Program of the United States concluded that it was carcinogenic in the male rat. No carcinogenic activity was noted in the female rat or mice of either sex. dLimonene and other hydrocarbons interact with α-2-Uglobulin, a protein found in the male rat but not in the female rat or in the mouse. Further, it is not found in humans. Evidence linking this interaction with kidney tumors in male rats has been cited (Concon, 1988). In the early 1990s, the JECFA concluded that male rat kidney tumors were male rat–specific and should not be considered in human risk assessment. The committee has assigned an ADI of 1.5 mg/kg to d-limonene.

Allyl isothiocyanate is a component of spice seasonings and condiments. It is most commonly known for the hotpungent character it imparts to mustard and horseradish. It is formed from the glycoside precursor sinigrin at the moistening and crushing of mustard seeds. It is a most potent irritant through skin contact and inhalation. High dietary concentrations can cause epithelial hyperplasia and ulcers of the stomach and minor inflammatory foci in the liver (dogs) and increase mitotic activity in mice and toxicity in rabbits (Cordier and Cordier, 1951; Rusch et al., 1955; Hagen et al., 1967). Ingestion of large amounts of isothiocyanate has been implicated in persons suffering from goiter in certain minor segments of the population. Its minor exposure in the normal human diet and the selflimiting factor account for an adequate margin of safety for its continued use in the human diet (Swaine, 1995). 8.9.2 Cinnamyl Anthranilate Cinnamyl anthranilate (cinnamyl-2-aminobenzoate) is a synthetic flavoring agent widely used to impart either grape or cherry flavor to beverages, candy, puddings, chewing gums, and baked goods, generally at concentrations of 1000 ppm or less. It was withdrawn in 1985 as a GRAS flavoring agent in the United States after the National Cancer Institute reported that it caused cancer in rats and mice when given at doses of 15,000 ppm or greater (Flavoring agent, 1981). Prior to its withdrawal approximately 1000 kg was used annually in the United States in artificial flavorings. 8.9.3 Furfural Furfural occurs naturally in many flavorings and is also a component of several essential oils at concentrations of 0.03%–1.45%. Although furfural is food-predominant and currently has GRAS status in the United States, the JECFA has not yet given an ADI for furfural. 8.9.4 Capsaicin Capsaicin is the nonvolatile pungent or hot principle found in the oleoresin of capsicum. Capsaicin activates perfusion of the gastric mucous membranes, movements of the intestinal fimbria, and reabsorption of glucose (Swaine, 1995).

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8.9.5 d-Limonene

8.9.6 Menthol Menthol, both as a synthetic chemical and as a naturally occurring constituent of peppermint oil (about 50%), is widely used as a flavoring in chewing gum, candy, and dentifrices. Menthol can cause sensitization reactions in the form of urticaria. More serious reactions, including heart fibrillation, have been reported after prolonged high consumption of candies and toxic psychosis from mentholated cigarettes (Thomas, 1962; Luke, 1962). 8.9.7 Monosodium Glutamate (MSG) Monosodium glutamate (MSG) is widely used as a flavor enhancer in several meat preparations. It is also responsible for the umami sensation (Yamaguchi, 1987), often called a fifth basic taste. MSG has often been the subject of controversy in the food industry. It causes glutamate hypersensitivity, more commonly known as the Chinese restaurant syndrome (CRS), a condition estimated to affect 0.2% of the U.S. population. The symptoms in hypersensitive individuals include tightness, warmth, tingling, and a feeling of pressure in the upper body. These symptoms, however, are transient. The mechanism underlying glutamate sensitivity appears to be esophagitis, similar to common heartburn; hypersensitive individuals are predisposed to this response if they ingest a large concentration of MSG, if they lack sufficient saliva flow to dilute MSG, or if they have a tendency to gastric esophageal reflux. Oser and coworkers (1975) studied the effects of MSG on four species of neonatal and infant animals. A uniform dose of 1 g/kg body weight was given to mice,

rats, dogs, and monkeys by intragastric or subcutaneous administration. Adverse effects on growth, appearance, or behavior were not noted in any of the species studied. These researchers concluded that the risk associated with MSG was extremely small and that except among sensitive people, food containing MSG would present no hazard to older children or adults. In another multigenerational study (Anantharaman, 1979), which used male and female mice fed 0%, 1%, and 4% MSG, no differences in sterility, fertility, gestation, viability, lactation indices, or pre- and postweaning performances of the offspring were found. Similarly, various salts of glutamates have been evaluated by utilizing the Ames test with and without metabolic activation to measure genotoxicity and no mutagenic activity was found (Ishidate et al., 1984). Heywood and Worden (1979) have comprehensively reviewed toxicological MSG differences among various species. Regulatory agencies in several countries deem MSG to be safe (JECFA, 1988). It is a GRAS additive and is used in some Standard of Identity foods as long as it is declared on the label. In Canada, it is considered a food ingredient, its use dictated by GMP. 8.9.8 Myristicin Oil of nutmeg and oil of mace contain less than 4% myristicin. Minor amounts are also present in black pepper, carrot, parsley, dill, and celery. Truitt and Ebersberger (1962) have reported that myristicin is a weak monoamine oxidase inhibitor. It also has narcotic and psychomimetic properties (Truitt et al., 1961; Truitt, 1967). Large doses of nutmeg or mace result in states comparable to alcoholic intoxication. Headaches, nausea, abdominal pain, delirium, hypotension, and stupor have been observed. Very high doses can cause liver damage and death. 8.9.9 Yeast The ingestion of high levels of nucleic acid components present in yeast and yeast extracts is associated with high blood levels of uric acid (Reed, 1981). Humans lack the enzyme urate oxidase. Elevated uric acid levels can lead to gout. Pharmacologically active amines, tyramine, for example, known to elevate blood pressure are formed during the fermentation of yeast. It is of concern only to individuals on a regimen of monoamine oxidase (MAO) inhibitors. 8.9.10

5′-Nucleotides

The two nucleotides of commercial significance as flavor potentiators found in nature are inosine monophosphate (IMP) and guanosine monophosphate (GMP). Most ma-

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rine animals appear to be good sources of IMP, especially when the product is dried, and mushrooms are usually good sources for GMP. Both these nucleotides appear to have low systemic toxicity as a result of their relatively high LD50 (>10,000–14,000 mg/kg body weight) values in mice and rats. In turn, near-lethal doses of IMP and GMP produced signs of depression, chronic convulsion, and dyspnea (Kojima, 1974). The extensive studies conducted by Kojima (1974) appear to indicate that IMP and GMP at normal levels of consumption present little if any risk to the short- and long-term health of normal humans. The toxicological aspects of flavoring agents such as protein hydrolysates, smoked foods, and Maillard reaction products are described elsewhere in this book.

8.10

ANTIMICROBIAL AGENTS

Perhaps no group of food additives is more economically valuable than the chemical antimicrobials. The most commonly used antimicrobial food additives in most parts of the world are benzoic acid and its salts, the parabens (p-hydroxybenzoates), sorbates, propionates, SO2 and sulfites, nitrates and nitrites, and acetates. Except the latter three, most of these substances are considered GRAS or affirmed as GRAS. The toxicological properties of these groups of chemicals are briefly described in the following. 8.10.1

Benzoic Acid and Its Salts

Benzoates are used widely for the preservation of foods with pH below 4.5 because of the low cost and ease of incorporation into the products. Benzoate is more effective against yeasts and bacteria than molds (Sofos and Busta, 1992). Benzoic acid and sodium benzoate are considered GRAS when used in accordance with GMP. A maximum level of 0.1% is permitted for use in foods in the United States. Furthermore, it is present naturally in cranberries, prunes, plums, cinnamon bark, and cloves (Reddish, 1957). The toxicological properties of benzoic acid and its salts have been extensively studied. Short-term feeding studies with mice fed 3 g sodium benzoate for 10 days showed a 10% reduction in their creatine output, probably as a result of depletion of the glycine pool (FAO, 1974). Shtenberg and Ignatev (1970) fed groups of 50 male and 50 female mice 80 mg benzoic acid/kg body weight/day, sodium bisulfite at 160 mg/kg/day, and a mixture of these two at the same levels by gavage. The highest mortality rates (60%) were observed in mice given the combination dose compared with the acid alone (32%). A 5-day period

of food restriction after 75 days produced an 85% mortality rate in both groups. Similarly, rats fed sodium benzoate at 16–1090 mg/kg/day over a 30-day period showed neither adverse effects on body weight, appetite, or morphological characteristics, nor histopathological abnormalities in the internal organs (FAO, 1974). The results of several long-term feeding studies were summarized in a report prepared by the FAO (1974). In one study, three groups of 20 male and 20 female rats were pair fed for 8 weeks diets containing 0%, 0.5%, and 1.0% benzoic acid and thereafter fed ad libitum over four generations. Two generations were fed for their entire life span; the third and fourth generations were evaluated at autopsy after 16 weeks. No adverse effects on growth rates, fertility, lactation, and life span were observed during the course of this study. The postmortem examination also did not reveal any abnormalities. In yet another experiment, 20 male and 30 female rats were fed a diet containing 1.5% benzoic acid with 13 male and 12 female rats as controls for 18 months. The mortality rates were five times higher in the test group. The test animals also showed reduced body weights and food intake. Repeat experiments on groups of 20 test animals and 10 controls taken from another rat study also showed similar findings (FAO, 1974). The results of extensive human feeding studies conducted during the early 20th century suggest that sodium benzoate is not deleterious to human health (Chittenden et al., 1909; Dakin, 1909). Benzoates do not appear to be accumulated in the body. They are absorbed from the intestine and detoxified and excreted as hippuric acid via the formation of benzoyl CoA intermediate (White et al., 1964). With the exception of fowls, almost all vertebrates excrete benzoic acid via the hippuric acid pathway. Benzoates have low toxicity in humans and animals (WHO, 1962, 1967). No mutagenic, teratogenic, or carcinogenic effects or other adverse clinical signs due to benzoic acid have been reported from animal feeding studies (Chipley, 1993). There is some evidence, however, that it may cause sensitization reaction in susceptible individuals (Michaelsson and Juhlin, 1973). On the basis of overwhelming data and considering the metabolic and large toxicological margin of safety in animals and humans, the FAO established the level of benzoate that causes no toxicological effect at 1% in the diet, or equivalent to 500 mg/kg body weight. The ADI for total benzoates in the human diet is established at 0–5 mg/kg body weight. 8.10.2

Parabens

Esters of p-hydroxybenzoic (PHB) acid used as antimicrobial agents in cosmetics and pharmaceutical and food products

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include methyl, ethyl, propyl, butyl, and heptyl esters. These are often known as parabens, paracepts, or PHB esters. The methyl and propyl parabens are listed as GRAS in the United States; the heptyl ester is also approved for use in malt beverages and noncarbonated soft drinks. Animal feeding studies have shown their noncarcinogenic and nonteratogenic nature. The parabens are rapidly hydrolyzed, conjugated in the body, and excreted in the urine. This property explains their low toxicity as well as their lack of carcinogenic or teratogenic effects. The principal adverse effect of the use of parabens in food is dermal sensitization reaction (dermatitis) in humans (Epstein, 1968; Wuepper, 1967). A cross-sensitization phenomenon among the parabens has been observed in most patients studied. In the United States, a maximal level of 0.1% is permitted in foods; however, the combined total of all preservatives cannot exceed 0.1% (U.S. FDA 1983). N-Heptyl paraben is permitted only in beer. The JECFA has established an ADI of 10 mg/kg body weight. 8.10.3

Sorbates

Sorbates, especially the acid and the highly soluble potassium forms, are widely used as antimicrobial agents throughout the world. They are effective against many yeasts and molds, as well as bacteria, and are among the least harmful preservatives allowed for food use. Sorbic acid is considered GRAS when used under GMP. Extensive studies of the toxicological properties of sorbates have involved feeding various species of animals (mice, rats, rabbits, and dogs) to determine acute, shortterm, and chronic toxicity levels and effects involving metabolic function, mutagenicity, carcinogenicity, teratogenicity, and reproduction (Concon, 1988; Sofos, 1989). Short-term feeding studies conducted with groups of 25 male and 25 female mice given 40 mg/kg body weight/day sorbic acid by oral intubation for 2 months showed no harmful effects (Shtenberg and Ignatev, 1970). Similar feeding studies in rats fed diets containing 1% or 2% sorbic acid for 80 days indicated no adverse effects on growth rates and no histopathological abnormalities in internal organs. Only the liver was enlarged slightly as compared with that of controls (FAO, 1974). Long-term toxicity feeding studies with rats and mice over a period of two generations with sorbic acid concentrations as high as 90 mg/kg body weight showed no abnormalities. In some instances, the growth rate of test animals increased significantly, apparently as a result of an increased caloric intake resulting from the metabolizable sorbic acid. No carcinogenic or mutagenic effects have been observed with sorbates alone (Dickens et al., 1968;

Litton Biometrics, 1974, 1977; Food and Drug Research Laboratory, 1975; Gaunt et al., 1975; Hendy et al., 1976; Lueck, 1980; Sofos and Busta, 1981). Overall, the concentrations of sorbates used in food preservation have been found to be nontoxic. Even when higher doses were evaluated, sorbate appeared to be a safer compound than other commonly used food additives (Walker, 1990). Sorbic acid and its salts, in fact, are considered to be among the safest antimicrobial agents used in foods. High levels of sorbate, however, may cause allergic-type responses in sensitive individuals by irritating mucous membranes and skin (Lueck, 1980; NAS, 1982; Sofos, 1989). This reaction, of course, may happen only to highly sensitive individuals using pharmaceuticals or cosmetic products preserved with sorbate. However, no allergenic activity from commercial foods preserved with sorbate has been documented (NAS, 1982; Sofos, 1995). Under normal metabolic conditions, sorbates are completely oxidized to carbon dioxide and water in the same way as other fatty acids, releasing 6.6 kcal/g energy. As a result of the extensive favorable toxicological and physiological aspects of sorbic acid, the FAO (1974) has assigned it the highest ADI of all food preservatives, 25 mg/kg body weight. 8.10.4

Propionates

Propionic acid, which occurs naturally in Swiss-type cheeses at levels as high as 1%, and its salts are used as preservatives in baked goods. Other applications involve inhibition of mold growth on the surface of cheeses, processed cheese products, fruits, vegetables, jams, jellies, preserves, malt extract, and tobacco. Propionic acid and its calcium and sodium salts are classified as GRAS when used according to GMP. In short-term feeding studies of mice and rats, they produced no adverse effects on growth, mortality rate, or hematological patterns and no histopathological effect (Harshbarger, 1942; Hara, 1965; Deshpande et al., 1995). In adult humans, daily oral doses of up to 6 g of sodium propionate produced no toxic effects; however, they did produce local antihistaminic activity (Heseltine, 1952; FAO, 1974). Since propionic acid is a normal constituent of food and an intermediary metabolite in humans and ruminants, the FAO (1974) has not established any guidelines on the ADI for humans. 8.10.5

Sulfur Dioxide and Sulfite

Sulfur dioxide (SO2) is one of the oldest antimicrobial agents used as a fumigant in products such as wines (Sofos

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and Busta, 1992). In addition to the gas, sulfite salts, such as sodium sulfite (Na2SO3), potassium sulfite (K2SO3), sodium metabisulfite (Na 2 S 2 O 5 ), and potassium metabisulfite (K2S2O5), may be used as preservatives. Sulfur dioxide and sulfites are GRAS substances, but levels of application are restricted to 0.035% in wines. The use of sulfites is restricted in the United States in foods low in thiamine (vitamin B1). The destruction of thiamine in foods treated with sulfite has been known for several years (Williams et al., 1935). They are thus prohibited as preservatives in meats and fish. They are, however, widely used in dehydrated fruits and vegetables, fruit juice concentrates, and especially wines. Ingestion of sulfites at levels present in foods does not result in accumulation in the body because they are rapidly oxidized to sulfate and excreted in urine. Sublethal, but higher than tolerable, doses result in physiological changes in rats, including polyneuritis, bleaching of incisors, visceral organ atrophy, bone marrow atrophy, renal tubular caste, limited growth, and spectacle eyes (Concon, 1988; Sofos and Busta, 1992). The destructive effects of sulfites on thiamine are partly responsible for their observed toxicity. Ingested levels of sulfur dioxide of 30–100 mg/kg body weight/day, depending upon species, were declared as causing no toxic effects (Select Committee on GRAS Substances, 1976). In addition, the committee indicated that the estimated human consumption of 0.2 mg/kg body weight/day was not a hazard to human health. These findings were reexamined and later endorsed by the U.S. FDA (1985). In general, no mutagenic, teratogenic, or carcinogenic effects were reported for sulfur dioxide in studies using rats and mice (Wedzicha, 1984; Ough 1993). The ADI established by the JECFA (1974) is 0.7 mg sulfur dioxide/kg body weight. Sulfites, however, have been associated with triggering of asthma attacks and other acute allergenic responses. Some are life-threatening or fatal in a small number of susceptible asthmatic humans (Ough, 1993). This property has resulted in restrictions in their use as food preservatives, labeling requirements, and concerns about foods sold without labels, such as salads in restaurants, which may be treated with sulfites to prevent browning (Walker, 1985; Sulfites in Food, 1986; Sofos and Busta, 1992). 8.10.6

Dimethyl and Diethyl Pyrocarbonates

The antimicrobial agents dimethyl and diethyl pyrocarbonates were once used as preservatives in wines, beer, and fruit juices. Inherently, they do not exhibit any toxic effects in both short-and long-term studies in rats. However, they can react with NH3 to form urethan, a known

carcinogen. Hence, their use in beverages was banned in the United States in 1972 (U.S. FDA 1972).

8.11

CONCLUSIONS

Food additives are an intricate part of our food supply. They have been used since prehistoric times to maintain and improve the quality of food products. They are intentionally added to food not only to prevent microbial spoilage, but also to maintain stability, oraganoleptic properties, and nutritional value of foodstuffs. If most were banned from use, dramatic changes in our food supply and subsequent eating habits would occur. At present, food additives undergo extensive toxicological screening before they are admitted for use. However, the majority of additives already in use are believed to be safe for the consumer at the levels applied in food, even though they have not been examined thoroughly for their toxicological properties. The substances involved are of natural origin and traditionally have been used since the early days of food processing. The search for new and safer additives to replace debatable ones, and for processing techniques that require fewer additives, continues. Evaluation of the safety of food additives is extensively provided for by regulation and legislation. The select few examples described in this chapter in relation to toxicological properties of food additives only highlight the difficulties in hazard characterization. In the future, special attention should be given to research methodology for immunotoxic and neurotoxic effects of food additives. Similarly, understanding their mechanisms of action would allow easier extrapolation from animal experimentation to humans. Progress in biochemistry as well as in cellular and molecular biology should enhance the knowledge base. Toxicological problems after long-term consumption of additives are not well documented. There is no conclusive evidence for the relationship between long-term consumption of food additives and the induction of cancer and teratogenic effects in humans. Efforts therefore to monitor exposure assessment, both at the national and at the international level, should also be continued.

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dyes, nonazo dyes, and preservatives in a population of perennial asthamatics. J. Allergy Clin. Immunol. 64:32–37. Wedzicha, B. L. 1984. Toxicology. In Chemistry of Sulfur Dioxide in Foods, pp. 312–365, Elsevier Applied Science, London. Weil, A. J. and Rogers, H. E. 1951. Allergic reactivity to simple aliphatic acids in man. J. Invest. Dermatol. 17:227–231. Weiss, B., Williams, J. A., Hicks, W. J., Margen, S., Abrams, S., Caan, B., Citron, L. J., Cox, C., McKibben, J., Ogar, D., and Schultz, S. 1980. Behavioral responses to artificial food colors. Science 207:1487–1489. Wells, A. 1989. The use of intense sweeteners in soft drinks. In Progress in Sweeteners, eds. T. H. Grenby, pp. 169–214, Elsevier Applied Science, New York. Wender, E. H. 1977. Food additives and hyperkinesis. Am. J. Dis. Child. 131:1204–1208. White, A., Handler, P., and Smith, E. L. 1964. Principles of Biochemistry, 3rd ed. McGraw-Hill, New York. WHO. 1962. Evaluation of the Toxicity of a Number of Antimicrobials and Antioxidants. Sodium Benzoate. Tech. Rep. Ser. No. 228. World Health Organization, Geneva. WHO. 1967. Specifications for the Identity and Purity of Food Additives and Their Toxicological Evaluation. Some Emulsifiers and Stabilizers and Certain Other Substances. Tech. Rep. Ser. No. 373. World Health Organization, Geneva. WHO. 1975. Toxicological Evaluation of Some Colors, Enzymes, Flavor Enhancers, Thickening Agents and Certain Other Food Additives. Food Additive Series No. 6. World Health Organization, Geneva. WHO. 1987. Principles for the safety assessment of food additives and contaminants in food. Environmental Health Criteria No. 70. World Health Organization, Geneva. Whysner, J., Wang, C.X., Zang, E., Iatropoulos, M. J., and Williams, G. M. 1994. Dose-response of promotion by butylated hydroxyanisole in chemically initiated tumors of the rat forestomach. Food Chem. Toxicol. 32:215–222. Wilder, O.H.M. and Kraybill, H. R. 1948. Summary of Toxicity Studies on Butylated Hydroxyanisole. American Meat Institute Foundation, Univ. of Chicago, Chicago, IL. Wilder, O.H.M., Ostby, P. C., and Gregory, B. R. 1960. Effect of feeding butylated hydroxyanisole to dogs. J. Agric. Food Chem. 8:504–506. Willheim, R. and Ivy, A. C. 1953. A preliminary study concerning the possibility of dietary carcinogenesis. Gastroenterology 23:1–19. Williams, G. M. 1977. The detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37:1845–1851. Williams, G. M. 1986. Epigenetic promoting effects of BHA. Food Chem. Toxicol. 24:1163–1166. Williams, J. I., Cram, D. M., Tausig, F. T., and Webster, E. 1978. Relative effects of drug and diet on hyperactive behaviors: An experimental study. Pediatrics 61:811–817.

Williams, R. R., Waterman, R. E., Keresztesy, J. C., and Buchman, E. R. 1935. Studies of crystalline vitamin B1. III. Cleavage of vitamin with sulfite. J. Amer. Chem. Soc. 57:536–537. Wiseman, R. D. and Adler, D. K. 1956. Acetic acid sensitivity as a cause of cold urticaria. J. Allergy 27:50–55. Wuepper, K. D. 1967. Paraben contact dermatitis. JAMA 202:579–581. Wursch, P. and Anantharaman, G. 1989. Aspects of the energy value assessment of the polyols. In Progress in Sweeteners, ed. T. H. Grenby, pp. 241–266, Elsevier Applied Science, New York.

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Yamaguchi, S. 1987. Fundamental properties of umami in human taste sensation. In Umami: a Basic Taste, eds. Y. Kawamura and M. R. Kare, pp. 41–59, Marcel Dekker, New York. Yokotani, H., Usui, T., Nakaguchi, T., Kanabayashi, T., Tanda, M., and Aramaki, Y. I. 1971. Acute and subacute toxicological studies of Takeda-citric acid in mice and rats. J. Takeda Red. Lab. 30:25–31. Zlotlow, M. J. and Settipane, G. A. 1977. Allergic potential of food additives. A report of a case of tartrazine sensitivity without aspirin intolerance. Am. J. Clin. Nutr. 30:1023– 1025.

9 Toxicants Resulting from Food Processing

9.1

INTRODUCTION

The primary objective of food processing operations is to improve the quality of foodstuffs to make them palatable. Nevertheless, some processing operations do induce the formation of materials that are potentially toxic and harmful to humans. Thus, a toxic substance may be formed by interaction between any endogenous and/or exogenous food components or their derivatives, or between these substances and outside agents, such as oxygen. Chemical degradation can also occur as a result of exposure to heat, light, enzymes, and other agents and in turn may result in the formation of toxic compounds. Thermal processing of foods is probably the most commonly used unit operation in the food industry. Heating operations are associated with cooking, frying, toasting, evaporation, sterilization, and similar processes. Even milder thermal operations, such as pasteurization, may bring about a variety of changes in the treated foods. The most noticeable adverse effects generally observed are a loss of heat-susceptible nutrients, especially vitamins and amino acids. Consequently, the nutritional quality of the food is often lowered. It is now well known that normal cooking of foods can induce the formation of many nonpyrolytic toxicants derived from amino acids and proteins. When foods are subjected to still higher temperatures (>200°C–300°C), pyrogenic compounds are produced. Many of these compounds are now known to be potent mutagens and carcinogens. Heating of fats and oils to high temperatures can result in oxidation and polymerization reactions, the products of which appear to be harmful to humans. Other processing operations, such as irradiation, used for food preservation, can form radiolysis prod-

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ucts, many of which are known to be potent toxicants to humans. The very large number of reactive endogenous and/or exogenous food components can readily lead to the formation of a number of derivatives. Several such derived toxicants have been identified and their toxicological properties studied. Yet, many more have not been assessed toxicologically. Similarly, many toxicological studies have been done with individual compounds. It is not only essential that such compounds be identified and their toxicity assessed; the toxicological properties of mixtures of such compounds must also be tested. In all likelihood, the toxicological profile of a foodstuff may be different in the presence of such mixtures; for additive, synergistic and/or antagonistic effects may come into play. In this chapter, some of the more important toxicants derived as a result of food processing operations are discussed. The categories of derived toxicants discussed include pyroorganic toxicants, nonpyrolytic toxicants derived from amino acids and proteins, Maillard reaction products, toxicants produced in rancid fats and oils as well as during their thermal degradation, toxic amino acids formed during alkali processing of proteins, toxicants such as nitrosamines produced by degradation or reaction with contaminants, and radiolysis products formed during food irradiation operations.

9.2

POLYCYCLIC AROMATIC HYDROCARBONS

The polycyclic aromatic hydrocarbons (PAHs) are formed from the incomplete combustion of organic materials.

There are two major sources of PAHs in the human food chain. The most important source entails the deposition and uptake of PAHs from polluted air on food crops. As a result, cereals, vegetables, fruits, and seed oils are important contributors to human intake of PAHs. This aspect of PAHs as industrial or environmental contaminants is discussed at greater length in Chapter 17. The other significant source is derived from the formation and deposition of PAHs on foods during heat processing using methods such as roasting, smoking, and grilling. The formation of PAHs is only significant at high temperatures. At temperatures below 400°C, only small amounts are formed; amounts increase linearly in the range 400°C–1000°C (Toth and Potthast, 1984; Concon, 1988). At these temperatures, a significant amount of charred or tarry products is formed. Benzo[a]pyrene (3,4-benzpyrene) has been identified as the active compound in charred material (Figure 9.1). Other carcinogenic PAHs identified as constituents of charred materials include dibenzo[a,h]an-

thracene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and benzo[b]fluroanthene (Badger, 1962). All are potent carcinogens. However, benzo[a]pyrene is probably the most potent and consequently has received the most attention. 9.2.1 Synthesis The formation of pyrogenic compounds such as PAHs is a complex process. This differs from other heat-induced processes in that the former is preceded by an initial, extensive breakdown of the molecular structures of organic compounds to simpler, reactive fragments. Combinations of these fragments to more stable compounds follow, provided the conditions preclude rapid formation of CO or CO2. The PAHs are most likely pyrosynthesized from degradative products consisting of four- or two-carbon units, such as butadiene or ethylene radicals (Badger, 1962; Hoffmann and Wynder, 1972). The ease of their formation at elevated temperatures follows from their thermodynamic stability. Some possible pathways showing the formation of benzo[a]pyrene are shown in Figure 9.2. 9.2.2 Occurrence

Benzo[a]pyrene

Dibenzo[a,h]anthracene

Benzo[a]anthracene

Dibenzo[a,h]pyrene

Benzo[k]fluoranthene

Dibenzo[a,i]pyrene

Benzo[b]fluoranthene

Figure 9.1 Polycyclic aromatic hydrocarbons (PAHs) commonly found in pyrolytic products.

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Intensive heat treatment leads to the production of PAH. In cooked foods, they are most typically formed from pyrolysis of fats at temperatures exceeding 400°C. This can occur if portions of the food or fat drippings encounter charcoal or very hot surfaces. Thus, there are a limited number of cooking applications that promote the formation of these compounds. The mechanism of formation suggests that PAHs may be produced from pyrolysis of other food components. However, in most cases they are not likely to result in edible products. Grilling or broiling of meat and fish on an open fire leads to PAH contamination in several different ways. First, the high temperatures lead to endogenous formation of PAH on the surface of the food. PAHs can also be formed during the combustion of the fuel used in the grilling. Finally and perhaps most significantly, PAHs are formed when melted fat drips down on the heat source. The PAHs so formed during pyrolysis of the fat are spread to the atmosphere and partially deposed on the surface of the meat (Larsen and Poulsen, 1987). The fat content of the meat is an important factor affecting the PAH level in foods (Lijinsky and Ross, 1967). This is probably most evident when a “clean” fuel, such as charcoal, is used. For example, hamburgers with high fat content when broiled close to the flame produce 43 ppb of PAHs, of which 2.6 ppb is benzo[a]pyrene. In contrast, the

H2C

CH2 Pyrosynthesis Pyrodegradation

Organic matter Pyrodegradation

Benzo[a]pyrene

Figure 9.2

Possible pathways showing the formation of benzo[a]pyrene.

lean product produces only 2.8 ppb PAHs and no benzo[a]pyrene. Reducing the amount of fat in grilled or broiled foods thus appears to reduce the level of PAHs. Thus, the contamination of grilled food with PAHs can be prevented or minimized by using charcoal as fuel, by avoiding open flames, and by employing special grill constructions that prevent the fat from dripping onto the heat source. Smoking is also a major contributory factor to the PAH levels found in smoked foods. The main purpose of smoking is to give products a special desirable taste and palatability. Curing smoke is normally produced from wood (sawdust) by the initial pyrolytic changes of lignin, hemicellulose, and cellulose, followed by secondary reactions leading to the formation of a variety of different chemical compounds. The most important compounds formed are phenols, carbonyls, acids, furans, alcohols, esters, lactones, and PAHs (Larsen and Poulsen, 1987). Low-molecular-weight PAHs, such as phenanthrene, anthracene, and pyrene, are frequently found in smoked foods at levels of 10 ppb. The higher-molecular-weight PAHs, such as benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene, dibenzo[a,c]anthracene, and dibenzo[a,h]anthracene, are found in much lower concentrations. Smoked fish and meat normally contain less than 1 ppb PAHs, and benzo[a]pyrene is most often found at levels of 0.1–0.5 ppb. Under special conditions, higher levels

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can be found, especially on the outside of heavily smoked products (Adrian et al., 1984). In addition to grilled and smoked products, foods processed by other means can also produce PAHs. For example, the soot and skin of coffee beans that had been roasted by direct contact with the combustion gases were found to be very rich (15–28 ppb) in benzo[a]pyrene (Yannai, 1980). Similarly, the bread crust, which receives a more severe heating during baking than the crumb, contains considerably more benzo[a]pyrene than does the latter. This difference is much greater if the bread is baked in a wood-fueled oven than in an electric oven. Several amino acids, especially tryptophan, glutamic acid, valine, proline, and lysine, may form potent mutagens when heated. Some of the tryptophan derivatives have been synthesized in sufficient quantities to permit animal testing and have been found to be very active mutagens (Concon, 1988). Levels of benzo[a]pyrene found in smoked and other foods are summarized in Table 9.1. The levels of this carcinogen vary, depending on the food and the manner of cooking. Generally, the closer the exposure to the source of the smoke or heat, the higher the levels of carcinogen formed. Although there is no clear evidence implicating foodstuffs containing benzo[a]pyrene and other PAHs as causes of cancer in humans, these foods may constitute a real health hazard.

Table 9.1

Benzo[a]pyrene Content of Selected Foodstuffs

Food

Benzo[a]pyrene (ppb)

Smoked fish Eel Herring Sturgeon Chubs White fish Kippered cod

Smoked meats Ham Mutton Close to stove Distant from stove Lamb Sausage (with casing) Cold smoked Hot smoked Salami Bacon

1.0 1.0 0.8 1.3 6.6 4.5

Food

Benzo[a]pyrene (ppb)

Barbecued meats (charcoal broiled) Hamburgers Pork chop Chicken Sirloin steak T-bone steak T-bone steak (flame-broiled) Ribs Other steaks

10.5 5.8–8.0

Miscellaneous foods Spinach Kale Yeast Tea Coffee Cereals Soybean Cheese (Provola)

7.4 12.6–48.1 1.8–40.4 3.9–21.3 0–15.0 0.2–4.1 3.1 4.1–6.2

11.2 7.9 3.7 11.1 57.4 4.4

0.7–55.0 107.0 21.0 23.0 2.9 0.7 0.8 3.6

Source: From Concon (1988) and Howard and Fazio (1983).

9.2.3 Metabolism The PAHs are oxidized by the cytochrome P-450 system in the liver to a complex mixture of phenols, dihydrodiols, and quinines. Arene oxides are formed as intermediate metabolites, which are spontaneously converted into phenols or further metabolized by epoxide hydrolase to dihydrodiols. These primary metabolites undergo further oxidative metabolism and conjugation reactions (Levin et al., 1982; Concon, 1988; Larsen and Poulsen, 1987). As with most known carcinogens, the PAHs are secondary carcinogens or procarcinogens. Their carcinogenic activity depends on specific metabolic transformation. The metabolism of PAHs can be exemplified by benzo[a]pyrene, whose metabolic transformation is rather complex, involving epoxidation and hydroxylation (Selkirk, 1977). A part of the metabolic pathways involved in proximate carcinogen formation is shown in Figure 9.3. The diol-epoxide I is the major metabolite of benzo[a]pyrene. It also binds readily with DNA and RNA (Concon, 1988). It is also the most mutagenic to mammalian cells as compared to the diol-epoxide II and all 13 other known benzo[a]pyrene metabolites and derivatives (Huberman et

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al., 1976; Concon, 1988). The evidence so far points to diol-epoxide I as the major proximate carcinogen of benzo[a]pyrene. The enzymes known collectively as aryl hydrocarbon hydroxylase (AHH) that are responsible for the carcinogenic or noncarcinogenic metabolic transformation of the PAH are affected by several factors. These enzymes, consisting of both constitutive and inducible components, may increase or decrease in activity depending on prior exposure to these carcinogens (Schlede et al., 1970). This relationship is significant because it has been estimated that in 1 year the average person in the United States may be exposed to about 6 g of benzo[a]pyrene (NAS, 1972), a dose that is several thousand times the amount needed to induce cancer in mice. There is also evidence that suggests that conjugation of the benzo[a]pyrene metabolites with glucuronic acid, presumably as a detoxification process, does not necessarily render the conjugates inactive (Kinoshita and Gelboin, 1978). β-Glucuronidase, which is abundant in the liver, spleen, kidney, and other secretory tissues, catalyzes the hydrolysis of this glucuronide. In the process, an active intermediate that can bind to DNA molecules with strong-

A

Mixed-function oxidase (MFO) D C O

B O

MFO

O

HO

F

MFO

E

OH HO

HO OH

OH

G

J

HO

HO

HO

HO

NADPH

NADPH HO

HO

HOH HOH

OH

HO

OH

OH

OH

HOH

OH

OH

HOH

HO

HO

HO

HO

HO

HO

HO

HO

OH

H

I

OH

K

OH

OH OH

L

OH

Figure 9.3 Metabolism of benzo[a]pyrene (BP) showing the formation of possible metabolites that may act as the proximate carcinogens or mutagens A, benzo[a]pyrene; B, (-) BP-trans-7,8-epoxide; C, epoxide hydratase; D, (-) BP-trans-7,8-diol; E, BP-diolepoxide I; F, BP-diolepoxide II; G, 7/8,9-BP-triol; H, 7,10/8,9-BP-tetrol; I, 7/8,9,10-BP-tetrol; J, 7,9/8-BP-triol; K, 7,9,10/8-BP-tetrol; L, 7,9/8,10-BPtetrol. The black box represents deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein binding that result in mutation and carcinogenesis.

er affinity than the parent molecule is formed. Thus, carcinogenesis distant from the site of contact may be mediated by glucuronide conjugates, which are well distributed throughout the body because of their greater water solubility.

9.2.4 Toxicity Fifteen PAHs have been identified as carcinogenic in animals through various routes of exposure (USDHHS, 1991). Some produce skin tumors in the two-stage mouse skin carcinogenesis system and produce local sarcomas at the injection site, and others have the ability to produce lung tumors after either intravenous injection or intratracheal instillation or inhalation (IARC, 1983). In almost all the studies, benzo[a]pyrene appeared to be the most potent

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carcinogen. Mammary tumors induced by intragastric dosed dimethylbenzo[a]anthracene have become an important model in the study of breast cancer. Only four PAHs (benzo[a]pyrene, benzo[a]anthracene, dibenzoanthracene, and methylcholanthrene) have been tested through the oral route and have shown positive indications of carcinogenicity (Nawrot et al., 1999). The target organs for these compounds were shown to be the rodent forestomach; mouse lung, liver, and blood vessels; and rat and mouse mammary glands. Oral administration of PAHs has also been found to result in reproductive, hematopoietic, and other systemic toxicity; however, the carcinogenic effects are considered the most significant (JECFA, 1991). The carcinogenic PAHs detected in foods have all been found to be genotoxic (USDHHS, 1993) and may be reasonably considered to be human carcinogens.

Other toxic effects of PAHs are described in detail in Chapter 17.

9.3

HETEROCYCLIC AROMATIC AMINES

Heterocyclic aromatic amines (HAAs) are generated in proteinaceous foods at normal cooking temperatures. Nagao and associates (1977a) first reported the presence of substantial mutagenic activity in smoke condensates and extracts of the charred surface of broiled fish and meat by using the Salmonella sp./mammalian microsome test of Ames and colleagues (1975). The Salmonella typhimurium tester strains normally used are the strains TA98 and TA1538, which detect frame-shift mutations, and to a lesser extent, the strains TA100 and TA1535, which detect base-pair substitution mutations. Nagao and coworkers (1977a) noted that the activity from the broiled fish and meat was especially pronounced in TA98 with metabolic activation in the presence of a liver homogenate from Arochlor 1254 (PCB)–treated rats (S9 mix). Furthermore, they were able to show that the activity was not caused by benzo[a]pyrene and other PAHs. Since these initial observations, pyrolysates (>300°C) from various proteins, carbohydrates, nucleic acids, and fats were tested in Salmonella sp. However, significant mutagenic activity toward TA98 with S9 was detected only in pyrolysates of proteins. Those from starch were directly mutagenic toward TA100 without metabolic activation, whereas a similar but very weak effect in TA100 was seen with pyrolysates of vegetable oil (Nagao et al., 1977b). Two potent mutagens, Trp-P-1 and Trp-P-2, were isolated in pure states from a pyrolysate of D ,L -tryptophan by monitoring mutagenic activity toward Salmonella typhimurium TA98 during their purification (Sugimura et al., 1977). These compounds were the first of a series of HAAs discovered in pyrolysates of amino acids, proteins, and proteinaceous foods (Sugimura et al., 1982, 1986; Felton et al., 1986; Becher et al., 1988). The recent literature on toxic products generated in foods during cooking or other heat treatments deals extensively with HAAs. 9.3.1 Chemical Characteristics and Nomenclature

ably from Maillard reaction products (pyridines and pyrazines) and creatinine. Examples include IQ, MeIQ, MeIQx, 4,8-DiMeIQx, 7,8-DiMeIQx, and PhIP. These are further subdivided into the quinolines, quinoxalines, and pyridines. The 2-aminopyridine type are formed at temperatures >300°C from protein pyrolysates; they include compounds such as AαC, MeαC, Trp-P-1, Trp-P-2, Glu-P-1, and Glu-P-2. The requirement for creatinine means that the 2-aminoimidazole-type HAAs are not formed from nonmuscle foods or invertebrates, including such proteinrich sources as cheese, tofu, beans, shrimp, and organ meats (Skog, 1993). In addition, new HAAs, such as benzoxazines and furopyridines, which include oxygen in their ring structures, are being investigated (Skog, 1993; Overvik and Gustafsson, 1990; Felton et al., 1992; Eisenbrand and Tang, 1993). The HAAs are classified into two types: the IQ type (Figure 9.4) and the non-IQ type (Figure 9.5). Their chemical names and abbreviations are listed in Table 9.2. Treatment with 2 mM nitrite does not affect the amino groups of the IQ-type HAAs but converts those of non-IQ-type HAAs to hydroxy groups (Sugimura et al., 1989). This conversion is associated with loss of mutagenicity (Tsuda et al., 1980, 1981, 1985). In contrast, treatment with hypochlorite results in loss of mutagenicity of both IQ- and non-IQ-type HAAs (Tsuda et al., 1983, 1985). On the basis of the differential effects of nitrite on the two types of HAAs and their behavior toward hypochlorite, Tsuda and colleagues (1985) determined the contributions of IQ-type and non-IQ-type mutagens to the total mutagenicity of the basic fractions derived from various pyrolyzed materials (Table 9.3). 9.3.2 Metabolism The conversion of HAAs to both activated and inactivated metabolites occurs initially through hydroxylation by cytochrome P-450, followed by conjugation of the hydroxyl group by phase II enzymes, mainly glucuronoyltransferase, sulfotransferase, and acetyl transferase (Wakabayashi et al., 1992; Lynch et al., 1992). The elucidation of species-specific patterns of metabolism and the disposition of HAAs are important in determining target organ and species susceptibility to tumor development. 9.3.3 Occurrence

The HAAs that have been the basis of most mutagenic studies fall into two broad categories based on their precursors and temperature of formation (Miller, 1985). The 2-aminoimidazole type (aminoimidazoazarenes) are formed at moderate temperatures (optimum 190°C– 200°C), prob-

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HAAs have been detected in all major protein sources in the Western-style diet when the foods are cooked at normal household cooking temperatures to a well-done, but not charred state (Overvik and Gustafsson, 1990). No

IQ

IQx

NH2

PhIP

NH2

CH3

N

N CH3

N

N

N

CH3

N NH2

NH2

NH2

MeIQ

MeIQx

N N

N

CH3

H3C

N

N

Figure 9.4

N

N

NH2

CH3

NH2

7,8-DiMeIQx N

N H3C

N

N

CH3

4,8-DiMeIQx

N

N

N

N

N

CH3

CH3

H3C

N

H3C

N

N

CH3

Structures of some IQ-type heterocyclic aromatic amines. See Table 9.2 for abbreviations.

HAAs were detected in raw beef or bacon prior to frying (Lynch et al., 1992; Gross et al., 1993), indicating that these compounds are produced during the cooking process. Temperature, duration of heating, method of heat transfer, and type of cooking medium all influence the extent of formation of HAAs in cooked foods (Nawrot et al., 1999). Frying and broiling generate a 10- to 50-fold greater mutagenic activity in meats than do boiling or baking. In contrast, microwave cooking of meat seems to produce only very low levels of HAAs and mutagenic activity (Gross et al., 1993; Barrington et al., 1990). HAAs are found in cooked foods at the parts per billion (ppb) level (nanograms per gram [ng/g]). Selected data on the content of individual HAAs in some cooked foods are shown in Table 9.4. Layton and coworkers (1995) found the mean daily intake of the major heterocyclic amines from meat to be 26 ng/kg body weight. The intake for an average person weighing 70 kg is therefore 1820 ng. HAAs are absorbed and are bioavailable from the normal diet, as indicated by the presence of PhIP and

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MeIQx in the urine of volunteers consuming a normal diet and the complete absence in the urine of postoperative patients receiving parenteral feeding (Ushiyama et al., 1991). 9.3.4 Toxicity There is an extensive database of toxicological studies on many HAAs found in cooked foods. The major findings from these studies are briefly described in the following sections.

Mutagenicity The pyrolysis products of amino acids, together with the muscle food mutagens, have been tested in a variety of mutagenicity assays in addition to the Ames test and have nearly always yielded positive results. HAAs by themselves are not mutagenic to Salmonella typhimurium strains; their mutagenic activity is exerted only in the presence of a metabolic activation system, viz., S9 mix prepared from the liver of rats treated with PCBs or other

Trp-P-1

Trp-P-2 CH3

CH3 N

N H

N NH2

N H

NH2

CH3

Glu-P-1

Glu-P-2 NH2

N

N

N

NH2

N N

N

CH3

Phe-P-1

Orn-P-1 CH3 N N

NH2

N

N

NH2 N

AαC

MeAαC CH3

N H

Figure 9.5

N

NH2

N H

N

NH2

Structures of some non-IQ-type heterocyclic aromatic amines. See Table 9.2 for abbreviations.

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Table 9.2 Amines

Abbreviations and Chemical Names of Heterocyclic Aromatic

Abbreviation

Chemical name

IQ MeIQ IQx MeIQx 4,8-DiMeIQx 7,8-DiMeIQx PhIP Trp-P-1 Trp-P-2 Glu-P-1 Glu-P-2 Phe-P-1 Prn-P-1 AαC MeAαC

2-Amino-3-methylimidazo[4,5-f]quinoline 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline 2-Amino-3-methylimidazo[4,5-f]quinoxaline 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline 2-Amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline 2-Amino-3,7,8-trimethylimidazo[4,5-f]quinoxaline 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole 3-Amino-1-methyl-5H-pyrido[4,3-b]indole 2-Amino-6-methyldipyrido[1,2-α:3′,2′-d]imidazole 2-Aminodipyrido[1,2-α:3′,2′-d]imidazole 2-Amino-5-phenylpyridine 4-Amino-6-methyl-1H-2,5,10,10b-tetraazafluoranthene 2-Amino-9H-pyrido[2,3-b]indole 2-Amino-3-methyl-9H-pyrido[2,3-b]indole

inducers. Cytochrome P-450s in S9 mix convert the heterocyclic amines to their hydroxyamino derivatives. Synthetic hydroxyamino derivatives of the HAAs show mutagenicity in the absence of a metabolic activation system (Sugimura et al., 1989). The specific mutagenic activities of amino acid pyrolysis products and muscle food mutagens and those of some well-known carcinogens are summarized in Table 9.5. The quinoline and quinoxaline HAAs (IQ, MeIQ, MeIQx, DiMeIQx) are the most potent mutagens, whereas PhIP and the α-carbolines (AαC and MeAαC) are the least potent. The specific mutagenicities of some of these HAAs in the Ames Salmonella sp. assay are much greater than those for such well-established genotoxic carcinogens as benzo[a]pyrene, aflatoxin B1, and MNNG (Table 9.5). Because of the great sensitivity of this assay for HAAs, it has been used to quantify HAAs in foods and biological media.

The HAAs have also been tested for genotoxicity in mammalian in vitro and in vivo assays and in insects. No consistent pattern of positive or negative results was obtained (Munro et al., 1993). In addition, the wide variations between individual HAAs in mutagenic potency observed in the Ames Salmonella sp. assay were not apparent in these studies. Carcinogenicity The carcinogenic potential of 10 of the HAAs found in cooked foods (IQ, MeIQ, MeIQx, PhIP, Trp-P-1, Trp-P-2, Glu-P-1, Glu-P-2, AαC, and MeAαC) was investigated in long-term feeding studies. In most studies, only one or two doses were tested, viz., the maximal tolerated dose (MTD) and a lower dose than the MTD. Groups of 20 to 50 F344 strain rats and CDF1 strain mice of both sexes were given a pellet diet containing 0.01% to 0.08% of the HAA and

Table 9.3 Proportions of Mutagenicity Due to IQ and Non-IQ Types of HAAs in the Basic Fractions of Various Pyrolyzed Materials Sample

IQ-type HAAs (%)a

Non-IQ-type HAAs (%)

Cigarette smoke condensate Broiled sardine Fried beef Broiled horse mackerel

6 88 75 48

85 3 24 42

a

HAA, heterocyclic aromatic amine; IQ, 2-amino-3-methyl-imidazo[4,5-f]quinoline. Source: From Sugimura et al. (1989).

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Table 9.4

Amounts of Some Heterocyclic Amines in Selected Foodsa

Food Broiled/grilled beef steak Fried ground beef Beef extract, food grade Beef flavors Broiled/grilled pork Broiled mutton Broiled grilled chicken Barbecued salmon <6 Minutes >6 Minutes Pan residues (pan-fried meat/fish)

IQ

MeIQ

MeIQx

4,8-DiMeIQx

PhIP

Trp-P-1

Trp-P-2

AαC

0.19 — <0.03 <2.0 — <0.03 <0.03

<0.03 — <0.03 <2.0 — <0.03 <0.03

2.11 2.2 3.10 7.2–21.2 0.07–0.4 1.01 2.33

<0.05 0.7 <0.05 4.2 0.1–0.2 0.67 0.81

15.7 21.5 3.62 <1.0 1.5–3.8 42.5 38.1

0.21 — <0.01 — — <0.01 0.12

0.25 — <0.01 — — 0.15 0.18

1.20 — <0.02 — — 2.50 0.21

ND ND —

ND ND —

<1.0 <1.0 29

ND ND 4.2

2.0–6.2 69–73 144

ND ND —

ND ND —

2.8–6.9 73–109 76.5

a

Amounts expressed in nanograms per gram (ng/g [ppb]). See, Table 9.2 for abbreviations of heterocyclic aromatic amines. ND, not detected. Source: From Nawrot et al. (1999).

Table 9.5 Mutagenicities of HAAs and Typical Carcinogens in Salmonella typhimurium Revertants/µg Compounda IQ MeIQ IQx MeIQx 4,8-DiMeIQx 7,8-DiMeIQx PhIP Trp-P-1 Trp-P-2 Glu-P-1 Glu-P-2 Phe-P-1 Orn-P-1 AαC MeAαC Aflatoxin B1 AF-2 4-Nitroquinoline 1-oxide Benzo[a]pyrene MNNGa N-Nitrosodiethylamine N-Nitrosodimethylamine

TA98

TA100

433,000 661,000 75,000 145,000 183,000 163,000 1,800 39,000 104,200 49,000 1,900 41 56,800 300 200 6,000 6,500 970 320 0.00 0.02 0.00

7,000 30,000 1,500 14,000 8,000 9,900 120 1,700 1,800 3,200 1,200 23 — 20 120 28,000 42,000 9,900 660 870 0.15 0.23

a

See Table 9.2 for abbreviations of heterocyclic aromatic amines. Om-P-1, AF-2, MNNG, N-methyl-N′-nitro-Nnitrosoguanidine. Source: From Sugimura et al. (1989).

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water ad libitum continuously for up to 2 years (Sugimura, 1985). Subsequently the results of multidose studies conducted with PhIP and IQ in the rat and monkey, respectively, were published (Hasegawa et al., 1993; Adamson et al., 1994). The results from these studies showed that almost all of the HAAs are multisite carcinogens in rodents that induce highly significant increases of tumors compared with those in controls, in most cases before 104 weeks of the study (Table 9.6). The liver was the primary target organ in mice, rats, and monkeys. PhIP, in contrast, induced a high incidence of tumors in the large intestine and mammary gland of the rat and immune tissue of mice. IQ, MeIQ, Glu-P-1, and Glu-P-2 also induced tumors of the large intestine in rats. The wide variation in potency observed in the mutagenicity studies was not observed in the carcinogenicity studies. The HAAs also appear to be comparable in potency to such moderate carcinogens as dimethyhnitrosamine. Thus, on the basis of the available literature data, the pyrolysis products of amino acids are potent liver carcinogens in rats and mice, especially in females, and glutamic acid and soybean pyrolysates also produce tumors in various other organs. The suspected role of dietary factors in causing human cancer indicated by epidemiological investigations suggests the possibility that HAAs in the diet may contribute to the cancer burden in the population (Nawrot et al., 1999). In particular, the imidazopyridine PhIP has come under scrutiny because the target organs of this carcinogen in the rat more closely resemble the pattern of carcinogenesis in human populations from developed countries (i.e.,

Table 9.6

Carcinogenicity of Heterocyclic Aromatic Amines

Chemicala

Species

IQ

MeIQ

Concentration (%)

Target organs

Rats

0.03

Mice Rats

0.03 0.03

Trp-P-2 Glu-P-1

Mice Rats Mice Rats Mice Mice Rats

0.01,0.04 0.04 0.06 0.015 0.02 0.02 0.05

Glu-P-2

Mice Rats

0.05 0.05

AαC MeAαC

Mice Mice Mice

0.05 0.08 0.08

Liver, small and large intestine, Zymbal gland, clitoral gland, skin Liver, forestomach, lung Large intestine, Zymbal gland, skin, oral cavity, mammary gland Liver, forestomach Liver, Zymbal gland, clitoral gland, skin Liver, lung, hematopoietic system Liver Liver Liver Liver, small and large intestines, Zymbal gland, clitoral gland Liver, blood vessels Liver, small and large intestines, Zymbal gland, clitoral gland Liver, blood vessels Liver, blood vessels Liver, blood vessels

MeIQx Trp-P-1

a

See Table 9.2 for abbreviations of heterocyclic aromatic amines. Source: From Sugimura et al. (1989).

colon and mammary gland) than that for many other HAAs. PhIP also appears to be the most abundant HAA in foods.

9.4

PREMELANOIDINS FROM MAILLARD REACTION

In various foods, a very complex series of reactions may develop, resulting in the formation of black or brown pigments and modifying other organoleptic properties such as smell and taste. Collectively, these reactions are known as the Maillard reaction and are essentially nonenzymatic in nature; they involve reactions of carbonylic compounds (e.g., aldehydes and ketones) such as reducing sugars (e.g., glucose and fructose) with compounds that have free amino groups, such as amino acids, peptides, and proteins. These reactions are enhanced by heating, particularly under alkaline conditions. The chemical properties of these reactions are extremely complex and still not fully understood in detail. The Maillard reaction (Maillard, 1912) occurs in stages; the final products are a mixture of insoluble darkbrown polymeric pigments known as melanoidins (Hodge, 1953). In the early stages of the reaction, a complex mixture of carbonyl compounds, aromatic substances, reduc-

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tones, and other components is formed. These products are water-soluble and mostly colorless and are known collectively as premelanoidins. The chemical structures of several well-characterized premelanoidins are shown in Figure 9.6. A schematic representation of the Maillard reaction is shown in Figure 9.7. The reaction can be divided into several main steps. An initial condensation of a reducing sugar with a compound having a free amino group is followed by the so-called Amadori rearrangement. This results in the formation of an Amadori compound, such as 1-amino-1-deoxy-2-ketose (Figure 9.7). At this stage, the compound may undergo one of three possible reactions: 1.

2.

A strong dehydration may occur (route 1). This is the most important of the three possibilities, and it gives rise to furfural and dehydrofurfural derivatives, notably 5-hydroxymethylfurfural. The aromas that are also produced as a result of thermal decomposition of sugars are not specific to the Maillard reaction. A splitting of the molecules into carbonyl compounds of which some lack nitrogen, such as acetol, diacetyl, and pyruvaldehyde, may be seen (route 2). The aromas also are not specific to these reactions.

(CH3)2N

O

CHO

HOH2C

O

OH

CHO O H3C

5-Hydroxymethylfurfural

Furfural (CH3)2N

OH

Dimethylaminohexosereductone

OH OH O

N

O O CH2

H 3C

OH

Morpholinohexosefurfural

Anhydrodimethylaminohexosereductone OH

OH N

N O CH2

Figure 9.6

Anhydropiperidinohexosereductone

O H3C

OH

Structures of some commonly occurring premelanoidins due to Maillard reaction in foods.

Aldose sugars

Amino compounds PREMELANOIDINS (SOLUBLE)

1-Amino-1-deoxy-2-ketose (Amadori compound)

N-substituted glycosylamine

Route 2 Scission

3.

A more moderate dehydration may take place (route 3). It gives rise to reducing substances that are a mixture of reductones and hydroreductones. These products may react with amino acids; they decarboxylate them and transform them into aldehydes that are characteristic of the amino acids involved. These reactions are known as Strecker degradation, which produces aromas characteristic of the Maillard reaction.

Dehydration Route 1

Route 3 Enolization 2,3

Enolization 1,2 Small carbonyl molecules

-2 H2O

-3 H2O Furfurals (COLOR, FLAVOR)

Reductones (REDUCING POWER)

Dehydroreductones Strecker Degradation Aldols, Aldimines, Ketimines Amines

Aldehydes (AROMA)

MELANOIDINS (INSOLUBLE)

Figure 9.7

Piperidinohexosereductone

Generalized scheme of the Maillard reaction.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

In the Maillard condensation, the sugars are a more important factor than the amino acids. There is a stronger reactivity with the pentoses than with the hexoses. The order of decreasing reactivity is ribose > xylose > arabinose > mannose > fructose > glucose. Reducing disaccharides, such as lactose and maltose, are even less reactive than the hexoses. However, the nature of the protein involved is also capable of modifying the sugar reactivity. One of the fundamental effects of the Maillard reaction is a drastic lowering of the nutritional quality of proteins. The reason is that amino acids, particularly lysine, are rendered unavailable; thus, the amino acid becomes limiting in an otherwise nutritionally adequate protein. The toxicological implication of the browning reaction was recognized by Kimiagar (1979), who reported changes in blood biochemical properties, increased relative organ weights, and brown-black pigmentation in liver

cells after feeding rats on 10% browned egg albumin for 12 months. Pintauro and associates (1983), however, did not find any toxicological effects in an 18-month rat feeding study using 3% browned egg albumin. They moreover explained the earlier observed changes as a result of nutritive or dietary imbalance. Furthermore, extracts of the brown egg albumin used by Pintauro and colleagues (1983) did not show any mutagenic activity in the Ames test. Several studies, however, suggest the toxic effects of premelanoidins in animal feeding studies. Adrian and Frange (1973) observed a marked reduction in digestibility and nitrogen retention of ingested proteins in diets to which these substances are added. Consequently, the protein efficiency ratio (PER) and the biological value (BV) also decreased. About 35% of premelanoidin nitrogen was retained in the tissues. However, this finding did not imply that the premelanoidin nitrogen was utilized for any useful metabolic function, since the growth rate of the animals was drastically reduced by over 20%–40%. Adrian and Frange (1973) also observed that premelanoidins inhibited digestive enzymes. That premelanoidins may be hepatotoxic has been indicated by the effects observed with spray-dried and roller-dried milk powder. The numbers of animal deaths due to hepatic necrosis in animals fed these products were 40% and 76%, respectively, as compared to less than 1% with liquid milk (Fink et al., 1968). Similarly, heat-dried meat containing 18% premelanoidin glycosylamine produced not only liver hypertrophy in experimental animals, but also hemorrhagic necrotic lesions and cirrhosis of liver (Ferrando, 1963, 1964; Ferrando et al., 1964). Maillard reaction products also have been implicated in certain types of allergic reactions. For example, the allergenicity of a lactoglobulin-lactose solution, as determined by skin reactivity measurements, increased from less than 100 to 1000 µg/mg after heating at 50°C at pH 7 for 48 to 72 hours. The allergenicity decreased slightly after heating (Bleumink and Young, 1968). Maillard reaction products have also found to be mutagenic and clastogenic in several short-term toxicity studies performed on model browning systems. When the testing was done directly on the incubated mixture without extractions, a tendency toward activity in tester strain TA100 without the addition of the S9 fraction was observed. This has been demonstrated with model browning systems with glucose and a variety of different amino acids (Table 9.7). Several studies (Jensen et al., 1983; Stich et al., 1981a, 1981b; Powrie et al., 1981; Shibamoto et al., 1981; Omura et al., 1983) indicate that a diversity of compounds with differing genotoxic activities in various shortterm test systems can be formed during the nonenzymatic browning of food and that their detection should not rely

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Table 9.7 Mutagenicity of Glucose (1 M) and Amino Acid (1 M) Model Browning Systems TA100

TA98

Amino acid

–S9

+S9

–S9

+S9

Glutamic acid Glutamine Lysine Arginine Phenylalanine Tryptophan Tyrosine Cysteine Cystine Methionine Proline Glycine Alanine Valine Leucine Isoleucine Serine Threonine Aspartic acid Asparagine Control

103 309 739 754 253 139 178 213 198 262 183 451 566 277 269 319 257 315 173 146 129

153 138 297 235 365 153 142 1,669 131 179 153 130 133 138 167 224 161 103 86 164 136

37 60 16 34 21 16 19 76 24 20 30 47 23 0 29 30 14 15 41 27 28

25 49 32 97 36 9 19 159 82 26 37 240 36 87 52 36 37 0 56 39 33

Source: From Omura et al. (1983) and Larsen and Poulsen (1987).

on one single test only. Furthermore, N-nitroso compounds, which are well-known carcinogens, can be formed during the nitrosation of certain Maillard reaction products, particularly the Amadori compounds. The Amadori compounds contain secondary amino groups readily nitrosated by the reaction with nitrite. It is quite possible that several N-nitroso compounds not detected by the analytical techniques can be readily formed during cooking and smoking of cured meat and fish by nitrosation of Maillard reaction products (Larsen and Poulsen, 1987). Thus, significant changes in nutritive value are caused by nonenzymatic browning reactions in foods. Furthermore, there is substantial evidence that at least some of the products of the Maillard reaction process may be toxic. This toxicity is evident over long periods and appears to be cumulative. Maillard reaction products also seem to possess potent mutagenic and clastogenic activity in mammalian in vitro systems. Whether these effects are mediated by active oxygen species remains to be seen, since the generation of active oxygen within the cells is now thought to have an important role in tumor promotion. It would be worthwhile to investigate further whether the Maillard reaction mutagens play any role in vivo.

9.5

LYSINOALANINE

Alkali treatment is increasingly used to isolate and process plant and animal proteins intended for food use. Alkaline processing conditions, as mentioned earlier, can promote the Maillard reaction, which renders the lysine residues in protein biologically unavailable. It also produces other undesirable changes, such as decreased digestibility and racemization and destruction of several amino acids (Concon, 1988). Alkali processing can also produce formation of derived amino acids, which may have potential toxic effects in humans. Examples of such derived amino acids formed during alkali treatment include lysinoalanine (LAL), ornithoalanine (OAL), and lanthionine (Figure 9.8) (Provansal et al., 1975). LAL has received particular attention as a possible nephrotoxic compound. It is formed by the condensation of dehydroalanine with the epsilon amino group of lysine (Bohak, 1964). The former, in turn, is produced by β-elimination of cystine, phosphoserine, or serine residues in the presence of alkali. The formation of LAL from cystine and lysine can take place more readily under milder conditions of pH and temperature; serine requires more severe conditions as well as longer reaction times (Karayiannis et al., 1979). LAL content of some representative food products is shown in Table 9.8.

COOH

COOH HC

NH

CH2

(CH2)4

CH NH2

NH2

Lysinoalanine

COOH

COOH HC

NH

CH2

(CH2)3

CH NH2

NH2

Ornithinoalanine

COOH HC

COOH

CH2

S

CH2

NH2

CH NH2

Lanthionine Figure 9.8 Structures of some unusual amino acids formed during alkali processing of proteins.

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LAL appears to be nephrotoxic in experimental animals. When alkali-treated soy protein is fed to rats at appropriate levels, cytomegalic changes are observed in the pars recta, the descending portion of the proximal tubules (Karayiannis et al., 1979; Newberne et al., 1968; Woodard and Alvarez, 1967; Woodard and Short, 1973). That this nephrotoxicity is due to LAL was shown by Woodard and Short (1977), who used purified LAL in their study. The renal lesions in rats fed with alkali-treated soy proteins for several weeks were morphologically similar to those seen in animals fed diets containing low levels of purified LAL for 4 weeks. At higher dose levels, more severe changes are noted, including epithelial necrosis, hypercellularity of the glomeruli, and thickening of Bowman’s capsule with hyperplasia of both visceral and parietal epithelial layers. LAL may not be the only nephrotoxic product produced during alkali treatment of proteins. Ornithoalanine, fructoselysine, and D-serine produce similar nephrotoxic effects in experimental animals (Concon, 1988). Several factors influence the development of nephrocytomegaly. The type of protein treated with alkali appears to be a primary determinant. The difference in the balance of amino acids in alkali-treated lactalbumin and soy protein, for example, may be responsible for the toxic effects. Although lactalbumin contains a much higher level of LAL, probably because of its higher levels of lysine and sulfur-containing amino acids and thus milder reaction conditions required, it does not induce nephrocytomegaly (Karayiannis et al., 1979). In contrast, soy proteins require much harsher processing conditions to produce LAL, and those conditions may cause other undesirable changes in the protein. Since supplementation with lysine, arginine, and threonine to the soy protein diets did not diminish the severity of nephrotoxicity, the decreases in these amino acids during alkaline processing may not be solely responsible for this effect. The composition of the diet may also influence the development of nephrocytomegaly. This appears to be the primary reason for the negative results reported by de Groot and coworkers (1976). They used a diet containing alkali-treated soy protein, similar in composition to that used by Karayiannis and associates (1979); lesions developed in only 20% of rats. The lack of toxicity may also be caused by decreased LAL absorption or protein digestibility. Determination of the LAL levels in the blood, urine, and feces indicated that the observed nephrotoxicity correlated with the extent of intestinal absorption of LAL. The toxicity of LAL or other unusual amino acids formed during alkali treatment of plant proteins may not be readily extrapolated to humans, particularly since spe-

Table 9.8

Lysinoalanine Content of Selected Foodstuffs

Food

LAL (µg/g)

Corn chips Pretzels Hominy Tortillas Taco shells Milk Infant formula Evaporated Skim, evaporated Condensed Simulated cheese

390 500 560 200 170

a

150–640 590–860 520 360–540 1,070

Food

LAL (µg/g)

Egg white solids (dried) Sodium caseinate Calcium caseinate Acid casein Hydrolyzed vegetable protein Whipping agent

160–1,820 430–6,900 370–1,000 70–90 40–500 6,500–50,000

Soya protein isolates

0–370

Yeast extract

120

LAL, lysinoalanine.

cies other than the rat were unaffected (Concon, 1988). In addition, under normal conditions of processing, only small amounts of LAL, which may not cause nephrotoxicity, may be formed. However, the formation of such amino acids during alkali treatment of proteins certainly appears to lower the protein nutritional quality.

9.6

OXIDIZED SULFUR-CONTAINING AMINO ACIDS

The nutritional quality of a food protein is determined primarily by the composition of the essential amino acids, the digestibility of the protein, and the utilizability or bioavailability of the absorbed amino acids. The latter plays a major role in determining protein quality. Several essential amino acids, such as the sulfur-containing amino acids and lysine, can exist either partially or completely in biologically unavailable forms in the protein, either in their native state or in proteins that have undergone some form of processing. Sulfur-containing amino acids are generally limiting in many food proteins, particularly in legumes. During processing and/or storage, they may be oxidized so that the nutritional quality of the food is reduced. A number of oxidized sulfur-containing amino acids have been found in food products. The absolute configurations of several oxidized sulfur-containing amino acids are shown in Figure 9.9. Upon oxidation, L-cysteine can be converted to L -cysteinesulfenic acid, L -cysteinsulfinic acid, and L-cysteic acid. The former is an unstable intermediate; the latter two are stable compounds. Other oxidized forms of L-cysteine include L-cysteine S-oxide (or L-

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cysteine monoxide), L-cystine S,S′-dioxide, and L-cystine S,S-dioxide (Figure 9.9). L-Methionine can be oxidized to L-methionine sulfoxide and L -methionine sulfone. The former can exist in four diastereoisomers because of the two asymmetrical centers of the α-carbon and the sulfur atom (Lavine, 1947). Methionine sulfoximine is formed when wheat flour is treated with nitrogen trichloride (agene) for bleaching and for improving baking quality. The use of agene was banned after methionine sulfoximine was shown to be a potent toxin in animal studies. The oxidation of the sulfur-containing amino acids in food proteins is affected by several factors, such as pH, temperature, type and concentration of oxidizing agents, and type of protein source. In normal food processing, heating without adding oxidizing agents is not likely to oxidize the sulfur-containing amino acids (Marshall et al., 1982). The oxidation of sulfur-containing amino acids results in a loss of bioavailability of these important essential amino acids in human nutrition. It is also unlikely that the oxidized forms can be utilized for protein synthesis. Oxidation of these amino acid residues may also affect their proteolysis and the release rates of free amino acids in the GI tract. Several animal studies have shown that the oxidized sulfur-containing amino acids are not toxic (Miller and Samuel, 1968, 1970; Miller et al., 1970; Daniel and Waisman, 1969). In contrast, methionine sulfoximine is shown to be a convulsant in canine feeding studies (Concon, 1988). It is a methionine antagonist, and thus its inhibitory effect on the growth of the bacteria Leuconostoc mesenteroides can be overcome by the essential amino acid.

COOH C

H2N

COOH

H

H2N

CH2 O

L-Cysteinesulfenic acid

H2N

H S

CH2

H2N

SOH

O

S

C

H2N

C

CH2

S

C

H

CH2

L-Cystine S,S'-dioxide

COOH

COOH

H

H2N S

O O

COOH

CH2

COOH

H

CH2

L-Cystine monoxide

C

SOH

COOH

H

H

O L-Cysteic acid

L-Cysteinesulfinic acid

O

H2N

C

CH2

COOH

COOH C

H

CH2

SOH

H2N

C

COOH

O

H2N

S

S

C

H2N

H

C

H

CH2

CH2

O

CH2 S

L-Cystine S,S-dioxide

O

CH3 L-Methionine sulfoxide

COOH H2N

C

COOH H2N

H

CH2 S

CH2

O

O

CH3 L-Methionine sulfone

Figure 9.9

9.7

H

CH2

CH2 O

C

S

NH

CH3 L-Methionine sulfoximine

Absolute configurations of oxidized sulfur-containing amino acids.

RANCID FATS AND OILS

Oxidation reactions in lipids involve the formation of unpleasant volatile compounds. These reactions may occur even in foods with less than 1% lipid content. The main substrates for oxidation are the unsaturated fatty acids, which generally oxidize faster in the free state than when they form part of a triglyceride or phospholipid. Other unsaturated substrates, such as cholesterol, may also undergo oxidation reactions.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Lipid oxidation leads to the development of various off-flavors and off-odors, generally called rancid (oxidative rancidity), which render foods containing them less acceptable and palatable. In addition, oxidative rancidity can also decrease the nutritional value of foods, and certain oxidative products are potentially toxic. There are three types of rancidity: hydrolytic, ketonic, and oxidative. Hydrolytic rancidity may be induced by heat, alkali, acid, or enzymes endogenous to the foodstuff or by microorganisms. It produces low-molecular-

weight free fatty acids, which contribute to the unpleasant flavor and odor. Ketonic rancidity is due to methyl ketones formed by the β-oxidation of fatty acids by microorganisms. Oxidative rancidity, in contrast, is due to the peroxidation of unsaturated fatty acids, which results in the formation of peroxides and other breakdown products. Once initiated, the free radical reaction continues unhindered unless an antioxidant is present in the system, or until the total unsaturated fatty acids in the oil are oxidized. The lipid peroxidation may be catalyzed or accelerated by exposure to light, iron and copper, presence of oxygen, and heat. Three types of reaction may be distinguished in the oxidation of lipids: 1.

2.

3.

Initiation reactions, which, from unsaturated fatty acids, lead to the formation of free radicals or lipid peroxides. These reactions have high activation energy, and the ease with which they occur is influenced not only by high tempera-

A general scheme summarizing the overall picture of lipid oxidation is shown in Figure 9.10.

Dimers, polymers, cyclic peroxides, hydroperoxy compounds

O2 RH Initiation

tures but more especially by light and trace metals. When the peroxide content increases, the so-called secondary initiation can be seen. The latter results essentially in the decomposition of the peroxides. Propagation reactions, which constitute the oxidation stage of unsaturated lipids by oxygen. They are characterized by an accumulation of lipid peroxides and require the intervention of free radicals, which in the case of pure lipids may lead to the formation of 10–100 molecules of peroxide. The activation energy of these reactions is very low. Termination reactions, in which free radicals associate to yield a wide variety of nonradical compounds.

ROO* R*

Cleavage RH Acyclic and cyclic compounds

ROOH

Aldehydes, ketones, hydrocarbons, furans, acids

OH ROOR ROR dimers

keto, hydroxy and epoxy compounds

RO*

Cleavage

Aldehydes

Alkyl radicals

Condensation

Semialdehydes or oxo-esters O2

O2 Hydrocarbons

Terminal ROOH Hydrocarbons, shorter aldehydes, acids, epoxides

Figure 9.10 Generalized scheme for autooxidation of lipids.

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Hydrocarbons, aldehydes, alcohols

The products formed during hydrolytic and ketonic rancidity development are probably nontoxic (Concon, 1988). In contrast, oxidative rancidity is probably accompanied by the formation of the same or similar toxic compounds formed in heated fats and oils. Peters and Boyd (1969) have observed that a biotin-deficient diet containing 14% cottonseed oil and 3% cod-liver oil is very toxic to rats when allowed to become rancid by standing at room temperature for 6 months or longer. In their study, listlessness, anorexia, oligodipsia, diuresis, diarrhea, proteinuria, marked loss in body weight, and loss in weight of all organs except the brain, adrenal glands, and GI tract occurred. The toxic rancidity reaction was not due to the biotin deficiency or the presence of raw egg white. No toxic reaction developed when the amount of cottonseed oil was decreased to 4%, cod-liver oil was eliminated, and vitamins A and D were supplemented. Rats tended to show more severe toxicity when fed during the winter months than during other seasons. Peters and Boyd (1969) concluded that the toxicity was due to the rancid fats alone and that the toxic factor was ether-extractable. Several other researchers have found similar toxic effects of oxidized fats and oils when fed to experimental animals in the diet at 10%–25% level (Cutler and Schneider, 1973; Addis et al., 1983; Alexander et al., 1987). The adverse physiological effects on animals seen in these studies included ophthalmia, digestive disturbances, reproductive failure, anemia, leukopenia, dermatitis, rapid weight loss, and high death rate. In some studies, these observations were related to the destruction of vitamins and other essential nutrients. Yannai (1980), after reviewing the pertinent literature, concluded that the adverse effects of rancid fats may be due to the following causes: (a) a condition of stress induced by the irritating action of the toxic factors, (b) interference with the normal nutritional properties of fat, (c) destruction of other nutrients, (d) interference with the function of intestinal flora, or (e) a lower food intake. In addition to being toxic, hydroperoxides are potentially carcinogenic. Fats can promote cancer in two ways: by possessing a carcinogenic potential themselves or by having the capacity to potentiate or promote other carcinogens. In addition, both fatty acid hydroperoxides and cholesterol oxidation products are atherogenic (Addis and Park, 1989). However, of the enormous number of lipid degradation products that have been isolated and characterized, only a few have been obtained in large quantities and subjected to toxicity tests.

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9.8

THERMAL DECOMPOSITION OF FATS AND LIPIDS

Thermal oxidation and polymerization reactions can occur in lipids during frying, baking, roasting, and similar processing operations, which are normally carried out at temperatures ranging from approximately 180°C to 250°C. The higher temperature range prevails more frequently during domestic cooking than in well-controlled industrial food processing plants. Thermal oxidation occurs because the oil is exposed to air and frying causes continuous stirring, which greatly increases the surface area of the oil, even in deep-fat frying operations. In contrast, in some operations, the prevailing temperature may be well above 200°C and the foods being fried continuously supply a “steam blanket,” which in turn shields the oil from the surrounding air, thus providing an anaerobic atmosphere in contact with the heated oil. The latter situation favors the so-called thermal polymerization process. The following four classes of compounds are produced from the oil during frying operations (Nawar, 1996): 1.

2.

3.

4.

Volatiles due to the oxidative reactions involving the formation and decomposition of hydroperoxides, which in turn lead to formation of such compounds as saturated and unsaturated aldehydes, ketones, hydrocarbons, lactones, alcohols, acids, and esters Nonpolymeric polar compounds of moderate volatility (e.g., hydroxy and epoxy acids) produced according to the various oxidative pathways involving the alkoxy radical Dimeric and polymeric acids and dimeric and polymeric glycerides from thermal and oxidative combinations of free radicals Free fatty acids from the hydrolysis of triacylglycerols in the presence of heat and water

Although many reports have demonstrated that fats subjected to high temperatures in the laboratory were definitely toxic when given orally to experimental animals, there is ample evidence of the apparent harmlessness of fats used in actual frying in food factories and restaurants, even after prolonged use. Generally, normal frying conditions do not generally produce these derived toxicants at sufficiently high levels to create significant toxic hazards. However, the potential does exist for the formation of deleterious derived substances in the heating or storage of fats and oils, and improper practices in the use of these oils may lead to excess production of these toxic substances. Additionally, the likelihood of cocarcinogenic or tumor-

producing activity of heated fats and oils is probably more pertinent to the question of the safety of normally heated or peroxidized fats and oils. Cutler and Schneider (1973), for example, pointed out that the consumption of highly peroxidized fats may provide a partial explanation of the higher incidence of mammary cancers in such places as Mumbai, India (46/105 persons), Hong Kong (41/105 persons), and Puerto Rico (33/105 persons), as contrasted, for example, to 19/106 in Singapore, 10/106 in Mozambique, and 22/106 in Uganda. The latter countries use more saturated fats in the form of coconut or palm oils. There is also some evidence that suggests that the consumption of heated fats is associated with the high incidence of gastric cancer in some countries (Dungal, 1958; Seelkopf and Salfelder, 1962). There is an extensive database on the effects of heated or oxidized fats fed to experimental animals. Most studies have shown the safety of fats heated under usual household or commercial practice (Marquez-Ruiz et al., 1992; Alexander et al., 1983; Chanin et al., 1988; Billek et al., 1978; Giani et al., 1985; Izaki et al., 1984). Adverse effects, such as depressed weight gains and feed consumption, were noticeable in these studies only when the fats were fed at abnormally high levels. In contrast, studies that have shown the deleterious effects have often used fats and oils that were heated continuously at abusive temperatures in the absence of food or fractions of heated oils were fed to animals at unreasonably high levels. For example, cyclic monomers that form in thermally abused oils cause acute toxicity when administered to rats in large doses. Similarly, malonaldehyde, which is one of the many breakdown products of the peroxidation of linoleic, arachidonic, and other fatty acids, is highly reactive toward amines and can in fact react with DNA in solution and in vivo via Schiff base formation. These properties of malonaldehyde and its structural similarity to known carcinogens suggest that this compound is also carcinogenic (Shamberger et al., 1974). Further evidence supporting the observed carcinogenicity of malonaldehyde has been reported by Mukai and Godstein (1976), who found this compound to be mutagenic in Ames testing. Heated fats may also influence enzyme activity in the liver. Lamboni and Perkins (1996) evaluated the effects of dietary heated fats from a commercial deep-fat frying operation on rat liver enzyme activity. The fats were partially dehydrogenated soybean oil used for 4 days and 7 days for frying foodstuffs in a commercial restaurant. These fats were then fed to rats through either free access to food or pair-feeding of graded doses. The rat enzymes evaluated were carnitine palmitoyltransferase-I (CPT-I),

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isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6PDH), and NADPH-cytochrome P-450 reductase and cytochrome P-450. Feeding rats commercially heated fats led to growth depression, suggesting that the heated oils do contain substances that prevent animals fed such fats from growing properly. The weight gain declined after 35 days of feeding of the experimental diet, indicating that there is a threshold value of accumulated deleterious compounds generated during the commercial deep-fat frying process that must be ingested to exert adverse nutritional effects. The animals also showed an increased activity of cytochrome P-450 that was due to the presence of xenobiotic materials. In contrast, lower activities were observed for CPT-I, ICDH, and G6PDH. Available literature does suggest that toxic compounds can be generated in fat, particularly during abusive heating and/or oxidation. However, moderate ingestion of foods fried in high-quality oils using recommended practices is unlikely to pose a significant health hazard to humans.

9.9

FOOD IRRADIATION

Research on the irradiation of food was begun in 1895 by German and French scientists using the newly discovered Roentgen X-rays. However, it was not until the 1960s, with the growth of research on the uses of atomic energy, that radiation sterilization and pasteurization were put to peaceful use in the preservation of food, sterilization of medical supplies, and structural changes of building materials. The food irradiation program began in the United States as a result of the “Atoms for Peace” program established by President Eisenhower in the early 1950s. The U.S. Congress approved irradiation of food as a food additive in 1958. By the early 1960s, only three countries, Canada, the United States, and the Soviet Union, had given clearance for human consumption of five irradiated foods, all treated with low radiation doses. The food industry had not yet made use of the approvals, and irradiated foods were still not marketed anywhere. Interest in food irradiation, nonetheless, grew worldwide. At the first International Symposium of Food Irradiation, held in Karlsruhe, Germany, and organized by the International Atomic Energy Agency (IAEA), representatives of 28 countries reviewed the progress made in research laboratories (IAEA, 1966). Questions about the safety for human consumption of irradiated foods were discussed and recognized as the major obstacle to commercial utilization of the new process. Because of this rec-

ognition, the International Project in the Field of Food Irradiation (IFIP) was created in 1970, with the specific aim of sponsoring a worldwide research program on the wholesomeness of irradiated foods. Animal feeding studies contracted by IFIP with various laboratories, mostly in the United States, United Kingdom, and France, involved irradiated wheat flour, potatoes, rice, iced ocean fish, mangoes, spices, dried dates, cocoa beans, and legumes. The results obtained in the framework of IFIP and in numerous national testing programs were repeatedly evaluated by the Joint FAO/IAEA/WHO Expert Committee on the Wholesomeness of Irradiated Food (JECFI), the internationally recognized arbiter in this field. In November 1980, this committee concluded that “the irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard; hence, toxicological testing of foods so treated is no longer required” (WHO, 1981). Because of this landmark decision, many national governments have approved the marketing of a growing number of irradiated foods. Currently, the food irradiation process is approved by 36 countries for more than 40 different food items, either on an unconditional or on a restricted (provisional) basis (Nawrot et al., 1999). Food irradiation involves the treatment of food with ionizing radiation for a variety of purposes, including delay of ripening and prevention of sprouting; control of insects, parasites, helminthe, pathogenic and spoilage bacteria, molds, and yeasts; and sterilization, which enables commodities to be stored unrefrigerated for long periods (WHO, 1994). The physical principle on which food irradiation is based is essentially the absorption of energy quanta of electromagnetic radiation by the treated food. The radiation employed is either the radiation continuously emitted by the isotopes 60Co and 137Cs during their radioactive decay or the radiation emitted discontinuously by X-ray sources or linear electron accelerators. The unique feature of the irradiation process is that it causes virtually no increase in the temperature of treated products and is therefore often termed a “cold” process. It thus offers an alternative for food processing when other, conventional methods cannot be used, particularly when increased temperatures are not desirable. Examples include treatment of frozen foods, fresh fruits, and vegetables; inhibition of sprouting of potatoes and onions; and insect disinfestations in grain and dried spices (van Kooij, 1988; WHO, 1988; Diehl, 1995; Nawrot et al., 1999). Irradiation, however, does not improve the nature and quality of the food at the time it undergoes this treatment. It does, however, improve its hygienic status and consequently permits a longer shelf life. Deception of the discerning consumer is therefore not likely.

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9.9.1 Types of Radiation The electromagnetic radiation suitable for treating foodstuffs has a wavelength between 103 and 10–1 nM. These are called ionizing radiations because they are capable of converting atoms and molecules to ions via the removal of electrons. Ionizing radiations can be energetic charged particles, such as electrons, or high-energy photons, such as X-rays or gamma rays. Not all types of ionizing radiation are suitable for irradiation of foods, either because they do not penetrate deeply into the irradiated material or because they make the irradiated material radioactive. The Codex General Standard for Irradiated Foods (Codex, 1984) recommends the following: 1. 2. 3.

Gamma rays from the radionuclides 60Co and 137 Cs X-rays generated from machine sources operated at or below an energy level of 5 MeV Electrons generated from machine sources operated at or below an energy level of 10 MeV

The electronvolt (eV) is the unit of energy used to measure and describe the energy of electrons and of other types of radiation. The energy of 1 eV is equivalent to the kinetic energy acquired by an electron on being accelerated through a potential difference of 1 V. Since eV is a very small unit of energy, it is often expressed as kiloelectronvolts (1 keV = 1000 eV) or MeV megaelectronvolts (1 MeV = 1 million eV). When ionizing radiation penetrates into a medium (e.g., the irradiated food), all or part of the radiation energy is absorbed by the medium. This is called the absorbed dose. It is measured by the International System of Units in terms of the gray (Gy), which is equivalent to the absorption of 1 J/kg of food. The dose accumulated per unit of time is called the dose rate. Gamma ray sources provide a relatively low dose rate (typically 100–10,000 Gy/h), whereas electron accelerators provide a high dose rate (104–109 Gy/sec). Therefore, to achieve a specified absorbed dose, irradiation with a gamma ray source may take several hours, whereas irradiation with an electron accelerator may take only seconds or minutes. The older and current Systeme International (SI) units of radiation dose and radioactivity are summarized in Table 9.9. 9.9.2 Effects on Food Components Irradiation of any material leads to the deposition in that material of energy that can lead to chemical reactions and changes. The chemical changes in the irradiated foods increase with increase in radiation doses. Consideration of

Table 9.9

Unit Definition Old unit Conversion

Units of Radiation Dose and Radioactivity Absorbed dose

Radioactivity

Gray (Gy) 1 Gy = 1 J/kg Rad 1 rad = 0.01 Gy 1 krad = 10 Gy 1 Mrad = 10 kGy

Becquerel (Bq) 1 Bq = 1 disintegration/sec Curie (Ci) 1 Ci = 3.7 × 1010 Bq = 37 GBq 1 kCi = 37 TBq 1 Mci = 37 PBq

the radiation-induced chemical changes is an important part of evaluation of the safety of consumption of irradiated foods. This section offers a general overview of the key nutrients found in foods and the possible changes caused to those nutrients by the irradiation process. For detailed description of the general mechanisms involved in the formation of radiolytic products in irradiated foods and food components, the readers are referred to several excellent reviews (Elias and Cohen, 1977, 1983; Elias, 1987; Josephson and Peterson, 1983; WHO, 1981; Diehl, 1995) Water Water is present in almost all foods, ranging from 5% to 15% in nuts, dried vegetables and cereal flours to as much as 80% to 90% in fruits and vegetables. The radiolysis of water is therefore of particular interest in food irradiation. The radiolytic products of water are listed in Table 9.10. The formation of hydrogen peroxide, a well-known oxidizing agent, might appear to be of great significance in irradiated foods. Actually, it is of less significance than the formation of the highly reactive intermediates. The hydroxyl radical resulting from the ionization of water is a powerful oxidizing agent and can react with any of the food’s other component nutrient molecules or additives/ingredients. The potential to form a great many different socalled radiolytic products is thus quite high, though the actual quantity formed is measurably extremely small (Swallow, 1991). Though these reactions occur with great rapidity (sometimes in fractions of a second), the radicals that result are detectable if they become trapped within the hard portions of foods (extremely dry, frozen, or dense ar-

eas, such as seeds, pits, or bone). Recognition of these entrapped radicals forms the basis of a means to detect whether or not a food has been irradiated. Carbohydrates The major effects of irradiation on the carbohydrates found in foods are the same as those caused by cooking and other types of processing treatments. These include shortening of large polysaccharide chains, degradation of starch and cellulose into simple sugars, and formation of sugar acids, ketones, and other sugars from monosaccharides. Generally, amino acids and proteins can protect carbohydrates from irradiation degradation. Irradiation at high doses causes softening of fruits and vegetables through its effects on plant cell walls and on the pectins that provide the structural rigidity to plant tissue. The use of irradiation in connection with fruits and vegetables that are to be consumed “whole in the skin” is therefore limited by postirradiation quality effects that can result at doses of about 1.0 kGy. Overall, low to medium doses (<1.0 to 10 kGy) of irradiation have mild effects on carbohydrates that do not significantly alter either carbohydrate functionality in foods or their nutritional value. Proteins With some 20 amino acids as constituents of the proteins, and with three reactive species of water hydrolysis, very complex interactions are possible. The irradiation of proteins at high doses is known to produce denaturation, formation of protein radicals due to interactions with water molecule radicals, and a host of reactions to the constituent amino acid subunits (Table 9.11). Low doses can cause a minor breakdown of food proteins into lower-molecularweight fragments and amino acids no more than does conventional steam heat sterilization, whereas very high doses (>100 kGy) can cause cleavage of amino acid side chains. Very little or no effect is seen on the biological value of proteins. Usually, application of the low doses is recommended in order to minimize potential for changes in protein functional properties. Lipids

Table 9.10 Radiolytic Products of Water •OH e–aq •H H2 H2O2 H3O+

Hydroxyl radical Aqueous (or solvated or hydrated) electron Hydrogen atom Hydrogen Hydrogen peroxide Solvated (or hydrated proton)

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In contrast to the radiation chemical characteristics of food proteins and carbohydrates, in which indirect radiation effects mediated through water play a major role, reactions of lipids with the reactive species of water radiolysis play only a minor role in most situations (Diehl, 1995). Irradiation of lipids induces oxidation, which can trigger the formation of lipid hydroperoxides. The development of rancidity, with undesirable odor and flavor production, oc-

Table 9.11 Irradiation Effects on Proteins and Lipids Component Proteins Amino acids

Polypeptides

Enzymes Lipids Triglycerides and fatty acids Phospholipids and sterols Lipids in foods

Nutritional effect

Possible quality and functional effect

Destruction due to deamination, decarboxylation, or breakage of side chain Production of amino acid radicals, ammonia Destruction due to deamination, decarboxylation, or breakage of side chain Production of amino acids due to peptide bond cleavage Production of amino acid radicals, ammonia Cross-linking Denaturation Formation of protein aggregates Activation, deactivation, or no effect Production of fatty acid esters, lactones, and ketones Production of fatty acid esters, aldehydes, and ketones Production of fatty acid esters, lactones, and ketones Production of fatty acid esters, aldehydes, and ketones

curs only at high doses. Other effects include lipid polymerization, typically seen on storage at some time after treatment with high doses (>100 kGy) of irradiation, and breakdown of lipids into hydrocarbons, aldehydes, esters, and ketones (Table 9.11). Generally, the removal of oxygen during irradiation inhibits the oxidation of lipids. This can be achieved by packaging the food or oil in an oxygen-free container or by irradiating under vacuum. The chemical changes that occur in lipids as a result of irradiation can also be minimized by applying the treatment to frozen samples. Vitamins In general, vitamins exposed to levels of irradiation show some losses due to destruction. Additionally, antioxidant vitamins, such as vitamin C and E, can combine with free radicals and lose their vitamin activity. Alternatively, free radicals and their products can attack and destroy vitamin structure or activity. However, certain vitamins have been found to be quite resistant to irradiation-induced destruction; these include vitamin B12, folacin, and pantothenic acid. In contrast, the water-soluble vitamins thiamine and ascorbic acid are the least resistant to effects caused by irradiation. From the available literature data it is clear that

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Formation of flavored compounds

Cross-linking Denaturation Activation, deactivation, or no effect Altered water-binding capacity Altered gelling, emulsifying, and foaming properties Formation of flavored compounds Activation, deactivation, or no effect Production of rancidity due to oxidation in the presence of oxygen Production of rancidity due to oxidation in the presence of oxygen Production of rancidity due to oxidation in the presence of oxygen Destruction of polyunsaturated fatty acid

vitamins are sensitive to degradative loss due to irradiation, just as they are to heating and other processes. The magnitude of sensitivity varies from vitamin to vitamin, product to product, and treatment condition to treatment condition. In order to retain vitamins in foods that are to be irradiated, low treatment doses combined with cold sample temperatures plus oxygen and light exclusion are often recommended. 9.9.3

Effect on Microorganisms

Most of the applications of food irradiation aim at certain biological effects, such as destruction of microorganisms (i.e., those causing food spoilage and/or those causing disease in humans). It is now universally accepted that the DNA in the chromosomes is the most critical target of ionizing radiation. Effects on the cytoplasmic membrane may play an additional role in some circumstances (Grecz et al., 1983; Diehl, 1995). Destruction of microorganisms by irradiation is affected by several factors. Microorganisms differ in their sensitivity to irradiation, depending on morphological variation, just as they differ in their sensitivity to heating, drying, and freezing. Generally, the order of resistance varies, with viruses > bacterial spores > bacterial cells >

yeasts and molds. Generally, the more simple the lifeform, the more resistant it is to irradiation. The resistance to irradiation is expressed as the D10 value, or dose required to reduce the microbial population in a given medium (or food) 10-fold. Thus, an organism with a D10 value of 0.5 kGy is more resistant to irradiation than one with a D10 value of 0.25 kGy, since twice the dose is required to destroy the same number of cells in the population. The D10 values for selected nonsporogenic and spore-forming bacteria are summarized in Table 9.12. No public health hazard related to microorganisms will arise from high-dose irradiation because this process results in a commercially sterile product. In contrast, when foods are treated with nonsterilizing doses of radiation, some microorganisms do survive. This finding has raised a number of concerns (HMSO, 1986, WHO, 1994; Diehl, 1995; Nawrot et al., 1999): 1.

2.

3.

4.

5.

Enhanced selective effects of radiation on the microbial flora in foods: harmless organisms are less resistant to radioactivity than certain pathogenic species. Therefore, more pathogenic microorganisms may survive. It is also possible that spoilage organisms may be preferentially destroyed by irradiation, thereby allowing pathogenic organisms such as Clostridium botulinum, C. perfringens, and Bacillus cereus to survive and grow unchecked. In the absence of spoilage organisms, the food would then appear fit for consumption on the basis of typical organoleptic properties yet contain increased numbers of pathogens and pose a hazard to human health. Enhanced mutations in the surviving population: this may convert nonpathogenic organisms into more virulent strains. Increased radiation resistance by repeated sublethal treatment with radiation: radiation resistance of certain microorganisms has been observed under specific laboratory conditions. Potential change in diagnostic characteristics of microorganisms as a result of irradiation: the species or strain may not be correctly identified. Enhanced toxin formation in toxin-producing bacteria or molds: increased production of aflatoxin has been reported when spores of Aspergillus flavus or A. parasiticus or cultures derived from such spores were irradiated.

Several studies reviewed by Diehl (1995) and Nawrot and associates (1999) show that microbiological safety of irradiated food is comparable with that of food preserved by other acceptable preservation methods, and

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that there is no indication of specific bacteriological hazards associated with radiation processing. On the basis of the existing body of scientific evidence, a WHO panel of experts concluded that there is no reason to suppose that irradiated food needs to be subjected to controls different from those regularly applied to food processed by conventional techniques (WHO, 1994). 9.9.4 Effects on Packaging Materials With the exception of such applications as sprout inhibition in potatoes or onions, insect disinfestations in bulk grain, or delay of postharvest ripening of fruits, irradiation of foods is usually carried out with packaged food items. The reasons for this include prevention of microbial reinfection or insect exposure, prevention of water loss, exclusion of oxygen, prevention of mechanical damage during transport, and improved handling and marketing (Diehl, 1995) The packaging material used must not release radiation-induced reaction products or additives to the food; nor should it lose its functional qualities such as mechanical strength, seal stability, or impermeability to water. Radiation resistance of various packaging materials has been extensively tested. Some are more radiation-resistant than others, but in the dose range of up to 10 kGy, almost all materials commonly used for food packaging are suitable because radiation effects are minor in this range. In the United States, packaging materials used in the irradiation of prepackaged foods must be approved for this purpose by the FDA. 9.9.5 Applications The application of radiation may be divided broadly into three categories: high-dose (>10kGy), medium-dose (1–10 kGy), and low-dose (<1 kGy). At high doses, food is essentially sterilized, just as in commercial canning. At medium doses, there is a pasteurization effect, whereby shelf life is extended and most pathogenic microorganisms are either destroyed or greatly reduced in numbers. At low doses, the product is disinfested of insects and other higher forms of life, and ripening of fruits and vegetables is delayed (Woods and Pikaev, 1994). General applications of food irradiation are summarized in Table 9.13. Approval for this technology has been granted in many countries. In the United States, approval must be sought by petitioning the FDA; at application evidence of scientific studies must be provided to the agency, substantiating the safety and wholesomeness of this process for a particular product. In 1963, the FDA approved the irradiation of wheat and wheat flour at doses between 0.2 and 0.5

Table 9.12 D10 Values of Selected Nonsporogenic and Spore-Forming Bacteria Bacterium

Medium

Irradiation temperature

D10 (Gy)

NONSPOROGENIC BACTERIA Vibrio parahaemolyticusa Pseudomonas fluorescens Pseudomonas putida Leuconostoc mesenteroides Campylobacter jujunia Aeromonas hydrophilaa Proteus vulgarisa Yersinia enterocolyticaa Shigella dysenteriaea Shigella flexneria Brucella abortusa Listeria monocytogenesa Listeria monocytogenesa Listeria monocytogenesa Listeria monocytogenesa Escherichia colia Escherichia colia Escherichia colia Escherichia colia Salmonella anatuma Salmonella enteritidisa Salmonella enteritidisa Salmonella newporta Salmonella oranienburga Salmonella panamaa Salmonella paratyphi Aa Salmonella paratyphi Ba Salmonella stanleya Salmonella typhimuriuma Salmonella typhimuriuma Salmonella typhimuriuma Salmonella typhimuriuma Salmonella typhosaa Staphylococcus aureusa Staphylococcus aureusa Lactobacillus species Streptococcus faecalisa Streptococcus faeciuma Deinococcus radiodurans Moraxella-Acinetobactera (from marine fish) Moraxella-Acinetobacttera (from beef)

Homogenized fish Low-fat ground beef Poultry meat Water on filter paper Ground beef Ground beef Homogenized oysters Ground beef Homogenized shrimp Homogenized shrimp Ground beef Poultry meat Phosphate buffer Trypticase soy broth Chicken feed powder Low-fat ground beef Mech. deboned chicken meat Mech. deboned chicken meat Mech. deboned chicken meat Ground beef Low-fat ground beef Mech. deboned chicken meat Liquid whole egg Liquid whole egg Ground beef Homogenized oysters Homogenized oysters Ground beef Ground beef Mech. deboned chicken meat Mech. deboned chicken meat Mech. deboned chicken meat Homogenized oysters Low-fat ground beef Mech. deboned chicken meat Ground beef Homogenized shrimp Buffer solution Nutrient broth Nutrient agar Nutrient broth

Ambient Ambient 10°C Ambient Ambient 2°C 5°C Ambient Frozen Frozen Ambient 12°C 0°C 0°C 0°C Ambient –5°C 5°C 10°C Ambient Ambient 10°C 0°C 0°C Ambient 5°C 5°C Ambient Ambient –30°C 0°C 10°C 5°C Ambient 0°C Ambient 5°C 5°C Ambient Ambient Ambient

30–60 120 80 120–140 140–160 140–190 200 100–210 200 410 340 490 180 210 440 430 420 280 230 670 700 620 320 320 660 750 850 780 550 900 450 390 750 580 470 300–880 750 900 3500 950–1900 4700

SPORE-FORMING BACTERIA Clostridium botulinum A-33a Clostridium botulinum A-62a Clostridium botulinum B-53a Clostridium botulinum B-51a Clostridium botulinum Ea (Alaska) Clostridium botulinum Ea (Iwanai) Clostridium perfringens Ca

Buffer solution Buffer solution Buffer solution Buffer solution Beef stew Beef stew Water

Ambient Ambient Ambient Ambient Ambient Ambient Ambient

D10 (kGy) 3.3 2.2 3.3 1.3 1.4 1.25 2.1

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Table 9.12 (continued)

Bacterium SPORE-FORMING BACTERIA (Continued) Clostridium perfringens Ea Bacillus cereusa Bacillus pumilus E-601 Bacillus subtilis 6051 Bacillus stearothermophilus

Medium Water Nutrient broth Dry Saline solution Buffer solution

Irradiation temperature

D10 (Gy)

Ambient Ambient Ambient Ambient 0°C

D10 (kGy) 1.2 3.2 3.0 0.64 1.0

a

Indicates a potential pathogen. Source: From Diehl (1995).

kGy for insect disinfestations. Irradiation of white potatoes at doses between 0.05 and 0.15 kGy to inhibit sprouting was approved in 1964. In July 1983, the FDA approved irradiation doses of up to 30 kGy to control microbial contamination in dried spices and dehydrated vegetable seasonings. In June 1984, that approval was extended to cover insect disinfestations as well. In 1985 two additional uses were approved: irradiation of dried enzyme preparations at doses of 10 kGy and treatment of pork carcasses and fresh pork cuts at doses between 0.l3 and 1.0 kGy, excluding vacuum packaged products. In May 1990, the FDA issued a final rule to permit the safe uses of ionizing radiation for the control of foodborne pathogens in poultry. The ruling permits irradiation up to 3 kGy on poultry (defined as any domesticated bird), fresh or frozen, and as whole carcasses, parts, or mechanically deboned. However, the poultry must be in a package that does not exclude air. In September 1992, the U.S. Department of Agriculture (USDA) issued their final regulations on poultry irradiation. These rules imposed a minimal dose of 1.5 kGy and a maximal dose of 3.0 kGy. Labeling requirements are that “Treated with Radiation” or “Treated by Irradiation” and “Keep Refrigerated” or “Keep Frozen” must be on the label. In addition, a green radura symbol must be on the label. Currently, there are petitions to the U.S. FDA for clearances of seafood and red meat for pathogenic bacteria control. All irradiated foods must bear the green “radura” symbol, signifying the treatment they have received. In the United States, the product label must also state that the product has been treated by ionizing radiation or by irradiation. This symbol was developed in the Netherlands and is internationally recognized by the World Health Organization and the International Consultative Group on Food Irradiation as the official symbol that indicates a product has been subjected to irradiation.

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9.9.6 Toxicological Considerations Treatment of foods with ionizing radiation does not induce any measurable radioactivity in the food, provided the energies and dose levels used do not exceed the generally recommended doses for commercial food processing. The potential for induced radioactivity only relates to electron or X-ray sources and is not an issue with radionuclide sources. Thus, foods subjected to ionizing radiation from 60 Co and 137Cs or accelerated electrons of 10 MeV or less and from X-rays of 5 MeV or less will not become radioactive (Diehl, 1995; Nawrot et al., 1999). The wholesomeness and potential toxicity of irradiated food and of irradiated food components have been studied in a large number of in vivo and in vitro investigations since 1961. Some 20 of 60 different foods have been extensively tested by using standard toxicological feed studied in a variety of species (rats, mice, dogs, monkeys, hamsters, pigs, and humans). These studies also included lifespan and multigeneration studies in laboratory animals. Short-term in vivo and in vitro mutagenicity studies have also been performed. Almost all these studies showed no adverse toxicological effects of feeding irradiated foodstuffs. At the 1976 and 1980 meetings, the Joint FAO/ IAEA/WHO Expert Committee on the Wholesomeness of Irradiated Food reviewed and evaluated the toxicity studies then available on a number of food commodities. These included chicken, fish and fish products, papaya, potatoes, strawberries, wheat, onions, rice, mango fruits, dates, cocoa beans, seasonings, and pulses that were irradiated at doses up to 10 kGy. The committee concluded that irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard (WHO, 1977, 1981). In 1981, the U.S. FDA’s Irradiated Food Task Group reviewed all available toxicological data (409 toxicity

Table 9.13 General Applications and Purposes of Food Irradiation

Objective Low-dose treatments (up to 1 kGy) Extension of storage life Improved/extended shelf life Insect disinfestations and parasite disinfection for quarantine purposes Medium-dose treatments (1–10 kGy) Improved shelf life Improved refrigerated storage

Prevention of food poisoning

Improving technological properties of foods Prevention of contamination of food to which the ingredients are added High-dose Treatments (10–45 kGy) Commercial sterilization without refrigeration Decontamination of certain food additives and ingredients

Means Inhibition of sprouting Delay in maturation and senescence Killing or sexual sterilization of insects, destruction of parasites such as Trichinella spiralis and Taenia saginata Pasteurization to reduce populations of bacteria, molds and yeasts Reduction of population of microorganisms capable of growing at refrigeration temperature Destruction of Salmonella, Shigella, Listeria, Campylobacter, Vibrio, Yersinia, and other non-sporeforming pathogen species Softening of tissue

Reduction of population of microorganisms in the ingredient Destruction of spoilage organisms and pathogens, including spore formers Destruction of spoilage organisms and pathogens, including spore formers

studies) concerning food treated with irradiation. Fortyfive of these dealt with subacute toxicity, 58 with subchronic toxicity, 126 with reproductive toxicity, 110 with chronic toxicity, and 102 with genetic toxicity of irradiated food. Only 5 of the 409 studies reviewed (3 chronic, 1 reproduction, and 1 teratogenic study) were considered by the reviewers to be properly conducted and reported, fully adequate by 1980 toxicological standards, and able to stand alone in the support of safety. All five studies indicated no adverse effects from the irradiated foods fed to test animals. The remaining studies, when examined either in isolation or collectively, supported the conclusion that the consumption of foods treated at low irradiation doses does not lead to cause adverse health consequences (FDA, 1986, 1987). Although the vast majority of all animal feeding studies have not indicated harmful effects of irradiated

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Food Potatoes, onions, garlic, shallots, yams, ginger root Fresh fruits and vegetables Cereals and pulses, flours, fresh and dried fruits, nuts, dried fish and meat, fresh pork

Dose (kGy) 0.05–0.15 0.25–1 0.15–0.7

Certain fruits and vegetables, sliced bread Meat, poultry, fish

1–3

Meat, poultry, eggs, egg powder, frog legs, frozen seafood, other foods carrying pathogenic microorganisms Grapes (increasing juice yield), dehydrated vegetables (reducing cooking time) Spices, dried vegetables, other food ingredients

3–10

1–5

2–7

3–10

Meat, poultry, seafood, prepared foods, sterilized hospital diets

30–50

Spices, enzyme preparations, natural gum

10–50

foods, some results have necessitated careful reevaluations. The most frequently cited studies in this regard are those performed at the National Institute of Nutrition in India, showing an increase in polyploid cells in the bone marrow of mice (Vijayalaxmi, 1976) and rats (Vijayalaxmi and Sadasivan, 1975) and in lymphocytes of monkeys (Vijayalaxmi, 1978) and malnourished children (Bhaskaram and Sadasivan, 1975) fed diets containing 70% freshly irradiated (0.75 kGy) wheat. These studies, particularly the study with malnourished children, were subjected to extensive evaluation by the National Food Agency of Denmark (Irradiation of Foods, 1986). The conclusion was that most of these data were insufficient, mutually contradictory, and at variance with well-established knowledge of biology, and that the studies failed to provide convincing evidence that feeding wheat irradiated at 0.75 kGy can induce polyploidy. In contrast, a rat feeding study carried

out at the Bhabha Atomic Research Center, Bombay, with freshly irradiated wheat in which the incidence of polyploidy was determined by counting 3000 cells from each animal showed no effect of consuming the irradiated wheat (George et al., 1976). Extensive attempts to replicate the studies that had presumably indicated increased polyploidy because of feeding freshly irradiated wheat were carried out at the request of the International Project in the Field of Food Irradiation (Tesh et al., 1977). In three assays with rats, flour milled from wheat, freshly irradiated at a dose of 0.75 kGy, caused no elevated polyploidy in bone marrow, no increase in dominant lethal mutations, and no response in the micronucleus assay. In 1992, WHO reviewed all relevant food irradiation safety studies carried out since the 1980 JECFI meeting, as well as many of the older studies. Those studies in which conclusions were reported that had potentially serious ramifications, such as radiation-induced polyploidy or nutrition destruction, were given particular and careful scrutiny. After reviewing all available data, the group reaffirmed the earlier findings and concluded that food irradiated under established good manufacturing practices (GMPs) can be considered safe and nutritionally adequate (WHO, 1994). However, Nawrot and colleagues (1999) have suggested that since the safety/wholesomeness of some irradiated foods was investigated more extensively than that of others, all petitions for a new application of the irradiation process should be evaluated on a case-bycase basis and be supported by sufficient existing and/or new data (if required) to address all safety aspects of the irradiated food under consideration.

9.10 NITRATE, NITRITE, AND N-NITROSO COMPOUNDS 9.10.1

Nitrates and Nitrites

Nitrate is present virtually in everything that we eat and drink and is essentially nontoxic at the levels found in foods and in drinking water. The toxicological significance of nitrate lies in its ready conversion to nitrite by nitrifying bacteria that may be present in foodstuffs, saliva, and the GI tract. Nitrite is also added directly to foods, particularly in the curing of meat and fish. Although nitrite is intrinsically toxic, it is as a precursor of N-nitroso compounds that it potentially causes the most severe problems. The populations that are particularly at risk to nitrite poisoning are those that lack the NADH-dependent methemoglobin reductase activity. This population includes infants less than 1 year of age and those with hereditary

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familial methemoglobinemia. Others who are genetically predisposed include those lacking erythrocyte glucose-6phosphate dehydrogenase activity. In the presence of NADH, the enzyme methemoglobin reductase maintains very low levels of methemoglobin (1.7%) in circulation. Methemoglobin is formed from the oxidation of iron in hemoglobin from the ferrous state to the oxidized ferric state. In this form, the pigment is unable to transport oxygen. Many types of oxidants can convert hemoglobin to methemoglobin. In neonates, levels of up to 5% are common in circulation. Above this value, however, mild cyanosis becomes evident; with increasing levels (>20%), marked cyanosis, fatigue, and dyspnea are observed. At levels over 40%, severe cyanosis, tachypnea, serious cardiopulmonary signs, tachycardia, and depression can occur. Ataxia, coma, and death occur above 60% (Concon, 1988). Most cases of methemoglobinemia involving infants occur because of high levels of nitrates in drinking water or consumption of high-nitrate spinach. The levels of nitrites in food as a derived toxicant are closely related to the levels of nitrates. The average nitrate content of some common foods in the United States and per capita daily intake are summarized in Table 9.14. Many vegetables contain high levels of nitrates. Beets, celery, lettuce, radishes, and spinach may contain more than 600 ppm nitrate nitrogen. These vegetables alone may contribute more than 86% of the total daily intake of nitrates. A large proportion of the total daily intake may also be derived from potatoes because of greater per capita consumption than of any other vegetable, even though potato contains only moderate levels of nitrates. Drinking water, especially that from shallow wells, may also contribute significantly to the level of nitrates in food. Shallow well water may contain as much as 100 mg nitrate/L (Simon et al., 1964). 9.10.2

N-Nitrosamines

As mentioned earlier, nitrates and nitrites can be converted to N-nitroso compounds in vivo. N-Nitroso compounds can be divided into two classes: the nitrosamines and the nitrosamides (and related compounds). Nitrosamines are N-nitroso derivatives of secondary amines, whereas nitrosamides are N-nitroso derivatives of substituted ureas, amides, carbamates, guanidines, and similar compounds (Mirvish, 1975). The general chemical structures of the three general types of nitrosamines significant in food toxicology are shown in Figure 9.11. The most common dialkylnitrosamines are dimethyl- and diethylnitrosamine (DMN and DEN, respectively). Both compounds have been widely studied and are found in foodstuffs. The alkyl group in these compounds may be symmetrical or asymmetrical and may contain

Table 9.14 Average Nitrate Contents of Common Foods in the United States and Per Capita Daily Intake Nitrate, mg/100 g Food

Content

Ingestion

Total vegetables Asparagus Beet Beans, dry Beans, lima Beans, snap Broccoli Cabbage Carrot Celery Corn Cucumber Eggplant Lettuce Melon Onion Peas Peppers, sweet Pickles Potato Potato, sweet Pumpkin/squash Spinach Sauerkraut Tomato and tomato products Breads All fruits Juices Cured meats Milk and milk products Water

1.3–27.6 2.1 276.0 1.3 5.4 25.3 78.3 63.5 11.9 234.0 4.5 2.4 30.2 85.0 43.4 13.4 2.8 12.5 5.9 11.9 5.3 41.3 186.0 19.1 6.2 2.2 1.0 0.2 20.8 0.05 0.071

8609.1 2.8 546.0 10.0 6.6 258.0 127.0 548.0 104.0 1600.0 77.0 7.8 14.8 1890.0 935.0 159.0 19.8 33.5 56.0 1420.0 26.4 38.0 420.0 33.2 198.0 198.0 130.0 10.7 1554.0 25.0 71.0

other functional groups. The parent compounds of dialkylnitrosamines are secondary amines. Nitrosamine formation can occur outside or inside the body; the principal precursors are the various amines and amides and nitrites (Mirvish, 1975, 1983). Thus, the fundamental requirements are a secondary amino nitrogen and nitrous acid. In reality, the nitrosating species is nitrous anhydride or, in the presence of thiocyanate or halides, nitrosylthiocyanate or nitrosylhalide. The precursors to nitrosamine formation occur widely in both the environment and biological systems (Table 9.15 and 9.16). Hence, low-level amounts of nitrosamine have been widely found in many foods and other environments. Nitrosamines can also be formed in vivo. In fact, the conditions in the alimentary tract from the mouth to the

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R1 R2

N N

O

Dialkylnitrosamine

R1

N N O C NH NH R2

Acylalkylnitrosamine

R1

N N O C O R2

Nitrosoguanidine Figure 9.11 General structures of three types of nitrosamines found in foodstuffs.

rectum are quite conducive to nitrosamine formation. First, oral bacteria may promote the reduction of nitrate to nitrite, so that the total nitrite load is increased (Tannenbaum et al., 1974, 1976). Second, nitrosamine formation can also be promoted in the mouth by oral bacteria. Third, saliva can promote the formation of cyanamides from secondary amines as well as primary amines. Both normal and hypoacidic conditions in the stomach also favor nitrosamine formation. The normal acidity of the stomach is ideal for nitrosation, whereas hypoacidity may allow some microorganisms to promote nitrate reduction. The dual occurrence of nitrate reduction and nitrosamine formation in human subjects with gastric hypoacidity who are gavaged with sodium nitrate and diphenylamine was reported by Sander and Schweinsberg (1972). The microflora in the small intestine promotes nitrification of ammonia and organic nitrogen compounds. Five strains of Escherichia coli from the human gut have been demonstrated to form nitrosamines from dimethylamine, diethylamine, piperidine, pyrrolidine, and other amines in the presence of sodium nitrite at neutral pH. In addition, strains of Bacteroides, Bifidobacterium, Clostridium, and Enterococcus spp., which do not reduce nitrate, have been shown to form nitrosamines on replacement of nitrate in the reaction medium with nitrite (Hawksworth and Hill, 1971). The nitrosation reaction is dependent on physicochemical factors. One of the most effective inhibitors of

Table 9.15 Nitrosamine Precursors Endogenous or Formed (Derived) in Foodstuffs Compound Creatine, creatinine Trimethylamine oxide Trimethylamine Dimethylamine Diethylamine Sarcosine Choline, lecithin Proline, hydroxyproline Pyrrolidine Piperidine Methylguanidine Citrulline Carnitine Dipropylamine Dibutylamine

Nitrosamine formeda

Food Meats, meat products, milk, vegetables Fish Fish Fish, meats, meat products, cheese Cheese Meats, meat products, fish Eggs, meats, meat products, soybean, corn Meats, meat products, other foodstuffs Meats, meat products, paprika Meats, meat products, cheese, black pepper Beef, fish Meats, meat products, vegetables Meats, meat products Cheese Cheese

NSA DMN DMN DMN DEN NSA DMN Npro, Npyr Npyr Npip MNC NCit DMN DPN DBN

a

NSA, nitrososarcosine; DMN, dimethylnitrosamine; DEN, diethylnitrosoamine; Npro, nitrosoproline; Npyr, nitrosopyrrolidine; Npip, nitrosopiperidine; MNC, methylnitrosocyanamide; DPN, di-N-propylnitrosoamine; DBN, di-N-butylnitrosoamine; Ncit, nitrosocitrulline. Source: From Concon (1988).

tions of cooking and/or processing. It must be noted, however, that the methods used in nitrosamine analysis in food may underestimate the level of these carcinogens. The reason is that, apart from the low recoveries inherent in the methods, determinations using separation of individual nitrosamines automatically exclude many others that may be present. Collectively, the latter may be quite significant. Thus, the amount of total nitrosamines in a food may be more pertinent in assessing the hazard from these carcinogens. Therefore, the methods of analysis should also focus on the determination of total nitrosamines rather than just each individual compound.

nitrosamine formation is ascorbic acid, which reacts with nitrite readily to form nitric oxide and dehydroascorbic acid (Mirvish et al., 1972). It thus competes for any nitrite present and hence reduces the availability of this reactant for nitrosamine formation. Other inhibitors of the nitrosation reactions include gallic acid, sodium sulfite, cysteine, and tannins. Examples of nitrosamine levels in foodstuffs are summarized in Table 9.17. Cured meats (especially fried bacon), followed by fish and cheese, represent the major sources of nitrosamines in the diet. The formation of these derived toxicants in foods is also dependent on the condi-

Table 9.16 Nitrosamine Precursors That Contaminate Foodstuffs Compound Atrazine Benzthiazuram Carbaryl Fenuron Ferbam Morpholine Propoxur Simazine Succinic acid 2,2′-dimethyl hydrazide Thiram Ziram Source: From Concon (1988).

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Chemical class

Nitrosamine derivative

Secondary amine Carbamate Carbamate Carbamate Amide Secondary amine Carbamate Secondary amine Amide Amide Amide

N-Nitrosoatrazine N-Nitrosobenzthiazuram N-Nitrosocarbaryl Dimethylnitrosamine Dimethylnitrosamine N-Nitrosomorpholine N-Nitrosopropoxur N-Nitrososimazine Dimethylnitrosamine Dimethylnitrosamine Dimethylnitrosamine

Table 9.17 Nitrosamine Levels in Foodstuffs Food Bacon, raw Bacon, fried

Bacon, frying fat drippings Luncheon meat Salami Danish pork chop Sausage Sausage, mettwurs Chinese Fish Sable, raw Salmon, raw Shad, raw Sable, smoked Salmon, smoked Salmon, sable, and shad, smoked and nitrate/nitrite cured Salted, marine fish Fish sauce Cheese Baby foods Shrimp, dried NPyr Shrimp sauce Squid

Canned meats Ham and other pork products Beef products Wheat flour

Nitrosaminea

Level, ppb

DMN, DEN, NPyr NPip NPyr DMN, NPyr NPyr

0 1–40 10–108 11–38 2–30 10–108

DMN, DEN DMN, DEN DMN, DEN DMN NPyr, NPip DMN

1–4 1–4 1–4 1–3 13–105 0–15

DMN DMN DMN DMN DMN DMN

DMN DMN, NPyr DMN DMN DMN 0–37 DMN NPyr DMN NPyr DMN DMN DMN DEN

4 0 0 4–9 0–5 4–26

50–300 0–2 1–4 1–3 2–10 0–10 0–10 2–8 0–7 1–3 0–5 1–2 0–10

a

DEN, diethylnitrosamine; DMN, dimethylnitrosamine; NPyr, nitrosopyrrolidine; NPip, nitrosopiperidine. Source: Modified from Concon (1988).

The biological activity of N-nitroso compounds has been studied extensively. The toxicity of nitrosamines was first recognized in 1937 by Freund, who reported two cases of accidental poisoning from inhalation of DMN. In 1956, Magee and Barnes reported that DMN was a potent hepatocarcinogen in rats. This report marked the beginning of worldwide interest in N-nitroso compound carcinogenesis. Since then, a large number of N-nitroso

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compounds have been tested for carcinogenicity. Among the 400-odd N-nitroso compounds assayed thus far for carcinogenicity, over 90% have yielded positive findings. The most widely tested N-nitroso compound, N-nitrosodiethanolamine (NDEA), has been shown to be carcinogenic in 40 species (Lijinsky, 1987). There are important distinctions between nitrosamines and nitrosamides: the former must be activated to carcinogens by oxidative enzymes (e.g., cytochrome P-450), whereas the latter are direct-acting carcinogens. Nitrosamines often produce tumors at a site(s) distant from the point of application. Selected N-nitroso compounds that may be found in foods and their corresponding target organs are listed in Table 9.18. A number of these compounds seem to have a broad organotropicity in a single species. The organ specificity depends on the chemical nature of the N-nitroso compound and may depend on the dose, route of administration, and animal species. Similarly, there are large interspecies differences in the organs affected by the same compound. N-Nitroso compounds have the ability to induce transplacental carcinogenesis. In rats, kidney tumors have been induced transplacentally by DMN; similar tumors of the lung and liver have been induced in mice (Concon, 1988). The effect observed in the offspring depends on the time during gestation when the N-nitroso compound is administered. Generally, embryotoxic effects are observed when the administration is on days 1–10, teratogenic effects on days 9–16, and carcinogenic effects from day 10 to delivery (Archer, 1982). Several factors influence the carcinogenicity of Nnitroso compounds. Potentiating factors include hormones and their levels, other carcinogens or toxicants, viral or bacterial infections, metals, and nutritional factors. Synergism is often observed in the presence of mycotoxins. In contrast, reducing agents such as ascorbic acid, cysteine, and tannins diminish the carcinogenic potential of N-nitroso compounds. No animal species tested thus far is resistant to DMN or DEN, the two nitrosamines commonly found in foodstuffs (Lijinsky and Taylor, 1977). Thus, the common consensus is that humans cannot be expected to be resistant to the nitrosamines (Mirvish, 1977, 1983). These compounds are particularly effective when exposure is through the oral route, at small doses, and over a long period, conditions particularly relevant to humans. Furthermore, these compounds are systematically organotropic and induce tumors in target tissues independently of the route of administration. Biochemical studies with human liver in vitro have produced evidence that nitrosamines are metabolized and interact with nucleic acids (Montesano and Magee, 1974). This finding suggests that human me-

Table 9.18 Target Tissues of Selected Nitrosamines Found in Foods Nitrosamines

Target tissues

Dimethylnitrosamine

Liver

Diethylnitrosamine

Kidney Lung Nasal cavities Liver

Di-N-propylnitrosamine

Di-N-butylnitrosamine

N-Nitrososarcosine N-Nitrosopyrrolidine

N-Nitrosopiperidine

N-Nitrosomorpholine

N-Methyl-N-nitrosourea

Kidney Lung Nasal cavities Esophagus Forestomach Larynx Trachea Bronchi Liver Esophagus Tongue Liver Lung Esophagus Bladder Forestomach Trachea Tongue Esophagus Liver Lung Nasal cavities Trachea Testes Liver Lung Nasal cavities Esophagus Larynx Trachea Testis Liver Lung Kidney, nasal cavities, ovaries, esophagus Trachea, larynx, bronchus Central nervous system Peripheral nervous system Intestines Kidney Forestomach Skin

Test species Rat, mouse, European hamster, guinea pig, rabbit, rainbow trout, newt, mink, mastomys (Praomys natalensis), aquarium fish (Lebistes reticulates) Rat, Syrian golden and European hamsters Syrian golden hamster Rat, rabbit Rat, mouse, Syrian golden hamster, Chinese hamster, guinea pig, rabbit, dog, pig, trout, grass parakeet, monkey, Brachydanio rerio Rat Mouse, Syrian golden hamster Rat, mouse, Syrian golden hamster, European hamster Rat, mouse, Chinese hamster Mouse, Chinese hamster Syrian golden and European hamsters Syrian golden and European hamsters European hamster Rat Rat Rat Rat, mouse, guinea pig Syrian golden and Chinese hamsters Rat, mouse Rat, mouse, Syrian golden and Chinese hamsters, guinea pig Mouse, Syrian golden hamster, guinea pig Syrian golden hamster Mouse Rat Rat Mouse, Syrian golden hamster Rat Syrian golden hamster Rat Rat, mouse, monkey, Syrian golden hamster Mouse, Syrian golden hamster Rat Rat, mouse Rat, Syrian golden hamster Rat, Syrian golden hamster Mouse Rat, mouse Mouse Rat Syrian golden hamster Rat, mouse, rabbit, dog Rat, dog Rat, Syrian golden hamster, rabbit Rat, mouse Rat, mouse Rat, mouse, dog (table continues)

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Table 9.18 (continued) Nitrosamines

Target tissues

Test species

N-Methyl-N-nitrosourea (continued)

N-Ethylnitrosourea

N-Methyl-N-nitrosourethane

N-Ethy-N-nitrosourethane N-Methyl-N′-nitro-Nnitrosoguanidinea

Subcutaneous tissues Glandular stomach, jaw, bladder, uterus, vagina Liver, lung, hematopoietic system Pharynx, esophagus, trachea, bronchus, oral cavity Stomach, pancreas, ear duct Central nervous system Peripheral nervous system Kidney Hematopoietic system Skin, intestines, ovary, uterus Lung Forestomach Esophagus Kidney, intestines, ovary Pancreas, subcutaneous tissues Forestomach, intestines Glandular stomach

Syrian golden and European hamsters Rat

Guinea pig Rat, mouse Rat, mouse Rat, mouse Rat, mouse Rat Rat, mouse Rat, mouse, Syrian golden hamster Rat, Syrian golden hamster Rat Rabbit Rat Rat, Syrian golden hamster

Forestomach Stomach Intestines Skin Subcutaneous tissues Lung

Rat, mouse Dog Rat, mouse, Syrian golden hamster, dog Mouse Rat Rabbit

Mouse Syrian golden hamster

a

Not found in foods as such, but one of similar structures may be derived from naturally occurring guanidines, such as methylguanidines. The latter has been detected in certain foods, e.g., meats, and is probably derived from creatinine. Source: From Magee et al. (1976) and Concon (1988).

tabolism of nitrosamines may also produce proximate carcinogens similar to those seen in almost every animal study. When the amounts of total nitrosamines in food, water, and other sources are added to those formed throughout the GI tract, the total nitrosamine load of modern populations would be considerable. Viewed from this perspective, each carcinogen, even in trace amounts, assumes considerable significance.

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Howard, J. W. and Fazio, T. 1983. Polycyclic aromatic hydrocarbons in foods. In Handbook of Naturally Occurring Food Toxicants, ed. M. Rechcigl, pp. 161–190. CRC Press, Boca Raton, FL. Huberman, E., Sachs, L., Yang, S. K., and Gelboin, H. V. 1976. Identification of mutagenic metabolites of benzo(a)pyrene in mammalian cells. Proc. Natl. Acad. Sci. (USA) 73: 607–611. IAEA. 1966. Food Irradiation. Proceedings of a Symposium, Karlsruhe, June 6–10, 1966. International Atomic Energy Agency, Vienna. IARC. 1983. Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol. 32: Polynuclear Aromatic Compounds, Part 1: Chemical, Environmental and Experimental Data. International Agency for Research on Cancer, Lyon, France. Irradiation of Food, 1986. Report by a Danish Working Group, Publication No. 120. National Food Agency, Soborg, Denmark. Izaki, Y., Yoshikawa, S., and Uchiyama, M. 1984. Effects of ingestion of thermally oxidized frying oil on peroxidative criteria in rats. Lipids 19:324–331. JECFA. 1991. Benzo(a)pyrene. The 37 Meeting of the Joint FAO/WHO Expert Committee on the Food Additives (JECFA). WHO Food Addit. Series 28:301–363. Jensen, N. J., Willumsen, D., and Knudsen, I. 1983. Mutagenic activity at different stages of an industrial ammonia caramel process detected in Salmonella typhimurium TA100 following preincubation. Food Chem. Toxicol. 21:527– 530. Josephson, E. S. and Peterson, M. S. 1983. Preservation of Food by Ionizing Radiation. Vol. I and II. CRC Press, Boca Raton, FL. Karayiannis, N. I., MacGregor, J. T., and Bjeldanes, L. F. 1979. Lysinoalanine formation in alkali-treated proteins and model peptides. Food Cosmet. Toxicol. 17:585–590. Kimiagar, M. 1979. Long-term feeding effects of Maillard brown products to rats. Diss. Abstr. Int. 40(6):2612B. Kinoshita, N. and Gelboin, H. V. 1978. β-Glucuronidase catalyzed hydrolysis of benzo(a)pyrene glucuronide and binding to DNA. Science 199:307–309. Lamboni, C. and Perkins, E. G. 1996. Effects of dietary heated fats on rat liver enzyme activity. Lipids 31:955–962. Larsen, J. C. and Poulsen, E. 1987. Mutagens and carcinogens in heat-processed food. In Toxicological Aspects of Food, ed. K. Miller, pp. 205–252. Elsevier, London. Lavine, T. F. 1947. The formation, resolution, and optical properties of the diastereoisomeric sulfoxides derived from Lmethionine. J. Biol. Chem. 169:477–491. Layton, D. W., Bogen, K. T., Knize, M. G., Hatch, F. T., Johnson, V. M., and Felton, J. S. 1995. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16:39–52. Levin, W., Wood, A., Chang, R., Ryan, D., Thomas, P., Yagi, H., Thakker, D., Vyas, K., Boyd, C., Chu, S.-Y., Conney, A., and Jerina, A. 1982. Oxidative metabolism of polycyclic

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hydrocarbons to ultimate carcinogens. Drug Metab. Rev. 13:555–580. Lijinski, W. 1987. Structure-activity relations in carcinogenesis by N-nitroso compounds. Cancer Metastasis Rev. 6:301–341. Lijinski, W. and Taylor, H.W. 1977. Feeding tests in rats on mixtures of nitrite with secondary and tertiary amines of environmental importance. Food Cosmet. Toxicol. 15:269–274. Lijinsky, W. and Ross, A.E., 1967. Production of carcinogenic polynuclear hydrocarbons in the cooking of food. Food Cosmet. Toxicol. 5:343–347. Lynch, A.M., Knize, M.G., Boobis, A.R., Gooderham, N.J., Davies, D.S., and Murray, S. 1992. Intra- and interindividual variability in systemic exposure in humans to 1-amino3,8-dimethylimidazo[4,5-f]-quinoxaline and 2-amino-1methyl-6-phenylimidazo[4,5-b] pyridine, carcinogens present in cooked beef. Cancer Res. 52:6216–6223. Magee, P. N. and Barnes, J. M. 1956. The production of malignant primary hepatic tumors in the rat by feeding dimethylnitrosamine. Br. J. Cancer 10:114–122. Magee, P. N., Montesano, R., and Preussmann, R. 1976. N-Nitroso compounds and related carcinogens. In Chemical Carcinogens, ed. C. E. Searle, pp. 491–625. American Chemical Society, Washington, D.C. Maillard, L. C. 1912. General reactions of amino acids with sugar: Its biological consequences. C. R. Soc. Biol. 72:590–601. Marquez-Ruiz, G., Perez-Camino, M. C., Ruiz-Gutierrez, V., and Dobarganes, M. C. 1992. Absorption of thermoxidized fats. II. Influence of dietary alteration and fat level. Grasas Aceites 43:198–203. Marshall, H. F., Chang, K. C., Miller, K. S., and Satterlee, L. D. 1982. Sulfur amino acid stability: Effects of processing on legume proteins. J. Food Sci. 47:1170–1174. Miller, A. J. 1985. Processing-induced mutagens in muscle foods. Food Technol. 39:75–113. Miller, D. S. and Samuel, P. D. 1968. Methionine sparing compounds. Proc. Nutr. Soc. 27:21A–22A. Miller, D. S. and Samuel, P. D. 1970. Effects of the addition of sulfur compounds to the diet on utilization of protein in young growing rats. J. Sci. Food Agric. 21:616–618. Miller, S. A., Tannenbaum, S. R., and Seitz, A. W. 1970. Utilization of L-methionine sulfoxide by the rat. J. Nutr. 100: 909–915. Mirvish, S. S. 1975. Formation of N-nitroso compounds: Chemistry, kinetics and in vivo occurrence. Toxicol. Appl. Pharmacol. 31:325–351. Mirvish, S. S. 1977. N-Nitroso compounds: their chemical and in vivo formation and possible importance as environmental carcinogens. J. Toxicol. Environ. Health 2:1267–1277. Mirvish, S. S. 1983. The etiology of gastric cancer: Intragastric nitrosamide formation and other theories. J. Natl. Cancer Inst. 71:630–655. Mirvish, S. S., Greenblat, M., and Choudari Kommineni, V. R. 1972. Nitrosamide formation in vivo. Induction of lung adenomas in Swiss mice by concurrent feeding of nitrite

and methylurea or ethylurea. J. Natl. Cancer Inst. 48:1311–1315. Montesano, R. M. and Magee, P. N. 1974. Comparative metabolism in vitgro of nitrosamines in various animal species including man. In Chemical Carcinogenesis Essays, eds. R. Montesano, L. Tomatis, and W. Davis, pp. 39–56. Scientific Publications No. 10. International Agency for Research on Cancer, Lyon. Mukai, F. H. and Goldstein, B. D. 1976. Mutagenicity of malonaldehyde, a decomposition product of polyunsaturated fatty acids. Science 191:868–869. Munro, I. C., Kennepohl, E., Erickson, R. E., Portoghese, P. S., Wagner, B. M., Easterday, O. D., and Manley, C. H. 1993. Safety assessment of ingested heterocyclic amines: initial report. Reg. Toxicol. Pharmacol. 17(2):S1–S109. Nagao, M., Honda, M., Seino, Y., Yahagi, T., and Sugimura, T. 1977a. Mutagenicities of smoke condensates and the charred surface of fish and meat. Cancer Lett. 2:221–226. NAS. 1972. Particulate polycyclic organic matter: Biological effects of atmospheric pollutants. National Research Council, National Academy of Sciences, Washington, D.C. Nawar, W. W. 1996. Lipids. In Food Chemistry, 3rd ed., ed. O. R. Fennema, pp. 225–320, Marcel Dekker, New York. Nawrot, P. S., Vavasour, E. J., and Grant, D. L. 1999. Food irradiation, heat treatment, and related processing techniques: safety evaluation. In International Food Safety Handbook, eds. K. van der Heijden, M. Younes, L. Fishbein, and S. Miller, pp. 287–316. Marcel Dekker, New York. Newberne, P. M., Rogers, A. E., and Wogon, G. N. 1968. Hepatorenal lesions in rats fed a low lipotrope diet and exposed to aflatoxin. J. Nutr. 94:331–343. Omura, H., Jahan, N., Shinohara, K., and Mujrakami, H. 1983. Formation of mutagens by the Maillard reaction. In The Maillard Reaction in Foods and Nutrition, eds. G. R. Waller and M. Feather, pp. 537–563. American Chemical Society, Washington, D.C. Overvik, E. and Gustafsson, J. A. 1990. Cooked-food mutagens: current knowledge of formation and biological significance. Mutagenesis 5:437–446. Peters, J. M. and Boyd, E. M. 1969. Toxic effects from a rancid diet containing large amounts of raw egg white powder. Food Cosmet. Toxicol. 7:197–207. Pintauro, S. J., Lee, T.-C., and Chichester, C. O. 1983. Nutritional and toxicological effects of Maillard browned protein ingestion in the rat. In The Maillard Reaction in Foods and Nutrition, eds. G. R. Waller and M. Feather, pp. 467–483. American Chemical Society, Washington, D.C. Powrie, W. D., Wu, C. H., Rosin, M. P., and Stich, H. F. 1981. Clastogenic and mutagenic activities of Maillard reaction model systems. J. Food Sci. 46:1433–1445. Provansal, M.M.P., Cuq, J.-L. A., and Cheftel, J.-C. 1975. Chemical and nutritional modifications of sunflower proteins due to alkaline processing: Formation of amino acid crosslinks and isomerization of lysine residues. J. Agric. Food Chem. 23:938–943.

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Sander, J. and Schweinsberg, F. 1972. Interrelationships between nitrate, nitrite and carcinogenic N-nitroso compounds. Zentralbl. Bakteriol. Hyg. B. 156:299–340. Schlede, E., Kuntzman, R., and Conney, A. H. 1970. Stimulatory effect of benzo(a)pyrene and phenobarbital pretreatment on the biliary excretion of benzo(a)pyrene metabolites in the rat. Cancer Res. 30:2898–2904. Seelkopf, C. and Salfelder, K. 1962. Animal experiments on the question of carcinogenicity and activity of certain epoxides in overheated fats. Z. Krebsforsch. 64:459–464. Selkirk, J. K. 1977. Benzo(a)pyrene carcinogenesis: A biochemical selection mechanism. J. Toxicol. Environ. Health 2:1245–1258. Shamberger, R. J., Andrione, T. L., and Willis, C. E. 1974. Antioxidants and cancer. IV. Malonaldehyde has initiating activity as a carcinogen. J. Natl. Cancer Inst. 53:1771–1773. Shibamoto, T., Nishimura, O., and Mihara, S. 1981. Mutagenicity of products obtained from a maltol-ammonia model system. J. Agric. Food Chem. 29;643–646. Simon, C., Manzke, H., Kay, H., and Mrowetz, G. 1964. The occurrence, pathogenicity and possibility of prophylaxis of methemoglobinemia caused by nitrate. Z. Kinderheilk 91:124–138. Skog, K. 1993. Cooking procedures and food mutagens: A literature review. Food Chem. Toxicol. 31:655–675. Stich, H. F., Rosin, M. P., Wu, C. H., and Powrie, W. D. 1981a. Clastogenicity of furans found in food. Cancer Lett. 13:89–95. Stich, H. F., Stich, W., Rosin, M. P., and Powrie, W. D. 1981b. Clastogenic actgivity of caramel and caramelized sugars. Mutat. Res. 91:129–136. Sugimura, T. 1985. Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process. Mutat. Res. 150:33–41. Sugimura, T., Nagao, M., and Wakabayashi, K. 1982. Mutagenic heterocyclic amines in cooked foods. IARC Sci. Publ. 40:251–267. Sugimura, T., Sato, S., Ohgaki, H., Takayama, S., Nagao, M., and Wakabayashi, K. 1986. Mutagens and carcinogens in cooked foods. Prog. Clin. Biol. Res. 206:85–107. Sugimura, T., Wakabayashi, K., Nagao, M., and Ohgaki, H. 1989. Heterocyclic amines in cooked food. In Food Toxicology: A Perspective on the Relative Risks, eds. S. L. Taylor and R. A. Scanlan, pp. 31–55. Marcel Dekker, New York. Swallow, J. 1991. Wholesomeness and safety of irradiated foods. In Nutritional and Toxicological Consequences of Food Processing, ed. M. Friedman, pp. 11–31. Plenum Press, New York. Tannenbaum, S. R., Sinskey, A. J., Weisman, M., and Bishop, W. 1974. Nitrite in human saliva. Its possible relation to nitrosamine formation. J. Natl. Cancer Inst. 53:79–84. Tannenbaum, S. R., Weisman, M., and Fett, D. 1976. The effect of nitrate intake on nitrite formation in human saliva. Food Cosmet. Toxicol. 14:549–552.

Tesh, J. M., Davidson, E. S., Walker, S., Palmer, A. K., Cozens, D. D., and Richardson, J. C. 1977. Studies in Rats Fed a Diet Incorporating Irradiated Wheat. Technical Report Series IFIP-R45, International Project in the Field of Food Irradiation. Karlsruhe, Germany. Toth, L. and Potthast, K. 1984. Chemical aspects of the smoking of meat and meat products. Adv. Food Res. 29:87–158. Tsuda, M., Nagao, M., Hirayama, T., and Sugimura, T. 1981. Nitrite converts 2-amino-α-carboline, an indirect mutagen, into 2-hydroxy-α-carboline, a non-mutagen and 2-hydroxy-3-nitroso-α-carboline, a direct mutagen. Mutat. Res. 83:61–68. Tsuda, M., Negishi, C., Makino, R., Sato, S., Yamaizumi, Z., Hirayama, T., and Sugimura, T. 1985. Use of nitrite and hypochlorite treatments in determination of the contributions of IQ-type and non-IQ-type heterocyclic amines to the mutagenicities in crude pyrolyzed materials. Mutat. Res. 147:335–343. Tsuda, M., Takahashi, Y., Nagao, M., Hirayama, T., and Sugimura, T. 1980. Inactivation of mutagens from pyrolysates of tryptophan and glutamic acid by nitrite in acidic solution. Mutat. Res. 78:331–339. Tsuda, M., Wakabayashi, K., Hirayama, T., and Sugimura, T. 1983. Inactivation of potent pyrolysate mutagens by chlorinated tap water. Mutat. Res. 119:27–34. USDHHS. 1991. Polycyclic aromatic hydrocarbons. Sixth Annual Report on Carcinogens-Summary. U.S. Dept. Health Human Services, Washington, D.C. USDHHS. 1993. Toxicological Profile for Polycyclic Aromatic Hydrocarbons (PAHs). U.S. Dept. Health Human Services, Washington, D.C. Ushiyama, H., Wakabayashi, K., Hirose, M., Itoh, H., Sugimura, T., and Nagao, M. 1991. Presence of carcinogenic heterocyclic amines in urine of healthy volunteers eating normal

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diet, but not of in patients receiving parenteral alimentation. Carcinogenesis 12:1417–1422. van Kooij, J. G. 1988. International trends in and uses of food irradiation. Food Rev. Int. 2:1 Vijayalaxmi and Sadasivan, G. 1975. Chromosomal aberration in rats fed irradiated wheat. Int. J. Radiat. Biol. 27:135–142. Vijayalaxmi. 1976. Genetic effects of feeding irradiated wheat to mice. Can. J. Genet. Cytol. 18:231–238. Vijayalaxmi. 1978. Cytogenic studies in monkeys fed irradiated wheat. Toxicology 9:181–184. Wakabayashi, K., Nagao, M., Esumi, H., and Sugimura, T. 1992. Food-derived mutagens and carcinogens. Cancer Res. Suppl. 52:2092S–2098S. WHO. 1977. Wholesomeness of Irradiated Food. Technical Report Series 604. World Health Organization, Geneva. WHO. 1981. Wholesomeness of Irradiated Food. Technical Report Series 659. World Health Organization, Geneva. WHO. 1988. Food Irradiation. A Technique for Preserving and Improving the Safety of Food. World Health Organization, Geneva. WHO. 1994. Safety and Nutritional Adequacy of Irradiated Food. World Health Organization, Geneva. Woodard, J. C. and Alvarez, M. R. 1967. Renal lesions in rats fed diets containing alpha protein. Arch. Pathol. 84:153. Woodard, J. C. and Short, D. D. 1973. Toxicity of alkali-treated soy protein in rats. J. Nutr. 103:569–574. Woodard, J. C. and Short, D. D. 1977. Renal toxicity of N6-(DL2-amino-2-carboxyethyl)-6-lysine (lysinoalanine) in rats. Food Cosmet. Toxicol. 15:117–119. Woods, R. J. and Pikaev, A. K. 1994. Applied Radiation Chemistry: Radiation Processing. John Wiley & Sons, New York. Yannai, S. 1980. Toxic factors induced by processing. In Toxic Constituents of Plant Foodstuffs, ed. I. E. Liener, pp. 371–427. Academic Press, New York.

10 Toxicants and Antinutrients in Plant Foods

10.1 INTRODUCTION Animal life on earth is primarily sustained by green plants with photosynthetic capacity to convert carbon dioxide and water into basic macronutrients, i.e., carbohydrates, protein, and fat. In fact, on a global basis over 65% of food protein and over 80% of food energy is supplied by plants, and in terms of gross tonnage, plant products directly contribute about 82% of the total world food harvest (Deshpande, 1992). The photosynthetic process of plants, however, is not confined to the production of basic macronutrients. It also includes the biosynthesis of a variety of organic compounds. Traditionally, the processes generating plant compounds have been categorized as either primary or secondary metabolism. Research in plant physiological characteristics since the 1980s, however, has clearly shown that such a distinction between primary and secondary metabolites is at best arbitrary. The once-popular view of a secondary metabolite as one that does not play an indispensable role in plant life at the cellular level is no longer valid. It is now widely recognized that plants do not haphazardly produce a large number of chemical compounds; rather, each metabolite is biosynthesized for a definite purpose, and all products are interrelated according to a complex process that conserves energy and scarce organic nutrients. Antinutritional or toxic compounds that occur naturally in many plants can be considered secondary metabolites. Most secondary metabolites are now known to be essential to plant life; many of them provide a defense mechanism against bacterial, viral, and fungal attack analogous to the immune system of animals. Many are also produced in large amounts as a direct result of some adverse environmental condition.

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The science of nutrition is not just the science of food and its relation to life and health. We must be concerned not only with what is required in a diet, but also with what is actually consumed. We should be as concerned with the problems caused by an excess of a food component as we are with the problems caused by the deficiency of an essential nutrient. In considering the physiological effects of food components, it should be noted that these effects are always related to the level of their intake. A useful concept is that for every food component, there are three ranges of intake: one associated with physiological inertness, a second with physiological function or benefit, and the third with potential hazard. Although it is arguable at what level a nutrient is physiologically inert, there is no doubt that certain levels of intake are insufficient to maintain normal body functions. The level of nutrient requirement associated with normal health, i.e., with physiological function and benefit, is well understood for most nutrients. We also know with certainty that the concept of “zero risk” cannot be considered valid anymore. One would probably not consider our food sources of energy as ever constituting a potential hazard, but there is a consensus among nutritionists that currently the most important problem of malnutrition in the United States and in many developed Western countries is obesity. This example clearly shows that the margin between the level of caloric intake consistent with normal physiological function and benefit and that creating a potential hazard is narrow. Thus, for every nutrient there is also a level of intake that constitutes a potential hazard. The margin between the level of function and the level of hazard varies considerably with each and must be determined in each case.

That the basic components (i.e., carbohydrates, protein, and fat) of human diet under normal conditions do not exert any adverse effects is taken for granted. Natural foods in everyday diets also contain a great number of other potentially toxic substances. However, this does not necessarily mean that the food is hazardous to human beings. A substance that is considered toxic has a more or less pronounced capacity to induce deleterious effects on the organism when tested by itself in certain doses. However, this capacity is not always realized under usual dietary conditions. Humans consume a multitude of toxic substances in their normal diet every day without showing any signs of intoxification. This is probably because natural toxicants usually exert their effects only when they are consumed under special conditions or when there are other potentiating substances available. In addition, the concentration of toxicants occurring naturally in the food is often so low that the item must be consumed in usually unrealistic amounts every day for an extended period to allow intoxification to occur. Furthermore, it should be noted that humans can handle small amounts of various toxicants. Similarly, most toxic effects of various chemicals that are potentially hazardous do not have an additive effect. There also seem to occur antagonistic reactions that make some ingredients interfere with and reduce the toxic effects of other components. All these facts prompt Liener (1989) to prefer the term antinutritional to toxicants to denote such hazardous food components, since the former is not very restrictive and may be liberally interpreted to mean nothing more nor less than an adverse physiological response produced in humans. Antinutritional and toxic factors that commonly are present in the human food chain can be classified into two broad groups: those that occur naturally (natural or inherent) as a result of intrinsic metabolism of the animal or plant and those that are formed (acquired) as a result of microbial growth, accumulated from the environment, or unintentionally introduced during handling, processing, and storage. In this chapter, only the naturally occurring antinutritional and toxic factors of important plant food sources are discussed. Because of the obvious limitations of space, coupled with the fact that several excellent reviews and books on various aspects of naturally occurring food toxicants are available, no attempt has been made to cover all of the natural toxic substances known to be present in plant materials. Therefore, only certain evolutionary, structural, biochemical, technological, nutritional, and toxicological aspects of most important antinutritional factors that occur in the human food chain are discussed.

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10.2 PROTEINASE (PROTEASE) INHIBITORS Protein inhibitors of proteinases (or protease inhibitors) are ubiquitous. They are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms. Their gross physiological function is the prevention of undesirable proteolysis, but detailed physiological functions have been only rarely elucidated. These inhibitors have attracted the attention of scientists in many disciplines. Nutritionists are concerned with their possible adverse effects on the nutritive value of plant proteins. The inhibitor-enzyme reactions have provided a simple model system for protein scientists to study protein-protein interactions as well as enzyme mechanisms. Because of their unique pharmacological properties, these inhibitors hold considerable promise in clinical applications in the field of medicine. Proteinase inhibitors were initially classified on the basis of protease inhibited, such as trypsin inhibitor. They can also be grouped on the basis of the class of protease inhibitor (Table 10.1). More recently, classification has also been based on similarities in the primary amino acid sequence and/or the disulfide bond location (Whitaker, 1997). Although the inhibition of proteolytic enzymes by extracts from animal tissues was first demonstrated in the 19th century (Fredericq, 1878), it was only in the 1930s that their presence in plant material was recognized. Read and Haas (1938) reported that an aqueous extract of soybean flour inhibited the ability of trypsin to liquefy gelatin. This report was soon followed by the first isolation of a

Table 10.1 Families of Plant Proteinase Inhibitors Serine protease inhibitors (serfins) Bowman-Birk (trypsin/chymotrypsin)a Kunitz (trypsin, others)a Potato I (chymotrypsin, trypsin)a Potato II (trypsin, chymotrypsin)a Cucurbit (trypsin) Cereal superfamily (amylase, trypsin)b Ragi I-2 family (amylase, protease)b Maize 22 kDa/thaumatin/PR (amylase, trypsin)b Cysteine protease inhibitors (cystatins and stefins) Cystatin superfamily Cystatin family Stefin family Fitocystatin family Metalloprotease inhibitors Carboxypeptidase a

Second enzyme listed binds less tightly. Double-headed inhibitor.

b

plant proteinase inhibitor from soybeans by Kunitz (1945, 1946). A year later, the first systematic study of plant proteinase inhibitors was made, by Borchers and Ackerson (1947). Because of the great and increasing importance of grain legumes as food and feed, research during the next period mainly concentrated on seeds from the legume family. A summary of our current knowledge of the distribution of these inhibitors in different food legumes is shown in Table 10.2. The proteinase inhibitors are nonglycosylated, water-soluble (albumin) proteins that account for about 0.2% to 2% of the total soluble protein of the legume seeds (Sgarbieri and Whitaker, 1982; Deshpande and Damodaran, 1990). Most are low-molecular-weight (4000- to 8000-Da) proteins. Two major families of proteinase inhibitors have been described in legumes: the BowmanBirk–type and the Kunitz-type inhibitors. They are distinct families of proteins, as evidenced by their molecular weights, compositions, and amino acid sequences. The im-

Table 10.2 Distribution of Protease Inhibitors in Legumes Species

Protease inhibitorsa

Arachis hypogaea Cajanus cajan Canavalia ensiformis Cicer arietinum Cyamopsis tetragonoloba Dolichos biflorus Dolichos lablab Glycine max Lathyrus odoratus Lathyrus sativus Lens esculenta Lupinus albus Phaseolus aconitifolius Phaseolus angularis Phaseolus aureus Phaseolus coccineus Phaseolus lunatus Phaseolus mungo Phaseolus vulgaris Pisum sativum Psophocarpus tetragonolobus Vicia faba Vigna unguiculata Voandzeia subterranea

T, C, Pl, K T T, C, S T, C T, C, S T T, C, Th T, C T T, C T T T T, C T, endopeptidases T, C T, C T, C, S T, C, E, S T T T, C, Th, Pr, Pa T, C T

a

C, chymotrypsin; E, elastase; K, kallikrein; Pa, papain; Pl, plasmin; Pr, pronase; S, subtilisin; T, trypsin; Th, thrombin. Source: Compiled from Liener and Kakade (1980) and Deshpande and Sathe (1991).

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portant properties of these two inhibitor classes are described in the following discussion. 10.2.1

Bowman-Birk Inhibitors

The Bowman-Birk inhibitor, also referred to as the acetone-insoluble inhibitor, was first recognized by Bowman (1944) and subsequently purified and characterized by Birk and coworkers (Birk, 1961; Birk et al., 1963). It is found in the seeds of all common agriculturally important legume species. These trypsin inhibitors generally contain between 60 and 85 amino acid residues in a single polypeptide chain (Norioka and Ikenaka, 1983), yielding a molecular weight of approximately 8000 Da. However, many members of this family exhibit strong self-association in solution and thus often appear considerably larger (usually dimer or trimer) in the absence of denaturing agents, such as urea (Birk, 1985). The amino acid composition of the Bowman-Birk inhibitors indicates a very high content of Cys (14 residues), all involved in disulfide bonds (Mossor et al., 1984). The inhibitors also have a relatively high content of Asp, Asn, and Ser. Met, Val, Tyr, and Phe are found in small quantities; Trp is generally absent. Because of their low molecular weight and a high content of disulfide bonds, Bowman-Birk inhibitors are generally considered to be heat stable (Mossor et al., 1984). The primary structures of Bowman-Birk trypsin inhibitors from various sources are invariably similar (Wilson, 1981; Norioka and Ikenaka, 1983). They are double-headed, i.e., generally capable of simultaneously and independently inhibiting two molecules of proteases (Laskowski and Kato, 1980). The two enzyme molecules may be the same (e.g., trypsin) or may be different (e.g., one trypsin and one chymotrypsin or elastase). This property is also reflected in their amino acid sequence. A high degree of homology is observed between the first half of the sequence, which contains the reactive site for one proteinase molecule, and the second half of the sequence, which contains the second reactive site. This internal homology suggests the evolution of the present-day doubleheaded inhibitors from an ancestral single-headed inhibitor by a partial gene duplication event (Wilson, 1981). 10.2.2

Kunitz Inhibitors

The Kunitz-type proteinase inhibitors typically contain 170 to 200 amino acid residues, with a molecular weight of about 20,000 Da (Liener, 1983). In soybeans, the inhibitor is a single polypeptide chain. They are single-headed, inhibiting one molecule of the enzyme (generally trypsin

or chymotrypsin) per molecule of the inhibitor. Although the Kunitz-type inhibitors are absent from many agriculturally important members of the legumes, such as Phaseolus, Pisum, and Vigna species, they are found in soybeans and winged beans. Since all naturally occurring trypsin inhibitors have bonds Lys-X or Arg-X in the cavity of their structure as a result of S-S bonds (Laskowski and Kato, 1980), they can also be classified according to the requirements for either Lys or Arg at their reactive sites. Thus, Kunitz inhibitors can be classified as arginine inhibitors, whereas BowmanBirk inhibitors are of the lysine type. The trypsin inhibitors that have Arg at the reactive site lose activity on modification of Arg, but not of Lys. Conversely, Lys-type inhibitors are still active on modification of Arg residues. The compositional and structural aspects of proteinase inhibitors, their physiological significance, as well as the mechanisms of interactions with various proteinases have been discussed in several excellent reviews (Laskowski and Kato, 1980; Liener, 1983; Sgarbieri and Whitaker, 1982; Liener and Kakade, 1980; Weder, 1986). The available literature data on proteinase inhibitors from various legumes are summarized in Table 10.3. Most

inhibitors show inhibiting effects against trypsin and chymotrypsin, but some also inhibit other enzymes, such as elastases, papain, plasmins, and thrombins. Also, in some species, isoinhibitors are frequently found. Examination of the compilation in Table 10.4 reveals that most legume species contain less than 50% of the trypsin inhibitory activity (TIA) of soybeans. Particularly low activities are present in most cultivars of Vicia, Pisum, and Lupinus species and a few cultivars of Phaseolus vulgaris. Legumes with at least 60% to 75% of the TIA in soybeans include Cajanus sp., Phaseolus lunatus, and Cicer sp. (Soni et al., 1978; Rackis et al., 1986). Although trypsin inhibitors from food legumes, particularly soybeans, are studied most widely, they are also found in several other food products, including the staple cereals and various meat products (Table 10.5). However, compared with legumes, these foods have attracted rather limited attention in this respect. The fact that proteinase inhibitors are so widely distributed among those very plants that constitute an important source of dietary protein throughout the world has stimulated a vast amount of research into their possible nutritional significance. Because of the important role of soy-

Table 10.3 Characteristics of Proteinase Inhibitors in Food Legumes MW, daltons

Legume Glycine max (soybean)

Phaseolus lunatus (lima bean) Phaseolus vulgaris (Great Northern) Phaseolus vulgaris (navy bean) Phaseolus vulgaris (pinto bean) Phaseolus aureus (mung bean) Vicia faba (broad bean) Cicer arietinum (chickpea) Vigna unguiculata (cowpeas)

a

Specificitya

21,700

T

8,000

T, C

9,000

T, C

8,086 I, 8,371 II, 8,884 IIIb 23,000 7,900 19,000

T, C

12,000

T, C T, C

11,000

T, endopeptidase of mung bean T, C

10,000

T, C

8,000

T

8,000

T, C

T, trypsin; C, chymotrypsin.

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Comments Has Trp, primary sequence of 181 amino acids is known, single-headed Kunitz type Double-headed Bowman-Birk type, contains seven disulfide bonds Double-headed inhibitor, has at least four to six isoinhibitors Chymotrypsin weakly inhibited by 1 and II, and strongly by IIIb, which has independent sites for the two enzymes Four possible isomers present Two isoinhibitors present, possibly different binding sites for trypsin and chymotrypsin Another inhibitor of MW 2000 also reported Also inhibited thrombin, pronase, and papain (slightly) Independent binding sites for trypsin and chymotrypsin, two to four isoinhibitors present as a result of proteolysis Binds with two moles of trypsin simultaneously Independent binding sites for trypsin and chymotrypsin

Reference Odani and Ikenaka (1972) Odani and Ikenaka (1972) Stevens et al. (1974) Birk (1976)

Whitley and Bowman (1975) Wang (1975) Beumgartner and Chrispeels (1976) Warsy et al. (1974) Belew et al. (1975), Smirnoff et al. (1979) Gennis and Cantor (1976) Gennis and Cantor (1976)

Table 10.4 Trypsin Inhibitory Activity Content of Selected Food Legumes in Relation to Soybeans

Botanical name Cajanus cajan Cicer arietinum Lens esculenta Lupinus spp. Phaseolus aconitifolius Phaseolus aureus Phaseolus lunatus Phaseolus mungo Phaseolus vulgaris Pisum sativum Vicia faba Vigna unguiculata a

Common name

Relative TIA (soy = 100%)a

Reference

Pigeon pea Chickpea Lentil Lupin Moth bean Green gram, mung Lima bean Black gram Dry beans Pea Fava/broad bean Cowpea

60 66 25 0 27 37 77 52 13–44 1.5–13 0.7–36 11.1–28

Soni et al. (1978) Soni et al. (1978) Soni et al. (1978) Valdebouze (1977) Soni et al. (1978) Soni et al. (1978) Hove and King (1979) Soni et al. (1978) Hove and King (1979) Valdebouz (1977) Valdebouz (1977) Valdebouz (1977)

TIA, trypsin inhibitory activity.

beans in animal feeding and their potential contribution to human nutrition, inhibitors from this particular legume have received special attention. In this regard, the effect of raw soybeans on growth has been extensively studied. Earlier reports found that the slight increase in apparent protein digestibility on feeding heated soybean meal to animals was too small to account for the pronounced improvement in their growth rate (Melnick et al., 1946; Mitchell et al., 1945). These studies on protein digestibility, however, were based on measuring fecal nitrogen excretion. Later on, Carroll and associates (1952) studied soy protein digestibility by measuring net protein absorption in

Table 10.5 Nonlegume Foods Containing Proteinase Inhibitor Activity Cereals Rice, wheat, corn, triticale, rye, barley, millet, sorghum, buckwheat, cereal-based products Roots and tubers Potato, sweet potato, yam, taro, cassava Vegetables Cabbage, tomato, lettuce, radish, onion, carrot, sweet corn Fruits and nuts Brazil nut, apple, banana, orange, raisin Animal foods Milk, cheese, egg, beef, lamb, pork, poultry, sweetbread, fish, meat-based products Miscellaneous Table sugar, butter, margarine, tea, coffee, drinking chocolate, milk chocolate Source: Compiled from Doell et al. (1981) and Rackis et al. (1986).

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rats. They observed that animals fed raw soy meal absorbed less than half the nitrogen of those fed heated meal. These studies suggested that feeding raw legumes results in a considerable loss of protein from the site of its absorption in the small intestine. Consequently, although fecal nitrogen is only slightly increased in animals fed raw vs. heated soybeans, less nitrogen is effectively utilized. The discovery of a heat-labile trypsin inhibitor suggested a hypothesis for this growth-depressing effect of soybeans. The inhibitor can interfere with protein digestion by complexing with trypsin and thus preventing the proteolysis of both endogenously secreted and dietary proteins. The fact that raw soybeans or the trypsin inhibitor itself could cause pancreatic hypertrophy and hyperplasia, an effect that is accompanied by an increase in the secretory activity of the pancreas (Liener, 1983), has ultimately led to a better understanding of the mode of action of the soybean trypsin inhibitor. The enzyme-inhibitor complex is neither degraded nor reabsorbed. Since pancreatic proteases are particularly rich in S-amino acids, this loss of essential amino acids from endogenous sources, in addition to the incomplete proteolysis of dietary proteins, contributes to the soy-induced growth depression. This relationship is further supported by the fact that supplementing raw soy meal with S-amino acids improves the growth rate to that of the heated meal (Borchers, 1961). Although part of this improvement undoubtedly results because soy meal itself is limiting in these amino acids, the growth response with their supplementation is much greater in animals fed a raw soy flour diet than the heated flour (Gertler et al., 1967). This finding is consistent with an increased loss of S-rich endogenous proteins due to

trypsin inhibitors. Thus, increasing the dietary supply of amino acids in terms of either protein quality or protein quantity supports better growth by compensating for endogenous losses. The mechanism whereby the trypsin inhibitor causes pancreatic hypertrophy and increases proteolytic enzyme concentrations may be explained in terms of the mechanism of the regulation of pancreatic secretion. Lyman and coworkers have shown that the levels of free intestinal trypsin and chymotrypsin monitor the secretion rate by a negative feedback inhibition involving a hormone, chloecystokinin-pancreozymin (Green and Lyman, 1972; Lyman et al., 1974; Schneeman and Lyman, 1975). This negative feedback mechanism is altered when trypsin is complexed with the inhibitor. This, in turn, releases the hormone, thus increasing the pancreatic juice flow. Although trypsin inhibitors contribute to the growth depression in animals fed raw legumes, other factors may also be involved. Kakade and colleagues (1973) observed that when the inhibitor-free raw soy meal was fed to rats, the degree of growth depression was only marginally less than that of the raw meal containing the inhibitor. This suggested that much, if not most, of the growth depression effect is due to the raw soybean protein itself. Studies in the late 1980s clearly showed that the native storage proteins of various food legumes are resistant to proteolysis (Deshpande and Nielsen, 1987; Nielsen et al., 1988; Deshpande and Damodaran, 1989a, 1989b). The prolonged presence in the intestine of protein of poor digestibility may act by a mechanism similar to that of trypsin inhibitors. It is now well established that most raw or partially cooked legumes are of low nutritional value, an observation that is attributed to the presence of the inhibitors as well as lectins. Fortunately, not only are the inhibitors found in low amounts in most legumes, but most of them are quite heat-labile. Hence, proteinase inhibitors pose few, if any, serious nutritional problems in properly processed legumes. The significance of proteinase inhibitors in human and animal nutrition has been comprehensively reviewed by several authors. The consensus is as follows: (a) Inhibitors of trypsin stimulate the biosynthesis of the enzymes of the pancreas, causing increased requirements of the necessary amino acids in animals. This leads to an increase in the transformation of Met to Cys in the pancreas. The increased requirement of S-amino acids, coupled with their deficiency in legume proteins, cannot be adequately compensated for by the dietary proteins. The enhanced secretion of pancreatic enzymes results in pancreatic hypertrophy in laboratory animals fed legumebased diets. (b) The trypsin inhibitors decrease the proteolysis of dietary proteins by forming trypsin-inhibitor com-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

plexes. These complexes are not broken down even in the presence of adequate amounts of enzymes. This effect further depresses animal growth, since such undigested complexes cannot be assimilated and hence excreted. Since the proteinase inhibitors are rich in Cys, this characteristic further lowers the availability of S-amino acids. Although there is little doubt that the proteinase inhibitors can produce adverse physiological effects in animals, the question naturally arises as to whether they have any physiological significance to humans. Their practical significance with respect to human nutrition is still speculative. TIA is invariably measured in vitro by its ability to inhibit bovine or porcine pancreatic trypsin, which is commercially available. The human trypsin exists in both a cationic form, which is the major component of human pancreatic juice, and an anionic form, which constitutes about 10% to 20% of the total trypsin activity (Weder, 1986). Whereas the less active anionic trypsin is fully inactivated by the soybean trypsin inhibitor, the predominant cationic species is only weakly inhibited (Figarella et al., 1974). That there is a causal relationship between the extent of in vitro trypsin inhibition and pancreatic hypertrophy in vivo has been largely assumed. The practical insignificance of trypsin inhibitors in humans as compared to that in rats was further elaborated by Liener (1977). He discussed the relationship between the size of the pancreas in various animal species and their susceptibility to pancreatic hypertrophy induced by raw soybean meal and trypsin inhibitors. Only animals whose pancreas weighed greater than 0.3% of their total body weight exhibited hypertrophy when fed raw soybeans. In humans, the size of the pancreas is only 0.09% to 0.12% of body weight. If the pancreatic size reflects its true functional activity, as suggested by Goss (1966), then the physiological response to raw soybeans or the inhibitors would likely differ in different animal species. Thus, it seems logical to conclude that despite the considerable body of evidence that implicates the proteinase inhibitors as contributory factors in the poor nutritive value of raw legumes in animals, their relevance to human nutrition remains uncertain. Deshpande (1992) critically reviewed the significance of trypsin inhibitors in human nutrition. Summarizing the various studies conducted during the 1981–1990 period (Table 10.6), he concluded that the inhibitor activity can readily be destroyed by more than 90% if the legumes are processed properly, i.e., by at least 30- to 60minute boiling water treatment or 15- to 20-minute autoclaving at 15 psi. The residual inhibitor activity of less than 10% may be attributable to nonspecific inhibition of trypsin by other dietary components such as phytates, tannins, and crude fiber as well as to our inability by the currently available techniques to detect low levels of inhi-

Table 10.6 Studies Reporting Effects of Processing on Proteinase Inhibitor Activities of Food Legumesa Country of origin

Legume

United States

Vigna unguiculata

India United States

Cicer arietinum, 8 desi and 7 kabuli types Arachis hypogaea

United States

Vigna unguiculata

United States United States

Glycine max Phseolus vulgaris, two cultivars

India

V. aconitifolia

Japan

Psophocarpus tetragonolobus

India United States

Cyamopsis tetragonoloba, four cultivars G. max, two cultivars

Japan

G. max

United States

P. vulgaris

United States/Japan United States

Lupinus angustifolius P. vulgaris, two cultivars

United States

P. tetragonolobus

Guatemala

P. vulgaris, three cultivars

Iraq England

V. faba, C. arietinum, V. unguiculata, V. radiata, and Lens culinaris V. faba, Pisum spp.

England

P. tetragonolobus

Sri Lanka United States

Mucuna utilis G. max

a

Inferenceb Over 92% TIA destroyed within 12 min of conventional heating (boiling in water) and 6 min microwaving Both TIA and CIA destroyed when heated for 60 min (only one time interval used) TIA completely destroyed by heat treatment for 180 min at 100°C or 45 min at 120°C At 100°C, heating cowpea flours of 19.4% and 25.5% initial moisture content, TIA decreased by over 90% within 6 and 1.75 min, respectively Autoclaving at 121°C for 30 min destroyed 98.9% TIA of soymeal Cooking for 35 min or frying at 180°C for 6 min inactivated TIA effectively, only75% CIA destroyed By ordinary cooking, pressure cooking after soaking in plain water or salt solution, and ordinary cooking of sprouts TIA “almost” eliminated from moth beans TIA in dry whole winged bean seeds completely inactivated after microwave heating for 5 min By autoclaving (1 kg/cm2, time not mentioned) of meals over 98% TIA destroyed By steaming or cooking for 20 min “as commonly practiced” TIA completely eliminated in immature seeds, soaking (24 hr) followed by cooking (20 min) essential for mature seeds TIA in soybeans of 24.3% moisture soaked for 1 hr inactivated completely after microwave treatment (2450 MHz) for 4 min, 6 min for unsoaked soybeans In conventionally cooked beans (soaked in water and boiled), loss of 97.2% TIA TIA completely lost on heating at 90°C for 8 min By canning of navy beans by two different methods >95% TIA destroyed: open-kettle cooking at 100°C for 10 min 92% TIA; TIA completely eliminated after 40 min cooking at 100°C Winged bean tuber TIA and CIA easily inactivated by wet heat after 2-min heating in boiling water By cooking of black, white, and red beans antitryptic activity eliminated, residual activity attributed to tannins to a large extent By heating of soaked seeds at 121°C for 30 min completely TIA in all destroyed By autoclaving (170 kPa for 10 min) TIA and CIA in both species completely destroyed; little effect of dry heat By infrared treatment, autoclaving, and boiling water treatments over 95% TIA destroyed; no effect of microwave (15% moisture meal, 10 min) and dry heat (100°C for 60 min) By cooking TIA completely destroyed In autoclaved soy flakes >90% TIA destroyed

Representative studies from the 62 research papers published during the 1981–90 period in the three major food science journals (J Food Sci, J Agric Food Chem, and J Sci Food Agric). These were selected to represent different food legume species and diverse processing conditions b TIA, trypsin inhibitor activity; CIA, chymotrypsin inhibitor activity. Source: Deshpande (1992)

Copyright 2002 by Marcel Dekker. All Rights Reserved.

bition. Generally, depending on the product desired, legumes are often cooked for 1 to 3 hours at atmospheric pressure. These cooking procedures should therefore effectively destroy the proteinase inhibitor activities of various food legumes. Moreover, legumes are often preprocessed by one of several different methods (soaking overnight is not uncommon) that alone may reduce the inhibitor activity to varying degrees. The possibility of residual inhibitor activity that would be nutritionally detrimental in thus-processed legumes therefore appears to be quite small. Unfortunately comprehensive studies concerning the actual dietary intake of trypsin inhibitors from various foods in the daily human diet are lacking. One such rare study estimated that the average British diet provides 330 mg TIA per person per day (Doell et al. 1981). In this study, diets comprising substantial amounts of egg, potato, brown bread, and some vegetables contained more TIA than those based on white bread and rice (polished?). Similarly, of the total of 294.6 mg of actual TIA ingested daily from the British household diet, eggs (93.5 mg), milk and milk products (56.8 mg), and potatoes (42.5 mg) alone contributed over 65% of the daily intake (Table 10.7). In contrast, the Asian soy-based foods included in this particular study contained only a marginal degree of residual TIA. This finding is in striking contrast to the food habits in Third World countries, where the per capita consumption of animal foods such as eggs and milk products is rather limited as a result of either nonavailability or various socioeconomic factors and religious beliefs. To illustrate the point further, let us assume that TIA accounts for 2% of total legume protein (Liener, 1976) and that legumes contain, on an average, 25% protein. If 90%

Table 10.7 Average Daily Protein and Trypsin Inhibitor Intake from the British Household Diet

Food group Milk and milk products Meat Fish Eggs Vegetables Potatoes Other Fruits and nuts Bread and cereals Other foods Total

Protein, g/person/day

Trypsin inhibitor, mg/person/day

17.9 19.5 2.9 3.7

56.8 19.8 1.5 93.6

2.8 3.8 0.7 18.7 1.0 71.3

42.5 37.8 11.8 25.3 5.5 294.6

Source: Doell et al. (1981).

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TIA is destroyed during cooking (the data presented in Table 10.6 certainly indicate so), an average serving of 100 g beans on a dry weight basis contributes 50 mg (100 mg × 0.25 × 0.02 × 0.1) residual TIA to daily dietary intake. To account for the 193-mg TIA intake in the average British household diet contributed by just the three food groups mentioned, one then needs to consume approximately 386 g beans (equivalent to almost 100 g protein per person per day, rather unrealistic statistics given the per capita legume consumption) on a dry weight basis. Because beans contain 65% moisture (as is, based on 1.5-g water uptake per 1 g of beans with optimal cooking [Deshpande and Cheryan, 1986]) on cooking, this amounts to a staggering portion of over 950 g cooked beans per person per day. Even if we follow the rather unrealistic conservative approach that only 50% of the original TIA is destroyed, this still amounts to approximately 77 g beans (or about 200 g cooked beans) per person per day. Globally, only the per capita consumption of legumes of certain population segments in India and a few selected African countries would rival these figures. The survey of Doell and coworkers (1981) raises yet another serious question. Free fatty acids, especially linoleic acid, appeared to inhibit trypsin in their study. Both fat and protein breakdown occur predominantly in the intestinal region. On a diet rich in fat, the likelihood of trypsin inhibition by fatty acids therefore should be of equal importance to that of proteinase inhibitors of legumes. Under such conditions, do the fatty acids stimulate endogenous trypsin secretion, thereby predisposing humans to pancreatic hypertrophy, as is often reported in laboratory animals fed “raw” legume flours (typically soy) or purified trypsin inhibitor from legumes, and is the rationale often speculated to have significance to human nutrition? The negative feedback control mechanism whereby the secretory activity of the pancreas is subject to control by the level of trypsin in the intestinal tract should then also be equally effective for a fat-rich diet. Deshpande (1992) further discussed the normal and worst-case scenarios of dietary intake of TIA from legumes from typical U.S. and Indian diets (Table 10.8). The average dry bean consumption in the United States has remained steady at about 8.5 g per person per day since the 1930s. A typical Indian diet generally contains about 50 g beans per person per day, although the average intake ranges from 35 to 135 g, depending on the population segment (Deshpande and Deshpande, 1991). Even if the per capita consumption of beans were to be doubled in the coming few years (the RDA guidelines do suggest an increased intake of legumes in U.S. diets), the dietary intake of residual TIA from properly processed legume-based foods should not exceed 0.01 g/day and 0.025 g/day in the

Table 10.8 Normal and Worse-Case Scenarios of Contribution of Proteinase Inhibitors from Legumes in the Human Diet Consumption, g/day/person United States Parameter a

Protein intake from legumes , g Contribution of proteinase inhibitors to dietb, g Actual intake if 50% destroyed during processing, g Actual intake if 90% destroyed during processing, g Probable loss of activity during gastric digestion in presence of other dietary constituents Extent of breakdown due to pepsin and other nonspecific proteinases during intestinal digestion Extent of residual inhibitor complexed to other dietary constituents, not digested and therefore excreted Probability of complexation with trypsin and chymotrypsin during intestinal transit time (maximum 2–3 hr, ?) of ingested food Rate and kinetics of complexation with enzymes under less than ideal conditions of other dietary components present at the same time Probability of human adaptation to presence of residual inhibitor in daily diet during minimum 7000-year history of legume domestication Pancreatic hypertrophy in humans Probability of deaths

India

10

20

50

100

150

2.5 0.05 0.025 0.005 ?

5.0 0.10 0.05 0.01 ?

12.5 0.25 0.125 0.025 ?

25 0.50 0.25 0.05 ?

37.5 0.75 0.375 0.075 ?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

Likely to be very high

??

?? ?? ?? Extremely unlikely

??

a

Assuming an average of 25% protein in food legumes. Based on proteinase inhibitors’ constituting, on an average, 2% of the total legume protein. Source: Deshpande (1992). b

United States and India, respectively, under normal circumstances. We then enter a completely gray area of their relevance to human nutrition as several questions remain to be answered (Table 10.8). The presence of other dietary constituents may accelerate gastric breakdown of these inhibitors; complexation with other proteins and minerals may change their potency, or such complexes may even be excreted. The kinetics of inhibitor binding with trypsin are altered in the presence of other dietary constituents compared with those observed under the ideal in vitro conditions in the laboratory. Similarly, legumes are high-fiber foods and thus have shorter residence times in the intestine than predominantly cereal- or meat-based diets (Hellendoorn, 1979). If these factors even marginally influenced the binding of trypsin to the residual inhibitor, the actual residual levels of TIA would be even lower and rather inconsequential. Deshpande (1992) considered yet another scenario in this respect. Soy protein products have long been an ac-

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cepted ingredient in various processed food products in the United States. What would be the dietary TIA intake from such adequately processed soy products? The average daily intake of soy products from various processed foods in Japan is estimated to be 9.5 g per person per day (Evans, 1980; Watanabe et al., 1974). According to Doell and colleagues (1981), exceptionally high intakes of TIA from this source are therefore unlikely in the Western diet. Furthermore, the method (a slight modification of the very widely used procedure of Kakade et al. [1969]) used by these researchers to estimate residual TIA of various foods in British diets also was not without limitations. According to Doell and coworkers (1981), the TIA of raw whole soybean (37.7% protein content) was 22.9 mg/g dry sample, i.e., equivalent to 2.29% on a seed weight basis or about 6.1% of protein content. On the basis of the data provided by these researchers (49.6 mg TIA/g protein), TIA still accounted for almost 5% of total soy protein. In contrast, most studies report that the total TIA in soybean

and other food legumes does not exceed 2% to 2.5% of total legume protein (see Liener, 1976; Sgarbieri and Whitaker, 1982; Rackis et al., 1986; and the references cited therein). The literature survey of Rackis and associates (1986) further suggests that most legume species in fact contain less than 50% of the TIA of soybeans (Table 10.4). Particularly low activities are present in most cultivars of fava bean, pea, and lupin and a few of the dry beans. Those with at least 75% of the TIA in soybeans include cowpea, dolichos bean, pigeon pea, and dry beans. Because most household preparations of other legume species include elaborate processing, unlike soybean, which is used primarily as an oilseed rather than a conventional food legume, the likelihood of their residual TIA contribution to human diet appears practically nonexistent. On the basis of these arguments, undue concern about the physiological significance to humans is rather unjustified, although there is little doubt that the proteinase inhibitors from legumes can produce adverse physiological effects in animals. It should also be noted that almost all animal studies that suggest pancreatic hypertrophy as a typical response to a legume-based diet were carried out with either purified inhibitors or raw, unheated legume flours. Both conditions are inconceivable and extremely unlikely in human nutrition. These arguments and rationale become even more complex because the TIA is measured invariably in vitro by its ability to inhibit bovine or porcine pancreatic trypsin under ideal laboratory conditions. In contrast, as mentioned earlier, the human trypsin exists in both a cationic form, which is the major component of human pancreatic juice, and an anionic form, which makes up about 10% to 20% of the total trypsin activity. Although the less active anionic form is inactivated by the soybean trypsin inhibitor in vitro, the predominant cationic species is only weakly inhibited. On the basis of the arguments presented by Deshpande (1992), the earliest cultivated species of food legumes probably contained much higher levels of antinutrients and Darwin’s proclamation that species continually adapt themselves to the evolving environment as a means for their survival, we could even speculate that the distribution of these two forms of trypsin in Homo sapiens is probably a direct result of their adapting legumes as a part of diet in the early history of our civilization. This argument certainly holds true because before learning and devising simple ways of eliminating antinutritive effects of proteinase inhibitors by such methods as soaking, heat treatment, and fermentation, our ancestors must have been consuming TIA at levels high enough to cause fits in any modern-day food scientist and human nutritionist! The two forms of trypsin in humans must therefore have evolved as a direct adaptive mechanism to such excessive intake of TIA in the nomadic diets.

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Deshpande (1992) considers the whole issue of nutritional concern over proteinase inhibitor intake from food legumes, scientific curiosity apart, as rather unjustified. Epidemiological surveys of dietary patterns from various regions of the globe prove the safety of food legume consumption in human nutrition. Although early studies suggested that feeding raw legumes to animals results in a considerable loss of protein from the site of its absorption in the small intestine, a direct consequence of the presence of trypsin inhibitors in legumes, they also proved that proper heat treatment of legumes largely alleviates these deleterious effects. Similarly, although these inhibitors constitute only 2% to 2.5% of total protein, they account for about 30% to 40% of the total cystine content of legume proteins (Kakade et al., 1969; Liener, 1976; Deshpande and Deshpande, 1991). Because their heat susceptibility under normal processing conditions has been proved beyond reasonable doubt, food scientists and nutritionists are looking at the possibility of breeding for higher levels of trypsin inhibitors as a means of increasing the Samino acid content and therefore improving the nutritional quality of legumes (see Deshpande, 1992, and the references cited therein). Moreover, proteinase inhibitors and other antinutrients in legumes are possibly involved in plant defense mechanisms against pests and diseases, and eliminating them through breeding approaches may create hitherto unknown physiological and agronomical problems in legume cultivation (Deshpande and Sathe, 1991). Proteinase inhibitors, therefore, seem to cause few, if any, deleterious effects in human nutrition.

10.3 AMYLASE INHIBITORS Naturally occurring inhibitors of pancreatic amylase were first discovered in aqueous extracts of wheat, rye, and kidney beans (Kneen and Sandstedt, 1943; Bowman, 1945). Jaffe and associates (1973) reported the presence of αamylase inhibitory activity in 79 of 95 legume cultivars tested; the most activity was found in a kidney bean (Phaseolus vulgaris) cultivar. Deshpande and colleagues (1982) also reported substantial α-amylase inhibitor activity in several cultivars of dry beans. The physiological role of α-amylase inhibitors in plants is not well understood. They are not active against the endogenous α- and β-amylases of legumes or those in malt, barley, or microbial amylases (Jaffe et al., 1973; Powers and Whitaker, 1977a). The bean inhibitors, however, inhibit insect larva α-amylase and, therefore, may have a physiological role in protecting the seeds against insect attact (Sgarbieri and Whitaker, 1982; Deshpande and Sathe, 1991).

Amylase inhibitors have been purified to homogeneity from kidney beans (Marshall and Lauda, 1975; Powers and Whitaker, 1977a). They constitute about 5% to 6% of the total water-soluble protein in kidney beans. Their molecular weights range from 45,000 to 49,000 Da, and all appear to be glycoproteins containing 8% to 10% carbohydrates. On SDS-PAGE, the protein gives four subunits of three size classes of molecular weights 15,000 to 17,000, 12,000 to 15,000, and 11,000 to 12,000 Da. The inhibitors have no Pro and two Cys residues and are relatively rich in Trp, Tyr, Val, and Gly. The kidney bean amylase inhibitors form a 1:1 complex with pancreatic α-amylase (Marshall and Lauda, 1975). However, the specific groups involved in the complex formation and the mechanism of inhibition are not known. The carbohydrate portion of the inhibitor appears to play a crucial role in this regard, since its periodate oxidation results in a complete loss of inhibitor activity (Powers and Whitaker, 1977b). Although the complex has no activity, it can still bind to maltose. This observation led these researchers to speculate that a binding site of α-amylase is still available in the inhibitor-enzyme complex, although by itself it is catalytically inactive. On a nutritional level, amylase inhibitors appear to affect the rate of mammalian starch digestion (Pace et al., 1978). However, this effect has been controversial, since Kneen and Sandstedt (1946) concluded that amylase inhibitors from wheat are inactivated by pepsin and, thus, are not of much nutritional significance. Some evidence has indicated that large amounts of the inhibitor may overcome gastric digestion in laboratory animals and humans (Puls and Keup, 1973). This has not been confirmed in several laboratory animal and most clinical studies (Savaiano et al., 1977; Carlson et al., 1983). Since amylase inhibitors are quite heat-stable, they have been detected in significant amounts in baked wheat flour products and some wheat-based breakfast cereals (Marshall, 1975). However, relatively few studies have been carried out on the physiological effects of these residual inhibitors. Evidence is conflicting as to whether amylase inhibitors reduce growth in animals. In one study in rats, wheat-derived amylase inhibitor reduced growth rate, whereas heat-inactivated inhibitor showed no adverse effect (Saunders, 1975). On the contrary, Savaiano and coworkers (1977) found kidney bean inhibitor to be quite ineffective in this regard. The source of the inhibitor may explain the discrepancy between the findings of these two studies. A decreased rate of starch hydrolysis due to the inhibitors in the small intestine would logically lead to a slower absorption of glucose and possibly cause certain metabolic changes. In rats and dogs, a wheat-derived amy-

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lase inhibitor added to a starch diet attenuated both a rise in blood glucose level and a fall in nonesterified fatty acids level that normally occur in such diets (Puls and Keup, 1973). In humans, a similar effect of the inhibitor on blood glucose levels was observed. Long-term feeding of this type of inhibitor led to a decreased rate of incorporation of 14 C starch into lipids of epididymal adipose tissue and of aortic tissue. Since proteinase inhibitors increase the pancreatic trypsin and chymotrypsin levels, one might expect amylase inhibitors to produce an analogous response. However, regulation of pancreatic amylase levels appears to be functionally different from that of the proteinases. The former is related to the quantity of digestion products absorbed (Lavau et al., 1974). Amylase inhibitors were also critically evaluated as to their effectiveness in the treatment of metabolic diseases, such as diabetes and adiposity (Fukuhara et al., 1982). On the basis of in vitro and early in vivo evidence that ingested kidney bean amylase inhibitors could block the digestion of starch in meals, starch blocker tablets containing these inhibitors were sold for weight control. However, Carlson and associates (1983) found several reasons for the apparent commercial lack of effect of starch blockers. These included the inactivation of the inhibitor by gastric acid and pepsin or by pancreatic proteinases, intraluminal conditions unfavorable to maximal inhibitions, and insufficient preincubation time for the inhibitor. However, the potential of new starch blockers for therapeutic purposes, such as in treating diabetes and obesity, merits long-term research.

10.4 LIPASE INHIBITORS In contrast to the large number of known proteinase and, to some extent, amylase inhibitors of plant origin, virtually no lipase inhibitors have been identified. Both Mori and associates (1973) and Satouchi and colleagues (1974) reported a protein from soybean cotyledons that decreased the lipolytic activity of a pancreatic lipase. Since all lipases primarily act at the oil-water interface of an emulsified oil droplet, the substrate concentration is dependent upon the total interfacial area. Inhibitors of pancreatic lipase may act as true inhibitors by forming the enzyme-inhibitor complex, or they may reduce the enzyme activity by adhering to the oil-water interface, thereby effectively reducing the substrate concentration. Subsequent studies on the soybean cotyledon lipase inhibitor revealed its mode of action to be adherence to the interface (Satouchi and Matsushita, 1976) and not enzyme inhibition in the

classic sense. Whether this protein would have any physiological significance remains to be explored.

10.5 LECTINS (PHYTOHEMAGGLUTININS) Lectins have been identified in many plant families, from slime molds, fungi, and lichens to flowering plants (Jaffe, 1980; Sharon and Lis, 1989; Liener, 1997). They are also found in animals, such as sponges, crustaceans, and mollusks; and in fish blood serum, amphibian eggs, and even mammalian tissue. Among several species of over 140 plant families, Toms and Western (1971) reported 79 hemagglutinin-positive species. According to Liener (1976), in over 800 different plant species in which lectin activity has been detected, more than 600 belong to the Leguminosae family. In many edible legumes, lectins thus are of widespread occurrence, and they constitute between 2% and 10% or more of the total seed protein. The presence of lectin was recognized as early as 1888, when Stillmark observed that the extreme toxicity of the castor bean could be attributed to a protein fraction, which was capable of agglutinating red blood cells (Stillmark, 1888). He coined the name ricin for this substance because it was derived from Ricinus communis. Lectins are a group of natural products that show some apparently unrelated characteristics. Their only common feature is that they are all proteins or glycoproteins. Their specificity and effects are measured in agglutination tests with treated or untreated erythrocytes, from which the term lectin (legere, “to elect, to choose”) is derived (Liener, 1997). In addition to agglutinating red blood cells, lectins exhibit a number of other interesting and unusual

biological and chemical properties, including interaction with specific blood groups, mitogenesis, agglutination of tumor cells, and toxicity to animals (Sharon and Lis, 1989). All of the effects manifest their ability to bind to specific kinds of sugars on the surface of cells. Their reaction mechanism is comparable to that of human antibodies, but lectins are not induced by or the result of an immune response. Several aspects of the chemical and biological properties of various plant lectins have been comprehensively reviewed (Liener, 1976, 1997; Pusztai, 1989; Sharon and Lis, 1989). Examples of the properties of some of these lectins are listed in Table 10.9. On the basis of their sugar-binding specificity, lectins are classified into several groups. Lectins binding D-mannose and D-glucose are found in the seeds of Pisum, Vicia, Lens, and Canavalia spp. and are mitogenic to lymphocyte (Jaffe, 1980). N-acetyl- D -galactosaminebinding lectins isolated from Glycine spp. and Phaseolus lunatus are specific for blood group A, and in the case of lima beans, they are also mitogenic. Arachis lectin is a Dgalactose-binding protein that agglutinates type B erytrocytes. Phaseolus vulgaris produces lectins with complex carbohydrate-binding sites (Pusztai, 1989). Thus, there are obviously no recognizable patterns between the chemical and biological properties of the lectins and their taxonomic distribution in the Leguminosae family. Lectins with the same specificity are therefore found in different genera, and within one genus, both specific and nonspecific lectins are found. There is a great variability in the biological effects of lectins, especially in the genus Phaseolus (Brown et al., 1982a, 1982b; Felsted et al., 1981a, 1981b). These biological effects are associated with different polypeptides. This

Table 10.9 Biochemical Properties of Selected Lectins

Botanical name

Common name

Molecular weight

Number of subunits

Arachis hypogaea Canavalia ensiformis Dolichos biflorus Glycine max Lathyrus odoratus Lathyrus sativus Lens esculenta Phaseolus lunatus Phaseolus vulgaris Pisum sativum Psophocarpus tetragonolobus Vicia faba

Peanut Jack bean Horse gram Soybean Sweet pea Chickling vetch Lentil Lima bean Kidney bean Pea Winged bean Fava bean

110,000 105,000 110,000 120,000 52,000 49,000 46,000 60,000 126,000 49,000 58,000 52,500

4 4 4 4 4 4 4 2 4 4 2 4

a

GalNAc, N-acetylgalactosamine; Man, mannose. Source: Compiled from Deshpande and Deshpande (1991) and Liener (1997).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Sugar specificitya GalNAc Man GalNAc GalNAc Man Man Man GalNAc GalNAc Man GalNAc Man

may explain the observed variability in agglutinating and mitogenic activities, since the polypeptide composition may vary in protein preparations from the same source. The ability to agglutinate erythrocytes necessitates polyvalent binding sites. Most lectins appear to have molecular weights in the range of 100,000 to 150,000 Da and are composed of tetramers. Some, such as lentil and lima bean lectins, appear to be dimers. With few exceptions (e.g., soybeans), each subunit has a sugar-binding site (Liener, 1997). The hybrid tetramer consists of erythrocyte-reactive subunits (E) and lymphocyte-reactive subunits (L) (Felsted et al., 1981b). The five possible isolectins would have the tetrameric structure: L4, L3E1, L2E2, L1E3, and E4, which explain the differences in the observed agglutinating and mitogenic activities of bean lectins. This property is lost if the lectins are dissociated into subunits. Most, if not all, lectins also contain up to 4% to 10% carbohydrates. Data regarding the biological functions of lectins still appear to be contradictory. The ability of lectins to bind to characteristic carbohydrate structures on the cell surface and the observed variability in lectin production in different genotypes of legumes have initiated a number of systematic investigations of the functions of lectins in host plants. Lectins appear to be important determinants of host-range specificity in Rhizobium sp.–legume symbiosis (Sharon and Lis, 1989). Thus, the lectin of Glycine sp. binds only to symbiotic strains of Rhizobium sp., although it is probably not essential for initiating the symbiotic relationship. Another biological function ascribed to the lectins is that of an insecticide. This function is interpreted in terms of an adaptive significance of the lectins in Phaseolus vulgaris for protecting seeds from attack by insect seed predators. One interesting aspect was discussed in connection with the characterization of a lectin isolated from the seeds of Phaseolus aureus, which possesses a strong enzymatic activity (Hankins and Shannon, 1978). By comparing data concerning the synthesis, distribution, and function within plants, it was postulated that legume lectins might, in general, be plant enzymes. However, the lack of detailed information on different aspects of the biological functions of lectins raises more questions that still need to be answered. Lectins were among the first toxic factors implicated in the toxicity of raw legumes to laboratory animals. Earlier studies in this regard were done in the laboratories of Liener. On the basis of observations that the antitryptic factor produced growth depression even when added to a ration containing a protein hydrolysate, Liener and associates (1949) suggested the presence in soybeans of a “substance other than the antitryptic factor which adversely affects growth.” Further investigations resulted in the isolation from raw soybean meal of a very potent hemag-

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glutinating protein fraction (Liener, 1951; Liener and Pallansch, 1952). When this fraction was added to a ration containing autoclaved soybeans, the rats gained weight at a rate equal to 75% that of the rats fed the basal ration (Wada et al., 1958). The primary effect of the hemagglutinin, as far as growth was concerned, appeared to be a depression of appetite. Liener (1958) also reported that the hemagglutinating activity was readily inactivated by pepsin. Since these earlier studies, several researchers have noted a marked reduction in protein digestibility and a growth inhibitory effect derived by depressing the appetite when purified lectins are added to the diet. As far as the mode of action is concerned, Jaffe (1980) postulated that the toxic effects of lectins when ingested orally may be due to their ability to bind to specific receptor sites on the surface of intestinal epithelial cells, thus causing a nonspecific interference with the absorption of nutrients. The fact that lectins are so widely distributed in food items commonly consumed by humans raises the important question as to whether they pose any significant risk to human health. Fortunately, most lectins are easily destroyed by the traditional methods of household cooking. As an example, the effectiveness of processing on the lectin content of food items containing soybeans as an ingredient is shown in Table 10.10. It is doubtful whether such low levels of lectin activity pose a risk to human health. Thus, there would be little cause for concern in human nutrition. Nevertheless, under special conditions, complete detoxification may not always be achieved, especially if ground seeds are used or industrial processes for quickcooking products are applied. Since lectins are resistant to inactivation by dry heat (Jaffe, 1980), practices such as the use of raw legume flours in baked goods should be viewed with caution. Of greater concern are the toxic effects that are associated with the inadvertent consumption of beans and

Table 10.10 Lectin Content of Processed Foods Containing Soybeans as an Ingredient Product Raw soybeans Unprocessed soy flour Defatted soy flour Textured meat analog Breakfast cereal Soy milk Cookie

Raw soy, % 100.0 47.0 4.3 0.4 5.2 0.5 0.7

Source: Compiled from Liener (1997).

dishes derived from them that have been improperly cooked or processed. Lectins appear to have been the causative agent in several cases of human intoxification. These symptoms are primarily manifested as gastrointestinal discomforts. An outbreak of what appeared to be food poisoning occurred in England in 1976. A party of schoolboys on holiday ate kidney beans that had been soaked in water but had not been cooked. All nine of the boys who ate the beans became acutely nauseated within 60–90 minutes and began to vomit, then to have diarrhea (Liener, 1977). Eating as few as four to five beans was sufficient to produce these reactions. Two of the boys were admitted to the hospital and needed intravenous infusion. However, recovery was rapid in all cases. Illness after the consumption of raw beans as part of a salad or in dishes such as a stew, casserole, or chili con carne cooked in a slow cooker is not uncommon. The times and temperatures involved in the slow cooking of kidney beans under household conditions may not be sufficient to destroy all of the lectin activity. Warning labels are often found on labels of dried kidney beans sold in the retail food markets in England and several other European countries, recommending that the beans be boiled for at least 10 minutes before consumption. Because of their unique capacity to bind in a specific fashion to sugars and other glycoconjugates, the lectins have broad applications in research and biomedical laboratories (Liener, 1997). Either in solution or in an immobilized form, lectins have proved extremely useful for the detection and identification of many diverse glycoconjugates. The identification of blood group substances, and membrane receptors and the detection of malignant cells are examples of clinical applications of lectins. Lectins could also be used to prevent graft rejection in bone marrow transplantation (Sharon and Lis, 1989). This potential derives from the property that the soybean lectin can be used to remove the mature T cells responsible for graft rejection.

10.6 PHYTATE Cereals and legumes contain significant amounts of phosphorus in the form of phytic acid (myo-inositol hexaphosphate). The terms phytic acid, phytate, and phytin refer to free acid, salt, and Ca2+/Mg2+ salt, respectively; however, these terms are often used interchangeably. The revised nomenclature for phytic acid in plant seeds is myo-inositol-1,2,3,5/4,6-hexakis (dihydrogen phosphate) (IUPACIUB, 1968). Phytic acid occurs primarily as a salt of mono- and divalent cations in discrete regions of cereal grains and legumes. It rapidly accumulates in seeds during the ripening

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period in the electron-dense aleurone particles or globoids (Reddy et al., 1982). Phytate accounts for up to 85% of the total phosphorus in many cereals and legumes. The phytate phosphorus and phytate contents of selected foodstuffs are summarized in Table 10.11.

Table 10.11

Food

Phytate Content of Selected Foodstuffs

mg %

Cereals Wheat 170–280 Rye 247 Maize 146–353 Rice 157–240 Barley 70–300 Oats 208–355 Sorghum 206–280 Buckwheat 322 Millet 83 Wheat bran 1170–1439 Legumes and vegetables Dry beans 269 Lima beans 152 Soybean 402 Lentil 295 Pea 117 Vetch 500 Chickpea 140–354 Pea 82 Potato 14 Green bean 52 Green pea 12 Carrot 0–4 Nuts and seeds Walnut 120 Hazelnut 104 Almond 189 Peanut 205 Cocoa bean 169 Pistachio 176 Rapeseed 795 Cottonseed 366 Spices and flavoring agents Caraway 297 Coriander 320 Cumin 153 Mustard 392 Nutmeg 162 Black pepper 115 Pepper 56 Paprika 71

Phytate phosphorus, % of total phosphorus 47–86 73 52–97 68 32–80 50–88 77–88 70 57 89–97 62 77 65 90 37 95 49–95 31 35 43 12 0–16 24 45 43 57 25 75 89 41 96 77 33 86 61 58 15 15

Source: Compiled from Concon (1988) and Deshpande and Sathe (1991).

The presence of phytate in processed foods has received considerable attention in recent years. Phytate is generally considered to be fairly heat-stable. Among the processing methods, germination and fermentation appear to be quite effective in decreasing the phytate concentrations, whereas soaking and cooking can remove more than 50% to 80% of the endogenous phytate in legumes and cereals (Deshpande and Damodaran, 1990; Deshpande et al., 1984a). The concern about phytate-mineral interactions is caused by the ability of phytate to form insoluble complexes with minerals at physiological pH values. Phytic acid has six reactive phosphates and meets the criterion of a chelating agent. In fact, a cation can complex not only within one phosphate or between two phosphate groups of the phytic acid, but also between two phytic acid molecules (Erdman, 1979). Thus, phytate is largely blamed for complexing dietary essential minerals in legumes and cereals and rendering them poorly available to monogastric animals. Zinc and copper appear to bind phytate in the physiological pH range more tightly than other minerals (Maddaiah et al., 1964). The possible interactions of phytate with minerals and with protein and starch are shown in Figure 10.1. The mechanism by which phytate affects mineral bioavailability is not clearly understood. Several studies, however, suggest the formation of insoluble phytate-mineral complexes in the intestinal tract, which prevent mineral absorption (Graf and Eaton, 1984; Wise, 1983;

HO O P

Ca2+

O

O- -O

O

Starch

O

-

-O

-O

O-

C

CH2-Protein

O

CH2 NH3

O

+Ca+

O P OH O

Protein

+

P O

O P OH O P O O

O-O

P O

HO CH2OH O O

O

n

Starch

Figure 10.1 Possible interactions of phytic acid with minerals, proteins, and starch.

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Erdman, 1979). The formation of these complexes is pHdependent. However, in most cases, the bioavailability of minerals in laboratory animals has been studied by using free ionic salts. The results obtained under such conditions may not represent the true bioavailability of minerals from similar sources to humans. The reduced bioavailability of minerals from phytate-rich sources depends on several factors, which include the nutritional status of animals/humans, the concentration of minerals and phytate in foodstuffs, the ability of endogenous carriers in the intestinal mucosa to absorb essential minerals bound to phytate and other dietary substance, the digestion or hydrolysis of phytate by phytase and/or phosphatase enzymes, the processing of products or methods of processing that include unit food processing operations (pH adjustment, level of refinement, and addition or removal of inhibitors/enhancers), and the digestibility of the foodstuffs (Reddy et al., 1982; Deshpande et al., 1984a; Deshpande and Deshpande, 1991). Phytate is not the only dietary component that influences mineral bioavailability. Other food components, such as dietary fiber, polysaccharides, oxalates, and polyphenols, are also known to influence the bioavailability of minerals adversely. In addition to complexing metal ions, phytate interacts nonspecifically with various proteins, enzymes, and carbohydrates (Figure 10.1). Phytate inhibits several important enzymes, such as trypsin, pepsin, α-amylase, and β-glucosidase. Sharma and colleagues (1978) studied the phytate inhibition of α-amylase derived from several plant sources. The inhibition of wheat α-amylase was found to be noncompetitive, with an apparent Ki of 1 mM. Later, Deshpande and Cheryan (1984) observed a 16% to 95% decrease in activity of a porcine pancreatic α-amylase at 0.5- to 6-mM phytate concentration. Conflicting mechanisms have been proposed for phytate inhibition of amylases. Cawley and Mitchell (1968) suggested phytate chelates Ca2+ ions, which are activators of amylases in sprouted wheat meal. On the contrary, Sharma and coworkers (1978) found that the addition of Ca2+ did not reverse the inhibition and that the inhibition was related to the direct interactions of phytate with the enzyme allosteric site. Subsequently, Deshpande and Cheryan (1984) suggested that the general complex-forming ability of phytate with enzyme proteins was the major reason for the inhibition of amylases. That phytate should inhibit proteolytic enzymes such as pepsin (Knuckles et al., 1985) is not surprising, since the acidic pH at which it is active can promote strong electrostatic linkages between phytate and the positively charged groups of protein. Indeed, the same can be said of enzymes whose pH optima are on the acid side of the

scale. In contrast, the more negative charges on proteins at neutral to alkaline pH may prevent formation of phytateprotein binary complexes unless an external povalent cation (such as Ca2+ or Mg2+) is present. Thus, phytate inhibition of proteolytic enzymes, such as trypsin and chymotrypsin, is insignificant compared to that of pepsin. In a study on the inhibition of trypsin activity in vitro by phytate, Singh and Krikorian (1982) reported 20% enzyme inhibition at 90 mM phytate in the assay system. On the basis of the data provided by these authors, this turns out to be an almost 3000:1 phytate-enzyme ratio. Deshpande and Damodaran (1989c) have argued that not only are such ratios of free phytate difficult to encounter under in vivo conditions, but also because of their high ionic strength, they may effectively salt out the enzyme from the assay system. Deshpande and Damodaran (1989c), using phytate-enzyme ratios that are more likely to be encountered under normal in vivo conditions, in fact observed a 5% to 7% increase in the activity of both trypsin and chymotrypsin. Only one other report in the literature suggests a positive influence of phytate on enzyme activity; Altschuler and Schwartz (1984) reported an enhancement of both mutant and wild-type enzyme activity of alcohol dehydrogenase from corn in the presence of phytate. The interaction of phytate with enzymes may be important in human nutrition, since it may result in decreased protein and starch digestibility of foods rich in phytate. Thompson and coworkers (Yoon et al., 1983; Thompson and Yoon, 1984) studied the effects of phytate on the in vitro starch digestibility by human saliva enzymes, as well as the blood glucose response (glycemic index) in healthy volunteers. A significant negative correlation between phytic acid (both concentration and intake) and the glycemic index was observed. Foods that were rich in phytate were digested at a slower rate and produced lower blood glucose responses than foods devoid of phytate. Button and associates (1985) found that the removal of phytate produces an increase in the rate of starch digestion both in vitro and in vivo, whereas the readdition of phytate yields an opposite effect. Thompson (1988) even suggested that there may be a therapeutic role for phytate in the management of diabetes and obesity and that complete elimination of phytate during processing may actually be undesirable. However, the levels at which phytate combines maximal health benefits with negligible adverse effects have not yet been determined. Although this seems to be a research area of considerable potential, the strategy of not eliminating phytate appears to be questionable, particularly in the developing countries, where the consumption of phytaterich legumes is high and at the same time malnutrition is prevalent.

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As compared to carbohydrate metabolism, phytate does not seem to influence dietary protein utilization to any significant extent. Several studies have clearly shown that although the digestibility of protein and the subsequent rate of release and absorption of amino acids are only marginally higher in low-phytate diets, from phytate complexed proteins they are not significantly different (Thompson and Serraino, 1986; Thompson, 1988; Reddy et al., 1988). Then there is also the question of how much “free” phytate would be available to inhibit the digestive enzymes. In addition, most in vitro enzyme inhibition studies use sodium phytate, whose solubility behavior is completely different from that observed for phytate from plant sources. Phytic acid is a highly unstable molecule and in nature is always present as a salt of Ca2+, Mg2+, or K+. When one considers its strong affinity to various cations and the type of interactions involved in its association with dietary protein as well as factors such as food processing, thermal degradation of inositol esters, and pH, little free phytate would be expected to be available to interact with enzymes in the digestive system and have any significant influence. In addition, it has been clearly shown that in the presence of minerals such as Ca2+ or Mg2+, the actual in vitro inhibition of enzymes would be much lower (Deshpande and Cheryan, 1984). Similarly, hosts of different enzymes are involved in the digestion process. Proteins in which some lysyl or arginyl side chains are complexed with phytate may not be effectively hydrolyzed by trypsin; under these conditions, enzymes such as chymotrypsin would prove more effective, since the latter shows specificity for large hydrophobic side chains. Until several questions regarding phytate-protein interactions under a wide variety of conditions and their influence on protein digestibility under simulated in vivo conditions are addressed and answers that are more definitive found, the adverse effects of phytate in human nutrition, given its nature and chemical properties, should be strictly addressed from the mineral bioavailability point of view. Rickard and Thompson (1997a) have suggested a protective role for phytic acid in the prevention of cancer of the colon, mammary gland, and other organs or tissues at various stages of the carcinogenic process. The ability of phytic acid to bind starch, proteins, and minerals has been implicated as part of the mechanism whereby phytic acid exerts its anticancer effects, although there is evidence that the hydrolysis products of phytic acid also play an important role. One of the breakdown products of phytic acid is inositol triphosphate (InsP3), which acts in cellular signal transduction and in the enhancement of natural killer (NK) cell activity. Phytic acid does not appear to act through a cytotoxic mechanism but through promotion

of the differentiation of the malignant cells to a more normal phenotype. In light of its healthful properties, Rickard and Thompson (1997a) suggested that the term antinutrient is an outdated label for food constituents like phytic acid.

10.7 TANNINS (POLYPHENOLS) Phenolic compounds (i.e., phenolic acids and their derivatives) are widely distributed in the plant kingdom. Although the pharmacological and therapeutic properties of many bioflavonoids (a class of phenolic compounds) are well documented in the literature, there is no evidence that they have a nutritional role in our diet. Plants provide nearly all the phenols to higher animals, since the latter cannot synthesize compounds with benzenoid rings from their aliphatic precursors. Phenolics are thus strictly exogenous food components of exclusively vegetable origin. The common dietary low-molecular-weight phenolics, owing to the evolution of efficient detoxification, are not considered toxicants under normal amounts and conditions. The condensed tannins of flavonoid origin (also referred to as polyphenols) are one possible exception. They are widespread in fruits and vegetables and in certain grains. The pigmented varieties of certain cereals and legumes contain 2% to 4% condensed tannins, although amounts as high as 7% to 8% have been reported for red high-tannin sorghum varieties (Mabbayad and Tipton, 1975). Humans also consume a number of other foods containing considerable amounts of condensed tannins, especially in beverages, such as cider, tea, cocoa, and red wine. Strong versions of these beverages may have as much as 1 g tannin per liter. The tannin content of several cereals and legumes is summarized in Table 10.12. The intake of dimeric flavans may be up to 400 mg/day in human diets (Kuhnau, 1976). The total tannin intake would be somewhat higher. The per capita consumption of red wines in some countries would guarantee an average intake of nearly that amount from that source alone (Singleton, 1981). Rao and Prabhavati (1982) reported a range of 1.5 to 2.5 g for the daily intake of dietary tannins in different regions of India. Tannins comprise a heterogeneous group of plant polyphenols all of which are able to combine with skin proteins in such a way as to render them resistant to putrefaction, or, in other words, to “tan” them into leather. More specifically, they are high-molecular-weight compounds (500 to 5000 Da) containing sufficient phenolic hydroxyl groups to permit the formation of stable cross-links with proteins. Although the presence of ortho dihydroxy phenols seems to be essential (Hathaway and Seakins, 1958;

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Table 10.12 Tannins of Cereals, Millets, and Legumes Source Cereals and millets Sorghums Wheat Corn Finger millet Legumes Pigeonpeas Chickpeas Soybeans Green gram Lima beans Black gram Dry beans Peas Winged beans Fava beans Cowpeas Adzuki beans

Tannins, % 0.04–7.87 0.12–0.41 0.13–0.14 0.36–1.17 0.03–1.00 0.03–0.22 0.045 0.03–0.47 0.77 0.31–0.86 0.00–2.00 0.43–0.47 0.403 0.75–1.92 0.19–2.12 0.29–0.37

Source: Compiled from Deshpande et al. (1986) and Deshpande and Damodaran (1990).

Bauer-Staob and Niebes, 1976) presumably to form hydrogen bonds with groups on the proteins, hydrophobic binding may be an important contribution to the stability of the complex. Tannins usually give rise to a dry, puckery, astringent sensation in the mouth. Because of their proteinbinding properties, tannins are of considerable importance in food processing, fruit ripening, and manufacturing of tea, cocoa, and wine. On the basis of structural types, tannins have been classified by Freudenberg (1920) into two groups: the hydrolyzable tannins and the condensed tannins. The condensed tannins are more widely distributed in higher plants. Most hydrolyzable tannins contain a central core of glucose or other polyhydric alcohol esterified with gallic acid (gallotannins) or hexahydroxydiphenic acid (ellagitannins). The latter is isolated as its stable dilactone, ellagic acid. These types of tannins are readily hydrolyzed by acids, bases, or certain enzymes. The condensed tannins, also referred to as procyanidins and, formerly, leucoanthocyanidins because many form cyanidin on acid hydrolysis, are mostly flavolans or polymers of flavan-3-ols (catechins) and/or flavan-3,4-diols (leucoanthocyanidins). Both are readily converted by dehydrogenating enzymes or even by very dilute mineral acids into flavonoid tannins at room temperature (Weinges, 1968). The condensed tannins are not readily degraded by

acid treatment but polymerize to form amorphous phlobaphenes or “tannin-reds.” Most physiological investigations of dietary tannins have been made with nonruminants. When fed at levels that commonly occur in cereals and legumes (up to 1% to 2%), tannins have depressed the growth rate, resulted in a poor feed efficiency ratio, and increased the amount of food required per unit weight gain (Price and Butler, 1980; Deshpande et al., 1984b). Other deleterious effects of tannins include damage to the mucosal lining of the gastrointestinal (GI) tract, alteration in the excretion of certain cations, and increased excretion of proteins and essential amino acids. High dietary levels (about 5%) can cause death (Singleton and Kratzer, 1969). The deleterious effects of tannins in the diet seem to be related to their interactions with dietary proteins. Tannin-protein complexes are believed to be responsible for growth depression, low protein digestibility, and increased fecal nitrogen level. Casein, bovine serum albumin, G1 protein from beans, and carob pod proteins resist proteolytic digestion when complexed with tannins (Feeney, 1969; Deshpande, 1985; Tamir and Alumot, 1969). Such complexes may not be dissociated at physiological pH and may pass out in the feces. The nitrogen content of the feces generally rises in proportion to the amount of tannin fed. Tannins also inhibit digestive enzymes, such as trypsin and amylases (Davis and Harbers, 1974; Deshpande and Salunkhe, 1982; Tamir and Alumot, 1969). Tannins have been implicated in carcinogenesis. Epidemiological studies conducted on a global basis suggest a possible correlation between unusual consumption of plant materials rich in condensed tannins (particularly high-tannin sorghums and the dark beers prepared from them, tea, red wines, and areca nuts in certain parts of the world) and the unusual frequency of cancer of the esophagus and mouth (Morton, 1970, 1972). Tannins and tannic acid have also been listed as tentative carcinogens of category I under the general carcinogenic policy of OSHA (OSHA, 1978). Several methods to overcome the antinutritional effects of tannins in the diet have been attempted. The basic principles involved include the physical removal of tannins by extraction or milling, addition of agents that complex with dietary tannins, use of agents that aid in the metabolic detoxification of tannins, and plant breeding approaches. Since tannins are primarily located in the seed coats, dehulling appears to be the simplest and least expensive approach to removing dietary tannins from food grains; however, the beneficial effects may be partially offset by some nutrient losses. Most food processing methods, such as soaking, cooking, and germination, result in an apparent loss of measurable tannins. A long-term approach, and the

Copyright 2002 by Marcel Dekker. All Rights Reserved.

most satisfactory one, would be to breed for varieties with fewer or no tannins. In recent years, several media reports comparing the dietary habits of French and U.S. people have ascribed certain beneficial effects such as lowering of the frequency of blood-related disorders to regular consumption of red wines in the French diet. Although moderate alcohol intake seems to have beneficial effects in human nutrition, the consumption of red wines is also accompanied by increased intakes of phenolics that should not be ignored in the French diet. The anticarcinogenic activities of polyphenols in foods and herbal medicines were reviewed by Miyamoto and colleagues (1997). Among the major tannin activities found are antioxidant and radical scavenging activities. These are the basic activities underlying the effects of tannin-rich medicinal plants that are effective in preventing and treating many diseases such as arteriosclerosis, heart dysfunction, and liver injury, as well as inhibiting lipid peroxidation. The inhibition of hepatotoxins and mutagens and the antitumor-promoter action of polyhphenols are also correlated with their antioxidant activity. In this regard, oligomeric ellagitannins, which consist of monomer units such as potentillin, tellimagrandin I and II, and related structures, seem to have potent anticancer activities.

10.8 CYANOGENIC GLYCOSIDES Cyanogenic glycosides are important natural toxicants in both animal and human nutrition and are widespread in the plant kingdom. Chronic toxicological effects occur in humans who consume cassava in tropical countries; cyanide production potential is of concern in several other food crops. Livestock poisonings are also associated with the consumption of forage sorghums. Cyanogenic glycosides have been identified in over 2000 species of higher plants belonging to 110 different plant families, including ferns, gymnosperms, and angiosperms (Poulton, 1983; Deshpande and Sathe, 1991). Among these families, the most notable for their cyanogenic ability are Rosaceae (150 species), Leguminosae (125), Graminae (100), Araceae (50), Compositae (50), Euphorbiaceae (50), and Passifloraceae (30). The distribution of some well-characterized cyanogens is summarized in Table 10.13. Cyanogenic glycosides are compounds that on treatment with acid or appropriate hydrolytic enzymes produce hydrocyanic acid (HCN). These compounds in higher plants are of two types: Cyanogenic glycosides usually contain glucose as their sugar component, although other mono- and disaccharides may be found. Cyanogenic lipids are another group of cyanide precursors, which contain,

Table 10.13 Glycoside Amygdalin Dhurrin Linamarin Lotaustralin Prunasin Cicianin

Distribution and Hydrolytic Products of Some Cyanogenic Glycosides in Food Plants Plants

Hydrolytic products

Almond, cherry, peach, plums, apples Sorghums Linseed, clovers, cassava, lima beans Linseed, cloves, cassava, lima beans Cherry, almond Vetches

HCN, gentobiose, and benzaldehyde HCN, glucose, and hydroxybenzaldehyde HCN, glucose, and acetone HCN, glucose, and 2-butanone HCN, glucose, and benzaldehyde HCN, vicianose, and benzaldehyde

Source: Compiled from Tewe and Iyayi (1989) and Poulton (1983).

instead of sugar, long-chain fatty acid moieties. Both are derivatives of α-hydroxynitriles (cyanohydrins), and both liberate a carbonyl compound and HCN when, respectively, the sugar or the fatty acid moieties are removed. The chemical structures of some well-characterized cyanogenic glycosides are shown in Figure 10.2. The cyanogenic glycosides are relatively stable chemical compounds at neutral pH. They can be hydrolyzed to their component parts (an aldehyde or ketone, sugar, and HCN) by acid at elevated temperatures. The production of HCN from cyanogenic glycosides is an enzymatic process, commonly known as cyanogenesis. One of the best-studied examples is that of linamarin, which is found in lima beans and cassava. In these plants, the presence of an endogenous enzyme linamarinase or linase causes its hydrolysis, with the production of HCN. The reaction proceeds via two steps (Figure 10.3). In the first, the linamarin is hydrolyzed by linamarinase to produce β-Dglucopyranose and 2-hydroxyisobutyronitrile or acetone cyanohydrin. The latter dissociates, catalyzed by a hydroxynitrile lyase, to produce acetone and HCN. Another well-studied example of cyanogenesis is the hydrolysis of amygdalin and prunasin (Figure 10.4). The process involves the stepwise removal of glucose by the action of two separate specific β-glucosidases and the subsequent action of a hydroxynitrile lyase, which catalyzes the dissociation of the α-hydroxynitrile. Since the cyanogenic glycosides accumulate in significant quantities in plant tissues, which also contain these enzymes, it is quite likely that the substrates are sequestered separately from their catabolic enzymes in one part of the plant cell or even in separate tissues (Conn, 1979). This indeed is true for sorghum leaves, where dhurrin is located exclusively in the vacuoles of epidermal cells and the degradative enzymes are found only in mesophyll tissue (Kojima et al., 1979). Cyanogenesis most frequently occurs when a cyanogenic plant tissue is crushed or otherwise disrupted (Conn, 1979). This may occur during the processing (grinding, drying, pounding) of the plant tissue during food prepara-

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tion and obviously takes place when the plant tissue is ingested directly and chewed by an animal. To the extent that the catabolic enzymes could be removed or inhibited in their action, or to the degree that the substrates could be removed or destroyed without the release of HCN, the cyanogenic phenomenon could be reduced in intensity or even prevented. The lethality of cyanide is due to its ability to inhibit respiration. Cyanide is a potent inhibitor of the cytochrome oxidase of the respiratory chain. Cyanide also inhibits several other metalloenzymes, including nitrate reductase, nitrogenase, and xanthin oxidase (all molybdoenzymes); alkaline phosphatase and carbonic anhydrase (zinc enzymes); plastocyanin and ascrobate oxidase (copper enzymes); and some selenoenzymes. Enzymes without metals, such as glutamate decarboxylase and α-amino butyric acid transaminase, can also be inhibited by cyanide (Deshpande and Sathe, 1991). The ability of cyanogenic glycosides to release cyanide implicates them in a number of diseases encountered in populations who depend primarily on food sources rich in these compounds. The consumption of cassava has been reported to cause neurological and endocrinological diseases in many tropical countries (Tewe and Iyayi, 1989). Ingested cyanide is rapidly absorbed from the upper GI tract. It also passes readily through the skin, and HCN gas is rapidly absorbed from the lungs. The amount of HCN produced by different plant species varies considerably (Table 10.14). Proper processing of foods, which usually involves soaking, cooking, and fermentation, is known to reduce the incidence of cyanide poisoning. The minimal lethal dose of HCN for humans has been estimated at between 0.5 and 3.5 mg/kg body weight. This is equivalent to 30 to 210 mg for a 60-kg adult. HCN is readily absorbed from the gastrointestinal tract and produces recognizable symptoms at both lethal and sublethal levels of ingestion. Sublethal doses of HCN may also be converted to thiocyanate, a well-known goitrogen. Although many food legumes contain only low levels of goitrogens, the conversion of cyanogens into goitrogens

(R)-Amygdalin CH2OH O OH

Linamarin H

C

H3C

N

CH2

O

O

CH2OH O OH

O

OH

HO OH

HO

CH3 C C

O

HO OH

OH

(R)-Lotaustralin

(R)-Prunasin H CH2OH O OH

C

H3C

N CH2OH O OH

O

C2H5 C

O

HO

HO

OH

OH

N

C

H

CH2OH CH2OH

O

OH HO

C

O OH HO

HO

O

O

H

C

C

N

OH

OH HO

(S)-Dhurrin

(R)-Taxiphyllin

(S)-Proteacin

C

H

N

C CH2OH O CH2OH O OH

O

HO OH

Figure 10.2 Chemical structures of some well-known cyanogenic glycosides.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

N

O OH HO OH

C

N

C

C C

C

H2 C H2 C

C

C

C

CC

C

C

C

CH3

C Enzyme

CH3

Acid

C C

H2C

C

C

C

Hydroxynitrile lyase

CH3

CH

C

CH3

C C

C

β-D-Glucose

Linamarin

CC

C

H2 C

C

CC

C

C

CH3

CH3

Acetone cyanohydrin

Acetone

Figure 10.3 Enzymatic hydrolysis of linamarin, the cyanogenic glycoside of lima beans.

Amygdalin CH2OH O OH

H O

N

CH2

OH β-Glucosidase

O

OH OH

OH

HO

C O

HO

C

CH2OH O OH

H2O

HO OH

(R)-Mandelonitrile H H CH2OH O OH

C

N

OH

O

β-Glucosidase

HO

H2O

HO

O

OH HO OH

OH

N

C

CH2OH

C

C

Prunasin

Hydroxynitrile lyase

H O

HCN

C

Benzaldehyde

Figure 10.4 Stepwise hydrolysis of amygdalin, a cyanogenic glycoside found in bitter almonds, yielding prunasin, (R)-mandelonitrile, benzaldehyde, and HCN.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Table 10.14 Plants

Yield of HCN Released from Food

Plant

HCN yield, mg/100 g

Bitter almond Seeds Young leaves Wild cherry, leaves Apricot, seeds Peach Seeds Leaves Sorghum Whole plant, immature Mature seeds Etiolated shoot tips Young green leaves Bamboo Stem, unripe Tops of unripe sprouts Linseed (flax) Seedling tops Linseed cake Bitter cassava Leaves Inner part of tuber Dried root cortex Whole root Fresh root bark Fresh stem bark Vicia sativa, seeds Lima bean varieties Java, colored Puerto Rico, black Burma, white Arizona, colored America, white

290 20 90–360 60 160 125 250 0 240 60 300 800 910 50 104 33 245 53 89 133 52

seed, and cassava. Nearly 80 naturally occurring glucosinolates have been discovered (Verkerk et al., 1998). The common skeletal structure for glucosinolates that is now widely accepted is shown in Figure 10.5. It has a sulfonated oxime grouping, and the sugar in almost all cases is D-glucose. The glucosinolate side chain may comprise aliphatic (saturated and unsaturated), aromatic, or heteroaromatic groupings and commonly includes hydroxy groups (which may occasionally be glycosylated) and terminal methylthio groups and their oxidized analogs, esters and ketones (Fenwick et al., 1989; Verkerk, et al., 1998). It is the side chain that determines the chemical nature of the products of enzyme hydrolysis and, thereby, their biological effects and potencies. The chemical properties and occurrence of glucosinolates and their breakdown products have been reviewed extensively by Rosa and coworkers (1997). Several important glucosinolates in plants consumed by animals and humans are listed in Table 10.15. The total glucosinolate contents of agriculturally important plants are summarized in Table 10.16. The presence of glucosinolates in brassica plants is always associated with an enzyme system, myrosinase (also called thioglucosidase or thioglucoside glucohydrolase, EC 3.2.3.1). The enzyme is located in cellular compartments separate from glucosinolates in the plant and is released when plant cells are damaged by cutting or chewing (Fenwick and Heaney, 1983). The glucosinolate-myrosinase system may have several functions in the plant. These include plant defense against fungal diseases and

312 300 210 17 10

Glucosinolate R

S

C6H11O5

N

O

C

Source: Compiled from Poulton (1983) and Tewe and Iyayi (1989).

H2O

may be a factor in causing goiter in certain parts of the world.

SO3-

Thiglucosidase (Myrosinase)

SH R

C NOSO3-

D-Glucose

10.9 GLUCOSINOLATES Glucosinolates are secondary plant metabolites found exclusively in cruciferous plants. These sulfur-containing glycosides occur at highest concentrations in the families Resedaceae, Capparaceae, and Brassicaceae. They are found in such common vegetables as cabbage, turnips, rutabagas, mustard greens, horseradish, radish, mustard

Copyright 2002 by Marcel Dekker. All Rights Reserved.

R

N

C

S

Isothiocyanate

R

C

N

Nitrile

R

S

C

N

Thiocyanate

Figure 10.5 Enzymatic breakdown of glucosinolate.

Table 10.15 Nomenclature and Structure of Common Glucosinolates in Edible Plants

Table 10.16 Total Glucosinolate Content of Agriculturally Important Crops

Common name

Species

Aliphatic glucosinolates Glucoiberin Progoitrin Sinigrin Gluconapoleiferin Glucoraphanin Glucoalyssin Glucocapparin Glucobrassicanapin Glucocheirolin Glucoiberverin Gluconapin Indole glucosinolates 4-Hydroxyglucobrassicin Glucobrassicin 4-Methoxyglucobrassicin Neoglucobrassicin Aromatic glucosinolates Glucosinalbin Glucotropaeolin Gluconasturtiin

Structure 3-Methylsulphinylpropyl 2-Hydroxy-3-butenyl 2-Propenyl 2-Hydroxy-4-pentenyl 4-Methylsulphinylbutyl 5-Methylsulphinylpentyl Methyl 4-Pentenyl 3-Methylsulphonylpropyl 3-Methylthiopropyl 3-Butenyl 4-Hydroxy-3-indolylmethyl 3-Indolylmethyl 4-Methoxy-3-indolylmethyl 1-Methoxy-3-indolylmethyl p-Hydroxybenzyl Benzyl 2-Phenethyl

Cabbage White Red Savoy Chinese cabbage Brussels sprouts Cauliflower Calabrese Turnip Radish Oil Red White Horseradish Mustard White Black Rapeseed

Glucosinolate content, mg/g 0.26–1.56 0.41–1.09 0.47–1.24 0.17–1.36 0.60–3.90 0.61–1.14 0.42–0.95 0.21–2.27 0.92–1.12 0.42–1.17 0.57–1.19 33.2–35.4 22.0–52.0 18.0–60.0 13.0–42.0

Source: Compiled from Deshpande and Sathe (1991) and Verkerk et al. (1998).

Source: Compiled from Deshpande and Sathe (1991) and Verkerk et al. (1998).

pest infestation, sulfur and nitrogen metabolism, and growth regulation (Verkerk et al., 1998). Myrosinase has been found in the seed, leaf, stem, and roots of glucosinolate-containing plants, and the activity appears to be higher in the young tissues of the plant. Glucosinolates that act as progoitrins are the source of organic nitriles, isothiocyanates, and SCN ions. Some of these compounds have been shown to be quite harmful if consumed in sufficient amounts by humans and animals. They contribute to the flavor of all brassica plants. These compounds are usually formed by the hydrolysis of glucosinolates by myrosinases (Figure 10.5); the enzyme system, however, does not become active until the wet raw plant material is crushed. Less frequently, the aglucon products from the glucosinolates may be formed by chemical hydrolysis or by the action of enzymes from nonplant sources, such as intestinal microflora. The isothiocyanate ion formed upon enzymatic hydrolysis is unstable and cyclizes to yield 1,5-vinyl-2-thiooxazolidine, commonly known as goitrin. Goitrogens act primarily by preventing iodine uptake in the thyroid gland and, hence, impairing the synthesis of the thyroid hormones triiodothyronine and thyroxine. Goitrogens in cassava are also believed to be re-

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sponsible for the differential distribution and severity of endemic goiter in some parts of Africa. The relative amounts of glucosinolates in a given species vary as a result of both genetic factors and agronomic practices. Similarly, any food processing method that causes disruption of cellular integrity causes at least a partial loss of glucosinolates by hydrolysis. Although heat processing inactivates myrosinases, some loss of glucosinolates may still occur through leaching into the cooking water. However, plant breeding remains an attractive alternative to achieve greater reduction in the glucosinolate content of cruciferous plants. In spite of their known toxicological effects, glucosinolates do seem to have certain beneficial health effects. The protective effect of cruciferous vegetables against cancer has been suggested to be due in part to the relatively high content of glucosinolates (Verkerk et al., 1998). This characteristic distinguishes them from other vegetables. Isothiocyanates that arise in plants as a result of enzymatic cleavage of glucosinolates by the endogenous enzyme myrosinase are attracting increasing attention as chemical and dietary protectors against cancer. These anticarcinogenic activities have been demonstrated in rodents (mice and rats) with a wide variety of chemical carcinogens (Table 10.17). The anticarcinogenic effects of isothiocyanates are attributed to a blocking effect involving the induction of

Table 10.17 Protection Against Chemical Carcinogenesis in Rat and Mouse Organs by a Variety of Isothiocyanates and Glucosinolates Protective isothiocyanates α-Naphthyl-NCS, β-naphthyl-NCS Phenyl-[CH2]n-NCS, where N = 0, 1, 2, 3, 4, 5, 6, 8, 10 PhCH(Ph)CH2-NCS, PhCH2CH(Ph)-NCS CH3[CH]n-NCS, where N = 5, 11 CH3[CH2]3CH(CH3)-NCS Sulforaphane, CH3S(O)[CH2]4-NCS 2-Acetylnorbornyl-NCS (three isomers) Protective glucosinolates Indolylmethyl glucosinolate (glucobrassicin) Benzyl glucosinolate (glucotropaeolin) 4-Hydroxybenzyl glucosinolate (glucosinalbin) Carcinogens employed 3′-Methyl-4′-dimethylaminoazobenzene 4-Dimethylaminoazobenzene N-2-fluorenylacetamide, acetylaminofluorene 7,12-Dimethylbenz[a]anthracene (DMBA) Benzo[a]pyrene Methylazoxymethanol acetate N-Nitrosodiethylamine 4-(Methylnitroamino)-1-(3-pyridyl)-1-butanone (NNK) N-Nitrosobenzylmethylamine (NBMA) N-Butyl-N-(4-hydroxybutyl)nitrosamine Tumor target organs Rat: Liver, lung, mammary gland, bladder, small intestine/colon, esophagus Mouse: Lung, forestomach Source: Compiled from Talalay and Zhang (1996) and Verkerk et al. (1998).

phase II enzymes in the small intestinal mucosa and liver (Zhang et al., 1992; Talalay and Zhang, 1996) and a suppressing effect involving suppression of tumor development by inducing programmed cell death, apoptosis (Smith et al., 1996). The evidence for anticarcinogenic effects of brassica vegetables in humans is strongly supported by evidence obtained with experimental animals. In a review by Steinmetz and Potter (1991), the overall conclusion from an analysis of 115 case-control studies was that a relatively high consumption of brassica vegetables was associated with a reduction in risk of cancer at many sites. For broccoli consumption in particular, there was a uniform protective effect, with no contrary evidence in any study. Consumption of brassicas, which might be expected to yield high levels of indoles and isothiocyanates, was particularly strongly associated with a lower risk of colon cancer. In view of this evidence concerning their beneficial biological activities in humans, factors inducing and di-

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recting indole glucosinolate metabolism in plants need to be studied in much greater detail.

10.10 FAVIC AGENTS (FAVISM) The occurrence of sporadic cases of acute hemolysis after ingestion of broad beans (also known as fava beans, Vicia faba) was first recorded in the medical literature around the mid-1850s (Chevion et al., 1983). Since then several clinical reports as well as epidemiological, genetic, and biochemical studies have contributed to the characterization of this disease, termed favism, and to the elucidation of its cause and pathogenesis. Fava beans are commonly grown and consumed in large quantities in the Middle East, Far East, and North Africa. Except for the presence of vicine and convicine, the two β-glycosides implicated in the cause of favism and relatively low concentrations of other antinutrients common in legumes, fava beans are an excellent source of dietary protein. Vicine and convicine have also been reported in sweet lupine (Lupinus albus). One study, however, has clearly shown that these glycosides are only associated with the Vicia species and are not present in any other food plants (Pitz et al., 1980). These authors reported the average dry seed concentrations of vicine and convicine to be, respectively, 0.72% and 0.27% in V. faba minor (fava beans), 0.71% and 0.19% in V. faba major (broad beans), and 0.75% and 0.08% in V. narbonensis, a wild type of fava bean. Both environmental and genetic factors seem to affect the concentration of vicine and convicine in the seed of Vicia faba. Fresh green seeds contain much higher levels of glycosides than do mature seeds. The glycosides are synthesized very early in the development of the seed, and their concentration within the seed decreases with the increasing maturity of the seed. Vicine and convicine are hydrolyzed by intestinal microflora to highly reactive free radical–generating compounds, divicine and isouramil, respectively. The chemical structures of these four compounds are shown in Figure 10.6. Divicine and isouramil have been strongly implicated as the causative agents in favism. Their free radicals also cause other adverse effects, including lipid peroxidation, altered fat and mitochondrial metabolism, and possibly diabetes (Marquardt, 1989). Some of these adverse effects can be overcome by increasing the dietary intake of such antioxidants as vitamins A, C, and E, as well as chelating agents such as EDTA. Favism occurs only in individuals with a deficiency of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that has an important function in red blood cell metabolism. It is essential to maintain adequate levels of the

Vicine O

OH

O

O

HN N

H2N

Divicine

NH2

OH HOH2C O

OH

Convicine O

O

H2N

N

N H

OH

NH2

NH2

Isouramil O

O

HN

OH

HN

OH HOH2C O

OH

HN OH

O

N H

NH2

Figure 10.6 Structures of vicine and convicine. Their active aglycones, divicine and isouramil, respectively, arise from the removal of their sugar (glucose) by a β-glucosidase.

reduced form of glutathione (GSH) and NADPH. GSH and NADPH help to prevent oxidative damage to erythrocytes. The red blood cells of individuals with a deficiency of erythrocyte G6PDH are thus susceptible to oxidative damage. G6PDH-deficient individuals are subject to hemolytic crises from a variety of substances; among them is primaquine, an antimalarial drug. G6PDH deficiency is common in Mediterranean and Middle Eastern population groups and is an inborn error of metabolism. By nature, vicine and convicine, in which an autooxidizable hydroxyl group at C-5 of the pyrimidine moiety is blocked by the β-glycosidic bond, are inert. However, their corresponding aglycones, divicine and isouramil, rapidly lower the glutathione levels of erythrocytes deficient in G6PDH, causing hemolysis. The clinical manifestations of favism include hemolytic anemia, hemoglobinuria, and jaundice, often accompanied by high fever. It usually begins suddenly after the inhalation of pollen or a few hours after ingestion of the beans. In severe cases, death may occur in 24 to 48 hours. Fava beans also contain 3,4-dihydroxy-L-phenylalanine (L -DOPA) (Kosower and Kosower, 1967). DOPA may be transformed to dopaquinone by the action of tyrosinase. The quinone is active in causing a decrease in GSH in erythrocytes in G6PDH-deficient humans, whereas LDOPA is not (Beutler, 1970). Indeed, L-DOPA is present in garden pea (Pisum sativum), which rarely if ever, causes acute hemolysis as seen in favism. It is, however, possible that L-DOPA and divicine or isouramil may act synergistically in causing a lowering of erythrocyte GSH level in

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these subjects (Razin et al., 1968). Ascorbic acid also seems to enhance the hemolytic property of divicine and isouramil. Favism is not known to occur in normal individuals and, hence, is not a serious problem. The only way of diminishing the risk of this disease in susceptible populations appears to be through effective genetic breeding. Since the glycosides appear to occur only in the cotyledon portions of the seed, dry milling of dehulled fava beans does not lower their concentration (Elkowicz and Sosulski, 1982). Wet-processing methods, on the contrary, remove over 97% of vicine and convicine. Cooking and sprouting of fava beans have a negligible effect on their total glycoside content; extracting whole or dehulled beans with 1% acetic acid or treatment with β-glucosidase has been shown to be highly effective (Hegazy and Marquardt, 1983; Arbid and Marquardt, 1985; Hussein et al., 1986).

10.11 LATHYROGENS (LATHYRISM) Consumption of the seeds of certain Lathyrus species results in a neurological disorder characterized by spastic paraparesis in humans and animals. This disease, lathyrism, is quite insidious and may be precipitated by fatigue and exposure to cold and wetness. The symptoms begin with back pain and weakness and stiffness of the legs. Muscular weakness, and in severe cases, paralysis of the legs follow, limiting the victim’s mobility to crawling. Males between 20 and 29 years old appear to be most sen-

sitive to lathyrogenic agents and are the ones usually affected most (Concon, 1988). Presently, lathyrism is confined mostly to the Indian subcontinent. Lathyrism has been known in humans at least since the days of Hippocrates (Seley, 1957) and still occurs in epidemic proportions in certain areas of the world during seasons of flood and drought, a time when other plants are destroyed and the species of L. sativus thrives and provides survival food (Roy and Spencer, 1989). The causative factor of this disease is still unknown. However, certain lathyrogens have been implicated and studied extensively. Two types of lathyrism are known: osteolathyrism and neurolathyrism. Osteolathyrism (also known as odoratism) is caused in rats and other animals by the ingestion of L. odoratus, L. hirsutus, and L. pusillus seeds; neurolathyrism is caused in humans by the prolonged consumption of seeds of L. sativus. A dipeptide (N-γ-glutamyl) aminopropionitrile was the first toxic principle to be detected in Lathyrus species. The β-aminopropionitrile (BAPN) (Figure 10.7) moiety of this dipeptide was shown to be responsible for osteolathyrism in experimental animals (DuPuy and Lee, 1954). BAPN, however, does not have any toxic effects on the nervous system. The human disorder is precipitated by excessive consumption of Lathyrus sp. seeds that contain the neurotoxin β-N-oxalylamino-L-alanine (BOAA) (Figure 10.7). Compounds possessing osteolathyrogenic potency can be broadly divided into four major groups: nitriles, urides, hydrazides, and hydrazines (Table 10.18). They show no structural relationship except that they all have, or

NC CH2

CH2

NH2

β-Aminopropionitrile (BAPN)

O HOOC C N CH2 H

CH COOH NH2

β-N-Oxalylamino-L-alanine (BOAA) Figure 10.7 Chemical structure of an osteolathyrogen β-aminopropionitrile (BAPN) and a neurolathyrogen β-N-oxalylamino-L-alanine (BOAA).

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potentially have, an available terminal primary amine group (Barrow et al., 1974). Osteolathyrogens interfere with the initial reaction in the formation of cross-links in connective tissues. The results are increasing solubility of collagen, bone deformities, and modification of arterial elastin with consequent reduction in the tensile strength of aorta. Neurolathyrogens, such as BOAA, induce convulsions in rodents and primates, and in the rat, vacuolar degeneration of dendrites and cell bodies of cirumventricular organs (areas where the blood-brain barrier is normally absent), including the area postrema, subcommissural organ, subfornical organs, and arcuate-median eminence region of the hypothalamus (Olney et al., 1976). Because of its structural similarity to glutamic acid, BOAA also interferes with the high-affinity transport of both aspartate and glutamate and induces selective spinal cord abnormality. Similar neurolathyrogens have also been identified in the seeds of Vicia spp. The acutely neurotoxic amino acids from Lathyrus and Vicia species are summarized in Table 10.19.

Table 10.18

Compounds Having Osteolathyrogenic Potency

Organic nitriles β-Aminopropionitrile (BAPN) and β-(γ-glutamyl)aminopropionitrile Aminoacetonitrile (AAN) Methyleneaminonitrile (MANN) β,β′-Iminodipropionitrile (IDPN) β-Hydrazinopropionitrile 2-Cyanopropylamine (2-CPA) Ureides Semicarbazide (SCA) Acetone semicarbazide Hydrazides Nicotinic and isonicotinic acid hydrazide Cyanoacetic acid hydrazide Benzhydrazide Glutamic acid hydrazide γ-L-Glutamylhydrazide Carbohydrazide Thiohydrazide Hydrazines Hydrazine hydrate Phenyl hydrazine Symmetrical and unsymmetrical dimethyl hydrazine Miscellaneous Cyanide β-Mercaptoethylamine Thiosemicarbazide Source: Compiled from Barrow et al. (1974), Roy (1981), and Roy and Spencer (1989).

Table 10.19 Naturally Occurring Neurotoxic Amino Acids and Their Derivatives in Lathyrus and Vicia Species Toxic amino acid and derivative β-Cyano-L-alanine and γ-glutamyl-β-cyano-L-alanine L-α,γ-diaminobutyric acid, L-2,4-diaminobutyric acid, and γ-N-oxalyl derivative β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), or β-N-oxalylamino-L-alanine, L-3-oxalylamino-2-aminopropionic acid (OAP)

Source Vicia sativa, V. angustifolia Lathyrus latifolius, L. sylvestris, V. aurantica L. sativus, L. cicera, L. latifolius

Source: Compiled from Roy (1981) and Roy and Spencer (1989).

Several methods have been suggested to remove BOAA from L. sativus seeds for human consumption (Mohan et al., 1966). These include cooking the seeds in an excessive amount of water followed by draining, soaking the seeds in cold water overnight, and steeping the dehusked seeds in hot water. More BOAA is removed from dehusked than from whole seeds; complete removal is not possible. A maximum of 80% of BOAA can be removed from the seeds by any of the methods. Roasting the whole seed at 150°C for 20 minutes also destroys over 85% of BOAA. The levels of BOAA have also been reduced to minimal amounts by careful selection and plant breeding techniques; a safe level has not yet been defined.

10.12 TOXIC AMINO ACIDS In addition to the toxic lathyrogenic derivatives of amino acids, several other uncommon amino acids occur in the

Table 10.20

human food chain. These are usually the structural analogs of the protein amino acids, which exert their toxic effects as antimetabolites. They are usually found as free or simple condensation products in the form of γ-glutamyl, acetyl, methyl, and oxalyl derivatives. A summary of some of the more commonly occurring unusual amino acids in food plants is given in Table 10.20. Nonprotein toxic amino acids are considered secondary metabolites. Their functions in the plant are not fully understood, although several studies have indicated that they form a part of a chemical defense mechanism of the plant against predators (Roy and Spencer, 1989; Evans, 1983). Some of these compounds are toxic to specific microorganisms, insects, birds, and mammals. Most of those that are toxic to humans give rise to chronic, rather than acute, symptoms of poisoning. This suggests that low concentrations of a particular toxin can have a cumulative harmful effect if they constitute a part of the normal diet over a prolonged period. Toxic amino acids, which are

Some Toxic Amino Acids Commonly Occurring in Plant Foods and Forages

Amino acid N-(3-hydroxypyridone-4)-2-aminopropionic acid (mimosine) 2-Amino-4-(guanidinooxy)butyric acid (canavanine) L-2-Amino-6-amidinohexanoic acid (indospicine) 5-Hydroxy-L-tryptophan (5-HTP) 2-Amino-3-methylaminopropionic acid L-3,4-dihydroxyphenylalanine (L-DOPA) 3-Methylenecyclopropylpropionic acid (hypoglycin A) Seleno-amino acids (methylselenocysteine, selenocystathionine, selenocystine) 3-Cyanoalanine and 4-Glutamylcyanoalanine δ-Acetyl ornithine Homoserine and o-oxalylhomoserine Lathyrine, γ-methyl glutamic acid, γ-hydroxynorvaline, and γ-hydroxyhomoarginine Homoarginine and pipecolic acid Source: Compiled from Roy and Spencer (1989) and Evans (1983).

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Source Many species of Mimosoidae (a subfamily of Leguminosae) Many species of Papilionoidae (a subfamily of Leguminosae) Tropical legume Indigofera spicata Griffonia simplicifolia Cycas spp. Vicia and some other legumes Plants of Sapindaeceae family Plants grown on selenium-rich soils Vicia spp. Lathyrus spp. Lathyrus spp. Lathyrus spp. Lathyrus and Vicia spp.

close structural analogs of protein amino acids, exert their toxicity by disrupting the enzyme systems. For example, the seleno amino acids are incorporated into enzymes in place of the normal sulfur amino acids, and canavanine replaces arginine in nuclear proteins. Mimosine is a potent inhibitor of cystathionine synthetase enzymes in the liver. Some uncommon amino acids also act as precursors of normal metabolites. Thus, 5-hydroxytryptophan gives rise to 5-hydroxytryptamine, a physiologically active amine in the mammalian brain. Others, such as L-DOPA, have been used in the treatment of Parkinson’s disease. As attempts are made to increase the food supply of the world by introducing plants not traditionally used for food and fodder, it is quite likely that the nonprotein amino acids may give rise to increased problems of toxicity in the future.

10.13 TOXIC FATTY ACIDS Either the normal fatty acids in foodstuffs that are of nutritional value are without exception saturated or unsaturated straight chains or they rarely exceed 20 carbon atoms in length. Furthermore, the double bonds in unsaturated fatty acids follow a specific pattern: position 9 in the monoenes, palmitoleic, and oleic acids; positions 9 and 12 in the diene, linoleic acid; positions 9, 12, and 15 in the triene, linolenic acid; and positions 5, 8, 11, and 14 in the tetraene, arachidonic acid. The specific structures of nutritional fatty acids suggest that any deviation from the norm may result in adverse effects, unless the animal system can successfully degrade the acid to nontoxic metabolites. Several unusual fatty acids have been shown to be toxic to humans. These include erucic acid, sterculic and malvalic acids (cyclopropene fatty acids), cetoleic acid, and in the presence of Refsum’s syndrome, phytanic acid (Concon, 1988). Cetoleic acid is of animal origin and is found in herring oil (Beare-Rogers et al., 1971). Its toxic effects are similar to those of erucic acid. Phytanic acid does not occur endogenously in plant foodstuffs. It is, however, a contaminant in ruminant fat and possibly dairy products (Hansen, 1965a, 1965b; Patton and Benson, 1966). Erucic acid and the cyclopropene fatty acids are the only ones in this group that are endogenous in plant foods. 10.13.1 Erucic Acid Erucic acid (C22:1, ω9) is found largely in the plant family Cruciferae, notably in Brassica spp. It accounts for as much as 20% to 55% of the fatty acid content of the oil derived from rapeseed (B. rapus and B. campestris) and mustard (B. hirta and B. juncea). As early as 1960, Roine and his collaborators reported myocardial effects in rats

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fed 50% to 70% of their caloric intake as rapeseed oil. Weanling rats fed high levels of rapeseed oil experience accumulation of fat in the heart muscles even after the first day of feeding; the fat droplets are scattered throughout the myocardium. Subsequent studies have shown that even an intake of 4 energy percent in the form of erucic acid can lead to certain morphological defects (Engfeldt, 1975; Ziemlanski, 1977). The mechanism for the development of the abnormalities shown in experimental animals on erucic acid diets is thought to be a blockage of the β-oxidation of the fatty acids in mitochondria (Engfeldt, 1975) Rapeseed oil also interferes with the ability of the mitochondria in heart muscles to oxidize substrates such as glutamate and thus impairs the rate of ATP synthesis (Houtsmuller et al., 1970). This does not occur with liver mitochondria. Thus, the fatty accumulation in the heart muscle is due to the failure of the myocardium to oxidize erucic acid and/or convert it to oleic acid. High levels of erucic acid in the diet also impair the growth of chickens (Salmon, 1969a); ducklings, guinea pigs, hamsters, and mice (Thomasson et al., 1970); pigs (Thoron, 1969); and turkeys (Salmon, 1969b). The retardation of growth in rats also can be accomplished by feeding other fats mixed with erucic acid (Beare et al., 1959; Thomasson and Boldingh, 1955). Because a large intake of erucic acid is necessary to induce myocardial damage in animals, the hazard of erucic acid toxicity in humans is probably minimal. The worldwide production and increasing use of rapeseed and mustard seed oils in margarine and the important role they play in the farming systems of certain countries, such as Canada and India and in northern Europe, however, have called for intensive studies in these areas. Plant breeding efforts during the past four decades have resulted in the identification and development of low erucic acid–content rapeseed varieties. 10.13.2 Cyclopropene Fatty Acids The oils or fat of every plant of the order Malvales that have been examined thus far, with one exception, contain cyclopropene fatty acids (Eckey, 1954; Phelps et al., 1965). The exception is cocoa butter from Theobroma cocoa (Hilditch and Williams, 1964). Two important acids in this group are sterculic (19 carbons) and malvalic (18 carbons) acids (Figure 10.8). From the food toxicological viewpoint, only the cyclopropene fatty acids in the oils of cottonseeds (Gossypium hirsutum) are of significance. Crude cottonseed oil may contain from 0.6% to 1.2% cyclopropene fatty acids in the form of sterculic and malvalic acids (Bailey et al., 1966). Processing may reduce these levels by 0.1% to

Table 10.21

H2 C

Saponin Content of Staple Plant Foods

Plant CH3(CH2)7C

C

(CH2)n

Malvalic acid:

n=6

Sterculic acid:

n=7

COOH

Figure 10.8 Cyclopropene fatty acids found in cottonseed oil.

0.5%. The cottonseed meal may contain about 0.01%, depending on the quantity of residual oil. Hydrogenation probably destroys some of the biological effects of these acids (Masson et al., 1957). Both sterculic and malvalic acids have been shown to possess carcinogenic activities. In their presence, the carcinogenic activity of aflatoxin B1 is also considerably enhanced (Lee, 1967; Sinnhuber et al., 1966). Sterculic acid also prevents the conversion of exogenous stearic acid to oleic acid. This results in increased levels of saturated fatty acids in body fat at the expense of monounsaturated fatty acids (Johnson et al., 1967). This increase in saturated fatty acids in body fat cannot be overcome by the addition of oleic or linoleic acids in the diet (Evans et al., 1963). One possible mechanism suggested is the inhibition of stearate desaturase by the cyclopropene group in these fatty acids (Johnson et al., 1967; Raju and Reiser, 1969). The importance of cyclopropene fatty acids to human health is unknown. Cottonseed oil has been used for several years in food preparation, apparently with no ill effects (Concon, 1988). However, experimental or epidemiological evidence to this effect is lacking. The carcinogenicity of these fatty acids together with their biological and biochemical effects give them greater toxicological significance than hitherto realized.

10.14 SAPONINS Saponins are a complex and chemically diverse group of compounds found primarily in many plants as well as in a number of marine animals. Their physiological effects are also as diverse as their chemical structures and properties, and not all of them are toxic. Saponins, however, occur in significant amounts in many commonly used food and forage plants (Table 10.21). Although many of these are herbs, spices, or medicinal plants and are consumed only in small amounts, some are staple items of the diet of a

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Chickpea (Cicer arietinum) Soybean (Glycine max) Dry bean (Phaseolus vulgaris) Mung bean (Phaseolus aureus) Broad bean (Vicia faba) Lentil (Lens culinaris) Green pea (Pisum sativum) Peanut (Arachis hypogaea) Asparagus (Asparagus officinalis) Spinach (Spinacea oleracea) Silver beet (Beta vulgaris) Sesame seed (Sesamum indicum) Oat (Avena sativa)

Saponin content, g/kg 2.3–60 5.6–56 4.5–21 0.5–5.7 3.5 1.1–5.1 1.1–1.8 0–16 15 47 58 3 1–1.3

Source: Compiled from Price et al. (1986), Fenwick and Oakenfull (1981), Curl et al. (1985), and Oakenfull and Sidhu (1989).

large part of the world population. Among the major ones are chickpeas in the Middle East and the Indian subcontinent, soybeans in much of Southeast Asia, and peanuts in central Africa. The major forage plants containing saponins include alfalfa, cloves, guar, sunflower, and lupine. Chemically, saponins have a triterpene or steroid backbone linked to one or more sugar groups (Price et al., 1987). There are thus two broad subdivisions of saponins: those with triterpenoid aglycones and those with steroid aglycones. Almost all the saponins in food and forage plants are of the triterpene class. The chemical structures of two well-characterized and biologically active saponins are shown in Figure 10.9. Within the same plant species, the saponins differ in composition as well as in quantity. For example, DuPuits and Lahontan alfalfa varieties contain 25 different saponins, but the chemical composition of the saponins in the two varieties is not the same (Birk and Peri, 1980). The aglycones of DuPuits saponins are mainly of the medicagenic acid type, whereas the saponins of Lahontan contain soybean sapogenins and monocarboxylic acids as aglycones. Also, DuPuits saponins lack D -galactose, which is present in Lahontan saponins. The composition of alfalfa seed saponins seems to be similar to that of soybean seeds (Birk and Peri, 1980). Saponins are commonly isolated by extraction of the plant material with hot water or ethanol. On complete hydrolysis, they yield sapogenins, which are the aglycones of steroids (C27) or triterpenes (C30). Among food plants, soy saponins are the most thoroughly studied saponins. They were first identified and characterized in 1964 (Willner et al., 1964). The sugars present on the intact soy saponins

H3C

Medicagenic acid 3-β-O-triglucoside CH2OH O OH

O

CH2 H3C

O

HO

OH OH

CH3

CH3

H

COOH

HO

O

HO

CH2OH O

OH

H

CH3

O H 3C

HO OH

H3C

COOH

CH3

Hederagenin H3C

CH3

COOH

HO CH3 HO H3C

CH2OH

Figure 10.9 Structural formula of the alfalfa saponin medicagenic acid 3-β-O-triglucoside, and the aglycone hederagenin of soyasapogenol.

include galactose, arabinose, rhamnose, glucuronic acid, xylose, and glucose. Despite their diverse chemical properties, saponins have a number of common and characteristic properties, including the following: 1. 2. 3. 4. 5.

Bitter taste Formation of stable foams in aqueous solutions Hemolysis of red blood cells Toxicity to cold-blooded animals, such as fish, snails, and insects Ability to interact with bile acids, cholesterol, or other 3-β-hydroxy steroids in aqueous or alcoholic solutions to form mixed micelles or coprecipitates

Saponins have had many industrial and commercial applications, including use as emulsifiers in soft drinks, shampoo, fire extinguishers, soap, and the synthesis of steroid hormones (Deshpande and Sathe, 1991). The wide range of chemical and physical properties of saponins is also reflected in the range of their physiological and pharmacological effects. Although they are extraordinarily physiologically active compounds, their reputation for toxicity is in general unfounded. Ingested saponins mostly remain

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within the GI tract, and it is only when they enter the bloodstream that their hemolytic activity causes damage (Oakenful and Sidhu, 1989). Their physiological activity appears to arise from two main causes: their strong physical interactions as surface-active agents with dietary and intestinal components and their ability to interact with the membranes of the mucosal cells. Among the effects of saponins on animals are growth inhibition in swine and poultry, reduced palatability of food, and increased excretion of cholesterol. Alfalfa protein concentrate (APC) prepared from low-saponin varieties yields better growth performance in rats than the one prepared from the high-saponin variety (Hegsted and Linkswiler, 1980). The reduced weight gains with high-saponin APC were attributed to lower feed intake and lower protein digestibility. In recent years, the ability of saponin-rich foods to lower plasma cholesterol concentration and thus the risk of cardiovascular diseases has attracted much attention. Kritchevsky and colleagues (1975) were among the first to suggest that the saponins in alfalfa were the cause of the lower plasma cholesterol levels in rats fed alfalfa-based diets. Malinow and coworkers (1985, 1987) have since provided conclusive evidence that the saponins in alfalfa are indeed responsible for the lower plasma cholesterol con-

centrations and that alfalfa fiber in the absence of saponins is not responsible for this response. The only reported experiment in which human subjects were given saponins directly is that described by Bingham and associates (1978), who gave a group of 174 arthritic patients tablets of a saponin-rich extract from Yucca schidigen in a treatment that included controlled diets, exercise, and physiotherapy. A control group was given a placebo, with the same treatment. Substantial reductions in plasma cholesterol concentration were observed in response to the saponins, particularly in individuals who had initially higher levels. There were also significant reductions in plasma triglyceride concentration and blood pressure. Foods containing saponins as a significant, but naturally occurring component have also been shown to be effective in lowering plasma cholesterol concentration in hyperlipidemic humans. Mathur (1968) found that giving hypercholesterolemic patients chickpeas as an isocaloric substitute for wheat flour and other cereals lowered their mean plasma cholesterol concentration from 206 mg/100 ml to 160 mg/100 ml. Other legumes have been found to be similarly effective (Effect of legume seeds, 1980). The mechanism of this effect seems to be, at least in part, that saponins inhibit absorption from the small intestine of cholesterol or bile acids. Bile acid and cholesterol metabolism are closely interrelated, since the former are synthesized from cholesterol in the liver. Both are secreted by the liver as bile to promote the digestion and absorption of lipids. Bile acids are normally very efficiently recycled. The ability of some saponins to form mixed micelles with bile acids interrupts this cycle. Bile acids are not reabsorbed from such micelles and consequently are excreted. Similarly, the formation of molecular complexes of saponins with cholesterol prevents its absorption. The net result is a higher loss by fecal excretion of cholesterol and bile acids than can be compensated from endogenous or exogenous sources, with consequent reduction in the cholesterol levels of blood plasma, liver, and other tissues. Further elucidation of the chemical structures of various saponins and their interactions with cellular and membranal components will undoubtedly lead to a better understanding of the role of saponins in human nutrition and medicine. It also appears quite likely that in the coming years, saponins may actually be deleted from the antinutritional factors in human nutrition.

10.15 POTATO GLYCOALKALOIDS Steroidal alkaloids are commonly found in Solanum spp., including potato and tomato. Solanine, a glycoside containing a steroid alkaloid nucleus (aglycone) with a side

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chain of sugars, was first discovered in potatoes in 1826 (Cheeke and Shull, 1985). The aglycone is called solanidine. Later on another glycoalkaloid, chaconine, was also discovered in potatoes. Solanine and chaconine have the same aglycone (solanidine) but differ with respect to the carbohydrate side chain (Figure 10.10). Since these early discoveries, several other alkaloids have been identified in potatoes. These were found to contain two different steroidal skeletons. Solanidane (solanidine type) contains the indolizidine system exemplified by solanines, chaconines, solanidine, leptines, leptinines, and demissidine. The second group, spirosolane (solasodine type), possesses oxa-azaspirodecane structure as represented by tomatidenol, and α- and β-solamarines (Jadhav et al., 1997). The hydrolysis products and sugar moieties of the glycoalkaloids are summarized in Table 10.22. Glycoalkaloids are toxic and thus are believed to be involved in pest resistance. These toxicants occur in potato tubers, peels, sprouts, and blossoms, and their concentration in tubers depends on cultivar, maturity, environmental factors, and stress conditions. Concentrations may vary because of fungal or bacterial infection and usually increase in response to wounding, apparently as a defense mechanism against potential disease. Glycoalkaloids, therefore, may function as phytoalexins (Jadhav et al., 1997). Green sprouts and green potato skins (peels) contain the highest concentrations of solanum alkaloids. The greening of potatoes occurs when the tubers are exposed to sunlight during growth or after harvest. The greening is due to chlorophyll; the increased concentration of solanum alkaloids in green potatoes occurs because similar environmental conditions promote the synthesis of both chlorophyll and glycoalkaloids (Jadhav et al., 1981). The two principal effects of solanum alkaloid poisoning are gastrointestinal tract irritation and impairment of the nervous system. Because of their relatively low gastrointestinal absorption, these alkaloids are much more toxic when administered parenterally then orally (Nishie et al., 1971). The glycosides are more toxic than the corresponding aglycones. Apathy, drowsiness, salivation, dyspnea, trembling, weakness, paralysis, and loss of consciousness are manifestations of the effects on the nervous system. Solanum alkaloids are cholinesterase inhibitors (Jadhav et al., 1981). Inhibition of this enzyme results in increased accumulation of acetylcholine in nerve tissue, thus impairing the neural function. This is manifested in such neurological signs as ataxia, convulsions, coma, muscle weakness, and involuntary urination. Gastrointestinal tract effects of solanum alkaloids include inflammation of intestinal mucosa, hemorrhage or

α-Solanine

CH3 CH3 N CH3 H

CH3 H CH2OH O HO CH2OH O OH

O

HO

H

H

H

O

O HO

O

OH

CH3 OH

OH

α-Chaconine CH3 CH3 N CH3 H

CH3 H HO

CH2OH O OH

O CH3 OH

O OH

H

H

H

O

O HO

O CH3 OH

Figure 10.10

OH

Molecular structures of potato glycoalkaloids.

ulceration, abdominal pain, constipation, and diarrhea. Teratogenic effects of potato alkaloids have also been suggested (Renwick, 1972). In addition to human poisoning, incidences of livestock poisoning have also been known in animals fed potato sprouts, peelings, and sunburned or spoiled potatoes. Potato vines have also caused toxicity, since the alkaloids are in highest concentration in green tissues.

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Solanum alkaloids are not destroyed by boiling, baking, frying, or drying at high temperatures (Jadhav et al., 1997; Cheeke and Shull, 1985). The relatively rare occurrence of solanine poisoning has been mainly due to three factors: Solanine is poorly absorbed, it is hydrolyzed to a considerable extent in the gut to the less toxic solanidine, and urinary and fecal excretion of its metabolites is rapid. Nevertheless, new potato varieties are now routinely

Table 10.22 Glycoalkaloids and Their Hydrolysis Products (Aglycone and Sugar Moiety)a Glycoalkaloid α-Solanine

Aglycone

Sugar moiety

Solanidine

D-Glu

-D-Gal< β-Solanine γ-Solanine α-Chaconine

Solanidine Solanidine Solanidine

β-Chaconine γ-Chaconine α-Solamarine

Solanidine Solanidine Tomatidenol

β-Solamarine

Tomatidenol

Leptine I

O(23)Acetylleptinidine

Leptine II Leptinine I

O(23)Acetylleptinidine Leptinidine

Leptinine II

Leptinidine

Commersonine

Demissidine

L-Rha -D-Gal-D-Glu -D-Gal L-Rha -D-Glu< L-Rha -D-Glu-L-Rha -D-Glu D-Glu -D-Gal< L-Rha L-Rha -D-Glu< L-Rha L-Rha -D-Glu< L-Rha D-Glu -D-Glu< L-Rha L-Rha -D-Glu< L-Rha D-Glu -D-Glu< L-Rha D-Glu

-D-Gal-D-Glu< D-Glu

Demissine

Demissidine

D-Glu

-D-Gal-D-Glu< D-Xyl a

Glu, glucose; Gal, galactose; Rha, rhamnose; Xyl, xylose.

screened before release, and glycoalkaloid levels must be below 20 mg/100 g (Jadhav et al., 1997; Plhak and Sporns, 1997). Levels of 14 mg/100 g are bitter; levels above 20 mg cause a burning sensation in the mouth and throat.

10.16 OXALATES Certain families and species of plants contain relatively large amounts of oxalic acid, mainly as the soluble sodium or potassium salts or the insoluble calcium salts. Some of the common plant foods containing appreciable amounts of oxalates (fresh weight basis) are spinach (0.3% to

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1.2%), rhubarb (0.2% to 1.3%), beet leaves (0.3% to 0.9%), tea (0.3% to 2.0%), and cocoa (0.5% to 0.9%). Lettuce, celery, cabbage, cauliflower, turnips, carrots, potatoes, peas, and beans also contain lesser amounts of oxalates. It is well known that humans excrete varying amounts of calcium oxalate crystals in the urine (6 to 45 mg/day, with a mean of about 20 mg/day in normal subjects). About two thirds of the urinary oxalate is derived from ascorbic acid and the amino acid glycine; the rest is from dietary oxalate and possibly from precursors, such as glycolic and glyoxylic acids. Like phytates, oxalates can decrease the availability of dietary essential minerals such as calcium. The adverse effects of oxalates in relation to calcium must be considered in terms of the oxalate/calcium ratio. On a milliequivalent basis, foods having a ratio greater than 1 may have serious effects on calcium availability (Table 10.23). Those with a ratio of 1 or below cause no difficulty in the availability of calcium as far as other calcium sources are concerned. In contrast to their role in calcium absorption, oxalates appear not to interfere in zinc absorption in zincdeficient rats (Welch et al., 1977). Somehow, a counteracting or protective mechanism may prevent the precipitation of zinc by oxalates. A metabolic disorder resulting in the production of renal stones and deposits of calcium oxalate in other tissues is often attributed to the excessive consumption of oxalates from foods. Under such conditions, urinary levels of oxalates and glycolates are greatly increased. This disorder is attributed to an inability to transaminate glyoxylate from glycine; this in turn results in increased formation of glyoxylate from glycolates and hence in oxalic acid (Fassett, 1973). However, the role of exogenous oxalates in the production of renal stones seems to be doubtful in most cases. Acute oxalate toxicity in humans is associated with corrosive gastroenteritis, shock, convulsions, low plasma calcium levels with correspondingly higher levels of oxalates, and renal damage. Acute oxalate poisoning in humans, however, is rare. Even for noticeable chronic toxic effects to occur, a rather improbable combination of circumstances (a very high intake of oxalate-containing foods plus a simultaneously low calcium and vitamin D intake over a prolonged period) is needed. In this connection, fatal rhubarb poisonings, which were thought to be due to the oxalate content, are probably caused by other factors even though the leaves may contain up to 1.1% oxalates (von Streicher, 1964). Also, the characteristic symptoms of oxalate poisoning, such as corrosive gastroenteritis, were absent in these fatal cases (Kalliala and Kauste, 1964; Tallquist and Vaananen, 1960).

Table 10.23

Foods Having an Oxalate/Calcium Ratio (MEQ/MEQ) > 1

Food

mg Oxalate/100 g

Average

Oxalate/CA2+

275–1336 270–730 300–700 320–1260 300–920 121–450 910–1679 500–900 50–150 20–141 300–2000 890 1100 300–1500 1586 1087

805 500 500 970 610 275 1294 700 100 80 1150

8.5 5.6 5.0 4.3 2.5 5.1 4.6 2.6 3.9 1.6 1.1 3.9 4.9 4.0 1.2 1.4

Rhubarb (Rheum rhaponticum) Common sorrel (Rumex acetosa) Garden sorrel (Rumex patientia) Spinach (Spinacea oleracea) Beet, leaves (Beta vulgaris, var. cicla) Beet, roots Purslane (Portulaca oleracea) Cocoa (Theobroma cocoa) Coffee (Coffea arabica) Potato (Solanum turberosum) Tea (Thea sinensis) New Zealand spinach (Tetragonia expansia) Pig spinach (Chenopodium spp.) Orache (Atriplex hortensis) Amaranth (Amarantus polygonoicles) Amaranth (Amarantus tricolor)

900

Source: Compiled from Gontzea and Sutzescu (1968) and Concon (1988). MEQ, milliequivalents.

It has been postulated that the anthraquinone glycosides may be responsible for fatal rhubarb poisonings (Von Streicher, 1964). Fresh rhubarb leaves may contain 0.5% to 1% anthraquinones; 10 to 20 g of fresh leaves has caused immediate poisoning in human volunteers (Schmid, 1951). Harmful oxalates in food may be removed by soaking in water. Consumption of calcium-rich foods, such as dairy products and seafood, as well as augmented cholecalciferol intake, are recommended when large amounts of highoxalate food are eaten (Schmid, 1951; von Streicher, 1964).

probably gave animals, including humans, the capability to detoxify these compounds very easily (Singleton and Kratzer, 1973). The first group includes phenolic acids such as caffeic, ferulic, sinapic, and gallic acids; their derivatives; flavonoids; lignin; hydrolizable and condensed tannins; and ellagic acids and their derivatives. The antinutritional effects of tannins were described earlier. Examples of the second group include gossypol, phlorizin, coumarins, myristicin, urushiols, and phenolic amines or catecholamines. Many of these compounds have specific pharmacological effects.

10.17 TOXIC PLANT PHENOLS AND ALCOHOLS

10.17.1 Gossypol

On the basis of frequency of occurrence, similarity in structure, and relative toxicity, phenolic compounds may be divided into two major groups: those having widespread and common occurrence in plant-derived foods and beverages, of which approximately 25 compounds have been identified, and more heterogenous groups of compounds, including a few dozen phenolic derivatives that are highly toxic or have potent pharmacological activities (Singleton and Kratzer, 1969). In the first group, although some compounds occur in relatively high concentrations in some foods, the majority are present in trace quantities. Because of the widespread consumption of these foods, it is presumed that these substances normally are devoid of acute toxicity at the levels usually found in food. Evolutionary adaptation

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The polyphenolic gossypol pigments are primarily confined to the genus Gossypium and a few other members of the order Malvales. The toxicity of cottonseed meal is associated primarily with its gossypol content. Although cottonseed is a by-product of the cotton fiber, the processing of cottonseed is a major industry in the cotton-producing areas of the world. Of the various products (oil, meal, linters, and hulls) obtained from cottonseed, oil is the most valuable. Earlier, cottonseed meal was used principally as a protein supplement for ruminant livestock. The naturally occurring gossypol pigments of the cottonseed are recognized toxicants to monogastric animals, and this property has limited the use of cottonseed meals in feeds for swine and poultry. However, it also represents a potentially excellent source of low-cost, high-quality protein products for food. The refined cottonseed flour may contain up to

60% protein and can become a valuable and abundant source of supplementary protein in human nutrition. Thus, the presence of gossypol in the meal is of toxicological importance. Concerted research efforts have led to glandless, gossypol-free cottonseed varieties. Gossypol was first isolated in a crude form in 1861, but it was not until 1915 that its toxic properties were generally recognized (Abou-Donia, 1989). In addition to gossypol, cottonseed contains several cyclopropenoid fatty acids, sterculate, and malvalate. These are, however, a component of cottonseed oil rather than the protein fraction of the seed. There are at least 15 gossypol pigments or derivatives in extracts of cottonseed oils and meals, but only about 8 have been isolated in more or less purified form and characterized (Concon, 1988; Deshpande and Sathe, 1991). They include gossypol (yellow), diaminogossypol (yellow), 6-methoxygossypol (yellow), 6,6′-dimethoxygossypol (yellow), gossypurpurin (purple), gossyfulvin (orange), gossycaerulin (blue), and gossyverdurin (green). Gossypol occurs in a greater amount in raw cottonseed than in cottonseed that has been subjected to moist heat treatment during processing. In contrast, gossypurpurin and gossyfulvin occur in the cooked seed. Gossycaerulin occurs almost exclusively in cooked cottonseed. Gossypol is converted to gossypurpurin during maturation and prolonged storage of the seed (Berardi and Goldblatt, 1980). The structure for gossypol suggested by Adams and colleagues (1938) is now universally accepted. It is a binaphthyl compound containing a number of hydroxyl and formyl groups. These groups interact to form aldehyde (I), hemiacetal (II), and enolic quinoid (III) types of compounds to yield three tautomeric forms of gossypol (Figure 10.11). Practially, all gossypol in cottonseed plant occurs in pigmented glands whose liquid portion contains 20% to 46% gossypol. It is primarily synthesized in the roots. In glandless varieties, the transport of gossypol from the roots to the seeds is prevented; thus, they contain little or no gossypol. Gossypol has a wide range of toxicological, pharmacological, physiological, and biochemical effects. It is more toxic to nonruminants than to ruminants, thus limiting the use of cottonseed protein to humans and animals. Swine, guinea pigs, and rabbits are the most sensitive to gossypol; rats are the least. The toxic effects of gossypol in monogastric animals have been classified in three levels. Acute doses cause circulation failure; subacute doses, pulmonary edema. Chronic toxicity results in symptoms of ill health and malnutrition. Most of these toxic effects occur by nonspecific interaction with some dietary and body components, such as iron and protein, and/or specific inhibition of certain enzymes (Abou-Donia, 1989). Gossypol

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has also caused reversible male antifertility in rats, hamsters, monkeys, and humans. From a nutritional viewpoint, the reaction of carbonyl groups of gossypol with the amino groups of the amino acids and proteins to form Schiff’s base–type derivative is significant. It renders the amino acids, particularly lysine via its ε-amino group, unavailable, thus lowering the nutritional value of the diet. The phenolic groups of gossypol may also combine with protein reversibly by hydrogen bonding and irreversibly by oxidation to quinines, which react with proteins. Several approaches have been used to reduce the gossypol content of cottonseed meals. One of the oldest techniques still used is controlled heating with moisture to react the gossypol with other seed components, especially lysine. The bound gossypol is less toxic. The toxicity of gossypol can also be reduced to varying degrees by adding metal ions, mainly iron, or chemically combining it with aniline, ammonia, and boric acid. The addition of large amounts of iron salts to cottonseed meal decreases the absorption of gossypol from the intestinal tract. The pigmented glands can be separated from the meal by a flotation procedure by using organic solvents. Gossypol exhibits a dose response, thus allowing the safe use of cottonseed meal in animal feedstuffs. FDA regulations specify that cottonseed products intended for human use in the United States contain no more than 0.045% free gossypol, and the Protein Advisory Group of the United Nations has set limits of 0.06% of free gossypol and 1.2% of total gossypol for human consumption in their program (Deshpande and Sathe, 1991). The widespread use of glandless cottonseed varieties, however, may render gossypol toxicity a subject of historical interest only. 10.17.2 Alkylated Catechols and Related Phenols Tropical plants of the family Anacardiaceae, which includes mango (Mangifera indica), cashew (Anacardium occidentale), and pistachio (Pistacia vera), contain a mixture of alkylated catechols that differ in the degree of unsaturation of the hydrocarbon substituent (Concon, 1988). Mango dermatitis is very similar to poison ivy. Although the poisonous compounds in mango have not been formally identified, they are presumed to be catechol derivatives. The active principle is located in the bark, stem, leaves, and peel of the fruit. The pulp itself is safe. Mango dermatitis is similar to other forms of what Merrill (1944) has called anacardiaceous dermatitis. For those who peel the fruit before eating it, mango dermatitis is manifested by acute erythematovesicular eruptions with some swelling in the lips, cheeks, chin, and sometimes

CHO

OH

OH

CHO

HO

A

OH

HO

OH

CH3 H3C H3C

CH3

HOHC

H3C

O

O

CH3

CHOH

HO

B

OH

HO

OH

CH3 H3C H3C

CH3

HOHC

H3C

OH

OH

CH3

CHOH

O

C

O

CH3 H3C

HO H3C

Figure 10.11

CH3

OH H3C

CH3

Three tautomeric forms of gossypol found in cottonseed meals: A, aldehyde; B, hemiacetal; C, quinoid.

hands. Sometimes stomatitis and acute gastrointestinal disturbance may also occur. The reaction generally appears within 6 to 24 hours after exposure, beginning with smarting, erythema followed by an intense burning sensation, and itching accompanied by vesiculation and sometimes edema. Adverse reactions to the toxic principle of mangoes appear to be dependent on individual susceptibility. 10.17.3 Cycasin and Related Azoxyglycosides The species of the plant family Cycadaceae thrive in the tropical and subtropical regions of the Pacific and Carib-

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bean islands, Mexico, Florida, Japan, and Southeast Asia. They are extremely hardy and are able to withstand adverse climatic conditions. When most other food crops are destroyed, for example, during the typhoon season, they become the principal source of subsistence. Although there are nine genera in Cycadaceae, at least four species from three genera, viz., Cycas circinalis, C. revoluta, Encepharlartos barkeri, and Macrozamia spiralis, have been found to contain toxic compounds. The toxic principles from cycads have been identified, isolated, and crystallized. These differed only in the sugar moieties, having the same aglycone. Cycasin has been isolated from C. circinalis and C. revoluta. The latter

species also yields several other azoxyglycosides, called neocycasins. These have the identical aglycone to cyacasin (Nishida, 1959; Nagahama, 1964). Macrozamin has been crystallized from M. spiralis. Cycasin and macrozamin differ only in that the former contains glucose and the latter primeverose (6-[β-D-xylosido]-D-glucose) (Figure 10.12). The azoxyglycosides of cycads are harmless when administered parenterally, and only when they are taken orally are their toxic effects evident (Nishida et al., 1956). Both malignant and benign tumors of the liver, duodenum, colon, rectum, and kidneys are induced. The toxicity of the glycosides is dependent on their hydrolysis in the GI tract to the aglycone form. The carcinogenicity of cycasin or cycad meal has been demonstrated in mice, hamsters, and guinea pigs. They are also probable human carcinogens (Concon, 1988). The toxic compounds in cycads are highly watersoluble. The powdered or grated endosperm can be rendered nontoxic by washing and thorough soaking (Whiting, 1963).

Cycasin CH3N

N

CH2

CH2OH

O

O OH

O

OH OH

Macrozamin CH3N

N

CH2

CH2

O

O

O

OH

O

OH

OH O

OH

OH OH

Figure 10.12 Toxic azoxyglycosides found in cycads. Cycasin contains glucose; macrozamin has primeverose (6-[β-Dxylosido]-D-glucose).

10.17.4 Safrole Safrole (4-allyl-1,2-methylenedioxybenzene) is widely distributed in the plant kingdom (over 50 species and varieties representing some 10 plant families). This phenolic compound commonly occurs in many spices and such essential oils as star anise oil, camphor oil, and mace, ginger, and cinnamon leaf oil. Nutmeg is one of the best sources of safrole (Belitz and Grosch, 1987). Safrole was widely used as a flavoring agent in soft drinks and other food products. However, its use for these purposes was discontinued after its hepatocarcinogenic activity in rodents was discovered. The chemical structures of safrole and its three structurally related analogs, isosafrole, dihydrosafrole, and estragole, are shown in Figure 10.13. The LD50 values in rats for safrole, isosafrole, and dihydrosafrole are, respectively, 1950, 1340, and 2260 mg/kg body weight. The carcinogenic properties of safrole were established by the induction of liver tumors in rats fed high levels of the compound for 2 years. Malignant liver tumors develop at high safrole concentration (0.5% level in the diet); at lower levels (0.1%) benign tumors are developed. The three safrole derivatives, however, are not of equal potency (Kinghorn, 1983). In rats, safrole and isosafrole appear to be hepatocarcinogens; dihydrosafrole is carcinogenic for the esophagus. However, the high levels at which these compounds are carcinogenic suggest that they pose very little, if any, health hazard under normal dietary conditions in human nutrition.

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O

O O

CH2

CH

O

CH2

Safrole

CH

CH

CH3

Isosafrole

O

OCH3 O

CH2

CH2

CH3

Dihydrosafrole

CH2

CH

CH2

Estragole

Figure 10.13 Chemical structures of safrole and its derivatives.

CH2

O

O

H3CO

CH2

O

O

H3CO

OCH3 CH2CH

CH2

Myristicin

CH2CH

CH2

O

Apiol

O

H3CO

CH2

H3CO CH2CH

CH2

Dill apiol O

CH2

O

H2C

O

O

O

O CH2 O

Sesamol

O

OH

Sesamin

O H2C O

O

O CH2

O

Sesamolin

O

O

CH2

O

CH2

O

O

O

Piperine H C H

C

C

H

O

C

C

Chavicine

N

H

C

C

C

C

C

H

H

H

H

O

N

CH2

O

O

Piperettine C H

Figure 10.14

C

H

H

H C H

C

C

C

H

Carcinogenic methylenedioxybenzenes from various foods.

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C O

N

Several other methylenedioxybenzenes are found in many food sources. These include myristicin and related compounds found in nutmeg and other spices; apiol, found in oleoresin of parsley (Petroselinum crispum and P. sativum) and celery (Apium graveoleus); sesamol, sesamolin, and sesamin in sesame (Sesamum indicum) seed oil; and alkaloids of black pepper (Piper nigrum), piperine, chavicine, and piperettine (Figure 10.14). These all have structures very similar to that of safrole. However, Miller (1983) has shown that in male B6C3F1 mice, myristicin, dill apiol, and parsley apiol did not produce a significant incidence of tumors after 13 months. It is quite likely that their activity may be weakened by the methoxy substituents meta and/or ortho to the allyl group. Tests using higher doses on the same or different stains of animal may reveal their carcinogenic potential. The potential carcinogenicity of black pepper has been reported by Concon and associates (1979, 1981). In addition, the tumorigenicity of sesamol has been evaluated by Ambrose and colleagues (1958), who reported that an increased incidence of benign proliferative lesions is observed in rats fed for several months a diet containing sesamol.

Table 10.24

10.18 VASOACTIVE (PRESSOR) AMINES Some foods contain substantial enough quantities of toxic substances to induce noticeable changes in the cardiovascular system (vasoactive) or in mental function (psychoactive). The number of substances in food that produce alterations in cardiovascular function is, in reality, quite small. Some of the known vasoactive or pressor amines found in plants are listed in Table 10.24. The amounts of amines listed may appear minimal. However, these compounds are highly active, so that if the amounts present in the quantity of foods normally eaten at one time were injected intravenously, disastrous consequences might be expected. The most prominent amines are tyramine, dopamine, and norepinephrine (Figure 10.15). Serotonin and histamine are also present in significant quantities in some foods. Most of the amines listed in Table 10.24 do not pose a significant health hazard since they are rapidly metabolized (oxidative deamination) by the enzyme monoamine oxidase (MAO) in the body. However, it was realized in the early 1960s that the pressor agent tyramine could produce very serious effects in individuals medicated with

Vasoactive Pressor Amines in Some Selected Foods Amine, µg/g or µg/mla

Plant substance

Serotonin

Tryptamine

Tyramine

Dopamine

Norepinephrine

Banana peel Banana pulp Plantain pulp Tomato Red plum Red blue plum Blue plum Avocado Potato Spinach Grape Orange Eggplant Pineapple juice Pineapple, ripe Pineapple, green Passion fruit Pawpaw

50–100 28 45 12 10 8 0 10 0 0 0 0 0 25–35 20 50–60 1–4 1–2

0 0 — 4 0–2 2 5 0 0 0 0 0.1 0 — — — — —

65 7 — 4 6 — — 23 1 1 0 10 — — — — — —

700 8 — 0 0 — — 4–5 0 0 0 0 — — — — — —

122 2 — 0 + — — 0 0.1–0.2 0 0 + — — — — — —

a

—, food was not tested for this amine; 0, level of the amine was below the detection threshold; +, material contained a trace of the amine. Source: Compiled from Bruce (1961), Foy and Parratt (1960, 1961), Hodge et al. (1964), and Udenfriend et al. (1959).

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NH2

NH2 HO

Phenyl ethylamine

Tyramine OH HO

HO

NH2

NH2

HO

HO

Dopamine

Norepinephrine OH

OH HO NH2

NH HO

HO

Octopamine

H3C

Epinephrine

OH

NH HO

CH2CH2N(CH3)2

H 3C

Synephrine Figure 10.15

HO

Hordenine

Chemical structures of some important vasoactive pressor amines commonly found in foodstuffs.

drugs that inhibit MAO (Kuhn and Lovenberg, 1982). MAO inhibitors (MAOIs) are often prescribed for people with depressive illnesses. Ingestion of foods containing large amounts of tyramine by individuals medicated with an MAOI can lead to sporadic bouts of hypertension, intense headache, and, in severe cases, intracerebral hemorrhage and death (Blackwell et al., 1967). Because of MAO inhibition, the ingested tyramine is not readily metabolized; nor is norepinephrine, which tyramine can release. Fortunately, the prevalence of hypertensive crises for individuals medicated with MAOIs after eating foods containing pressor amines is quite low, reportedly 8.4% (Bethune et al., 1964).

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Another vasoactive amine causing severe headaches is phenyl ethylamine, which is present in chocolates, cheeses, and red wines (Sandler et al., 1974). As little as 3 mg of phenyl ethylamine has been shown to provoke migraine headache in 50% of subjects compared to 15% after placebo challenge. The mechanism by which these amines cause headache is unknown. Both amines are present in human brain and may function as modulators of catecholamine formation (Concon, 1988). Tyramine causes a release of norepinephrine from its binding sites, and its hypertensive effects may be related to this process. Phenyl ethylamine causes dramatic changes in blood flow through the brain.

Other amines not listed in Table 10.24 include synephrine and octopamine, both found in lemons and bananas, and hordenine, found in barley, particularly on germination. All these compounds are hypertensive agents and psychostimulants and in large doses cause convulsions and respiratory failure (Concon et al., 1979). Vasoactive amines can also be formed by metabolic transformation of precursors that are endogenously present in plant foodstuffs. Thus, dihydroxyphenylalanine (DOPA) is present in broad beans (Vicia faba) (Hodge et al., 1964). This compound has been implicated in hypertensive episodes that followed the consumption of broad beans by patients on MAOIs. DOPA appears to have been found only in the pods and seeds of certain legumes. It is decarboxylated to dopamine, which is responsible for the pressor activity.

10.19 PSYCHOACTIVE SUBSTANCES The psychoactive agents in foods are generally divided into nonnitrogenous and nitrogenous compounds. The first category includes phenylpropenes, such as myristicin from Myristica fragrans, a tree that yields the spices nutmeg and mace. After the ingestion of 5–15 g (several teaspoons) of powdered nutmeg, many people report acute confusional states with visual hallucinations and distortions of time and space. The psychoactive episode produced by nutmeg is frequently followed by abdominal pain and, in some cases, depression and stupor. Poisoning and death due to fatty degeneration of liver have also been documented after nutmeg ingestion (Kreig, 1964). Other substances that probably also contribute to the psychoactive effects of nutmeg include eugenol, geraniol, safrole, borneol, and elemicine (Kuhn and Lovenberg, 1982). The nitrogenous group of psychoactive substances includes phenyl ethylamines, tropanes, tryptamines, and xanthines. The chemical structures of some of the important toxicants in this class are shown in Figure 10.16. Mescaline (3,4,5-trimethoxyphenethylamine) is found in peyote cactus or peyotl, the dried crown of which is often eaten in parts of Mexico. Dioscorine is a tropane alkaloid found in several species of yam. It is a central nervous system depressant and convulsant (Concon, 1988). In humans, dioscorine produces a burning sensation in the mouth and throat, abdominal pain, vomiting, diarrhea, and speech disturbances. These symptoms are followed by vertigo, salivation, lachrymation, sensation of heat, exosphthalmos, deafness, and delirium. In severe cases, death may occur (Corkill, 1948). Yams of Dioscorea du-

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metorum and D. hispida have been implicated in many cases of poisoning. Another psychoactive compound found in carrots and celery is carotatoxin. This acetylenic alcohol (Figure 10.16) resembles the powerful central nervous system (CNS) stimulant cicutoxin, a very poisonous substance from the water hemlock (Cicuta masculata) (Anet et al., 1953). Carotatoxin is highly neurotoxic to mice. The hallucinogenic tryptamines are pharmacologically a potent class of compounds (Figure 10.17) and are found in a variety of mushrooms, the most famous of which is perhaps Psilocybe mexicana. Several case reports from Great Britain in the clinical literature have described anxiety and panic attacks as well as schizophrenia-like symptoms after ingestion of psilocybin mushroom (Hyde et al., 1978; Benjamin, 1979). The hallucinogenicity of these mushrooms has been attributed primarily to psilocybin (N,N-dimethyl-4-hydroxytryptamine-O-phosphate) and psilocin (N,N-dimethyl-4-hydroxytryptamine). Another mushroom, fly agaric (Amanita muscaria), contains bufotenine (N,N-dimethyl-5-hydroxytryptamine), which is also a potent hallucinogenic agent.

CH2CH2NH2 OCH3

Mescaline H3CO OCH3

O O

N CH3

(C9H17)C

Dioscorine CH3

C C C CH2

CH CH

CH2

OH

Carotatoxin Figure 10.16

Some psychoactive compounds found in foods.

HO NH2

NH2

N H

N H

Tryptamine

Serotonin HO NH

N H

N N H

H 3C

N-Methyl tryptamine

H3C

Bufotenin

OH

O

PO3H2

N N H

H3C

CH3

N CH3

H3C

N H

Psilocin

CH3

Psilocybin H3CO N

N N H

H3C

CH3

Dimethyltryptamine Figure 10.17

N H

H3C

CH3

5-MethoxyN,N-dimethyltryptamine

Chemical structures of tryptamine and its derivatives.

10.20 METHYLXANTHINES The most common methylxanthines found in our food supply are caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine). Theophylline (1,3dimethylxanthine) is only a minor dietary constituent, although it is ingested commonly as a therapeutic agent. These compounds are all methylated derivatives of xanthine, a dioxypurine, and differ only in the number and placement of their methyl groups (Figure 10.18). Caffeine is the most prevalent methylxanthine in our diet. It is found in as many as 60 plant species throughout

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the world. Historically, caffeine from natural sources has been consumed and enjoyed from very early times; tea is the oldest recorded caffeine-containing beverage. Perhaps the universal consumption of beverages that contain these substances indicates that they are of no significance. Nonetheless, these are toxicants and their effects are not generally appreciated. Caffeine content of various beverages is summarized in Table 10.25. On the basis of the beverage consumption data from the Market Research Corporation of America (MRCA), the mean caffeine intake from coffee for adult users ranged from 2.74 to 3.98 mg/kg body weight per day (Barone and Roberts, 1996).

O H3C

Table 10.25

CH3 N

N

Caffeine O

N

O

O

H N

N

Theophylline N

Beverage or food Prepared coffee (drip, vacuum, percolated) Instant coffee Decaffeinated coffee, instant Decaffeinated coffee, ground Tea Cola drinks Cocoa

N

CH3

H3C

Caffeine Content of Various Beverages Caffeine content, mg/cup 90–500 60–100 1–4 2.6 60–75 40–60 0

Source: Compiled from Concon (1988) and Emerson and Chappel (1999).

N

CH3

O H

CH3 N

N

Theobromine O

N

N

CH3

Figure 10.18 Chemical structures of methylxanthines commonly found in foods.

For tea, the comparable caffeine intake range was 0.9–1.4 mg/kg body weight per day and for colas, 0.23–0.46 mg/kg/day. The methylxanthine content of beverages varies considerably. Caffeine is the only methylxanthine in coffee; tea contains caffeine, theobromine, and theophylline in decreasing order of concentration, respectively; in cocoa, the predominant methylxanthine is theobromine, with a smaller amount of caffeine and a trace of theophylline. Caffeine is the only methylxanthine added to the soft drinks at concentrations of 15–29 mg/6-oz serving (NSDA, 1982; Emerson and Chappel, 1999). Methylxanthines are vasoactive compounds. Jick and associates (1973) suggested that long-term coffee drinking might be associated with an increased risk of myocardial infraction. Caffeine also has other effects on the cardiovascular system, including decreases in heart rate (Coltan et al., 1968) and increases in blood pressure and cardiac output, probably because of its ability to increase (by inducing the release) the urinary concentration of the pressor amines epinephrine and norepinephrine (Atuk et

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al., 1967; Bellet et al., 1969). In a double-blind random cross-over design, Robertson and colleagues (1978) demonstrated that the administration of 250 mg caffeine to noncoffee drinkers increased plasma rennin activity by 57%, and plasma norepinephrine and epinephrine activity were increased by 75% and 207%, respectively. Blood pressure also rose 14/10 mm Hg 1 hour after caffeine ingestion. The acute toxicity of methylxanthines has been thoroughly characterized in several animal species (Table 10.26). The most common identified cause of death is respiratory failure after convulsion. Dogs are more sensitive to the toxic effects of theobromine than other animal species, presumably because of their slower elimination rate. In humans, methylxanthine toxicity manifests itself as a disturbed state of consciousness, followed by convulsions, vomiting, and coma. Death has been reported to follow ingestion of massive doses. The possible mutagenicity and carcinogenicity of coffee, tea, or the related methylxanthines have been extensively reviewed (IARC, 1991; Haynes and Collins, 1984; Aeschbacher, 1991; Mohr et al., 1993). Carcinogenicity studies of coffee, tea, cocoa powder, and caffeine in a variety of animal models under various experimental conditions and dose levels have been reported and are summarized in Tables 10.27 and 10.28. These studies provide convincing evidence that these compounds are not carcinogenic in laboratory animals. Evidence continues to accumulate that behavioral differences exist between caffeine consumers and abstainers. These differences may be attributed to effects of methylxanthines at the adenosine receptor site, since ingestion of caffeine from dietary sources could potentially produce blood caffeine levels high enough to compete with endogenous adenosine at its receptor site.

Table 10.26

Acute Toxicity of Methylxanthines and Their Derivatives LD50, mg/kg

Compound Methylxanthines Caffeine

Theobromine Theophyllinec Methylxanthine dervatives Caffeine Caffeine and sodium benzoate Theobromine Theobromine and sodium acetate 1-Allyl theobromine 1-Butyl theobromine 1-Isoamyl theobromine Theophylline Theophylline ethylenediamine (aminophylline) 7-Allyl theophylline 7-Butyl theophylline 7-Isoamyl theophylline

Species

Oral

i.p.

Clinical signs

Human Rat Mouse Hamster Guinea pig Rabbit Cat Dog Rat Mouse Rat Mouse

150–200a 200 127 230 230 246 125b 145b 950 1350b — 332

— 200 220b — 235 — 190b — — 789 206 217

Convulsions, emesis, coma Convulsions, respiratory failure Convulsions Convulsions Convulsions, stupor Convulsions Convulsions Convulsions — — Delayed convulsions, titanic spasm —

Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse

127 878 950 1356 191 667 772 332 391

220b 525 — 789 102 230 222 217 257

Convulsions

Convulsions Emesis Convulsions Profuse salivation, emesis, convulsions

Mouse Mouse Mouse

315 617 723

299 272 211

Convulsions Convulsions, emesis

Emesis

a

Fatal dose. Median lethal dose. c Theobromine sodium acetate was used because of poor solubility of theobromine. Source: Compiled from Tarka (1982) and Tarka and Shiveley (1987). b

There is also a long history of concern about the safety of beverages containing caffeine. In 1958, caffeine was placed on the U.S. Food and Drug Administration (US FDA) Generally Recognized as Safe (GRAS) list. However, as part of its 1978 review of all GRAS substances, this agency recommended that additional research be done to resolve any uncertainties about the safety of caffeine (FASEB, 1978). In 1980, the FDA proposed that caffeine no longer be considered a GRAS substance, but that it be placed in an interim food additive status pending the completion of additional studies. The areas that raised most concern were the potential reproductive, teratogenic, mutagenic, carcino-

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genic, and behavioral effects of the methylxanthine. Several of these aspects of methylxanthine toxicity have been reviewed (Tarka and Shively, 1987; Concon, 1988). At present, the overwhelming evidence available provides ample assurance that sane dietary intakes of methylxanthines have no substantial deleterious effects on health. Reports and allegations of serious bad effects have consistently not been sustained by careful research studies. The task of assessing whether there are slight and inconstant deleterious effects of dietary caffeine on health or performance is difficult because assessment of all slight effects in people is often difficult.

Table 10.27

Carcinogenesis Assays of Coffee, Tea, and Cocoa

Test material Assam leaf tea (tannin fraction) Assam leaf tea (aqueous extract) Instant coffee

Dose

Route

Duration

Animals, no.

8 mg/Wk

Subcutaneous

45–77 Wk

15 M, 15 F

12 mg/Wk

Subcutaneous

69–70 Wk

15 M, 15F

0%, 1%, 2.5%, 5% W/W 0%, 25%, 50%, 100%

Diet

150 M, 150 F

0 and 120 mg/kg/Day 0%–5%

Drinking water Diet

During gestation, lactation,a 720 days post weaning For 5 Wk before mating; during gestation, during lactation, and for 2 yr after weaningc 103 Wk

Instant coffee, regular coffee, decaffeinated coffee, decaffeinated coffee plus caffeine

0%, 6%

Cocoa powder

0%, 1.5%, 3.5%, 5%

Brewed coffee

Brewed coffee Instant coffee solids

a

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NIH black rat, 1–2 months NIH black rat, 1–2 months Weanling Swiss mice

Results Local malignant histiocytomas No increase in tumor incidence

55 M 55F

Sprague-Dawley rats, 5–6 weeks

Benign liver adenomasb, growth impaired and survival improved at higher dose levels Tumor incidence significantly increased only at lowest dose level

C 25 M, T 25 M

C57 BL/6J mice

No increase in tumor incidence

2 yr

C 41 M, 41 F T 144 M, 144 F

Sprague-Dawley rats, 21 days

Diet

2 Yr

C 40 M, 40 F T 40 M, 40 F per group

Sprague-Dawley rats, 100 g body weight

Diet

During gestation, lactation, and 104 weeks post weaning

90 M, 90 F (50 M, 50 F additional control group)

Sprague-Dawley rats

Urinary bladders of 94 M and 99 F treated and 29 M and 29 F control examined histologically; no hyperplasia or bladder tumors observed In decaffeinated coffee groups lower body weight and improved survival rate vs. in controls, no increased tumor incidence in any treatment group No evidence of carcinogenicity

Drinking water

Maternal females were fed 1% instant coffee in the diet during gestation and lactation. Two hepatocellular carcinomas were observed in one male mouse fed 1% coffee and in one male mouse fed 2.5% coffee. c Maternal females were fed 50% coffee in the drinking diet. Source: Emerson and Chappel (1999). b

Species and age

Table 10.28

Carcinogenesis Studies of Caffeine

Dose

Route

Duration

Drinking water

No. of animals

Species and age

C 50 M, 50 F T 50 M, 50 F 32 M, 32 F

ICR mice

0%, 0.04%, 0.1%, 0.2% 0 and 100 mg/kg Body weight 0%, 0.1%, 0.2%

Gavage

M 60 weeks F 104 weeks Lifetime

Diet

117 Weeks

C 30 M T 30 M

0%, 0.1%, 0.2%

Drinking water

104 Weeks

C 50 M, 50 F T 50 M, 50 F

Wistar rats

0%, 0.2%

Drinking water

12 Months

C 40 F T 40 F

Wistar rats

0%, 0.02%, 0.43%, 0.093%, 0.2%

Drinking water

104 Weeks

C 50 M, 50 F (two groups) T 50 M, 50 F

Sprague-Dawley rats

0%, 0.25%, 0.5%

Drinking water

43 Weeks

37–43 F

C3H mice

Sprague-Dawley rats Sprague-Dawley rats

Results Tumor incidence not significantly affected by caffeine No effect on tumor incidence of caffeine Tumor incidence not significantly different in treated rats and controls Tumor incidence for any organ site not different in treated animals and controls Number of pituitary adenomas significantly greater in treated rats than in controls No significant increase in tumor-bearing rats or tumors of specific sites in treated rats vs. controls Incidence of mammary tumors, time to tumor appearance not affected by caffeine; number of mammary tumors in affected mice increased significantly at 0.05% dose level

Source: Emerson and Chappel (1999).

10.21 PYRROLIZIDINE ALKALOIDS The pyrrolizidine alkaloids form a group of some 200 different compounds. Plants containing pyrrolidizines have been responsible for numerous outbreaks of poisoning of livestock causing serious economic damage. In recent years, they have been identified as causing human deaths in less developed countries through contamination of cereal crops and harvested seed. They also have been suspected of causing illness after intentional ingestion as vegetables and in the form of herbal remedies. Pyrrolizidines are found mainly in the families of Compositae, Boraginaceae, and Leguminosae, but also in Apocyanacae, Ranunculaceae, and Scrophulariaceae. Some species may essentially contain only a single pyrrolizidine alkaloid but most contain between five and eight. The pyrrolizidine structure is based on two fused five-membered rings that share a bridgehead nitrogen atom, forming a tertiary alkaloid. In nature the rings are most frequently substituted with a hydroxymethylene

Copyright 2002 by Marcel Dekker. All Rights Reserved.

group at position C-1 and a simple hydroxyl group at position C-7, forming a structure known as necine base. The bases most commonly encountered are heliotridine and retronecine, which differ only in their configuration about C-7. The chemical structures of several important representatives are also shown in Figure 10.19. The two most significant sources of exposure of humans to pyrrolizidine alkaloids are the accidental contamination of foodstuffs and the intentional ingestion of plants containing pyrrolizidine alkaloids in the form of culinary vegetables or herbal medicines. Serious and wide-scale incidents of illness have been reported through contamination of cereal grains with the seeds of pyrrolizidine alkaloid-containing plants (Crews, 1998). Similarly, many plants that contain these alkaloids are deliberately consumed as food or herbal remedies in all parts of the world. For example, in Japan, plants of Petasites, Symphytum, and Tussilago spp. are eaten as vegetables, although in other countries the major uses are medicinal. As a result of increasing interest in “alternative” therapies and herbal medicines in Europe and the United States, preparations of

A

B

CH2OH

HO

O

O

O

CH3

O

CH3

O

H 3C

O

H3C

CH2OH

HO

O

O

CH2

CH2

N N O

C

H3C

D

HO

H3C

CH3 HO

O

O

O

O

CH3

CH3

HO

CH3

O O

O

H3C

CH2

HO

CH3

O

OCH3 N

N

E

CH3

O

H

F

O

H3C

H O

OH CH3

O

O

HO

CH3

H3C

O

O

CH3 O

CH2

O

O

N

N

CH3

G

H3C

H HO

CH2OH

N

CH3CO

O

CH2OCO

CH3

C

CH

OH

OH

CH3

N

Figure 10.19 Structures of carcinogenic pyrrolizidine alkaloids: A, retrorsine; B, isatidine; C, monocrotaline; D, lasiocarpine; E, petasitenine; F, senecionine; G, dehydroretronecine; H, acetyllycopsamine.

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pyrrolizidine alkaloid–containing plants have become widely available commercially and have been publicized for their health-giving properties. Comfrey (Symphytum officinale) in particular has long been a popular herb. Consumers of comfrey could be ingesting up to 5 mg of these alkaloids per day (Speijers and Egmond, 1999). The toxicity and metabolism of pyrrolizidine alkaloids have been extensively studied and reviewed (Winter and Segall, 1989; Segall et al., 1991; Cheeke and Huan, 1995; Crews, 1998). Several have been shown to be hepatotoxic and hepatocarcinogenic in animal studies, and they represent a potential cause of cancer in humans (WHO, 1988). Consumption of herbs containing pyrrolizidine alkaloids might be associated with the high incidence of chronic human liver disease, including primary liver cancer, in Asian and African countries, especially as they may act synergistically with the hepatotoxic agents aflatoxin and hepatitis B virus (Arseculeratne et al., 1981). Some toxicity data are available, such as data on the activation of the liver, vascular lesion in the lung accompanied by pulmonary hypertension, chronic liver diseases in animal experiments, and acute venoocclusive effects and liver cirrhosis in humans; neurologic effects have also been reported. Children seem to be particularly vulnerable. However, further studies on the exposure and toxicity of many pyrrolizidine alkaloids are required to make a meaningful risk assessment possible. The risk of large-scale poisoning through cereal contamination, however, remains serious in view of the continuing practice of consuming poor-quality grain in times of drought and famine.

10.22 PHYTOESTROGENS Estrogens are steroidal compounds produced in the mammalian body that serve to maintain female sexual characteristics; of them the main human estrogen is 17β-estradiol (Figure 10.20). Phytoestgrogens are a group of nonsteroidal compounds produced by a range of plants that, although lacking the classic steroid ring structure (Figure 10.20), have properties similar to those of 17β-estradiol. In some plant-derived foods, phytoestrogen concentrations are high, and thus their mode of action and possible implications to human health are of interest. Estrogenic activity has been reported in grasses and feedstuffs as well as in fruits, vegetables, cereals, and oils. Well-known examples include apples, cherries, carrots, garlic, parsley, potatoes, barley, corn, oats, rice, wheat, and oils from soy, coconut, peanuts, and olives. Phytoestrogen levels of selected foods are summarized in Table 10.29. The compounds that are most likely to be responsible for the estrogenic activity of plants and plant products

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Steroid skeleton

OH

17β-Estradiol

HO

Figure 10.20 Classic steroid ring structure of estrogen and the main human estrogen, 17β-estradiol.

Table 10.29 Foods

Phytoestrogen Levels (µg/g Sample) in Selected

Food Soybean seeds, dry Soy flour Black soybean seeds, dry Tofu Soybean seeds, fresh, raw Green split peas, dry Clover sprouts Kala chana seeds, dry Soybean hulls Cowpea seeds, dry Small white bean seeds, dry Garbanzo seeds, dry Pink bean seeds, dry Small lima bean seeds, dry Yellow split peas, dry Mung bean seeds, dry Great Northern bean seeds, dry Pinto bean seeds, dry Red bean seeds, dry Green beans, fresh, raw Round split peas, dry Alfalfa sprouts Large lima bean seeds, dry a

Isoflavonesa

Coumestrol

1953.0 1777.3 1310.7 278.8 181.7 72.6 30.7 19.0 18.4 17.3 15.6 15.2 10.5 9.2 8.6 6.1 6.0 5.6 3.1 1.5 ND Trace Trace

NDb ND ND ND ND ND 280.6 61.3 ND ND ND ND ND ND ND ND ND 36.1 Trace ND 81.1 46.8 14.8

Isoflavones: daidzein + genistein + formononetin + biochanin A. ND, not detected. Source: Compiled from Concon (1988) and Rickard and Thompson (1997b).

b

are the isoflavones, the coumestans, and the resorcyclic acid lactones (Figure 10.21). Genistein, genistin, daidzen, biochanin A, formononetin, and pratensein are the isoflavones most common in food plants. The two most important coumestans are coumestrol and 4′-O-methyl coumestrol; zearalenone and zearalenol are the two resorcyclic acid lactones most likely to occur in plant products. The latter two are produced by various species of Fusarium molds found in food and fodder plants (de Nijs et al., 1996). Thus, zearalenone and its metabolites may also find their way into the human food chain. The biological potencies of various estrogens have been compared in their affinity for estrogen receptors. The comparative binding affinity for receptors in rat uterine cytosol is 17β-estradiol > coumestrol > zearalenone > genistein > daidzein > biochanin A > formononetin (Verdeal et al., 1980; Aldridge and Tahourdin, 1998). Competition for binding sites in steroid-binding globulin is 17βestradiol > genistein > formononetin > coumestrol > zearalenone (Martin et al., 1978). The physiological effects of estrogens include hypertrophy of the vagina, uterus, and mammary glands in female mammals and hypertrophy of the accessory glands and development of female secondary characteristics in male mammals. The degree to which these signs develop

Isoflavones

depends on the amount of estrogen present, the duration of exposure to them, and the species of the animal. All these effects are usually temporary and disappear with a change to a diet free of estrogens, unless the animals have been exposed to a high level of these compounds for a prolonged period. Estrogens are also implicated in the induction of cancer; the carcinogenic risk is related to the extent to which estrogens are present in food as well as their inherent biological potency (Stob, 1983; Quattrucci, 1987; Aldridge and Tahourdin, 1998). However, since most of the naturally occurring estrogenic substances show only weak activity, it is doubtful that the normal consumption of foods known to contain estrogens would provide sufficient amounts of these substances to elicit a physiological response. Beneficial effects have also been ascribed to the consumption of phytoestrogens, largely on the basis of observed differences in chronic disease between Asian and Western populations. Apart from the beneficial effect on plasma cholesterol concentration and possibly coronary heart disease, other benefits appear less well documented. Soy-containing foods also seem to have beneficial physiological effects in postmenopausal women. Further research on the beneficial effects of phytoestrogens in selected population segments is thus definitely warranted.

Coumestans

R4 R3 R1

R2

O

HO

O

O

O

O

OR

R1

R2

R3

R4

Coumestrol

Daidzein

H

OH

OH

H

4-Methoxycoumestrol

Daidzin

H

O-Glu

OH

H

Genistein

OH

OH

OH

H

Genistin

OH

O-Glu

OH

H

Formononetin

H

OH

OCH3

H

Biochanin A

OH

OH

OCH3

H

Pratensein

OH

OH

OCH3

OH

Prunetin

OH

OCH3

OH

H

R=H

Resorcyclic Lactones OH

O

H

CH3

O HO

O Zearalenone

Figure 10.21

Structure of some typical plant estrogens.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

R = CH3

10.23 ALLERGENS Food allergy and food intolerance are terms that are frequently used interchangeably, but they are not the same reaction. Food intolerance refers to any kind of reproducible, unpleasant reaction to a specific food (Angus, 1998). A subgroup of food intolerance is food allergy. A reaction can only be called allergic if it is caused by an immunological response, often involving immunoglobulin E (IgE) antibodies that bind to the food. Allergy is thought to account for less than 20% of all adverse food reactions. Allergens are common constituents of foods and, in the case of soy protein and some tree nuts, are used increasingly in the diet. In contrast to antinutritional and toxic factors associated with foodstuffs, allergens display their effects only in those individuals who have an altered reactivity (allergy) to otherwise innocuous substances. Allergy symptoms usually include flushing of the face, skin disorders, respiratory problems, and gastrointestinal disturbances. The intensity of reaction depends on the degree of hypersensitivity of the individual consuming the allergen rather than the quantity consumed (Perlman 1980; Taylor et al., 1989; Angus, 1998). In true food allergies, the known allergens are usually naturally occurring proteins, glycoproteins, or polypeptides found in the food (Taylor et al., 1987, 1989). The allergenicity can be associated with the type of structure of the proteins and the peptides as primary, secondary, or tertiary. In the case of tertiary structures, allergenicity often disappears on denuatration, whereas it is retained in the case of primary structures. Furthermore, the protein has to be large enough to be recognized by the immune system as a foreign compound. As a general rule, the allergenicity of molecules with a molecular mass lower than 5000 is low, unless they are bound to endogenous proteins. On the other hand, substances with a molecular mass higher than 70,000 are not absorbed and do not have contact with the immune system. Some common allergenic foods are listed in Table 10.30. A few food allergens have been purified and characterized (Table 10.31). Soy and other vegetable and tree nut proteins are among the most common causes of hypersensitivity reactions. Peanut is named as the vegetable protein with the greatest allergenic potential among legumes (Perlman, 1980). Furthermore, sensitization to one legume plant protein may sometimes cause sensitization to another plant of legume origin (Brandon et al., 1986). The increased use of soy protein products in prepared foods and feeds has raised concern about the possible immunological consequences of a high intake of soy protein in humans (Heppell et al., 1987).

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Table 10.30

Common Allergenic Foods

Infants Cow’s milk Eggs Legumes (peanuts and soybeans) Wheat Adults Legumes (peanuts and soybeans) Crustaceans (shrimp, crab, lobster) Molluscs (clams, oysters, scallops) Fish Tree nuts (almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, walnuts) Eggs Wheat Miscellaneous (coconut, sunflower seed, nut oils)

Only a few studies detail the size of the soy allergy problem, its incidence and prevalence, the mechanism of the allergy reaction, and the factors responsible. Immunoassays have been used to identify the soy protein fractions responsible for the allergic reactions in humans. The two storage globulins of soybeans, glycine (11S) and βconglycinin (7S) and their subunits, as well as proteins from the 2S fractions, such as the Kunitz inhibitor, are involved in the allergic reactions in humans (Table 10.31). It is yet unclear whether soy oil can provoke allergic reactions in soy-sensitive individuals. Processing conditions can often influence the concentration of antigens and aller-

Table 10.31

Allergenic Food Proteins

Cow’s milk: casein, β-lactoglobulin, α-lactalbumin, others Egg whites: ovomucoid, ovalbumin, ovotransferrin (conalbumin) Egg yolks: lipoprotein, livetin Peanuts: peanut I, lectin-reactive glycoprotein, arachin, conarachin Soybeans: β-conglycinin (7S fraction), glycinin (11S fraction), 2S fraction, Kunitz trypsin inhibitor, unidentified 20-kD protein Codfish: allergen M (parvalbumin) Shrimp: antigen II Green peas: albumin fraction Rice: glutelin fraction, globulin fraction Cottonseed: glycoprotein fraction Tomato: several glycoproteins Source: Compiled from Taylor et al. (1989) and Deshpande and Sathe (1991).

gens by altering the immunochemical structure of proteins and by influencing the digestibility of antigenic proteins (Pedersen, 1986). The epitopes of soy agents may be affected in a variable manner according to the conditions encountered during food processing. The antigenic activities of both glycinin and β-conglycinin can be eliminated by physicochemical denaturation of these proteins (Sissons et al., 1982). The literature on tree nut allergens has been reviewed by Angus (1998). Allergenic responses are seen in sensitive individuals who consume almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, walnuts, coconut, and sunflower seeds. In many cases, the allergens are found to be IgE-binding proteins. In the case of food allergy, late reactions seldom occur. The clinical symptoms of allergic food reactions are listed in Table 10.32. The oropharynx and gastrointestinal tract are the initial sites of exposure to food antigens. Symptoms such as edema and itching of the mouth often occur. However, these reactions may be transient and are not necessarily followed by other symptoms. In some individuals, certain fruits, nuts, and vegetables cause oral symptoms only; in others a more extensive reaction is seen. The quantity of the offending food also plays a role in the gravity and extent of reaction, although in principle a small amount of a certain food can readily cause a response. Sometimes the allergic reaction develops only if the food intake is followed by exercise. This is referred to as exercise-induced food allergy. Hypotension and shock are life-threatening consequences of a food-allergic reaction. Generally, the reaction is accompanied by other anaphylactic symptoms such as abdominal cramps, nausea, vomiting, diarrhea, dyspnea, urticaria, and angioedema.

Table 10.32

Symptoms of Food Allergy

Skin symptoms Itching, erythema, angioedema, urticaria, increase of eczema Respiratory symptoms Itching of eyes, nose, throat; tearing and redness of the eyes; sneezing; nasal obstruction; swelling of throat, shortness of breath; cough Gastrointestinal symptoms Nausea, vomiting, abdominal cramps, diarrhea Systemic symptoms Hypotension, shock Controversial symptoms Arthritis migraine, glue ear, irritable bowel syndrome

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Similar to food allergy, food intolerance comprises many different clinical disease entities, with different symptoms. Often, the clinical picture is difficult to distinguish from that of an allergic reaction. The distinction intolerance/allergic, therefore, cannot always be made on the basis of the individual’s history alone. The manifestation of food allergy and food intolerance can vary from innocent symptoms, such as rhinorrhea, to life-threatening symptoms, such as shock. The diagnosis is made on the basis of clinical as well as laboratory data, according to the following general procedure: 1.

2.

3.

4. 5. 6. 7. 8.

History of the patient (complaints, possible associations with food intake, family history, atopic manifestations) Overview of food intake, recorded by a dietician (often, people have already excluded food products of their own accord) Physical examination (signs of eczema, asthma, rhinitis, abdominal disorders, and nutritional state) Blood examination (food allergens, inhalant allergens) Skin tests Exclusion of all potentially suspected foods (trial diet) Challenge test, for one or a few food products or additives Gradual reintroduction of food products

Some of the diagnostic tests are rather time-consuming and costly. In addition, they cause some risk or discomfort to the patient. The approach is modified according to the type of reaction involved, age, and other characteristics of the patient. Skin test results and specific IgE determinations may be unreliable. For many foods, the identity of the allergenic moieties is unknown and information about their stability is lacking. Examples include the allergens of some fruits and vegetables. Negative results do not rule out a possible allergy for the tested allergen, and, positive results do not automatically imply that the particular food does indeed cause the symptoms. The gold standard for the diagnosis of food allergy remains reintroduction or challenge after a period of exclusion. If the diagnosis is correct and compliance is maintained, exclusion of the suspicious food(s) shuld result in improvement in the patient’s condition, and challenge or reintroduction should lead to relapse of symptoms. Food intolerance, in contrast, is not IgE-mediated and cannot be detected by skin tests and specific IgE determinations. It can only be demonstrated by exclusion and challenge. The preferred approach to the management of food allergy is

prevention. Once the treatment is started, strict avoidance of all offending foods is needed. In sensitive individuals allergic responses to use of these ingredients in food products have created special problems in the food industry. The industry is becoming increasingly aware of the special attention required when manufacturing nut-containing and other foods in the same factory. A large number of food manufacturers have altered their production processes to reduce the risk of crosscontamination. Warnings on food labels are also being increasingly used to alert customers to the presence or absence of nuts in prepared food products.

10.24 ANTIVITAMINS A number of substances have been reported to act on the availability of vitamins and are commonly referred to as antivitamins. An antivitamin can be defined as any compound that under certain conditions can actually or potentially produce toxic symptoms such as in a deficiency of a corresponding vitamin, whether administered parenterally, ingested, or occurring along with the vitamin in food (Concon, 1988). According to this definition, a substance that degrades a vitamin is also considered an antivitamin even though deficiency symptoms have not been demonstrated because of its presence in the diet. These substances may possess only the potential to produce deficiency symptoms, especially when present in large amounts, and must be examined when vitamin intakes from food sources are considered. The definition also includes nutrients that when taken in excess increase the physiological requirement of other vitamins. The deleterious effects of antivitamins may be due to one or several of the following factors: They may have similar chemical structures, they compete with the vitamin in various metabolic reactions, they may react with the vitamin per se and make it unavailable, or they may give rise to symptoms resembling vitamin deficiencies. Somogyi (1978) has proposed dividing the antivitamins into two major groups: (a) structurally similar compounds that compete with the vitamins as a result of the resemblance of their chemical structure and (b) structuremodifying chemicals that destroy or decrease the effects of a vitamin by modifying the molecule or by forming a complex. Most antivitamins occurring in the natural food chain belong to the latter group. The first antivitamin to be described seems to be thiaminase, which was reported in the mid-1930s (Green, 1937). Antithiamine factors are found in many fish species and in certain species of crab and clam. A thiamine inactivity effect has been described

Copyright 2002 by Marcel Dekker. All Rights Reserved.

in some fruits and vegetables, such as blueberries, black currants, red beets, Brussels sprouts, and red cabbage. The thiaminase enzyme splits thiamine at the methylene linkage (Figure 10.22). The enzyme contains a nonprotein coenzyme, structurally related to hemin, the red pigment component of hemoglobin. The coenzyme is the actual antithiamine factor. Cooking destroys thiaminases in fish and other sources. Antithiamine factors can also be of plant origin. A study of the effects of tannins in tea leaves, tea infusions, and betel nuts on thiamine in volunteers showed that they were responsible for thiamine destruction (Janssen, 1997). The interaction of these substances with thiamine is oxygen-, temperature-, and pH-dependent. Other antithiamine factors include ortho-catechol derivatives. A well-known example is present in bracken. The so-called fern poisoning in cattle is attributed to this factor. Possibly there are two types of heat-stable antithiamine factors in this fern, one of which is identified as caffeic acid (3,4-dihydroxycinnamic acid) (Figure 10.23). Caffeic acid can also be formed on hydrolysis of chlorogenic acid by intestinal microflora. Chlorogenic acid is found in green coffee beans, green apples, and sunflower seeds. Other ortho-catechols, such as methylsinapate (Figure 10.23), which occurs in mustard or rapeseed, also have antithiamine activity. Another example of an antivitamin is ascorbic acid oxidase. This copper-containing enzyme mediates the oxidation of free ascorbic acid (vitamin C) to dehydroascor-

H 3C

Thiamine

CH2

N H3C

CH2CH2OH

NH2 N S N

Thiaminase

NH2 N H3C

H3C

CH2CH2OH

CH2OH N N

S

Figure 10.22 Degradation of thiamine (vitamin B1) by thiaminase.

HO

CH

Methylsinapate

CHCOOH

HO

Caffeic acid

H3CO

CH

CH

COOCH3

HO OCH3

HO

COOH

O HO

CH

CHCO

HO

Figure 10.23

OH OH

Chlorogenic acid

Antiathiamine factors of plant origin.

bic acid and then to diketogulonic acid, oxalic acid, and other oxidation products. The enzyme is present naturally in many fruits and vegetables, including cucumbers, pumpkins, lettuce, cress, peaches, bananas, tomatoes, potatoes, carrots, and green beans. Its activity varies with the type of fruit or vegetable. As an enzyme, ascorbic acid oxidase can be inhibited effectively by blanching of fruits and vegetables. A variety of plants and mushrooms contain pyridoxine (vitamin B6) antagonists; most are hydrazine derivatives. A pyridoxine antagonist, linatine (γ-glutamyl-1amino-D-proline), was identified in linseed meals. This water-soluble, heat-labile compound readily hydrolyzes to the hydrazine derivative 1-aminoproline, the actual antipyridoxine factor (Figure 10.24). Antipyridoxine factors have also been found in wild mushrooms, the common commercial edible mushroom, and the Japanese mushroom shitake. Commercial and shitake mushrooms contain agaritine. It is hydrolyzed in the mushroom by γ-glutamyl-transferase to the active agent 4-hydroxymthylphenylhydrazine (Figure 10.25). The hydrolysis of agaritine is accelerated if the cells of the mushrooms are disrupted. The mechanism underlying the antipyridoxine activity is believed to be condensation of the hydrazines with the carbonyl compounds pyridoxal and pyridoxal phosphate, the active form of the vitamin, which results in the formation of inactive hydrazones.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Other examples of antivitamins include a pantothenic acid inhibitor purified from peas (Kratzer et al., 1954; Smashevskii, 1966). Perhaps the best-known example in this category is avidin, a protein that occurs in raw egg white, which acts as a biotin antagonist.

Linatine HOOC

N

γ-Glutamyl

NH

H2O

1-Amino-D-proline Glutamic acid HOOC

N NH2

Figure 10.24 Hydrolysis of linatine, an antipyridoxine factor found in linseed to the active factor, 1-amino-D-proline.

Agaritine HOH2C

NHNH

CO

CH2

CH2

CH

COOH

NH2

γ-Glutamyl transferase

HOH2C

NHNH3

HOOC

CH2

CH2

CH

COOH

NH2

4-(Hydroxymethyl)phenylhydrazine

Glutamic acid

Figure 10.25 Hydrolysis of agaritine, an antipyridoxine factor found in common edible mushrooms, to the active agent. 4-hydroxymethylphenylhydrazine.

10.25 MISCELLANEOUS ENDOGENOUS TOXICANTS A number of toxicants, which do not fall into any of the categories described, are also found in some specific foods. Some of these are briefly described in the following sections. 10.25.1

D-Mannoheptulose

Avocados are the only known major source of the compound D-mannoheptulose (Figure 10.26). In both human and animal subjects, consumption of avocados has been shown to result in hyperglycemia or a depression in plasma insulin level and the insulogenic index. Coore (1963) has shown that mannoheptulose completely but reversibly suppresses the stimulating effect of D-glucose on insulin secretion. This sugar is potent enough to inhibit the rat pancreas from secreting insulin at doses that do not induce hyperglycemia. 10.25.2 Umbellulone Umbellulone (Figure 10.26) is found in California bay laurel (Umbellularia californica), whose oil contains from 40% to 60% of this compound. It possesses an atropinelike effect on the nerves and muscle fibers of frog heart. Contact with the oil, or even exposure to the vapors, can cause

Copyright 2002 by Marcel Dekker. All Rights Reserved.

skin irritation and headache, and in some cases the effect can be serious enough to cause unconsciousness (Concon, 1988). California bay laurel should not be mistaken for the conventional bay leaf (Laurus nobilis), which is devoid of this toxic effect. 10.25.3 Glycyrrhizin Licorice, widely used in candies and other confections as a flavoring agent, contains a glycoside, which constitutes about 5% to 10% of the root. The glycoside consists of two glucuronic acid moieties and a steroidal aglycone glycyrrhetinic acid. The calcium or potassium salt of this glycoside is called glycyrrhizin (Figure 10.26). Because of its steroidal character, the compound possesses marked physiological activity. Licorice has been reported to have a deoxycorticosterone activity, as shown by the usual water and sodium retention and potassium loss that follow consumption (Conn et al., 1968). Excessive consumption of licorice candy can also cause severe hypertension, hypokalemia, aldosteronopenia, suppressed plasma rennin activity, and cardiac enlargement (Conn et al., 1968; Koster and David, 1968). 10.25.4 Menthol Peppermint oil contains approximately 40% menthol. This terpene is widely used as a flavoring agent in candies,

B

CH2OH

A

O

H

CH2OH H3C

OH

OH

CH3 OH

OH H

H3C

H O O H3C

C

C

(C6H8O6)

O

CH3 H 3C CH3

H O CH3

CH3 H

CH3

OH

CH3 O

C

D

(CH2)7

HO H3C

H

NH

CH3

E

O CH3

Figure 10.26

Miscellaneous toxicants found in plants: A, mannoheptulose; B, umbellulone; C, glycyrrhizin; D, menthol; E, Carpaine.

chewing gum, liqueurs, and other products. Besides sensitization reactions in the form of urticaria, excessive consumption of menthol-containing products may cause heart fibrillation and toxic psychosis. Symptoms in such cases disappear when the incriminating products are avoided. 10.25.5 Carpaine Papayas (Carica papaya) contain an alkaloid, carpaine (Figure 10.26). It has a powerful cardiac and diuretic action (Concon, 1988). Carpaine depresses arterial, followed by ventricular contractions of the heart without interference to the conduction system. The depression of cardiac action causes hypotension. At higher concentrations, carpaine causes vasoconstriction, which is not reversed by epinephrine.

10.26 REMOVAL OF TOXICANTS AND ANTINUTRIENTS Traditionally, several methods have been used to remove the toxicants and antinutrients present in plant foods in or-

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der to improve their nutritional quality and utilization. To accomplish this goal, several approaches may be considered. Breeding plant varieties containing low or no levels of toxicants is one such approach. Such an approach, however, requires long-term efforts, since the type and number of antinutrients and toxicants present in plant foods are rather large and diverse with respect to their chemical and biochemical nature. Such efforts will also need to consider agronomic consequences of genetic manipulation, including crop yield, soil tolerance, light and water requirements, and resistance to pests and diseases. The physical and chemical means of removing undesirable antinutrients include several processing methods, such as soaking, cooking, germination, fermentation, selective extraction, membrane filtration, irradiation, and enzymatic treatments. In many instances, the use of only one method may not effectively remove all the antinutrients present, and thus a combination of two or more methods may be required to accomplish the desired level of removal. The effectiveness of some of these methods in removing the plant antinutrients is briefly described in the following discussion. Soaking usually forms an integral part of such processing methods as cooking, germination, fermentation,

and roasting. Discarding soak water before further processing can remove several water-soluble antinutrients, such as protease inhibitors, phytates, lathyrogens, goitrogens, cyanogenic glycosides, and polyphenols. The extent of their removal depends upon the soaking temperature, the type of soaking medium, the seed type, the length of soaking, and the solubility characteristics of the soluble antinutrients. Soaking media frequently include water, salt (or combination of salts) solutions, and dilute aqueous alkali solutions. Salt and alkali help leach the solubles into the soaking medium by increasing the cell membrane permeability. However, such loss of antinutrients during soak-

Table 10.33

ing is also associated with loss of desirable nutrients, such as proteins, minerals, and vitamins. Cooking, or heat processing, is probably the oldest known method of processing plant foods for human consumption. Cooking may be done at atmospheric pressure and temperature or at high-pressure and high temperature (autoclaving). The primary purpose of cooking is to render the food palatable and to develop its aroma. The cooking water may or may not be discarded, depending on cultural and personal preferences. Cooking generally inactivates heat-sensitive factors, such as enzyme inhibitors, lectins, volatile compounds such as HCN, and some of the off-fla-

A Summary of the Effects of Antinutrients and Toxicants of Plant Origin

Antinutrient/toxicant

Effects

Proteinase inhibitors Amylase inhibitors

Pancreatic hypertrophy, dietary loss of S-amino acids Amylase inhibition; may hinder carbohydrate utilization Growth depression, fatal

Proper thermal processing Proper thermal processing

Cyanogens

Reduced mineral bioavailability, altered protein solubilition, enzyme inhibition Reduction in protein digestibility and utilization, inhibition of several enzymes Cyanide poisoning; action as progoitrogens

Goitrogens

Inhibition of iodine binding to thyroid gland

Lathyrogens Favism

Neurotoxic, nervous paralysis of lower limbs; fatal Hemolytic anemia

Allergens Saponins Estrogens

Several allergic reactions Bitter taste, foaming, hemolysis Growth inhibition, interference with reproduction

Toxic amino acids

Structural analogs of protein amino acids; act as antimetabolites, potent inhibitors of several enzyme systems Impairment of nervous system; cholinesterase inhibitors; ataxia, convulsions, coma, muscle weakness, fatal Loss of lysine, lowered utilization of dietary proteins, malnutrition, nonspecific interactions with iron and certain enzyme systems

Traditional household processes that remove phytate to a variable degree Traditional household processes that remove polyphenols to a variable degree Proper processing, traditional household methods, breeding for low levels Proper food processing, leaching, breeding for low levels Breeding for low levels, leaching, roasting Breeding for low levels, wet-processing methods, treatment with β-glucosidases Avoidance of foods eliciting allergic responses Leaching with hot water or ethanol Breeding for low levels, reversible effects, avoidance of prolonged consumption of foods containing high levels Breeding for low levels

Phytohemagglutinins (lectins) Phytate Polyphenols (tannins)

Potato glycoalkaloids

Gossypol

Safrole Oxalates

Carcinogenic Renal stones, gastroenteritis, shock, convulsions, low plasma calcium levels, renal damage

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Methods of removal

Proper thermal processing

Breeding for low levels

Use of glandless cottonseed varieties, controlled moist heat processing; treatment with organic solvents, aniline and boric acid, chelation using iron Lower dietary intake Leaching, avoidance of prolonged consumption of high-oxalate foods

vor components. The heat-stable factors, such as estrogens, saponins, phytate, polyphenols, and allergens, may not be affected to any significant extent by cooking. Even among the heat-labile antinutrients, complete inactivation may not always be possible. When the soaking and cooking medium (such as water) is not discarded, a significant amount of heat-stable antinutrients and toxicants remain practically unchanged. On the contrary, if the medium is discarded, a significant amount of heat-stable antinutrients can be removed from plant foods. Excessive heat processing, however, should be prevented, since it adversely affects the protein quality of foods. Germination mobilizes reserve nutrients required for the growth of plant seedlings and, therefore, may help in the removal of at least some of the antinutrients, such as phytates and raffinose oligosaccharides (see Chapter 7), which are thought to function as reserve nutrients. Significant reductions in phytate, lectin activity, and raffinose sugars are reported on germination of various legumes (Deshpande et al., 1984a). Beneficial effects of germination in terms of reduction in enzyme inhibitory activities, however, remain controversial. Similarly to those in germination, most of the changes occurring during the fermentation of foods are of a catabolic nature, and they help in the hydrolysis of such components as proteins and carbohydrates. Fermentation of foods can result in significant reduction in the quantity of certain antinutrients. The removal of raffinose oligosaccharides of legume during fermentation, for example, is primarily due to the α-galactosidase activity present in legume seeds as well as in the microorganisms involved in the process. Depending upon the type of legume as well as fermentation, phytic acid is also hydrolyzed during fermentation to a variable degree. In addition to the traditional household processes for preparing plant foods for human consumption, enzymatic methods have been used to remove certain antinutrients of plant origin, including phytates and raffinose sugars. Endogenous enzymes, such as linamarinase, as well as externally added β-glycosidases are often used to remove the cyanogenic glycosides of various legumes. The HCN thus produced is water-soluble and volatile and can be easily removed by heating and/or discarding the soaking water. In addition, processes such as ultrafiltration, irradiation of foods, addition of antibiotics or bacteriostats, extrusion cooking, and protein texturization have proved useful in removing certain toxicants and antinutrients of plant foods. Table 10.33 presents a summary of the commonly occurring antinutrients in plant foods used for human consumption, their antinutritinal effects, and methods of their removal.

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10.27 SUMMARY A number of toxic and/or antinutritional factors occur naturally in foods of plant origin. Even those that naturally occur in the animal products usually are derived directly or indirectly from vegetable sources. Food and fodder sources from the plants of the Leguminosae family seem to have the greatest diversity of the toxicants and antinutritional factors. Most of the antinutritional or toxic factors have a pronounced capacity to induce deleterious effects in test animals, when tested by themselves in certain doses. Fortunately, under normal dietary conditions, the concentration of toxicants in the food is too low to cause any adverse effects. The very fact that humans have been using these plants as staple foods over the centuries also suggests that their physiological processes are well suited to handle small amounts of various toxicants in the normal diet. In addition, none of these food plants forms the sole source of nutrients in diets in most parts of the world. We are not likely to have an “ideal food source” that would take care of all our nutritional requirements. The fact that balanced nutrition results from a wise choice of foods of different food groups also minimizes the contribution of any one antinutritional factor from a particular food source. Nevertheless, there is the ever-present danger of adverse circumstances’ forcing major changes in the dietary pattern for a short period. The occurrence of lathyrism in seasons of droughts and floods in certain tribal parts of India is a classic example in this regard. Humans are currently exploring several unconventional food sources to meet the increasing demands and shortages of food supplies. This also means an increased potential risk for the adverse effects of compounds hitherto unknown in the human food chain. Because of the growing concern about the possible direct relationships between diet and disease, we are thus constantly challenged to respond to the general need for exploring the possible adverse consequences of antinutrients and food toxicants and for identifying the factors that contribute to the formation and inactivation of such compounds in our food supply. The basic mechanisms of toxic action of antinutritional factors and toxicants have permitted rational research approaches to develop new ways to minimize their adverse effects. Such approaches include inactivation of deleterious compounds to prevent them from interacting with living cells, proper processing and handling methods to eliminate them altogether from our diet, breeding of new plant varieties that are both nutritious and safe to consume, and identification of dietary constituents that protect against the adverse action of these compounds. The con-

tinuation of our current research efforts in this field can only ensure the certainty of an adequate, wholesome, and balanced diet for all the world’s people.

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11 Fungal Toxins

11.1 INTRODUCTION The toxicity of certain fungi, such as mushrooms, has been recognized for a long time. However, potential human and animal health hazards of other toxigenic fungi were not recognized until the 1850s, when a distinct disease called ergotism was shown to be associated with the ingestion of rye and other cereals infected with the fungus Claviceps purpurea. This was followed by Russian reports of other mycotoxicoses affecting humans. Thus, human stachybotryotoxicosis was linked with the ingestion of bread infected by Fusarium graminearum, and alimentary toxic aleukia (ATA) was associated with the ingestion of overwintered grains infested with Fusarium poae and Fusarium sporotrichioides (Sarkisov, 1954). Although several sporadic cases of mycotoxicoses in domestic animals were reported before 1960, the incidence of turkey X disease in England was the turning point in mycotoxin research and stimulated enormous interest in the study of mycotoxins in the scientific community (Sargeant et al., 1961). More recently, the scientific community was shocked by the discovery of a new group of mycotoxins, the fumonisins, produced by Fusarium moniliforme. Some considered the fumonisins the aflatoxins of the 1990s, because of the dramatic effects of ingestion by horses and pigs and the possibility that they may be involved in human disease (Sydenham et al., 1991; Dutton, 1996). Mycotoxins are secondary fungal metabolites with diverse structures and toxicological properties that induce a variety of toxic effects in humans and animals when foods contaminated with these compounds are ingested.

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The toxic effects include acute toxicity, carcinogenicity, mutagenicity, teratogenicity, and estrogenic effects on animals at normal levels of exposure. Biological conversion products of mycotoxins are also referred to as mycotoxins. Although mycotoxins are fungal metabolites, all fungal metabolites are not necessarily mycotoxins. For example, fungi also produce antibiotics, such as penicillin; however, they are not considered mycotoxins. The inherent toxic effects in higher organisms are thus important to the determination of whether a chemical is a mycotoxin. Although many hundreds of such toxic mycotoxins have been identified, only about 20 to 30 have been shown to be contaminants of human or animal food (Watson, 1985). The term mycotoxicosis is applied to pathological conditions that result from the ingestion of foods or feeds contaminated with fungal toxins. Forgacs and Carll, who defined it as “poisoning of the host following entrance into the body of toxic substances of fungal origin,” introduced the term in 1962. During the last four decades, research on mycotoxins has revealed potential hazards of mycotoxin contamination of many important agricultural products. The occurrence of mycotoxins in agricultural commodities depends on such factors as region, season, and conditions under which a particular crop is grown, harvested, and stored. Crops grown in warm and moist weather in the tropical and subtropical countries are much more prone to mycotoxin contamination than those in temperate zones. However, certain toxigenic fungi such as Fusarium species can proliferate at low temperature and produce toxins. Over 100 fungal species have been shown to produce mycotoxins associated with naturally occurring diseases in

animals and humans throughout the world (Rechcigl, 1983; Sharma and Salunkhe, 1991). Although toxigenic fungi and their spores are ubiquitous, mycotoxicosis is primarily a problem in areas that have high rainfall and relative humidity and temperatures that favor fungal growth and mycotoxin production. In addition to specific growth conditions, the fungal spoilage of crops and their grains is enhanced by drought, insect damage, cracking or breaking of kernels during harvesting, and presence of excessive chaff in the harvested grain. During the entire postharvest period, food crops are essentially in a state of storage, and fungal growth on them is preventable only by careful regulation of moisture content, temperature, and other environmental conditions. Mature fruits and vegetables are also highly susceptible to invasion of toxigenic fungi, because they are high in moisture and nutrient content. In addition, many fruits are more easily injured as they approach full maturity and therefore are vulnerable to fungal attack. Postharvest

losses of fresh fruits and vegetables due to microbial decay range from 20% to 50% of total production, depending on the geographic region. Thus fungal infection of foods not only results in economic losses but also is a potential health hazard to humans and animals. Some economically important mycotoxins that contaminate human foods worldwide are listed in Table 11.1. Species of toxigenic fungi can be found in all major taxonomic groups of fungi. Most of the mycotoxins known have been recognized as metabolic products of genera such as Aspergillus, Penicillium, and Fusarium. In addition, other genera, such as Claviceps and other Ascomycetes, are known to produce various mycotoxins. Important toxigenic fungi of several taxonomic groups and their mycotoxins are listed in Tables 11.2 to 11.4. Information on the natural occurrence of mycotoxins in foods and feeds is summarized in Table 11.5. In view of the highly toxic nature of many of the mycotoxins, it is fortunate that they occur in food sporadically and often only in small amounts, at least in the

Table 11.1 Commonly Encountered Toxigenic Fungi, Their Toxins, and Important Agricultural Commodities They Damage Toxin

Fungus

Aflatoxins

Aspergillus flavus A. parasiticus

Ochratoxins

A. ochraceus, Penicillium viridicatum Fusarium spp. (e.g., F. roseum) Fusarium moniliforme Fusarium spp. (e.g., F. tricinctum)

Zearalenone Fumonisins Trichothecenes T-2 toxin Vomitoxin Rubratoxins Citrinin

Tremorgens

Patulin Ergotoxins

P. rubrum P. purpurogenum P. viridicatum P. citrinum A. ochraceus P. cyclopium P. palitans Aspergillus spp. Penicillium spp. Aspergillus spp. Claviceps purpurea C. paspali

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Commodities damaged Groundnuts, tree nuts, cereals, cottonseed, soybeans, spices, fruits, feeds such as groundnut meal Legumes, cereals, coffee beans Fungally infected corn, wheat, barley, etc. Corn, other cereals Fungally infected corn, wheat

Fungally infected corn Barley, wheat, rye, oats, rice

Fungally infected feeds, groundnuts, rice

Fungally infected fruits, such as apples, plums, peaches, pears, apricots Fungally infected grains and grasses

Table 11.2 Important Mycotoxins Produced by Aspergillus Species

Table 11.3 Mycotoxins Produced by Penicillium Species

Mycotoxin

Mycotoxin

Producing organism

Citreoviridin Citrinin Penicillic acid Rubratoxins Griseofulvin Luteoskyrin Islanditoxin Mycophenolic acid Decumbin Viridicatin Cyclopiazonic acid Patulin Brevianamide A Penitrems Verruculogen

P. citreoviride P. citrinum P. puberculum P. rubrum P. janczewski P. islandicum P. islandicum P. brevicompactum P. decumbens P. viridicatum P. cyclopium P. patulum P. viridicatum P. cyclopium P. verruculosum

Aflatoxins Sterigmatocystins Ochratoxins Fumigatin Aspergillic acid Kojic acid Terreic acid Helvolic acid Fumagillin Tryptoquivaline Fumitremorgins

Producing organism A. flavus, A. parasiticus A. vesicolor A. ochraceus A. fumigatus A. flavus A. fumigatus A. terreus A. fumigatus A. fumigatus A. clavatus A. fumigatus

developed areas of the world. However, the potential for wide-scale problems exists should the appropriate circumstances arise. Low-level contamination of foods by highly biologically active chemicals presents a major difficulty in assessing their true significance for humans. Legislation is usually set on a national basis and is mainly restricted to the aflatoxins, of which aflatoxin B1 is accepted as a potent liver carcinogen. Some countries also have introduced limits for other selected mycotoxins (FAO, 1997). Various international organizations now pay increased attention to mycotoxins in the human food chain. One development in this regard was the establishment in 1994 of the WHO Collaborating Center for Mycotoxins in Food (WHO-CCMF), located in the Albert Ludwigs University School of Medicine in Freiburg, Germany. A particular task of this center is to elucidate the role of mycotoxin-contaminated food in human health and disease. In 1997, the National Institute of Public Health and the Environment, Bilthoven, The Netherlands, was appointed as a Community Reference Laboratory (CRL) for mycotoxins in animal products. This chapter encompasses both human food and animal feeds, and since humans consume meat, which may be contaminated through animal feeds, the word feed is not used in the text. Toxicological aspects of only the most important and well-studied mycotoxins are described in the following sections.

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Table 11.4 Mycotoxins Produced by Fusarium Species and Miscellaneous Fungi Mycotoxin Fusarium species toxins Deoxynivalenol, nivalenol T-2 toxin Fumonisins Moniliformin Zearalenone Trichothecenes Monoacetoxyscirpenol Miscellaneous fungal toxins Psoralens Ergotoxins Mushroom toxins Alternariol, alternariol monomethyl ether, tenuazonic acid

Producing organism F. graminearum, F. culmorum, F. crookwellense F. poae, F. sporotrichioides F. moniliforme, F. proliferatum Fusarium spp. F. graminearum, F. culmorum, F. crookwellense Fusarium spp. Fusarium roseum Myrothecium spp., Sclerotinia sclerotiorum Claviceps purpurea Amanita spp. Alternaria alternata, A. tenuis

Table 11.5

Natural Occurrence of Mycoxotins in Foods and Feeds

Toxin

Commodity contaminated

Alternariol Emodin Sterigmatocystin Sporidesmins Patulin

Penicillic acid Fumonisins Ochratoxin

Psoralens Citrinin Aflatoxin M1 Aflatoxin B1

T-2 toxin Vomitoxin Diacetoxyscirpenol Zearalenone

Pecans, sorghum Chestnuts Pecans, coffee Grass litter, grass Apples, apple cider, apple juice, pears, grape juice, bananas, pineapples, grapes, peaches, apricots Corn, dried beans Corn, corn-based foods and feeds Corn, barley, wheat, oats, rye, green coffee beans, beans, groundnuts, hay, animal products (pork and poultry), poultry feed, Celery, parsley Groundnuts, wheat, oats, barley, rye Milk, dried milk products, buffalo milk, yogurt, fresh and processed cheese Groundnuts, corn, groundnut oil, groundnut oilcake, peanut butter, cottonseed meal, pecans, pistachio, figs, Aleppo pinenuts, hazelnut, walnut, almonds, legumes Barley, corn, sorghum Corn, barley Feed corn, mixed feed Corn, corn flakes, sorghum, feedstuffs, sesame meal, hay, barley,

11.2 MYCOTOXINS OF ASPERGILLUS SPECIES 11.2.1

Aflatoxins

Aflatoxins are a group of extremely toxic metabolites produced by the common fungi Aspergillus flavus, A. parasiticus, and A. nominus. These fungi are ubiquitous and, under favorable conditions, can grow on a wide variety of agricultural commodities. Although A. flavus is associated with most food and feed contamination with aflatoxins, only a few strains of A. flavus actually produce this toxic metabolite. This mold is taxonomically related to the larger Aspergillus flavus-oryzae group, which is widely distributed, in the soil and in many foodstuffs (Concon, 1988). Historical Aspects Aflatoxins were first identified as etiological agents for animal disease in the early 1960s, after an outbreak of deaths of turkeys in England and elsewhere. The disease, termed turkey X disease because of the then-unknown

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Reporting country United States United States Southwest Africa Australia, New Zealand United States, France, Germany, Canada, Sweden United States United States, India, China, South Africa United States, Canada, Denmark, Finland, France, Norway, Poland, Sweden, United Kingdom, Yugoslavia, Bulgaria, Japan United States, Italy Canada, India Iran, Germany, India, South Africa, United States, France Worldwide

Canada, United States, Russia, India United States, Japan, South Africa Germany, United States United States, Canada, Russia, India, Japan, South Africa, Germany, Yugoslavia, Scotland, France, Finland

cause, was characterized by acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy (Blount, 1961). Similar symptoms and lesions in poultry were reproduced later (Asplin and Carnaghan, 1961). The cause of these effects was traced to the Brazilian groundnut meal that was used in a component of the poultry ration (Allcroft and Carnaghan, 1963). These effects were not confined to poultry: in other animals that consumed rations containing groundnut meal exported from African countries similar pathologic signs concurrently developed (Loosmore and Markson, 1961). An intense investigation into the cause of these outbreaks then began. The causative agents as well as the responsible fungal species were rapidly identified by several workers (Nesbitt et al., 1962; Sargeant et al., 1963). The identified compounds were named aflatoxins after one of the fungi from which they are produced, Aspergillus flavus, and were given names descriptive of their metabolites relative to the solvent front on thin-layer chromatography (TLC) plates as well as their fluorescent color. The outbreak of turkey X disease, clearly described and documented, had been preceded by a number of less

well-described episodes of epizootics in a number of animal species (Paget, 1954; Schoental, 1961; Burnside et al., 1957). Thus, aflatoxins appear to have existed for a considerable time before the epizootic outbreak in England in 1960. However, that dramatic episode of the hepatotoxic disease, which initially destroyed more than 100,000 turkeys, demonstrated the seriousness of the problem facing the food animal industry and ultimately led to the recognition that aflatoxin is both an economic and a public health problem in many areas of the world. Interest in the health effects of aflatoxins might have been confined to the veterinary establishment had it not been for the fact that groundnut meal was being considered by the World Health Organization as a protein supplement for malnourished children in third world countries (Stoloff, 1977). Equally important were reports that aflatoxins were not only acutely toxic, but also carcinogenic in experimental rats (Schoental, 1961; Dickens and Jones, 1963a). As more commodities, such as corn, cottonseed, and rice, were discovered to contain aflatoxins, the potential health problems surrounding these mycotoxins generated much wider concern. Structural Diversity and Chemical Characteristics Aflatoxins consist of a group of approximately 20 related fungal metabolites. They are produced by Aspergillus flavus, A. parasiticus, and A. nominus and can occur in a wide range of important raw food commodities such as cereals, nuts, spices, figs, and dried fruit. Originally, the toxic factors isolated from feed were separated chromatographically into four distinct compounds: aflatoxins B1, B2, G1, and G2 (Figure 11.1) (Nesbitt et al., 1962; Sargeant et al., 1961). The molecular formulae indicated that aflatoxins B2 and G2 (AFB2 and AFG2) were dihydro derivatives of the parent AFB1 and AFG1, respectively (Nesbitt et al., 1962; Cheung and Sim, 1964; van Soest and Peerdeman, 1964). AFB1 and AFB2 fluoresce blue under UV light with RF values of 0.4 and 0.36, respectively, on thinlayer silica plates developed in chloroform-methanol. The other two, with slight lower RF values of 0.34 and 0.31, fluoresce turquoise green and are designated AFG1 and AFG2, respectively. Aflatoxins contain a coumarin nucleus fused to a bifuran and either a pentanone (AFB1 and AFB2) or a sixmembered lactone (AFG1 and AFG2). AFB1 and AFG1 were more toxic to ducklings, rats, and fish than either AFB2 or AFG2; AFB1 was the most toxic (Wogan et al. 1971; Abedi and Scott, 1969). A similar pattern holds for its carcinogenic potency, AFB1 > AFG1 > AFB2 (Wogan et al., 1971; Ayres et al., 1971).

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Allcroft and Carnaghan (1963) reported that extracts of milk from cows fed aflatoxin-containing groundnut meal, when administered to ducklings, induced liver lesions that were identical to those caused by AFB1. However, extracts of toxic milk by TLC showed no AFB1 (the main aflatoxin in the groundnut meal) present. Subsequently, DeIongh and associates (1964, 1965) showed by silica gel TLC that the toxic factor was a blue-violet fluorescing compound that had an RF value well below that of AFB1. They produced chromatographic evidence that the lactating rat was able to convert AFB1 into this “milk toxin” and also that the factor was produced by cultures of A. flavus on crushed groundnuts. Allcroft and coworkers (1966) later suggested the trivial name aflatoxin M for this milk toxin. Further chromatographic studies in different solvent systems resolved this milk toxin into two fluorescing components, a blue-violet one of RF 0.34 and a violet one of lower RF value (0.25). These were subsequently designated aflatoxins M1 and M2, respectively (Figure 11.1). AFM1 and AFM2 are hydroxylated aflatoxin derivatives, which were subsequently isolated from lactating rats, rat liver, and sheep urine, liver, and kidneys, as well as from moldy groundnut and corn. AFM1 was established to be as toxic as but less carcinogenic than AFB1 (Purchase, 1967; Canton et al., 1975). AFGM1 and AFGM2 (Figure 11.1), the hydroxylated derivatives of AFG1, were isolated from A. flavus cultures (Heathcote and Dutton, 1969). However, these are very minor natural metabolites. The 2-hydroxyaflatoxins (Figure 11.1) produced by A. flavus have been reported under several names by various scientists. They are known as AFB2a and AFG2a (Dutton and Heathcote, 1966, 1968) but have also been described as AFB1 hemiacetal (Buchi et al., 1966; Pohland et al., 1968), aflatoxin-W (Andrellos and Reid, 1964), and hydroxydihydroaflatoxin B1 (Ciegler and Peterson, 1968). AFB2a and AFG2a are relatively nontoxic (Dutton and Heathcote, 1966). In view of the susceptibility to hydration of the 2-3 double bond in the terminal furan ring of aflatoxins B1 and G1, the theoretical possibility arose that both aflatoxins M1 and GM1 could be hydrated in dilute acid to form the corresponding hemiacetals. The hemiacetal of AFM1 might also be formed in vitro by liver homogenates. Subsequently, these two new aflatoxins were isolated and characterized, were found to be dihydroxy aflatoxins, and were designated as M2a and GM2a. A possible precursor in the biosynthesis of aflatoxins was isolated from rice and wheat inoculated under the laboratory conditions. It was described as 6-methoxy-7(2′-hydroxyethyl)difurocoumarin and named parasiticol (Stubblefield et al., 1970) or AFB3 (Figure 11.1) (Heath-

O

O

O O

O

O

O

O O

O

OH

OCH3

O

O

OCH3

O

Aflatoxin B1

O

O

O

O

O

O

OCH3

O

O

O

O

O

O

O

OCH3

Aflatoxin M2a

HO

O

O

O

O

O

O

O

O

HO

O

Aflatoxin GM2a

O

OCH3

O CH2

O

O

Aflatoxin B2a

O

OCH3

Aflatoxin GM2

O

CH2OH

OCH3 Aflatoxin B3

Figure 11.1 Chemical structures of naturally occurring aflatoxins B1, B2, G1, G2, M1, and M2, and their metabolites.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O

O

O

OH

OCH3

O

OCH3

O

O O

O

O

OH

O

Aflatoxin GM1

O

O

OH

O

Aflatoxin G2

Aflatoxin B2

HO

OCH3

OCH3

Aflatoxin M2

O O

O

O

Aflatoxin M1

OH

O

O

OCH3

O

O

OH

O

Aflatoxin G1

O

O

O

HO

O

O

OCH3 Aflatoxin G2a

cote and Dutton, 1969). AFB3 is a natural metabolite of A. flavus and A. parasiticus. Rhizopus species are also capable of metabolizing AFG1 to form AFB3 (Cole and Kirksey, 1971). Ducklings appear to be as sensitive to AFB3 as to AFB1, whereas AFB3 is much less toxic to chick embryos than AFB1 (Stubblefield et al., 1970). Several important metabolites of aflatoxins have been identified. Reduction of the pentanone of AFB1 by microorganisms yields aflatoxicol (AFL or AFR0) (Figure 11.2) (Detroy and Hesseltine, 1970). The importance of AFL increased dramatically when it was demonstrated to be produced by animals and its highly toxic and carcinogenic activities became apparent (Patterson and Roberts, 1971; Schoenhard et al., 1974). Animals also metabolize AFB1 by O-demethylation to produce AFP1 (Figure 11.2). It has been detected in the urine of several animal species (Palmgren and Ciegler, 1983). AFP1 is less toxic than AFB1 . In vitro metabolism by liver microsomes from monkeys and humans produces a major metabolite of AFB1, AFQ1. It contains a hydroxyl group, which is in the β-

O

OH

O

H

position to the carbonyl group of the cyclopentanone-ring in AFB1 (Figure 11.2) Detoxification procedures using ammonia produce two major derivatives of AFB1: AFD1 and AFD2 (Lee et al., 1974; Cucullu et al., 1976). The six-membered lactone ring in AFB1 is disrupted by treatment with ammonia (Figure 11.3). Physical and Chemical Properties Some of the physical and chemical properties of various aflatoxins are summarized in Table 11.6. They are crystalline substances, freely soluble in moderately polar solvents such as chloroform, methanol and dimethyl sulfoxide, and they dissolve in water to the extent of 1 to 20 µg/ml (Watson, 1998). They fluoresce under UV irradiation, although AFB1 and AFG1 need derivatization to enhance their fluorescence to a level similar to that of AFB2 and AFG2. This forms the basis for their detection by TLC or high-performance liquid chromatography (HPLC). On TLC plates the four substances are distinguished on the basis of their fluorescent color: B stands for blue and G for green or turquoise, and subscripts relate to their chromatographic mobility. AFB1 is usually found in the highest concentration. Crystalline aflatoxins are extremely stable in the absence of light and particularly UV radiation, even at tem-

Aflatoxicol (AFL or AFR0) O

O

O

OCH3

OH O

O

O

O

Aflatoxin P1

O

OCH3

Aflatoxin D1 O

O

OH

OH O

O

O H

O

Aflatoxin Q1

O

OCH3

OH O

O

OCH3

Aflatoxin D2

H

Figure 11.2 Some important metabolites of aflatoxins.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Figure 11.3 Proposed major reaction products from the ammoniation of aflatoxin B1.

Table 11.6 Physical and Chemical Properties of Some Aflatoxins Ultraviolet absorption maxima in methanol, ε Aflatoxin B1 B2 G1 G2 M1 M2 Aflatoxicol

Molecular formula

Molecular weight

Melting point, °C

265 nm

360–362 nm

C17H12O6 C17H14O6 C17H12O7 C17H14O7 C17H12O7 C17H14O7 C17H14O6

312 314 328 330 328 330 314

268–269 286–289 244–246 237–240 299 293 230–234

12 400 12 100 9 600 8 200 14 150 12 100 (264) 10 800 (261)

21 800 24 000 17 700 17 100 21 250 (357) 22 900 (357) 14 100 (325)

peratures in excess of 100°C. Solutions prepared in chloroform or benzene are stable for years if stored in a cold and dark place. The purity and concentration of reference solutions can be calibrated by using molar absorptivity data (Scott, 1990). The lactone ring makes aflatoxins susceptible to alkaline hydrolysis, and processes involving ammonia or hypochlorite have been investigated as means for their removal from food commodities. However, questions concerning the toxicity of the breakdown products have restricted the use of this means of eradicating aflatoxins from food and animal feeds. If alkaline treatment is mild, acidification reverses the reaction to re-form the original aflatoxin. AFB1 and AFG1 are converted to AFB2a and AFG2a by acid catalytic addition of water across the double bond of the furan ring. Oxidizing reagents react, and the molecules loss their fluorescence.

Representative sampling is of utmost importance in aflatoxin determination. Many mycotoxins are very inhomogeneously distributed in the commodities to be inspected, with the result that it is very difficult to draw a representative sample. Samples are further reduced in size to obtain test portions that usually vary in weight from approximately 20 to 100 g, a range resulting from a compromise between homogeneity requirements and practical considerations. Test portions then undergo the further steps outlined in Figure 11.4. All analytical procedures include three steps: extraction, purification, and determination. Extraction is usually

Sampling

Extraction

Analytical Methods The major aflatoxins of concern are B1, B2, G1, G2, M1, and M2; AFB1 is the most potent of these six naturally occurring aflatoxins. Because of the diversity of toxicological manifestations and the economic losses caused by exposure to aflatoxins, humans and susceptible animals must be protected from undue exposure to these toxins to safeguard their health. Numerous chemical and biological methods for the detection and quantification of AFB1 and related compounds have been proposed since the 1970s. The basic steps in aflatoxin and other mycotoxin determinations are outlined in Figure 11.4. The early observations of Sargeant and colleagues (1961), concerning the chemical properties of toxic components in suspected groundnut meal, form the basic technology for the separation and detection procedures, an extraction of aflatoxins with chloroform and detection of their fluorescence under UV exposure.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Cleanup

Concentration

Ultimate separation

Detection and quantitation

Confirmation of identity

Figure 11.4 Basic steps involved in the analytical procedures for mycotoxin determination.

done with (combinations of) organic solvents and water. The most common system for extraction has been a mixture of chlorohydrocarbon (e.g., chloroform) and water. This system is gradually being replaced by the methanolwater or acetonitrile-water system. Purification of the extract to remove lipids and other substances is usually done by passing the extracts through chromatography columns or prepacked cartridges (SepPak, Bond-Elut, Aflatest, Multifunctional, etc.). The latter are commercially available with many types of adsorbents and in many formats that may suit the needs of the analyst. The most recent advance in cleanup of extracts containing mycotoxins is the use of immunoaffinity cartridges. These columns are composed of monoclonal antibodies, specific for the toxin of interest, which are immobilized on Sepharose and packed into small plastic cartridges. These can be incorporated in fully automated sample preparation systems that take the sample from the extraction stage through to completion of HPLC. In addition to HPLC, other chromatographic techniques can be used for the ultimate separation and quantitation: TLC and gas-liquid chromatography (GLC). TLC was very popular in the 1960s and 1970s; though still very valuable and widely applied in the developing countries, it was largely superseded during the 1980s by HPLC. The latter is an attractive alternative to the TLC fluorodensitometry and visual analytical procedures, because of its high resolution potential, rapidity of separations, and potentially improved quantitative accuracy and precision. Its limitations are that large numbers of samples cannot be quantitated as rapidly as with TLC and that interfering substances contained in extracts obtained from naturally contaminated foods and feeds impede the measurement of aflatoxins. However, for the detection of aflatoxins in smaller quantities of urine, blood, or other tissue samples, HPLC offers distinct advantages. GLC has limited applications in mycotoxin analysis because it requires volatile components, whereas most mycotoxins are nonvolatile. Besides the chromatographic techniques, the immunoassay techniques are worth mentioning. In particular, enzyme-linked immunosorbent assay (ELISA) has become an important technique in mycotoxin methodology (Deshpande, 1995). The simplicity of the ELISAs and the large number of samples that can be handled in 1 day have made these tests important, especially for screening and semiquantitative determinations. Their lack of selectivity might prevent their use as quantitative tools, and therefore they are less suitable for regulatory analysis. Another point that requires attention is their specificity. Although most of the antisera seem to be quite specific, the possibility of cross-reactions cannot be ruled out fully. It is therefore a good laboratory practice to confirm positive findings ob-

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tained with immunoassays by using methods of analysis based on other principles. Currently, certified reference materials have become available for AFM1 in milk powder; for aflatoxins B1, B2, G1, and G2 in peanut butter; and for AFB1 in peanut meal and compounded animal feed. Many methods of analysis for aflatoxin in foods and feeds are published each year. An appropriate method must be selected for each need. Factors that should be considered before selecting a method include number of analyses needed, time, location, cost, equipment, safety, waste disposal, and, above all, the experience of the analyst. The simple, specific, and rapid immunoassays will play an increasingly important role in monitoring foods and feeds for aflatoxins and other mycotoxin contamination. Currently, these methods appear to have their greatest value when used in conjunction with existing TLC and LC methods. Biosynthesis Similarly to many other toxic secondary metabolites produced by fungi, aflatoxins are synthesized by the polyketide route, wherein head-to-tail condensations of acetate units proceed via poly-β-keto-thiol ester intermediates (Applebaum and Marth, 1981). In this biosynthetic pathway, the chain is initiated by acetyl CoA, and malonyl CoA is the source of additional carbon units. Relative to that of other polyketide-derived mycotoxins, the synthesis of aflatoxins has been particularly difficult to elucidate. It is now known that aflatoxins are derived from a C 20 polyketide (Coulombe, 1991). In the biosynthetic scheme for AFB1 (Figure 11.5), the initial intermediate is norsolorinic acid, which is converted to averufin in a two-step process via the intermediate averantin. The next biosynthetic step involves the ring opening of averufin, followed sequentially by dehydration, epoxidation, and epoxide rearrangement to form versicolorin A via versiconal A hemiacetal acetate. Versicolorin A is converted to the last major intermediate, sterigmatocystin, via an oxidoreductase. The o-methoxy group common to nearly all of the aflatoxins arises from the methyl donor S-adenosylmethionine (SAM) by catalysis of S-adenosyl methyltransferase. It converts sterigmatocystin to O-methylsterigmatocystin (Coulombe, 1991). The formation of the bisfuran ring system present in versicolorin A, sterigmatocystin, and AFB1 appears to involve the hydrolysis of the ester bond in versiconal hemiacetal acetate with elimination of an acetyl group, followed by ring closure. This proceeds via oxidation of the terminal hydroxyl group to the aldehyde, resulting in the hemiacetal structure.

OH O

O

OH

O O

10 CH3COOH

O

O OH

O

O

O

O

H 3C O

OH

HO

O O

CYCLIZED DECAKETIDE

OH

O

OH

OH

O

NORSOLORINIC ACID

OH

O

OH

CH3

H3C

AVERANTIN

O

AVERUFIN

OH

HO

OH

O

OH

O

O

OH

OH

O

OH

O

H3C

O

HO

OH

O O

VERSICONAL HEMIACETAL ACETATE O

O

OH

O

VERSICOLORIN A

O

O O

O

OH O

O

O

OCH3

AFLATOXIN B3

Figure 11.5 Proposed biosynthetic scheme for aflatoxin B1.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O

O

STERIGMATOCYSTIN

OCH3

The biosynthesis of other fungally-produced aflatoxins as well as their metabolic relationship are less clear and often the subject of conflicting reports in the literature. The formation of the tetrahydrobisfuran AFB2 and the dihydrobisfuran AFB1 probably arises independently by a branched-chain pathway beginning at versiconal hemiacetal through versicolorin A and versicolorin C, respectively. AFG1 and AFG2 probably also arise independently by a branched-chain pathway. There is good evidence that AFM1 and AFM2 are synthesized from the analogous B toxins via monooxygenase enzymes (Dutton et al., 1985). That many filamentous fungi can enzymatically hydroxylate tertiary carbon atoms is well documented in the literature (Sharma and Salunkhe, 1991).

Table 11.7 Enzyme Systems and the Types of Biotransformation Reaction Involved in the Metabolism of Aflatoxin B1

Biotransformation

AFQ1

As described in Chapter 5, biotransformation plays an important role in the biological activity and disposition of aflatoxins. Bioactivation of aflatoxins is a necessary step in the most dramatic of their toxic and carcinogenic effects. Several detoxification mechanisms involving biotransformation are also known. AFB1 represents by far the most toxic of the aflatoxins, and almost all of the available information on the bioactivity of aflatoxins in animals has focused on AFB1 and its metabolites. It is also usually the aflatoxin found in the highest concentrations in contaminated food and feed. Therefore, most of the discussion that follows focuses on AFB1; other aflatoxins are discussed whenever possible. For a detailed treatment of this topic, the readers are referred to several excellent reviews (Heathcote and Hibbert, 1978; Neal, 1987; Coulombe, 1991; Eaton et al., 1994; Watson, 1998). An overall scheme for the primary metabolism of AFB1, based on results obtained from in vitro studies using hepatic subcellular fractions isolated from a range of animal species, is shown in Figure 11.6. Not all metabolites have been identified in all species, and significant quantitative differences in the formation of the various products may exist. The enzyme systems and the types of biotransformation reaction involved in the metabolism of AFB1 are summarized in Table 11.7. A requisite step in the toxic and carcinogenic action of AFB1 is its conversion to one or more metabolites in various tissues of exposed animals. As in the case of other “procarcinogens,” the majority of metabolic conversions of AFB1 are catalyzed by cytochrome P-450s, which are a group of mixed-function oxidases present in the liver and other tissues. AFB1 is also transformed in various tissues by cooxidation reactions via prostaglandin H synthetase, although this pathway is generally of lesser importance

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AFB1 metabolitea AFB2a

AFP1

AFM1

AFL

AFL-M1

AFL-H1

AFB1-epoxide

AFB1 dihydrodiol a

Enzyme systems and the type of reaction Microsomal metabolite of AFB1, doubtful enzymatic formation, occurs nonenzymatically through hydration of furan double bond in absence of cofactors Mixed-function oxidase–catalyzed odemethylase reaction in microsomes, major urinary metabolite in monkeys Hydroxylated metabolite, NADPHdependent mixed-function oxidase, major metabolite in milk and urine of animals fed AFB1-contaminated diets NADPH-dependent hepatic microsome– mediated hydroxylation of AFB1, major metabolite produced by primate microsomal metabolism Reversible reduction of AFB1 by reductase in the cytosol fraction, NADPH required as a cofactor, major metabolite of avian species Cytosol-catalyzed reduction of AFM1, or microsomal mixed-function oxidase–catalyzed hydroxylation of AFL Cytosol-catalyzed reduction of AFQ1 or by cytochrome P-450-catalyzed hydroxylation of AFL, major metabolite of AFB1 by humans or rhesus monkey Not isolated from biological systems or synthesized chemically, formation deduced from production of AFB1dihydrodiol as acid hydrolysis product of metabolically or chemically generated AFB1–nucleic acid adducts Formed by enzymatic or nonenzymatic hydrolysis of AFB1-epoxide

AF, aflatoxin; NADPH, reduced nicotinamide-adenine dinucleotide phosphate.

(Coulombe, 1991). It has long been known that species susceptibility to the effects of AFB1 depends, in large part, on the metabolic fate of this compound after exposure. Most of the metabolites shown in Figure 11.6 are less toxic than AFB1, whereas one possesses more toxicity. From a toxicological viewpoint, the most important reputed toxic intermediate of AFB1 is the AFB1-2,3-epoxide (or the 8,9-epoxide in IUPAC nomenclature). The epoxide undergoes rapid hydrolysis to form AFB1-2,3-dihydrodiol (and its Tris adduct, if this buffer is present) (Lin et al., 1978; Neal et al., 1981). AFB1-2,3-dihydrodiol can exist in a resonance form as a phenolate ion that is capable of

O

OH

O

Glucuronide conjugate

O

Aflatoxicol H1

O OH O

OH O

O

Aflatoxin M1-P1

O

O

O

OH

O

O O

O

Aflatoxin Q1

O

Aflatoxin P1

OCH3

O

Glucuronide conjugates

O

Aflatoxin M1

OH

O

O

O OH O

OH O

O

O

O

OCH3

O

O

OCH3

DNA adducts O O

O

OCH3 O

AFLATOXIN B1

O

OH

Aflatoxicol M1 O

GSH conjugate

OH

O

Aflatoxin B1-2,3-epoxide

O O O

O

O O

Protein adducts O

O

OCH3

OCH3 OH

O O

O O

O

Glucuronide conjugates

HO O HO HO

O

O

OCH3

O

O

Aflatoxin B2a

Aflatoxin B1-2-3-dihydrodiol

Figure 11.6 Overall scheme for the primary metabolism of aflatoxin B1 in animals.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

OCH3

O Aflatoxicol

OCH3

forming Schiff base adducts with protein amino groups, particularly lysine (Figure 11.7). The diol is most likely to be involved in the binding of AFB1 to protein observed in vivo (Garner et al., 1979; Appleton et al., 1982). The AFBlysine adduct is the principal adduct found in plasma albumin after in vivo AFB1 exposure (Sabbioni et al., 1987). This type of reaction could be involved in the mechanisms of acute toxicity of aflatoxin. It is the metabolite responsible for alkylation of cellular nucleic acids and subsequent carcinogenic and mutagenic activity. Only a very small portion of administered AFB1 is generally present in the unmetabolized form in either the tissues or secretions of animals. The major hydroxylated metabolites of AFB 1 formed by cytochromes P-450 are AFM1, AFP1, AFQ1, and AFB2a (Figure 11.6, Table 11.7). Additional metabolites, which are generally formed in smaller quantities, depending on various conditions, include AFL M1 and AFL H1. These stable metabolites are considered to be detoxified relative to AFB1, are more polar, and, as such, are

O

O

O HO

O

HO

OCH3

O

AFB1-2,3-dihydrodiol

O O

O

H

H

O

O HO

-

O

OCH3

Phenolate resonance form

Schiff base formation with protein amino groups

Figure 11.7 Schiff base formation between protein amino groups and aflatoxin B1-2,3-epoxide.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

more easily excretable. The cyclopentanol AFL is not a product of oxidative metabolism, but rather a result of the reductive metabolism of AFB 1 catalyzed by soluble NADPH-dependent reductases (Wong and Heish, 1978). For the secondary metabolism (phase II reactions, see Chapter 5), the formation of sulfate and glucuronic acid conjugates of AFB1, and a range of primary microsomal metabolites (phase I products) of AFB1, including AFP1, AFM1, and AFQ1, have been described (Wei et al., 1978). Sulfate or glucuronide conjugates are excreted in the urine and bile of animals treated in vivo with AFB1 (Dalezios et al., 1971; Bassir and Emafo, 1970). AFB1 epoxide can be conjugated with reduced glutathione, a process requiring the presence of the appropriate glutathione S-transferase (GSTase) enzymes. The glutathione conjugate has been chemically characterized and its possible role in modifying AFB1 toxicity described (Degen and Neumann, 1978; Moss et al., 1983). The subsequent stages in the mercapturic acid pathway have been examined in in vitro systems (Figure 11.8). The initial stage is catalyzed by γ-glutamyl transpeptidase, which is particularly abundant in the brush borders of the proximal tubules of the kidney. Examination of bile of AFB1-treated rats indicates that the majority of AFB1 conjugated with glutathione (GSH) in the liver emerges into the common bile duct in the form of AFB1-GSH conjugate, despite the presence of high levels of γ-glutamyl transpeptidase on the canalicular surfaces of the bile ducts through which the AFB 1-GSH conjugate is secreted (Moss et al., 1984). When a large proportion of the liver hepatocytes contain elevated amounts of γ-glutamyl transpeptidase, as a result of feeding hepatocarcinogenic substances such as AFB1, the conjugated AFB 1 in the bile primarily comprises AFB1-Cys-Gly. The subsequent stages in the formation of the AFB 1 -mercapturate, dependent on the activity of dipeptidase and N-acetyl transferase, have been demonstrated in vitro in rat kidney–derived systems (Moss et al., 1985). The mercapturate of AFB1 has been detected in the urine of rats and marmoset monkeys after the acute administration of AFB1. The fate of AFB1 is dependent on the relative activity of several biotransformation pathways, in addition to other factors such as DNA repair rates (Figure 11.9). The amount of the mycotoxin that exerts carcinogenic or toxic effects depends on the amount converted to various metabolites as well as on the biological activity of those metabolites. With respect to carcinogenicity, AFB1-2,3-epoxide is the key active metabolite. As indicated in Figure 11.9, hydroxylated metabolites of AFB1 (AFM1, AFP1, AFQ1) are assumed to represent detoxification products. Detoxification of the reactive epoxide also may occur through conjugation with GSH. Hydrolysis of AFB1-2,3-epoxide forms

Aflatoxin B1-2,3-epoxide O

O

O O

O

Macromolecular binding

Microsomal mixed-function oxidase O

O

O

O O

OCH3

AFLATOXIN B1

O

OCH3

Epoxide hydratase

Glutathione S -transferase GSH

AFB1-2,3-dihydrodiol

AFB1-N-acetylcysteine O CH3 CO

O

O

O COOH HO

O

CHCH2CH2CONH

O

NH2

CHCH2S

O

O

OCH3

HO CONHCH2COOH

NH CHCH2S

O

O

AFB1-GSH

OCH3 Glycylglycine

COOH γ-Glutamylglycylglycine Acetyl CoA

γ-Glutamyltranspeptidase

N -Acetyltransferase O

O

O

O

CoA O

O OH

OH

Dipeptidase

NH2 CHCH2S COOH

O

O

OCH3

AFB1-Cys

Figure 11.8 Mercapturic acid pathway of phase II biotransformation of aflatoxin B1.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

NH2 CHCH2S

O

CONHCH2COOH

O

OCH3

AFB1-Cys.Gly

AFLATOXIN B1 Hydroxylated Metabolites

Epoxide

GSH Conjugate Dihydrodiol

Protein Binding

TOXICITY

DNA Products

CANCER

Activation products

Detoxification products

Figure 11.9 Schematic representation of the role of various biotransformation pathways in the disposition, toxicity, and carcinogenicity of aflatoxin B1.

a dihydrodiol that probably still is capable of causing toxic effects via binding to proteins but presumably is a less potent carcinogenic species than the epoxide. The metabolism of the aflatoxins other than AFB1 has received rather less research interest because of their lower potencies and rate of occurrence. The metabolic pathways described in the literature for AFG1 are shown in Figure 11.10. The doubt concerning the metabolic formation of AFB2a from AFB1 applies also to the formation of AFG2a from AFG1 (Patterson and Roberts, 1970). The formation of a 4-hydroxylated AFG1 is catalyzed by microsomal mixed-function oxidases (Patterson, 1973). AFG1 is both acutely toxic and carcinogenic, and the proposed mechanism of metabolic activation, epoxidation of the terminal furan ring, similar to the proposed mechanism of activation of AFB1, is based on chemically and metabolically catalyzed formation of DNA adducts (Garner et al., 1979). The principal pathways reported for the metabolism of AFB2 by animal species are given in Figure 11.11.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Lacking the double bond in the terminal furan ring, and given the apparent importance of this feature in the metabolic activation of AFB1 and AFG1, it is of some interest to account for the albeit rather lower, but nevertheless well-authenticated toxicity of AFB2 (Wogan et al., 1971). AFB2 is converted to AFB1 in both rat and duck liver, and the rates of these reactions appear to correlate with the toxic potential of AFB2 in these species. The formation of AFB2a from AFB2 cannot be accounted for in terms of artifactual hydration, as in the case of AFB1 and AFG2a from AFG1, and presumably represents a direct hydroxylation of AFB2. Hydroxylation of AFB2 producing AFM2 and reduction of the cyclopentenone carboxyl forming dihydroAFL has also been described (Patterson, 1973). It is important to note that not all of the biotransformations occur in every species and that the profile of metabolites formed from AFB1 varies with species, tissue type, and age of the animal. Many of the same dietary factors or agents known to modify the metabolism of other

O

O

O

O

OH

O

O

OCH3

AFGM1

O

O

O

O O

O

O

O

O O

O

OCH3

O

AFG1

OCH3

AFG1-2,3-epoxide

O

O

O

HO

O

O

O

O

OCH3

AFG2a

Figure 11.10

Pathways of aflatoxin G1 metabolism in animal systems.

carcinogens similarly affect AFB1 biotransformation patterns as well as in vivo biological effects. The prior or concurrent administration of a wide variety of modulating agents, such as flavone and indole compounds, phenolic antioxidants, polyhalogenated hydrocarbons, phenobarbital, and 3-methyl-cholanthrene, as well as dietary factors, such as protein level, protein quality, and dietary fat consumption, are known to affect the metabolism of aflatoxins. Occurrence in Foods After the finding that aflatoxins were potent hepatocarcinogens, numerous surveys were carried out on the natu-

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ral occurrence of these mycotoxins in foods. Among foods and foodstuffs, groundnut and groundnut products, which are widely grown and utilized for food and feedstuffs in tropical and subtropical countries, would seem to be significantly contaminated with aflatoxins. The contamination of groundnut and groundnut products with aflatoxins has been reported in the United States, Africa, Asia, the Indian subcontinent, as well as other countries. Aflatoxin has also been found in other kinds of edible nuts, including almonds, hazelnuts, and pistachio nuts, although the levels have been relatively low. Aspergillus flavus is able to infect several kinds of cereals, in particular corn, and produces aflatoxins before

O

O

O OH

O O

O

OCH3 AFM2

O

O

O

O

O

OCH3

O

O

AFB1

O

OCH3

AFB2

O

OH

O

O

O

O

O

O

OCH3

HO

Dihydro AFL

Figure 11.11

O

O

O

OCH3 AFB2a

Pathways of aflatoxin B2 metabolism in animal systems.

harvest. The levels and frequency of aflatoxin contamination are significantly high, and corn heavily contaminated with AFB1 may induce acute hepatitis in humans. AFB1 and related mycotoxins are often detected in cereals such as corn, barley, wheat, sorghum, oats, millet, rice, beans, cowpeas, peas, soybeans, sesame, sweet potatoes, cassava, and other local edible grains and foods (Stoloff, 1976; Newberne and Butler, 1969; Coulombe, 1991). Although their contents are low relative to the levels found in groundnuts and corn, these commodities are important foods worldwide. The aspergilli may infect these commodities in the field or in storage. Factors that promote the growth of this fungus include a relatively high water activity in the field (0.84 to 0.86), high temperature, drought stress, mechanical damage during harvest, insect infestation, rain during harvest, and moisture accumulation during storage (Stoloff, 1976). Drought may act to increase the susceptibility of groundnuts to aflatoxins by reducing the ability of the plant to produce phytoalexins (Dorner et al., 1989).

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Drought conditions in the midwestern and southeastern United States in the late 1980s resulted in increased aflatoxin contamination in corn (Brosten, 1989). Dairy products are sometimes contaminated with AFB1 and AFM1. The consumption of AFB1-contaminated feeds by lactating animals results in the extraction of metabolites such as AFM1. The relative amount of AFM1 excreted is related to the amount of AFB1 in the feed, and about 0.1% of AFB 1 ingested is excreted into milk as AFM1 . Kiermeier and Mashalev (1977) reported that AFM1 was detected in 8 dried milk products of 166 samples tested in the range of 0.7–2 ppb, which corresponds to about 0.08–0.26 µg/L. Meat and meat products are also contaminated with aflatoxins when farm animals are fed with aflatoxin-contaminated rations. Furthermore, many meat products such as “country style” hams and certain sausages are traditionally mold-ripened. As suggested by Leistner (1984), undesirable Penicillium spp. and other fungi grow quite frequently on meat products, especially on fermented sau-

sages (salami) and raw hams. Natural inoculation has been the basis for the traditional methods, but now there is a trend to control the ripening process by using selected starter nontoxic strains. Aflatoxins have also been shown to occur in spices, including cayenne pepper, Indian chili powder, dried chili peppers, black pepper, capsicum peppers, and nutmeg (Jones 1977). Natural occurrence of aflatoxins in cereals and cereal products is summarized in Table 11.8. Toxicology Aflatoxins are both acutely and chronically toxic. AFB1, in fact, is one of the most potent hepatocarcinogens known (Fishbein, 1979). Hence, the long-term chronic exposure to extremely low levels of aflatoxins in the diet is an important consideration for human health. In the temperate, developed areas of the world, acute poisoning in animals is rare and in humans is now extremely unlikely. Extremely high levels of aflatoxins in the imported groundnut meal caused the outbreak of the turkey X disease in England. This alerted industry and governments to the potentially devastating effects of mycotoxins, particularly the aflatoxins. Acute Toxicity in Animals The original identification of the aflatoxins as important naturally occurring toxins resulted from their acutely toxic and carcinogenic effects in sensitive species fed contaminated feedstuffs. The acutely toxic properties were recognized as a result of the investigation into turkey X disease in domestic flocks in England caused by feeding of contaminated groundnut meal. The carcinogenic properties were detected by a subsequent experimental feeding of the contaminated meal to rats and also by recognition that outbreaks of hepatoma in rainbow trout were the result of feeding of cottonseed meal contaminated with aflatoxins. These early observations therefore highlighted the potential hazards of dietary exposure to levels of these toxins existing in naturally contaminated foodstuffs, which could provoke an acutely toxic reaction in sensitive species. The greater potential hazard from prolonged human exposure to the much lower levels, which could provoke a carcinogenic response in sensitive species, was evident. Since the outbreak of turkey X disease in the 1960s, recognition of sources of aflatoxin contamination and the factors affecting its production have received very extensive research attention, which has led to a general lowering of levels of contamination. However, samples of feedstuffs containing levels of aflatoxins in excess of 1 to 2 ppm are

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still being identified (Neal, 1998). This level of contamination is capable of presenting a chronic hazard to susceptible species. There have been many experimental animal studies using aflatoxins, too numerous to detail in this review. The toxicity of aflatoxins has been demonstrated in many domestic and experimental animals. A common feature is its potent hepatotoxicity. In fact, liver is a target organ for toxicity in almost all species studied thus far. Toxic effects have also been reported for other organs, including kidney, lung, colon, myocardium, and nasal epithelium. One of the most important facts to emerge from the experimental animal studies has been the recognition of a wide species variation in sensitivity to these toxins. In the vertebrate species tested, there is at least a 10-fold variation in the susceptibility to the acute effects of AFB1, and no species tested thus far is totally resistant. Differences in age, sex, strain, and route of administration are important factors in the toxic potency of AFB1. For example, male rats are generally more sensitive than females of the same age, and both very young and old mice and rats are the most susceptible to AFB1 (Newberne and Butler, 1969; Hayes et al., 1977). Oral administration of AFB1 is more effective than intraperitoneal administration in PortonWistar rats, although the reverse is true in Swiss mice (Busby and Wogan, 1984). Species relatively resistant to acute AFB1 include the mouse and hamster, with oral LD50 values of 9.0 and 10.2 mg/kg, respectively. The duck, monkey, and rainbow trout are relatively sensitive, with values of 0.34 to 0.56, 3.0, and 0.81, respectively (Patterson, 1973; Bauer et al., 1969). Rats are not particularly sensitive to acute aflatoxicosis, and oral LD50 values vary from 1.2 to 17.9 mg/kg (Hayes et al., 1977; Patterson, 1973). Poultry are sensitive to acute AFB1, although chicks are more resistant than are ducklings and young turkeys. Patterson (1973) observed that the rates of AFB1 metabolism as well as the profile of metabolites dictate the relative susceptibility to acute aflatoxicosis. For example, sensitive species generally metabolize an LD50 dose of AF1 rapidly, and liver preparations from those same species generally were shown to convert AFB1 to AFL more efficiently. Animals exposed to AFB1 often exhibit malaise, loss of appetite, and lower growth rates. Histopathological effects due to acute AFB1 are most dramatic in liver and are observed in all vertebrate animal species exposed to AFB1. The principal hepatic lesions are hemorrhagic necrosis, fatty infiltration, and bile duct proliferation. The hepatic zone in which these lesions occur varies. In ducklings and adult rats, the periportal zone is most commonly affected; in pigs, guinea pigs, and dogs, centrilobular lesions predominate. Hemorrhagic necrosis has also been observed in

Table 11.8 Natural Occurrence of Aflatoxins in Selected Agricultural Commodities

Commodity

Country

Corn Yellow corn Corn White corn (feed grade) Corn Corn Corn Corn Corn Corn Corn Corn Wheat Wheat Wheat flour Spaghetti Sorghum Sorghum Oats Oats Barley Millet Millet Millet Rye Rice Rice Rice and rice products Boiled rice Rough rice Raw rice Rice Garri (manihot flour) Red pepper Yam flour Beans

United States United States United States Nigeria Nepal China Philippines Thailand Japan India Kenya Philippines United States France France Canada Uganda France Sweden France France Uganda Thailand Nigeria France Philippines Thailand Philippines Philippines Brazil Nepal

Cassava Yam Sweet potato Cocoa Pistachio nut

Philippines Philippines Philippines

Nigeria Nigeria Nigeria Japan

Japan

Source: Compiled from Ueno (1985, 1987).

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Aflatoxins, µg/kg, ppb 3–27 4–308 10,000 100–1000 8.8–37.5 B1 89.5 400 93 B1 131–340 B1 6250–15,600 3200–12,000 45.9 2–19 0.25–180 0.25–150 13 1–1 000 0.25–100 2 600 0.25–100 0.25–10 1–100 248 1.4 0.25–100 16 98 30 0.6 400 5–10 40 1 600 700 400 1.3–26.9 B1 0.4–6.9 B2 467.5 88.8 60.6 50.6 2.0–800 B1 0.4–180 B2 0.6–51.4 G1 0.2–16.3 G1 1.8–39.3 M1

Remarks 2.7% Positive 34.6% Positive Associated with aflatoxicosis in chickens 100% Positive 51% Positive 36% Positive (<30 ppm) 35% Positive Commercial samples Acute hepatitis Acute hepatitis 84% Positive 0.4% Positive 47% Positive 28% Positive Isolated sample 37.7% Positive 25% Positive 42% Positive 7% Positive 16.4% Positive

2% Positive 38% Positive 20% Positive

1.8% Positive 1.8% Positive 100% Positive 39% Positive 84% Positive 70% Positive

other organs, such as the kidney, heart, spleen, and pancreas. In addition, gastrointestinal hemorrhaging has been observed in poultry acutely intoxicated by AFB1. Many of these effects were shown to begin 1 to 2 days after exposure to AFB1. Structure-activity studies have identified features important in the acute effects of aflatoxins. The acute toxicity of AFB1 and its congeners in ducklings and rats followed the order B1 > G1 > B2 > G2 (Wogan et al., 1971). Aflatoxins with an unsaturated terminal furan ring, such as AFB1 and AFG1, had similar acute potencies in rats and ducklings, whereas AFB2 and AFG2, which contain a saturated terminal furan, were much less potent in ducklings and were nontoxic to rats at a dose of 200 mg/kg. That AFG1 was less acutely toxic than AFB1 also indicated that substituents fused to the lactone ring are also involved in determining the toxicity of aflatoxins. Acute Toxicity in Humans That humans are sensitive to acute aflatoxin poisoning, aflatoxicosis, appears to be beyond doubt. Though epidemiological studies have suggested that aflatoxins may be responsible for human diseases, there are several welldocumented episodes of acute poisoning in humans. Much of the information available today indicates that human exposures to high levels of aflatoxins in food are much more frequent in third world countries whose climates and prevalent agricultural practices favor mold infestation of foodstuffs. Thus, most of the recorded outbreaks of acute aflatoxicosis have occurred in Southeast Asia and Africa. Campbell and Stoloff (1974) extrapolated from primate data the doses of AFB1 that would be required to cause acute aflatoxicosis in humans. Recognizing the uncertainties associated with species extrapolation, they predicted that persons who consume food containing 1.7 ppm for a short time could experience severe liver damage, that a single dose of 75 mg/kg could result in death, and that a daily dose of 0.34 mg/kg would not result in apparent acute aflatoxicosis. The involvement of aflatoxins in acute poisoning of humans is very well documented as a result of an episode in India. In two adjacent western states (Gujarat and Rajasthan), a food-borne toxicosis started during late October 1974 in rural areas where the staple food was corn. During this month, there were unseasonal rains, which drenched the standing corn crop; a total of 397 patients were affected and 106 died (Krishnamachari et al., 1975). Clinical features were characterized by jaundice, vomiting, and anorexia and followed by ascites, which appeared rapidly within a period of 2–3 weeks. The liver was enlarged and tender in only a few cases. Death was usually sudden and

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in most cases preceded by massive gastrointestinal bleeding. Twice as many males as females were affected. Liver histopathologic analysis revealed extensive bile duct proliferation with periductal fibrosis. During the outbreak and for several weeks afterward, a large number of dogs exhibited ascites and icterus and died within 2–3 weeks of the onset. Affected corn grains obtained from afflicted households showed the presence of A. flavus, and chemical analysis revealed the presence of aflatoxins in the range 6.25–15.6 ppm. Since an adult consumes about 350 g of corn daily, the patients would have ingested 2–6 mg of aflatoxin daily for several weeks. These findings indicated that the hepatitis was due to the uptake of AFB1. A subsequent outbreak of fatal hepatitis in Kenya was also attributed to aflatoxin-contaminated corn (Ngindu et al., 1982). Several other case reports have also been reviewed in the literature (Campbell and Stoloff, 1974). Establishing causality in these situations is very difficult, but the circumstantial evidence, the measurably high levels of aflatoxins in food and human tissues, and the histologic findings are all persuasive. Aflatoxins have been implicated in subacute and chronic effects in humans. These effects include primary liver cancer, chronic hepatitis, jaundice, hepatomegaly, and cirrhosis through repeated ingestion of low levels of aflatoxin. Aflatoxins may also play an important role in causing a number of other human diseases, including Reye’s syndrome (Shank et al., 1971), kwashiorkor (Hendrickse et al., 1982; Hendrickse, 1991; Coulter et al., 1986), and hepatitis (Krishnamachari et al., 1975; Ngindu et al., 1982). Aflatoxins can also suppress the immune system (Pier, 1991). Mutagenicity As in the case of other procarcinogens, the binding of the AFB1-epoxide to cellular DNA is thought to be the initiating event in the AFB1-mediated mutagenesis and carcinogenesis. The level of AFB1-DNA adducts formed in a species or a tissue is often an accurate indicator of susceptibility to the carcinogenic effects of AFB1. Activated AFB1 binds exclusively with guanyl residues in DNA, and the AFB1-N7-Gua adduct is by far the most predominant form. Additional adducts have been isolated; of them the “ring-opened” derivative of the AFB1-N7-Gua adduct, the formamidopyrimidine, or AFB1-FAPyr, is the most common (Figure 11.12). The relevance of AFB1-DNA adduct formation to carcinogenesis has been the subject of intense study. The mutagenicity of AFB1 has been repeatedly demonstrated in systems employing bacteria (Lowery et al., 1983), yeast (Niggli et al., 1986), and human and other mammalian

O

O

O HO O

H2N

O

N

HN N

OCH3

O

N

AFB1-N7-GUA

O

O

O HO O N

HN H2N

N

O CHO

NH2

O

OCH3

AFB1-FAPyr

Figure 11.12 Structures of the major aflatoxin B1-DNA adducts. DNA, deoxyribonucleic acid.

cells (Billings et al., 1985; Kaden et al., 1987), among others. On a molar basis, AFB1 is one of the most potent mutagens known. In the Salmonella typhimurium test, it induces 8527 revertants per microgram, followed in potency by AFL (1940 revertants), AFG1 (285), AFM1 (275), AFL-H1 (170), AFQ (99), AFB2 (18), AFP1 (10), AFG2 (9), and AFB2a (2) (Wong and Hsieh, 1976). Carcinogenicity AFB1 is known to be carcinogenic in a wide variety of animals, such as the rat, mouse, duck, monkey, and rainbow trout. The major target organ involved after chronic exposure of AFB1 is the liver; tumors of other organs appear but are less prevalent. As is the case with acute toxicity, there exist significant species differences with respect to susceptibility. Other aflatoxins have proved to be less potent carcinogens than AFB1. The order of potency, as demonstrated in trout and rat feeding studies, generally follows that seen in acute studies from the same species. The approximate relative carcinogenic potency ranking follows

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the order AFB1 > AFL > AFM1 >AFQ1 > AFG1; AFB2 and AFG2 are inactive (Ayres et al., 1971; Schoenhard et al., 1981; Hendricks et al., 1980; Coulombe, 1991). These studies again support the observations that the presence of an intact 2,3 double bond is a requirement for carcinogenicity and that substitution of the cyclopentanone ring of AFB1 to the dilactone of AFG1 results in a significant reduction in the biological activity of aflatoxins. The ecological process in which populations are compared in terms of average aflatoxin exposure and liver cancer rates has produced the most convincing evidence of an association between aflatoxins and hepatocellular carcinoma in humans. In some geographical areas, most notably sub-Saharan Africa, India, and Southeast Asia, primary hepatocellular carcinoma (PHC) is often the most common form of malignancy seen. In these areas, the consumption of aflatoxin-contaminated foods is also much more prevalent. In addition to tropical climates that favor growth of the fungus, harvesting, handling, and storage practices common in these regions, taken together, appear to promote widespread aflatoxin contamination. Several field studies conducted in Uganda (Alpert et al., 1971), the Philippines (Campbell and Salamat, 1971; Bulatao-Jayme et al., 1982), Thailand (Shank, 1977), Kenya (Peers and Linsell, 1973; Autrup et al., 1987), Mozambique (Van Rensburg et al., 1974), and Swaziland (Peers et al., 1976) have demonstrated a linear dose-response relationship between consumption of aflatoxin and human liver cancer. A high consumption of aflatoxin was consistently associated with a high incidence of PHC. Hsu and coworkers (1991) and Bressac and associates (1991) provided additional evidence of the role of AFB1 in human liver cancer. These researchers showed that 50% of human liver tumor tissue samples from regions in southern China and southern Africa associated with high dietary AFB1 level and PHC incidence contained a single point mutation in the tumor-suppressor gene p53. Confounding a firmer conclusion regarding the link between aflatoxin consumption and human PHC is the observation that hepatitis B virus (HBV) antigen is coincident in those geographical areas with high incidences of PHC. HBV infection is often assumed to be the major factor for PHC, since it has been demonstrated that the majority of PHC cases are characterized by chronic or previous HBV infection (Wild and Montesano, 1991; Neal, 1998). The extent to which the association between liver cancer and aflatoxin can be explained by an association between hepatitis B and aflatoxin is not clear. This relationship has been addressed in several studies (Peers et al., 1987; Yeh et al., 1989; Campbell et al., 1990) that, in general, conclude that an effect of aflatoxin in addition to hepatitis B is

functioning. According to Lutwick (1979), it is possible that AFB1 may act as cofactor to promote HBV-mediated chronic infection, cirrhosis, and liver cancer by suppressing cell-mediated immunity. More rigorous epidemiological studies on the role of AFB1 and human cancer may require the discovery of relatively HBV-free, high-dietaryaflatoxin populations, if such groups exist. A hypothetical scheme for the development of PHC is shown in Figure 11.13. The activation process of AFB1 mediated by the cytochrome P-450 system is regulated by genetic and dietary factors, and the reactive AFB1 epoxide attacks some promoter/regulation locus of HBV integrated into the host DNA. This leads to the expression of the protooncogenic property of HBV. Impairments of the immunosurveillance system by AFB1 and other mycotoxins such as trichothecenes, and tumor-promoting agents in foods, may cause the acceleration of tumorigenicity of the hepatic cells. AFB1 has been implicated as a factor in human liver cancer and classified as a probable human carcinogen by the International Agency for Research on Cancer (IARC). The current IARC classification for AFM1 is 2B (possibly carcinogenic to humans) (IARC, 1993). Teratogenicity Knowledge of the potential of mycotoxins for inducing birth defects has largely been obtained from laboratory

experimentation. In the initial study involving a mammal, Le Breton and associates (1964) reported that AFB 1 (300–400 µg per rat) given to pregnant rats caused increased prenatal death and hemorrhage at the uteroplacental junction. Fetal growth retardation followed chronic administration of smaller doses. Butler and Wigglesworth (1966) soon reported on the effects of oral aflatoxin doses of approximately 5.6 mg/kg on one of the gestation days 6–22 in Wistar rats. Animals treated before day 17 showed little effect, but fetuses from rats treated on day 17 were stunted. No malformations were reported, but the researchers described some fetuses as having “loose, wrinkled skin and unduly large heads.” Histological examination of fetuses and placentas yielded negative findings. When additional dams were treated with a more crude aflatoxin preparation, maternal liver damage was correlated with decreased fetal weight. Aflatoxin was detected in the fetal livers, however, so a direct toxic effect on the fetus could not be ruled out. Mammals other than the rat and hamster have been largely ignored in tests of aflatoxin teratogenicity. However, studies with nonmammalian species (e.g., chick embryos) have confirmed the toxicity of aflatoxins to developing systems. They also support the view that AFB1 is teratogenic only in specific organisms. Such data imply that AFB1 often displays neither specificity of action nor preferential concentration in specific tissues. At least one of these characteristics is probably necessary for the pro-

Hepatic cells HBV

(Integration into host DNA)

AFB1

Selection and hepatic injury (Activation) Mutation of protooncogenic HBV Dietary factors (Promotion) Immunosurveillance PRIMARY HEPATOCELLULAR CARCINOMA (PHC)

Figure 11.13

Multifactorial causation of aflatoxin-mediated primary hepatocellular carcinoma (PHC).

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duction of malformations rather than the more general toxic effects, such as growth retardation. As a ready reference, the toxic symptoms of aflatoxins in various animal species are summarized in Table 11.9. Nutritional Modulation of Toxicity and Carcinogenicity Even before aflatoxins were isolated and identified as toxins and carcinogens, a dietary effect on their carcinogenicity had been reported. Investigators studying dietary deficiency of methyl group donors or lipotropes (the combined deficiencies of choline and methionine, sometimes with additional deficiency of folate) reported the appearance of hepatocellular carcinomas in deficient rats and much lower incidence in methyl-supplemented controls (Copeland and Salmon, 1946; Newberne et al., 1966). Both control and deficient diets contained groundnut meal, later shown to be contaminated with aflatoxins, to which the deficient rats were highly susceptible. The mechanism by which the deficiency of methyl group donors acts is not known, but several potential mechanisms have been demonstrated. Other dietary and nutritional effects on AFB1 toxicity and carcinogenicity in laboratory animals are known. High dietary levels (20% or more by weight) of corn oil increase AFB1 hepatocarcinogenesis in rats (Newberne et al., 1979). Extensive liver damage is seen in rats fed lowprotein diets (4%) compared to those fed 20% protein diets (Madhavan and Gopalan, 1965). The low-protein-diet animals had fatty livers, bile duct proliferation, and periductal fibrosis, whereas only minor changes were seen in the high-protein-diet group. Newberne and Wogan (1968) also found a higher incidence of liver tumors in a shorter time in rats that consumed a 9% protein diet than those fed 22% protein. Diets low in vitamin A tend to show a lower incidence of aflatoxin-induced liver cancer (Newberne and Rogers, 1972). Different dietary fats may also alter the potency of aflatoxins (Concon, 1988). AFB1 hepatocarcinogenesis is also invariably influenced negatively by increased intake of selenium in the diet (Lei et al., 1990). Some compounds, such as ethionine and the cyclopropenoid fatty acids and malvalic and sterculic acids from cottonseed, are cocarcinogens to aflatoxins (Newberne et al., 1966; Lee et al., 1966; Sinnhuber et al., 1966). In contrast, urethane, diethylstilbestrol (DES), and phenobarbitone are anticarcinogenic and reduce the tumor incidence or retard tumor development (Concon, 1988). Control and Detoxification Contamination of grains with mycotoxins results in substantial losses to agriculture (Stoloff, 1976; CAST 1989).

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Every year a significant percentage of the world’s grain and oilseed crops is contaminated with aflatoxins. Unfortunately, discontinuing the feeding of aflatoxin-contaminated grain is not always practical, especially when alternate food and feedstuffs are not readily available or affordable. The prevalence of aflatoxins in a variety of foods destined for human consumption is a major concern. Interrelationships such as shown in Figure 11.14 exist among humans, animals, and aflatoxin-contaminated products. Detoxification should only be considered as a last resort, since the simplest and most practical way of reducing the contamination of foodstuffs by aflatoxins and related metabolites is to prevent the growth of those fungal species that produce them. Phillips and associates (1994) have grouped aflatoxin reduction strategies into the following five categories: 1. 2. 3. 4. 5.

Food and feed processing Biocontrol and microbial inactivation Structural degradation after chemical treatment Dietary modification of toxicity Reductions in bioavailable aflatoxin by selective chemisorption

Processing methods such as thermal inactivation, irradiation, solvent extraction and mechanical separation, density segregation, and adsorption from solution have been suggested for reducing aflatoxin content in foods. Aflatoxins are resistant to thermal inactivation and are not destroyed completely by boiling water, autoclaving, or a variety of food and feed processing procedures (Christensen et al., 1977). Aflatoxins may be destroyed partially by conventional processing procedures such as oil- and dry-roasting of groundnuts to be used as salted nuts, in confections, or in peanut butter (Lee et al., 1969; Marth and Doyle, 1979). A considerable reduction in aflatoxin levels has been associated with the limewater treatment (nixtamalization) of corn to produce tortillas (Ulloa-Sosa and Schroeder, 1969). However, subsequent studies have shown that much of the original aflatoxin is re-formed on acidification of the products (Price and Jorgensen, 1985). Although some destruction of aflatoxins has been achieved by conventional processing procedures, heat and moisture alone do not provide a very effective method of detoxification. Irradiation of contaminated products with shortwave and long-wave UV light and gamma irradiation has produced conflicting data. Although proven useful, solvent extraction methods for the detoxification of aflatoxincontaminated oilseed meals appear to be impractical and cost-prohibitive (Shantha, 1987). A new preharvest strategy for the prevention of aflatoxin contamination of groundnuts and cottonseed is the

Table 11.9 Toxic Effects of Aflatoxins in Various Animal Species Animal Poultry ducklings

Ducks

Coturnix quails Chicken

Turkey poults

Fish, rainbow trout Rodents, rats

Mice Guinea pigs Hamsters Ferrets Rabbits

Canines, dogs Ruminants, cattle

Sheep Primates, Rhesus monkey

Humans

Histopathology Extensive biliary proliferation in liver and fatty degeneration of peripheral parenchymal cells after intubation with 15 µg aflatoxin; proliferation of bile duct epithelial cells, vacuolation of focal parenchymal cells, slight to moderate fibrosis; liver infarction with 10–40 µg aflatoxin per day; mitochondrial necrobiosis and regressive changes, reticular fiber proliferation; hemorrhagic necrosis caused by aflatoxin M1 at high doses; at low doses, bile duct proliferation and extensive changes in the liver cells and renal tubular necrosis Decreased liver weights with sublethal doses of aflatoxin B1; renal parenchymal hemorrhage; liver atrophy, liver tumors with doses of aflatoxin B1; renal parenchymal hemorrhage; liver atrophy, liver tumor development after 14-month feeding; lowest tumorigenic dose is 0.03 ppm Bile duct proliferation, slight to moderate fibrosis, hepatic cell vacuolation Hydropic and fatty liver cells, cell vacuolation, slightly increased cell size, mild proliferation of ductules, hemorrhages in liver, muscle necrosis with diffused increase of sarcolemnal nuclei; decrease in RNA and vitamin A and fats with 10 ppm aflatoxin B1 for 3 weeks; decreased hatchability of eggs Liver, kidney, and myocardial congestion; duodenal catarrhal enteritis; retrogressive and regenerative changes in liver parenchyma; swollen liver cells; vacuolation in some cells; necrosis in perisinusoidal region; karyoorrhexis; karyolysis; grayish white surface and internal nodules throughout liver with fibrosis and bile duct proliferation Hyperplasia of bile duct epithelium and cholangitis; bile duct proliferation; hepatoma Liver enlargement, brownish yellow irregular nodular surfaces, red and greenish cysts, yellowish focal lesions, macroscopic grayish lesions with zones of hemorrhages in lungs; periportal liver necrosis; hyperplastic foci and preneoplastic lesions; atrophy of testicles, aspermatogenesis, retardation of fetal growth; teratogenesis; hepatocarcinoma, kidney tumors, carcinoma of glandular stomach; adenocarcinoma of colon; malignant sarcomas and fibrosarcomas at injection (subcutaneous) site Resistant to acute toxic effects even with high levels, but development of hepatomas, subcutaneous sarcomas; adenomatous pulmonary tumors Centrilobular necrosis, biliary proliferation within 48 hr; lesions in kidneys, adrenals, pancreas, and GI tract; severe edema and liver cirrhosis, hepatomas; kidney tubular reflux Liver lesions, teratogenesis Yellowish, hemorrhagic liver, fatty infiltration, centrilobular necrosis, cellular vacuolation in liver, bile duct proliferation, bile duct and hepatic cell tumors Lethal dose 65 ppm aflatoxin/kg body weight fed for 2 consecutive days; developed anorexia, retarded growth, weight loss, and death by fifth week of administration in 5-month-old rabbits at 40 µg/day Hepatic lesions, hepatitis like disease Severe tenesmus, liver fibrosis, ascites, visceral edema, centrilobular necrosis, ductal cell hyperplasia, occlusion of centrilobular veins; bile duct proliferation, chronic endophlebitis of centrilobular and hepatic veins, karyomegaly of some parenchymal cells, liver cirrhosis; epithelial nephritis and ulceration of abomasums; at levels of 0.7–1.0 µg aflatoxin/kg of feed decreased weight gains; gross evidence of liver damage at ≥ 0.7 µg/kg of feed, enlarged liver cell nuclei Relatively resistant, but hepatic parenchymal cell neoplasia, nasal carcinoma and nasal chondroma may develop; decreased fertility Fatty liver and cirrhosis; biliary fibrosis, severe fatty changes in parenchymal cells, soft enlarged livers, enlarged yellow kidneys with fat accumulation; viral hepatitis–like necrotic lesions; lethal to Macaca irus monkey at 50 µg aflatoxin B1/kg body weight Potent hepatocarcinogen, childhood cirrhosis, fatty infiltration of liver cells leading to cellular degeneration, fibrosis, hepatomegaly; acute poisoning lethal; acute hepatitis; Reye’s-like syndrome; subacute and chronic effects, e.g., primary liver cancer, chronic hepatitis, jaundice, hepatomegaly, cirrhosis; potent mutagen

Source: Modified from Concon (1988).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Animal waste

Compounded animal feed containing groundnut meal

4 µg Tc/cow/day

Cattle

0.02 mg/kg AFB1a Meat and meat products

Aspergillus flavus

1.4 ng/kg AFB1c Milk

Groundnut crop

Aflatoxin-contaminated nuts

0.07 µg/liter Mc

Groundnuts 0.015 mg/kg Tb

Human food

Storage

AFB1

Aflatoxin B1

a

U.K. Fertilizers and Feeding Stuffs Regulations

M

Aflatoxin M1

b

U.S. Peanut Industry Guidelines

T

Total aflatoxins

c

Approximate calculations

Figure 11.14

Schematic representation of the interrelations of humans, animals, and aflatoxin B1.

use of nontoxigenic strains of A. flavus and A. parasiticus fungi to compete with and exclude toxin-producing strains. Initial studies have shown that bioprevention can reduce preharvest aflatoxin contamination in these crops significantly (Cole et al., 1989; Cole and Cotty, 1990). Numerous chemicals, including acids, bases, aldehydes, bisulfite, oxidizing agents, and various gases, have been tested for their ability to degrade and inactivate aflatoxins. Although several have been found to be effective, most are impractical or potentially unsafe because of the formation of toxic residues or the perturbation of nutrient content, flavor, color, odor, texture, or functional properties of the product (Phillips et al., 1994). Two chemical approaches to the detoxification of aflatoxins that have received considerable attention are ammoniation and reaction with sodium bisulfite. The ammoniation process has been used successfully for many years in the United States, France, and Africa but has not yet been sanctioned by the U.S. Food and Drug Administration. Current research supports the use of ammoniation in an effort to reduce markedly the risk posed by aflatoxin contamination of grains and oilseeds. However, no safe

Copyright 2002 by Marcel Dekker. All Rights Reserved.

method currently exists to be used to eliminate the aflatoxin problem in the human food chain. 11.2.2

Sterigmatocystins

Sterimatocystin (ST) is a carcinogenic metabolite produced by fungi such as Aspergillus versicolor, A. sydowi, A. nidulans, Bipolaris spp., Chaetomium udagawae and C. thielavioideum, and Emericella spp. These molds are capable of producing ST in relatively large amounts on a given substrate. For example, on corn meal, up to 0.75 to 1.2 g of mycotoxin may be produced by these molds (Holzapfel et al., 1966). A. versicolor can produce as much as 1.3 g ST per 100 g of dried mycelia (Davies et al., 1960). The ability of these molds to produce larger amounts of ST may pose a greater degree of hazard from ST toxicosis, compared to that of aflatoxin, even though ST is a much weaker carcinogen than the latter (Holzapfel et al., 1966). ST has been found in moldy grain, green coffee beans, and cheese, although information on its occurrence in foods is limited. However, it appears to occur much less

frequently than the aflatoxins. In addition, analytical methods for its determination are less sensitive, and it is quite likely that small concentrations in food commodities may not always be detected. Since ST was found to be a contaminant in brown rice stored in warehouses under natural conditions (Manabe and Tsuruta, 1975), several reports have cited the presence of ST in cereals and green coffee beans. The U.S. FDA did not detect ST in an analysis of more than 500 samples in 1974–1975 (Stoloff, 1976). Vesonder and Horn (1985) detected 7.75 µg/g of ST in dairy cattle feed associated with acute clinical symptoms of bloody diarrhea and death. ST is an intermediate in the biosynthesis of aflatoxins and is much like aflatoxins in its chemical structure and biological activity. However, it is not very toxic when compared to AFB1. ST was the first naturally occurring compound identified to contain the furfuran ring system (Hamasaki and Hatsuda, 1977). A bishydrofuran ring fused to a substituted anthraquinone characterizes it. The terminal bond of ST, which is important for its mutagenic and carcinogenic activity, is similar to AFB1. Its chemical structure and those of some related compounds are shown in Figure 11.15. The most economically important member of this group is ST from A. versicolor. Other species that produce this toxin include aspertoxin (3-hydroxy-6,7-dimethoxydifuroxanthone) (Rodricks et al., 1968a, 1968b; Waiss et al., 1968), o-methylsterigmatocystin (Burkhardt and Forgacs,

1968), and dihydro-o-methylsterigmatocystin (Cole and Kirksey, 1970) from A. flavus; and 5-methoxysterigmatocystin (Holker and Kagal, 1968), 6-demethylsterigmatocystin (Elsworthy et al., 1970), dihydrosterigmatocystin (Hatsuda et al., 1972), and dihydrodemethylsterigmatocystin (Hatsuda et al., 1972) from A. versicolor. The major differences among the various sterigmatocystins are the presence or absence of unsaturation in the difurano ring system (similar to AFB1 and AFB2) and the substitution pattern on positions 6, 7, and 10 of the xanthone ring system and/or position 3 of the difurano system. Similarity in structure of ST and AFB1 suggests that these mycotoxins have a common biogenetic pathway or that the aflatoxins may be derived from ST and/or vesicolorin-type precursors (Holker and Underwood, 1964; Holker and Mulheirn, 1968; Rodricks, 1969; Biollaz et al., 1970). A. parasiticus can convert ST to AFB1 (Singh and Hsieh, 1976). The physical properties of ST are summarized in Table 11.10 together with those of several other mycotoxins described in this chapter. This particular mycotoxin crystallizes as pale yellow needles and is readily soluble in methanol, ethanol, acetonitrile, benzene, and chloroform. It reacts with hot ethanolic KOH and is methylated by methyl sulfate and methyl iodide. Methanol or ethanol in acid produces dihydroethoxysterigmatocystin. Although ST is primarily a hepatotoxic agent, its hepatotoxicity and tumorigenic potency are considerably

R1

O

OR2

R4 O O

Figure 11.15

O

OR3

R1

R2

R3

R4

H

H

CH3

H

Sterigmatocystin

H

CH3

CH3

H

O-Methylsterigmatocystin

OCH3

H

CH3

H

5-Methoxysterigmatocystin

H

H

H

H

Demethylsterigmatocystin

H

CH3

CH3

OH

Aspertoxin

Chemical structures of sterigmatocystin and related mycotoxins.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Table 11.10

Physical Properties of Selected Mycotoxins

Mycotoxin

Molecular formula

Molecular weight

Melting point, °C

Sterigmatocystin Cyclopiazonic acid Ochratoxin A

C18H12O6 C20H20N2O3 C20H18ClNO6

324 336 403

246 246 169

Citrinin

C13H14O5

250

179

Patulin Deoxynivalenol T-2 toxin Diacetoxyscirpenol Fumonisin B1 Fumonisin B2 Zearalenone

C7H6O4 C15H20O6 C24H34O9 C19H26O7 C34H59NO15 C34H59NO14 C18H22O5

154 296 466 366 721 705 318

111 131–135 150–151 162–164 Powder Powder 164

Moniliformin Tenuazonic acid

C4HO3Na C10H15NO3

120 197

Oil

Altenuene

C15H16O6

292

190–191

Alternariol Alternariol monomethy ether

C14H10O5 C15H12O5

258 272

350 267

less than those of AFB1. For example, the oral LD50 in mice exceeds 800 mg/kg. The 10-day LD50 in Wistar rats is 166 mg/kg in males, 120 mg/kg in females, and 60 to 65 mg/kg for intraperitoneal administration in males. The intraperitoneal 10-day LD50 for vervet monkeys is 32 mg/kg (Ueno and Ueno, 1978; Van der Watt, 1974). Its hepatocarcinogenic activity is approximately one tenth that of AFB1 (Van der Watt, 1974). As with aflatoxins, mice are less susceptible to ST than are rats. Chronic symptoms of ST poisoning include induction of hepatomas in rats, pulmonary tumors in mice, and renal lesions and alterations in the liver and kidneys of African green monkeys. Rats fed 5 to 10 mg/kg ST for 2 years showed a 90% incidence of liver tumors (Ohtsubo et al., 1978). Toxic effects of ST-fed laboratory animals have included kidney and liver damage and diarrhea (Ciegler and Vesonder, 1983). Cattle that had bloody diarrhea and loss of milk production and in some cases died were found to have ingested feed containing A. versicolor and high levels of ST of about 8 mg/kg (Vesonder and Horn, 1985). Several in vitro mutagenicity tests suggest that ST is, like AFB1, a mutagenic agent that covalently binds to

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Ultraviolet absorption, nm 15 200 (325) benzene 20 400 (282) methanol 36 800 (213) 6400 (332) ethanol 22 280 (222) 8279 (253) 4710 (310) ethanol 14 600 (275) ethanol Maximum at 218 ethanol Maximum at 187 cyclohexane None Low Low 29 700 (236) 13 910 (274) 6020 (316) ethanol 5000 (218) 12 500 (277) acid methanol 11 500 (240) 14 500 (280) methanol 30 000 (240) 10 000 (278) 6600 (319) ethanol 38 000 (258) ethanol

DNA at approximately 20% to 30% of the level observed with AFB1 (Purchase and Van der Watt, 1968). The toxicity of ST analogs on primary cell culture was greater for the compounds containing the ∆1,2-furobenzofuran ring system than those for the compounds containing a saturated furobenzofuran ring system (Englebrecht and Altenkirk, 1972). These researchers also showed that a carbonyl group unsaturated in the α,β position and an unsaturated bond in the ∆1,2 position are essential to their carcinogenicity. Also, a methoxy group at position 6 enhances, whereas the one at position 7 decreases the toxicity of these compounds. ST does not appear to be teratogenic; however, conclusive data are lacking (Hood and Szczech, 1983) Wannemacher and colleagues (1991) have reviewed the acute toxicity, carcinogenicity, and metabolism of ST in relation to aflatoxins and other hepatotoxic mycotoxins. 11.2.3

Ochratoxins

Ochratoxins (OTs) are metabolites produced by many Aspergillus and Penicillium species. The Aspergillus species

lites of the culture of A. ochraceus. The toxicity of the esters of OT-A is similar to that of OT-A, whereas those of OT-B are not toxic (Ueno, 1987). Mellein and 4-hydroxymellein, which are structurally related to the dihydroisocoumarin moiety of OT-A, have been isolated from A. ochraceus and other strains.

include A. ochraceus, A. sulphureus, A. sclerotinium, A. alliaceus, A. melleus, A. ostianus, and A. petrakii. The Penicillium species include P. purpurescens Sopp, P. commune Thom, P. viridicatum Wes, P. palitans, P. cyclopium Westing, and P. variabile (van Walbeek et al., 1969; Ueno, 1987; van Egmond and Speijers, 1998). The production of ochratoxins depends on environmental conditions such as water activity and temperature. For A. ochraceus high temperatures (12°C–37°C) are necessary, whereas frigophilic penicillia (4°C–31°C), particularly P. viridicatum, also produce ochratoxins in areas with a colder climate. The latter is a common storage fungus in areas such as Canada, Eastern Europe, Denmark, parts of South America, and the United Kingdom.

Occurrence in Foods The frequency and level of OT-A contamination of agricultural commodities are summarized in Table 11.11. OTA often occurs in stored cereals and has been found in other foods, including coffee, beer, dried fruit, wine, cocoa, and nuts. Speijers and van Egmond (1993) have comprehensively reviewed the literature on the worldwide occurrence of ochratoxins. Sensitive and reliable analytical methods now routinely detect ochratoxins at less than 1-µg/kg levels (ppb).

Chemical Characteristics The ochratoxins are a group of seven closely related fungal metabolites (Figure 11.16). OT-A, the 7-carboxy-5chloro-8-hydroxy-3,4-dihydro-3R-methyl isocoumarinamide of L-β-phenylalanine, is the most important and most common. It is also the most toxic of ochratoxins and is produced in highest yield (Neshiem, 1969). OT-B is a less toxic metabolite and lacks the C-5 chlorine; the methyl and ethyl esters of both OT-A and OT-B are minor metabo-

Physical and Chemical Properties OT-A is a colorless crystalline compound, which exhibits blue fluorescence under UV light. It is highly soluble in polar organic solvents, is very slightly soluble in water, and dissolves in aqueous sodium bicarbonate. On acid hydrolysis, it yields phenylalanine and an optically active

OR

O

OH

O

O CH2

CH

NH

O

C

CH3 R'

Figure 11.16

R"

R

R'

R"

H

Cl

H

Ochratoxin A (OT-A)

H

H

H

Ochratoxin B (OT-B)

Et

Cl

H

Ochratoxin C (OT-C) (OT-A ethyl ester)

Me

Cl

H

OT-A methyl ester

Et

H

H

OT-B ethyl ester

Me

H

H

OT-B methyl ester

H

Cl

OH

4-OH OT-A

Chemical structures of ochratoxin A and related fungal metabolites.

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Table 11.11

Natural Occurrence of Ochratoxin A (OT-A) in Agricultural Commodities

Commodity

Country

Samples, no.

Contamination, %

Corn Wheat (red spring) Corn Wheat Sorghum Groundnut Wheat (red winter) Wheat (red spring) Animal feeds and tissues Coffee beans Moldy bread Porcine kidney Porcine serum Porcine serum Nuts Beans Corn

United States Canada India India India India United States United States Canada United States Italy Poland Poland Sweden Germany Bulgaria Bulgaria

4 21 24 24 18 291 286 496 267 1 122 388 279 150 24 22

100 30–50 8 12.5 11 1 2.8 1.1 7.1 50 38 16 5 16.7 27.3

OT-A, µg/kg 110–150 20–100 30–50 50–70 50–200 25–35 15–115 50–200 22–360 80 000 ≥1 1–450 ≥2 0.2–8.6 (ng) 25–27 25–35

Source: Compiled from Ueno (1987).

lactone acid, ochratoxin α. OT-A is a moderately stable molecule and survives most food processing to some extent (Scott, 1996). In biological systems, it binds to serum albumin. Metabolism OT-A is metabolized to isocoumarin and hydroxylated metabolites in both in vivo and in vitro systems. The half-life of the toxin is about 55 hours after either oral or intravenous administration in rats, and approximately 56% of the toxin is excreted via urine and feces, both as the free metabolite and hydrolyzed as ochratoxin α 120 hours after dosing (Galtier et al., 1979). Biotransformation pathways for OT-A metabolism are shown in Figure 11.17. It is likely both that OT-A is directly toxic and that other toxicities result from metabolism. In vitro studies, including primary cultures of hepatocytes, have provided evidence that toxicological effects may be linked to biotransformation processes. On biotransformation, OT-A is converted to 4(R)-4-hydroxyochratoxin A (4R), the major metabolite, and 4(S)-4-hydroxy-ochratoxin A (4S), the minor metabolite (Figure 11.17). In structure-activity toxicity studies in vitro in eukaryotic cell systems (HeLa cells) and in rats and mice in vivo using OT-A, its opened lactone ring form and several analogs have shown that the acute toxicity of this mycotoxin is attributable to its isocoumarin moiety. The lac-

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tone carboxyl group may also be involved in its toxicity (Neal, 1998). Toxicological Characteristics In acute toxicity studies, LD50 values vary greatly in different species (Table 11.12). Dogs appear to be especially susceptible to OT-A poisoning. Kuiper-Goodman and Scott (1989) have reviewed the subacute and subchronic effects of several feeding trials lasting up to 90 days or more. OT-A is a potent kidney toxin, and both swine and poultry are very sensitive to it (Jacques, 1988). It acts principally on the first part of the proximal tubules in the kidney and induces a defect in the anion transport mechanism on the brush border of the proximal convoluted tubular cells and basolateral membranes, thus leading to the release of membrane-bound enzymes (Endou et al., 1986). However, its dechloro derivative, OT-B, is nontoxic. A nephrotoxic effect has been demonstrated in all mammalian species tested to date (Scudamore, 1998). Dietary concentrations as low as 200 ppb of OT-A over a 4-month period in pigs produce nephropathy that is characterized by degeneration of the proximal tubules, intestinal fibrosis, and hyalinization of some glomeruli (Krogh et al., 1979). OT-A-induced nephropathy of chickens indicates similar signs, with enlarged and pale kidneys (Ellings et al., 1975). The disease has been experimentally produced in several other species (Carlton and Krogh, 1979).

OH

O

HOOC

Ochratoxin α

O H CH3 Cl OCHRATOXIN A COOH CH2

C H

O

HN

OH

O (4R)-4-Hydroxyochratoxin A

C

O OH

O

COOH

O

CH3 CH2

Cl

HN

C H

C

O H CH3 Cl

COOH CH2

C H

HN

O

OH

C

O O H COOH

CH2OH 10-Hydroxyochratoxin A

OH

H

Cl

CH2

C H

HN

O

OH

C

O O H CH3

(4S)-4-Hydroxyochratoxin A

Figure 11.17

Cl

HO

H

Biotransformation pathways for OT-A metabolism in animals.

Although no true cases of mycotoxic nephropathy in humans have been yet reported, it is quite likely that this potent nephrotoxin, which produces renal disease in several species, can also induce renal disorders in humans on exposure. Because of the distinct similarity between the so-called Balkan nephropathy and the ochratoxin-induced porcine renal disease, OT-A has been suggested to be a toxin associated with Balkan endemic nephropathy (Krogh, 1974; van Egmond and Speijers, 1998). This endemic fatal renal disease in humans occurs in certain rural areas of Bulgaria, Romania, and Yugoslavia. OT-A has been found in human blood at levels of about 1–2 µg/kg in about 50% of the samples in various Western European countries, and in mother’s milk at subµg/kg levels (van Egmond and Speijers, 1998). The significance of the detection of OT-A in human body fluids is not yet clear.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Experimentally, OT-A has been found to be a potent inhibitor of protein synthesis, an immunosuppressive agent, a teratogen, a mutagen, and a carcinogen in laboratory animals (Ueno, 1987; Dirheimer, 1996). IARC (1993) has classified OT-A as a 2B carcinogen (possibly carcinogenic to humans). OT-A is both embryotoxic and teratogenic in a variety of species, including mice, rats, hamsters, chicks, and Japanese quail, but not in pigs (Kuiper-Goodman and Scott, 1989; Scudamore, 1998). The central nervous system is one of the most susceptible targets and is affected at the time of early oncogenesis. This mycotoxin must therefore be viewed with suspicion as a potential human teratogen. The presence of OT-A in foodstuffs is clearly undesirable, although few countries seemed to have introduced statutory control by the late 1990s (FAO, 1997). Despite

11.2.4

Table 11.12 Lethal Dose in 50% of Sample Values for Ochratoxin A in Various Species

Fumigatin (Figure 11.18a) was first noted to be a product of A. flavus isolated from Indian soil that was responsible for pH color changes in the culture broth of the strain (Anslow and Raistrick, 1938). It was also produced by A. fumigatus (Anslow and Raistrick, 1938; Pettersson, 1963). Fumigatin is one of a group of toluquinone compounds (Wilson, 1971). It has marked in vitro inhibitory properties against several gram-negative and gram-positive bacteria. The effective range is approximately the same for both types. Not much is known about the animal toxicity of fumigatin. Since it is structurally similar to quinines and hydroquinones, fumigatin is thought to have toxicity similar to that of the quinones.

LD50 values, mg/kg body weighta Species Mouse Rat Rat neonate Dog Pig Chicken

Oral

Intraperitoneal

Intravenous

46–58.3 20–30.3 3.9 0.2 1 3.3

22–40.1 12.6

25.7–33.8 12.7

Source: Compiled from Kuiper-Goodman and Scott (1989) and Scudamore (1998).

the uncertainties, the FAO/WHO Joint Expert Committee on Food Additives has established a provisional tolerable weekly intake (PTWI) level of 112 ng OT-A/kg body weight (WHO, 1991). A working group of Nordic countries proposed a much lower tolerable daily intake of OT-A of 5 ng/kg body weight (NNT, 1991). O

(a) HO

(b)

CH3

Fumigatin

11.2.5

Aspergillic Acid

Aspergillic acid (Figure 11.18b) was first discovered and named by White (1940) and White and Hill (1943). It is

(c)

CH3

N

CH3

N

CH3 H3C

H3CO

N CH3

O

N

O

CH3

O

OH

Aspergillic acid (hydroxy pyrazinone form)

H 3C

O

(e)

CH2OH

O

H3C

Aspergillic acid (hydroxamic acid form)

Fumigatin

(d)

CH3

OH

CH3

OH

O HO

CH3 O Kojic acid

COOH

(f)

O

CH3 OCOCH3

Terreic acid CH3

(g) HOOC

O

Fumagillin (CH

OCOCH3

O CH3

CH)4COO

O

CH3 H3CO

CH2 H 3C

Figure 11.18

H C

C

CH3

O

Chemical structures of miscellaneous mycotoxins produced by various Aspergillus species.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Helvolic acid

the first of a number of closely related pyrazine fungal metabolites reported. It is a major metabolite of certain strains of A. flavus and other Aspergillus species. Aspergillic acid can exist in either the hydroxamic acid form (2hydroxy-pyrazine-1-oxide, Figure 11.18b) or the 1-hydroxy-2-pyrazinone form (Figure 11.18c). Toxicity of aspergillic acid is related to the hydroxamic acid functionality. Little effect on toxicity is observed for differences in the 3- and 6-position side chain substituents. It is acutely toxic to mice at LD50 values of 100–150 mg/kg, intraperitoneally, but has no chronic effects at sublethal dosages; convulsions are the major manifestation of toxicity (White and Hill, 1943). Chelation of physiologically important ions, such as calcium, by aspergillic acid appears to be the likely mechanism of its toxic action (Wilson, 1971, MacDonald, 1973). 11.2.6

Kojic Acid

Kojic acid (Figure 11.18d) is a relatively common metabolite of several species of Aspergillus, including A. flavus and A. parasiticus. Additionally, certain species of Penicillium, such as P. citrinum and P. rubrum, also produce kojic acid as a secondary metabolite. The name of this acid is derived from koji, the mold starter used in Asian food fermentations. Its chemical structure can be defined as 5hydroxy-2-(hydroxymethyl)-4H-pyran-4-one. It is characterized by a γ-pyrone nucleus substituted at positions 2 and 5 with a hydroxymethyl and a hydroxy group, respectively. Kojic acid is classified as a convulsant mycotoxin, but a relatively large quantity is required to produce severe intoxication or death in animals. For example, a group of mice given kojic acid at dosage levels of 250 to 1250 mg/kg exhibited only mild intoxication but complete immobilization lasting for 3 hours without any lethal effects (Wilson, 1966). The LD50 in 17-g mice was 40 mg through intraperitoneal injection (Morton et al., 1945). Natural cases of kojic acid toxicosis either in animals or in humans have not yet been reported. 11.2.7

Terreic Acid

Wilkins and Harris discovered terreic acid, 2,3-epoxy-6hydroxy-toluquinone (Figure 11.18e), produced by A. terreus, in 1942. It has in vitro activity against several bacteria and fungi as well as a protozoan (Trichomonas vaginalis) that infects the human vaginal tract. The intravenous LD50 in mice has been reported to be 70–119 mg/kg (Cole and Cox, 1981).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

11.2.8

Helvolic Acid

Previously known as fumigacin, helvolic acid (Figure 11.18f) is a toxic secondary metabolite found in some isolates of A. fumigatus, Cephalosporium caerulens, and Emericellopsis terricola (Cole and Cox, 1981; Dhumal and Salunkhe, 1992). Repeated intraperitoneal injections to 20-g mice caused peritonitis and superficial liver lesions, with minimal effects on kidneys (Wilson, 1971). Helvolic acid is absorbed from the subcutaneous tissues and gastrointestinal tract and is excreted in an active form in the urine and bile. 11.2.9

Fumagillin

Fumagillin (Figure 11.18g), a metabolite of A. fumigatus, possesses antibacteriophage and amebicidal properties against Entamoeba histolytica (McCowen et al., 1951, Wilson, 1971). However, because of its toxic side effects, such as the peeling of the skin from the palms and soles and sensory disturbances in the hands, feet, and ears, fumagillin has not been approved for use in humans. Fumagillin is not a highly toxic substance since orally dosed mice can tolerate doses up to 2000 mg/kg body weight (Dhumal and Salunkhe, 1992). 11.2.10 β-Nitropropionic Acid Many species of the genus Aspergillus, including A. flavus and A. parasiticus, produce β-nitropropionic acid as a secondary metabolite. It is also known as hiptagenic acid or bovinocidin. The LD50 value for mice is about 250 mg/kg; prominent signs of toxicity are rapid respiration followed by apnea, incardination, marked dilatation of subcutaneous and visceral blood vessels, and mottled liver (Wilson, 1971). 11.2.11 Gliotoxin Gliotoxin, an epidithiodioxopiperazine mycotoxin, is a metabolic product of A. fumigatus, A. terreus, A. chevalieri, Trichoderma lignorum, Penicillium obscurum, and P. terlikowskii (Cole and Cox, 1981; Frame and Carlton, 1988). It possesses potent immunosuppressive activity in vitro (Mullbacher and Eichner, 1984). It is acutely toxic to rabbits (LD50 = 45 mg/kg), mice (50 mg/kg), rats (50 to 65 mg/kg) and hamsters, in which the mortality rate is normally more than 50% after the oral administration of doses ranging from 15 to 35 mg/kg body weight (Cole and Cox, 1981). Clinical signs of toxicity are usually nonspecific; they include decreased activity, tachypnea, and prostration

and, in some cases, diarrhea, low blood pressure, hematuria, and congestion of the liver and kidneys. 11.2.12 Fumitremorgins The group of fumitremorgins includes three toxins: fumitremorgins A, B, and C. All of them are produced by A. fumigatus; the first two are also produced by certain strains of A. caespitosus (Cole and Cox, 1981). Fumitremorgin A is a potent neurotropic mycotoxin, which on intravenous injection causes tremors and generalized tonic-clonic convulsions in experimental animals (Nishiyama and Kuga, 1989). A dose as small as 0.1 mg/kg can induce tonic-clonic convulsions in rabbits and also in mice. Similarly, fumitremorigins B and C are tremorgenic in mice (1 mg per mouse, intraperitoneally) and in day-old cockerels (25 mg/kg, orally), respectively (Cole and Cox, 1981). 11.2.13 Maltoryzine The mycotoxin maltoryzine has been isolated from A. oryzae; it has an intraperitoneal LD50 value of 3 mg/kg in mice, in which it causes muscular paralysis and swollen yellow liver. It has been implicated in at least two cases of feed poisoning in cattle (Iizuka and Iida, 1962; Iizuka, 1974).

11.3 MYCOTOXINS OF PENICILLIUM SPECIES Penicillium, like Aspergillus, comprises many toxigenic species. They are as common as Aspergillus molds, and under favorable conditions are found in practically all types of food products. Penicillia have been found in food products implicated in animal mycotoxicoses. For example, moldy corn toxicosis of various forms seems to be associated with Penicillium species, such as P. rubrum, P. cyclopium, P. viridicatum, and P. urticae (Concon, 1988). Some of the important mycotoxins produced by Penicillium spp. are described in the following sections. 11.3.1

Patulin

Patulin was discovered in a long, intensive search for new antibiotics. It was first isolated in crystalline form from P. claviforme and given the name clavicin. Identical materials from other fungal species were isolated by a number of investigators, each of whom assigned a name based on the fungal source. Therefore, the literature contains many synonyms for patulin, including clavacin, expansin, myocin, penicidin, leukopin, and tercinin. Woodward and Singh

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(1949) finally suggested the name patulin with elucidation of the correct chemical structure in 1949. Patulin-producing fungi belong principally to the genera Penicillium and Aspergillus (Table 11.13). P. expansum is probably the most common producer of patulin in the environment. The optimal temperature for the growth of this fungus on whole wheat is 10°C and between 20°C and 25°C for the biosynthesis of patulin. In a screening for production of antifungal and antibacterial molecules by 850 strains of fungi, patulin was produced by 58 fungi, including Mucorales and Fungi Imperfecti. Natural substrates that allow production of patulin by Penicillium spp. include malt, barley, rice, wheat straw, grass silage, soil containing root bark, leaves of apple tree, root residues from other fruit trees, and apples. Occurrence in Foods Brian and associates (1956) first identified food products naturally contaminated with patulin by using antimicrobial methods. Patulin was found at concentrations in excess of 1000 ppm in the sap of apples naturally contaminated with P. expansum. Several researchers have reported the natural contamination of apple products with patulin (Drillean and Bohuon, 1973; Eyrich, 1975; Harwig et al., 1973). Thus, contamination of apple juice and other apple products with patulin has occurred when unsound apples were used. The

Table 11.13 Patulin-Producing Fungi and Synonyms for Patulin Fungal species P. expansum Penicillium spp. P. claviforme P. patulum P. melinii P. urticae P. equinum P. novae-zeelanliae P. leucopus P cyclopium, P. griseofulvum P. equinum P. divergens P. lapidosum Aspergillus clavatus A. giganteus A. terreus Byssochylamys nivae Mold species

Name given to substance Expansine, clavacin Penicidin Claviformin, clavatin Patulin, clavatin Clavacin Clavacin Clavacin Clavacin Patulin Patulin Patulin Patulin Patulin Patulin Clavacin, patulin Gigantic acid, claviformin Clavacin Clavacin Myocin c

increased mechanization of apple harvesting has increased the possibility of including unsound apples in processing. The results of a limited survey of cider mills revealed that patulin contamination depended on the proportion of decayed apples used in making fresh apple cider (Wilson and Nouvo, 1973). Frank (1977) reported finding patulin in spontaneously molded pears, peaches, apricots, bananas, pineapples, and grapes. Buchanan and colleagues (1974) also reported detection of patulin in mechanically damaged pears and grapefruit. Patulin has been found in commercially available apple products. Scott and coworkers (1972) found 1 ppm patulin from 1 of 12 commercially available apple cider samples in Canada. Kiermeier (1985) has summarized the analytical data from more than 67 reports during 1980–1983; of 356 samples analyzed, 16 apple juice and related products were contaminated with more than 50 ppb of patulin. Patulin has been reported in spontaneously molded baked goods, including bread, at concentrations of 100 to 300 ppb (Reiss, 1972, 1973). It has also been found in unroasted cocoa beans, grape juice, and commercial tomato paste. Food commodities that have been contaminated with patulin-producing fungi are listed in Table 11.14. The established tolerance level of patulin is zero in all foods in Belgium and 50 ppb in apple juice in Norway, Sweden, and Switzerland (Schuller et al., 1982, 1983). In most countries, patulin is not usually covered by statutory regulation, but quality is sometimes controlled by the setting of a “guideline” or a “recommended” maximal conTable 11.14 Food Commodities Contaminated with PatulinProducing Fungi Wheat flour Refrigerated dough products Cereals and legumes

Pecans Fruits (apricots, crab apples, persimmons, pears, grapes, apples) Fruit juices Meat Poultry feed Cheese, Swiss Cheese, Cheddar Bread

Aspergillus terreus, A. clavatus, P. patulum, P. cyclopium A. terreus, P. urticae Penicillium expansum, P. urticae, A. terreus, A. clavatus, Byssochlamys nivea P. expansum P. expansum, B. nivea

B. nivea P. expansum, P. urticae, P. melinii, P. claviforme P. patulin, P. cyclopium Penicillium spp. Penicillium spp. P. patulum, P. cyclopium

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centration, commonly set at 50 ppb in most countries. On the basis of reproduction and long-term carcinogenicity studies in rats and mice, the Joint Expert Committee on Food Additives of the World Health Organization has allocated a provisional tolerable weekly intake of 7 µg patulin/kg body weight (WHO, 1991). Chemical Characteristics Patulin is a β-unsaturated lactone, 4-hydroxy-4H-furo(3,2C)pyran-2(6-OH)one, with an empirical formula of C7H6O4 and a molecular weight of 154 (Figure 11.19a). Its chemical structure was elucidated by Woodward and Singh (1949) and later confirmed by Dauben and Weisenborn (1949). Patulin is optically inactive in spite of having an asymmetrical carbon atom in its structure. It can be isolated as colorless to white crystals from ethereal extracts. It is soluble in water and polar organic solvents. It undergoes all the chemical reactions expected of a secondary alcohol, including esterification to form benzoate, cinnamate, and monoacetate. It reacts as a simple carbonyl compound after ring opening at the hemiacetal function and can form semicarbazone, oxime, and phenylhydrazone. Patulin is generally stable in acidic solutions at pH 2 or below. It is, however, unstable in alkali and is slowly decomposed in distilled water and methanol at ambient temperatures. Patulin is stable in grape and apple juice at 22°C and in dry corn but is unstable in orange juice, flour, baked bread, wet corn, ground sorghum, cheese, or apple juice fermented with Saccharomyces spp. The instability of patulin and its disappearance from various food commodities have been attributed to patulin reactivity with sulfhydrylcontaining amino acids in proteins (Ciegler, 1977). Metabolism Distribution and metabolism studies of patulin are limited, and no metabolic products have yet been identified. It is quite likely that the metabolic fragments or conjugated metabolites of patulin either are bound to the cell membranes or become incorporated into the cellular components. Results of metabolic studies using 14C-patulin show that it is excreted principally via the feces and urine. The major retention and storage site is the erythrocyte (McKinley and Carlton, 1991). Patulin inhibits aerobic respiration in several systems (Singh, 1967; Stott and Bullerman, 1975). The available evidence supports the hypothesis that patulin acts on respiration and that a modified form of patulin may be the real toxic agent. The exact site of patulin action on respiration is not known. It is quite possible that such a site may occur in the respiratory chain before the terminal electron

transport chain of aerobic respiration. Inhibition of anaerobic bacterial growth also supports a prior site of action. Cytological effects of patulin can be interpreted on the basis of respiratory inhibition. Inhibitors of respiration, such as cyanide and dinitrophenol, also inhibit mitosis, but anabolic systems, such as nucleic acid synthesis and protein synthesis, are relatively insensitive to the effects of patulin. Toxicology Patulin possesses wide-spectrum antibiotic properties and has been tested extensively in human subjects to evaluate its ability to treat the common cold. However, its effectiveness has never been proved and its use to treat medical conditions has not been pursued because it irritates the stomach, causing nausea and vomiting. No natural outbreak of disease in animals or humans has been definitely attributed to patulin contamination. However, in several disease outbreaks in cattle, known patulin-producing fungi were isolated from suspect feedstuffs. The LD50 values of patulin have been determined in various animal species (McKinley and Carlton, 1991). The ranges of LD50 values by routes of administration were oral, 25 to 46; subcutaneous, 10 to 33; intraperitoneal, 5 to 15; and intravenous, 15 to 25 mg/kg body weight. Clinical signs after patulin administration include restlessness, increased activity, and dyspnea within 5 minutes of subcutaneous administration; and locomotor difficulties, abdominal pain, and dyspnea after oral, intraperitoneal, and intravenous administration. Convulsions preceding death were reported after intravenous administration. In acute and short-term studies patulin causes gastrointestinal hyperemia, distention hemorrhage, and ulceration. Patulin is also toxic to the rabbit, guinea pig, dog, nonhuman primate, cat, chicken, pigeon, sheep, frog, and fish. It is also toxic to a wide variety of microorganisms and cell types in vitro. Patulin injected in large amounts over a 2-month period was carcinogenic, resulting in induction of sarcomas at the injection site (Dickens and Jones, 1961). In longterm studies at lower dose levels, these effects were not observed. Patulin has been shown to be immunotoxic and neurotoxic. The International Agency for Research on Cancer (IARC, 1986) concluded that no evaluation could be made of the carcinogenicity of patulin to humans and that there was inadequate evidence in experimental animals. Similarly, patulin has not yet been found to be teratogenic in mammals, although it can be fetotoxic and is teratogenic to the chick. Since it is both a highly toxic inhibitor of RNA polymerase and possibly carcinogenic as well, patulin exposure should obviously be avoided

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regardless of whether or not it is a likely teratogen in humans. 11.3.2

Citreoviridin

The mycotoxin citreoviridin (Figure 11.19b) was first isolated from P. citreoviride growing on rice and later from many other species of Penicillium (Hirata, 1947; Ueno, 1985). Yellowed rice infected by fungi is the major source of this mycotoxin. Citreoviridin is a neurotoxin that causes paralysis in the extremities of laboratory animals, followed by convulsions and respiratory arrest (Ueno and Ueno, 1972). The LD50 values of citreoviridin in male mice were reported to be 11 (subcutaneous), 7.5 (intraperitoneal), and 29 (oral) mg/kg body weight. Acute signs of toxicity include early onset of progressive paralysis in the hindlegs and flank followed by vomiting, convulsions, and respiratory arrest. Similar signs of poisoning are seen in other species, including cats and dogs. Some of these toxic manifestations may be attributed to inhibition of mitochondrial ATPase activity by citreoviridin (Gause et al., 1981). 11.3.3

Penicillic Acid

Penicillic acid (Figure 11.19c) was first isolated from a mold culture in 1913 as part of a study of corn deterioration (Alsberg and Black, 1913). It is produced by a large number of Penicillium species and also by some species of Aspergillus (Cole and Cox, 1981). Toxigenic strains of these fungi produce substantial amounts of penicillic acid in corn, rice, barley, sorghum, and oats. It also occurs naturally in Swiss cheese (Bullerman, 1976). Although it has significant activity against many gram-negative and a few gram-positive bacteria, its potential as an antibiotic diminished when it was found to be too toxic in clinical use (Oxford, 1942). The LD50 of penicillic acid in mice by oral, intravenous, and subcutaneous routes was found to be 600, 250, and 110 mg/kg, respectively (Murnaghan, 1946). Major manifestations of toxicity include CNS signs and liver lesions. The mechanism of toxicity of penicillic acid may involve the inhibition of sulfhydryl group–containing enzymes. It also shows synergism with other mycotoxins in exhibiting and enhancing their toxic effects. The presence of penicillic acid as a contaminant in foods and feeds is of concern because of its carcinogenic properties (Dickens and Jones, 1963b). In contrast to its carcinogenic potential, penicillic acid also seems to possess antitumor activity (Suzuki et al., 1971).

O

(b) Citreoviridin

OCH3

O

CH3

CH3

HO OH

O

O

OH

O

(a) Patulin

CH3

OCH3

OCH3

O

HOOC

HO C

CH3

OH

H3CO

H3C

O CH3

O

O O

O

H3CO

O

O CH3

Cl

CH2

CH3

(d) Griseofulvin

(c) Penicillic acid

OH

OH

O

CH3 CH3

(e) Citrinin

OH

O

H3C

H3C

O

OH

(f) Luteoskyrin OH

CH2OH O H N

O C2H5 N H HN

O

OH

O

O

OH

H N

COOH O

O O

OCH3

N CH3

(g) Islanditoxin Cl

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CH3

Cl

(h) Mycophenolic acid

CH3 O N

H3C

OHO

CH3 O

HO

CH3

OH

O

NH

(i) Decumbin

H3C(CH2)5

(j) Cyclopiazonic acid

OH OH

O O

O

H3C

OCH3 OH

O

O

OH

O

O O O

O

HO

OH

O H3CO

CH3

OH O

(k) Rubratoxin A

(m) Viomellein

O

H3C(CH2)5

OH OH O

O

O OCH3

H3C

OH O

O

O

O

O

O

O O O

OH

O H3CO

OH O

(l) Rubratoxin B

(n) Xanthomegnin

O

Figure 11.19

CH3 O

Chemical structures of mycotoxins produced by Penicillium species.

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11.3.4

Griseofulvin

Griseofulvin (Figure 11.19d) is a metabolic product of P. griseofulvum and several other species of Penicillium. It is used as a systemic therapeutic agent for cutaneous fungal infections. In spite of the clinical effectiveness of griseofulvin and the ability of humans and animals to tolerate relatively large doses given daily for several weeks, the serious nature of toxic response observed has restricted its therapeutic use somewhat. Toxic symptoms of griseofulvin poisoning include angioneurotic edema, erythema, urticaria, vesicular and macular eruptions, and photosensitivity of the skin. Other acute toxicity signs include nausea, vomiting, diarrhea, vertigo, blurred vision, headache, transient leukopenia, granulocytopenia, punctate basophilia, and monocytosis (Dhumal and Salunkhe, 1992). Griseofulvin is carcinogenic and has been found teratogenic in rats and cats (Scott et al., 1975). 11.3.5

Citrinin

Citrinin (Figure 11.19e) was first isolated from P. citrinum by Hetherington and Raistrick (1931). It is produced by many species of Penicillium and Aspergillus. The most important fungus is P. citrinum, which was reported as one of the causative fungi of “yellow rice toxicosis” in Japan (Saito et al., 1971). Other Penicillium species that produce citrinin include P. fellutanum, P. lividum, P. implicatum, P. jeneni, P. citreoviride, P. steckni, P. expansum, P. velutinum, P. canescens, P. notatum, P. viridicatum, P. palitans, and P. claviforme. The aspergilli that produce this mycotoxin include A. terreus, A. niveus, A. candidus, Clavariopsis aquata, and Blennoria spp. Citrinin is (3R-trans)-4,6-dihydro-8-hydroxy-3,4,5trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid (Figure 11.19e). TLC and HPLC can isolate it. Nuclear magnetic resonance has been employed to confirm its structure in the presence of other mycotoxins. Citrinin crystallizes as lemon-colored needles, melting at 172°C. It is sparingly soluble in water but soluble in dilute sodium hydroxide, sodium carbonate, or sodium acetate; in methanol, acetonitrile, and ethanol; and in most other polar organic solvents. It is likely to be degraded by heat and alkali. Citrinin has been found in Indian groundnuts infected with A. flavus, P. citrinum, and A. terreus (Subramanyam and Rao, 1974). Citrinin-producing strains of Penicillium spp. also have been found as contaminants of sausage (Ciegler et al., 1972). Citrinin often occurs in conjunction with ochratoxin A, another mycotoxin capable of altering renal function. The mycotoxin is found mainly in rice and other cereals, such as barley, wheat, rye, and oats.

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Citrinin is rapidly absorbed irrespective of the route of administration and localized in the liver and kidneys. Under normal conditions, citrinin and/or its metabolites possibly undergo enterohepatic circulation with ultimate disposition through the urine, with the liver as the primary organ of metabolism (Reddy and Berndt, 1991). Similarly to OT-A, citrinin is a potent nephrotoxin. A renal disease, porcine nephropathy, was associated with the feeding of moldy cereals to farm animals (Larsen, 1928). This disorder of the kidney function was later reproduced by feeding contaminated barley and rye to pigs (Nielsen and Hesselager, 1965) and by feeding purified citrinin (Krogh et al., 1973). The nephropathy is characterized by degeneration of the tubules, with subsequent cortical fibrosis along with functional impairment of tubular activity. Similar nephrotoxic action of citrinin is observed in guinea pigs, turkeys, ducklings, beagle dogs, and swine. The mouse is relatively resistant, whereas the pig is relatively sensitive to the nephrotoxic effects of citrinin. The LD50 of citrinin was found to be about 50 mg/kg for oral administration to rat, 35 to 58 mg/kg (intraperitoneally) to the mouse, and 19 mg/kg (intraperitoneally) to the rabbit. Scott (1977) has reviewed the toxicologic characteristics of citrinin. Citrinin alone is not tumorigenic in rats, except that it synergistically increases the renal tumor induced by a nephrotoxic chemical, N-(3,5-dichlorophenyl)succinimide (DDPS) (Shinohara et al., 1976). Citrinin is an embryocidal, fetotoxic, and mildly teratogenic compound (Reddy and Berndt, 1991). Mice exposed to citrinin (30 or 40 mg/kg) on gestation day 6, 7, 8, or 9 experienced reduced fetal weight gain, increased fetal lethality, and increased maternal lethality rates. The combination of citrinin with OT-A increased the occurrence of gross, soft tissue, and skeletal malformations (Mayura et al., 1984). Little appears to be known of citrinin’s mechanism of action, however, and much more extensive testing would be required to establish its teratogenicity or its lack thereof in mammalian species. 11.3.6

Luteoskyrin and Islanditoxin

Luteoskyrin, a yellow anthraquinonelike pigment synthesized by P. islandicum Sopp, contaminates rice, maize, and other cereals, especially in Eastern countries, where human malignant and nonmalignant hepatomas are more frequent. P. islandicum was first isolated in 1912 from skyr, a kind of yogurt produced in Iceland, and the pigments isolated from this fungus were named after the domestic name. Luteoskyrin, a yellow lipophilic toxin, is a substituted bis-polyhydroxy-dihydro-anthraquinone (Figure

11.19f). Islanditoxin is a hydrophilic toxic cyclic peptide (Figure 11.19g). The toxicity of luteoskyrin varies according to the dose and diet, strain, sex, and age of the animal species. For a 10-g mouse, the LD50 is 1.5 mg, subcutaneously, and 2.2 mg, orally. It is known to be toxic to mice and rats, inducing chronic liver injuries, such as malignant and nonmalignant hepatomas (Ueno et al., 1971; Dhumal et al., 1991). Liver cirrhosis has been noted in rats fed a cereal diet contaminated with the fungus. This toxin is also cytotoxic to HeLa cells and to rat liver, kidney, and lung cells over a concentration range of 0.32 to 1.0 µg/ml (Umeda et al., 1972). Luteoskyrin is lipophilic and relatively slow-acting, and the liver damage caused by it is characterized by centrilobular necrosis and diffuse fatty metamorphosis of the liver cells. The mitochondria that control energy production and the nuclei as the information center are both affected by luteoskyrin. It also binds to DNA, causes pigment damage, and alters the activity of DNA-dependent RNA polymerase (Dhumal et al., 1991). Luteoskyrin, therefore, is a potent cytotoxic and mutagenic agent. Islanditoxin is also extremely hepatotoxic, causing severe liver damage, hemorrhaging, and death. LD50 values for 10-g mice range from 4.75 µg, subcutaneously, to 65.5 µg, orally. It interferes with carbohydrate metabolism by causing the disappearance of glycogen granules in the injured liver. 11.3.7

Mycophenolic Acid

Mycophenolic acid is produced by many species of Penicillium, among which P. brevicompactum and P. stoloniferum appear to be most common (Cole and Cox, 1981). It contains a five-membered lactone ring fused to a benzene moiety (Figure 11.19h). Mycophenolic acid is a relatively less toxic fungal metabolite. Its oral and intravenous LD50 values for mice have been reported to be 2.5 g/kg and 550 mg/kg, respectively (Carter et al., 1969). The compound exhibits antibacterial, antifungal, antiviral, and antitumor activity (Wilson, 1971; Cole and Cox, 1981). In food, it has so far been reported only in blue cheese and starter cultures of P. roqueforti (Lafont et al., 1979). 11.3.8

Decumbin

Decumbin (Figure 11.19i) is a toxic metabolite of P. decumbens, isolated by Singleton and coworkers (1958) from corn spoiled in storage. The compounds, brefeldin isolated from the culture filtrate of P. brefeldianum (Harri et al., 1963) and cyanein obtained from that of P. cyaneum

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(Betina et al., 1965) and P. simplicissimum (Betina et al., 1966), were later found to be identical to decumbin. Administration of decumbin at levels of 250, 300, and 400 mg/kg to fasted rats resulted in 0%, 80%, and 97% mortality rates, respectively. The acute signs of toxicity began with a rapid onset of anorexia followed by diarrhea, lethargy, labored breathing, cyanosis, stupor, and subsequent death in about 24 hours (Betina et al., 1966). Decumbin is an inhibitor of the synthesis of nucleic acids and proteins in microbial and animal cells. 11.3.9

Cyclopiazonic Acid

Cyclopiazonic acid, a toxic indole tetramic acid (Figure 11.19j), is a toxic metabolite of P. cyclopium and P. puberulum. It can also be produced by certain strains of A. flavus (Yates et al., 1987). It has been detected in naturally contaminated mixed feeds, corn, groundnuts, cheese, and other foods and feeds. Cyclopiazonic acid only appears to be toxic when present in high concentrations. It is toxic to rats; the oral and intraperitoneal LD50 values are 36 and 2.3 mg/kg body weight, respectively (Ohmomo et al., 1973). It is also toxic to mice, chickens, rabbits, dogs, and pigs (Yates et al., 1987). The clinical signs of toxicity include inactivity, diarrhea, anorexia, rough hair coats, tremors, and death. Histopathological examinations have shown degenerative changes and necrosis in the liver, spleen, kidney, pancreas, salivary glands, and muscles. Its effects on the hepatic endoplasmic reticulum and HeLa cells suggest that it is an inhibitor of protein synthesis (Hinton et al., 1985; Zaera et al., 1983). Cyclopiazonic acid can cooccur with aflatoxins (Takashi et al., 1992) and may enhance the overall toxic effect when it does (Cole, 1986; Scudamore, 1998). The lack of authentic human exposure data precludes an assessment of possible health effects. However, “Kodua” poisoning in India resulting from ingestion of contaminated millet seeds has been linked to this toxin. It has pharmacological properties similar to those of the antipsychotic drugs chlorpromazine and reserpine in mice and rabbits. Near-lethal doses of 11 to 14 mg/kg body weight induce continuous involuntary tremors and convulsions. It may be able to produce similar neurotoxic effects in humans. 11.3.10 Rubratoxins Burnside and associates (1957) reported a disease in pigs and cattle caused by the consumption of fungally infected corn. Of 13 cultures of fungi isolated from such toxic corn,

only 2 were shown to cause illness and death when fed to experimental animals. One of the cultures was found to be P. rubrum, which produced a mycotoxin. Townsend and colleagues (1966) first described the rubratoxins as pure compounds, isolated from crude preparations of P. rubrum culture filtrate. The compounds were named rubratoxin A and B on the basis of the difficulty of isolation of the toxic fractions; the former was more easily isolated. The major and most toxic constituent isolated was rubratoxin B (Moss et al., 1967). Rubratoxin A (Figure 11.19k) is a dihydro-derivative with one of the anhydride groups reduced to lactol. Rubratoxin B is a cyclic bisanhydride (Figure 11.19l). The rubratoxins are unusual compounds in that they possess relatively stable anhydride groups. The aldehyde lactol group in rubratoxin A is also an unusual structural feature (Moss, 1971). Rubratoxin B is the major metabolite, and the head-to-tail, head-to-tail coupling of two C13 units produces it. Penicillium rubrum Stoll has been isolated from a variety of cereal and legume products, corn, bran, sunflower seeds, and peanut pods (Scott, 1965). However, the role of these feeds in natural outbreaks of mycotoxic disease remains undetermined (Newberne, 1974; Engelhardt and Carlton, 1991). Both the rat and the mouse excrete rubratoxin B or its metabolite(s) fairly rapidly after initial accumulation in the liver and kidneys. This probably accounts for the development of lesions in these two organs. The long elimination half-life for rubratoxin B in the plasma suggested that enterohepatic circulation was taking place (Unger and Hayes, 1979). The absence of detectable amounts of glucuronide or sulfate conjugates in the plasma and urine indicated that most of the conjugated rubratoxin produced by the liver was being excreted in the bile. Rubratoxin B interferes with several hepatocellular biochemical mechanisms. Decreases in hepatic ATP content (Hayes et al., 1978), ATPase activity (Unger et al., 1978), and hepatic-reduced glutathione content (Watson and Hayes, 1982) could all contribute to hepatocellular degeneration and necrosis. Rubratoxin B also has a strong affinity for sulfhydryl groups (Phillips and Hayes, 1979). Inactivation of membrane or enzymic sulfhydryl groups due to binding of the toxin could initiate membrane damage, leading to cellular degeneration and necrosis. The differences in target organ, lesion distribution, and lesion severity in the animal species evaluated may be related to differences in metabolism among the various species and/or in the biochemical susceptibility of cell populations in the various organs of the different species to the toxic effects of rubratoxin B.

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The LD50 values for rubratoxin A and B are 6.6 and 3.0 mg/kg, respectively. Rubratoxin B has been shown to be mutagenic as well as teratogenic (Hood et al., 1973; Evans et al., 1975). 11.3.11 Viomellein and Xanthomegnin Several fungi, such as P. viridicatum and A. ochraceus, produce the mycotoxins viomellein and xanthomegnin (Figure 11.19m and 11.19n). In mice fed these mycotoxins for 10 days identical lesions develop, predominantly in the liver. These include necrotizing cholangitis, focal hepatic necrosis, and hyperplasia of the biliary epithelium, with only minor changes in the kidneys (Carlton et al. 1976; Ueno, 1987). Hald and coworkers (1983) surveyed these mycotoxins in a barley batch associated with field cases of mycotoxic porcine nephropathy. The data revealed the presence of 1 ppm viomellein, along with 1.9 mg OT-A and 0.8 ppm citrinin. These findings indicate the natural cooccurrence of these nephrotoxic and hepatotoxic mycotoxins in barley. Systematic surveys of the natural occurrence of these mycotoxins in foods are required for their evaluation in food toxicology.

11.4 MYCOTOXINS OF FUSARIUM SPECIES 11.4.1

Zearalenone

Zearalenone, also known as F-2 toxin, is produced by several species of Fusarium. These include F. tricinctum, F. gibbosum, F. roseum, and three subspecies of the latter, F. roseum culmorum, F. roseum equisetti, and F. roseum graminearum (Gibberella zeae in sexual stage) (Urry et al., 1966; Concon, 1988; Dhumal and Salunkhe, 1992). Under favorable growth conditions, these Fusarium species have been found to produce this mycotoxin in corn, barley, mixed feeds, wheat, oats, and sorghum and have caused toxicosis in livestock in various countries. Generally, zearalenone concentrations are well below 1 ppm in processed cereal foods (Morehouse, 1985). In the United States and Canada, contamination of grains by zearalenone is frequently encountered. In these countries, the mycotoxin is most commonly found in cornbased products such as breakfast cereals and cornmeal. In Japan, high concentrations of this mycotoxin sometimes occur as a cocontaminant with deoxynivalenol and nivalenol (Jelinek, 1987). However, no cases of F-2 toxicosis in humans have been reported. Zearalenone is only partly decomposed by heat. Approximately 60% remains unchanged in bread, and about 50% survives the production of noodles (Matsuura and

Yoshizawa, 1981). In dry milling of corn, concentrations in the main food-processing fractions including flour and grits are reduced by 80% to 90%, although increased concentrations are found in bran and germ (Bennett et al., 1976). Zearalenone is a phenolic resorcyclic acid lactone (Figure 11.20a). In fungal cultures, a number of closely related metabolites are also formed. However, there is only limited evidence that these occur in foodstuffs. There is experimental evidence for some transmission of zearalenone and α- (Figure 11.20b) and β-zearalenols (Figure 11.20c) into the milk of sheep, cows, and pigs fed high concentrations (Mirocha et al., 1981). Zearalenone is a white crystalline compound that exhibits blue-green fluorescence when excited by longwavelength UV light (360 nm) and a more intense green fluorescence when excited with short-wavelength UV light (260 nm). It is slightly soluble in hexane and progressively more so in benzene, acetonitrile, methylene chloride, methanol, ethanol, and acetone. It is also soluble in aqueous alkali. Zearalenone and its derivatives produce estrogenic effects in farm animals (pigs and cattle) as well as in laboratory animal species. Its uterotropic activity is exhibited in a similar manner to that of β-estradiol. It binds with the cytosolic estrogen receptor of uterine tissue, followed by translocation to a nuclear receptor and induction of messenger RNA (mRNA) synthesis (Kawabata et al., 1982). Metabolic studies have shown that the hepatic zearalenone-reductases catalyze the transformation of zearale-

none into α-and β-zearalenols. The estrogenic activity of α-zearalenol is much higher than that of the parent compound (Ueno and Tashiro, 1981). Pigs appear to be the species most affected by the consumption of feed contaminated with zearalenone. The estrogenic syndrome in pigs is characterized by swollen, edematous vulva and enlarged mammary glands in females; shrunken testes in young males; a dramatic increase in the weight of the uterine horn of gilts; and possible abortion in pregnant sows and gilts. In sows consuming feed with 25 ppm zearalenone infertility, constant estrus, pseudopregnancy, small litter sizes and birth weights, and fetal malformations can develop (Chang et al., 1979). Although swine have been found to be the most sensitive domesticated animal to zearalenone, calves have been reported to show earlier sexual maturity; dairy cows have been reported to have vaginitis, prolonged estrus, and infertility (Palti, 1978); and sheep are reported to become sterile (Towers and Sprosen, 1992). The effective dose for sheep may be approximately 1 ppm. Poultry appear to be more resistant to the effects of zearalenone than are swine. The acute toxicity of zearalenone is quite low. The LD50 values (milligrams per kilogram body weight) for oral and intraperitoneal administration are, respectively, >2000 and >500 in the mouse, >4000–10,000 and >5500 in the rat, and >15,000 for oral administration in chickens (Scudamore, 1998). Subacute and subchronic toxicity studies of up to 14-week duration indicated that most effects were due to the estrogenicity of zearalenone (KuiperGoodman et al., 1987). In mice, it caused atrophy of semi-

(b) α-Zearalenol

(a) Zearalenone OH

O

OH

CH3

HO

O

HO

OH

O O

CH3 O

O

OH

O

O

CH3 O

HO

OH (c) β-Zearalenol

Figure 11.20

Chemical structures of mycotoxins produced by Fusarium species.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

H

O Na (d) Moniliformin

nal vesicles and testes, squamous metaplasia of the prostate gland, osteoporosis, myelofibrosis of bone marrow, cytoplasmic vacuolization of the adrenal gland, hyperkeratosis of the vagina, and endometrial hyperplasia. Makela and associates (1995) compared the estrogenic potency of zearalenone with that of other plant-derived estrogens in MCF-7 or T-47D breast cancer cells and concluded that, in comparison with 17-β-estradiol, it is one of the most potent natural xenoestrogens. Zearalenone has a potential for prenatal toxicity and possibly for teratogenicity. Evidence for genotoxicity has been contradictory, but Pfohl-Leszkowicz and coworkers (1995) showed it to be genotoxic in mice. The International Agency for Research on Cancer (IARC, 1993) concluded that there was limited evidence in experimental animals for the carcinogenicity of zearalenone. Because of its anabolic action on promoting growth hormone and insulin levels, zearalenol, a derivative of zearalenone, has been extensively used as a growth promoter in ruminants (Knight, 1981). Probably because of its low acute toxicity, so far there are no reports of adverse effects of zearalenone and its derivatives in humans. Nevertheless, because chronic low doses have adverse biological effects as a result of their potent anabolic properties and their possible presence in milk, in edible tissues, and in products like beer, zearalenone may cause human health problems after long-term exposure (Ueno, 1985; WHO, 1979; Scudamore, 1998). 11.4.2

Moniliformin

Moniliformin, a sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione (Figure 11.20d), has been isolated from F. moniliforme, a fungus that causes southern leaf blight in the United States, and also from F. fusarioides (Cole et al., 1973; Rabie et al., 1978). Data on the occurrence of moniliformin in food are scarce. Thiel and colleagues (1982) showed that levels up to 12 mg/kg occurred in corn intended for human consumption in the Transkei. More recently analysis of imported corn-milled products destined for incorporation into animal feeding stuffs in the United Kingdom showed that 60% of the samples were contaminated with concentrations up to 4.6 mg/kg (Scudamore, 1998). Moniliformin has also been shown to occur in other cereals, such as rice and wheat. There is only limited information on its degradation during processing. Moniliformin is a potent inhibitor of mitochondrial pyruvate and α-ketoglutarate oxidation. The reported oral LD50 value for moniliformin in day-old cockerels is 4.0 mg/kg (Cole and Cox, 1981). The intraperitoneal LD50 for female and male mice was, respectively, 20.9 and 29.1

Copyright 2002 by Marcel Dekker. All Rights Reserved.

mg/kg. In contrast, the oral LD50 values for female and male rats were 41.6 and 50.0 mg/kg, respectively (Ueno, 1985). The poisoned cockerels showed ascites with edema of the mesenteries and small hemorrhages in the gizzard and intestines. The clinical signs in rats were characterized by a rapid progressive muscular weakness, respiratory distress, terminal coma, and death. Moniliformin does not seem to have any teratogenic effects in experimental laboratory animals (Hood and Szczech, 1983). 11.4.3

Trichothecenes

The ability of fungi to produce a large number of secondary metabolites is striking in the case of the trichothecenes. Several families of imperfect, saprophytic, and plant pathogenic fungi, such as Fusarium, Trichothecium, Myrothecium, Cephalosporium, Stachybotrys, Trichoderma, Cylindrocarpon, and Verticimonosporium spp., produce trichothecenes (Ueno, 1983). More than 80 trichothecenes and trichothecene metabolites have been identified. Furthermore, several interesting trichothecenes have been detected in the biotransformed products catalyzed by animal and microbial systems. In the United States, serious outbreaks of toxicosis among farm animals fed fungally infested corn have been reported. The most toxic fungus frequently isolated from the fungally infected corn in Wisconsin was F. tricinctum, out of which a much more toxic strain, T-2, was selected to produce the toxin. Hence, the toxin was named T-2 toxin. This has become a common name for this trichothecene (Bamburg et al., 1968). The naturally occurring trichothecenes, especially T2 toxin, cause a variety of mycotoxicoses, such as bean hull poisoning in horses in Japan, fungally infested corn toxicosis in various livestock species in the United States, and alimentary toxic aleukia (ATA) of humans and animals in Russia (Joffe, 1986). Interest in trichothecene mycotoxins grew after a widespread outbreak of ATA, or septic angina, in the Soviet Union, which began during World War II and lasted up to 1947. The disease was called septic angina, since it was characterized by a progressive leukopenia and often led to a stage that had some of the signs of sepsis (Joffe, 1986). The disease killed thousands of people and was linked to the ingestion of millet, rye, wheat, and other small grains contaminated with Fusarium spp. The name ATA emphasizes the progressive leukopenia and the characteristics that ingestion of grains (alimentary) and secretion of the toxin by the fungi are necessary for an outbreak of this disease. Deoxynivalenol (vomitoxin) and nivalenol have been associated (together with acetyl deoxynivalenol and T-2 toxin) in an outbreak

of disease in Kashmir, India, in 1987 (Bhat et al., 1989). Some 50,000 people were affected after consumption of bread made from rain-damaged wheat that contained several trichothecenes. Overwintering conditions under low fluctuating temperatures appeared to promote Fusarium sp. mycotoxin production in these grains. The seasonal occurrence of ATA, its endemic nature, and the composition of the affected population suggested the importance of climatic and ecological factors in producing toxins that were found in field grains naturally infected by Fusarium species (Joffe, 1986). The years marked by low temperatures, considerable precipitation, and high humidity were generally associated with outbreaks of the disease in people and animals. Chemical Characteristics Trichothecene mycotoxins have a common sesquiterpene nucleus, which consists of cyclopentane, cyclohexane, and a six-membered oxyrane ring with four methyl groups. The skeleton contains an epoxide ring at C-12,13 and a double bond at C-9,10, characterized as 12,13-epoxytrichothec-9-ene. The numbering system is shown in Figure 11.21. In the early stages of trichothecene research, Ueno and coworkers (1973) classified these mycotoxins into four groups according to their chemical properties and the producing fungi. The types A, B, and C are simple trichothecenes; type D contains macrocyclic trichothecenes. The first (type A) is represented by T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and others (Figure 11.22). These are produced by F. sporotrichioides, F. sporotrichioides var. tricinctum, and F. poae. Type B trichothecenes are characterized by the presence of a ketone (carbonyl function) group in the C-8 position. This group includes nivalenol, deoxynivalenol, and fusarenon-X and is produced by F. nivale and F. episphaeria (Figure 11.23). The

16

O 1

10

2

11

9

13 6

8 7

O

3

12 5

type C group, represented by crotocin (Figure 11.24) produced by Cephalosporium crotocingigenum, contains a second epoxide at C-7,8 or C-9,10. The type D group includes trichothecenes containing a macrocyclic ring between C-4 and C-5 with two ester linkages. Subsequently, Ueno (1987) added two more groups to this classification. The fifth group (type E) is represented by the macrocyclic trichothecenes in which the macrocyclic ring is opened; the sixth group (type F) is represented by verrucarin K (Figure 11.24), in which 12,13epoxide function is changed to a vinyl linkage, and thereby the oxygen atom in the epoxide ring is removed. These compounds are presumed to be intermediates produced during the biosynthesis of macrocyclic trichothecenes. All natural trichothecenes have the same stereochemical characteristics: α at C-3, C-7, and C-8; and β at C-4 and C-15 for type A and B trichothecenes. The full systematic chemical name of T-2 toxin, therefore, is 3α-hydroxy-4β,15-diacetoxy-8α-(3-methylbutyryloxy)12,13-epoxytrichothec-9-en, and fusarenon-X is 3α-7α15-trihydroxy-4b-acetoxy-12,13-epoxytrichothec-9-en-8one. The chemical structures of various trichothecenes are shown in Figures 11. 22–11.24. The group A trichothecenes are highly soluble in ethyl acetate, acetone, chloroform, methylene chloride, and diethyl ether. The highly hydroxylated type B mycotoxins are relatively polar; they are soluble in methanol, acetonitrile, and ethanol. Physical and Chemical Properties All trichothecenes containing an ester group are hydrolyzed to their respective parent alcohols when treated with alkali. Thus, T-2 toxin and neosolanil are converted to T-2 tetraol and diacetoxy-and monoacetoxy-scirpenol to scirpentriol. Many of the alcohols are unaffected even by hot dilute alkali. Trichothecenes are thus chemically stable and can persist for long periods once formed. Prolonged boiling in water or under highly acidic conditions causes a skeletal rearrangement due to opening of the epoxide ring. Because of the hindered nature of the epoxide and stability of the ring system, reactions of trichothecenes usually proceed in a manner predictable from sound chemical principles. For example, strong oxidizing agents easily oxidize primary and secondary hydroxyl groups to the aldehyde and ketone derivatives (Scudamore, 1998).

4

Occurrence in Food 15 14

Figure 11.21

The numbering system of trichothecene.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Of the 80-odd trichothecenes identified thus far, only a few are detected in cereals and foods. The major naturally occurring mycotoxins of this group are T-2 toxin, diacetoxyscirpenol, deoxynivalenol, nivalenol, and satratoxins. In

H T-2 Toxin

H

H3C O H

HT-2 Toxin

H OH

O

O

CH3

H

H

O OH

O

O

OAc CH2 CH3 OAc

H H3C

H

H3C O H

O

OH H

H H3C

CH3

CH2 CH3 OAc

H

Diacetoxyscirpenol H

H

H3C Neosolaniol

H

H

O

H

H

H

H

H3C T-2 tetraol

H

OAc CH2 CH3 OAc

HO H

CH2 CH3 OAc

H

H

O

CH2 CH3 OH

OAc H

OH

H 3C O H

OH

O

O

H

OH

O

H

HO

H

O

OH

O

H

H

H

H 3C

H OAc

O

OAc H

H H3C

H O

CH3

CH2 CH3 OAc

H

Acetyl T-2 toxin

Figure 11.22

Chemical structures of type A trichothecenes.

H H3C

H

H

H

O

H3C OH

O O

OH

H CH2

CH3

Deoxynivalenol

H

H

H H3C

O

H

O

OAc

H

O

OH

O

OH

O O

OAc

CH2

CH2 CH3

H

OH Fusarenon-X

Chemical structures of type B trichothecenes.

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H

OH

H

Figure 11.23

CH3

HO

H

Nivalenol

HO

OH

O

OH

H3C

H O

CH2 HO

H O

HO

CH3 OAc

Diacetylnivalenol

H

H

H H3C

H3C

O O O

O O

H O

H2C

H3C

CH3

O

CH3

H O O

O

Crotocin O

H3C

O Satratoxin H

H3C

OH

OH

H

H

CH3

O O

H O

H2C O

CH3

Verrucarin A

O O H O HO H3C

H O

Figure 11.24

Chemical structures of type C and macrocyclic trichothecenes.

Canada, the United States, South Africa, and England, deoxynivalenol is the sole trichothecene present in corn, wheat, barley, and other cereals (Scott, 1984; Eppley et al., 1984; Marasas et al., 1977). Ueno (1987) reviewed extensively the literature on the contamination of cereal samples from various countries and found both nivalenol and deoxynivalenol in wheat, barley, malt barley, and corn. More than 80% of the samples studied contained both these trichothecenes. Trichothecenes contaminants in cereal flours are readily carried over into foodstuffs such as bread, snack foods, and cake. Residues of trichothecenes, however, are not known to occur in animal products such as meat, milk, and eggs. In contrast, T-2 toxin and diacetoxyscirpenol are found much less frequently. The macrocyclic trichothecenes (types D, E, and F) are rarely found in human food, although their presence in airborne fungal spores may contribute to some forms of sick building syndrome (Croft et al., 1986). Metabolism Metabolic pathways of T-2 toxin in various systems are shown in Figure 11.25. T-2 toxin or its metabolites are primarily excreted from liver via bile into the intestine. It is primarily excreted in feces. In guinea pigs, metabolites of

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T-2 toxin include HT-2 toxin, 4-deacetylneosolaniol, 3′hydroxy T-2 triol, 3′-hydroxy HT-2, and several unknowns, after an intramuscular injection (Pace et al., 1985), and T-2 triol, 3′-hydroxy HT-2, T-2 tetraol, the glucuronide conjugate of HT-2, and several unknowns, after a dermal treatment (Kemppainen et al., 1987). Thus, T-2 toxin appears to be metabolized into several hydrophilic compounds, although the trichothecene skeleton itself is not modified by the metabolic action. Toxicological Characteristics The acute toxicity of the trichothecenes varies considerably. T-2 toxin and the macrocyclic mycotoxins are by far more toxic than deoxynivalenol and nivalenol but fortunately are not often found in foods. The symptoms of acute toxicity of trichothecenes appear similar across various species; they include vomiting; inflammation; diarrhea; cellular damage of the bone marrow, thymus, spleen, and mucous membranes of intestine; feed refusal; anemia and leukopenia; and depression of circulating white blood cells. This group of mycotoxins is acutely cytotoxic and strongly immunosuppressive (Scudamore, 1998). The experimental lethal doses, a major characteristic of the severe toxicity of trichothecenes in various species, are summarized in Table 11.15. When given orally or in-

H3C

H3C O

O OH

O

AcO

CH2 CH3

O

OAc AcO

H3C

Neosolaniol

H3C

OH

O

OAc

HO

H3C O

O

OH

O

OAc

O

CH2 CH3

CH3

O

AcO H3C

T-2 toxin

OH

CH2 CH3

CH3

3'-Hydroxy T-2

O OH

O

HT-2 toxin

H3C O

OAc

HO

O OH

O

CH2 HO

CH3

OH

O AcO

15-Deacetylneosolaniol

H3C

H3C

CH3

Glucuronide conjugate

O O

H3C O

OH OH

HO AcO

CH2 CH3

O OH

O

OH

O

CH2 CH3

HO

H3C

H3C O

CH2 CH3

CH3

O

AcO H3C

H3C

O OH

O HO

H3C O

CH2 CH3

O O

HO

H3C

OH

Glucuronide conjugate

Metabolic pathways of T-2 toxin in various systems.

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OH OH

O

T-2 tetraol

Figure 11.25

CH2 CH3

CH3 3'-Hydroxy HT-2

OH HO

OH

CH2 CH3

CH3 3'-Hydroxy T-2 triol

OH OH

O

T-2 triol

4-Deacetylneosolaniol

O

Table 11.15 Lethal Dose in 50% of Sample Values of the Major Trichothecenes

Trichothecenes T-2 toxin

Diacetoxyscirpenol

Nivalenol Fusarenon-X Deoxynivalenol Crotocin Roridin A Verrucarin A

Verrucarin B Verrucarin J Satratoxin H Satratoxin G a

Animal species

Routeb

LD50, mg/kg

Mouse Rat Swine Mouse Rat Rabbit Swine Mouse Mouse Rat Mouse Mouse Mouse Mouse Rat Rabbit Mouse Mouse Mouse Mouse

i.p. p.o i.v. i.p. p.o i.v. i.v. i.p. p.o. p.o. p.o. p.o. i.p. i.p. i.v. i.v. i.v. i.p. i.p. i.p.

5.2 5.2 1.21 23.0 7.3 1.0 0.37 4.1 4.5 4.4 46 1 000 1.0 1.5 0.87 0.54 7.0 0.5 5.69 1.29

LD50, lethal dose in 50% of sample.

b

i.p., intraperitoneal; p.o., oral; i.v., intravenous. Source: Compiled from Ueno (1985, 1987) and Scudamore (1998).

layed-type hypersensitivity (DTH), antibody formation, and other immunological responses are markedly affected by the trichothecenes. Trichothecenes are therefore classified as immunodepressors (Obara et al., 1984). Alimentary toxic aleukia (ATA) is the most common human trichothecene mycotoxicosis. T-2 toxin is thought to have contributed to the epidemic of ATA in Russia, which was responsible for widespread disease and many deaths. Continuous exposure to trichothecenes results in skin rashes, which may proceed to necrotic lesions. The trichothecenes have not been shown to be mutagenic or carcinogenic but do inhibit DNA and protein synthesis. Although trichothecenes have not been found to be mutagenic in bacterial testing and the IARC has concluded that the carcinogenic potency of these mycotoxins has not been adequately demonstrated, their occurrence in food is still of some concern. This is particularly true because of the immunotoxic effects that these toxins may have. Similarly, the exact mechanism of trichothecene toxicity is not yet clear because the known mechanisms do not explain the severe acute toxicity of these mycotoxins. Further biochemical studies are required at the molecular level, and the exact biochemical entity interacting with trichothecenes needs to be elucidated. This entity may turn out to be a basis for the development of an antidote for trichothecene poisoning. Regulation

traperitoneally, the trichothecenes are acutely toxic at low concentrations. Dosed animals become listless or inactive, and diarrhea and rectal hemorrhaging develop. Necrotic lesions may develop in the mouthparts; the mucosal epithelium of the stomach and small intestine erodes, accompanied by hemorrhage, which may develop into severe gastroenteritis, followed by death (Pier et al., 1980; Scudamore, 1998). The acute toxicity of T-2 toxin suggests that it is rapidly distributed to the target organs (Ueno, 1984). It also appears that all types of cells are susceptible to the toxic effects of trichothecenes; the rapidly proliferating cells, such as epithelial cells and lymphocytes, are more sensitive. Deoxynivalenol, the commonly occurring trichothecene in foods, causes vomiting in pigs at relatively low concentrations. However, pigs are very sensitive to its presence and reject contaminated feed, thereby effectively limiting any further toxic effects. Bone marrow, spleen, and thymus are very susceptible to the toxic action of trichothecenes; hence, various immune functions may be modulated as a result of intoxication by these mycotoxins. In mice and other animals, de-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

After the observations that Canadian grains are significantly contaminated with deoxynivalenol and that this mycotoxin is carried over to finished cereal flours and foodstuffs, a guideline for deoxynivalenol in soft wheat was proposed in 1983 (Scott, 1984). These guidelines, set by the Canadian Health Protection Branch, are summarized in Table 11.16. After toxicological studies on deoxynivalenol, the tentative tolerable daily intake for adults was set at 3 µg/kg body weight, and the maximal deoxynivalenol level of 2 ppm in raw, unclean soft wheat is expected to be reduced to approximately 1.2 ppm in the flour portion of finished foods.

Table 11.16 Wheat

Canadian Guidelines for Deoxynivalenol in Soft

Unclean soft wheat Unclean soft wheat for infant foods Unclean soft wheat for bran manufacture Imported nonstaple foods

≤ 2.0 ppm ≤ 1.0 ppm ≤ 2.0 ppm ≤ 1.2 ppm (on flour or bran basis)

11.4.4

Fumonisins

The fumonisins are a family of closely related mycotoxins produced primarily by F. moniliforme and F. proliferatum. These two are the most prevalent molds associated with corn grown in all regions of the world, although they are more common in the tropical than in the temperate regions. Other Fusarium species that produce these polar metabolites include F. nygamai, F. anthophilum, F. dlamini, and F. napiforme. The fumonisins, which occur mainly in corn and in commercial corn-based human foods, are considered the aflatoxins of modern times (Scudamore, 1998). Gelderblom and associates (1988) first isolated fumonisins B1 (FB1) and B2 (FB2) from cultures of F. moniliforme MRC 826. Soon thereafter, Bezuidenhout and colleagues (1988) elucidated their structures. During the fall of 1989 and the winter of 1990, corn screenings of the 1989 U.S. crop caused numerous outbreaks of poisoning in horses and pigs. In the short time since the discovery and major outbreaks, there has been an explosion in the number of studies on these mycotoxins. The intense interest in the toxins and the fungi has arisen for a couple of reasons. First, fumonisins are found in measurable concentrations in corn grown all over the world. Second, the fungal toxins from Fusarium spp. have been epidemiologically associated with esophageal cancer in humans. These toxins cause a variety of toxic effects in various animals and probably play a role in human disease as well. Epidemiological studies suggest that fumonisins could be responsible for human esophageal cancer in South Africa and China (Norred, 1993). However, it has not been determined whether fumonisins are causally related to development of this cancer. A report from India described an acute but self-limiting food-borne disease outbreak in villagers who consumed moldy corn containing up to 64.7 mg fumonisins/kg body weight (Bhat et al., 1997). It is not known whether lower mycotoxin concentrations, chronically consumed, cause other detrimental effects in humans. Occurrence in Food Most corn and corn-containing foods and feeds are likely to be contaminated with fumonisins at detectable levels. The degree of contamination is highly dependent on environmental conditions. Environmental stresses such as heat and drought may be factors that increase the colonization of the Fusarium spp. and their production of fumonisins. Sound whole corn kernels may contain 6–10 mg/kg fumonisins; as much as 63–140 mg/kg has been detected in moldy corn (Rheeder et al., 1992). Foods with substantial

Copyright 2002 by Marcel Dekker. All Rights Reserved.

contamination (>250 µg/kg) include corn meal, corn bread mix, corn grits, and corn flour. The greatest likelihood of high levels of occurrence (>5 mg/kg) is in animal feeds, particularly those containing damaged corn and corn crack-out, such as corn screenings. Chemical Characteristics Contrary to most of the other mycotoxins, the fumonisins do not have cyclic structures. Their structures are based on a long hydroxylated hydrocarbon chain (Figure 11.26). Two hydroxyl groups are esterified to two propane-1,2,3tricarboxylic acids. FB1 differs from FB2 in that it has an extra hydroxyl group at the 10 position. Some 11 fumonisins have thus far been identified; FB1 and FB2 are the most commonly found in moldy corn. Fumonisins contain four free carboxyl groups and an amino group. Their physical structures make these toxins highly water-soluble. In fact, their insolubility in many organic solvents partly explains the difficulty in their original identification. Fumonisins are quite stable and are not broken down by moderate heat (Bordson et al., 1995). However, no fumonisins were detected in tortilla flour made by treatment with calcium hydroxide (nixtamalization), and it has been suggested that this process degrades fumonisins (Sydenham et al., 1991). However, caution is required as the breakdown products of fumonisins are just as toxic as the parent compounds (Hopkins and Murphy, 1993). Toxicological Characteristics Exposure of animals to feedstuffs naturally contaminated with fumonisins has been clearly identified as the causative agent in the incidence of equine leucoencephalomalacia (ELEM), an abnormality of the brain in horses. It has subsequently been established experimentally, by using cultures of F. proliferatum containing principally FB2 or FB3 at levels of 75 mg/kg diet fed to ponies. ELEM was observed after 150 days of feeding. The liver was also affected (Marasas et al., 1988; Bane et al., 1992). Affected animals commonly lose appetite, become lethargic, and experience neurotoxic effects after a period of ingesting contaminated feed. Autopsy showed edema in the brain and liquefaction of areas within the cerebral hemispheres. In severe cases, gross liver lesions may be seen with fibrosis of centrilobular areas. In pigs, fumonisins cause pulmonary edema and hydrothorax; thoracic cavities are filled with a yellow liquid (Bane et al., 1992). There may also be respiratory problems and fetal mortality. In chickens, fumonisins cause the so-called spiking disease characterized by neurotoxic effects in young chickens that are receiving a changing diet (Ledoux et al., 1992).

COOH

O

Fumonisin B1

COOH O

O

O

NH2

OH

OH

OH

COOH

COOH

Fumonisin B2

COOH

O

COOH O

O

O

H

NH2

OH

OH

COOH

COOH

Figure 11.26

Chemical structures of fumonisin B1 and B2.

On a weight basis, fumonisins are actually far less acutely toxic than aflatoxins. However, fumonisins commonly occur in concentrations of parts per million in corn (Shephard et al., 1996), at up to 300 mg/kg (Fazekas and Tothe, 1995), whereas aflatoxins are usually measured at concentrations of parts per billion (micrograms per kilogram) in foods. On the basis of animal data, mainly involving feeding to rats, fumonisins do not appear to be strong initiators of carcinogenesis. When rats were fed material from F. moniliforme cultures, primary hepatocellular carcinomas were produced (Gelderblom et al., 1988; Gelderblom and Snyman, 1991). A level in the diet of 250 ppm fed for 3 weeks was necessary to initiate cancer. These results were later reproduced using purified fumonisins, FB1, FB2, and FB3 (Gelderblom et al., 1993, 1994). However, experimental carcinogenicity studies have been hampered by lack of pure standards.

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In longer-term feeding studies with fumonisins, the development of hepatocarcinogenesis in rats is accompanied by a significant incidence of cholangic carcinoma (Gelderblom et al., 1991). Since hepatocarcinogenesis in rats by fumonisins is accompanied by toxic hepatitis and cirrhosis, it has been suggested that although poor initiators, fumonisins may be more powerful promoters, acting at dietary levels much lower than those required for initiation and complete carcinogenesis. This would correlate with the fact that fumonisin test results have been found to be negative in Ames tests (Park et al., 1992), in vitro rat hepatocyte DNA repair assay results (Gelderblom et al., 1989), and micronucleus test results (Gelderblom et al., 1995). The IARC (1993) concludes that there is limited evidence in experimental animals for the carcinogenicity of FB1 and that toxins derived from F. moniliforme are possibly carcinogenic to humans (group 2B). More extensive

studies are required to generate a better picture of the chronic toxicity of the fumonisins. Lebepe-Mazur and coworkers (1995a, 1995b) showed that FB1 affected the fetus in pregnant rats, causing low litter weight and fetal bone development as compared with those of controls. Developmental toxicity has also been noted in Syrian hamsters (Floss et al., 1994). Flynn and associates (1996), on the basis of studies with cultured rat embryos, concluded that rat embryos are highly sensitive to FB1 toxicity. No definitive studies have been done to establish conclusively whether fumonisins are developmental or reproductive toxins in farm animals or humans at the levels encountered in naturally contaminated foodstuffs. Human exposure to fumonisins through the ingestion of contaminated food has been estimated in several countries. In Canada, between 1991 and 1995, consumption averaged about 0.109 µg/kg/day (Kuiper-Goodman et al., 1995), and in the Transkei, South Africa, 14 µg/kg/day (Thiel et al., 1992). Despite very high levels of human exposure to these mycotoxins, there are no details in the literature of any resulting toxicities. It appears, therefore, that although there are animal data of toxicities at low levels of exposure, humans are not very sensitive to the acutely toxic effects of fumonisins. One of the principal concerns in terms of hazards to human health, however, as mentioned earlier, is the possible involvement of exposure to fumonisins in the development of esophageal cancer. Such a possibility has been indicated by epidemiological studies in South Africa, where there are high incidences of esophageal cancer in areas of high contamination of the staple foodstuff, corn, with fumonisins (Rheeder et al., 1992). In these areas, contamination of food with other mycotoxins is also quite low. Studies in areas of China with high incidences of esophageal cancer have shown correlations with exposure to fumonisins (Chu and Li, 1994). In certain areas of northern Italy, where there is one of the highest incidences of esophageal cancer in Europe, locally grown corn forms a substantial portion of the diet. It has been found to be contaminated with fumonisins and possibly to constitute a significant risk factor to human health (Visconti et al., 1996). There is a strong structural similarity between FB1 and sphinganine. This has led to the hypothesis that fumonisins exert their toxic effects through a disruption of sphingolipid metabolism or inhibition of a function of sphingolipids (Riley et al., 1994). The fumonisins are potent inhibitors of sphinganine N-acetyl transferase, an enzyme involved in sphingolipid biosynthesis and turnover (Figure 11.27). Sphingolipids have important roles in membrane and lipoprotein structure, cell-to-cell communication, interaction between cells and the extracellular matrix, and regulation

Copyright 2002 by Marcel Dekker. All Rights Reserved.

of growth factor receptors and as second messengers for a wide range of factors, including the tumor necrosis factor and interleukin-1. The resulting accumulation of free sphinganine is growth-inhibitory and cytotoxic to cells. The loss of complex sphingolipid synthesis would be expected to alter cell behavior and could also lead to cell death, on the basis of findings with mutants in serine palmityltransferase, the initial enzyme of the sphingolipid biosynthesis. Regulation The U.S. FDA has utilized informal guidance levels for fumonisins in animal feed since 1993. The levels proposed earlier were based on those recommended by the Mycotoxin Committee of the American Association of Veterinary Laboratory Diagnosticians (AAVLD). The U.S. FDA had not determined a definitive “safe level” for fumonisin residues at that time because the then-available data did not provide enough information to allow determination of a safe level for the toxin residues in milk, meat, and eggs. The agency concluded that limiting exposure to fumonisin in animal feed will protect consumers from unsafe residues, on the basis of the following toxicological and biochemical properties of fumonisins and the metabolism studies conducted to date: 1.

2.

3.

Fumonisins, which are water-soluble, do not bioaccumulate in animal tissues and thus are less likely to leave residues than lipid-soluble mycotoxins. Because fumonisins are structurally related to endogenous substrates (sphinganine and sphingosine), the mycotoxins are susceptible to be detoxified into less toxic metabolites. Residues exist usually in organ tissues, kidney and liver, which humans consume less than muscles.

These guidance levels for fumonisins in livestock feed were as follows: horse (nonroughage portion), 5 ppm; porcine (total ration), 10 ppm; and poultry (total ration) and beef cattle (nonroughage portion), 50 ppm total fumonisins. In June 2000, the U.S. FDA revised guidelines for industry for fumonisin levels in human foods and animal feeds. These guidelines are considered adequate to protect human and animal health and, according to the agency, are achievable in human foods and animal feeds with the use of good agricultural and good manufacturing practices. The revised guidelines are summarized in Table 11.17. Tolerance levels for fumonisins have not been set for foods intended for human consumption in most other countries. However, Switzerland has set a level of 1 ppm fumonisins in cereal grains.

Palmityl CoA + Serine

Sphinganine N-Acyltransferase Fatty acyl CoA

Ceramide N-Acyltransferase

Sphingolipid turnover products

Fatty acyl CoA

Sphingosine

Site of inhibition by fumonisins Figure 11.27

The disruption of sphingolipid pathway by fumonisins.

Table 11.17

U.S. Food and Drug Administration Guidance Levels for Fumonisins in Human Foods and Animal Feeds

Foods Human foods Degermed dry milled corn products (e.g., flaking grits, corn grits, corn meal, corn flour with fat content <2.25%, dry weight basis) Whole or partially degermed dry milled corn products (e.g., flaking grits, corn grits, corn meal, corn flour with fat content 2.25%, dry weight basis) Dry milled corn bran Clean corn intended for masa production Cleaned corn intended for popcorn Animal feeds Equines and rabbits Swine and catfish Breeding ruminants, breeding poultry, and breeding minkb Ruminants 3 months old raised for slaughter and mink raised for pelt production Poultry raised for slaughter All other species or classes of livestock and pet animals a

Dry weight basis. Includes lactating dairy cattle and hens laying eggs for human consumption. Source: From Fed. Reg. 65:109, June 6, 2000. b

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Total fumonisins (FB1 + FB2 + FB3) 2 ppm 4 ppm 4 ppm 4 ppm 3 ppm 5 ppm (No more than 20% of diet)a 20 ppm (No more than 50% of diet)a 30 ppm (No more than 50% of diet)a 60 ppm (No more than 50% of diet)a 100 ppm (No more than 50% of diet)a 10 ppm (No more than 50% of diet)a

11.5 MYCOTOXINS OF ALTERNARIA SPECIES

Alternaria toxins can be classified into three main structural categories: the dibenzo-α-pyrones, which include alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altenuisol (AS), altenusin, dehydroaltenusin; the tetramic acid class, which includes tenuazonic acid (TA); and a third class of compounds that includes altertoxins I, II, and III (ATX-I, II, and III). The toxins that occur most frequently are AOH, AME, and TA. The chemical structures of various mycotoxins produced by Alternaria are shown in Figure 11.28. In addition to cereal crops, Alternaria species and their mycotoxins have been reported to occur in oilseeds such as sunflower and rapeseed, apples, olives, tomato products, and several other fruits and vegetables.

Alternaria species can infect plants in the field and in storage and often cause considerable loss of fruits and vegetables through decay. Alternaria spp. is the principal contaminating fungus in wheat, sorghum, and barley; in some years nearly 100% of these grains are infected (Christensen, 1974). Many Alternaria spp. are hostspecific. For example, A. citrii infects citrus fruits; A. solani solanaceous vegetables, such as potato and tomato; and A. longipes is the causal pathogen of brown spot of tobacco. Under favorable conditions, they can produce certain mycotoxins.

O

O

O OH

O

O

H3C

OH

OH

O

HO HO

HO H OH

CH3 Alternariol (AOH)

CH3

H

OCH3

OH CH3

OCH3

Altenuene (AS)

Alternariol monomethyl ether (AME)

O

O

O

OH

CH3O

CH3 COOH

OH

OH

HO

OCH3 Altenusin

Altenuisol

O

OH

HO

HO HO

H 3C

OH

O

CH3

OCH3

Dehydroaltenusin

OH

O

O

O HO CH3 OH

OH OH CH3

N H

O

O

CH3 OH Tenuazonic acid (TA)

Figure 11.28

O

Altertoxin I (ATX-I)

Chemical structures of mycotoxins produced by Alternaria species.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

OH

O

Altertoxin II (ATX-II)

In contrast to knowledge of toxins produced by other genera of filamentous fungi, relatively little is known regarding the toxicological properties of Alternaria spp. metabolites. That Alternaria spp. are important toxinproducing fungi in foods and feeds was first reported by Joffe (1960), who demonstrated that Alternaria species isolated from moldy grains that had been incriminated in human toxicosis in the Soviet Union in the 1940s were toxic to experimental animals. Many of the symptoms observed in the human outbreaks, such as leukocytosis, leukopenia, hemorrhage, edema, and mortality, were observed in experimental animals fed Alternaria sp.–infected grain cultures. Joffe (1960) also showed that some of the isolates were also toxic when topically administered to rabbits. Alternaria spp. isolates grown in a sterilized corn-rice culture and then fed to various animal species (rats, chicks, turkey, poults, and ducklings) were consistently more lethal than were cultures of Aspergillus, Fusarium, and Penicillium spp. (Christensen et al., 1968). There are few data available on the toxicity of purified Alternaria sp. metabolites. Alternaria spp. toxins exhibit both acute and chronic effects. The LD50 values for AME, AOH, ALT, and ATX-I in mice are 400, 400, 50, and 0.2 mg/kg body weight, respectively. Those for TA are 162 and 115 mg/kg by the intravenous route for male and female mice, respectively (Scudamore, 1998). TA has been the most studied of the Alternaria spp. toxins. It has two primary toxic actions: an emetic action, which likely accounts for the ability of this compound to produce salivation and anorexia, and a cardiovascular action, which has resulted in circulatory failure and produced resulting tachycardia, hemoconcentration, and hemorrhagic gastroenteropathy. TA also inhibits protein synthesis by suppressing the release of newly formed proteins from ribosomes into supernatant fluid (Shigeura and Gordon, 1963). AOH and AME also show fetotoxic and teratogenic effects in mice, including a synergistic effect when a combination of the toxins was administered (Pero et al., 1973). Most Alternaria spp. mycotoxins exhibit considerable cytotoxic activity, including mammalian toxicity. Those of the ATX type are of particular concern because of their mutagenic activity (Chu, 1981; Stack and Prival, 1986). Stack and Prival (1986) also showed that ATX-III exhibits mutagenic activity that is approximately one tenth that of AFB1; ATX-I and ATX-II showed less mutagenicity.

11.6 MYCOTOXINS OF CLAVICEPS SPECIES Despite the suffering of people in many regions of Europe in the Middle Ages from severe and repeated epidemics of

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peripheral gangrene and convulsions due to the consumption of ergot-infested rye products, it was not until about 1850 that a distinct association between the ingestion of rye infected with Claviceps purpurea and ergotism was established. Ergotism, like ATA, is another striking example of the devastating effects of mycotoxins. C. purpurea is among the 30 or more species of the genus Claviceps and is by far the most widespread and common as a causative agent of ergotism in humans and domestic animals; other important species include C. paspali, C. cinerea, and C. fusiformis. Many of these species can form alkaloids that are poisonous when consumed in relatively large amounts. The fungus invades cereal grains and pasture grasses under high-moisture conditions, producing sclerotia, approximately 2 to 20 mm long and purple-black in color. They contain many toxic alkaloids. By plain carelessness and ignorance, the sclerotia are ground with the grains and mixed with the flour. With the shift in consumption to wheat bread and better regulation of food contamination, ergotism has slowly ceased to be a major public health problem. Outbreaks, however, still occur, as a result of improper storage of rye. If rye is stored damp, ideal conditions obtain for the growth of these parasitic molds. In the 20th century, major outbreaks of ergotism occurred in Russia in 1926, in Ireland in 1929, and in France in 1953. In Africa, an outbreak was recorded in 1979 in Ethiopia (Huxtable, 1992). Chemical Characteristics The toxic or biologically active principles of ergot have been extensively studied. Basically, these are 3,4-substituted indole alkaloids and are of two types: (a) lysergic acid (ergotamine) and steroisomeric isolysergic acid (ergocristine) amide-type derivatives, and the (b) clavine alkaloids. The alkaloids of C. purpurea are principally the first type, and only trace amounts of the clavine alkaloids are produced. The main alkaloids of this mold are peptides of lysergic acid (mevalonylindole) or isolysergic acids in cyclol form. On alkaline hydrolysis, phenylalanine, leucine, isoleucine, or valine is obtained, depending on the alkaloid (Concon, 1988). Proline is a constituent of the type I alkaloids. Hydrolysis also yields the α-keto-acids, pyruvic, dimethylpyruvic, and α-ketobutyric acids and ammonia. Lysergic acid possesses the trans configuration of the hydrogen atoms at C-8 and C-5 of the acid moiety; isolysergic acid has the cis configuration. The lysergic acid derivatives are recognized by the suffix -ine (e.g., ergotamine); the isolysergic acid derivatives by the suffix inine (e.g., ergotaminine). Altogether there are nine classes of peptide alkaloids, and about 24 major peptide alkaloids in the ergot from C. purpurea have been reported

(Groger, 1972; Concon, 1988). The chemical structures of various ergot alkaloids are shown in Figures 11.29 and 11.30. Occurrence in Foods Ergot alkaloids are primarily found as contaminants in rye, wheat, and barley. The common contaminants include ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, and ergocristine. In general, ergocristine and ergotamine are most prevalent in these grains (van Egmond and Speijers, 1998). Heat treatment of grains, such as baking of bread, leads to a reduction of the ergot alkaloids. The reduction varies from 50% to 100%, depending on the type of ergot alkaloid and the heating conditions (Schoch and Schlatter, 1985). Toxicological Characteristics Cerletti (1959) characterized the toxic effects of ergot alkaloids into three major types as follows: 1. 2. 3.

Peripheral effects such as vasoconstriction and contraction of the uterus (oxytocic effects) Neurohumoral effects, e.g., antagonism between epinephrine and serotonin CNS effects such as inhibition of the vasomotor activity of the brain thalamus

Various ergot alkaloids possess similar activity in these three areas, but they vary in degree. For example, the ergotamines and ergotoxines have similar activities, but the latter appear to be more potent (Concon, 1988). Ergometrine is principally an oxytocic agent and does not possess antagonistic effects on epinephrine. It affects the CNS only at high doses. In contrast, the isomeric isolysergic acid alkaloids are relatively weakly active. The effects seen in ergot poisoning, such as in ergotism, are generally the results of the combined action of all the lysergic acid alkaloids, including other compounds in the sclerotia, such as amines and the clavine alkaloids. The acute effects of ergot alkaloid poisoning include tachycardia, hypertension, confusion, thirst, abdominal colic, vomiting, diarrhea, and hypothermia of the skin. Symptoms of chronic poisoning include disturbances in the GI tract, angina pectoris, hypotension or hypertension, and the characteristic symptoms seen in ergotism, i.e., gangrene or mental confusion and/or convulsions (Stecher et al., 1960; Concon, 1988; van Egmond and Speijers, 1998). Speijers and associates (1993) evaluated the subacute toxicity in rats with ergotamine, ergometrine, and ergocryptine, and subchronic toxicity with ergotamine. The subchronic toxicity study with ergotamine revealed a no

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observed adverse effect level (NOAEL) of 20 mg ergotamine tartrate/kg diet/day. This is equivalent to 0.9 mg ergotamine/kg body weight/day. The results of the subacute studies indicated toxic effects including vasoconstrictive effects on peripheral arterial vessels, effects on kidney function, decreased thyroid function (decreased serum thyroxin levels), effects on the carbohydrates, and effects on ovaries. Several outbreaks of ergotism have documented the toxic effects of ergot alkaloids in humans. The ergot poisoning of the people in the Middle Ages was characterized by unbearable pain, tissue necrosis, detachment of limbs, and death. The dreadful suffering was described as St. Anthony’s fire, which consumed the limbs that turned black (necrosis and gangrene) before detachment. Although ergotism is now rare in developed countries, it sporadically still occurs in developing countries. An outbreak of ergot poisoning occurred in the Wollow region of Ethiopia as a result of infestation of local barley with C. purpurea (King, 1979; Pokrovskii and Tuteljan, 1982). There were 93 cases of gangrenous ergotism, of which 47 ended in death. The following symptoms have been reported: weakness, formication, “burning”-type paresthesia, nausea, vomiting, and diarrhea. Other symptoms included dry gangrene, feeble or absent peripheral pulses, and loss of limbs, especially the lower ones. More than 50 infants died as a result of failure of lactation in affected mothers. A similar, but mild, incident of ergot poisoning occurred in 78 persons in India after infection of pearl millet with C. fusiformis (Krishnamachari and Bhat, 1976). Although there were no deaths, the affected persons experienced nausea, vomiting, and giddiness with prolonged drowsiness. Ergotism in domestic animals is characterized by either a nervous system disorder or gangrene of the extremities. The common signs of acute ergot poisoning in horses and sheep include vertigo, incoordination, and convulsions, followed by posterior paralysis (Burfening, 1973). Cattle poisoned with ergot have been reported to exhibit primarily a gangrenous form of ergotism. Lameness due to swelling of hindlimbs and loss of extremities, along with anorexia, digestive disturbances, and ataxia, develop in affected animals. Pigs exposed to ergot-contaminated feed show reduced growth rate, disturbances of the reproductive system, and agalactia (Burfening, 1973). Another disease syndrome associated with ergot, known as paspalum staggers, is seen in cattle, sheep, and horses that consume paspalum grasses infected with C. paspali. No data on the mutagenicity, teratogenicity, and carcinogenicity of ergot alkaloids are available (van Egmond and Speijers, 1998). However, poisoning with ergot alkaloids has long been known to cause reproductive failure in

O H

C

N

H3C

OH

H CH3 O

O

CH3 OH

H CH

N

H

C

H

N

O N

N N

H

O

O

CH3

N

CH2C6H5

H

H

N

O

O

CH3

CH2C6H5

H

H

Ergocristine

Ergotamine

HN

HN

O H

C

N

H3C

OH

H CH3 O

O

CH3 OH

H CH

N

H

C

H

N

O N

N N

H

O

O

CH3

N

CH2C6H5

H

H

N

O

O

CH3

CH2C6H5

H

H

Ergocristinine

Ergotaminine

HN

HN

O H

C

H N

CH3

OH

CH2 O

O N

H

H

C

OH

H CH3 O

N

N

N N

H

O

O

CH3

N

CH2C6H5

H

H

N

O

O CH3

CH2CH(CH3)2

H

H

Ergosine

Ergostine

HN

HN

O H

C

H N

CH3

H3C OH

O

CH2 O

H

CH3 OH

H CH

C

N

O

N

N

H

H

N N

CH3

H

H

HN

Figure 11.29

N O

O

Chemical structures of ergot alkaloids of the type I group.

O

O

CH3

CH2C6H5

Ergostinine

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N

H

H

Ergocornine HN

CH2CH(CH3)2

O H

C

O H

NH2

C

N

N CH3

CH3

H

H

Ergine

HN

H

O

H

CH3

C

N H

CH2OH

Erginine

HN

H

N

O

H

CH3

C

N H

CH2OH

N CH3

CH3

H

H

HN

HN

Ergometrine (ergobasine or ergonovine) Figure 11.30

NH2

Ergometrinine (ergobasinine or ergonovinine)

Chemical structures of ergot alkaloids of the type II group.

herbivorous animals and in humans; thus, ergots can induce abortion, stillbirth, agalactia, malnutrition, or clinical disorders in the progeny. Alkaloids such as ergotamine are used clinically in the treatment of migraine and postoperative trauma, and cases of intoxication from such usage are known (Macguire and Cassidy, 1990). Ergot alkaloids are potent oxytocins; some of them, such as ergonovine, have been used therapeutically to induce labor. However, oxytocin itself has replaced ergonovine in current practice. The pharmacological actions of the ergot alkaloids are variable and complex: peripheral vasoconstriction, depression of vasomotor centers, peripheral adrenergic blockade, secretion of prolactin, and stimulation of uterine smooth muscles.

characterized by a linearly annulated structure (Figure 11.31) formed by the fusion of a furan ring in the 2 and 3 positions with carbon atoms 6 and 7, respectively, of the coumarin structures. Photosensitizing psoralens occur as natural constituents of many plants, principally in the botanical families Umbelliferae, Rutaceae, Leguminosae, and Morraceae (Pathak et al., 1962). The fungus Sclerotinia sclerotiorum growing on the celery plant has been reported to result in the biosynthesis of two phototoxic derivatives, xanthotoxin and 4,5′,8-trimethylpsoralen (Figure 11.31), in the affected area of this plant (Scheel et al., 1963). These compounds possessing phototoxic properties have been associated with bullous dermatitis suffered by workers handling celery parasitized by the fungus. These compounds are not detected in healthy celery tissue.

11.7 MISCELLANEOUS MYCOTOXINS

11.7.2

11.7.1

Slaframine (Figure 11.31) as a mycotoxin has been isolated from the fungus Rhizoctonia leguminicola, a common pathogen of red clover. The most consistent clinical sign of slaframine toxicosis seen in ruminants is excessive salivation or slobbering (Crump, 1973). Since it is a para-

Psoralens

A number of naturally occurring furanocoumarines have phototoxic activity, and much research has been done on the simplest member of this group, psoralen. Psoralens are

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Slaframine

(a)

(c)

(b)

H

O

O

Psoralen

CH3

OCH3

H

H

H

O

O

O

H

Xanthotoxin

H

H3C

O

O

O

H

H

O

CH3

4,5',8-Trimethyl psoralen

OH

O

(d) O

(e)

C

Cl

OH

CH3 O H3CO

Slaframine

N

N H3CO

H2N

N

CH3 O

S S

Sporidesmin N CH3

CH3

(f)

(g) OH

O

OH

OH

O

OH OH

H3C

OH

H3C

O

O

2-Hydroxyemodin

Emodin Figure 11.31

OH

Structures of some miscellaneous mycotoxins.

sympathomimetic alkaloid, it also produces lacrimation, diarrhea, frequent urination, and bloat. Pharmacologically, this compound exerts a stimulatory effect on the pancreas and stimulates the synthesis of digestive enzymes.

tually the ability of the liver to remove phylloerythrin from blood is impaired.

11.7.3

Emodin (Figure 11.31) is produced by several Penicillium species (Ueno, 1984). This anthraquinone is chemically composed of versicolorin A, in which emodin is fused with a bisfuranoid ring. It is also present naturally in rhubarb. In Ames test results, emodin is mutagenic to TA 1537 and is transformed into several hydroxylated metabolites, among which 2-hydroxyemodin (1,2,3,8-tetrahydroxy-6-methylanthraquinone) (Figure 11.31) is a directacting mutagen (Tanaka et al., 1987; Ueno, 1987). Free radicals derived from 2-hydroxyemodin attack DNA, leading to mutation in bacterial cells (Ueno, 1987)).

Sporidesmin

Sporidesmin is produced by the fungus Pithomyces chartarum on certain pasture grasses in warm and humid weather (Richard, 1973). Sporidesmins are a complex mixture of closely related toxic metabolites (sporidesmins A through G), which are characterized by a 3,6-epidithia2,5-dioxopiperazine moiety and an indolopyrrolopyrazine moiety (Figure 11.31) (Cole and Cox, 1981). Sporidesmin is principally toxic to the liver, producing severe occlusive damage to the bile duct system. Even-

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11.7.4

Emodin

11.8 CONTROL IN FOODS AND FEEDS Mycotoxins are relatively stable compounds, and once formed, they can persist in foods and feeds for a long time. The deleterious effects of fungal spoilage on many grain, fruit, and vegetable crops result in serious economic consequences. Consumption of contaminated foods with mycotoxins produced during fungal growth is a serious health problem. The toxicity syndromes or mycotoxicoses have posed direct or indirect health hazards to humans the world over. Mycotoxin contamination is a field problem, a storage problem, or a combination of both. Preventive management of mycotoxins in crops has been attempted through recommended practices for growing, harvesting, handling, storage, processing/manufacturing, and sampling and analysis. Significant contamination, however, still may occur in spite of these preventive measures. Mycotoxigenic fungi can be prevented in fieldgrown crops through proper management of crop residues, moisture and plant nutrition, crop rotations, and use of resistant varieties. Crop residues often provide the basic inoculum for the fungi when susceptible crops are grown. Proper management of crop residues, therefore, may be an important factor in reducing mycotoxin contamination in the field. Cultivars have been developed with resistance against many important plant pathogens, including resistance to Fusarium head blight. The use of such cultivars will certainly minimize, if not eliminate, trichothecenes such as nivalenol and deoxynivalenol in the food supply. Grain and oilseed crops that are susceptible to fungal damage should be harvested at their optimal maturity when the moisture content is the lowest. Delay in harvesting of many crops influences mycotoxin contamination, creating loss of yield and quality. During harvesting, damage to the crop should be prevented. The crop should also be dried further to reduce the moisture content to levels that prevent mold growth. Maintenance of foods below 0.7 water activity and at low temperatures is generally effective to control fungal spoilage and mycotoxin production. Good storage practices alone cannot always prevent the problem of mycotoxin formation, because many are sometimes produced before or immediately after harvest. Processing of agricultural products into foods for humans should involve careful selection of raw materials, and high-quality control practices should be employed from the source to final preparation of the food. Commodities like nuts, kernels, or oilseeds should be inspected for any damage before processing because they create highly favorable conditions for fungal growth.

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If preventive measures have failed, the last possibility to prevent occurrence of mycotoxins in the food is to eliminate the toxins. In principle, mycotoxin-contaminated consignments of foods and feeds may be decontaminated by removing the mycotoxin (segregation) or by converting the mycotoxin to a nontoxic form (degradation). The latter may be achieved by physical, chemical, or biological means. Attempts have been made to degrade mycotoxins in food and feed by applying physical treatments such as heat, microwave, gamma rays, x-rays, UV light, and adsorption. In most cases, heat treatment does not work, and neither does irradiation (van Egmond and Speijers, 1998). Various chemical agents, such as hydrogen peroxide, sodium and calcium hydroxide, chloramines, ammonia, and sodium hypochlorite, have been used to destroy mycotoxins in foods and feeds. Most of these chemical procedures to degrade mycotoxins have been developed for aflatoxins in feedstuffs. In this regard, ammonia seems to be particularly effective. In addition to the physical and chemical processes to get rid of mycotoxins, biological methods have been employed. For example, procedures are under development to degrade aflatoxins in feedstuffs with the help of the bacterium Flavobacterium auranthiacum. The toxicity of mycotoxins is also strongly influenced by dietary nutritional components such as protein, fat, vitamins, and trace elements. A well-balanced diet, therefore, goes a long way in minimizing the toxic effects of mycotoxins. Despite these various preventive measures, the present methods of control, inactivation, or elimination of the presence of mycotoxins in foods and feeds are both costly and impractical in most situations. Thus in this particular case, prevention is certainly better than cure.

11.9 REGULATORY ASPECTS Several countries have established measures to safeguard the health of consumers and the economic interests of producers and traders. Many factors influence the decisions made by authorities to establish limits for certain mycotoxins (van Egmond and Speijers, 1998). These include the following: 1.

2.

The availability of toxicological data, which is the basis for risk assessment: Without toxicological information, there can be no hazard assessment. The availability of survey analytical data: These may indicate which commodities should be

3.

4.

5.

6.

considered for regulatory action, and they provide a basis for exposure assessment, the other major ingredient of risk assessment. The distribution of mycotoxins over commodities: If such a distribution is nonhomogeneous (e.g., aflatoxins in groundnuts), there is a good chance that the mycotoxin concentration in the batch to be inspected will be incorrectly estimated, as a result of the difficulties in representative sampling. The availability of methods of analysis: The enforcement of mycotoxin regulations is ultimately based on the ability of analysts to identify and quantify these toxins accurately. Legislation in other countries with which trade contracts exist: Unnecessary strict regulative actions may create difficulties for importing countries in obtaining supplies of essential commodities, whereas for exporting countries difficulties may arise in finding markets for their products. Sufficient food supply: The regulatory philosophy should not jeopardize the availability of some commodities at reasonable prices, especially in developing countries.

At present, there are at least 77 countries known to have specific regulations or detailed proposals for regulations on mycotoxins (van Egmond and Speijers, 1998). Most of the existing mycotoxin regulations concern aflatoxins, and in fact, all countries with mycotoxin regulations have tolerances for aflatoxins in foods and/or animal feedstuffs. Less frequently, specific regulations also exist for patulin, ochratoxin A, deoxynivalenol, nivalenol, zearalenone, T-2 toxin, chetomin, stachybotryotoxin, phomopsin, and ergot. FAO (1997) has provided an update on worldwide mycotoxin regulations.

11.10 CONCLUSIONS The current status of information on mycotoxins and mycotoxicoses continues to raise several questions because of their widespread occurrence in fields, fruits, vegetables, food and feed crops. The recent discovery of a hitherto unknown class of mycotoxins, fumonisins, serves to remind us that the problem needs our continuous vigilance. The task force has to evaluate and determine the major needs for research and development activities in the foreseeable future. The following guidelines probably will be useful in establishing the research priorities in this field.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

1. 2.

3.

4. 5. 6.

7.

8. 9. 10.

Surveillance of foods and feeds for the presence and quantity of mycotoxins Surveillance of human and animal populations to evaluate harmful consumption levels of specific mycotoxins Evaluation of control methodology of mycotoxins, involving the technologies of documentation and detoxification Development of regulations to control contamination in the international trade Genetic development of plants resistant to toxigenic fungi Development of technology to create/improve analytical and proper sampling devices so that they are simple, inexpensive, and yet accurate Toxicity evaluation of newly developed or discovered mycotoxins individually and in combination with other commonly occurring mycotoxins An integrated worldwide assessment of the economic and health aspects of mycotoxins A need to study and to evaluate the economic impact (loss) of each mycotoxin A need to develop prevention programs for food and feed infestation and subsequent infection to consider the following aspects: storage, antifungal treatments, detoxification, dehydration of produce and products, irradiation, microbial inactivation, integrated mycotoxin control involving academia, government, industry and international cooperation

Since many mycotoxins may occur simultaneously in foods and feeds, there is obviously a need to evaluate the interactions of these chemicals. The demonstrated effects in animals of mycotoxins will continue to serve as reliable predictors of the possible toxic effects in humans. Studies of the mechanisms of action at the molecular level, target tissues and cellular compartments, detoxification mechanisms, and absorption and transport should make the correlation between animal and human effects much closer.

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12 Food-Borne Infections

12.1 INTRODUCTION Food-borne infection or disease is defined as any illness resulting from ingestion of contaminated food. Pathogenic bacteria, fungi, parasites, viruses, marine phytoplankton, and cyanobacteria cause microbial food-borne diseases. Food-borne illnesses are among the most widespread diseases of the contemporary world. In most cases, severity of these diseases ranges from infections without apparent clinical manifestations, to mild illness, to severe illness, to death. Fortunately, in most cases, the clinical picture is mild and self-limiting, but because of their high frequency of occurrence, their socioeconomic impact is very significant. Their importance as a vital public health problem is often overlooked because the true incidence is difficult to evaluate and the severity of the health and economic consequences is often not fully understood. For most foodborne diseases only a small proportion of cases reach the notice of health services, and even fewer are investigated. It is believed that in industrialized countries less than 10% of the cases are reported, and in developing countries reported cases probably account for less than 1% of the total (Kaferstein et al., 1999). Despite these limitations in reporting, available data give evidence of a tremendous public health problem (Table 12.1). In fact, both in the United States and in other developed countries, the actual and the reported incidences of food-borne diseases increased during the 1990s. There are several reasons for this. Better methods of detection and identification together with better epidemiological capacity have contributed to a rise in reported cases. Life-style factors may also be responsible for a rise in actual inci-

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dence. People are eating out more, traveling more, and choosing exotic foods more frequently than in the past. Vegetables and fruits available during the winter increasingly are bought from other countries with different food sanitation standards and perhaps different strains of organisms (Osterholm, 1991; Jones, 1992). Similarly, as people get further away from learning about correct food handling, they have a greater likelihood of handling food correctly. As a result of current food trends of increased food purchasing from restaurants and delis and in prepared and refrigerated forms, food is handled by more people, treated and distributed in stages, and held before being sold. All these factors increase the chance of its becoming contaminated or being held at improper temperatures. The popularity of organically grown foods may also contribute to the rising outbreaks and incidences of food poisoning. These products contain no additives or preservatives and are often grown without the applications of pesticides, thereby allowing infection to take place. Consolidation of many small food-processing operations into larger ones could also contribute to an increase in foodborne disease. Although larger operations are likely to be more conscious of sanitation than small ones, one foodhandling mistake reaches large numbers of people and results in a massive outbreak (Jones, 1992). Changes in animal husbandry practices have also contributed to this trend. The concentration of animals into larger production units and of animal slaughter into fewer and larger plants increases the possibility of cross-contamination among meat carcasses. A further worrisome factor is development of resistance to antibiotics of pathogenic

Table 12.1 Food-borne Illness in the United States Cause Bacteria Salmonella non-typhi Salmonella typhi Campylobacter spp. Staphylococcus aureus Streptococcus (group A) Shigella spp. Escherichia coli Brucella, spp. Vibrio (non-cholerae) Vibrio cholerae/vulnificus Listeria monocytogenes Yersinia enterocolitica Clostridium perfringens Clostridium botulinum Bacillus cereus Miscellaneous organisms Viruses SRSV Hepatitis A Other viruses Parasites Trichinella spiralis Giardia lamblia Toxoplasma gondii Taena spp. Fish parasites Chemicals/toxins Ciguatera toxin Chemical poisons Plant poisons Scombroid toxin Paralytic shellfish poison

Cases

Deaths

3,000,000 600 4,000,000 8,900,000 5,000,000 300,000 200,000 50,000 30,000 13,000 25,000 20,000 650,000 100 84,000 107,000

2,000 36 2,100 7,120 175 600 400 0.1 300–900 1–2 1,000 2–3 6–7 2–3 0 11

181,000 48,000 6,000,000

0 150 6

100,000 7,000 2,300,000 1,000 1,000

1,000 0 450 10 0

27,000 96,000 7,000 31,000 260

2.1 5.4 5.9 0 0.3

Source: Compiled from Forsythe and Hayes (1998) and CAST (1994).

microorganisms, which further contributes to increased incidence and decreased treatability of food-borne disease. Food-borne diseases are any illnesses that result from ingestion of contaminated food. Bacteria, fungi, parasites, marine phytoplankton, viruses, and cyanobacteria cause microbial food-borne diseases. Virulence of the pathogen dose and resistance/susceptibility of the host dictate the outcome. Bacterial pathogens are the most commonly identified cause of outbreaks of food-borne illnesses. Bacterial pathogens can be easily transmitted and can multiply rapidly in food, making them difficult to control. The mere presence of pathogens is not usually sufficient to cause food-borne disease. In fact, many foods test positive for a variety of pathogens. However, the dose re-

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quired to cause disease can vary markedly for different organisms. Many require populations of at least 10,000 and sometimes as many as 1 million before any symptoms appear. In contrast, some may require populations as small as 100 to 1000. Furthermore, individuals vary in their susceptibility to these microorganisms just as they vary in susceptibility to a variety of other disease organisms. Food-borne diseases can be classified as infections, toxicoinfections, or intoxications (Figure 12.1). The characteristics of these three classes are now briefly described. 12.1.1

Food-Borne Infections

A food-borne infection occurs when pathogenic microorganisms in ingested food establish themselves in the human host’s body. They are able to grow or colonize the intestines, often invading the mucosa or other tissues and thereby causing invasive infections. All classes of foodborne pathogens (viruses, bacteria, parasitic protozoa, and other parasites) include infectious agents (CAST, 1994). Pathogenic bacteria that are not obligate intracellular parasites, e.g., Salmonella and Shigella spp., invade intestinal cells and multiply, thereby causing salmonellosis and shigellosis, respectively. Since infections are a consequence of the growth of a microorganism in the human body, the time from ingestion until symptoms occur (i.e., the incubation period) generally is rather long, compared to that of most foodborne intoxications, which manifest their symptoms within hours. Fever is the most commonly seen clinical manifestation of food-borne infections, although many have chronic sequelae associated with them. These include, among others, endocarditis, chronic incapacitating diarrhea, Guillain-Barré syndrome (GBS), and immunemediated disorders such as reactive arthritis, Reiter’s syndrome, rheumatoid arthritis, and septic arthritis (Archer and Kvenberg, 1985; CAST, 1994). 12.1.2

Food-Borne Toxicoinfections

Food-borne toxicoinfections result when a microorganism from contaminated ingested food grows in the intestinal tract and produces a toxin or toxins that damage the tissues or interfere with normal organ or tissue function. Examples of toxicoinfective bacteria include Vibrio cholerae, Bacillus cereus, Clostridium perfringens, C. botulinum (infant botulism), and enterotoxigenic Escherichia coli. The onset times for toxicoinfections are frequently, but not necessarily, longer than those for intoxications but shorter than those for infections. The predominant clinical manifestation of food-borne toxicoinfections is diarrhea. Some organisms, such as enterotoxigenic Escherichia coli, may

Figure 12.1 Classification of food-borne diseases.

produce hemorrhagic colitis, hemolytic uremic syndrome (HUS), and other debilitating sequelae. 12.1.3

Food-Borne Intoxications

Food-borne intoxications, the most common cause of food poisoning outbreaks, occur when during their growth, specific pathogenic bacteria release toxins into food that subsequently is consumed. The time in which symptoms develop after consumption of foods containing microbial toxins often is useful in differentiating intoxications from infections. Generally, intoxications are manifested more rapidly after consumption of contaminated food than are infections because time for growth and invasion or elaboration of the toxin in vivo is not required (Concon, 1988; CAST, 1994). Bacteria capable of causing food-borne intoxications include Staphylococcus aureus, Bacillus cereus, and Clostridium botulinum. Some agents of important food-borne diseases and their salient epidemiological features are listed in Table 12.2. Globally, Salmonella and Campylobacter spp. and Staphylococcus aureus are by far the most common causes of food poisoning. Similarly, although almost all types of foods have been associated one time or another with food poisoning outbreaks, over 70% of the cases in which the food has been identified have involved reheated or cold precooked meats (principally beef, pork, ham, and lamb) or poultry (chicken, turkey, and ducks), stews, minced meats, meat pies, or salads containing various types of

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meats. Thus, foods of animal origin are the most predominant causes of food-borne disease outbreaks. Other foods implicated in food poisoning outbreaks include milk, cheese, ice cream, orange and apple juices, cantaloupes, and vegetables. Some examples of the types of pathogens routinely detected in certain raw foods worldwide are listed in Table 12.3. Characteristics of diseases caused by various foodborne organisms and toxins produced by them are summarized in Table 12.4. Factors contributing to outbreaks of food-borne disease, as identified by the World Health Organization (WHO) Center for Control of Food-borne Infections and Intoxications, are summarized in Table 12.5. The most susceptible population groups are infants and children, the elderly, and immunocompromised people. Factors increasing the risk of food-borne infection or the severity of illness are summarized in Table 12.6 Between 6.5 million and 81 million cases of foodborne illness and as many as 9100 related deaths occur each year in the United States alone. Annual costs due to medical treatment and loss of productivity range from $5 billion to $22 billion (Subcommittee on Human Resources and Intergovernmental Relations, 1997). The wide range in the estimated number of food-borne illnesses and related deaths is due primarily to the considerably uncertainty about the number of cases that are never reported to the Centers for Disease Control (CDC). For example, many intestinal illnesses that are commonly referred to as stomach flu are caused by food-borne pathogens. People

Table 12.2 Some Agents of Important Food-Borne Diseases and Salient Epidemiological Featuresa Transmissionb Important reservoir/carrier

By waterb

By foodb

Person-topersonb

Multiplication in foodb

Soil

–

+

–

+

Cattle, goats, sheep Chickens, dogs, cats, cattle, pigs, wild birds Soil, mammals, birds, fish

– +

+ +

– +

+ –c

–

+

–

+

Agents Bacteria Bacillus cereus Brucella spp. Campylobacter jejuni Clostridium botulinum Clostridium perfringens Escherichia coli Enterotoxigenic Enteropathogenic Enteroinvasive Enterohemorrhagic

Soil, animals, humans

–

+

–

+

Humans Humans Humans Cattle, poultry, sheep

+ + + +

+ + + +

+ + • +

+ + + +

Listeria monocytogenes

Environment

+

+

–d

+

Mycobacterium bovis Salmonella typhi

Cattle Humans

– +

+ +

– ±

– +

Salmonella paratyphi Salmonella non-typhi

Humans and animals

±

+

±

+

Shigella spp. Staphylococcus aureus

Humans

+ –

+ +

+ –

+ +

Vibrio cholerae, O1 Vibrio cholerae, non-O1 Vibrio parahaemolyticus Vibrio vulnificus Yersinia enterocolitica

Humans, marine life Humans, marine life Seawater, marine life Seawater, marine life Water, wild animals, pigs, dogs, poultry

+ + – + +

+ + + + +

± ± – – –

+ + + + +

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Examples of incriminated foods Cooked rice, cooked meats, vegetables, starch puddings Raw milk, dairy products Raw milk, poultry Fish, meat, vegetables (home preserved), honey Cooked meat and poultry, gravy, beans Salad, raw vegetables Milk Cheese Raw or undercooked ground meat products, raw milk Cheese, raw milk, cole slaw, meat products Raw milk Dairy products, meat products, shellfish, vegetable salads Meat, poultry, eggs, dairy products, chocolate Potato, egg salads Ham, poultry, egg salads, cream-filled bakery products, ice cream, cheese Salad, shellfish Shellfish Raw fish, crabs, other shellfish Shellfish Milk, pork, poultry

Viruses Hepatitis A virus Norwalk agents Rotavirus Protozoa Cryptosporidium parvum Entamoeba histolytica Giardia lamblia Toxoplasma gondii Helminths Ascaris lumbricoides Clonorchisw sinensis Fasciola hepatica Opisthorchis viverrini O. felinum Paragonimus spp. Taenia saginata T. solium Trichinella spiralis Trichuris trichiura

Humans Humans Humans

+ + +

+ + +

+ – +

– – –

Shellfish, raw fruit and vegetables Shellfish salads •

Humans Humans Humans, animals Cats, pigs

+ + + •

+ + ± +

+ + + –

– – – –

Raw milk, raw sausage (nonfermented) Vegetables and fruits Vegetables and fruits Undercooked meat, raw vegetables

Humans Freshwater fish Cattle, goats Freshwater fish

+ – + –

+ + + +

– – – –

– – – –

Soil-contaminated food Undercooked/raw fish Watercress Undercooked/raw fish

Freshwater crabs Cattle, swine

– –

+ +

– –

– –

Undercooked meat Undercooked meat

Swine, carnivora Humans

– •

+ +

– –

– –

Undercooked meat Soil–contaminated food

Source: From WHO (1984) and Kaferstein et al. (1999). a Almost all acute enteric infections show increased transmission during the summer and/or wet months, except infections due to Rotavirus spp. and Yersinia enterocolitica, which show increased transmission in cooler months. b +, yes; ±, rare; –, no; •, no information. c Under certain circumstances, some multiplication has been observed. Its epidemiological significance is not clear. d Vertical transmission from pregnant women to their fetus(es) occurs frequently.

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Table 12.3 Prevalence of Pathogens or Potential Pathogens in Foods Organism Aeromonas hydrophila

Aeromonas spp. Bacillus cereus

Campylobacter (thermophilic)

Campylobacter coli Campylobacter jejuni

Clostridium botulinum

Clostridium perfringens

Escherichia coli (enterotoxigenic) Escherichia coli O157:H7

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Food Seafood Raw milk Poultry Raw meats Cooked meats Pork Beef Produce Lamb Pork Beef Chicken Meat additives Raw milk Pasteurized milk Dairy products Raw rice Pasta and flour Seafood Pork carcasses Beef carcasses Veal carcasses Turkey carcasses Chicken carcasses Pork carcasses Pork Beef carcasses Beef Lamb Turkey Chicken Raw milk Fresh mushrooms Bacon Liver sausage Infant foods Corn syrup Honey Pork Cooked pork Beef Chicken Seafoods Cheese Raw milk Raw beef kidneys Beef Pork

Percentage positive 19–100 33 16–100 100 10 6–27 11–33 95 59 4–7 11–63 0–7 39 9 35 0–63 100 0 1 17 23 43 74 38 13 0–24 50 0–5 1–20 56–64 8–89 0.4–1.2 2 0.1 2 0 20 2 0–39 45 22 0–54 2.4 0 0 0.1–0.5 3.7 1.5

Table 12.3 (continued) Organism

Food

Percentage positive

Poultry Lamb Raw beef Ground beef Ground pork Raw red meats Ground beef Ground pork Ground veal Chicken Turkey Cured meats and fermented sausages Seafood Raw milk Pasteurized milk Ice cream Raw whole egg Produce and vegetables Beef Veal carcasses Pork Pork products Turkey carcasses Turkey sausage Chicken Shellfish Fish Raw milk Raw beef Pork Pork sausage Raw chicken Seafood Bakery itemsa Shellfish Seafood Beef Pork Processed pork products Chicken Raw milk Pasteurized milk Ice cream Raw vegetables

1.5 2 17 36.4 10.6 0–43 77 95 100 13–56 12–18 0–20 11–26 1.6–4.2 0 0.25 5 0 0–2.6 4.1 0–18 3–20 69 100 0–100 3.7–33 0 0.5–4.7 16 13 33 41–73 38 9.8 7.4–33 2.8–46 2 2.5–49 7–37 11–25 2.7–48 1 22 46

Escherichia coli O157:H7 (continued)

Escherichia coli (verotoxigenic)

Listeria monocytogenes

Salmonella serovars

Staphylococcus aureus

Vibrio cholerae Vibrio parahaemolyticus Yersinia enterocoliticab

a

Oatmeal raisin cookies, apple muffins, cream puffs, long johns. Many strains of Yersinia enterocolitica isolated from foods are avirulent. Source: From CAST (1994). b

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Table 12.4 Characteristics of Diseases Caused by Food-Borne Organisms and Toxins

Organism/toxin Viruses Hepatitis A

Norwalk and Norwalklike Bacteria Aeromonas hydrophila

Fatality: case rate, %d

Toxic or infectious illness dosee

Incubation period

Disease severity Moderate to severe

Weeks to months

?

0.3

Unknownf

F

1 To 7 wk, usually 25 days 1 To 2 days

Mild to moderate

1 To 2 days

—

0.0

Unknownf

Unknown

—

Mild, selflimiting Mild, selflimiting

Days to weeks 1 Day

Yes

—

?

—

0.0

105 to 1011 CFU

Illness Typea F

Durationb

Sequelaec

T/F (diarrheal), T (emetic)

8 To 16 hr 0.5 To 5 hr

Brucella abortus

F

—

Moderate to severe

Weeks

Yes

—

?

Campylobacter jejuni C. coli Clostridium botulinum

F

1 To 7 days

Days

≥500 CFU

12 To 36 hr

2% to 10% —

0.05

T, T/F

Mild to moderate Severe

7.5

Up to about 109 LD50/mg toxin in miceh

Clostridium perfringens

T/F

8 To 16 hr

Mild, selflimiting

1 Day

—

<0.1

106 to 1010 CFU

Coxiella burnetti

F

Days to weeks Days to weeks

—

?

T/F

Mild to severe Moderate to severe

Yes

Escherichia coli O157:H7 (enterohemorrhagic)

2 To 4 weeks 3 To 7 days

Yes

2.0

Estimated to be 101 to 103 CFU

Bacillus cereus

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Days to months

Comments Contaminated shellfish and foods prepared by infected workers Contaminated shellfish and foods prepared by infected workers No confirmed food-borne cases; strong indirect evidenceg Emetic illness associated with cooked rice and pasta prepared in food service establishments; emetic toxin heat-resistant U.S. incidence declining; usually food-borne in raw goat milk and cheese Associated with raw milk, poultry, beef, pork, shellfish T/F for infants only; most illness due to home-canned or fermented foods, occasionally mishandling in food service; most due to vegetables (peppers, pimentos), meat, fish Most outbreaks from meat and poultry products and beans; foods mishandled in food service establishments Raw milk; controlled by milk pasteurization Most outbreaks associated with insufficiently cooked ground beef in food service establishments and nursing homes; also associated with raw milk

E. coli (enteropathogenic)

(F?)

1 To 6 days

Mild to moderate

Days to weeks

?

≤0.1h

Estimated 106 to 1010 CFU for adults

E. coli (enteroinvasive)

F

1 To 3 days

Mild to moderate

Days to weeks

?

≤0.1h

108 CFU for adults with no underlying illness

E. coli (enterotoxigenic)

T/F

1 To 3 days

Mild to moderate

Days

?

≤0.1h

106 To 108 CFU for adults with no underlying illness

Listeria monocytogenes

F

4 Days to several weeks

Mild to severe

Days to weeks

?

0 to 30j

?

Mycobactgerium bovis M. avium M. tuberculosis Salmonella typhi S. paratyphi

F

4 To 6 wk

Severe

Weeks to months

?

—

106 CFU for adults

F

7 To 28 days

Severe

Weeks to months; possible relapses

Yes

6.0

<103 To 109 CFU

Salmonella spp. serovars

F

6 to 48 hr

Mild to severe

Days to weeks

2% To 3%

<0.1

1 To about 109 CFUk

Shigella spp.

F

1 To 7 days

Moderate to severe

Days to weeks

2% to 3%

0.1

101 To 106 CFU

Unlikely as significant cause of food-borne illness in U.S., Canada, or W. Europe; most cases in young children in tropical areas with poor hygienic standards Unlikely as significant cause of food-borne illness in U.S. or Canada; most cases in young children in tropical countries with poor hygienic standards Most cases in tropical countries with poor hygienic standards; U.S. cases rare; most associated with foreign travel; most common cause of traveler’s diarrhea; highest incidence in children <2 yr in developing countries Most severe form of illness in unborn, newborn, and immunocompromised; pregnant women transmit to fetus Currently not food-borne in U.S. Outbreaks frequently waterborne; contaminated shellfish or foods handled by carriers and not subsequently heated; rare in U.S. Commonly meat, poultry, milk, eggs, but numerous other foods (e.g., chocolate, peanuts) involved Poor personal hygiene of infected food handlers responsible for most cases

(table continues)

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Table 12.4 (continued)

Organism/toxin Bacteria (continued) Staphylococcus aureus

Illness Typea

Incubation period

Disease severity

Durationb

Sequelaec

Fatality: case rate, %d

Toxic or infectious illness dosee

T

2 To 7 hr

Mild to severe (rarely)

<1 Day to several days

—

<0.02

<1 µg

Streptococcus group A

F, T/F

1 To 2 days

<0.03

?

T/F

1 To 3 days

Days to weeks Days

?

Vibrio cholerae (O1)

Mild to moderate Mild (rarely) to severe

—

<1.0m

106 CFU

V. cholerae (non-O1)

F, T/F

1 To 3 days

Mild to moderate

Days

—

<1.0l

106 to 108 CFU

V. parahaemolyticus

F

1 To 3 days

Mild, selflimiting

Days

Yes

<1.0l

105 to 107 CFU

V. vulnificus

F

Median 16 hr

Severe

Days to weeks

—

0 To 6l

Yersinia enterocolitica

F

1 To 3 days

Mild to moderate, selflimiting; chronic

Days to weeks

2% To 3%

0.03

Estimate 1 CFU for persons with elevated serum iron concentration ?

F

1 To 2 weeks

Moderate to severe

4 Days to 3 weeks

Yes

—

<30 Cysts

F

2 To 4 weeks

Mild to severe

Weeks to months

—

5 Cysts

Parasitic protozoa Cryptosporidium parvum Entamoeba histolytica

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Comments High-protein foods; foods handled frequently during preparation (e.g., meat, salads); tolerates salty foods (e.g., ham); enterotoxin heatresistant — Outbreaks frequently associated with seafood; many other foods involved; rare in U.S. Outbreaks associated with seafood, mainly shellfish in southern waters Most cases associated with seafood (some crosscontamination) All known cases associated with seafood, especially raw oysters; most victims male and have chronic liver or blood-related disorders Sometimes mimics appendicitis; linked to chronic reactive arthritis and Reiter’s syndrome; associated with pork, milk, or milk products; carried by swine Water, marine fish; possibly associated with raw milk and raw vegetables Contaminated vegetables; rare or nonexistent in U.S.

Giardia lamblia

F

5 To 25 days

Mild to moderate

Weeks to years

Yes

—

10 Cysts

Toxoplasma gondii

F

—

Mild to severe

—

?

—

1 Cyst

Other parasites Anisakid nematodes

F

—

—

?

—

1 Larva

Marine fish

Diphyllobothrium spp.

F

—

Mild to severe Mild to moderate

—

?

—

1 Larva

Taenia saginata T. solium

F

—

Moderate to severe

—

Yes

1 Cyst

Trichinella spiralis (nematode)

F

—

Moderate to severe

—

Yes

Significant for T. solium neurocysticercosis —

Transmitted by freshwater fish; severe illness in highly immunocompromised individuals Beef, pork

1 To 500 larvae

Primarily from undercooked pork, game meat, bear meat, walrus meat

T

Minutes to 6 hr

Mild to severe

Several days

—

1% to 4%

≥100 µg

Ciguatoxins

T

Minutes to 24 hr

Mild to severe

Up to several months

—

40–70 ng

Diarrhetic shellfish poisons (DSP)

T

—

Mild

—

—

May be as high as 13% Low

Shellfish only from NE or NW U.S. and North American coasts; also in Central America and Asia Tropical fish only

≥32–77 µgm

Domoic acid

T

—

Moderate to severe

Hours to permanent

—

Unknown

≥60 mgn

Neurotoxic shellfish poisons (brevetoxins) Histamine, histaminelike (scombroid)

T

—

—

Several days

—

Low

>80 µg

T

Minutes to 6 hr

Mild to severe

≤1 Day to 2 days

—

0.01%

≥50 mg Histamineo

Toxins Paralytic shellfish poison (PSP)

Rarely food-borne, primarily water-related cases; salads and ice implicated Pork, insufficiently cooked hamburger

Asia, Europe; two outbreaks on North American East coast Amnesic shellfish poisoning associated with mussels and clams Shellfish in southern states; may be associated with red tide (including prebloom) Mainly mackerel, tuna, mahimahi, blue fish

(table continues)

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Table 12.4 (continued)

Organism/toxin Toxins Tetrodotoxin

Illness Typea T

a

Incubation period

Disease severity

Minutes to 3 hr

Moderate to severe

Durationb Hours

Sequelaec —

Fatality: case rate, %d

Toxic or infectious illness dosee

May be as high as 50%

May be similar to levels for PSP

Comments Puffer fish only; no recent U.S. cases

T, intoxications; F, infection; T/F, toxicoinfection. Excluding sequelae that may last 6 mo to 30 yr. c Sequelae only recently being investigated for certain diseases, including reactive arthritis, Reiter’s syndrome, Guillain-Barré syndrome, ankylosing spondylitis, rheumatoid arthritis, septic arthritis, and cardiac manifestations. —, sequelae are not thought to occur and/or there is no evidence or reason to believe that they do; ?, occurrence of sequelae is unknown. d From Todd (1989). e For intoxications, toxic dose estimates are indicated. For infections and toxicoinfections, doses are those causing infection (often asymptomatic) or illness, probably the latter. ?, lack of available information or information so speculative as to permit little confidence; CFU, colony-forming units. f Hepatitis A and Norwalk virus cannot be cultivated and counted; therefore, the infectious dose is unknown. g From Kirov (1993b). h LD50, lethal dose for 50% of the population. i ≤0.1 Is total fatality/case rate for all E. coli illnesses except E. coli O157:H7. j For infants, fetuses, or adults with underlying illnesses or compromised immune systems; for healthy adults, fatality rate is nil. k Low number related to ingestion of contaminated foods that are protective to the pathogen. l There are about 50 known cases of V. vulnificus septicemia each year, which cause about 30 deaths. These persons have predisposing factors to infections. There are probably many others with mild infections for which the mortality rate is likely to be low or nil. Thus, the upper limit is probably much higher than would be calculated if mild cases were considered. m From Todd et al. (1993). n From Todd (1993). o From Todd and Holmes (1993). Source: From CAST (1994). b

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Table 12.5 Factors Contributing to Outbreaks of Food-Borne Diseases Poor general hygiene Consumption of raw ingredients Use of contaminated ingredients Contamination by infected persons Cross-contamination Use of contaminated equipment Failures in processing Preparation too early in advance Inadequate heating Inadequate hot holding Inadequate refrigeration Excessive storage time Contamination during final preparation Inadequate heating before reuse

do not usually associate these illnesses with food because the onset of symptoms occurs 2 days or more after the contaminated food was eaten. Furthermore, most physicians and health professionals treat patients who have diarrhea without ever identifying the specific cause of the illness. In severe or persistent cases, a laboratory test may be ordered to identify the responsible pathogen. Finally, physicians may not associate the symptoms they observe with a pathogen that they are required to report to the state or local health authorities. In the absence of more complete reporting, researchers can only broadly estimate the number of illnesses and related deaths.

The USDA’s Economic Research Service estimated 1992 costs for several bacterial and parasitic pathogens (Table 12.7). Costs per case differ dramatically, in that disease severity is exceedingly diverse. Food-borne toxoplasmosis was the most costly disease, at $2.6 billion annually, followed by salmonellosis, campylobacteriosis, diseases caused by E. coli O157:H7, and listeriosis. These diseases are estimated to cost in total $5 billion and $6 billion annually (Weiss et al., 1993). It is, however, quite difficult to put a monetary value on losses caused by chronic illnesses with a food-borne source. Such effects occur in 2% to 3% of all infections. The range of estimates for annual cases of food-borne diseases is 6.5 million to 81 million annually, or 130,000 to 2.4 million new cases of chronic disease annually. Thompson (1986) surveyed rheumatoid arthritis sufferers and asked what they would be willing to pay for an arthritis cure. The answer was an average of 22% of household income. Although rheumatoid arthritis is a more severe outcome than most chronic food-borne illnesses, such cost information makes an important addition to the acute illness costs of treating food-borne diseases. In this chapter, important food-borne infections caused by pathogenic bacteria and certain parasitic organisms are described. Although it is not a bacterial infection, because of the controversy surrounding it, bovine spongiform encephalopathy (“mad cow disease”) is also discussed. Food-borne bacterial intoxications and toxicoinfections are described in Chapter 13.

Table 12.6 Factors That Increase the Risk of Food-Borne Infection or the Severity of Illness Factors Microbial factors Type and strain of pathogen ingested Quantity of pathogens ingested Host factors Age less than 5 years Age greater than 50 or 60 years (depending on pathogen) Pregnancy Hospitalization Concomitant infections Consumption of antibiotics Excessive iron level in blood Reduced liver/kidney function (alcoholism) Possession of certain human antigenic determinants duplicated or easily mimicked by microorganisms

Reasons Some pathogens and strains more virulent than others Increased severity of illness and/or shortening of onset time potential result of higher numbers ingested Lack of developed immune systems, smaller infective dose by weight required Immune systems falling, weakened by chronic ailments, as early as 50 to 60 years of age Altered immunity during pregnancy Immune systems weakened by other diseases or injuries, or at risk of exposure to antibiotic-resistant strains Overloaded or damaged immune systems Alteration of normal intestinal microflora Iron in blood serving as nutrient for certain organisms Reduced digestion capabilities, altered blood iron concentrations Predisposition to chronic illnesses (sequelae) (table continues)

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Table 12.6 (continued) Factors

Reasons

Microbial factors Surgical removal of portions of stomach or intestines Immunocompromised individuals including those on chemotherapy or radiation therapy; recipients of organ transplants taking immunocompromising drugs; persons with leukemia, AIDS,a or other illnesses Stress Poor hygiene Diet-related factors Nutritional deficiencies either through poor absorption of food (mostly ill or elderly persons) or unavailability of adequate food supply (starving persons) Consumptions of antacids Consumption of large volume of liquid, including water Ingestion of fatty foods (e.g., chocolate, cheese, hamburger) containing pathogens Other factors Geographic location

Reduction in normal defensive systems against infection Immune system inadequate to prevent infection

Body metabolism changes allowing easier establishment of pathogens, or lower dose of toxin required for illness Increased likelihood of ingestion of pathogens Inadequate strength to build up resistance and/or consumption of poor-quality food ingredients, which may contain pathogens Increased pH of stomach Dilution of acids in stomach and rapid transit through stomach Protection of pathogens against stomach acids by fat

Likelihood of exposure to endemic virulent strains, limited food and water supply, varied distribution of organisms in water and soil

a AIDS, acquired immunodeficiency syndrome. Source: Adapted from CAST (1994).

Table 12.7 Medical Costs and Productivity Losses Estimated for Food-Borne Pathogens, 1992

Cases, no.

Deaths, no.

Medical and productivity costs, million $

1,920,000 2,100,000 7,668–20,448 1,526–1,581

960–1,920 120–360 145–389 378–433

1,188–1,588 907–1,016 216–580 209–233

2,090 131 894 210

42 0 0 0

Food-borne pathogena Bacteria Salmonella spp. Campylobacter jejuni or coli Escherichia coli O157:H7b Listeria monocytogenes Parasites Toxoplasma gondii Trichinella spiralis Taenia saginata Taenia solium Total a

2,628c 0.8 0.2 0.1d $5 Billion to $6 billion/y

Analysis assumes that 100% of human illnesses are food-borne for Campylobacter spp., E. coli O157:H7, Trichinella spp., and Taenia spp. and assumes that 96% of Salmonella spp., 85% of Listeria spp., and 50% of Toxoplasma spp. cases are food-borne. b From Marks and Roberts (1993). c Productivity losses are high for survivors when mental retardation or blindness results from toxoplasmosis. These costs exclude premature deaths (50% of cases also may have a food-borne origin). d Estimates do not include costs for cystericercosis, which may have an indirect food-borne transmission. Source: From Weiss et al. (1993).

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12.2 BACTERIAL PATHOGENICITY Bacterial pathogenicity is a multifactorial and dynamic process that requires coordinate production and repression of many bacterial gene products. Though each bacterial pathogen interacts with the human host in a unique way, common events occur in many infections. These include the following: 1. 2.

3. 4. 5. 6.

Primary attachment to host cells or tissue Invasion into host cells or tissues (in the case of invasive pathogens) or increased adherence by extracellular pathogens Avoidance of and/or resistance to host immune defenses Acquisition of nutrients Multiplication Evacuation from the host to either a new host or an environmental reservoir.

Invariably, at one or more of these stages, bacterialproduced toxins and/or the host immune response results in damage to host tissues or organs and gives rise to a pathological condition. Essential to the establishment of a complete understanding of the host-pathogen interactions that are necessary for the manifestation of the disease are the identification and detailed characterization of virulence factors produced by pathogens at each stage of the infection process. It is beyond the scope of this chapter to describe in detail many host-pathogen interactions. Only the secretory mechanisms by which bacterial toxins and/or virulence factors are transferred from the organism to the host are described. As a reference, the various biochemical, immunological, and genetic strategies that have been successfully employed to identify bacterial pathogenicity factors are summarized in Table 12.8. To understand the basis of food-borne poisoning and intoxication, it is essential to study the underlying mechanism of pathogenesis. Pathogenic bacteria synthesize a diverse array of virulence determinants. This arsenal of bacterial weapons comprising virulence proteins has a wide variety of activities that require them to be targeted to specific locations within the host. For example, proteins involved in attachment of the microorganism to the host cell must be localized on the bacterial surface; some bacterial toxins are secreted into surrounding fluids, and others are injected directly into the cytoplasm of the eukaryotic host cell. Thus, in pathogenic bacteria, secretion itself is a key virulence determinant. Without the means to target proteins selectively, these bacteria are harmless. Since these mechanisms are mentioned in various contexts while describing the major food poisoning– and intoxication-

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causing bacterial pathogens, a brief overview of mechanisms involved in virulence factor delivery is given in this section. To maintain the integrity of subcellular organelles, cells have developed specific mechanisms to target proteins from their site of synthesis in the cytoplasm to each noncytoplasmic location. This target requires the movement of proteins into and across the lipid bilayers of cell membranes. In most cases, proteins destined to leave the cytoplasm are tagged with a signal sequence at the amino terminus. This signal sequence, or peptide, as it is usually referred to, serves to target them to the cellular secretion (Sec) machinery that includes a heterotrimeric complex of integral membrane proteins. Signal sequences and the components of this heterotrimeric complex have been conserved in all three domains of life (Pohlschroder et al., 1997). The secretory mechanisms are somewhat different in gram-positive and gram-negative bacteria, probably because of their different chemical compositions. Gram-negative cell walls generally have a higher lipid content than gram-positive cell walls. If lipid bilayers are considered as a separate subcellular compartment, then gram-positive bacteria have three destinations to which proteins can be targeted: the cytoplasm, the membrane, and the extracellular environment. In contrast, the gram-negative bacteria can target proteins to five distinct locations: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular environment. In gram-positive bacteria, the Sec machinery is sufficient for targeting to the extracellular environment since the secreted proteins must pass through only one lipid bilayer. The outer membrane of gram-negative bacteria complicates the protein secretion to the extracellular environment. The five different locations mentioned for targeting proteins entail at least five different mechanisms, termed types I through V, and these appear to be highly conserved among the gram-negative bacteria. Coincidentally, gram-negative bacteria also include most of the human pathogens. The various gram-negative secretion systems can be divided into those that utilize the Sec machinery for translocation across the inner membrane (Sec-dependent) and those that do not (Sec-independent). The Sec-dependent secretion systems include types II and V. The former involves a two-step mechanism in which proteins are first targeted to the periplasm by the Sec machinery and then secreted from the cell by a complex reaction requiring a dozen or so additional proteins. The type V systems, also known as autotransporters, utilize the normal pathway for outer membrane targeting. Autoproteolysis releases a secretory protein domain into the environment.

Table 12.8

Techniques and Strategies for Identifying Pathogenicity Factorsa

Technique/strategy Classical biochemical approaches Chemical modification screens Zymography

Receptor/ligand affinity screens Immunological methods Subtractive hybridization

Differential display Reverse genetics Large-scale screenings

Targeting of exported proteins

Coordination of regulation screens Host mimicry screens Recombination-based in vivo expression technology Signature-tagged mutagenesis

Direct selections

Advantages Doesn’t require genetic manipulation of pathogen; can identify factors essential for viability Doesn’t require genetic manipulation; can identify essential factors; not necessary to know enzymatic activity or function Doesn’t require genetic manipulation; can identify essential factors; direct detection of purified or partially purified factor Doesn’t require genetic manipulation; direct purification of factor Doesn’t require genetic manipulation; targets factors expressed during infection Doesn’t require genetic manipulation; can identify essential factors; doesn’t require knowledge of function Doesn’t require genetic manipulation; can identify essential factors Doesn’t require genetic manipulation; can identify essential pathogenicity proteins Can be comprehensive

Many pathogenicity factors exported; simultaneously generates null mutation in pathogenicity gene Directly identifies pathogenicity genes coregulated with known ones Directly identifies pathogenicity genes and may point to role during infection Identifies pathogenicity genes that are transiently induced or induced at a low level in host tissues Targets pathogenicity genes that play essential roles during infection; allows multiple mutant strains to be screened per host animal Directly identifies strains with mutations in pathogenicity genes

Complementation approaches

Directly identifies genes necessary and in some cases sufficient to confer pathogenic property

Selection for nongrowing bacterial mutants

Directly identifies genes necessary for multiplication in vitro under hostlike conditions (e.g., within tissue culture cells) Directly identifies pathogenicity genes expressed in host tissues

In vivo expression technology

Limitations Requires knowledge of enzymatic activity or function; can be laborious Targets only a subset of pathogenicity factors (e.g., those exposed on outer surface) Requires knowledge of enzymatic activity for which substrate is available; enzymatic activity must be stable and in most cases, renaturable; can be expensive Requires knowledge of either ligand or receptor Targets only factors that are immunogenic, many of which may not be pathogenicity factors Lacks sensitivity and can be laborious; only subset may encode pathogenicity factors Lacks sensitivity and can be laborious; only subset may encode pathogenicity factors Requires some amino acid sequence of protein factors Laborious; usually requires in vitro model of infection (e.g., tissue culture); cannot identify redundant pathogenicity factors Only subset of pathogenicity factors is exported proteins; some protein-receptor fusions toxic Requires knowledge of in vitro growth conditions that induce expression of pathogenicity genes Requires knowledge of in vitro conditions that mimic host parameter(s) Not all pathogenicity genes transcriptionally induced in host; only subset of infection-induced genes encode pathogenicity factors Can be laborious; requires large infectious dose in some disease models; cannot identify redundant pathogenicity factors Requires knowledge of phenotype associated with pathogenicity factor; for some direct selections only subset of mutations in pathogenicity genes Depending on strategy, requires efficient means of transformation into pathogen or simple in vitro model of disease (e.g., adherence to tissue culture cells) Only subset of mutations in genes specifically required for in vivo growth May not identify pathogenicity genes transiently expressed during infection or expressed at low levels (table continues)

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Table 12.8

(continued) Advantages

Limitations

Rapid and simple method of identifying pathogenicity genes Targets pathogenicity genes that play essential roles during infection; allows multiple mutant strains to be screened per host animal Rapid and simple method of identifying pathogenicity genes

Requires mutagenesis studies to confirm roles in pathogenicity Laborious; requires prior genome sequence information; cannot identify redundant pathogenicity factors Requires prior knowledge of pathogenicity factors to serve as query sequences; requires mutagenesis studies to confirm roles in pathogenicity Requires prior genome sequence information; expensive; requires mutagenesis studies to confirm roles in pathogenicity

Technique/strategy Genome walking Genomic analysis and mapping by in vitro transposition Computational screens

Transcriptional profiling and use of microarrays

Rapid and comprehensive method of identifying pathogenicity genes

a

This table lists most of the techniques and strategies discussed in the text and is not all-inclusive. Only a brief description of some of the major advantages and disadvantages of each is given. Source: Compiled from Camilli et al. (2001).

The Sec-independent secretion systems include types I, III, and IV. The type I systems utilize a complex of three proteins that span both the inner and outer membranes of the gram-negative bacteria. The proteins are directly secreted into the media. The type IV systems are not well characterized at this time. They appear to be closely related to systems used for the conjugal transfer of deoxyribonucleic acid (DNA) from one bacterium to another. The type III systems were identified only recently and are fascinating devices that actually allow injection of bacterial proteins into the cytoplasm of the host cell. Sometimes they are also called contact-dependent to reflect this capacity for injection. The biochemical characteristics of these various secretory pathways are briefly described in the sections that follow. 12.2.1

General Secretory Pathway

In the general secretory pathway, proteins destined for noncytoplasmic locations are synthesized with a signal sequence that targets them for translocation. This amino-terminal signal is later cleaved during the export process (Blobel and Dobberstein, 1975). Many signal sequences from both prokaryotic and eukaryotic proteins have been shown to consist of 15–26 amino acids. There is no one conserved sequence, but all possess common features (von Heijne, 1985). They contain a stretch of 10–12 hydrophobic amino acids preceded by one or two positively charged residues and followed at the carboxy-terminal end by a cleavage site for leader peptidase. This peptidase removes the signal sequence from the preprotein to yield the mature

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protein. This run of hydrophobic residues resembles a transmembrane domain. Mutations that block signal sequence processing do not prevent translocation but leave the mutant precursor protein tethered to the inner membrane with the amino terminus of the signal sequence facing the cytoplasm (Fikes and Bassford, 1987). The bacteria secrete the so-called Sec proteins to catalyze insertion into and translocation across the inner membrane. Three proteins, viz., SecA, SecE, and SecY, constitute the essential core of the secretion machinery. Other proteins involved in this process include SecB, SecD, SecF, SecG, and YajC (Akimaru et al., 1991; Duong and Wickner, 1997a). The Sec machinery thus comprises soluble cytoplasmic proteins and peripheral and integral cytoplasmic membrane proteins (Figure 12.2). In order to be exported from the cytoplasm, a protein must first be recognized by the secretion machinery constituted by these various proteins. Multiple partially redundant mechanisms ensure the accuracy of precursor recognition. The secretion-specific chaperone SecB binds to the mature portions of exported proteins and maintains them in an unfolded, export-competent state (Smith et al., 1997). This contributes to recognition because SecB also binds SecA (den Blaauwen et al., 1997). SecA functions as a dimer to direct the precursor to the membrane and energize the translocation reaction. The complex of SecA, precursor protein, and SecB interacts on the cytoplasmic face of the heterotrimeric SecYEG complex in the inner membrane. The latter likely forms a protein-conducting channel through the inner membrane. Once SecA enters the membrane, SecB is released into the cytoplasm, and the precursor is partially translocated through the SecYEG

Figure 12.2 General secretory pathway. SecA and SecB bind nascent polypeptide, shown by the thick black line. The signal sequence is represented by the small shaded rectangle. SecA delivers the polypeptide to the SecYEG complex in the inner membrane. SecA undergoes a dramatic conformational change that drives sequences of the precursor protein across the membrane. SecDFYajC stabilizes the inserted form of SecA. The signal sequence is subsequently cleaved by signal peptidase and the protein is secreted into the periplasm. OM, outer membrane; IM, inner membrane.

complex. This allows access of signal peptidase to the precursor, which then cleaves the signal peptide. SecD, SecF, and YajC seem to stabilize the membrane-bound form of SecA (Duong and Wickner, 1997b). Adenosine triphosphate (ATP) hydrolysis causes SecA to release the partially translocated precursor and deinsert from the membrane. Additional ATP binding and hydrolysis by SecA repeat the insertion/deinsertion process through the inner membrane (Driessen et al., 1998). As mentioned earlier, the components of the secretion machinery are conserved throughout biological forms. However, SecB has only been found in the family Enterobacteriaceae (Pohlschroder et al., 1997). 12.2.2

Sec-Dependent Secretion Systems

Type V Secretion (Autotransporters) The type V systems are the simplest of the Sec-dependent secretion systems. These are also known as autotransporters. The prototype of this type of transport system is the immunoglobulin A (IgA) protease from Neisseria gonorrhoeae; the proteolysis of IgA by IgA protease is a major contributor to virulence (Koomey and Falkow, 1984). Autotransporters are first exported to the outer membrane, where a proteolytic event releases the final product into the medium (Figure 12.3).

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The secreted IgA protease is first expressed in the cytoplasm as a large multidomain protein consisting of an amino-terminal Sec-dependent signal sequence, the 106kDa mature portion of the protein, a 30-amino-acid γ-protein, a 15-kDa secreted α-protein, and a 45-kDa carboxy terminal β-protein, which remains inserted in the outer membrane (Pohlner et al., 1987). The signal sequence targets the protein for translocation from the cytoplasm by the Sec machinery. Concomitant with translocation across this membrane, the signal peptide is cleaved and the remainder of the protein is released into the periplasm. The carboxy-terminal β-domain is targeted to the outer membrane, where it forms a β-barrel pore or channel through which the rest of the protein can pass in its unfolded state through the outer membrane onto the cell surface. The protein then undergoes autoproteolysis to cleave the β-domain from the rest of the protein. The β-protein remains in the outer membrane, while the mature α- and γ-domains are released into the extracellular environment. Subsequent autoproteolytic cleavages release the mature protein from the small α- and γ-proteins (Klauser et al., 1993). Although their exact function is unknown, the latter two could play a role as chaperones. Thus, the two main features of this secretion process are the presence of an amino-terminal signal sequence that directs export of the secreted protein from the cytoplasm

Figure 12.3 Type V secretion, or the autotransporters. The protein to be secreted has four domains: α, β, γ, and protease. The βdomain forms a pore in the outer membrane through which the other domains pass into the external milieu. Autoproteolytic cleavage releases the α- and γ-domains from the protease domain. OM, outer membrane; IM, inner membrane.

via the Sec machinery and the presence of β-domain for secretion from the periplasm across the outer membrane. The sequences translocated through the β-domain are generally in an unfolded state, and the translocation occurs in a linear fashion. The simplicity of these systems is striking, especially in contrast to other systems described later. Type II Secretion System (Two-Step Secretion) The type II secretion systems are commonly used for the extracellular secretion of proteins from gram-negative bac-

teria. They are found in Klebsiella oxytoca, Erwinia chrysanthemi and E. carotovora, Pseudomonas aeruginosa, Aeromonas hydrophila, Xanthomonas campestris, and Vibrio cholerae. The type II secretion systems consist of about 12–14 proteins that are encoded by a cluster of genes (Pugsley, 1993; Russel, 1998) (Figure 12.4). The presence of proteins such as PulG, H, I, and J is believed to form a piluslike structure in the envelope. These proteins, the pseudopilins, contain consensus prepilin peptidase cleavage sites and are processed by the PulO protein so

Figure 12.4 Type II, or two-step secretion. PulA is translocated through the inner membrane by the Sec machinery and secreted out of the cell by an apparatus that comprises 14 Pul proteins. OM, outer membrane; IM, inner membrane; ATP, adenosine triphosphate.

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that they can assemble properly. Additional components required for secretion include PulC, F, K, L, M, and N, all of which are located in the inner membrane and have unknown functions (d’Enfert et al., 1989). Like that via type IV autotransporters, secretion via type II systems requires the presence of a classical signal peptide recognized by the Sec machinery and cleaved by signal peptidase I in most cases, or signal peptidase II in the case of lipoproteins. This signal is necessary and sufficient to get the secreted protein into the periplasm. Type I Secretion Systems (Adenosine Triphosphate–Binding Cassette Transporters) In terms of number of components, the type I systems are quite simple. Only three proteins are required for Sec-independent secretion. Examples of the proteins secreted by the type I secretion systems include α-hemolysin from E. coli, Proteus vulgaris, metalloprotease from Serratia marcescens, and alkaline protease from Pseudomonas aeruginosa (Koronakis et al., 1987; Letoffe et al., 1993; Duong et al., 1992). These systems include a component that is a member of the ATP-binding cassette, or ABC, family of transporter proteins (Fath and Kolter, 1993) (Figure 12.5). The type I systems secrete proteins and other substrates via a mechanism in which transfer across both the inner and outer membranes occurs in a single step and requires only three proteins. These three components include an ABC

transporter; a membrane fusion protein, or accessory factor; and an outer membrane component. The secreted protein, while still in the cytoplasm, is recognized by and binds to the ABC protein via a carboxy-terminal signal. Substrate binding inhibits the adenosine triphosphatase (ATPase) activity of the ABC protein and promotes its interaction with the membrane fusion protein. This interaction then stimulates interaction of the membrane fusion protein with the outer membrane component, so that the secretion apparatus is assembled in an ordered fashion (Letoffe et al., 1996). The mechanism by which the substrate passes from the cytoplasm to the environment is not clear. The most likely scenario is that the complex of the ABC protein, membrane fusion protein, and outer membrane factor forms a channel through which the proteins pass, in one step, from the cytoplasm to the external medium. Type IV Secretion Systems (Conjugal Transfer Systems) The type IV secretion systems are used by the plant pathogen Agrobacterium tumefaciens for the transfer of oncogenic T-DNA and proteins to plants, and certain type IV components are used by Bordetella pertussis for the secretion of pertussis toxin (Figure 12.6). On the basis of the sequence homologies, these secretion systems appear to be novel adaptations of the conjugal transfer system used for the horizontal transfer of plasmids. Winans and associates (1996) have reviewed these systems.

Figure 12.5 Type I secretion, or ABC exporters. A complex of HlyB, HlyD, and TolC comprises a bridge that traverses the inner and outer membranes and allows secretion of hemolysin. ABC, adenosine triphosphate binding cassette; ATP, adenosine triphosphate; OM, outer membrane; IM, inner membrane.

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Figure 12.6 Type IV secretion, or conjugal transfer systems. The T-complex, made up of protein and DNA, is secreted from Agrobacterium tumefaciens and into plant cells by a piluslike complex of Vir proteins. DNA, deoxyribonucleic acid; ATP, adenosine triphosphate; IM, inner membrane; OM, outer membrane.

Type III Secretion Systems (Contact-Dependent Secretion) Like the type I systems, the type III systems secrete proteins via a Sec-independent mechanism. They allow not only secretion but also injection of virulence factors directly into the cytosol of eukaryotic host cells. Type III systems are known in animal pathogens such as Yersinia pestis and other yersiniae, Shigella flexneri, and Salmonella typhimurium, as well as plant pathogens such as Pseudomonas syringae and Erwinia species. The Yersinia Ysc system is the best characterized of these systems (Figure 12.7). This is described in detail later in this chapter, and only the salient features are mentioned here. Hueck (1998) has comprehensively reviewed the type III secretion systems. Yersinia spp. secretes a collection of virulence proteins called Yersinia outer proteins (Yops). The Yop secretion (Ysc) proteins mediate Yop secretion in a Secindependent manner. Yops require both secretion out of the bacteria and injection or translocation into the host cells to exert their toxicity. This translocation of Yops is mediated by a subset of the Yops, YopD and YopB. The process of Yop injection occurs in two steps: secretion out of the bacterium and translocation into the eukaryotic host. These two steps require more than 20 proteins to transfer

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toxins directly from pathogen to host. Little is known about the exact function of many of these Ysc proteins. The direct transfer requires that the cell coregulate secretion and translocation. The bacterial cell senses contact with the host cell via the YopN (LcrE) protein. This protein plugs the secretion pore on the cytoplasmic side of the secretion apparatus. When the bacterium contacts a host cell or Ca2+ is removed, the secretion channel is unplugged. The next step, translocation, is facilitated by the formation of a translocator through which the effector Yops are injected into the eukaryotic cell. YopB and YopD are thought to form a channel through which the effector Yops can enter the host cytosol. A group of proteins, called the chaperones, is required for the translocation of Yops into host cells. These chaperones are bacterial cytoplasmic proteins that demonstrate specificity for one or two substrates and are usually named Syc for specific Yop chaperone. Several of these chaperone proteins have been identified and characterized. The five mechanisms for protein secretion described earlier that are conserved among gram-negative bacteria appear to be quite diverse. Some work independently of the Sec apparatus (e.g., types II and V); others (types I, III, and IV) do not. The various systems require anywhere

Figure 12.7 Type III, or contact-dependent secretion. Yops are secreted from Yersinia species and translocated into host cells by a complex of more than 20 proteins. Left to right: secretion in the absence of Ca2+, inhibition of secretion in the presence of Ca2+, and contact-dependent secretion and translocation. OM, outer membrane; IM, inner membrane.

from one special component in the case of the autotransporters, to 20 or more components, as in the case of the contact-dependent secretion systems. Despite this diversity, there are at least two major themes emerging. One, these secretion systems all possess a putative pore-forming protein that allows passage of the secreted substrate through the outer membrane, thereby overcoming the barrier problem posed by lipid bilayers. Second, the multiple components of complex secretion systems form a macromolecular machine that spans the gram-negative cell envelope, and such structures likely resemble surface structures such as pili or flagella. Understanding the simplicity and beauty of these secretion systems gives us vital insights on the pathogenic mechanisms devised by various microorganisms for infecting humans and animals.

12.3 SALMONELLA SPECIES (SALMONELLOSIS) The salmonellae, well known for causing enteric typhoid fever and gastroenteritis in humans, infect humans and virtually all known wild and domestic animals, including birds, reptiles, and insects. These infections result in significant morbidity and mortality rates. Salmonella spp.

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gastroenteritis is a type of food poisoning that is usually characterized by the abrupt onset of nausea, fever, vomiting, diarrhea, and abdominal cramps 8–24 hours after consumption of contaminated food or water. It is usually a self-limiting infection for which antibiotics are contraindicated. The second form of Salmonella spp. infection is enteric fever, which connotes a disease with greater systemic involvement. Those of typhoid fever typify the clinical symptoms of enteric fever. In enteric fevers, Salmonella spp. become disseminated via the lymphatic vessels and phagocytic cells to the bloodstream after penetration of the small intestinal epithelium. Unlike in the treatment of gastroenteritis, enteric fevers require appropriate antibiotic therapy. Globally, Salmonella spp. continue to be the leading cause of food-borne bacterial diseases in humans; in fact, food-borne salmonellosis tends to dwarf all illnesses associated with other food-borne pathogens. Its worldwide occurrence has prompted both national and international surveillance. Since both humans and animals are reservoirs and many animals are sources of human food, the salmonellosis problem appears to be a permanent element of human existence. Fortunately, with the exception of the enteric fevers, salmonellosis is neither a highly lethal disease nor a particularly serious one. Over 2 million cases of

salmonellosis occur annually in the United States. Our inability to limit salmonella infections in the human food chain is a major reason for the large number of human infections. Typhoid fever caused by the exclusively human pathogens S. typhi and S. paratyphi was a major cause of death throughout the world in the 19th and early 20th centuries. As a result of improvements in sanitation, the incidence of typhoid fever has dropped dramatically in developed nations; however, it remains a significant problem in the developing world. In contrast, human gastroenteritis caused by nontyphoidal salmonellae is globally increasing because of zoonotic contamination of food. Antibiotic resistance of both typhoidal and nontyphoidal serotypes is also increasing and has magnified the public health problem of salmonella infections. The association of Salmonella spp. with human intestinal ulceration, later identified as typhoid fever, was first documented in the early 19th century in France. Prior to this, human enteric or typhoid fever was often confused with typhus, a rickettsial disease. These two diseases were pathologically distinguished by P. Ch. A. Louis in France in 1829 and by William Jenner in the United States in 1850 (Scherer and Miller, 2001). Gaffkey was the first to isolate the typhoid bacillus (S. typhi) from human spleens in 1884. Subsequently, Daniel Salmon, a veterinary pathologist, isolated S. choleraesuis from the intestines of pigs infected with hog cholera in 1885. The genus was subsequently named after him. In 1896, Pfeiffer and Kalle introduced the first heat-killed bacterial typhoid vaccine, and Widal determined that convalescent patient sera could agglutinate S. typhi. This technique provided a useful clinical tool for the identification of salmonellae. An antigenic scheme based on the serological detection of somatic (O) and flagellar (H) antigens within the Salmonella spp. group was first used for the classification by White in the 1920s and subsequently expanded by Kauffmann into the Kauffmann-White scheme, which in 1994 recognized more than 2300 serovars (Popoff et al., 1994). In this section, some of the microbiological and biochemical characteristics of Salmonella spp., the epidemiological factors of salmonellosis, general disease course, and underlying pathogenesis mechanisms are briefly described. 12.3.1

Organism

The genus Salmonella is a member of the family Enterobacteriaceae. They are facultatively anaerobic gram-negative rods. Most species are motile by peritrichous flagella. Nonflagellated variants, such as S. pullorum and S. gallinarum, and nonmotile strains resulting from dysfunctional flagella also occur. Salmonellae grow readily on simple

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media, but they almost never ferment lactose or sucrose. They form acid and sometimes gas from glucose and mannose. They usually produce H2S. Salmonellae are oxidasenegative and catalase-positive. The organisms grow optimally at 37°C; the growth temperature range is 2°C–54°C. Above the maximal growth temperature, Salmonella spp. die quickly. Thus, they are relatively heat-sensitive, being killed at 60°C in 15–20 minutes. Salmonellae also exhibit psychrotrophic properties, as reflected in their ability to grow in foods stored at 2°C to 4°C (D’Aoust, 1991b). Moreover, preconditioning of cells to low temperatures can markedly increase the growth and survival rates of salmonellae in refrigerated food products (Airoldi and Zottola, 1988). Although the time varies with substrate and the influence of factors such as pH and aw, strains may survive for days to weeks at chill temperatures. Salmonellae do not tolerate salts very well. They are generally inhibited in the presence of 3%–4% NaCl. However, bacterial salt tolerance increases with increasing temperature in the range of 10°C to 30°C. Foods with water activity ≤0.93 do not support the growth of salmonellae (D’Aoust, 1989). Although there is no growth below 0.93, Salmonella survives; the time of survival increases as aw decreases. In low-moisture foods, such as pasta, peanut butter, and chocolate, survival is measured in months (Cox, 2000a). The optimal pH for growth is between pH 6 and 8, although they have the ability to proliferate at pH values ranging from 4.5 to 9.5. Increasing temperature increases sensitivity to low pH, as does the presence of food additives such as salt or nitrite. Tolerance or adaptation to low pH is significant with respect to virulence, increasing the likelihood of surviving gastric acidity, or the acidic intracellular environment of phagocytic cells. The classification of the Salmonella spp. group is complex because the organisms are a continuum rather than a defined species. It has progressed through a succession of taxonomical schemes based on biochemical and serological characteristics and on principles of numerical taxonomy and DNA homology (Table 12.9). As mentioned earlier, the Kauffmann-White scheme was the first realistic attempt to classify salmonellae systematically on the basis of scientific parameters. In this scheme, the genus can be classified into five biochemically defined subgenera (I to V), wherein individual serovars were given species status (Kauffmann, 1966). In this classification scheme, members of the Arizona group (S. arizonae) were included in subgenus III. The genus has also been classified in terms of 16 discriminating biochemical tests into three species as S. typhi (single serovar), S. choleraesuis (single serovar), and S. enteritidis (all other Salmonella spp. serovars) (Ew-

ing, 1972). In this scheme, members of the Arizona group are treated as a distinct genus. On the basis of DNA hybridization studies, the formal taxonomic classification includes the genus Salmonella with one single species (S. choleraesuis) and seven subspecies (ssp.), each with its own phenotypic characteristics and history (Table 12.9). This scheme also utilizes numerical taxonomic principles, in which a statistical comparison of morphological and biochemical attributes of the strains (phenetic analysis) is made to evaluate the taxonomical proximity of test strains. Almost all (>99%) of the salmonellae that cause disease in humans are in subspecies 1 (choleraesuis or enterica) and can be isolated from warm-blooded animals; the other groups are predominantly isolated from cold-blooded animals and the environment (Le Minor et al., 1986). Subsequent modification of this scheme included a change from S. choleraesuis to S. enterica while retaining the name of seven recognized subspecies (Le Minor and Popoff, 1987). This was done to prevent confusion with the serovar choleraesuis. In practice, the formal species and subspecies names are rarely used. The simplified nomenclature considers the serotype

names as specific names as if they were a genus and species designation. Using yet another criterion of multilocus enzyme electrophoresis (MLEE), members of S. enterica ssp. bongori were classified into a new species (Reeves et al., 1989). The new species was designated S. bongori. The WHO Collaborating Center for Reference and Research on Salmonella (Institut Pasteur, Paris) now recognizes S. enterica and S. bongori as two distinct species, which currently include 2356 and 19 serovars, respectively (Table 12.10) (Popoff et al., 1994; D’Aoust, 1997). As the correct taxonomic classification for Salmonella subspecies is rather confusing, the common species name that prevailed before reclassification of the species is still widely used. Thus Salmonella choleraesuis ssp. choleraesuis (or group 1), serovar typhi, is simply referred to by its common name, S. typhi. Whereas salmonellae are initially detected by their biochemical characteristics, groups and species are identified by antigenic analysis. Like other Enterobacteriaceae, salmonellae possess several O lipopolysaccharide (LPS) antigens (from a total of more than 60) found on the exter-

Table 12.9 Various Classification Schemes Used for Salmonella Species Diagnostic basisa Biochemical Biochemical

Phenetic analysis and DNA homology

Phenetic analysis and DNA homology MLEE

a

Features Five subgenera (I–V) Serovar = species status Three species S. typhi S. choleraesuis S. enteritidis Arizona treated as separate genus Single species (S. choleraesuis) Seven subspecies S. choleraesuis (or enterica, group 1) S. salamae (group 2) S. arizonae (group 3a) S. diarizonae (group 3b) S. houtenae (group 4) S. bongori (group 5) S. indica (group 6) Type strain = S. choleraesuis Single species (S. enterica) Seven subspecies as previous entry Type strain = S. typhimurium LT2 Two species S. enterica (six subspecies) S. bongori

DNA, deoxyribonucleic acid; MLEE, multilocus enzyme electrophoresis.

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Serovar designation

Reference

S. typhimurium

Kauffmann (1966)

S. enteritidis serovar S. typhimurium

Ewing (1972)

S. choleraesuis ssp. choleraesuis serovar S. typhimurium

Le Minor et al. (1986)

S. enterica ssp. enterica serovar S. typhimurium

Le Minor and Popoff (1987)

Reeves et al. (1989)

Table 12.10 Salmonellaa

Species of the genus

Species Salmonella enterica ssp. enterica ssp. salamae ssp. arizonae ssp. diarizonae ssp. houtenae ssp. indica Salmonella bongori Total a

No. of serovars 1,405 471 94 311 65 10 19 2,375

Compiled from Popoff et al. (1994) and D’Aoust (1997)

nal surface of the bacterial outer membrane, and different H antigens associated with the peritrichous flagella in one or both of two phases. Some have the capsular K antigens, referred to as Vi, which may interfere with agglutination by O antisera and are associated with invasiveness. It is a homopolymer of N-acetylgalactosaminouronic acid. The Vi antigen occurs only in S. typhi, S. paratyphi C, and S. dublin (Le Minor, 1981). Agglutination tests with absorbed antisera for different O and H antigens form the basis for serological classification of salmonellae. Loss of O antigen is associated with a change from smooth to rough colony form. Smooth variants are strains with well-developed serotypic LPS antigens that readily agglutinate with specific antibodies, whereas rough variants exhibit incomplete LPS antigens that show weak or no agglutination with Salmonella spp. somatic antibodies. Agglutination by antibodies specific for the various O antigens is used to group salmonellae into six serogroups: A, B, C1, C2, D, and E. Although these groupings can help identify pathogenic bacteria as Salmonella spp., cross-reactivity between groups does not allow for definitive identification of the serotype. For example, both S. enteritidis and S. typhi express O antigens of group D. The O antigens are numbered 1 to 67, though noncontiguously, as some antigens have been removed from the typing scheme since they were assigned to organisms that were originally but now no longer are within the genus Salmonella. Some serovars are easily defined with respect to H antigens as they produce only one form or phase. The H antigens are heat-labile proteins. Individual Salmonella spp. strains may produce one (monophasic) or two (diphasic) H antigens. Some strains are genotypically triphasic, capable of producing a third phase H antigen, which may be encoded chromosomally or more often extrachromo-

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somally (plasmide-borne), although phenotypically these strains appear diphasic. An alternative H antigen produced in phase 1 is referred to as a R-phase H antigen. Phase 1 H antigens are typically described by lowercase letters (a, b, c) or, beyond the 26th such antigen, by the letter z and a consecutive number (z1, z2 . . . z83, and so on) (Cox, 2000a). The first phase 2 H antigens identified are described, like O antigens, by numerals (1, 2, 3), although many serovars express H antigens typical of phase 1 as phase 2 antigens (e.g., e, n, x). Organisms may lose H antigens and become nonmotile. Capsular antigens commonly encountered in members of the family Enterobacteriaceae are limited to the Vi antigen, which is necessary for the immunological identification of underlying serotypic LPS. Vi antigen may be lost partially or completely. Further classification of serotypes is based on the antigenicity of the flagellar H antigen and other more specific genetic and molecular methods, such as bacteriophage typing, pulsed field gel electrophoresis, and restriction fragment length polymorphism (RFLP) analysis. It is worth noting that the efficacy of any technique varies with the serovar. The current method of choice for a growing number of serovars is phage typing. Typing sets have been developed for serovars enteritidis, heidelberg, paratyphi B, typhi, typhimurium, and virchow. Further strain analysis becomes necessary when a particular phage type (PT) of a serovar becomes dominant, such as enteritidis PT4 in Great Britain. Four species of salmonellae that cause enteric fever can be identified in the clinical laboratory by biochemical and serological tests. These species should be routinely identified as to species because of their clinical significance. They include S. paratyphi A (serogroup A), S. paratyphi B (serogroup B), S. choleraesuis (serogroup C1), and S. typhi (serogroup D). The 2000-odd other salmonellae that clinical laboratories serogroup as A, B, C1, C2, D, E, etc. (the serogroup list goes further in the alphabet and on to numbers), are sent to public health reference laboratories for serological identification. This system allows public health officials to monitor and assess the epidemiological characteristics of Salmonella spp. infections on a statewide and nationwide basis. Representative antigenic formulas of some selected salmonellae are shown in Table 12.11. 12.3.2

Pathogenesis

Salmonellae almost always enter via the oral route, usually with contaminated food or drink. The mean infective dose to produce clinical or subclinical infection in humans is 105–108 salmonellae, but perhaps as few as 103 S. typhi organisms. Newborns, infants, and elderly and immunocom-

Table 12.11 Representative Antigenic Formulas for Selected Salmonellae

Table 12.12

Human Infectious Doses of Salmonella Species

Food O group D D D A C1 C1 B D F E D C2 a

Serotype

Antigenic formulaa

S. typhi S. typhi (R-phase) S. dublin S. paratyphi A S. choleraesuis S. virchow S. typhimurium S. enteritidis S. rubislaw S. orion S. pullorum S. munchen

9, 12 (Vi):d:9, 12 (Vi):j:z66 1, 9, 12(Vi):g,p:1, 2, 12:a:1,5 6, 7:c:1,5 6,7:r:1,2 1, 4, 5, 12:I:1,2 1, 9, 12:g,m:11:r:e,n,x 3,10:y:1,5 1,9,12:-:6,8:d:1,2

O antigens: boldface numerals, serogroup-specific antigen; (Vi), Vi antigen if present; phase 1 H antigen, lowercase letter; phase 2 H antigen, numeral.

promised individuals are more susceptible to salmonella infections than healthy adults (D’Aoust, 1989). Immunocompromised people are approximately 20 times more likely to contract symptomatic salmonellosis than the general population, even when presented with a low infectious dose (Scherer and Miller, 2001). Among the host factors that contribute to resistance to salmonella infection are gastric acidity, normal intestinal microbial flora, and local intestinal immunity. Low gastric acid production, especially in infants and elderly people, allows intestinal colonization and systemic spread of salmonellae (D’Aoust, 1991a). Similarly, antibiotic treatment of subjects prior to their encountering salmonella infection enhances the severity of the disease through an antibiotic-mediated clearance of native gut microflora. It reduces the level of bacterial competition for nutrients and attachment sites in the intestinal tract of the host (Isberg and Nhieu, 1994). The infective dose also depends on the type and chemical composition of the food vehicle responsible for infection (Table 12.12). Foods associated with low infective doses include high-fat-content foods such as chocolate, cheese, and meat. Entrapment of salmonellae within the hydrophobic lipid micelles may protect the organism against the bactericidal action of gastric acidity. The characteristic that only a few Salmonella spp. cells can develop into a variety of clinical conditions and deteriorate into septicemia and even death highlights the unpredictable pathogenicity of this large and heterogeneous group of human bacterial pathogens. Thus, even low levels of salmonellae in a finished food product have the potential to lead to serious public health consequences.

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Eggnog Goat cheese Carmine dye Imitation ice cream Ice cream Chocolate Chocolate Chocolate Peanut butter Hamburger Cheddar cheese Cheddar cheese Paprika potato chips

Serovar S. meleagridis S. anatum S. Zanzibar S. cubana S. typhimurium S. enteritidis S. eastbourne S. napoli S. typhimurium S. mbandaka S. Newport S. Heidelberg S. typhimurium S. saintpaul S. javiana S. rubislaw

Infectious dose 106–107 105–107 105–1011 104 104 <102 102 101–102 ≤101 101–102 101–102 102 100–101 ≤4.5 × 101

Source: Compiled from D’Aoust (1994, 1997) and Cox (2000a).

The presence of viable salmonellae in the human intestinal tract indicates that the organisms have overcome nonspecific host defenses. A list of a number of host immune mechanisms and corresponding bacterial properties that allow salmonellae to evade or interact with the host system is presented in Table 12.13. Host immune mechanisms relevant to Salmonella spp. infection include gastric acidity, peristalsis, complement, opsonins, antimicrobial peptides, cilia, mucin, lysozyme, and the intestinal cell glycocalyx (Scherer and Miller, 2001). Those after invasion include phagocytosis, antimicrobial activity within phagosomes (antimicrobial peptides, nitrates, oxygen radicals, acidity), and secretion of chemokines and cytokines in response to signature bacterial molecules such as LPS. The major bacterial factor responsible for stimulation of these various defense mechanisms is thus LPS or endotoxin. However, other bacterial factors have been shown to induce expression of inflammatory molecules in vitro. These include flagellar filament proteins FliC (either monomers or filaments) and FljB (Ciacci-Woolwine et al., 1998). Salmonellae that survive the low pH of the stomach quickly colonize the lumen of the small intestine, where they predominantly localize to Peyer’s patches. Passage of Salmonella spp. through the intestinal epithelial barrier most likely occurs through specialized microfold (M) enterocytes, which overlay Peyer’s patches (Brandtzaeg, 1989). Proteinaceous appendages develop on the surface of salmonellae upon contact with epithelial cells. The ATPase and translocase enzymes may contribute to the

Table 12.13

Host Immune Defenses and Salmonella Species Infection

Host immune mechanismsa Stomach acid Extracellular matrix (glycocalyx) barrier Antimicrobial peptides Lysozyme Defensins and other cAMP Complement Phagocytosis Mannose-binding protein Lectins Complement

Bacterial properties/factors

Envelope modifications Outer membrane proteins, lipoproteins Lipid A modifications Transporters Vi antigen Inhibition of phagocytosis Macropinocytosis/spacious phagosomes Unique phagosome trafficking

Cytokine and chemokine production Intracellular survival Immune cell chemotaxis PMN transmigration

a

Stimulators of innate immunity Lipid A Flagellin Type III secretion

cAMP, cyclic adenosine monophosphate; PMN, polymorphonuclear.

formation of these structures. The inability of mutants defective in the assembly of these appendages to enter cultured epithelial cells suggests that these bacterial organelles are essential for invasion (Ginocchio et al., 1994). These appendages are short-lived and shed concomitantly with the appearance of membrane ruffles on colonized epithelial cells. After bacterial attachment, signal transduction between the pathogen and host cell culminates in an energy-dependent Salmonella spp. invasion of enterocytes and M cells. Invasive salmonellae enter M cells via a process that induces membrane ruffling and endocytosis and gain access to the underlying lymphatic tissue either by transcytosis across the M cells or by lysis of the M cell (Clark et al., 1994; Penheiter et al., 1997). Salmonella spp. first destroy the enterocyte microvilli and then induce the formation of large membrane ruffles in the enterocytes. Bacteria are internalized when the ruffles fuse to form phagosomes. The invasive mechanism triggers two profound changes in enterocytes and M cells: a Ca2+ influx and cytoskeleton rearrangement in the targeted host cells. The influx of Ca2+ ions causes host cell actin polymerization into microfilaments in the vicinity of the invading pathogen. Adherent salmonellae can also stimulate phospholipase C activity in the host cells (D’Aoust, 1997). This enzyme activation results in the breakdown of lipids in the plasma membrane of host cells and in the intracytoplasmic release of inositol triphosphate, which in turn triggers the solubilization of Ca2+ bound in the cytoplasmic matrix. The gene products

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for this alternative mechanism of increasing the intracellular Ca2+ ion concentration in the epithelial cells remain unidentified. The appearance of membrane ruffles coincides with the disappearance of the bacterial appendages and with the pathogen-directed aggregation of host plasma membrane proteins. These are predominantly class I major histocompatibility complex heavy-chain proteins. The latter protein aggregates are incorporated into the membrane of infected pinocytotic vacuoles to camouflage the intravacuolar presence of a foreign bacterium, thereby preempting host-cellmediated immune responses (D’Aoust, 1997; Scherer and Miller, 2001). After a time, the enterocytes eventually repair the brush border and return to normal. The ability to invade epithelial cells may enable Salmonella spp. to colonize and cross epithelial barriers with greater efficiency. Once bacteria have crossed through the intestinal epithelium, they enter phagocytic cells (macrophages) in the underlying lymph tissue. Salmonellae are able to survive and replicate within macrophages, a feature that is correlated with pathogenesis in the mouse typhoid model of S. typhimurium infection (Fields et al., 1986). Phagosomes containing wild-type S. typhimurium have been termed spacious phagosomes (SPs) because they are unusually large (approximately 2–6 µm) and because bacteria appear to be swimming freely within them. The strategies that salmonellae use to survive within macrophages have not yet been fully elucidated, but re-

sults to date suggest two general mechanisms: (a) salmonellae traffic to the phagolysosome and synthesize factors, including those that remodel the bacterial surface, that promote resistance to microbicidal activities within the phagosome; or (b) they alter host cell processes that modify the salmonellae-containing vacuoles and promote bacterial survival (Scherer and Miller, 2001). Bacterial replication within the vacuoles begins within hours after internalization. The infected vacuoles translocate from the apical to the basal pole of the host cell, where salmonellae are released into the lamina propria (Isberg and Nhieu, 1994). Once released, the salmonellae can migrate into deeper layers of tissues. This movement into deeper tissues is apparently facilitated by the binding of thin aggregate fimbriae on the outer surface of Salmonella spp. cells to host plasminogen. This zymogen form of the latter would be converted into its proteolytic (plasmin) form on the bacterial surface, thereby providing salmonellae with an effective means to breach host tissue barriers and facilitate transcytosis into deeper tissues (Sjobring et al., 1994). Salmonella spp. infection of macrophages results in cytotoxicity, in the form of either necrosis (generalized cell death) or apoptosis (programmed cell death). Necrotic death is characterized by cytoplasmic swelling, membrane disruption, disappearance of nuclear chromatin, and eventual lysis of the cell. It usually produces an inflammatory response in the area. Apoptotic death, in contrast, is characterized by membrane blebbing, cell shrinkage, and chromatin condensation and fragmentation. The ingestion of apoptotic cell fragments by neighboring cells prevents the inflammatory response seen in necrosis. The interaction of Salmonella spp. with their hosts is complex and requires many different bacterial factors. Approximately 4% of the Salmonella spp. genome has been estimated to be required for virulence in the BALB/c mouse model system (Bowe et al., 1998). A number of virulence factors map to regions of the genome known as Salmonella spp. pathogenicity islands (SPIs). They contain large segments of DNA that appear to have been acquired by horizontal transmission from an exogenous source, as the ratio of GC to AT base pairs in these regions differs from that of the rest of the Salmonella spp. chromosome (Ochman and Groisman, 1996). To date, five such islands have been described, at least two of which are specific for Salmonella species. A list of many proposed virulence factors and their in vitro and in vivo phenotypes is provided in Table 12.14. For a detailed description of the genetic regulation of virulence, major transcriptional regulators, and factors required for invasion of epithelial cells, the readers should refer to several excellent reviews (Miller, 1991; D’Aoust, 1997; Scherer and Miller, 2001). Only the salient features are described here.

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Transcriptional regulons required for Salmonella spp. pathogenesis are made up of a two-component regulatory system, viz., PhoP and PhoQ. Together they control expression of more than 40 genes (Miller et al., 1989). PhoP/PhoQ is required for virulence in mice and humans, survival within macrophages, growth on succinate as a sole carbon source, and growth in the presence of magnesium limitation. PhoQ is a sensor histidine kinase that phosphorylates PhoP, a response regulator, in response to environmental conditions (Gunn et al., 1996). PhoPphosphate activates expression of a set of genes arbitrarily designated as pags (for PhoP-activated genes), which promote Salmonella spp. survival within host tissues. Proteins encoded by a pag include nonspecific acid phosphatases, cation transporters, outer membrane proteins, and enzymes important for lipopolysaccharide (LPS) modification. In contrast to the pag genes that are expressed under adverse environmental conditions such as low pH, nutrient deficiency, and presence of host antibacterial factors (e.g., defensins), the PhoP/PhoQ system also regulates the expression of prg (for PhoP-repressed gene) genes found on SPI-1, which are induced under nonstress conditions (Behlau and Miller, 1993). Thus, conditions that activate pag genes generally repress prg expression. Prg products are required for epithelial cell invasion and spacious phagosome formation. In addition to regulating a large number of genes, the PhoP/PhoQ operon is autoregulated, as full expression requires both PhoP and PhoQ. Invasion of epithelial cells by salmonellae requires a large number of gene products that allow both adhesion and invasion. A number of these proteins constitute a specialized secretion system, known as type III secretion system. These products include adhesion factors that comprise at least four different fimbriae, including type I fimbriae (fim), thin aggregative fimbriae or curlie (agf), plasmid-encoded fimbriae (pef), and long polar fimbriae (lpf); the type III secretion apparatus encoded by SPI-1 and consisting of structural proteins encoded in the inv, spa, and prg loci; and putative effector proteins including SptP, AvrA, SopE, SopB, SopD, and Ssp/SipA. Virulence factors required for systemic infection of salmonellae include another type III secretion system encoded by genes in SPI-2. Gene sequences in SPI-2 are conserved throughout the genus, with the exception of S. bongori (Ochman and Groisman, 1996), but are not found in other enteric bacteria. This pathogenicity island is believed to contribute to Salmonella spp.–specific aspects of infection. The presence of virulence plasmids within the genus Salmonella is limited and has been confirmed in S. typhimurium, S. dublin, S. gallinarum-pullorum, S. enteriti-

Table 12.14

Salmonella Species Virulence Factorsa

Protein/locus

Function

agf

Thin aggregative fimbriae (or curli)

aro Crp, Cya fim

Aromatic amino acid synthesis cAMP receptor, adenylate cyclase Type I fimbriae

GalE

UDP-galactose-4-epimerase, LPS synthesis Transcriptional regulator of SPI1 Type III secretion components (SPI1) Long polar fimbriae

HilA InvABCDEFGHIJ lpf

MetL MgtC OmpR/EnvZ pef

Methionine biosynthesis (homocysteine production) Unknown Transcriptional regulators (osmolarity) Plasmid-encoded fimbria

PhoP/PhoQ

Transcriptional regulators (pH, Mg2+)

PmrAB PrgHIJK

Transcriptional regulators (pH) Type III secretion components (SPI1) Purine biosynthesis Resistance to complement

pur Rck RpoS Sip/SspBCD

Transcriptional regulator (stationary phase, stress response) Translocase (SPI1)

SlyA SopB

Transcriptional regulator Inositol phosphate phosphatases

SopD

Unknown

SopE

Guanine exchange factor

SpaOPQRS

Type III secretion components (SPI1) Unknown (mgtC) Type I secretion system? Unknown

SPI3 SPI4 SPI5

Phenotype in vivob

Phenotype in vitro Adherence to cultured mouse small intestinal epithelial cells

Adherence to (and invasion of) HeLa cells Galactose sensitive and rough phenotype Epithelial cell invasion defect Secretion, translocation, and invasion defects Adherence to (and invasion of) Hep-2 cells

Slightly attenuated on oral inoculation

Avirulent in mice and humans (S. typhi) Avirulent in mice and humans (S. typhi) Slight decrease in LD50 on oral inoculation Avirulent in mice; disease caused in humans by S. typhi mutant Attenuated by oral inoculation

Resistance to host nitric oxide

Adherence to murine Peyer’s patch; slight decrease in LD50 on oral inoculation Attenuated in mice

Intracellular survival Intracellular survival

Avirulent Avirulent

Adherence to murine small intestine Intracellular survival, CAMP resistance, stimulation of cytokine secretion

Reduced fluid accumulation in infant mice Avirulent in mice; PhoP– mutants immunostimulatory compared to wildtype; S. typhi PhoP null mutants avirulent in humans

Polymyxin resistance Invasion defect

Attenuated by oral inoculation Avirulent

Prevention of complementmediated lysis Avirulent in mice and humans (S. typhi) Invasion and translocation defect; decreased macrophage cytotoxicity Intracellular survival

Attenuated by oral inoculation

Fluid secretion and inflammation in cow ileal loops Fluid secretion and inflammation in cow ileal loops Slight invasion defect (S. dublin); actin rearrangements when expressed exogenously Secretion, translocation, and invasion defects Macrophage survival Survival within macrophages

Attenuated by oral inoculation Avirulent Effectors of gastroenteritis in mice and cows (table continues)

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Table 12.14 Protein/locus

(continued) Function

SptP

Protein tyrosine phosphatases

SpvABCD

Unknown (sopB)

SpvR

Transcriptional regulator of spvABCD Type III secretion apparatus of SPI2 SPI2 translocase? Transcriptional regulators of SPI2 Unknown

SsaBCDE,G-V SseBCD SsrAB TolC

Phenotype in vivob

Phenotype in vitro Actin rearrangements when exogenously expressed

Growth within reticuloendothelial system Intracellular survival Intracellular survival Intracellular survival Increased sensitivity to complement and detergents

Slight decrease in colonization of liver and spleen Growth within reticuloendothelial system

Avirulent Avirulent Avirulent Attenuated by oral inoculation

a

cAMP, cyclic adenosine monophosphate; UDP, uridine diphosphate; LPS, lipopolysaccharide; CAMP, cationic antimicrobial peptide. Mouse, unless specified. Source: Compiled from D’Aoust (1997) and Scherer and Miller (2001). b

dis, S. choleraesuis, and S. abortusovis (D’Aoust, 1997). The plasmids vary between 30 and 60 MDa in size and contain an essentially identical piece of DNA, known as the Salmonella plasmid virulence or spv region. The region contains at least five genes, spvRABCD. Transcription is regulated by both the spvR gene product and the stima factor RpoS, influenced in turn by factors such as the host intracellular environment, low pH, iron limitation, and nutrient limitation concurrent with reaching the stationary growth phase (Cox, 2000a). The gene products of spvABCD appear to enhance intracellular multiplication and systemic dissemination. Minor sequence differences in spvR markedly affect transcription of spvABCD, in turn influencing the invasiveness of different serovars. Several choleralike toxins and enterotoxins have also been described in Salmonella spp. Diarrheagenic enterotoxin figures prominently as a Salmonella spp. virulence factor responsible for the onset of diarrheal symptoms in human cases of salmonellosis. Whereas early studies suggested a serological relationship among the Salmonella spp. enterotoxin, cholera toxin (CT), and the heatlabile toxin (LT) of enterotoxigenic Escherichia coli, more recent serological and nucleic acid studies indicate they are distinct entities (Cox, 2000a). However, the Salmonella spp. enterotoxin appears to be structurally similar to CT, with a molecular mass of 90 to 110 kDa and consisting of A and B subunits that act, respectively, to stimulate host cell adenylate cyclase and produce a pore through which the former enters. Increased levels of cellular cyclic adenosine monophosphate (cAMP) lead to a net massive increase in concentration of sodium and chloride ions and a consequent accumulation of fluid in the intestinal lumen.

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Salmonella spp. strains also produce a thermolabile, membrane-bound proteinaceous cytotoxin, which is serologically and genetically distinct from Shiga toxins of Shigella spp. and E. coli. The virulence attribute of cytotoxin stems from its inhibition of protein synthesis and lysis of host cells, thereby promoting the dissemination of viable salmonellae into host tissues. Host cell lysis may also result from chelation of divalent cations by the toxin, causing disruption of host cell membranes (Peterson and Niesel, 1988). Acquisition of iron is critical to survival and growth of microorganisms; iron must be obtained from the host during infection as it is complexed in a range of proteins, or the little available free iron must be scavenged. Like many other members of Enterobacteriaceae, Salmonella spp. produce two types of sequestering molecules, or siderophores, to acquire iron. The first, a high-affinity siderophore known as enterochelin or enterobactin, is a phenolate, composed of a cyclic trimer of dihydrobenzoic acid and L-serine; the second, a hydroxamate called aerobactin, is synthesized from a citrate molecule and two lysine derivatives. These siderophores sequester ferric ions from the environment (intestinal lumen, serum) and after binding to an outer membrane protein are translocated to the cytoplasm, where Fe3+ is reduced to Fe2+, which is released from the siderophore. Strains producing enterochelin are generally more virulent than those producing aerobactin (Cox, 2000a). Several serotypes of salmonellae are becoming increasingly resistant to multiple clinically relevant antibiotics. The antibiotic resistance of S. typhi was first discovered in 1950, when strains resistant to chloramphenicol were iso-

lated in Great Britain, only 2 years after the successful use of chloramphenicol in treatment of typhoid fever (Colquhoun and Weetch, 1950). At present, S. typhi isolates resistant to six different antimicrobial agents prevail in highly endemic typhoid areas, particularly China, Pakistan, and India. These strains of S. typhi carry a 120-kb plasmid that encodes resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim. In addition, many strains have also acquired resistance to ciprofloxacin, a fluoroquinolone antibiotic, which is one of the preferred antibiotics for treatment of typhoid fever (Hampton et al., 1998). Multiantibiotic-resistant (MAR) typhoid has become a significant cause of death in children; the mortality rate of children infected with MAR S. typhi ranges from 7% to 16%, compared to a rate of 2% for children infected with susceptible strains of Salmonella spp. (Gupta, 1994). Resistance of nontyphoidal salmonellae is also a growing health problem. Particularly troubling is the penta-resistant strain of S. typhimurium definitive phage type 104 (DT104). This strain first emerged in Great Britain in 1984 and was reported in 1997 to have been isolated in the United States (CDC, 1997; Scherer and Miller, 2001). This strain has been isolated from numerous species of both wild and farm animals and is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R type ACSSuT). In addition, there have been reports of resistance to two other antibiotics, trimethoprim and fluoroquinolones, in Great Britain (Threlfall et al., 1996). The veterinary use of antibiotics, such as ciprofloxacin and trimethoprim, to treat DT104 infections in cattle is believed to be responsible for the acquisition of resistance to these and other antibiotics by salmonellae. Interestingly, resistance to ciprofloxacin has not been observed in the United States yet, possibly because fluoroquinolones are only licensed for use in poultry, in which DT104 may not yet be established as a pathogen. Reports documenting the emerging resistance to fluoroquinolones by salmonellae are worrisome, as ciprofloxacin is the antibiotic of choice for treating human salmonellosis (Scherer and Miller, 2001). Resistance to this drug will leave few available options. In addition to the problem of treatment of DT104 infections, there are reports indicating that DT104 may be more virulent for humans. In a study performed in the United Kingdom, 41% of patients infected with DT104 were hospitalized, and 3% of patients who had culture-confirmed salmonellosis died, compared to an average death rate of 0.1% (Wall et al., 1994).

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12.3.3

Symptoms, Diagnosis, and Treatment

Salmonella spp. cause two distinct clinical syndromes: typhoid fever (enteric or systemic) and gastroenteritis. Some strains of typically gastroenteric serovars are also capable of causing the enteric disease. Focal infections of the vasculature (endocarditis), bone (osteomyelitis), and joints (arthritis) as well as a variety of other organs can also occur but with much less frequency. Also, these conditions are often associated with specific immune defects. Systemic disease is usually associated with strains of serovars that exhibit a narrow range of hosts; those exhibiting strict human species specificity cause enteric fever. These serovars include S. typhi, S. paratyphi, and S. sendai in humans; S. dublin in cattle; S. choleraesuis in swine; and S. pullorum in poultry. Serovars associated with specific animal populations are also capable of causing disease in humans. The two predominant agents of human gastroenteritis, S. typhimurium and S. enteritidis, also infect a wide range of zoonotic hosts, including poultry, cattle, sheep, pigs, horses, rodents, and primates. A list of common serotypes, their hosts, and characteristics of the disease is provided in Table 12.15. Enteric Fever S. typhi causes typhoid fever, an enteric fever quite distinct from the typical gastrointestinal syndrome associated with most Salmonella species. Although S. paratyphi is also a causative agent of human enteric fever, it generally produces a milder form of the disease. Both serovars are solely human pathogens and do not exist in animal reservoirs.

Table 12.15 Hosts

Serotype S. arizonae S. choleraesuis S. dublin S. enteritidis S. gallinarum S. pullorum S. hadar S. hartford S. marina S. paratyphi S. stanley S. typhi S. typhimurium

Major Salmonella Species Serotypes and Their

Primary host

Disease in humans

Reptiles Swine Cows Wide range Chickens Ducks Poultry Unknown Iguanas Humans Unknown Humans Wide range

Gastroenteritis Gastroenteritis, bacteremia Gastroenteritis, bacteremia Gastroenteritis None None Gastroenteritis Gastroenteritis Gastroenteritis Enteric fever Gastroenteritis Enteric fever Gastroenteritis

The infectious dose for typhoid is not well documented. However, it is considered to be 1–2 logs lower than that required for most Salmonella spp. (Cox, 2000b). Infection occurs on ingestion of food or water contaminated with human waste. Onset of disease usually occurs 5 to 56 days post infection, although 10–20 days (average 14 days) is more typical. It depends on the inoculum and the immune status of the individual. An asymptomatic chronic carrier state commonly follows the acute phase of enteric fever. Although infection begins in the gastrointestinal tract, the organisms enter the lymphatic system, moving to the mesenteric lymph nodes, and begin to multiply within macrophages (Figure 12.8). After multiplication, they move into the bloodstream, circulating throughout the body and localizing in various organs, especially the spleen, liver, and gallbladder. In the bloodstream, the bacterial cells are ingested by macrophages, in which they continue to multiply; eventually they kill the macrophages (Cox, 2000b). At this stage, large numbers of S. typhi cells are released into the bloodstream, leading to septicemia and onset of the symptoms typical of enteric typhoid fever.

Progress to this first clinically overt phase of disease is slow. Typhoid fever is characterized by high fever; gastrointestinal symptoms, including diarrhea and constipation; and sometimes a characteristic rash (rose spots) from which bacteria can be cultured. During the early phase of disease, the organism can also be isolated easily from blood and urine. The fever may persist for several weeks, during which the organism reaches the gallbladder and multiplies in the bile. The organism-rich bile flows into the small intestine, causing enteritis and ulceration at Peyer’s patches in the terminal ileum, cecum, and ascending colon, leading to diarrhea characterized by formation of loose to watery stools. The ulcers may hemorrhage, leading to blood in the stools and, potentially, perforation of the intestine and consequent peritonitis. During the second phase of disease, the organism is more readily isolated from feces. Complications arising from typhoid fever may be both intra- and extraintestinal; they include perforation of the terminal ileum or appendix, paralytic ileus, hepatitis,

Figure 12.8 Typhoid infection by salmonellae. Bacteria are ingested via contaminated food or water and are transported through the gastrointestinal tract to the lumen of the small intestine. There they enter M cells and enterocytes overlaying Peyer’s patch via a process requiring membrane ruffling. Bacteria traverse M cells and enter the lymphatic pocket underneath, where they are phagocytosed by macrophages. In typhoid fever, bacteria spread to reticuloendothelial tissues via the lymphatic system and induce a chronic inflammatory response. Active antimicrobial therapy is required. E, epithelial cell (a, apical; b, basal); M cell, microfold cell; M, macrophage; B, B cell; T, T cell; P.P., Peyer’s patch; TNF-α, tumor necrosis factor alpha; IFN-γ, gamma interferon.

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hepatic failure, bronchopneumonia, typhoid abscess, myocarditis, encephalopathy, and meningitis, particularly in neonates. Other sequelae may also develop, including reactive arthritis in individuals of particular histocompatibility (human leukocyte antigen [HLA]) types (Cox, 2000b; Scherer and Miller, 2001). Such autoimmune conditions may involve cross-reactivity with either LPS or some stress proteins. The preferred treatment for typhoid fever is in the form of antibiotic therapy. As described earlier, antibiotics include chloramphenicol, ampicillin, amoxycillin, and sulfonamides. The evolution of multiple drug–resistant (MDR) strains has caused concern over therapy, and newer drugs including furazolidone, fluoroquinolones, and latergeneration cephalosporins are employed in such cases. Most individuals recover without therapy from what is a chronic and potentially relapsing systemic febrile illness. However, if the disease is untreated, complications such as intestinal perforation and hemorrhage can occur in a small percentage (0.5%–1.0%) of patients (WHO, 1998). Approximately 10% of immunocompromised individuals die of typhoid fever if proper antibiotic therapy is not initiated. Compared to that of other Salmonella spp., mortality due to typhoid fever is high, ranging between 2% and 10%. The corresponding rate for food-borne gastroenteric salmonellosis is only about 0.1%–0.2%. Typhoid fever remains a disease characterized by significant morbidity and mortality rates in developing nations. Incidence rates are difficult to estimate because the disease is difficult to diagnose definitively without a microbiological laboratory. The World Health Organization (WHO, 1998) estimates about 16–17 million cases occur annually, resulting in about 600,000 deaths. Approximately 400 cases are reported in the United States per year (CDC, 1990). These cases are usually attributed to recent travel in endemic areas or spread by infected food handlers. Large outbreaks, like the 50,000–60,000 cases reported annually in Tajikistan between 1996 and 1997, continue (Pang et al., 1998). In addition, antibiotic-resistant strains have been isolated in most endemic areas, particularly Southeast Asia, India, Pakistan, and the Middle East (Mirza et al., 1996). Recent outbreaks of multiantibioticresistant typhoid have also been reported in Great Britain and have been linked primarily to travel to endemic areas (Hampton et al., 1998; Scherer and Miller, 2001). Gastroenteritis Human infection with nontyphoidal salmonellae that result in enterocolitis usually is associated with food-borne transmission in developed countries. It is caused by many serotypes of Salmonella spp., the most common of which

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in the United States are S. enteritidis and S. typhimurium (Altekruse et al., 1997). The infection occurs via ingestion of contaminated food or water. However, unlike cases of systemic typhoid fever, gastroenteritis cases are usually due to contamination of food with animal rather than human waste. Undercooked meat, seafood, and eggs are common causes of salmonellosis. Contamination of fresh produce with animal waste is also a significant problem (Tauxe, 1997). The disease onset is approximately 8–72 hours after ingestion of the contaminated food. Organisms that survive the acidic conditions of the stomach begin to colonize and invade intestinal epithelial cells and the M cells at Peyer’s patches (Figure 12.9). Nausea and vomiting, diarrhea, abdominal pain, and fever characterize the disease. In most cases, the disease is self-limiting and symptoms resolve within 5–7 days without treatment. However, some infections may result in bacteremia or other complications and require treatment. Except in these rare cases, antibiotic treatment for salmonellosis is usually not advised. It only prolongs the presence of bacteria in the stool. This asymptomatic persistence of salmonellae in the gut likely results from a marked antibiotic-dependent repression of native gut microflora that normally competes with salmonellae for nutrients and intestinal binding sites. Salmonella spp. gastroenteritis is life-threatening in a small percentage (<1%) of total cases. The susceptible population groups include infants, the elderly, and immunocompromised individuals. Worldwide, the incidence of acute gastroenteritis due to Salmonella spp. is estimated at 1.3 billion cases per year, resulting in about 3 million deaths (Pang et al., 1995; Scherer and Miller, 2001). In the United States, approximately 2–4 million cases of Salmonella spp.–related gastroenteritis occur each year, causing about 500 deaths annually, most in children or immunocompromised people (CDC, 1998a). In the 1990s, S. enteritidis surpassed S. typhimurium as the most prevalent source of gastroenteritis. In 1980 and 1995, the former accounted for only 8% and 25% of total cases of salmonellosis, respectively. In 1998, however, it accounted for approximately 80% of current cases of salmonellosis, primarily due to contaminated shell eggs (USDA, 1998). The major increase of S. enteritidis cases in humans is due to its ability to cause ovarian infections in egg-laying hens, thus contaminating the contents of intact shell eggs, which cannot be conveniently pasteurized (Altekruse et al., 1997). This serotype is easily transmitted vertically from breeding flocks to egg-laying hens and is difficult to eliminate because of its ability to survive in rodents and in manure.

Figure 12.9 Nontyphoidal infection by salmonellae. Nontyphoidal serotypes are usually contained within Peyer’s patches. They induce an acute inflammatory response characterized by cytokine/chemokine secretion and neutrophil infiltration. The infection usually is self-limiting. E, epithelial cell (a, apical; b, basal); M cell, microfold cell; M, macrophage; N, neutrophil; B, B cell; T, T cell; IL-8, interleukin-8.

The characteristics of clinical diseases induced by salmonellae are summarized in Table 12.16. 12.3.4

Sources

Salmonellae are quite ubiquitous in the natural environment (Figure 12.10). Both animal- and, to a lesser extent, plant-derived animal feed materials are important in the persistence of Salmonella spp. in the food production envi-

Table 12.16

Clinical Symptoms of Diseases Induced by Salmonella Species

Feature Incubation period Onset Fever Duration of disease Gastrointestinal symptoms Blood cultures Stool cultures

ronment. A major reason for this persistent presence of salmonellae are the intensive husbandry practices used in meat, fish, and shellfish industries and the recycling of offal and inedible raw materials into animal feeds (D’Aoust, 1997). Beef, pork, and poultry carcasses are frequently contaminated at slaughter with feces. If the intestine of meat birds is ruptured when the pluck is removed, the rib cage can become contaminated with feces. Similarly, stuffing of large birds (e.g., turkeys) is readily prone to

Enteric fever

Septicemias

7–20 Days Insidious Gradual, then high plateau, with typhoidal state Several weeks Often early constipation; later bloody diarrhea Positive in 1st and 2nd weeks of disease Positive from 2nd week on; negative earlier in disease

Variable Abrupt Rapid rise, then spiking septic temperature Variable Often none

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Positive during high fever Infrequently positive

Gastroenteritis 8–72 Hours Abrupt Usually low 2–5 Days Nausea, vomiting, diarrhea at onset Negative Positive soon after onset

Figure 12.10

Sources and transmission of salmonellae in the environment.

contamination with salmonellae. Although the musculature may reach appropriate temperature during cooking, temperatures of the stuffing may only rise to 43°C (109°F), enabling the bacteria to multiply extensively. In view of the high rates of salmonella contamination in poultry, it is not surprising to find poultry meat so often the cause of human salmonellosis. Reilly (1988) found in Scotland that poultry was responsible for 224 outbreaks affecting 2245 people between 1980 and 1985; this represented 52% of the total number of salmonellosis cases in which the food could be identified. In Denmark (1995 data) it was estimated that approximately 40%–50% of all salmonellosis cases were associated with eggs, whereas 10%–15% and 15%–20% were associated with pork and poultry, respectively (Forsythe and Hayes, 1998). Until the 1980s hens’ eggs had not been an important cause of salmonellosis in humans, whereas egg products had long been recognized as serious sources of infection by salmonellae. Outbreaks of salmonellosis were regularly traced to frozen, liquid, and dried whole eggs and similar products. However, pasteurization, carried out principally to kill salmonellae, is now mandatory and has proved extremely effective, as very few cases that have emanated from these sources are now being reported. For example, for liquid whole egg, the British require pasteurization at 64.4°C for 2¹⁄₂ minutes and in the United States 60°C for

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3 minutes is deemed necessary; times and temperatures vary for different types of egg products (ICMSF, 1980). In recent years, eggs have become the primary cause of human salmonellosis specifically associated with S. enteritidis phage type 4 (PT4). The organisms can enter the egg through the shell or through transovarian contamination of egg yolk membrane and/or albumen. According to the USDA, transovarian egg contamination occurs in about 1 of 10,000 eggs produced in the United States, in spite of the fact that Salmonella spp. isolation rates from the internal organs of chicken can be approximately 25% (Hopper and Mawer, 1988; Hammack and Andrews, 2000). This low level of contamination, however, still results in approximately 4.5 million contaminated eggs and hence the exposure of large numbers of people to this pathogen, propagating the continuing pandemic of human S. enteritidis phage type 4 (Europe) and 8 (North America) infections associated with the consumption of raw or lightly cooked shell eggs and egg-containing products. S. typhimurium DT104 strain is the second most common Salmonella spp. after S. enteritidis PT4 in human beings in England and Wales. As mentioned earlier, it carries multiple resistances to several antibiotics (R type ACSSuT). Both S. enteritidis PT4 and S. typhimurium DT104 show high tolerance to heat and acid and are capable of prolonged survival in the environment.

It is quite difficult to establish and maintain Salmonella spp.–free supply flocks and distribution of noncontaminated feeds to the various sectors of the vertically integrated poultry industry. The inability to prevent contamination of the housing with rodent, insect, and avian carriers of Salmonella spp. and the feeding habits and close proximity of multiplier breeder, layer, and broiler birds in rearing facilities encourage the rapid and widespread dissemination of vertically and horizontally introduced salmonellae in poultry houses (D’Aoust, 1997). Animal by-products, with Salmonella spp. contamination levels of up to 80%, are widely used in animal feeds (Forsythe and Hayes, 1998). This practice significantly increases the chance of home-reared animals’ becoming infected. The use of these products in pet foods has been an additional hazard and some cases of salmonellosis have been traced to domestic pets. Milk and Salmonella species have always been linked. The organism is found in 60% of raw milk samples tested (McManus and Lanier, 1987). Infection may persist in dairy herds with shedding of the agent in feces and milk, especially where liver fluke is endemic. In spite of this association with raw milk, the aging of cheese made from raw milk for over 60 days was thought to inhibit the growth of this pathogen. Hence, the FDA requires that cheese made from raw milk be aged a specific length of time. However, an outbreak of salmonellosis that affected nearly 2000 people in Canada was traced to cheddar cheese made from raw milk. Subsequent studies show that Salmonella spp. from raw milk can survive in cheddar and other cheeses aged at 13°C for 112–210 days (Johnson, 1987). Molluscan shellfish, including clams, oysters, and mussels, have all been implicated in outbreaks of human salmonellosis and pose a significant risk, as filter feeding in contaminated water leads to concentration of the organism in the tissues of the animal. Often these products, especially oysters, are consumed raw. Depuration may not completely eliminate the organism from molluscan shellfish, and this is of particular concern. In recent years, outbreaks of human salmonellosis have also been traced to contaminated fruits and vegetables. The fertilization of crops with untreated sludge or sewage effluents potentially contaminated with antibioticresistant Salmonella spp., the irrigation of garden plots and fields, the washing of fruits and vegetables with contaminated waters, and the repeated handling of products by local workers are some of the underlying causes. Processed foods can also become contaminated with Salmonella spp. The contamination arises from the failure of some food production process or from inadvertently cross-contamination of a processed food with a contami-

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nated foodstuff by a food preparer. The former occurs, for example, when the pasteurization process fails because of mechanical defects or when a finished ingredient has contact with a raw ingredient without further processing. An example of the latter is an outbreak of salmonellosis associated with ice cream in the midwestern United States in 1994. In that outbreak, ice cream premix, which underwent no further processing, was transported in tanker trucks that had been used immediately before to transport nonpasteurized eggs. This outbreak resulted in 277 culture-confirmed cases of Salmonella spp. with an estimated 224,000 nonreported cases (Hennessy et al., 1996). Chronic carriers can spread Salmonella spp. infections, especially those who work in food-related industries. Nontyphoidal serotypes on average persist in the gastrointestinal tract, depending on the serotype, from 6 weeks to 3 months (Scherer and Miller, 2001). However, persistence beyond 6 months is rare. In only about 0.1% of nontyphoidal Salmonella spp. cases are the organisms shed in stool samples for periods exceeding 1 year, the clinical definition of chronic carriage. Approximately 2%–5% of untreated typhoidal infections result in a chronic carrier state (WHO, 1997). Bacteria can persist in several reservoirs, including the urinary tract and bile duct. The most famous chronic carrier is Mary Mallon, otherwise known as Typhoid Mary (Leavitt, 1996). A New York City cook, she was held responsible for transmitting typhoid fever to at least 22 individuals (3 of whom died) between the years 1900 and 1907, although she herself never had any symptoms. After being apprehended in 1907 by public health officials, she was confined to an isolation cottage for 3 years. Although she was released with the stipulation that she never cook again, she was unable to keep this promise and is thought to be responsible for at least 25 more cases of typhoid fever at a Manhattan maternity hospital, where she was employed as a cook in 1915. She was subsequently confined to isolation until her death in 1938. Besides human transmission, a range of foods have also been implicated in the transmission of S. typhi, including desiccated coconut, unpasteurized liquid whole eggs, raw milk, a range of soft cheeses made from raw milk, ice cream, cold cooked red meats and poultry, shellfish, and salad vegetables (Cox, 2000b). Direct consumption of contaminated water has also led to outbreaks of typhoid fever. 12.3.5

Outbreaks

Worldwide, food-borne disease associated with Salmonella spp. is considered to be second only to that involving Campylobacter spp. (Cox, 2000a). The trend also seems to be rising. To some extent, this may be attributed to better

reporting and surveillance rather than a real increase in disease. Nevertheless, a significant proportion of the reported cases represent an actual increase. Events such as the ongoing pandemic of egg-borne S. enteritidis clearly have an impact on current disease statistics. The case rate for human salmonellosis varies immensely, from <1 to >300 per 100,000 population. It is profoundly influenced by geographic, demographic, socioeconomic, meterological, and environmental factors. The increased occurrence of nontyphoidal salmonellosis observed in the industrialized world may in part be due to centralization of food processing and the increased range of food distribution. Traditionally, Salmonella spp. outbreaks have been linked to focused gatherings such as weddings or other social events. However, large-scale distribution of prepared food from industrial production facilities has resulted in much more diffuse patterns of infection that can be difficult to trace epidemiologically. The FDA estimates that the cost of salmonellosis from all causes in 1995 was $350 million to $1.5 billion in the United States. These figures are based on 41,222 reported cases of salmonellosis, and an estimated 20–100 unreported cases for each reported case. The Centers for Disease Control and Prevention (CDC) reports that from 1976 to 1994, the proportion of Salmonella spp. isolates that were S. enteritidis rose from 5% to 26%. If these figures are extrapolated to include 1995, then it can be estimated that the total cost of S. enteritidis in 1995 alone was $91–$390 million. Major outbreaks of human salmonellosis are listed in Table 12.17. These exemplify the multiplicity of foods as well as Salmonella spp. serovars that have been implicated in human disease. 12.3.6

Prevention and Control

Because of the close links among the environment, feeds, food animals, and humans, control of Salmonella spp. with particular regard to food-borne gastroenteritis is quite difficult. It requires vigilance at two levels, food production and food processing. A range of management strategies, including the hazard analysis critical control point (HACCP) approach, have been developed or devised to control Salmonella spp. in food production environments, especially in the production of poultry, a major vehicle of transmission of S. enteritidis. These strategies include provision of Salmonella spp.-free stock and feed; stringent biocontrol, particularly of rodents; vaccination with attenuated Salmonella spp. strains; and use of probiotic preparations (e.g., competitive exclusion). Since Salmonella spp. infection induces mucosal immunity, efforts have also been made to develop multiva-

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lent Salmonella spp. vaccines that can induce immunity not only to Salmonella spp. but also to heterologous antigens. The development of effective multivalent vaccine strains is dependent on several factors (Chatfield et al., 1994). A mere introduction of antibiotic resistance genes on plasmids carried by the vaccine strain may not be an effective strategy. Heterologous antigens must either be integrated into the chromosome in single copy or be expressed on plasmids that do not utilize selectable antibiotic resistance markers. An alternative approach is to express antigens on balanced lethal plasmids that are required for bacterial survival in the host but do not acquire antibiotic selection for maintenance in vivo (Schodel et al., 1994). Using this approach, a number of studies have shown that immunity to foreign antigens, including tetanus toxin, can be attained in mice (Chatfield et al., 1992; VanCott et al., 1996). Three vaccines are currently available for typhoid fever, none of which is 100% effective, even when tested on endemic populations (CDC, 1998b). The live attenuated Ty21a strain manufactured by the Swiss Serum and Vaccine Institute and orally administered requires at least four doses to achieve 51%–76% protective efficacy (Levine et al., 1987). The heat- and phenol-inactivated typhoid vaccine manufactured by Wyeth has similar efficacy and requires at least two doses by injection. However, this vaccine has a variety of severe local and systemic side effects that limit its use. Finally, the cell-free, parenteral Vi-antigen vaccine produced by Pasteur Merieux (ViCPS) requires only a single dose but most likely has a shorter duration of protection. Although there is little requirement for human vaccines for nontyphoidal salmonellae, as the infections are usually self-limiting, various vaccines for use in animals have been developed. Such vaccines would be quite useful if they could eliminate the source of most Salmonella spp. infections without increasing the potential for additional antibiotic resistance. Both aro-negative (Hassan and Curtiss, 1997) and crp/cya mutant (Coloe et al., 1995) S. typhimurium strains have proved effective vaccine strains in chickens. These strains offer long-term protection against infection with both S. typhimurium and S. enteritidis. In addition, immunization with the crp/cya strain in ovo up to 7 days before hatching protected chicks from infection (Coloe et al., 1995). This is particularly important in light of the ability of S. enteritidis to colonize intact shell eggs. Although Salmonella spp. may never be eliminated completely, significant reduction should be achieved through the application of appropriate control strategies within a well-developed and implemented HACCP-based food safety plan from the commencement of production through to consumption (Cox, 2000a).

Table 12.17 Year

Major Outbreaks of Human Salmonellosis Country

Vehicle

Serovar

No. of casesa

S. typhimurium PT8 S. typhi PT34 S. typhimurium PT2a, S. braenderup S. typhimurium PT32a S. eastbourne S. derby S. newport S. typhimurium PT9 S. typhimurium S. heidelberg S. enteritidis PT4 S. indiana S. typhimurium PT204 S. napoli S. oranienberg S. typhimurium PT10 S. goldcoast S. enteritidis PT4 S. virchow S. typhimurium S. typhimurium S. typhimurium S. champaign Salmonella spp. S. typhimurium PT12 S. typhimurium PT193 S. chester S. javian, S. oranienberg S. enteritidis S. agona S. poona S. heidelberg S. enteritidis S. enteritidis S. saintpaul S. javiana S. rubislaw S. bovismorbificans S. enteritidis S. mbandaka S. typhimurium PT1 S. agona

8,845 507 1,790b

1953 1964 1967

Sweden Scotland United States

Pork Canned corned beef Ice cream

1968 1973 1973 1974 1976 1976 1976 1977 1981 1981 1982 1982 1984 1984 1984 1984 1985 1987 1987 1988 1988 1989 1989 1989 1989

Scotland Canada/United States Trinidad United States Australia Spain United States Sweden Netherlands Scotland England/Wales Norway Canada France/England International England/Wales United States China Norway Japan Japan England England United States United States

Raw pork Chocolate Milk powder Potato salad Raw milk Egg salad Cheddar cheese Mustard dressing Salad base Raw milk Chocolate Black paper Cheddar cheese Liver pate Aspic glaze Ham Pasteurized milk Egg drink Chocolate Cuttlefish Cooked eggs Cold meats Roast pork Cantaloupe melon Mozzarella cheese

1990 1990 1991 1991 1991 1993 1993

United States United States United States/Canada United States Germany France Germany

Bread pudding Turkey meat Cantaloupe melon Mexican fajitas Fruit soup Mayonnaise Paprika potato chips

1994 1994 1996 1997 1998

Finland, Sweden United States Australia Australia United States

Alfalfa sprouts Ice cream Peanut butter Pork rolls Toasted oat cereal

a

Confirmed cases unless stated otherwise. Estimated number of cases. Source: Compiled from D’Aoust (1997) and Cox (2000a). b

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472 217 3,000b 3,400b >500 702 339 2,865 600b 654 245 126 2,700 756 766 274 16,284 1,113 361 330 10,476 538 206 295 164 1,100b 851 400b 673 600 751 1,000b

492 200,000b 200b 770b 209

Controlling and preventing outbreaks of salmonellosis involve three general approaches: breaking the cycle, educating the public, and preventing contamination. Breaking the cycle involves controlling rendering so that animal feeds containing recycled animal by-products are free of viable salmonellae, shortening the interval between the time an animal leaves the farm and its slaughter because this reduces environmental buildup of organisms in stockyards and abattoirs, using proper cooking temperatures, and pasteurizing milk. Sanitary measures must be taken to prevent contamination of food and water by rodents or other animals that excrete salmonellae. Public education about how Salmonella spp. organisms are transmitted is vital to an effective control program. Education should also be concerned with the proper handling and preparation of foods in the home and should be included in the school curriculum. Infected poultry, meats, and eggs must be thoroughly cooked. Carriers must not be allowed to work as food handlers and should observe strict hygienic precautions. The consumer should always regard unpasteurized foods of animal origin as a source of infection and act accordingly. By doing so, the consumer can drastically reduce the chance of contracting salmonellosis. Making consumers aware that a cooked product can be recontaminated when handled improperly may prevent contamination. Foods should be covered and thawed under refrigeration because holding foods exposed to air at ambient temperatures allows for microbial buildup if contaminated as well as possible airborne contamination. Growth at temperatures between 4°C and 10°C (39°F and 50°F) has been observed among several non-host-adapted serovars. This finding raises concern regarding the safety of food stored under refrigeration and then consumed without subsequent heating adequate to kill salmonellae. Although not a popular practice or practical in all areas, periodic examination of employees who handle food to determine whether they are carriers of Salmonella spp. is nevertheless wise. Because of the rising number of infections due to S. enteritidis, the FDA has issued special guidelines for handling and cooking poultry eggs. Consumers should avoid eating raw eggs and foods containing raw eggs. Raw eggs should be handled and stored in the same manner as other raw foods of animal origin. Eggs should be stored at 7.2°C (45°F) or below. Raw eggs ought not to be considered “health foods,” particularly for the hospitalized, the elderly, the immunocompromised, and perhaps pregnant women. Cracking single eggs for use, rather than “pooling” several eggs, greatly lessens the chance of illness. Recipes calling for raw eggs (e.g., homemade ice cream, Caesar salad, Hollandaise sauce, mayonnaise) should be

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considered potentially hazardous if they are not heated sufficiently to kill Salmonella spp. Commercially produced mayonnaise and sauces are safe since they are prepared with pasteurized eggs and are adequately acidified to prevent the growth of S. enteritidis. Whenever possible, pasteurized, liquid eggs should be substituted for raw eggs if they are destined for high-risk individuals. In addition, the FDA has issued the following guidelines for consumers to cook eggs and foods containing eggs. Eggs should be cooked until the yolk and white are firm. There may be some risk in eating eggs lightly cooked, e.g., soft-cooked, soft-scrambled, or “sunny-sideup.” Eggs should be cooked throughout to 60°C (140°F) or above. The following cooking times should be used: 1.

2. 3.

4.

5.

Scrambled eggs should be cooked at least 1 minute at 121°C (250°F) in order to raise the temperature of the eggs to 73.9°C (165°F). Poached eggs should be cooked for 5 minutes in boiling water. Eggs fried sunny-side-up should be cooked in a frying pan at 121°C (250°F) for a minimum of 7 minutes uncovered or a minimum of 4 minutes covered. Eggs fried over-easy should be cooked with the frying pan at 121°C (250°F) for a minimum of 3 minutes on one side and 2 minutes on the other side. Boiled eggs, in the shell, should be cooked while completely submerged in boiling water for 7 minutes.

The FDA uses the word should throughout because there are uncontrollable variables, such as the starting number of organisms in the eggs. The preceding guidelines should be sufficient to inactivate any Salmonella spp. present in raw eggs and should be considered the minimal cooking times necessary for the inactivation of Salmonella spp. Higher levels of Salmonella spp. take longer to kill than lower levels. Also, in the case of boiled eggs, these guidelines assume near-sea-level conditions. High-altitude cooking requires longer boiling because water boils at a lower temperature than at sea level. In addition to the specific guidelines suggested by the FDA, the following general guidelines are useful to minimize the incidence of human salmonellosis: 1.

2.

Ensure animal feeding stuffs are Salmonella spp.–free and imported feeds are suitably heattreated. Eliminate salmonellae in poultry breeding stocks.

3. 4.

5. 6.

7. 8. 9.

Improve hygiene standards in abattoirs and broiler houses. Avoid cross-contamination risks, particularly of cooked by raw foods, in processing factories and kitchens. Ensure adequate heating of foods, followed by rapid cooling where foods are to be stored. Refrigerate foods at below 5°C where possible and avoid leaving foods at ambient temperatures for lengthy periods. Ensure food handlers are not Salmonella spp. carriers. Control rodents, birds, and pests in and around factory premises. Increase Salmonella spp. surveillance, particularly of cooked foods.

annually, surpassing the incidence of salmonellosis (Kvenberg and Archer, 1987; Jones, 1992). A 15-month study in eight hospitals in different parts of the United States isolated this microorganism from stool samples more frequently than Salmonella and Shigella species combined (Doyle, 1985). Currently, Campylobacter spp. are isolated more frequently than Salmonella spp. in human gastroenteritis patients (Rowe and Madden, 2000). In these cases, C. jejuni is the major species responsible; the incidence of C. coli is approximately 7%–10%. Several other Campylobacter species, including C. lari, C. hyointestinalis, C. concisus, and C. upsaliensis, have also been implicated as causative agents of enteritis. All these enteric campylobacters may also cause blood and systemic infections. C. fetus subsp. fetus usually causes opportunistic septicemic infections in humans. 12.4.1

12.4 CAMPYLOBACTER JEJUNI (CAMPYLOBACTERIOSIS) Campylobacter jejuni and C. coli have exploded in recent years from relative obscurity to a recognized major cause of food-borne human diarrhea in the United States and throughout the world. They mainly cause enteritis and occasionally systemic infection. C. jejuni and C. coli cause infections that are clinically indistinguishable, and laboratories generally do not differentiate between the two species. Failure to recognize their significance has been the result, in large part, of their requirements for optimal growth, which caused them to be overlooked in procedures used routinely to isolate human pathogens. Theodor Escherich in the 1880s is credited for making the first recorded observation of spiral bacteria in the feces of patients with infantile diarrhea (Rowe and Madden, 2000). He was, however, unsuccessful in culturing them and regarded them as nonpathogenic. In 1946, Levi reported an outbreak of gastroenteritis transmitted by raw milk and suspected a bovine strain of Vibrio spp. as the cause of the disease (Levy, 1946). King achieved the first isolation of Campylobacter spp. in 1957 when he successfully isolated “vibrios” from blood samples of humans with diarrhea (King, 1957). A major advance in the culture of these organisms occurred when Skirrow (1977) developed a selective medium that obviated the need for a laborious filtration stage, making possible the routine isolation of these organisms. The organism was previously identified as Vibrio fetus subsp. jejuni. It is now widely recognized that campylobacters are responsible for more cases of food-borne acute infective diarrhea in developed countries than any other bacteria. Campylobacteriosis is estimated at over 4 million cases

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Organism

The Campylobacter (derived from the Greek words kampylos and bacterion, meaning “curved rod” and “little rod,” respectively) species resemble the vibrios morphologically and, therefore, were once considered as members of the genus Vibrio. However, because of marked differences in DNA homology and growth requirements, these bacteria were placed in the present genus (Christie, 1980). The family Campylobacteraceae at present includes 20 species and subspecies within the genus Campylobacter (Table 12.18). The use of DNA hybridization studies led to a major reorganization of campylobacters in 1991 with the creation of the genus Arcobacter, now comprising four species. Organisms that were formerly classified in the genus Wolinella (W. curva, W. recta) are now included in the genus Campylobacter (Nachamkin, 1997). The genus Arcobacter now contains several former Campylobacter species as well as some new species (Table 12.18). Similarly, some species from the genus Campylobacter are now classified in the genus Helicobacter. Campylobacter is the type genus within the family Campylobacteraceae. Campylobacters are curved, S-shaped, or spiral rods. They are gram-negative, non-spore-forming rods that may form spherical or coccoid bodies in old cultures or cultures exposed to air for prolonged periods. They are highly motile by means of a single polar flagellum at one or both ends, giving rise to a characteristic corkscrewlike motion. The principal distinguishing physiological feature of this genus is that they are microaerophilic with a respiratory type of metabolism. Thus, oxygen is required for energy production but can only be tolerated at levels below normal atmospheric pressure. This property was partly re-

Table 12.18

Reservoirs and Disease-Associated Species in the Family Campylobacteraceae Disease, sequelae, or comments

Organism Campylobacter C. jejuni subsp. jejuni C. jejuni subsp. doylei C. fetus subsp. fetus C. fetus subsp. venerealis C. coli C. lari C. upsaliensis C. hypointestinalis C. mucosalis C. hyoilei C. sputorum biovar sputorum C. sputorum biovar bubulus C. sputorum biovar faecalis C. concisus C. curvus C. rectus C. showae C. helveticus C. hyoilei C. gracilis Arcobacter A. butzleri A. cryaerophilus A. skirrowii

A nitrofigilis

Reservoirs Humans, other mammals, birds Unknown Cattle, sheep Cattle Pigs, birds Birds, dogs Domestic pets Cattle, pigs Pigs Pigs Humans Cattle Cattle, sheep Humans Humans Humans Humans Domestic pets Pigs Humans Cattle, pigs Cattle, pigs, sheep Cattle, sheep, pigs

Human Diarrhea, systemic illness, GBSa Diarrhea Systemic illness, diarrhea Diarrhea Diarrhea Diarrhea Rare, proctitis, diarrhea Rare, diarrhea Proliferative enteritis Isolated from oral cavity and abscesses Isolated from oral cavity and abscesses

Animal Diarrhea in primates

Abortion Infertility

Diarrhea Proliferative enteritis Proliferative enteritis

Isolated from genital tract Enteritis

Periodontal disease Periodontal disease Periodontal disease, pulmonary infections Periodontal disease Diarrhea Proliferative enteritis Infections of head, neck, and other sites Diarrhea, other illness Diarrhea, bacteremia

Diarrhea, abortion Abortion Abortion, diarrhea; isolated from genital tract of bulls

Isolated from plants

a

Guillain-Barré syndrome. Source: Compiled from Alderton et al. (1995), Skirrow (1994), Vandamme et al. (1995), and Nachamkin (1997).

sponsible for the genus’s remaining undetected until relatively recently as it could tolerate neither fully aerobic nor anaerobic conditions, i.e., those normally employed to isolate organisms from animals and humans. Optimal oxygen concentrations have been cited as being 3%–6% (Rowe and Madden, 2000). Elevated levels (2%–10%) of CO2 are also recommended, and the growth of some species is dependent on the presence of hydrogen, which is recommended in the atmosphere used to incubate any clinical samples. Campylobacters are mesophilic, associated with warm-blooded creatures. Growth temperatures range from about 30°C to 45.5°C; the optimal is 43°C. Although campylobacters do not replicate at temperatures less than

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30°C, they survive refrigeration and freezing. They are heat-labile and thus should not survive in food products cooked to adequate temperatures. Campylobacters are susceptible to drying and low pH and are killed readily at pH 2.3 (Blaser et al., 1980). Organisms have been shown to remain viable and multiply at 37°C and to survive better in feces, milk, water, and urine held at 4°C than in material held at 25°C. The maximal periods of viability of Campylobacter spp. at 4°C were 3 weeks in feces, 4 weeks in water, and 5 weeks in urine (Blaser et al., 1980). Campylobacters are chemoorganotrophs, which neither ferment nor oxidize carbohydrates. Instead energy is derived from either amino acids (e.g., aspartate and glutamate) or tricarboxylic acid (TCA) cycle intermedi-

ates. The amino acids are deaminated to provide TCA cycle intermediates for subsequent oxidation, but no complex molecules, such as proteins, are utilized. Between the two commonly implicated species in human diarrheal conditions, C. jejuni can hydrolyze hippuric acid, whereas C. coli cannot. Thus, the hippurate hydrolysis test is of great importance in food microbiology. Because of the selective media containing antimicrobials and inhibitory compounds and incubation conditions for growth (42°C), an abbreviated set of tests is usually all that is necessary for identification. C. jejuni and the other campylobacters pathogenic for humans are oxidase-negative and catalase-positive. Nitrate reduction, hydrogen sulfide production, hippurate tests, and antimicrobial susceptibilities can be used for further identification of species. Two serotyping schemes have been developed for use in epidemiological tracing of campylobacteriosis. The Lior serotyping scheme is based on heat-labile antigens and utilizes slide agglutination of live bacteria with serotype-specific rabbit antisera preabsorbed with homologous heated and heterologous unheated cross-reactive antigens (Lior et al., 1982). Flagellin is considered to be one of the antigens involved in this serotyping scheme. This scheme can detect over 100 serotypes of C. jejuni, C. coli, and C. lari. The Penner-Hennessy serotyping scheme is based on heat-stable O antigens and involves the passive hemagglutination of erythrocytes, sensitized with boiled saline bacterial extracts, by serotype-specific rabbit sera (Penner and Hennessy, 1980). Lipopolysaccharides (LPSs) are the dominant heat-stable antigen involved in this scheme. The O serotyping scheme detects over 60 types of C. jejuni and C. coli. There is, however, no central or commercial supply of antisera for either type of serotyping scheme. This lack causes problems in standardization and comparison of results. Phage typing can differentiate strains within a serotype and is a simple enough technique to apply to a large number of strains simultaneously (Khakhria and Lior, 1992). 12.4.2

Pathogenesis

Little is known about the mechanism by which C. jejuni causes human disease. C. jejuni can cause an enterotoxigeniclike illness with loose or watery diarrhea or an inflammatory colitis with fever and the presence of fecal blood and leukocytes and occasionally bacteremia that suggests an invasive mechanism of disease. A major problem in elucidating the pathogenesis of Campylobacter spp. infection has been the lack of suitable animal models (Fox, 1992; Nachamkin, 1997). However, as in many other pathogens, the pathological changes can be multifactorial

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in nature, and a combination of determinants are involved. The main factors described are motility, adhesion, invasion, iron acquisition, and toxin production. Campylobacter spp. infection is acquired by the oral route from food, drink, contact with infected animals, or anal-genital-oral sexual activity. C. jejuni is susceptible to gastric acid. However, the infective dose of C. jejuni does not appear to be high: <1000 organisms are capable of causing illness (Black et al., 1988). The organisms multiply in the small intestine, invade the epithelium, and produce inflammation that results in the appearance of red and white blood cells in the stools. The campylobacters have single, bipolar flagella. In animal models using isogenic mutants, fully active flagella are essential virulence factors for the invasion-translocation process (Grant et al., 1993; Wassenaar et al., 1991). The rapid motility coupled with spiral morphological features gives the organism a selective advantage in penetrating and colonizing the thick viscous mucus barrier of intestinal cells. The flagella are highly immunogenic and can undergo both phase and antigenic variations, which help them to evade the immune response of the host. There are two flagellin genes (flaA and flaB) in tandem in all strains, although only one of these genes may be expressed. The flagellins have a molecular weight of approximately 62 kDa and are the major protein surface antigen. The antigenicity of campylobacter flagellin is complex, and epitopic analysis indicates that common as well as serotype-specific linear epitopes are expressed. The common epitopes are antigenically cross-reactive with flagellins from most Campylobacter species, including C. fetus fetus, C. lari, and Helicobacter pylori (formerly C. pylori). Antigenic variations occur between these flagellins, and a microheterogeneity in charge properties due to posttranslational modification, probably glycosylation, also demonstrates some antigenic variation. Additionally there is a reversible transition between flagellated and aflagellated variants (Newell et al., 1996). Flagella, in particular flagella with type A flagellin, do appear to be necessary for invasion and internalization but may or may not act as adhesion factors. Several strains of C. jejuni have been shown to invade in vitro cell lines (de Melo et al., 1989; Konkel and Joens, 1989). Strain 81-176 invades an intestine-derived cell line, Caco-2, and appears within membrane-bound vacuoles (Russell and Blake, 1994). Carbohydrate moieties, probably a glycoprotein, are important for adhesion and invasion since pretreatment with D-glucose, D-mannose, and L-fucose can prevent it (Russell et al., 1993). A variety of outer membrane proteins that bind to eukaryotic cells have also been described. These proteins may be important in internalization of the organism

(Konkel et al., 1993; Panigrahi et al., 1992). Campylobacter spp. may also possess fimbriae whose synthesis is enhanced by bile salts. Nonfimbriated mutants, however, are still able to adhere to and invade another intestinal cell line, INT407, and colonize ferrets but with ameliorated disease symptoms, a property that suggests some role in virulence. Other proteins may also be important in the pathogenesis of Campylobacter spp. infection. A group of surface extractable antigens phosphatidylethanolamine binding proteins (26–32 kDa), called the PEB proteins (PEB 1–4), have been isolated and partly characterized. PEB1 has been cloned from C. jejuni 81-176 and sequenced and may have adherence properties (Pei and Blaser, 1992). It shows homology with Enterobacteriaceae glutamine-binding protein (GlnH), lysine/arginine/ornithine-binding protein (LAO), and histidine-binding protein (HisJ). Although evidence of epithelial cell invasion in vivo is at best sketchy, host cell invasion, which occurs within a very short time, has been observed experimentally in macaque monkeys and in the colon of patients (Rowe and Madden, 2000). In addition to epithelial cell invasion, the organism may overcome the gut barrier by translocation (passing between cells) possibly via M cells in Peyer’s patches (Walker et al., 1988). Campylobacter species have been observed to associate preferentially to intercellular junctions. The ability to invade is strain-dependent. Using HEp-2 cells, no correlation was found between invasiveness and the type of symptoms observed, showing that host factors such as immune status are important. Invasion has been shown to be more efficient when cells of human origin are used in tissue culture studies. C. jejuni and C. coli do not produce iron-chelating agents or siderophores. However, they can utilize exogenous siderophores, including enterobactin and ferrichrome, from other bacteria as iron carriers (Baig et al., 1986). A transport system encoded by the ceu operon may be involved in this process. Most strains of C. jejuni and C. coli produce a cell-associated hemolysin and possess a mechanism to transport hemolytic products into the cell and release iron from the complexes. The organism also elaborates an iron-containing superoxide dismutase (SOD), which provides protection against oxidative stress during invasion. Encoded by the sodB gene, the enzyme may also have some role in intracellular survival of the organism (Pesci et al., 1994). A sodB mutant had a 12-fold decrease in ability to survive in INT407 cells. C. jejuni possesses an iron-responsive regulatory fur (ferric uptake regulator) gene, which, if similar to fur genes in other bacteria, is involved in the synthesis of SODs and outer membrane siderophore receptors as well as regulating other genes involved in pathogenesis.

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Campylobacter spp. appear to produce several types of cytotoxins, including a Shiga-like toxin (SLT) (Moore et al., 1988), a cytolethal distending toxin (CLDT) (Johnson and Lior, 1988), C. jejuni toxin (CJT, similar to Vibrio cholerae toxin) (Daikoku et al., 1990), hepatotoxins (Kita et al., 1990), and heat-labile toxin (similar to E. coli LT toxin) (Rowe and Madden, 2000). The reported frequency of enterotoxin elaboration among isolates varies greatly (Table 12.19). Some of the cytotoxins, which have a molecular weight range of 50,000–70,000, are as yet poorly characterized. However, on tissue culture cell lines their effects include cell rounding with nuclear condensation, loss of cell monolayer adhesiveness, and cell death within 24–48 hours. Cover and colleagues (1990) studied the presence of cytotoxic activity in fecal filtrates of patients with Campylobacter spp. enteritis; they found cytotoxic activity in both patients with disease and healthy asymptomatic subjects, raising doubts about the clinical relevance of the toxin and its role in pathogenicity. The role of CLDT in pathogenesis is unknown, but the toxin was reported to be active in CHO, Vero, HeLa, and HEp-2 cells but not in Y-1 cells. Forty-one percent of over 700 strains produced the toxin, and it produced a hemorrhagic response in rat ligated ileal loops. C. jejuni infection of rabbit ileal loops caused an elevation of cAMP, prostaglandin E2, and leukotriene B4 levels in tissue and fluids. This fluid caused an elevated cellular cAMP level in Caco-2 cells and was inhibited by antiserum against prostaglandin E2 (Everest et al., 1993). These findings suggest that inflammatory mediators elicited by C. jejuni may be involved in the pathogenesis of infection. No mechanism by which C. jejuni and C. coli evade the host immune response has been identified; however, in some experimental models virulence is associated with persistence in the bloodstream, survival in extraintestinal organs, and resistance to macrophage ingestion (Newell et al., 1996). One potential mechanism for bacterial avoidance of host immune responses could be antigenic variation of the flagellin proteins. C. fetus fetus has a well-defined microcapsule, comprising surface array proteins (S-layer proteins), which is responsible for resistance to the complement-mediated bactericidal activity of normal human serum and phagocytosis by polymorphonuclear (PMN) cells (Nachamkin et al., 1992). These S-layer proteins, which form a crystalline subunit structure on the outer surface of the bacterium, have a range of molecular weights (97–149 kDa) and are encoded by about eight homologs of the sapA gene. The Slayer proteins are highly immunogenic during infection. There is significant antigenic variation between each of these proteins, and antigenic switching occurs throughout

Table 12.19 Incidence of Enterotoxin Production Among Strains of Campylobacter jejuni and Campylobacter coli Strains, no. Clinical isolates 25 62 22 44 32 80 12 316 44 372 39 202 22 22 15 Carriers 47 30 6 8 77

Origin

Enterotoxin–positive, %

Method of detectiona

Belgium United States South Africa Belgium Mexico Algeria Diverse origin Canada Costa Rica Diverse origin United States Sweden India United States United States

100 94 77 75 65 65 50 48 47 45 36 32/19c 32 0 0

CHO GM1 ELISAb Y–1 CHO CHO, RILT CHO CHO, GM1 ELISA, RILT CHO, Y–1 Y–1 CHO GM1 ELISA CHO, GM1 ELISAc CHO, GM1 ELISA CHO, GM1 ELISA CHO, GM1 ELISA

12 60 16 0 0

CHO, GM1 ELISA CHO RILT CHO CHO, GM1 ELISA, RILT

India Algeria Mexico Mexico India

a

CHO, elongation of Chinese hamster ovary cells; GM1 ELISA, enzyme-linked immunosorbent assay with ganglioside GM1 as the solid phase and antiserum against enterotoxin CT or LT as primary antiserum; Y–1, rounding of mouse adrenal tumor cells; RILT, fluid accumulation in the rat ileal loop test. b The primary antiserum used with homologous serum against enterotoxin of C. jejuni. c Different results were obtained with the two methods. Source: Compiled from Wassenaar (1997) and Rowe and Madden (2000).

infection, apparently allowing the persistence of this organism in host tissues. 12.4.3

Symptoms, Diagnosis, and Treatment

Symptoms of campylobacteriosis are not very distinctive and thus are not easily differentiated from those resulting from other enteric pathogens. They vary from brief insignificant enteritis to an enterocolitis with abdominal pain and profuse diarrhea that may be grossly bloody. Headache, malaise, and fever may accompany other symptoms. Vomiting is unusual. Onset of the disease is also usually somewhat slower than that of some of the other enteric pathogens, with an incubation period of 2 to 5 days. The disease is usually self-limiting to a period of 3–5 days but occasionally may recur for periods of up to 2 weeks. Also, abdominal pain and cramping can persist for up to 3 months. Most cases resolve without antimicrobial therapy, and the fatality rates are low. Although it affects all ages, prevalence is highest during the first year of life followed

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by a second peak during young adulthood (15–29 years). Bacteremia occurs in less than 1% of cases, at the highest rate in the elderly (Skirrow et al., 1993). C. jejuni and C. coli are susceptible to a variety of antimicrobial agents, including macrolides, fluoroquinolones, aminoglycosides, chloramphenicol, and tetracycline. Erythromycin has been the drug of choice for treating C. jejuni gastrointestinal infections, but ciprofloxacin is a good alternative drug (Nachamkin, 1997). Early therapy of campylobacteriosis with either of these antibiotics is effective in eliminating the organism from stool and may also reduce the duration of symptoms associated with infection. In recent years, increasing evidence has shown an association of Campylobacter spp. infection with GuillainBarré syndrome (GBS), reactive arthritis, and Reiter’s syndrome. The GBS disorder is rare and characterized by acute inflammatory demyelinating polyneuropathy. It can vary greatly in severity from the mildest cases, in which clinical treatment is not sought, to a complete paralysis

that puts the patient close to death. Studies in the 1990s also showed an association of Campylobacter spp. infection with an illness clinically similar to GBS, known as acute motor axonal neuropathy, which occurs primarily in northern China and may occur in other parts of the world (McKhann et al., 1993). Mishu and Blaser (1993) estimate that the annual incidence of GBS preceded by C. jejuni infection ranges from 0.17 to 0.51 cases per 100,000 population and accounts for 425 to 1272 (10% to 30%) of all GBS cases per year in the United States. Kuroki and coworkers (1993) determined that a certain serotype of C. jejuni, O:19, accounted for 83% of 12 strains isolated from patients with GBS, compared with 1.7% of over 1000 sporadic strains. Of the 10 O:19 strains from GBS patients, all belonged to a specific lectin type, type 8, compared with only 1 of the sporadic O:19 strains. This study strongly suggested that O:19 strains were unique and that some specific virulence factor(s) that could induce GBS was associated with these strains. Core oligosaccharide lipopolysaccharides isolated from O:19 as well as other serotypes (O:4, O:1) of Campylobacter spp. have regions homologous to the human gangliosides GM1, GD1a, and GD1b (Aspinall et al., 1994; Yuki et al., 1993, 1994). Host genetic factors such as HLA type may also play an important role in the pathogenicity of GBS (Yuki et al., 1991). Miller-Fisher syndrome, similar to GBS, is an acute neuropathic disorder characterized by paralysis of the eye muscle, absence of reflexes, and facial weakness (Rowe and Madden, 2000). GBS is usually a monophasic illness, with a rapid initial onset, progressive weakness over 1 to 4 weeks, and recovery over subsequent months (Ropper et al., 1991). Although most GBS patients recover after several weeks or months, others are bedridden permanently or have fatal complications. Some patients also suffer from relapses. According to Hughes (1990), the prognosis of GBS varies

Table 12.20 Sources Sample Sewage River water Poultry Beef Pork Lamb Seafood Cooked meats Salads

and up to 13% die and a further 20% are left significantly disabled and unable to work after a year. Reactive arthritis and Reiter’s syndrome are characterized by sterile inflammation of joints from infections originating from nonarticular sites and are mediated by T cells. They may be triggered by Campylobacter spp. or other pathogens, such as Salmonella or Yersinia spp. Although viable organisms are not present, bacterial antigens are probably transported to the joints within phagocytic cells. These rare complications are strongly associated with the human lymphocyte antigen B27 (HLA-B27). 12.4.4

Sources

Campylobacters are ubiquitous in foods of animal origin. Contamination of poultry and raw milk has been the main cause of infections in humans. Contamination rates of raw poultry products may reach nearly 90%. Most clinically healthy poultry, swine, and cattle excrete C. jejuni in their feces, and more than 106 cells/g may be present. Surveys show that 20% to 100% of retail chickens are contaminated (CFSAN, 1998). Lammerding (1988), in a survey of different raw foods in Canada, found that 74% of turkeys and 38% of chickens harbored campylobacters; lower isolation rates for C. jejuni and C. coli were found for beef (23%) and pork (17%); veal (43%) was more heavily contaminated. Subsequently, Fricker and Park (1989) in a detailed study covering a 2-year period again found the highest recovery of these campylobacters from poultry. Data on recoveries of campylobacters from environmental and food sources are summarized in Table 12.20. Food poisoning outbreaks involving poultry are usually due to the campylobacters’ surviving cooking. Another important source of infection is unpasteurized milk. In this case, the bacteria originate from the bovine feces or possibly infected udders. Lactoperoxidase

Recovery Rates of Campylobacter Species from Environmental and Food

Sample examined, no.

Sample positive, no.

Sample positive, %

436 345 758 127 158 103 89 89 106

424 105 421 30 29 16 13 2 0

96.6 30.4 55.5 23.6 18.4 15.5 14.6 2.3 0.0

Source: From Fricker and Park (1989) and Forsythe and Hayes (1998).

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naturally present in milk tends to kill off the campylobacters. However, because of the low infective dose, enteritis outbreaks emanating from unpasteurized milk are often reported. Other reservoirs for infection include rabbits, rodents, wild birds, sheep, horses, and domestic pests (Nachamkin, 1997). Dogs probably account for nearly 5% of human sporadic cases and puppies, in particular, have been implicated. Contaminated vegetables and shellfish may also be sources of infection. Nonchlorinated water may also be a source of campylobacteriosis. 12.4.5

Outbreaks

Campylobacters are the most common cause of sporadic bacterial enteritis in the United States (Borczyk et al., 1987; Finch and Riley, 1984; Tauxe, 1992; CFSAN, 1998). The CDC estimates the overall infection rate to be 1000 per 100,000 population, accounting for over 2 million cases annually. Sporadic cases occur most often in the summer months and usually follow ingestion of improperly handled or cooked food, primarily poultry products (Stern, 1992). Raw milk has been implicated in over 50% of large outbreaks of campylobacteriosis that occur in the United States. In a 10-year review of outbreaks from 1981 to 1990, 20 outbreaks occurred each year, and most of the outbreaks occurred in children who had made field trips to dairy farms (Wood et al., 1992). Campylobacters are readily killed by pasteurization. 12.4.6

Control

Survival of the organism in food is fairly poor. Most processing techniques, such as heat, acid, salt, and drying, destroy it. Gastric pH of 2.3 rapidly kills the organism. Nonetheless, because of the low infective dose, campylobacters are important enteric pathogens and control methods are necessary. These include milk pasteurization, introduction of campylobacter-free poultry flocks, thorough cooking of poultry, separation of raw and cooked meats and poultry, and chlorination of water. Similarly, proper production, processing, and preparation of foods of animal origin reduce the level of contamination, but because the organism is ubiquitous, proper cooking is the only practical procedure. Microwave cooking may not completely remove contamination from meat. Interest has focused on low-level irradiation as an effective technique for killing C. jejuni, particularly in cut-up poultry. This procedure, when coupled with modern impervious packaging techniques, provides a commercially acceptable

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method for supplying consumers with a C. jejuni–free product. Protective immune responses can be produced after campylobacter infection. Evidence for this response includes a progressive decrease in the illness/infection ratio in children in underdeveloped countries with endemic infections; high antibody levels and asymptomatic infections after multiple exposures to campylobacter-contaminated foodstuffs; and development of protective immunity to rechallenge in experimentally infected volunteers and nonhuman primates (Black et al., 1988; Calva et al., 1988). Passive immunization may also be effective as campylobacter-specific secretory immunoglobulin A (sIgA) antibodies in breast milk apparently protect infants from disease (Nachamkin et al., 1994). These sIgA antibodies are primarily directed against flagellin. Epidemiological evidence suggests that protective immunity controls the symptoms of disease but does not necessarily prevent intestinal colonization (Hartug et al., 1995; Newell et al., 1996). Vaccines against campylobacter enteritis are currently under development. A killed-whole cell vaccine induces protection in a mouse model using the heat-labile LT toxin of E. coli as the mucosal adjuvant (Baqar et al., 1995a, 1995b). The same vaccine has been successfully tested for safety and immunogenicity in nonhuman primates. Potential candidates for subunit vaccines include the flagellins and other surface acid–extractable proteins.

12.5 SHIGELLA SPECIES (SHIGELLOSIS) Members of the Shigella species cause bacillary dysentery or shigellosis. Hippocrates, who noted the seasonal pattern of the disease, introduced the term dysentery. The illness is characterized by frequent passage of stools containing blood and mucus accompanied by painful abdominal cramps. Shigellosis is a disease of the poor, primarily affecting young children in the developing world. Epidemic outbreaks also occur in industrialized nations after accidental breaches in hygiene or sanitation. Shigellosis affects only humans and, on occasion, subhuman primates. This narrow host range is a consequence of the fact that the causative organisms are exceedingly host-adapted to the higher primates; the basis for this selectivity is not clearly understood. Along with salmonellosis, it is one of the more common intestinal infections occurring throughout the world. In the United States there are about 15,000 cases of shigellosis and 30,000 cases of salmonellosis reported annually (O’Brien et al., 1988), although the actual incidence is estimated to be 200,000 and 2 million cases, respectively. The lower number of shigella cases can be explained in part by the fact that all of the 30-

odd serotypes that make up the genus Shigella have a host range limited to primates. Furthermore, though the infectious dose of shigellae for humans is low (100–1000 cells), the organism can easily be controlled by the classical public health procedures of safe water supplies and effective waste disposal (Levine et al., 1973). In contrast, the higher incidence of salmonellosis can certainly be attributed to over 1500 serotypes that make up the genus Salmonella. With the low infectious dose required to cause disease coupled with oral transmission via fecally contaminated food and water, shigellosis often follows in the wake of many natural (earthquakes, floods, famine) and humangenerated (war) disasters. The Japanese bacteriologist Shiga conclusively identified the causative agent of bacillary dysentery in 1898 in his report of a severe epidemic in Japan in 1896, during which a mortality rate of 24% was observed in nearly 90,000 cases. Shiga found a distinct gram-negative rod to be involved, and in recognition of this feat, the genus was ultimately named in his honor. The prototype organism, Shigella dysenteriae type 1, is often called Shiga’s bacillus. This discovery was rapidly followed by isolation of the same organism in different parts of the world (Flexner, 1900; Kruse, 1900). However, it required an additional 40 years to sort out the microbiological characteristics and to define and characterize the four species and multiple types now included in the genus (Table 12.21). Flexner (1900) was the first to identify a toxic agent in the cultures of the organism and concluded that shigellosis was due to “a toxic agent rather than to an infection per se.” Vaillard and Dopter (1903) and Dopter (1905a, 1905b) soon confirmed his findings. Todd (1904) also noted that S. paradysenteriae Flexner (S. flexneri) filtrates caused diarrhea but not paralysis in experimental animals. Thus, within 5 years of the initial observation of Flexner, several researchers demonstrated that Shiga spp. bacillus produced a soluble, antigenic protein toxin, precipitable by ammonium sulfate and destroyed by heating, that caused delayed-onset neurological symptoms and death in rabbits but not in guinea pigs. These studies also showed the presence of a bacterial endotoxin responsible

Table 12.21 Present designation S. dysenteriae S. flexneri S. boydii S. sonnei

Pathogenic Shigella Species Group and type

Mannitol

Ornithine decarboxylase

A B C D

– + + +

– – – +

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for producing fever or hypothermia in a few hours, with hyperemia, hemorrhage, and necrosis of various parenchymal organs and death. By 1950, it was conclusively established that Shigella dysenteriae type 1 produced a potent protein toxin in addition to the LPS endotoxin (see Chapter 13). The role of the former in the pathogenesis of dysentery and its mechanism of action were still open to question. Interest in this field was regenerated by the occurrence in 1969–71 of a massive epidemic of diarrhea and dysentery in Mexico and Central America that was due to Shigella dysenteriae type 1, after several decades of dormancy as a pathogen in the region (Mata et al., 1970). At around the same time, it was conclusively proved that clinical cholera was due to the action of a heat-labile protein toxin produced by the causative organism, Vibrio cholerae. This was the first time a gram-negative bacterial toxin was implicated in the pathogenesis of diarrheal disease (Finkelstein, 1973). The epidemic outbreak provided researchers with fresh isolates of highly virulent organisms, and an opportunity to observe the clinical presentation of the disease. Keusch and associates (1970, 1972) finally showed that the epidemic Shigella spp. strain produced an enterotoxin in vitro, which was active in ligated rabbit ileal loop (LRIL). They suggested that the enterotoxin might cause the diarrhea and/or dysentery of shigellosis. Subsequently, Shigella species were also found to be toxigenic (Keusch and Jacewicz, 1977a; O’Brien et al., 1977). Compared to other enteric diseases, food-borne shigellosis has often been a neglected area of study. This section describes briefly the causes and pathogenicity of shigellosis in humans via food-borne transmission. 12.5.1

Organism

Members of the Enterobacteriaceae family, the shigellae are nearly genetically identical (over 80% nucleotide homology) to Escherichia coli and closely related to Salmonella and Citrobacter species (Ochman et al., 1983; Maurelli and Lampel, 1997). The shigellae are gram-negative, nonsporulating, oxidase-negative facultative anaerobes. Although nonmotile, the genes encoding the flagellar apparatus are present (Tominaga et al., 1994); the significance, however, is not known. Shigella spp. are identified on the basis of their capacity to ferment various sugars and on the antigenic specificity conferred by LPS O-side chains (i.e., somatic antigen), which accounts for their specific serotype. The genus comprises four different species (Table 12.21): S. flexneri (6 serotypes), S. dysenteriae (16 serotypes), S. sonnei (1 serotype), and S. boydii (15 serotypes). An important biochemical characteristic that distinguishes

shigellae from other enteric bacteria is their inability to ferment lactose or utilize citric acid as a sole source of carbon; they do not produce H2S and, except for S. flexneri 6, do not produce gas from glucose. They may also be divided into those that ferment mannitol and those that do not (Table 12.21). Of the four species, S. dysenteriae type 1, or the Shiga bacillus, is the most virulent; other infective serotypes are type 3 and type 4. These are, however, rare types. All other serotypes of S. dysenteriae are rarely involved in human disease (Christie, 1980). S. flexneri type 2 is the most common cause of shigellosis in the United States and developing countries. S. sonnei is most common in Japan, the United States, and Western Europe. The Shigella spp. lipid A of the LPS moiety is identical to that of E. coli and mediates the endotoxicity of LPS; the core region consists of an oligosaccharide similar but not identical to that of E. coli (Sansonetti et al., 2001). The O-side chains are composed of repeated sugar subunits, which vary in their composition, thereby contributing to the serotypic diversity. The similarity between the oligosaccharide units explains the high level of cross-reactivity among the S. flexneri serotypes, which extends in some cases to E. coli strains. In contrast, S. sonnei does not cross-react with E. coli (Brahmbhatt et al., 1992). 12.5.2

Pathogenicity

Shigella spp. and enteroinvasive E. coli (EIEC, see Chapter 13) are the principal agents of bacillary dysentery and as such belong to the group of enteric pathogens that cause disease by overt invasion of epithelial cells in the large intestine. In the case of Shigella spp., the invasive process remains localized to the colonic and rectal mucosa, thereby causing major inflammatory destruction that accounts for a dysenteric syndrome. In many cases, however, shigellosis causes only a watery diarrhea similar to that observed with noninvasive pathogens. Shigella spp. infections are almost always limited to the gastrointestinal tract; bloodstream invasion is quite rare. Shigellae are highly communicable: the infective dose is on the order of 102–103 organisms, whereas it usually is 105–108 for salmonellae and vibrios. The essential pathological process is invasion of the mucosal epithelial cells (e.g., M cells) by induced phagocytosis, escape from the phagocytic vacuole, multiplication and spread within the epithelial cell cytoplasm, and passage to adjacent cells. Microabscesses in the wall of the large intestine and terminal ileum lead to necrosis of the mucous membrane, superficial ulceration, bleeding, and formation of a “pseudomembrane” on the ulcerated area, which consists of fibrin, leukocytes, cell debris, a necrotic mucous mem-

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brane, and bacteria. As the process subsides, granulation tissue fills the ulcers and scar tissue forms. Some of the virulence factors involved in these processes are described in the following. Shigella spp. virulence is multigenic, involving both chromosomal and plasmid-encoded genes (Table 12.22). In vitro cultured cells (e.g., HeLa cells, Henle cells, Hep-2 cells) have been extensively used to study the capacity of Shigella spp. to penetrate the cells that are not professional phagocytes (LaBrec et al., 1964). Cellular microbiological techniques coupled with molecular genetic analysis of bacterial pathogenicity have shown that Shigella spp. are characterized by expression of an “invasive phenotype” whose effect depends on the target cells: entry/escape into cytoplasm, intracellular motility, and cell-to-cell spread in the presence of epithelial cells. The pathogenic factors involved in Shigella spp. invasion of epithelial cells have been extensively studied and reviewed (Goldberg and Sansonetti, 1993; Parsot and Sansonetti, 1996; Menard et al., 1996; Sansonetti et al., 2001). Shigella spp. can enter several cell lines in vitro, regardless of the species and organ of origin. Entry into Epithelial Cells Shigella spp. enter the epithelial cells via a “triggering” mechanism. This process is closely related to macropinocytosis and results in bacterial internalization in a vacuole that initially is loosely associated to the bacterial body. The organism secretes a set of homologous invasion proteins on contact with their cellular targets and induces important but localized rearrangements of the cell cytoskeleton at the site of interaction (Sansonetti et al., 2001). Actin polymerization is essential for bacterial entry since it is abrogated by cytochalasins. Cellular extensions rise to 10 µm over the cell surface, forming a flowerlike structure. These projections merge and engulf the bacterial body within 5 to 10 minutes (Adam et al., 1995). The newly formed actin filaments support these membrane projections. These filaments are oriented with their fast-growing “barbed” end facing the inner face of the cell cytoplasmic membrane and bundled by plastin, which is necessary to stabilize the projections and required for efficient bacterial entry. A 180-kb plasmid in S. sonnei and a 220-kb plasmid in S. flexneri are essential to the invasion process. These plasmids contain most of the identified virulence genes of these microorganisms (Sansonetti et al., 1981, 1982). A 30-kb locus in these plasmids contains all the genes necessary for entry. This region is divided into two divergently transcribed operons that encode two classes of proteins. The mxi-spa locus comprises about 20 genes specifying a

Table 12.22

Virulence-Associated Loci of Shigella Species

Locus (i) Chromosomal rfa, rfb stx virR (hns) iuc sodB vacB Plasmid icsB ipgC ipaB ipaC ipaD mxi/spa icsA (virG) virB virF ipa

Product

Role in virulence

Enzymes for core and O-antigen biosynthesis Shiga toxin Histonelike protein (H-NS or H1)

Correct polar localization of IcsA Destruction of vascular tissue Repressor of virulence gene expression; binding to virB promoter at 30°C, preventing activation by virF Acquisition of iron in host Inactivation of superoxide radicals; defense against oxygendependent killing in host Controls expression of icsA and ipaB genes

Synthesis of aerobactin and receptor Superoxide dismutase Transcriptional activator 57-kDa Protein 17-kDa Protein 62-kDa Protein 43-kDa Protein 38-kDa Protein 20 Proteins 120-kDa Cell-bound and secreted protein Transcriptional activator 30-kDa Regulatory protein AraC family of transcriptional activators Transcriptional activator

Lysis of double membrane for intercellular spread Chaperone for IpaB and IpaC Invasion; lysis of vacuole; induction of apoptosis Invasion Invasion Secretion of Ipa and other virulence proteins Actin polymerization for intracellular motility and intercellular spread Temperature regulation of virulence genes Temperature regulation of virulence genes; control of virB gene transcription Operon activating transcription of ipa, mxi, and spa operons of entry region

Source: Compiled from Maurelli and Lampel (1997) and Sansonetti et al. (2001).

type III secretory apparatus (Andrews et al., 1991; Allaoui et al., 1993). These genes are expressed and assembled at 37°C and activated on contact with target cells (Parsot and Sansonetti, 1996; Menard et al., 1994a). The mxi (membrane expression of invasion plasmid antigens) genes comprise an operon that encodes several lipoproteins (MxiJ and MxiM), a transmembrane protein (MxiA), and proteins containing signal sequences (MxiD, MxiJ, and MxiM) (Allaoui et al., 1992; Andrews et al., 1991). MxiH, MxiJ, MxiD, and MxiA have homology with proteins involved in secretion of virulence proteins (Yops) in Yersinia spp. described later in this chapter. The surface presentation of Ipa antigens (spa) genes encode proteins that have significant homologies with proteins involved in flagellar synthesis in E. coli, Salmonella typhimurium, Bacillus subtilis, and Caulobacter crescentus (Maurelli and Lampel, 1997). In Shigella spp., a complex formed by the IpaB and IpaD proteins regulates secretion of the Ipa proteins. Optimal secretion occurs during the exponential phase of growth. The Mxi-Spa system of Shigella spp. secretes about 15 proteins that share such common features as the lack of signal peptide and the capacity to aggregate in an extracellular milieu in large supramolecular structures

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(Parsot et al., 1995). The invasion plasmid antigens (ipa) locus, which essentially consists of an operon, encodes four proteins—IpaB (62 kDa), IpaC (42 kDa), IpaD (37 kDa), and IpaA (70 kDa)—which are secreted by the type III Mxi-Spa system on contact of bacteria with host cells. It also encodes IpgC, an 18-kDa cytoplasmic chaperone that binds IpaB and IpaC in the bacterial cytoplasm, preventing their aggregation and proteolytic degradation (Menard et al., 1994b). The deletion of ipaB, ipaC, and ipaD genes leads to complete inactivation of the bacterial entry phenotype, indicating their essential role in entry (Menard et al., 1993). In contrast, inactivation of the ipaA gene leads only to partial inactivation of entry (Tran Van Nhieu et al., 1997). The Ipa products are associated with the outer membrane of shigellae. Once secreted, IpaB and IpaC form a complex interacting with the epithelial cell membrane, which is probably responsible for transducing the signal that leads to entry of shigellae into the host cells via bacterium-directed phagocytosis. Although the Ipa proteins have no typical signal sequence, these proteins are secreted into the extracellular medium. Contact of the bacterium with epithelial cells causes increased secretion of the cytoplasmic pool of Ipa products (Andrews et al., 1991;

Menard et al., 1994b). The surface-expressed and cell-free Ipas probably play complementary roles in generating the signals required for uptake by the host cell. Intracellular Motility Shigella spp. was the first pathogen identified as an intracellular spreading bacterium inside infected cells. Listeria monocytogenes, responsible for food-borne listeriosis, also utilizes a similar mechanism, which is described in detail later in this chapter. The organisms escape from the phagocytic vacuole and use cytoplasmic cytoskeletal components to propel themselves inside the first infected cell before they reach the cell membrane and induce cellular protrusions. Engulfment of these cellular extensions, which contain bacteria, by the neighboring cells leads to cell-to-cell spread of the pathogen (Theriot, 1995; Dramsi and Cossart, 1998). A plasmid-encoded virulence gene, virG or icsA (intracellular spread), which is not required for invasion, encodes a protein that catalyzes the polymerization of actin in the cytoplasm of the infected cells (Bernardini et al., 1989; Makino et al., 1986). The IcsA protein (also named VirG) is unusual in that it is expressed asymmetrically on the bacterial surface and is present only at one pole. Although the mechanism of unipolar localization of IcsA is unknown, it is dependent on synthesis of a complete LPS complex (Sandlin et al., 1995). IcsA is an outer membrane protein. Translocation across both membranes and surface anchoring is independent of the Mxi/Spa type III secretion apparatus and is mediated by an autotransporter secretion pathway. Shigella spp. intracellular motility is associated with formation of actin tails at one pole of the bacterium. By using video microscopy and microinjection of labeled actin monomers, it has been shown that the actin tail remains stationary in the cytoplasm, trailing behind the moving bacterium, and that the rate of incorporation of actin monomers correlates directly with the speed of bacterial movement, suggesting that continuous actin polymerization at one pole of the bacterium is itself sufficient to generate the motile force (Theriot et al., 1992). Shigella spp. movement inside the cytoplasm is random and rapid (6–60 µm/minute). It occurs optimally at the stage of bacterial division. Several cytoskeletal proteins have been detected in Shigella spp. actin tails. These include T-plastin, α-actinin, vasodilator stimulated phosphoprotein (VASP), vinculin, Mena, Arp2/3, and neural Wiskott-Aldrich syndrome protein (N-WASP) (Sansonetti et al., 2001). However, the contribution of these proteins to actin-based motility has been demonstrated for only a few.

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Cell-to-Cell Spread When contact between a moving organism and the inner face of the cytoplasmic membrane occurs, a protrusion is formed that is phagocytosed by the adjacent cell. This process involves interaction with components of the cellular junction, allowing bacterial passage via an actin-driven protrusion into the adjacent cell. Expression of cadherins is a prerequisite to allowing phagocytosis of these protrusions by the adjacent cells (Sansonetti et al., 1994). Once absorbed by the adjacent cell, the bacteria are trapped inside a pocket surrounded by a double membrane, which is subsequently lysed. This process requires the intervention of IcsB, a 57-kDa protein encoded by a gene located upstream from the ipa genes in the entry locus. Thus, in the context of an epithelial lining, the invasive phenotype of Shigella spp. leads to an efficient process of intracellular colonization. It encompasses entry into nonphagocytic cells, escape to the cytoplasm, intracellular growth, intracellular motility, and cell-to-cell spread. This multifactorial process is a spectacular example of integration of several defined steps that leads to progression of infection in a sanctuary that is relatively protected from humoral and cellular effectors of the innate and adaptive immune response (Figure 12.11). In contrast to the genes of the virulence plasmid that are responsible for invasion of mammalian tissues, most of the chromosomal loci associated with Shigella spp. virulence are involved in regulation or survival within the host. These genes can be classified in regulatory genes such as virR, and structural gene-encoding factors such as LPS and toxins (Table 12.22). Upon autolysis, all shigellae re-

Figure 12.11 Shigella spp. entry, escape into the cytoplasm, and intracellular and cell-to-cell spread, with reference to the effectors required for each step of invasion of a polarized epithe-

lease their toxic LPS. This endotoxin probably contributes to the irritation of the bowel wall. The virR gene encodes the H-NS (H1) protein (Dorman et al., 1990). H-NS is a major component of a family of histonelike proteins that control DNA supercoiling, thereby regulating expression of numerous genes. Another chromosomal gene, vacB, regulates the invasive properties of Shigella spp. (Sasakawa, 1995). Toxins Shigella spp. produce various toxins, among which Shiga toxin is the best characterized. Among the numerous species and serotypes of Shigella spp. only S. dysenteriae type 1 produces this toxin. Five years after S. dysenteriae was identified as the causative agent of bacillary dysentery, Conradi (1903) demonstrated that intravenous administration of Shiga bacilli autolysates could paralyze and kill rabbits. He concluded that a neurotoxin was present in the microbial extract. That this “neurotoxin” was in fact distinct from the LPS endotoxin of S. dysenteriae was the subject of considerable research over the following four decades. The confusion stemmed in part from the prevailing convention that classified bacterial toxins on the basis of topographical characteristics (exo- versus endotoxins, see Chapter 13) rather than chemical composition (LPS versus protein toxin) as is done today (Keusch et al., 1986). Thus, Shiga toxin was called an endotoxin because its “neurotoxic” activity was cell-associated rather than cell-free. By the late 1930s, Boivin and Mesrobeanu (1937a, 1937b, 1937c, 1937d) had shown that the Shiga neurotoxin was indeed a protein and, hence, distinct from the LPS of gram-negative bacteria. Furthermore, the toxin appeared to be a product unique to Shiga’s bacillus; no conclusive evidence for neurotoxin production by other Shigella species was found. However, by the 1970s, it was demonstrated that both S. flexneri and S. sonnei produce these same toxins (Keusch and Jacewicz, 1977a; O’Brien et al., 1977). However, the biological activity produced, per milligram (dry weight) of bacterial cell pellet, by these latter two species in liquid culture is at least 103–104-fold less than that produced under identical conditions by S. dysenteriae type 1 (O’Brien et al., 1977; O’Brien and LaVeck, 1982). Some of the biochemical and pharmacological properties of Shiga toxin are briefly described in the following. Shiga toxin is a holotoxin composed of five B subunits of 7 kDa each, and one A subunit of 32 kDa. The A subunit is the toxic part of Shiga toxin that becomes enzymatically active on proteolytic cleavage, releasing a 27kDa A1 fragment corresponding to the toxic moiety and a 4-kDa corboxy-terminal A2 portion (O’Brien et al., 1992).

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The pI of the intact, biologically active toxin is 7.02, whereas the B subunit has a pI of 5.67. Most of the A subunit appears as A1 in culture supernatants, suggesting that the toxin is cleaved by endogenous proteases as it exits the bacterial cell (O’Brien et al., 1988). The B subunit is the portion of the molecule that binds to the eukaryotic receptor. It has specific affinity to the disaccharide Galα1-4galβ found in glycolipids such as the globotriaosylceramide Gb3. Shiga toxin is an N-glycosidase that cleaves adenine off one specific adenosine of the 28S component of the 60S ribosomal subunit, thus irreversibly destroying ribosomal functions (Endo et al., 1988). Specifically it prevents peptide chain elongation. Therefore, Shiga toxin is a potent inhibitor of protein biosynthesis, in both eukaryotic and prokaryotic systems (Thompson et al., 1976; Olenick and Wolfe, 1980). Shiga antitoxin responses are elicited in humans after natural and experimental infection with shigellae (Keusch et al., 1976). Shiga toxin is also immunogenic in rabbits, monkeys, and mice (O’Brien et al., 1988). Purified preparations of Shiga toxin exhibit three physiological activities: lethality (“neurotoxicity”), cytotoxicity, and enterotoxicity. As mentioned earlier, Shiga toxin was first recognized by its ability to paralyze and kill rabbits (Conradi, 1903), and, consequently, the material was considered a neurotoxin. Although both rabbits and mice exhibit neurological dysfunctions when given Shiga toxin, this substance is not a true neurotoxin. Its primary effect is on the endothelium of the small blood vessels of the central nervous system rather than on nerve cells directly (Howard, 1955; Bridgewater et al., 1955). Evidence from clinical studies also indicates that adults with shigellosis rarely show signs of neurological disorders (Barrett-Connor and Connor, 1970). However, the reported incidence of convulsions in children with shigellosis varies from 10% to 45%. Studies in animals have shown a remarkable variation (>10,000-fold) in sensitivity to the neurotoxic lethal action of Shiga toxin (Table 12.23). In the very sensitive rabbit, a characteristic progression of symptoms occurs, the timing and duration affected by the dose of toxin. After an intravenous dose of one or two times the LD50, the animal appears well for about 48 hours, when forelimb paresis first becomes apparent. The muscle weakness causes the forelimbs to splay out and the animal has some difficulty in moving about. This increases in severity over the next 24–48 hours, begins to affect the hind limbs, and progresses to paralysis. By then, the rabbit is obviously sick, prostrated, usually with loss of muscle tone so that the head cannot be held up, although sometimes there is opisthotonus. Depending on the dose, the animal may die or recover in a few days, usually with no sign of residual

Table 12.23 Lethal Dose for Neurotoxic Activity of Shiga Toxin for Several Animal Species Species Rabbit Rhesus monkey Hamster Mouse Rat Guinea pig

Table 12.24 Shiga Toxin

Sensitivity of Various Tissue Culture Cells to

Relative dose/kg body weight 1 5 40 700 5,000 >10,000

Source: Compiled from Cavanagh et al. (1956) and Keusch et al. (1986).

paresis. In the mouse, flaccid paralysis also occurs, although in this species it characteristically appears first in the hind limbs. No paralysis is noted in hamsters, and instead bilateral pleural effusions associated with congestion and pulmonary edema have been described (Keusch et al., 1986). Minimal abnormality of the gastrointestinal tract is noted in the rat, and no central nervous system (CNS) lesions, while the guinea pig appears to be totally resistant (Table 12.23). The function of Shiga toxin in eliciting diarrhea in the course of shigellosis due to S. dysenteriae type 1 has not been convincingly demonstrated in humans, although its enterotoxicity in the rabbit ligated-loop model would indicate that it has this function (Butler et al., 1986). Gb3 is mainly detected in the nonepithelial fraction of colonic sections. Hence, it is not clear how the toxin could interact with the luminal pole of epithelial cells to cause diarrhea. On the other hand, Shiga toxin is cytotoxic to intestinally derived epithelial cells in vitro and may thereby participate in the cytotoxic effect to the epithelium, through release of the toxin by extracellular or intracellular bacteria. In any event, considering the localization of Gb3, it is likely that the toxin has subepithelial targets. Vicari and colleagues (1960) first reported that the toxin is lethal to a variety of tissue culture cells (human cervix carcinoma [KB], human liver, monkey kidney). The cytotoxic effects were dose-dependent and could be abrogated if the toxin was preincubated with antitoxin. Simultaneous but separate addition of toxin and antitoxin to cell culture fluids did not protect cell monolayers. The cytopathogenic events progressed as follows: transparent cytoplasm, thickened plasma cell membrane, appearance of large eosinophilic granules around a pyknotic nucleus, and cell death. Some representative data on the cytotoxic effects of Shiga toxin are summarized in Table 12.24. HeLaS3 cells are the most responsive to toxin of all tissue culture lines tested to date. The resistance of most tissue culture cells to Shiga toxin may reflect the absence of specific toxin re-

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Cell line

Origin

HeLa S3 Vero

Human cervical African green monkey kidney Human epidermoid carcinoima of the larynx Human cervical carcinoma

HEp-2

HeLa cells adapted to suspension culture Human melanoma WI-38 H-Vero CHO BHK Y-1 adrenal cells L cells

Melanoma Human lung fibroblasts African green monkey kidney Chinese hamster ovary Baby hamster kidney clone 15 Mouse adrenal cell Mouse connective tissue

Relative dose required to kill 50% of cells 1 10 10,000

Refractorya

Refractory Refractory Refractory Refractory Refractory Refractory Refractory

a

Less than 50% kill at any toxin dose. Source: From O’Brien et al. (1988).

ceptors. Binding of toxin to HeLa or rat liver cells does in fact appear to be the first step in cytotoxicity of Shiga toxin. This is mediated by a cell surface receptor containing oligomeric N-acetyl- D -glucosamine (Keusch and Jacewicz, 1977b). Three lines of evidence support this contention (Table 12.25): First, enzyme treatment of HeLa cells reduces cell sensitivity to cytotoxin in cells exposed to proteolytic enzymes or lysozyme, but not various other glycosidases. This suggests that the receptor is a glycoprotein containing a lysozyme-sensitive substrate. Second, in competitive inhibition studies with soluble mono- or oligosaccharide haptens, of 28 hapten inhibitors tested, only the lysozyme substrate of N-acetyl-D-glucosamine oligomers of 3 or 4 units in length (chitotriose or tetraose) were effective inhibitors of toxicity (Keusch and Jacewicz, 1977b). Finally, the sugar binding protein (lectin) wheat germ agglutinin (WGA), which has specificity for chitotriose, is effective in blocking cytotoxin action, whereas lectins with different specificity, including phytohemagglutinin (PHA) and concanavalin-A (ConA), were not. These data collectively indicate that the presence of specific sugar-containing receptors is necessary for cyto-

Table 12.25

Properties of the Shigella Species Toxin Receptor Test system

Treatment

a

Pronase Trypsin Neuraminidase Galactose oxidase β-Galactosidase Lysozyme Ganglioside N-acetyl-D-glucosamine Chitobiose Chitotriose Chitotetraose PHA ConA WGA

Intact HeLa cells

Rat liver cell membrane

NTb ↓ Binding No effect No effect ↑ Binding ↓ Binding No effect No effect No effect ↓ Binding ↓ Binding No effect No effect ↓ Binding

↓ Binding ↓ Binding No effect No effect ↑ Binding ↓ Binding No effect NT NT NT NT No effect No effect ↓ Binding

a

PHA, phytohemagglutinin; ConA, concanavalin A; WGA, wheat germ agglutinin. NT, not tested. Source: Compiled from Keusch and Jacewicz (1977b) and Keusch et al. (1986). b

toxicity and that the receptor contains a chitotrioselike moiety. Shiga toxin has enterotoxic activity as is evidenced by fluid secretion in ligated segments of rabbit ileum and jejunum. The mechanism by which the toxin causes fluid secretion is still debatable but might conceivably involve either cAMP or cyclic guanosine monophosphate (cGMP), or possibly even serotonin release (Keusch et al., 1986). In macaque monkeys inoculated intragastrically either with a wild-type or with a stxA (knockout mutant lacking expression of the catalytic A subunit of Shiga toxin) mutant of S. dysenteriae type 1 dysentery developed. However, animals infected with the wild-type strain consistently showed the presence of blood in their dysenteric stools. In support of this difference in clinical symptoms, the histopathological analysis showed severe alterations of the capillaries in the lamina propria of colonic tissues from animals infected with the toxin-producing strains (Fontaine et al., 1988). These observations indicate that Shiga toxin may primarily behave as a toxin for the vascular endothelium, thereby adding a component of ischemic and hemorrhagic colitis to the dysentery caused by the invasive phenotype. Epidemiological observations confirm this finding, showing that the patients infected with S. dysenteriae type 1 have more blood in their stools than patients infected with other Shigella species (Stoll et al., 1982; Sansonetti et al., 2001). The classical neurotoxic effect of Shiga toxin is also likely to be due to brain capillary damage.

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S. dysenteriae type 1 causes yet another severe condition, the hemolytic uremic syndrome (HUS). Although the pathogenesis of this acute (often fatal) renal failure is not fully understood, evidence points to Shiga toxin’s penetrating the blood vessels of the intestinal mucosa and reaching the kidneys via the bloodstream as a major etiological factor (Sansonetti et al., 2001). The glycolipid Gb3 is found in high concentrations on human kidney endothelial cells, which are exquisitely sensitive to Shiga toxin (Obrig et al., 1993). Histopathological analysis shows thrombosis and destruction of the glomerular capillaries, and sometimes, renal cortical necrosis (Koster et al., 1978). It is possible that LPS acts in synergy with Shiga toxin to destroy renal blood vessels (Heyderman et al., 1994). Shigellae produce toxins other than Shiga toxin. Two enterotoxins described in 1995 may account for the early diarrheal phase often observed during shigellosis. Shigella enterotoxin 1 is a chromosomally encoded, irondependent toxin of 55 kDa that is primarily expressed by S. flexneri 2a (Noriega et al., 1995). Shigella enterotoxin 2 is a plasmid-encoded protein of 63 kDa (Nataro et al., 1995). Miscellaneous Virulence Factors All Shigella spp. strains express siderophores and their corresponding outer membrane receptors (Table 12.22). Siderophores are low-molecular-weight molecules with

high affinity for ferric iron that they can extract from their physiological carriers, transferrin and lactoferrin. Aerobactin is a hydroxamate siderophore, which S. flexneri and S. sonnei use to scavenge iron, whereas S. dysenteriae and S. sonnei produce enterochelin siderophores. Some isolates can express both types (Lawlor et al., 1987). When the iuc locus, which contains the genes for aerobactin synthesis and transport, is inactivated, the aerobactin mutants retain their capacity to invade host cells but are altered in virulence, as measured in animal models. These results suggest that aerobactin synthesis is important for bacterial growth within the mammalian host (Lawlor et al., 1987; Nassif et al., 1987). 12.5.3

Symptoms, Diagnosis, and Treatment

There are essentially two patterns of illness caused by Shigella spp. The first is the classical bacillary dysentery, characterized by fever, severe intestinal discomfort with intestinal cramps and tenesmus, as well as permanent emission of bloody, mucopurulent stools. This clinical form is characteristic of infections caused by S. dysenteriae type 1 and S. flexneri. Second is a more benign episode of bloody watery diarrhea that may last several days, which is typically associated with S. sonnei. There are, however, severe cases of S. sonnei infection and mild cases of S. flexneri infection; therefore, the intrinsic capacity to cause dysentery seems to be present in all strains, regardless of species and serotype (Bennish and Wojtyniak, 1991; Sansonetti et al., 2001). The dysentery stage of shigellosis is often preceded by watery diarrhea. This stage reflects the transient multiplication of bacteria as they pass through the small bowel. Jejunal secretions probably are not effectively reabsorbed in the colon, as a result of transport abnormalities caused by bacterial invasion and destruction of the colonic mucosa. The diarrheal stage also reflects the production of one or several “classical” enterotoxins that have been recognized in S. flexneri. The more severe, dysentery stage of shigellosis correlates with extensive bacterial colonization of the colonic mucosa. The organisms invade the epithelial cells of the colon, spread from cell to cell, but penetrate only as far as the lamina propria. Foci of individually infected cells produce microabscesses, which coalesce, forming large abscesses and mucosal ulcerations. As the infection progresses, dead cells of the mucosal surface slough off, thus leading to the presence of blood, pus, and mucus in the stools. Shigellosis can also result in acute complications. The most frequent causes of death among hospitalized children during shigellosis are septicemia, primarily seen

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in malnourished children, and hypoglycemia (Struelens et al., 1985). Other complications arising from the disease include severe dehydration, intestinal perforation, toxic megacolon, seizures, Reiter’s syndrome, and HUS (Bennish, 1991). Reiter’s syndrome, a form of reactive arthritis, is a postinfection sequela to shigellosis and is strongly associated with the HLA-B27 histocompatibility group (Simon et al., 1981). HUS is a rare but potentially fatal complication associated with infection by S. dysenteriae type 1. The syndrome is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. HUS is also produced by strains of E. coli O157:H7, which produce high levels of Shiga toxins (see Chapter 13). Malnutrition is a severe consequence of serious cases of shigellosis (Sansonetti et al., 2001). It has been recognized as a major cause of chronic malnutrition in children in developing countries. The pathogenesis of this condition is probably multifactorial, associating lack of appetite and limited malabsorption, the latter having a weak impact as the small intestine is not affected by Shigella spp. However, it is likely that the persistence of intestinal inflammation with colonic protein loss and the systemic effects of elevated levels of tumor necrosis factor α (TNF-α) account for this condition. The incubation period for shigellosis is 1 to 7 days; however, typical symptoms appear within 2–3 days. Strains of S. dysenteriae type 1 cause the most severe disease; S. sonnei produces the mildest; S. flexneri and S. boydii infections can be either mild or severe. Despite the severity of the disease, shigellosis is self-limiting. If untreated, clinical illness usually persists for about 1 to 2 weeks, although it may persist as long as a month, and the patient recovers (Maurelli and Lampel, 1997). For diagnosis of shigellosis, fresh stool, mucus flecks, and rectal swabs are used for culturing the organism. Large numbers of fecal leukocytes and some red blood cells often are seen microscopically. Serum specimens, if desired, must be taken 10 days apart to demonstrate a rise in titer of agglutinating antibodies. Normal persons often have agglutinins against several Shigella species. However, serial determinations of antibody titers may show a rise in specific antibody. Serological testing is rarely used to diagnose shigellae infections. As mentioned earlier, shigellosis is a self-limiting disease in normally healthy patients, and full recovery can occur even without the use of antibiotics. Although stool fluid losses are not as massive as with other bacterial diarrheas (e.g., cholera), oral intake of fluids is still advisable. Intravenous rehydration may be required in very young and elderly patients.

Several antibiotic therapies are recommended to suppress acute clinical attacks of dysentery and shorten the duration of symptoms. Ciprofloxacin, ampicillin, tetracycline, trimethoprim-sulfamethoxazole, and chloramphenicol are most commonly inhibitory for shigellae. However, antibiotics may fail to eradicate the organisms completely from the intestinal tract. Multiple drug resistance can be transmitted by plasmids, and resistant infections are widespread. Improvements in sanitary and hygienic conditions can help contain secondary spread of shigellosis; the single most effective means of preventing secondary transmission is hand washing (Maurelli and Lampel, 1997). Despite many years of intensive effort, an effective vaccine against shigellosis still has not been developed. 12.5.4

Sources

The only natural hosts of shigellae are humans and monkeys. Most of the disease transmission thus occurs via person-to-person contact; the bacteria are able to survive on the skin. Shigella is also often transmitted by contaminated food and water. When food is involved, the disease is associated with conditions of poor personal hygiene and general sanitation and/or with food that is not subsequently cooked or is held at temperatures that allow the organisms to grow. Fecally contaminated water and unsanitary food handling methods are thus the most common causes of contamination. Foods implicated as vehicles for shigellosis include salads (potato, tuna, shrimp, macaroni, and chicken), raw vegetables, milk and dairy products, and poultry. However, Shigella spp. are not associated with any specific foods. In over 70% of outbreaks in the United States, salads have been implicated as the primary vehicle of transmission. Salads, as a rule, are prepared with ingredients that inevitably are touched to a large degree by food handlers. Many of the ingredients require chopping, causing contamination from the cutting board. In addition, salads require no further heating before being served. Shigellosis in the United States is primarily a foodborne and not a waterborne disease. This is because the water supply of most communities is under sanitary control. Most outbreaks occur in food service operations. Prime months for this infection are the late spring to early fall. 12.5.5

Outbreaks

The most common Shigella species in the developing world are S. flexneri and S. dysenteriae type 1. They accounted for 66% and 16% of hospitalized cases of

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shigellosis in Bangladesh, respectively, in the early 1980s (Stoll et al., 1982). S. flexneri is primarily responsible for the endemic form of the disease, whereas S. dysenteriae accounts for the epidemic form (Sansonetti et al., 2001). S. boydii is rarely encountered and seems essentially associated with cases of shigellosis on the Indian subcontinent. In industrialized countries, Shigella spp. epidemic outbreaks are dominated by S. sonnei. The transition from S. flexneri to S. sonnei is associated with economic development. The exact incidence of shigellosis is difficult to assess. Analysis of the reliable epidemiological data selected on a worldwide basis indicate that there are about 145 million cases every year, 99% of which take place in the developing world. Children under the age of 5 years are the principal victims (Kotloff et al., 1999). Mortality due to shigellosis reaches between 500,000 and 1.5 million every year and is particularly associated with epidemics that develop in a dramatic public health context (e.g., wartimes refugee camps, floods, droughts). During epidemics, which are often due to S. dysenteriae, attack rates have been calculated to range from 1% to 50%, and the mortality rate from 6 to 70 per 1000 people. Examples of selected food-borne shigellosis outbreaks in the United States since the 1960s are shown in Table 12.26. One of the striking features of these foodborne outbreaks by shigellae is that contamination of foods usually did not occur at the processing plant; rather, the source could be traced to a food handler. These incidents often occur by improper food handling, from individuals to small town gatherings and picnics and to largerscale outbreaks such as those on cruise ships and in institutions. 12.5.6

Control

“Food, fingers, feces, and flies” transmit shigellae from person to person. Since humans are the main recognized host of pathogenic shigellae, control efforts must be directed at eliminating the organisms from this reservoir by (a) sanitary control of water, food, and milk; sewage disposal; and fly control; (b) isolation of patients and disinfection of excreta; and (c) detection of subclinical cases and carriers, particularly food handlers. The infectivity of shigellae is quite high, and a small infective dose may be all that is necessary to cause the disease. Mass chemoprophylaxis for limited periods has been tried, but resistant strains of shigellae tend to emerge rapidly. Establishing specific critical control points for preventing Shigella sp. contamination of foods is not always suitable for the HACCP concept (Maurelli and Lampel,

Table 12.26

Selected Food-borne Outbreaks of Shigellosis

Year

Food vehicle

1964 1964 1965 1968 1974 1975 1968 1965 1973 1970 1971 1971 1973 1973 1965 1973 1970 1970 1966 1973 1973 1987 1989 1990 1991 1994 1994 1994

Potato salad Potato salad Potato salad Potato salad Potato salad Potato salad Potato and chicken salad Shrimp salad Shrimp salad Tossed salad Chicken salad Fruit salad Tuna fish salad Fish salad Macaroni salad Chopped turkey Chocolate pudding Poi Milk Rice balls Multiple vehicles Food, water, food handlers German potato salad Contaminated fresh vegetables Homemade moose soup Spring onions Contaminated lettuce Green onions from Mexico

Shigella spp. serotype S. flexneri 4a S. flexneri 2a S. flexneri 4a S. flexneri 4a S. sonnei S. flexneri S. sonnei S. flexneri S. flexneri 2a S. sonnei S. sonnei S. sonnei S. sonnei S. sonnei S. flexneri 2a S. flexneri 2a S. sonnei S. sonnei S. flexneri 2a S. flexneri 2a S. sonnei S. sonnei S. flexneri 2a S. sonnei S. sonnei S. flexneri 2a S. sonnei S. flexneri 6

Cases, no. 100 13 196 60 15 119 3 250 150 350 22 22 137 399 20 172 334 200 99 26 248 >6,000 >200 214 25 >600 110 17

Source: Compiled from Concon (1988) and Maurelli and Lampel (1997).

1997). An infected person such as a food handler with poor personal hygiene usually introduces this pathogen into the food supply. In some cases, this may occur at the manufacturing site, but more likely it happens at a point between the processing plant and the consumer. Another factor is that foods such as vegetables (lettuce is a good example) can be contaminated at the site of collection and shipped directly to market. This perhaps explains why salads have been a main vehicle for many reported outbreaks of shigellosis in the United States. Although HACCP is a method for controlling food safety and preventing foodborne outbreaks, pathogens such as shigellae that are not indigenous to but rather are introduced into foods are most likely to be undetected. Although research since the 1980s has come a long way in elucidating the mechanisms of pathogenesis of

Copyright 2002 by Marcel Dekker. All Rights Reserved.

shigellosis, several questions remain to be answered. According to Sansonetti and coworkers (2001), these include the following: 1. 2.

3.

4.

Understanding the colonic specificity of Shigella spp. Understanding the dramatically high infectiousness of the pathogen (approximately 100 CFU absorbed orally) Deciphering the signaling pathways that, on expression of the invasive phenotype, lead either to entry into epithelial cells or to programmed cell death of macrophages Confirming that translocation through the epithelium occurs essentially via M cells, since inflammation occurring at distant sites may also facilitate entry

5.

6.

Identifying the specificities of the signaling cascades that cause the particularly severe inflammation observed during shigellosis Understanding the bases of immune protection against the disease and developing live oral or subunit parenteral vaccines

2.

3. 12.6 LISTERIA MONOCYTOGENES (LISTERIOSIS) Listeria monocytogenes was presumably first isolated at the beginning of the 20th century as a gram-positive rod in tissue specimens of infected patients. In 1911, the Swedish microbiologist Hulphers (1911) isolated a bacterium from necrotic foci of a rabbit liver. The description of this isolate, which he named Bacillus hepatis, closely resembles the present-day description of L. monocytogenes. In 1926, the species name monocytogenes was given by Murray and colleagues to describe a new bacillus with potent monocytosis-producing activity in rabbits and guinea pigs (Murray et al., 1926). In 1927, in honor of Lord Joseph Lister, who discovered antisepsis, Pirie named Listerella hepatolytica the bacillus responsible for an epizootic among gerbilles (African jumping mouse) in South Africa (Pirie, 1940). After it was learned that B. monocytogenes and L. hepatolytica were the same organism, the name was changed to Listerella monocytogenes. In 1929, Nyfeldt reported the first unambiguous isolations of these bacteria from humans. In 1939, it was discovered that the name Listerella was given to a group of slime molds in 1906. Hence, in 1940, the proposed name change by Pirie (1940) from Listerella monocytogenes to Listeria monocytogenes was accepted. The Judicial Commission on Bacteriological Nomenclature and Taxonomy later approved the name change (Opinion 12, 1954). Although Listeria spp. infections of humans were thus known since 1929, like Yersinia enterocolitica, this microorganism was not considered a major problem until more recently. Three major food-borne outbreaks occurred in the early 1980s: one in the Maritime Provinces of Canada and two in the United States. Since then, listeriosis has emerged as one of the major food-borne diseases with hundreds of cases being reported every year (Rocourt and Brosch, 1992). The emergence of listeriosis appears to be a result of complex interactions among various factors reflecting changes in social patterns. According to Rocourt and Cossart (1997), these factors include the following: 1.

Medical progress and consequent demographic changes, such as the increased proportion of im-

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munocompromised and elderly persons in the general population Changes in primary food production, e.g., largescale production of raw materials, modifications in food processing technology, expansion of the agrifood industry, and development of cold storage systems Changes in food habits and in handling and preparation practices, e.g., increased consumer demand for convenience foods that require essentially little or no cooking before consumption

Listeriosis is often considered an atypical food-borne disease of major public health concern because of its severity and nonenteric nature, a high case-fatality rate (around 20% to 30%), a frequently long incubation time, and the ability of the organism to survive and multiply at cold temperatures. However, if one refers to human listeriosis, the name monocytogenes is somehow misleading, because monocytosis is a rare feature of human infection (Fsihi et al., 2001). The so-called monocytosis-producing agent of L. monocytogenes is a lipid that produces monocytosis in rabbits and rodents but not in humans (Stanley, 1949). In this section, some of the characteristic features of the organism and the disease are described. 12.6.1

Organism

On the basis of DNA-DNA hybridization, MLEE, and 16S ribosomal ribonucleic acid (rRNA) sequencing, the genus Listeria consists of six different species divided into two lines of descent (Figure 12.12). The first group includes L. monocytogenes and the closely related species L. innocua, L. ivanovii (subspecies ivanovii and subspecies londoniensis), L. welshimeri, and L. seeligeri. The second group includes L. grayi; L. murrayi was included in this species in 1997 (Rocourt and Cossart, 1997). The various Listeria species can be distinguished by their hemolytic phenotypes and their abilities to reduce nitrate and ferment specific sugars. Listeria spp. possess the following biochemical traits: catalase +, oxidase –, urease –, and Methyl Red/Voges-Proskauer (VP) test (+/+) (Ryser and Marth, 1991). Among these various species, only L. monocytogenes infects both humans and animals causing meningitis, sepsis, and abortion (Gellin and Broome, 1989; Lorber, 1997). L. ivanovii is restricted to sheep and cattle, in which it causes septicemic disease, neonatal sepsis, and abortion, but no brain infection (Ivanov, 1962). The other species are generally considered nonpathogenic, although L. seeligeri and L. welshimeri have each been reported as the

Figure 12.12

Phenotypic identification of Listeria species. CAMP, Christie, Atkins, Munch-Peterson test.

causative agent of human infections (Rocourt et al., 1986; Andre and Genicot, 1987), and L. innocua has been implicated in a case of ovine meningoencephalitis (Walker et al., 1994). L. monocytogenes, the causative organism of listeriosis, is a facultatively anaerobic, motile (at 20°C–25°C), short gram-positive rod of regular appearance. The optimal temperature of growth is typically about 35°C–37°C, but strains can exhibit wide growth temperature ranges of 1°C to 45°C; in fact, the majority of strains may well grow at about 1°C (Junttila et al., 1988). L. monocytogenes is also capable of growing at low pH and high NaCl concentrations. However, the organism also exhibits a surprising resistance to heat. It has been suggested that the organism survives the minimal pasteurization heat treatment (72°C for 15 seconds) required by many countries for raw milk (Fernandez Garayzabel, 1987). Apparent differences in heat sensitivity have been highlighted, depending on the methodologies employed. Thus, Bradshaw and colleagues (1987) claimed that normal pasteurization treatments were sufficient to destroy L. monocytogenes. Doyle and coworkers (1987) have questioned this conclusion, stressing the need to use suitable enrichment procedures in order to facilitate the recovery of metabolically injured cells. This

Copyright 2002 by Marcel Dekker. All Rights Reserved.

point is illustrated in Table 12.27. In this study, L. monocytogenes was only isolated after 48-hour refrigeration of the milk after pasteurization. All virulent strains of L. monocytogenes are hemolytic, producing β-hemolysis on blood agar plates (horse or sheep). The organism is engulfed by macrophages but breaks out of the phagosome by the action of hemolysin and phospholipase C. It then causes actin molecules to ac-

Table 12.27 Counts of Listeria monocytogenes in Milk Stored at 4°C after Pasteurizationa

Immediately after pasteurization Storage at 4°C

a

Days

Count, per milliliter

0 1 2 3 4 5

0 0 40 150 800 2500

Initial count 108/ml, pasteurized at 72°C for 15 seconds. Source: Fernandez Garayzabel (1987).

crete at one end of the cell. This action pushes the organism into an adjacent cell, where the cycle of engulfment and release continues. At least 16 serovars of L. monocytogenes, based on both flagellar and somatic (carbohydrate-containing) surface antigens, have been recognized (Fsihi et al., 2001). Three serotypes, 4b, 1/2a, and 1/2b, are responsible for about 90% of human infections (Schuchat et al., 1991; Lamont et al., 1988). Serovar 4b strains are responsible for 33% to 50% of sporadic human cases worldwide and for all major food-borne outbreaks since 1981 (Rocourt and Cossart, 1997). In contrast, most isolates recovered from food in numerous countries are of serogroup 1/2. The organism is carried asymptomatically in the intestinal tract at carriage rates of 10%–29% in human population samples. 12.6.2

Pathogenesis

Bacterial virulence or pathogenicity factors include all bacterially produced substances that allow an organism to cause illness or infection. L. monocytogenes must survive the acidity of the stomach, penetrate the intestinal lining, and grow in the host before it can produce the illness. Like other enteroinvasive pathogens, L. monocytogenes possesses a variety of virulence factors that damage the host’s tissue, thus allowing the organism to invade the bloodstream and produce illness. Those virulence factors that contribute to the disease-causing capability of L. monocytogenes include one or more hemolysins, a monocyte-producing agent, various cell wall/cell membrane constituents, toxic oxygen species, and several undefined toxins. With the exception of transmission from mother to fetus and sporadic cases due to direct contact with infected animals, human infection with L. monocytogenes begins with consumption of contaminated food. Since the organism elicits clinical features in infected animals similar to those of human listeriosis, various experimental animal models including mice, rat, rabbits, and guinea pigs have been widely used to understand the pathogenicity of L. monocytogenes. L. monocytogenes must cross the intestinal barrier in animals infected by the oral route. Reports in the literature vary as to the first phase of infection. In some studies, the organism is shown preferentially to target Peyer’s patches, the lymphoid follicles of the gut, as demonstrated by bacteriological and histopathological analyses of orally and intragastrically inoculated mice (MacDonald and Carter, 1980; Marco et al., 1992). However, its preferential site of translocation in the intestine is yet to be proved conclusively. The organisms are then internalized by resident macrophages, in which they can survive and replicate.

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They are subsequently transported via the blood to regional lymph nodes. The listeriae are readily phagocytosed by Kupffer’s cells in the liver and spleen (Rocourt and Cossart, 1997). After an initial phase of bacterial killing that results in the disappearance of the majority of the inoculum, the organism can spread to adjacent hepatocytes, where they may induce apoptosis (Rogers et al., 1996). Infected hepatocytes can trigger recruitment of neutrophils, which eliminate cellular debris as well as most surviving bacteria, while a sterilizing T-cell response readily appears. Depending on the T-cell response induced in the first days after initial infection, further dissemination via the blood to the brain or, in the pregnant animal, the placenta may subsequently occur. Thus, L. monocytogenes infection is not localized at the site of entry but involves entry and multiplication in a wide variety of cell types and tissues. Tissue culture assays of bacterial invasion reveal that L. monocytogenes is capable of penetrating hepatocytes (Dramsi et al., 1995; Gregory et al., 1997), fibroblast, epithelial (Ireton et al., 1996), and endothelial cells (Parida et al., 1998). Thus, it is one of the most invasive bacterial human pathogens known. It has presumably evolved specific strategies that allow entry into different cell types but retain some tropism for particular organs. The various determinants of virulence as well as their known corresponding eukaryotic receptors involved in the listerial infectious process are listed in Table 12.28. Some of these mechanisms are briefly described in the following. Detailed reviews on the biochemical properties, gene expression, and functions of these proteins have been published (Rocourt and Cossart, 1997; Fsihi et al., 2001). Entry and Spread into Mammalian Cells As mentioned, L. monocytogenes has the potential to infect a variety of eukaryotic cells including phagocytic and nonphagocytic cells, both in vitro and in vivo. This process involves several different steps, and the virulence factors involved in each of these steps have been identified. The first step in the infection is prevented by the addition of cytochalasin D, a drug that inhibits actin polymerization and, hence, the active participation of the mammalian cell. The organism can induce phagocytosis in nonphagocytic cells (Ireton and Cossart, 1997). The surface proteins internalin A (InlA) and InlB, encoded by inlA and inlB genes belonging to a multigene family, are required for the entry of the organism into epithelial cells. Both these proteins are members of the superfamily of leucine-rich repeats(LRR-) containing proteins known to be involved in protein-protein interactions (Kobe and Deisenhofer, 1995; Kajava, 1998). LRR are sequence motifs (20 to 29 resi-

Table 12.28

Some Virulence Determinants in Listeria monocytogenes

Determinant

Properties

Function

LLO

58-kDa, Thiol-activated, cholesterol-requiring poreforming cytolysin; pH-dependent hemolytic activity

InlA

84-kDa Leucine-rich protein; E-cadherin eukaryotic receptor, covalently linked to peptidoglycan via LPXTG motif 67-kDa Leucine-rich protein, surface exposed via 3 GW modules

InlB

ActA PI-PLC

PC-PLC

67-kDa Protein, polar localization; anchored to bacterial surface via hydrophobic C-terminal region Phosphatidylinositol-specific phospholipase C; 33-kDa basic protein; active at pH 5.5–7.5

PrfA

PC-preferring phospholipase C; 29-kDa protein; active at pH 5.0–8.0; activated on cleavage by zinc-dependent metalloprotease Mp1 27-kDa DNA-binding protein (CAP/FNR family)

ClpC InlC

27-kDa Stress protein (Clp/ATPase family) 30-kDa Leucine-rich protein

Aids organism escape from phagosome; induces phosphorylation of MAP kinases; induces PtdIns hydrolysis in HUVEC cells Facilitates organism entry into cultured epithelial cells (Caco-2); no in vivo role identified Entry into cultured HepG2, HeLa, Vero, Hep-2, HUVEC, and fibroblast cells; role in multiplication in hepatocytes in vivo; stimulation of PI3-kinase activity Actin polymerization in infected cells Escape from primary vacuole in murine macrophages; escape from double-membraned vacuole; amplification of LLO-related PtdIns hydrolysis Escape from primary vacuole in human epithelial Henle 407 cells, escape from double-membraned vacuole and cell-to-cell spread Transcriptional activator of most Listeria spp. virulence genes Intracellular growth in macrophage No defined role; reduced virulence in mice of inlC mutants

a

HUVEC, human umbilical vein endothelial cell; MAP, mitogen-activated protein; PtdIns, phosphoinositides; PI3-kinase, phosphoinositide 3-kinase, LLO, listeriolysin O; InlA, internalin; GW, glycyl-tryptophan; PC, phosphtidylcholine; DNA, deoxyribonucleic acid; CAP/FNR, catabolite activator protein/fumarate and nitrate reduction transcriptional regulator; ATPase, adenosine triphosphatase Source: Compiled from Rocourt and Cossart (1997) and Fsihi et al. (2001).

dues) with a defined periodicity of leucine residues that have now been found in over 60 proteins. The LRR regions of InlA and InlB are essential and sufficient for the entry process, suggesting that in both cases these repeats interact with the corresponding InlA and InlB receptors. Analysis of spontaneous rough mutants has led to the identification of a second protein, p60, associated with invasion. Its gene was named iap, for invasion-associated protein (Kohler et al., 1990). The inlA gene encodes an 800-amino-acid protein with a signal sequence that has two repeat regions and a Cterminal cell wall–associating motif highly conserved in a number of gram-positive bacterial membrane-anchored proteins. This motif consists of an LPTTG pentapeptide followed by a stretch of 20 hydrophobic amino acids and a short tail of charged residues (Lebrun et al., 1996). This motif is required for covalent anchoring of these proteins in the bacterial cell wall. When this region is deleted or absent in natural isolates, the InlA protein is completely released and the invasion capacity of the strain severely impaired. The inlA gene also confers invasiveness to the noninvasive species L. innocua (Rocourt and Cossart, 1997); hence, its product was named internalin. The release

Copyright 2002 by Marcel Dekker. All Rights Reserved.

of internalin begins during the exponential phase of growth, when the cell wall–associated form is most abundant. The InlB protein, which is 630 amino acids long, contains a signal sequence and a region of leucine-rich repeats analogous to those of internalin (same length of 22 amino acids and with the same consensus sequence). However, it lacks a membrane anchor. Its 232 C-terminal amino acids are required for surface association (Braun et al., 1997). This region contains tandem repeats of 80 amino acids that begin with the dipeptide GW (glycyltryptophan) and, therefore, are often referred to as the GW molecules. Whereas internalin can be easily detected at the surface of L. monocytogenes, InlB is not detected by immunofluorescence, indicating that it is partially embedded in the cell wall. The loose association of InlB with the bacterial surface suggests that this soluble form of the protein may play a role during infection. E-cadherin, a key adhesion molecule in epithelial cells, has been identified as the receptor for InlA in Caco-2 cells (Mengaud et al., 1996). It is a transmembrane glycoprotein whose intracellular domain interacts via catenins with the active cytoskeleton. The receptor is involved in Ca2+-dependent homophilic interactions that mediate cell-

cell adhesion and maintain tissue integrity in differentiated epithelia. The protein gC1qR, the receptor for the globular part of C1Q, was identified in 2000 as the corresponding receptor for InlB protein (Braun et al., 2000). The bacterial surface associations of InlA and InlB proteins of L. monocytogenes are schematically shown in Figure 12.13. The p60 protein encoded by the iap gene consists of 484 amino acids with a signal sequence but no membrane anchor (Kohler et al., 1990). The C-terminus of the protein contains a peculiar sequence of 19 Thr-Asn pairs. In cultures of wild-type L. monocytogenes, p60 is present both on the cell surface and in the culture supernatant; however, it is present only on the surface of the rough mutant. This protein has bacteriolytic activity and may be a murein hydrolase involved in septum formation (Wuenscher et al., 1993). The exact role of this protein in invasion is unclear, although it is known that it primarily affects entry into fibroblasts. The entry of mammalian cells by L. monocytogenes induces phosphorylation of a 37-kDa host cell protein,

thus exploiting mammalian signal transduction pathways to promote its uptake. Tyrosine kinase inhibitors prevent L. monocytogenes invasion of mammalian cells (Tang et al., 1994; Velge et al., 1994). Similarly, inhibitors of the actin polymerization process, such as cytochalasin D, and genistein, which competes with the binding of ATP to protein tyrosine kinases and thereby inhibits the activity of these enzymes, inhibit the entry but not the adherence of L. monocytogenes in the epithelial cells. Thus, tyrosine phosphorylation of host proteins and an intact actin cytoskeleton are needed for listeria invasion. By using a combination of pathway-specific inhibitors, genetically inactivated cell lines, and bacterial mutants, identification of the mammalian pathways activated during infection as well as the listerial factors triggering these events are progressively emerging. Phosphatidylinositol 3-kinase (PI 3-K) is required for Listeria spp. entry into mammalian cells, and its activity increases during the infection process (Ireton et al., 1996). Activation of PI 3-K in mammalian cells requires binding of its regulatory subunit p85 to tyrosine phospho-

Figure 12.13 Bacterial surface associations of the ActA, InlA, and InlB proteins of Listeria monocytogenes. Independently of their final localization, all these proteins are synthesized initially as precursors with NH2-terminal leader peptides. A, After removal of the leader peptide by a membrane-associated signal peptidase, the protein is translocated across the cytoplasmic membrane. B, A hydrophobic domain targets sorting of ActA to the membrane. InlA possesses in its C-terminal part the conventional LPXTG motif for anchoring gram-positive bacteria in the cell wall. C, D, A “sortase” enzyme is probably involved in the proteolytic cleavage. E, The GW motifs present in the carboxy-terminal part of the InlB proteins are required for the cell wall association of InlB with specific components of the cell wall of gram-positive organisms. GW, glycyl-tryptophan (From Fsihi et al. [2001].)

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rylated proteins (Fsihi et al., 2001). Three adaptor proteins in L. monocytogenes, viz., Gab1, Shc and c-Cbl, are tyrosine phosphorylated during infection in Vero cells (Ireton et al., 1999); however, which tyrosine kinase(s) is (are) involved in this pathway remains to be elucidated. Other signaling pathways involved in the infection process include tyrosine phosphorylation of the four isoforms (extracellular signal regulated kinases [ERK-1 and ERK-2], cJun N-terminal protein kinase [JNK], and p38) of the mitogen-activated protein (MAP) kinase (Tang et al., 1998). Listeriolysin (LLO, discussed later) has been identified as a bacterial factor that induces ERK-1 and ERK-2 phosphorylation in these epithelial cells (Tang et al., 1996; Weiglein et al., 1997). The MAP kinase kinases MEK-1 and MEK-2 are the upstream activators that tyrosine-phosphorylate ERK-1 and ERK-2 in the MAP kinase pathway. Overall, it appears that Listeria spp. invasion requires the MEK-1/ERK-2 signaling pathway. During the infection process, L. monocytogenes triggers both release of lipid inflammatory mediators such as platelet-activating factor (PAF) and prostaglandin I2 (PGI2) and hydrolysis of phophoinositides (PtdIns) (Sibelius et al., 1996a). Although LLO is identified as the bacterial inducer for the PAF-PGI2 response, another released bacterial factor is required for full PtdIns response. This factor is found to be phosphatidylinositol- (PI-) phospholipase C (PI-PLC). It acts as a key cooperative agent in LLO-induced PtdIn metabolism during the signaling process (Sibelius et al., 1996b). Various interactions with the host signaling pathways that occur during Listeria spp. infection are shown in Figure 12.14. Escape from the Primary Vacuole Once Listeria spp. are internalized in membrane-bound vacuoles (or phagosomes) that have an acidic pH of 5.6, they have to escape rapidly from this hostile compartment before its fusion with lysosomes for intracellular survival and growth. Lysis of this primary vacuole is largely mediated by the pore-forming ability of listeriolysin O (LLO), although in some cell lines, the process also involves PIPLC or broad-substrate phospholipase C (PC-PLC). The lysis of the primary vacuole usually occurs within less than 30 minutes. Once released from the vacuole, the listeriae can grow and replicate in the cytosol with a doubling time of about 1 hour (Fsihi et al., 2001). Listeriolysin O LLO belongs to the class of thiol-activated toxins, which include streptolysin O of Streptococcus pyogenes, pneumolysin of Streptococcus pneumoniae, and perfrin-

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golysin of Clostridium perfringens (Tweten, 1995). These toxins are active only on cholesterol-containing membranes, where cholesterol likely acts as the receptor in the membrane. The mode of action of these structurally related toxins involves interaction with cholesterol in target cell membranes in which they oligomerize to form transmembrane pores (cholesterol sequestration), leading to cell lysis. The activity of these pore-forming toxins is usually determined by their lytic activity on erythrocytes; hence, they are often called hemolysins. LLO has been purified from culture supernatants. It is a 58-kDa protein encoded by the hly gene located in the virulence gene cluster of L. monocytogenes. LLO and ivanolysin (the homolog of LLO in L. ivanovii) are the only members of the thiol-activated toxins produced by intracellular bacteria (Fsihi et al., 2001). It has maximal activity at pH 5.5, the pH encountered in the phagosome. At neutral pH of the cytosol, it is less active; therefore, the deleterious effects of LLO on cellular membranes when the organism is free in the cytosol are prevented. LLO has a critical role in mediating the escape of L. monocytogenes from the phagosome. LLO-negative mutants are defective in their ability to breach the phagosomal membrane and to multiply in the cytoplasm of the host cell (Gaillard et al., 1986; Portnoy et al., 1988). As mentioned earlier, LLO stimulates MAP kinase pathways and PtdIns metabolism. Phospholipases L. monocytogenes secretes two phospholipases of the C type (PLC): a phosphatidylinositol- (PI-) specific PLC (PI-PLC) and a phophatidylcholine- (PC-) preferring PLC (PC-PLC). PLCs are virulence factors in several bacterial pathogens, such as B. cereus, S. aureus, C. perfringens, and Pseudomonas aeruginosa. These enzymes cleave phospholipids between the glycerol moiety and the phosphate group and generally lead to alterations in cell membrane composition and function (Titball, 1993; Songer, 1997). The two PLCs of L. monocytogenes have overlapping roles in escape from the primary vacuole and cell-tocell spread (Smith et al., 1995; Goldfine et al., 1998). PI-PLC is a 33-kDa extracellular protein encoded by the plcA gene (Mengaud et al., 1991). It is more basic than its structurally related counterparts from B. cereus and B. thuringiensis. It specifically cleaves PI and/or glycosyl-phosphatidylinositol (GPI) anchors found in many eukaryotic membrane proteins. Like LLO, it is active at acidic pH. PC-PLC, although active on a broad range of phospholipids, shows a higher specificity for phosphatidylcholine (lecithin) and is sometimes also referred to as lecithinase. Encoded by gene plcB, PC-PLC is secreted as an inactive precursor of 33 kDa that is processed, in broth

Figure 12.14 Interaction of invasive Listeria monocytogenes with host signaling pathways. The various host responses triggered on L. monocytogenes infection have been reported in different cell lines and, unless experimentally proved, should not be extrapolated to all cells. PGI2, prostaglandin I2; PAF, platelet-activating factor; MEK, MAP kinase kinase; MAPK, mitogen-activated protein (MAP) kinase. (From Fsihi et al. [2001].)

culture, to an active mature form (29 kDa) by a zinc-dependent metalloprotease (Mp1) (Poyart et al., 1993). Intra- and Intercellular Spreading Once escaped from the phagosomes, L. monocytogenes must efficiently invade its preferred tissues by direct cellto-cell spread. This process is the result of intracellular movement, protrusion formation, phagocytosis of the protrusion by a neighboring cell, formation of a double-membrane-bound vacuole, and lysis of the vacuole. These steps are now described. The role of actin filaments in mediating the intracellular movement of L. monocytogenes and its cell-to-cell spread was described in 1990 (Mounier et al., 1990; Dabiri et al., 1990). The actA gene has been identified as the primary and probably the only bacterial factor required for actin-based motility (Kocks et al., 1992). The gene encodes a 610-amino-acid surface protein (ActA) anchored in the bacterial cytoplasmic membrane by its hydrophobic C-terminal region. The N-terminus of ActA is absolutely essential for the bacterium to induce actin assembly on the surface. ActA is a major virulence determinant since ActA

Copyright 2002 by Marcel Dekker. All Rights Reserved.

mutants are three orders of magnitude less virulent than wild-type organisms in a mouse model of infection (Brundage et al., 1993). Although these mutants were invasive, escaped from the phagosome, replicated, and formed intracellular microcolonies, they were not covered with actin, did not move intracellularly, and did not spread from cell to cell. Actin is an abundant globular 43-kDa nucleotidebinding (ATP or ADP) protein (G-actin) found in all eukaryotic cells. Its intracellular concentrations in nonmuscle cells range from 10 to 200 µM. The actin monomer can spontaneously polymerize under certain conditions, forming filaments of F-actin (Carlier and Pantaloni, 1997). Polymerization in vitro starts with the formation of thermodynamically unstable dimers or trimers (nucleation), which can then rapidly grow into filaments by monomer addition. The nucleation step is rate-limiting during actin polymerization (Fsihi et al., 2001). Numerous actin-binding proteins control actin polymerization in living cells (Table 12.29). The function of some of these proteins is shown in Figure 12.15. Fsihi and coworkers (2001) have published a detailed description and biochemical properties of these proteins.

Table 12.29

Cytoskeletal Components Associated with Actin Polymerizing Intracellular Listeria Speciesa

Protein

Localization

Function within cell

Interaction with ActA

α-Actinin Tropomyosin Fimbrin Vinculin Talin Villin Ezrin Profilin

Cytoplasmic tails Tail Tail Tail Tail Tail Tails in protrusions Bacterial pole

– – – – – – – –

VASP Mena Arp2/3 complex Cofilin Gelsolin Capping protein Rac Coronin

Bacterial pole Bacterial pole Tail Tail Bacterial pole Tails ? Tails

F-actin cross-linking F-actin pointed end binding F-actin bundling Component of focal adhesions Actin nucleation Barbed end capping Cortical actin organization G-actin binding; nucleotide exchange; actin polymerization Component of focal adhesions Component of focal adhesions Actin nucleation F-actin depolymerizaation Barbed end capping; F-actin severing Barbed end capping Small GTPase Actin organization during phagocytosis

a

+ + Likely – ? – ? –

VASP, vasodilator stimulated phosphoprotien; GTPase, guanosine triphosphatase.

Figure 12.15 Different steps in the actin polymerization-depolymerization cycle. Actin monomers are shown as chevrons, and different control points for the cycle are indicated by thick arrow. T, adenosine triphosphate actin; D, adenosine diphosphate actin. (From Fsihi et al. [2001].)

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Early electron microscopy studies of thin sections of Listeria spp.–infected macrophages showed that free bacteria in the cytosol are surrounded with a coat of filamentous actin. This actin cloud is then rearranged into the comet-shaped actin tail of up to 40 µM in length (Tilney and Portnoy, 1989; Mounier et al. 1990). The actin comet tail is stationary in the cytosol and left behind by moving bacteria. The length of the tail is proportional to the speed of movement; faster-moving bacteria have longer tails. Their speed ranges from 0.1 to 1 µM/second (Rocourt and Cossart, 1997). When the organism reaches the plasma membrane, it puts out a long protrusion. A neighboring cell, giving rise to a two-membrane-bound vacuole, then internalizes these protrusions. After lysis of this vacuole, a new cycle of replication, movement, and spreading of the bacteria begins. The entire cycle is completed in about 5 hours. If cytochalasin D is added after entry, listeriae do not spread within the cytosol; they replicate and form microcolonies in the vicinity of the nucleus. Hence, actin polymerization is es-

sential to intracellular movement and cell-to-cell spread of the organism. A model summarizing the actin-based motility of listeriae is depicted in Figure 12.16. The overall intracellular life cycle of L. monocytogenes is shown in Figure 12.17. The strategy of direct cell-to-cell spread allows the organism to evade the extracellular humoral immune system (e.g., antibodies, complement, polymorphonuclear cells) of the host or other bactericidal components. This may explain why antibodies play no role in recovery from infection or protection against secondary infection (Portnoy et al., 1992). The genes encoding the virulence factors, viz., internalin, LLO, PI-PLC, PC-PLC, and ActA, involved in the different steps of the infectious process are clustered in two regions of the chromosome (Michel and Cossart, 1992). Their expression is modulated by the same environmental conditions: repressed at low temperatures and maximally expressed at 37°C. Other factors influencing their expression include the growth phase of the organism and

Figure 12.16 Model of the molecular mechanism underlying actin polymerization by Listeria monocytogenes. Host cell proteins implicated in the process are indicated. VASP, vasodilator stimulated phosphoprotein. (From Fsihi et al. [2001].)

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Figure 12.17 Intracellular life cycle of Listeria monocytogenes. Successive steps are indicated. The striped material around the bacteria and in the tails represents F-actin.

the culture medium. Many of these effects are coregulated by the prfA gene encoding a pleiotropic regulatory protein, PrfA. 12.6.3

Symptoms, Diagnosis, and Treatment

Human listeriosis remains a rare disease compared to other reportable food-borne illnesses. Most cases of human listeriosis are sporadic, and although the source and route of infection in most cases often remain unidentified, foodborne transmission appears to be fairly common. The clinical features of listeriosis vary widely and often are confused with those of other illnesses. The infective dose of L. monocytogenes depends on many factors, including the immunological status of the host and the strain (Rocourt and Cossart, 1997). Epidemiological data of food-borne outbreaks of listeriosis suggest an infective dose of more than 100 CFU/g of food. L. monocytogenes is a low-grade pathogen; the ingestion of low numbers of viable cells causes no clinical manifestations in healthy adults. Certain groups of humans

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are more susceptible to the organism. These include infants, the elderly, persons immunocompromised by drugs (e.g., corticosteroids, cytotoxic drugs) or disease (hematological malignancies such as leukemia, lymphoma, and myeloma as well as solid malignancies), and pregnant women. Listeriosis occurs 300 times more frequently in people who have acquired immunodeficiency syndrome (AIDS) than in the general population (Jurado et al., 1993). Cases of human listeriosis cannot always be classified into one of the nine categories listed in Table 12.30. Combinations of two or more manifestations may occur simultaneously or in succession. The onset of these disorders is usually preceded by gastrointestinal symptoms such as nausea, vomiting, and diarrhea. The onset time to gastrointestinal symptoms is unknown but is probably greater than 12 hours. That for more serious forms of listeriosis may range from a few days to up to 3 weeks. Human listeriosis is generally characterized by meningoencephalitis, bacteremia, and, rarely, diffuse or focal encephalitis (brain abscesses). Compared to meningitis

Table 12.30

Manifestations of Listeriosis in Humans

Listeriosis during pregnancy Listeriosis of the newborn (granulomatosis infantiseptica) Meningitis, meningioencephalitis, and encephalitis Cutaneous form Septicemia with pharyngitis and mononucleosis Oculoglandular form Cervicoglandular form Granulomatosis septica and typhoid-pneumonic form Other forms Source: From Ryser and Marth (1991).

caused by Streptococcus pneumoniae, Neisseria meningitides, and Haemophilus influenzae, Listeria spp.–associated meningitis has one of the highest mortality rates (Mylonakis et al., 1998). Listeric encephalitis exhibits two phases. The first one lasts about 10 days and is characterized by headache, backache, vomiting, conjunctivitis, and rhinitis. The second or acute phase begins about 10 days later with a high fever, which is followed by disturbances of the central nervous system. Visible lesions are found almost exclusively in the pons and medulla oblongata of the brain; however, lesions in the ganglions of the brain stem, cerebellum, and spinal cord are relatively rare. Death normally ensues within 2–3 days if the victim is not appropriately treated with antibiotics. Listeriosis is of special concern in pregnant women; infections, although not severe, produce “flulike” symptoms often associated with myalgias, arthralgias, headache, and backache and may cause premature labor, stillbirth, or early death of the newly born infant. Pregnant women are predisposed to development of listerial bacteremia especially during the third trimester of gestation when the cell-mediated immunity is thought to be impaired (Weinberg, 1984). Although usually not severe for the mother, listeriosis during pregnancy can be devastating for the infant. Deficiencies in local immunoregulation at the placental level probably contribute to perinatal infections (Redline and Lu, 1987). L. monocytogenes is one of the few bacteria that can cross the transplacental barrier to infect the fetus, thus resulting in premature labor, fetal death, or severe neonatal sepsis. Neonatal infection acquired in utero (early-onset listeriosis) may lead to the fatal syndrome of granulomatosis infantisepticum, characterized by disseminated abscesses and granulomas in the liver, spleen, lungs, kidneys, brain, and skin (Spencer, 1987). Infection acquired during delivery can also lead to meningitis in newborns (late-onset neonatal listeriosis).

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The overall case fatality rate for systemic or invasive listeriosis is usually about 20% to 30% for both epidemic and sporadic cases. Mortality rate is usually higher (38% to 40%) among immunocompromised people, elderly patients, and those suffering from central nervous system infections. Up to 11% of neonates and 30% of adult survivors of central nervous system infections suffer from residual symptoms (Bula et al., 1995). The severity of illness, the incidence of residual symptoms, and the high case fatality rate make listeriosis a costly food-borne disease. Listeriosis can only be positively diagnosed by culturing of the organism from blood, cerebrospinal fluid, or stool, although the latter is difficult and of limited value. Although some individuals may recover spontaneously from listeric infections, early antibiotic therapy is usually required to prevent permanent disabilities and possible death. Many antimicrobial drugs inhibit listeriae in vitro. These include penicillin, ampicillin, tetracycline, erythromycin, chloramphenicol, and cephalothin. These antimicrobials are often used in combinations. 12.6.4

Source and Modes of Transmission

Listeria spp. are widely distributed in the environment because of their ability to survive for long periods in many different environments and to tolerate cold temperatures. L. monocytogenes can be isolated from soil, silage, water, vegetation, a wide range of foods, and the feces of many animals, including humans. Many healthy humans are carriers, as are healthy wild and domestic animals. Sheep, goats, and cows are the most common sources of human infection (Donnelly et al., 1987). Food vehicles of L. monocytogenes include leafy vegetables fertilized with manure containing the organism, chocolate milk, raw milk, raw fish, raw meat and chicken, soft cheeses, and fermented sausage. Food can become contaminated at any step of the food chain, and cold storage does not inhibit the growth of listeriae. The fact that the organism survives pasteurization heat treatments, repeated freezing and thawing, direct sunlight, and longwave ultraviolet (UV) light indicates that it is not easily destroyed by common processing techniques (Doyle, 1988). It also survives in shredded vegetables, even when chlorine-treated and stored under modified atmosphere (Beuchat et al., 1990). This is quite remarkable for a bacterium that does not form spores. Incidence of listeria contamination of various foods is summarized in Table 12.31. L. monocytogenes has been shown to survive the manufacturing process for dry and creamed cottage cheese. Soft cheeses have a pH that allows the growth of this organism to large populations. During cheese making,

Table 12.31 Incidence of Listeria monocytogenes Contamination of Various Foods Food

Contaminated samples, %

Soft cheese Pate Vegetables Potatoes, fresh Radishes, fresh Cook-chill meals Raw fish Red meat Poultry Sausages Minced beef

4 5 6 26 33 17 25 75 82 87 95

Source: Compiled from Snyder and Poland (1990) and MacGowan et al. (1994).

the organism becomes concentrated in the curd and relatively few cells remain in the whey. Although the pH eventually drops from 6.8 to 4.8 or less, the warm milk is a suitable medium for growth of L. monocytogenes during the first several hours of curd formation. In some soft cheeses, the organism may survive or even multiply during the various stages of ripening, whereas most cells are

Figure 12.18

killed in cottage cheese making because, after the curd is cut, it is heated to 55°C over 90 minutes before the whey is drained. With a frequency of about 2% to 10%, populations of L. monocytogenes ranging from 10 to 107 CFU/g thus are of greatest concern. The organism was found in cheddar cheese made from contaminated raw milk even after the required 60 days of aging. Both the chocolate and the sugar in chocolate milk aid the growth of L. monocytogenes (Rosenow and Marth, 1987). Although the origin and mode of transmission of listeric infection are quite complex, some of the routes of infection are shown in Figure 12.18. 12.6.5

Outbreaks

L. monocytogenes was not considered a major problem until 1981, when the first food-borne transmission of listeriosis was demonstrated during an outbreak in Canada with the simultaneous use of a case-control study and strain typing. Since that time, numerous epidemiological investigations have established contaminated food as the primary vehicle of transmission of listeriosis. Over 30 major outbreaks of listeriosis since the 1980s are attributed to this source alone; dairy products and vegetables account for the lion’s share.

Routes for transmission of Listeria monocytogenes to humans. (From Ryser and Marth [1991].)

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The first confirmed food-borne outbreak of listeriosis occurred in 1981 in the Maritime Province of Nova Scotia in Canada. The outbreak involved 41 patients and was traced to locally prepared contaminated coleslaw. The cabbage used was grown in fields fertilized with manure from a flock of sheep subsequently found to carry listeriosis. Before use, the cabbage was held in cold storage, which allowed the microorganisms to proliferate (Schelch et al., 1983). Several in the United States followed the Canadian outbreak of listeriosis. The first was in Boston, Massachusetts, in 1983 over a period of 2 months, in which 49 patients were hospitalized with septicemia or meningitis. Of these cases, 42 (86%) occurred in adults and 7 (14%) in mother-infant pairs (Table 12.32). Fourteen of 49 individuals died: a mortality rate of 29%. A case-control study implicated a specific brand of pasteurized milk as the vehicle. The organism was found in a bulk tank of milk from one of the supplying farms. Since the organism is fairly heatresistant, if there was a very large initial population of organisms, a few could have survived pasteurization and subsequently proliferated (Fleming et al., 1985). Forty of 49 isolates were available for serotyping, and 32 (80%) of them were of serotype 4b, later defined as the epidemic strain. Two years later, Todd (1988) calculated the total cost of this outbreak at $1.89 million ($1.37 million, deaths; $387,000, hospitalization; $70,000, investigation;

Table 12.32 Characteristics of Adult and Perinatal Listeriosis Cases Identified in Massachusetts between June 30 and August 30, 1983 Number of cases (%) Case profile

Adult

Perinatal

Total cases Total fatalities Sex, M/F ratio Clinical syndrome Meningitis Septicemia Death in utero Underlying condition Cancer Cirrhosis/alcoholism Diabetes Corticosteroid therapy Renal transplant Myelofibrosis Chronic hepatitis Intravenous drug abuse

42(86) 12(29) 27/15

7(14) 2(29) 2/5

13(31) 29(62) —

3(42) 2(29) 2(29)

20(48) 7(17) 5(12) 4(9) 2(5) 2(5) 1(2) 1(2)

— — — — — — — —

Source: From Fleming et al. (1985).

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$61,000, financial/legal costs), or $38,614 per case, excluding legal settlements. A second major outbreak of listeriosis occurred in Los Angeles and the surrounding Orange County in June 1985. In this outbreak, the consumption of Jalisco brand Mexican-style cheese was linked to a massive listeriosis outbreak, which later proved to be among the deadliest of all known outbreaks of food-borne disease recorded in the United States and prompted a flurry of research activity in North America and Western Europe. In 1988, Linnan and 14 other members of the investigative team (Linnan et al., 1988) published their findings concerning 142 cases of listeriosis that were linked to consumption of Jalisco brand cheese in Los Angeles County between January 1 and August 15, 1985. An additional 160 cases occurred elsewhere in California (Orange, San Diego, and Fresno Counties) and in other states (Colorado, Texas, Oregon, and Connecticut). Half of these cases were concentrated in the Hispanic communities. During the 7.5month epidemic period, 93 reported listeriosis cases (65.5%) involved pregnant women or their offspring and 49 (34.5%) involved nonpregnant adults (Table 12.33). Forty-eight of the 142 victims died, an overall mortality rate of 33.8%. Thirty deaths occurred among the 87 early fetal/neonatal cases; however, no late fetal/neonatal or maternal deaths were reported. The calculated annual crude incidence rate of ~12 cases/million population is still approximately twice the national average (Mascola et al., 1989). After identification of the epidemic L. monocytogenes strain as belonging to serotype 4b, all 105 clinical isolates available for study were phage-typed and compared to the strain isolated from Jalisco brand Mexican-style cheese. Results showed that 86 of 105 (82%) clinical isolates were serotype 4b; the remaining 19 non–serotype 4b isolates originated from victims whose illnesses were presumably not related to consumption of the contaminated cheese. The organism could have originated in infected raw milk or postprocessing contamination of the cheese, as the organism was found in the plant. A number of other listeriosis outbreaks have occurred since this incident in the United States as well as in Europe (Table 12.34). In the majority of the outbreaks, meat and dairy products were implicated as vehicles of transmission. Epidemiological data gathered from the sources of these outbreaks suggest that some foods are more hazardous than others. The high-risk category includes ready-to-eat foods stored at refrigeration temperatures for a long period, thereby enabling listeriae to grow, and foods contaminated with a high population of L. monocytogenes (>100 CFU/g or ml). This observation was confirmed by an analysis of sporadic listeriosis cases

Table 12.33 Clinical and Demographic Data on 142 Listeriosis Cases Occurring in Los Angeles Country, California, Between January 1 and August 15, 1985 Fetal or neonatal Variable No. of patients Mean age Race or ethnic group: number (%) Hispanic White Black Asian Fatalities (%) Epidemic phage type (%) Mean birth weight (kg) Septicemia (%) Meningitis (%) Septicemia + meningitis (%) Other positive culture (%)

Early

Late

87 32 weeks gestation

6 38 weeks gestation

— — — — 30 (34) — 2.54 88 2 6 4

Maternal

— — — — 0 — 3.15 17 67 17 0

Nonpregnant adults

93 26 yr

49 58 yr

81 (87) 10 (11) 0 2 (2) 0 75 — 52 0 0 48

14 (29) 26 (53) 7 (14) 2 (4) 18 (37) 27 — 71 14 14 2

Source: From Linnan et al. (1988).

Table 12.34 Year 1949–57 1956 1978–79 1979 1980 1981 1981 1983 1983–87 1985 1986 1986–87 1986–87 1987 1987 1989 1989 1989–90 1992 1993 1995 a

Selected Food-Borne Outbreaks of Human Listeriosis Location

Cases, no.

Vehicle

Halle, East Germany Soviet Union Western Australia Massachusetts, U.S. Auckland, New Zealand East Cambria, UK Nova Scotia, Canada Massachusetts, U.S. Vaud, Switzerland Los Angeles, Calif., U.S. Linz, Austria Los Angeles, Calif., U.S. U.S. Los Angeles, Calif., U.S. Philadelphia, Pa., U.S. Spokane, Wash., U.S. San Francisco, Calif., U.S. UK France France France

~100 19 12 20 22 11 41 49 122 142b 20 33 Unknown 11 Unknown 1 1 300 279 39 33

Raw milk, sour milk, cream, cottage cheese Pork Raw vegetables Raw vegetables, milk Shellfish, raw fish Cream Coleslawa Pasteurized milk Vacherin Mont d’Or cheesea Mexican-style cheesea Raw milk, vegetables Raw eggs Uncooked hot dogs Butter Salami Cooked ground beef Cooked Cajun pork sausage Contaminated pate Contaminated pork tongue in aspic Potted pork (“rillettes”) Soft cheese

Vehicle of contamination positively identified. Estimated number of cases as high as 300.

b

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(Pinner et al., 1992; Schuchat et al., 1992). In many of these outbreaks, recalling the implicated food, advising the general population through the mass media to avoid eating contaminated product, and taking appropriate action to prevent L. monocytogenes contamination at product processing and handling facilities terminated the outbreaks. Since the early 1990s, the food processing industry has made tremendous progress in reducing the prevalence of L. monocytogenes in processing plant environments and in high-risk foods. Preventive measures, especially the HACCP approach, have been developed and implemented in the food processing plants. These efforts are proving beneficial since there appears to have been large reduction in the incidence of illness (44%) and death (48%) between 1989 and 1993 (Tappero et al., 1995). 12.6.6

Control

Available evidence indicates that L. monocytogenes can multiply at pH above 5.5 and survive for long periods in refrigerated foods, as well as multiply in them. Some organisms have been reported to survive temperatures used in high temperature-short time (HTST) pasteurization. Furthermore, the organism has the ability to repair the effects of metabolic injury at low temperatures. Control measures, therefore, should be able to surmount these problems associated with L. monocytogenes. The following guidelines in this regard may be useful: 1.

2. 3. 4.

5.

Ensure that milk is adequately pasteurized and there is no postpasteurization contamination. Do not consume after manufacturer’s expiration date. Use only L. monocytogenes–free milk in the manufacturing of soft cheeses. Thoroughly wash vegetables consumed raw before storing in a refrigerator. Avoid undercooking of any food of animal origin, especially poultry, which should be heated to a minimum internal temperature of 72°C. Where chill storage of preheated foods, such as poultry, is required: a. Ensure higher internal temperatures (minimum 75°C) are reached, b. Limit the shelf life of all products, heated or unheated, where growth of L. monocytogenes is feasible to a maximum of 5 days, particularly for ground beef or sausage during refrigerated storage. c. Ensure storage temperatures are maintained at a maximum of 4°C for such products.

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12.7 STREPTOCOCCAL INFECTIONS The streptococci are a heterogeneous group of bacteria responsible for a wide variety of diseases that range from caries in human teeth to meningitis in fish. They are widely distributed in nature. Some are members of the normal human flora; others are associated with important human diseases attributable in part to infection by streptococci, in part to sensitization to them. However, despite their heterogeneity, they have numerous characteristics in common and many similarities in pathogenic processes. The pathogenic properties include stimulation of an intense inflammatory response by activating the complement cascade, production of toxins and tissue-damaging enzymes, and initiation of immune-mediated diseases such as rheumatic fever or glomerulonephritis. Some of the characteristics of medically important streptococci are listed in Table 12.35. Because of their heterogeneity, no one system suffices to classify them. Twenty species, including Streptococcus pyogenes (group A), S. agalactiae (group B), and the enterococci (group D), are characterized by a combination of features: colony growth characteristics, hemolysis patterns on blood agar (α-hemolysis, β-hemolysis, or no hemolysis), antigenic composition of group-specific cell wall substances, and biochemical reactions. The hemolytic classification is based on the ability of many streptococci to hemolyze red blood cells in vitro to varying degrees. Complete disruption of erythrocytes with release of hemoglobin is called β-hemolysis. This is recognized as a clear zone, 2 to 4 mm in diameter, surrounding a Streptococcus spp. colony when grown on blood agar plates. Incomplete lysis of erythrocytes with the formation of green pigment is called α-hemolysis. S. pneumoniae (pneumococcus) types are further classified by the antigenic composition of the capsular polysaccharides. 12.7.1

Organism

Individual cocci are spherical or ovoid and are arranged in chains. The cocci divide in a plane perpendicular to the long axis of the chain. The members of the chain often have a striking diplococcal appearance, and rodlike forms are occasionally seen. The lengths of the chain vary widely and are conditioned by environmental factors. Streptococci are gram-positive. However, as culture ages and the bacteria die, they lose their gram-positivity and appear to be gram-negative; this can occur after overnight incubation. Streptococci share a fermentative metabolism in which lactic acid is the major end product. They lack the ability either to synthesize heme or to incorporate heme into enzymes. Despite these characteristics, most species

Table 12.35

Characteristics of Some Medically Important Streptococci Groupspecific substancea

Organism

Hemolysisb

Habitat

Important laboratory criteriac

Streptococcus pyogenes

A

β

Throat, skin

PYRd test result positive; inhibited by bacitracin

S. agalactiae

B

β

Female genital tract

Enterococcus faecalis (and other enterococci)

D

None, α

Colon

S. bovis (nonenterococci)

D

None

Colon

S. anginosus (S. intermedius, S. constellatus, S. milleri group)

F (A, C, G) and untypable

β

Throat, colon, female genital tract

Usually not typed or untypable Usually not typed or untypable

α, None

Throat, colon, female genital tract

Hippurate hydrolysis; CAMP result positivee Growth in presence of bile; hydrolysis esculin; growth in 6.5% NaCl; PYR result positive Growth in presence of bile; hydrolysis of esculin; no growth in 6.5% NaCl; degradation of starch Small (“minute”) colony variants of β-hemolytic species; group A bacitracin-resistant and PYR result–negative Carbohydrate fermentation patterns

α, None

Mouth, throat, colon, female genital tract

Optochin-resistant; colonies not soluble in bile; carbohydrate fermentation patterns Susceptible to optochin; colonies soluble in bile; quellung reaction positive Obligate anaerobes

Viridans streptococci (many species)

S. pneumoniae

None

α

Throat

Peptostreptococcus (many species)

None

α, None

Mouth, colon, female genital tract

a

Lancefield classification. Hemolysis observed on 5% sheep blood agar after overnight incubation. c PYR, L-pyrrolidonyl-2-naphthylmide; CAMP, Christie, Atkins, Munch-Peterson test. d Hydrolysis of L-pyrrolidonyl-2-naphthylamide. e Christie, Atkins, Munch-Peterson test. b

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Common and important diseases Pharyngitis, impetigo, rheumatic fever, glomerulonephritis Neonatal sepsis and meningitis Abdominal abscess, urinary tract infection, endocarditis Endocarditis; common blood isolate in colon cancer Pyrogenic infections, including brain abscesses

Not well defined

Dental caries (S. mutans), endocarditis, abscesses (with many other bacterial species) Pneumonia, meningitis, endocarditis Abscesses (with multiple other bacterial species)

of streptococci are tolerant to oxygen and readily grow in ambient air. Since their metabolism also allows them to grow equally well in the complete absence of oxygen, they are traditionally referred to as facultative anaerobes. Their growth and hemolysis are aided by incubation in 10% CO2. Whereas most pathogenic hemolytic streptococci grow best at 37°C, group D enterococci grow well at between 15°C and 45°C. Enterococci also grow in high (6.5%) sodium chloride concentrations, in 0.1% methylene blue, and in bile-esculin agar. Some streptococci produce a capsular polysaccharide comparable to that of pneumococci. Most group A, B, and C strains (Table 12.35) produce capsules composed of hyaluronic acid. The capsules are most noticeable in very young cultures. They impede phagocytosis. The streptococcal cell wall contains proteins (M, T, R antigens), carbohydrates (group-specific), and peptidoglycans. Hairlike pili project through the capsule of group A streptococci. The pili consist of M protein and are covered with lipoteichoic acid, which is important in the attachment of streptococci to epithelial cells. Over the years, the classification of streptococci into major categories has been based on a series of observations, including the following: 1. 2.

3. 4.

Colony morphological characteristics and hemolytic reactions on blood agar Serological specificity of the cell wall groupspecific substance (Lancefield classification) and other cell wall or capsular antigens Biochemical reactions and resistance to physical and chemical factors Ecological features

Many of these schemes are overlapping and are nonexclusive; as a result, classification can be confusing, can place several unrelated species in the same class, and can cause one species to be referred to by several different names. For example, S. pyogenes is also commonly known as β-hemolytic streptococcus and as group A streptococcus. The Lancefield (1962) classification scheme does provide a useful framework for considering the pathogenic range of the streptococci. Thus hemolytic streptococci can be divided into serological groups (A–U), and certain groups can be subdivided into types. The basis of serological grouping is group-specific cell wall antigen, which is a carbohydrate contained in the cell wall of many streptococci. The serological specificity of the group-specific carbohydrate is determined by an amino sugar. For group A streptococci, this is rhamnose-N-acetylglucosamine; for group B, rhamnose-glucosamine polysaccharide; for group C, rhamnose-N-acetylgalactosamine; for group D, glycerol teichoic acid containing D-alanine and glucose;

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and for group F, glucopyranosyl-N-acetylgalactosamine. Groups A and D streptococci can be transmitted to humans via food. Using this scheme, some of the medically important streptococci can be classified as follows: Group A: Streptococcus pyogenes Most streptococci that contain the group A antigen are S. pyogenes. They are β-hemolytic. S. pyogenes is the main human pathogen associated with local or systemic invasion and poststreptococcal immunological disorders. Thus they are responsible for a large number of different diseases of humans. Group B: Streptococcus agalactiae Group B streptococci are members of the normal flora of the female genital tract and an important cause of neonatal sepsis and meningitis. They typically are β-hemolytic. Groups C and G Groups C and G resemble the group A streptococci but cause a number of different diseases in animals, e.g., horses, cows, cats, dogs, and pigs. Group C S. dysgalactiae causes mastitis in cows, and group G S. equi causes an often-fatal infection of the throat (“strangles”) in horses. These streptococci can also occur sometimes in the nasopharynx in humans and may cause sinusitis, bacteremia, or endocarditis. Group D: Enterococcus faecalis (Enterococcus faecium, Enterococcus durans) Several species of group D streptococci formerly classified in the genus Streptococcus have recently been reclassified within the genus Enterococcus and commonly infect both the urinary tract and the valves of the heart (endocarditis). Enterococci are also part of the normal enteric flora. They are usually nonhemolytic and occasionally α-hemolytic. The group D antigen is a teichoic acid. S. bovis is among the nonenterococcal group D streptococci. They are part of the enteric flora, occasionally cause endocarditis, and sometimes cause bacteremia in patients with colon carcinoma. These streptococci are nonhemolytic. S. anginosus (also known as S. milleri, S. intermedius, S. constellatus) are part of the normal flora. They may be β-, α-, or nonhemolytic. Groups E, F, G, H, and K–U Streptococci of groups E–H and K–U occur primarily in animals.

Group N Streptococci Group N are rarely found in human disease states but produce normal coagulation (“souring”) of milk. Among species not classified by the Lancefield scheme are large collections of species known as the viridans streptococci, which are commonly found as residents of the oral cavity. Although usually harmless commensuals, viridans species include the organisms responsible for dental caries (S. mutans and several other closely related species), and most viridans species can be associated with endocarditis. Also of significance are S. pneumoniae, an important agent of pneumonia and otitis media in children, and S. iniae, a pathogen of fish and a rare cause of cutaneous infections in humans.

Table 12.36 pyogenesa

Fibronectin Fibrinogen Vitronectin Albumin Plasmin(ogen) Collagen Factor H H-kininogen CD46 C4b-binding protein Immunoglobulin A Immunoglobulin G Laminin a

12.7.2

Pathogenesis

Despite their heterogeneity in the types of hosts they infect and the types of diseases they cause, streptococci essentially use one of three basic mechanisms to cause disease: invasion and spread through tissue (strep throat, impetigo, ecthyma, cellulitis, necrotizing fasciitis, myositis), production of toxins (scarlet fever, toxic shock syndrome), and provocation of an autoimmune response (rheumatic fever, acute glomerulonephritis, certain forms of psoriasis) (Bisno, 1990; Bisno and Stevens, 1996; Charles and Larsen, 1986). Regardless of the ultimate class of disease, all streptococcal infections share certain steps in common. Transfer of the streptococcus usually initiates the infection from person to person (humans are the only significant reservoir of S. pyogenes), probably via aerosol transmission of infected droplets or through direct contact with an infected individual or with environmental surfaces that have been contaminated by an infected individual (Wannamaker, 1970). The disease can be established after entry of only a few organisms. Once streptococci are in contact with a new host, their adherence with an epithelial cell of the skin or pharynx appears to be mediated by stereospecific interactions between a microbial adhesin and its cognate host receptor molecule (Hultgren et al., 1993). The best-characterized adherence mechanisms of streptococci involve recognition and binding of host molecules that are, in turn, associated with the surface of a host epithelial cell or tissue. The host proteins that participate in this type of interaction are often those that are components of the extracellular matrix (Patti et al., 1994). Numerous extracellular matrix proteins, including laminin, various types of collagen, fibrinogen, vitronectin, and fibronectin, are known to bind to S. pyogenes (Table 12.36).

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Host Proteins Bound by Streptococcus

This is only a partial list of the many host proteins that are known to bind to the cell surface of S. pyogenes. In many cases, streptococcal receptors for the host proteins have been identified. Streptococcal binding structures may recognize a single or multiple host proteins, and multiple streptococcal surface products may bind a single host protein.

Streptococcal adhesin that binds to an extracellular matrix component is a fibronectin-binding protein known as protein F or Sfb (Hanski and Caparon, 1992; Talay et al., 1991). It is a cell wall–associated protein that binds soluble fibronectin essentially irreversibly with high affinity (apparent Kd ~1.0 nM). It has a conventional signal sequence at its N-terminus and a cell wall attachment domain at its C-terminus. The latter domain is distinguished by the characteristic pentapeptide LPXTG motif. The minimal fibronectin-binding domain (repetitive domain-2 [RD2]) is 44 amino acids in length and consists of the 25 carboxy-terminal amino acids of one repeat and the 19 amino acid residues from the amino terminus of an adjacent repeat. It binds to the amino terminal 29 kDa fibrin and heparin-binding domains of fibronectin. The second fibronectin-binding domain (upstream region [UR]) in protein F binds to the N-terminal 70-kDa domain of fibronectin that includes the fibrin- and heparin-binding domains along with an adjacent collagen-binding domain (Okada et al., 1997; Ozeri et al., 1996). The UR domain is responsible for the very high binding affinity of the whole protein F molecule. After attachment to host tissues, the next step common to all streptococcal diseases is the requirement for multiplication of the organisms in a tissue environment. This is a largely unexplored topic of streptococcal pathogenesis. Interaction with the atmosphere appears to play an important role in regulating the virulence properties of S. pyogenes. After attachment and multiplication on the surface of a host cell, the next step common to all strepto-

coccal infections is the communication between the signaling pathways of both the bacterium and the host cell. Adherence has been implicated as a mechanism by which the streptococci modulate the proinflammatory responses of epithelial cells since adherent and nonadherent streptococci induce different patterns of cytokine and prostaglandin responses in several different types of epithelial cells (Courtney et al., 1997; Wang et al., 1997). In spite of preliminary investigations, the precise role that invasion into cells contributes to streptococcal pathogenesis has yet to be established. However, the fact that it can occur and is dependent on specific streptococcal proteins makes a strong case for a highly structured communication between S. pyogenes and epithelial cells. The three basic mechanisms of streptococcal pathogenesis in causing disease are briefly described in the following. Invasion and Multiplication in Tissue (Strep Throat, Cellulitis) The first major class of diseases caused by S. pyogenes are those that result from the organism’s ability to invade and multiply extracellularly in host tissues. Streptococcal infection in tissue provokes an intense inflammatory response (e.g., polymorphonuclear leukocytes [PMNs]) by the host. The streptococci have almost no ability to survive the microbicidal defenses of the PMN; therefore, their principal virulence mechanisms involve avoidance of recognition, ingestion, and killing by these host cells. One mechanism that S. pyogenes uses to prevent recognition by PMNs involves the surface M protein. The M proteins represent a related family of proteins that share a common structure. They appear as hairlike projections of the streptococcal cell wall. They are fibrous rodlike molecules that are highly α-helical homodimers arranged in a coiled-coil conformation. This structure basically consists of two α-helices that coil around one another (Fischetti, 1989). The structure allows for a large number of sequence changes to occur while maintaining function, and the M protein immunodeterminants thus can readily change. The basic structure of an M protein consists of three distinct blocks of tandem repetitive sequences referred to as the A, B, and C repeat domains. This basic structure is conserved between different M proteins. However, they can differ quite dramatically in their primary sequences. The degree of variability generally decreases from the Nterminus to the C-terminus. The N-terminus and the A and B repeat domains vary extensively among M proteins, whereas the C repeats and the cell wall attachment domains are highly conserved (Fischetti, 1989).

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The classic work of Rebecca Lancefield (1962) identified the M protein as having a role in avoidance of phagocytosis. She demonstrated that streptococci rich in surface M protein had the capacity to survive and multiply in the presence of PMNs in human blood, while those isolates deficient in M protein were killed. The PMNs recognize S. pyogenes via complement deposited on the streptococcal cell wall, and the presence of M protein, although not preventing deposition of complement, somehow alters an essential characteristic of the deposited material, either in the pattern of deposition or in the quantity of complement deposited (Fischetti, 1989). Most, if not all, M proteins implicated in avoidance of phagocytosis can bind the host protein fibrinogen. Immunity to infection with group A streptococci is related to the presence of type-specific antibodies to M protein. Because there are more than 80 types of M protein, a person can have repeated infections with group A S. pyogenes of different M types. Not all M proteins are equally efficient in the avoidance of recognition by PMNs. In these strains, the polysaccharide capsule makes a major contribution to this property (Dale et al., 1996). The capsule of S. pyogenes is a glycosaminoglycan and is a relatively simple homopolymer of hyaluronic acid consisting of repeating subunits of glucuronic acid and N-acetylglucosamine. Although the mechanism by which the capsule of S. pyogenes interferes with recognition by PMNs is not yet established, mechanisms involving steric interference of phagocyte recognition of complement deposited on the cell surface and recruitment of complement inhibitory factors to the capsule surface to block the deposition of complement have been described for different capsular types of S. pneumoniae (Brown, 1985). S. pyogenes also possesses a number of additional products that act to protect it from interacting with phagocytic cells, including at least two proteases and a hemolysin. The first of these is a serine protease known as the streptococcal C5a peptidase (SCPA), whose mature form becomes associated with the streptococcal cell wall (Wexler et al., 1985). SCPA is likely to contribute to the early stages of streptococcal pathogenesis by inhibiting the recruitment of PMNs to an initial site of streptococcal multiplication in tissue. The second protease that protects streptococci from interacting with phagocytic cells is the streptococcal cysteine protease (SCP or SpeB), which differs from SCPA in that it is freely secreted from the streptococcal cell and has very broad substrate specificity (Kapur et al., 1993a, 1993b; Burns et al., 1996). It can degrade host extracellular matrix proteins and can also cleave and release active

forms of streptococcal surface proteins, including SCPA, from the cell wall. Killing the phagocytic cells themselves provides an obvious advantage in a bacterium’s attempts to evade recognition, uptake, and killing by these host defense cells. S. pyogenes makes and secretes a number of additional gene products that can affect the viability and function of PMNs. Most notable among these is streptolysin S (SLS), so named because, unlike the thiol-dependent pore-forming hemolysin streptolysin O (SLO), it is active in the presence of oxygen and is primarily responsible for the zone of β-hemolysis produced by colonies of S. pyogenes on the surface of blood agar (Ginsberg, 1970). Under suitable inducing conditions, almost all strains of groups A, C, and G and some strains of groups B, E, H, and L streptococci produce SLS (Duncan and Marchlewicz, 1981; Wannamaker and Schlievert, 1988). Strains of group A streptococci generally produce larger amounts than strains of other groups. SLS is cell-bound. It is induced and released by several unrelated classes of substances, including serum or serum constituents (e.g., albumin and α-lipoprotein), RNA, detergents, and a variety of aniline dyes. The SLS toxin is composed of a polypeptide moiety associated to a carrier and is only active in the carrierbound state. The apparent molecular weights of SLS and its carrier oligonucleotide were estimated at 15,000 and 7100, respectively (Lai et al., 1978). The peptide that constitutes the specific active principle of SLS contains 32 amino acids. Its amino acid composition is peculiar as compared to that of many bacterial protein cytolysins since six amino acids, histidine, arginine, cysteine, valine, methionine, and isoleucine, are lacking. Its amino acid composition is somewhat similar to that of mellitin, the cytolytic polypeptide of bee venom, which also lacks six of the usual amino acids (Yunes et al., 1977). SLS is a very potent membrane-damaging agent. However, it does not appear to involve the formation of large pores. It lyses a wide variety of living cells; its lytic spectrum is somewhat broader than that of SLO: it is lytic or cytotoxic not only for eukaryotic cells but also for wallless forms of some bacteria, notably protoplasts and Lforms of various species (Bernheimer, 1972). Intracellular organelles such as mitochondria and lysosomes are also disrupted by SLS. No role for SLS in streptococcal infections of humans has been clearly elucidated. The fact that it is nonantigenic suggests that it may continue to exert its toxicity unimpeded during repeated episodes of streptococcal infection. It is known that SLS is a potent cytotoxin for PMNs.

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SLO is the oxygen-sensitive hemolytic agent produced by most strains of group A and some strains of groups C and G streptococci in the extracellular fluid, especially those causing human infection. SLO closely resembles the hemolysins produced by a variety of other gram-positive bacteria, notably the pneumococci, the tetanus bacillus, and other Clostridium spp. and Listeria monocytogenes (Wannamaker and Schlievert, 1988). They are antigenically related as shown by cross-neutralization and immunoprecipitation. Their cytolytic and other biological effects are lost by oxidation and restored by SHcompounds and other reducing agents. These toxins are inactivated by cholesterol and certain related sterols. This group of oxygen-labile cytolytic toxins has been named sulfhydryl-activated toxins (Bernheimer, 1976) or thiolactivated cytolysins (Smyth and Duncan, 1978). A wide range (53,000 to 75,000) of estimated molecular weights for SLO has been published. Multiple forms appear to be present. A characteristic feature of SLO is its reversible activation by reducing agents. Some biochemical characteristics of SLO are summarized in Table 12.37. SLO acts on cell membranes. However, unlike that of most other bacterial lysins, the rate of lysis of erythrocytes is a nonlinear function of SLO concentration (Bernheimer, 1974). An initial short lag phase is followed by a constant-rate phase during which the hemolytic rate is proportional to the square of the concentration of SLO. Cholesterol appears to be the binding site for SLO in nucleated mammalian cells as well as erythrocytes (Duncan and Buckingham, 1980). It is active only on cells whose membranes contain cholesterol. Mice, rats, guinea pigs, rabbits, and cats are rapidly killed by intravenous injection of activated SLO (Alouf, 1986). The values of the LD50 by the intravenous route have been determined in mouse (0.2 µg), guinea pig (6 µg), and rabbit (2 µg). A variety of physiopathological manifestations are observed on administration of lethal or sub-

Table 12.37

Some Characteristics of Streptolysin Oa

Molecular weight (SDS-PAGE) Isoelectric point HU/µg proteinb LD50 (intravenous), µg protein 20-g Mouse 250-g Guinea pig 2-kg Rabbit Minimal lethal dose for 9-day-old embryonated hen egg, µg protein a

67,000 ± 5,000 7.8 400,000 0.2 6 3 2.10 × 10–2

Sodium dodecyl sulfate-pol ya crylam ide gel electrophoresis; HU, hemolytic unit. b 1 HU = 2.5 ng protein or 1.5 × 10–2 pmole.

lethal doses of SLO in these animals. These include generalized tonic and clonic motor convulsion with the head in extreme extension, pulmonary edema, hemorrhage, blood pressure drop, respiratory failure, and micturation. The almost instantaneous lethal effects of SLO on laboratory animals have been attributed to the direct cardiotoxicity of SLO. The possible mechanism(s) of the cardiotoxicity of SLO is not well understood. Although the potentiality of SLO to produce a wide range of cytotoxic effects is clear, its contribution to the pathogenesis of streptococcal infections and their complications is uncertain. If damage by SLO is an important factor, the presence of large amounts of a potent natural inhibitor (nonesterified cholesterol) in epidermal tissue may account for the absence of rheumatic fever after cutaneous infections with group A streptococci (Kaplan and Wannamaker, 1976). An anti-SLO serum titer in excess of 160–200 units is considered abnormally high and suggests either recent infection with streptococci or persistently high antibody levels due to an exaggerated immune response to an earlier exposure in a hypersensitive person. Once the streptococci overcome the initial host defenses, they begin to spread in the tissue and the tissue is damaged. The degree of spread and the nature of the damage are characteristic of specific diseases and can range from a very localized mildly inflammatory lesion to complete necrotic destruction of tissue. It is likely that these processes involve damage generated both by the direct action of some streptococcal product(s) on host tissues and by the host’s own inflammatory response to those products. S. pyogenes secretes several different products into its environment (Table 12.38), including numerous high-

Table 12.38

Extracellular Products of Streptococcus pyogenesa

Glucuronidase Nicotinamide adenine dinucleotide glycohydrolase (NADase) Amylase Phosphatase Hyaluronidase Bacteriocins Pyrogenic exotoxin serotype A, B, C, and F Streptolysin S (SLS) Streptolysin O (SLO) Cysteine protease Deoxyribonuclease (DNase) A, B, C, D Streptokinase Streptococcal superantigen (SSA) Lipoteichoic acid (LTA) Glyceraldehyde phosphate dehydrogenase (GAPDH) a

This is only a partial list of many products secreted by S. pyogenes.

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molecular-weight proteins with known enzymatic and/or toxic activities (Alouf, 1980; Ginsberg, 1972). Many of these secreted enzymes have degradative activities that may also assist in multiplication of the streptococci by helping the cells to acquire nutrients or may directly participate in damage to tissues. These include hyaluronidase and up to four different types of DNases. This group of enzymes may also assist the streptococci in spreading through tissue through their abilities to degrade highmolecular-weight polymers, such as DNA, that become viscous when released by damaged cells. Other secreted enzymes include a nicotinamide-adenine dinucleotidase (NADase), glucuronidase, and esterase and various peptidases, neuraminidases, and phosphatases. Capture of host-derived proteolytic activity may also contribute to streptococcal spread through a tissue and invasion across tissue barriers. A key component of this activity is the streptococcal-derived plasminogen activator streptokinase (Boyle and Lottenberg, 1997). Almost all streptococcal strains isolated from human infections secrete streptokinase. Unique among plasminogen activators, streptokinase itself has no enzymatic activity. Rather, it binds and forms an equimolar complex with plasminogen that induces a conformational change in plasminogen such that the resulting complex becomes a potent plasminogen activator. Activated plasmin is a serine protease whose physiological role is primarily in fibrinolysis, but it is actually a very potent protease of broad specificity. For example, it can degrade proteins of the extracellular matrix, including fibronectin and laminin, and can activate other host proteases. Activation of plasminogen by streptokinase is an important virulence property of streptococci. Toxin-Mediated Diseases (Scarlet Fever, Toxic Shock Syndrome) The defining characteristic of a streptococcal disease that is mediated by a toxin is that the most serious damage to the host occurs at sites distinct from the primary site of infection. These diseases, however, can also be associated with a very local and mild or even nonapparent streptococcal infection. Children in particular are more vulnerable to toxin-mediated disease in the absence of severe local disease. For group A streptococci, the classic examples of toxin-mediated diseases are scarlet fever and the streptococcal toxic shock syndrome (Schlievert et al., 1996). The streptococcal erythrogenic toxins are typically associated with the red rash produced during scarlet fever but are now referred to as the streptococcal pyrogenic exotoxins (SPEs) (Kim and Watson, 1970). A majority of group A streptococci make SPEs whether associated with scarlet fever or not, and production of the erythematous

rash is but one of many potentially important biological properties of SPEs. SPE is a toxic, immunogenic, low-molecular-weight single-chain protein released in the extracellular medium by group A streptococci. Three serologically distinct toxin types, A, B, and C, have been identified and characterized biochemically and biologically. These toxins may be produced simultaneously or separately, depending on strain. Approximately 95% of group A streptococcal strains make SPEs in any of the various combinations (Schlievert et al., 1979). Type A toxin consists of a protein and 4% to 8% of hyaluronic acid. Its molecular weight is 8000 with a pI of 5.0 (Schlievert et al., 1977). Toxin B is heterogeneous in charge. The three isomers each have a molecular weight of about 17,500 and pI of 8.0, 8.4, and 9.0, respectively. Only pI 8.4 toxin B is pyrogenic (Alouf, 1986). Toxin C is antigenically different from A and B toxins and has a molecular weight of about 13,000 and pI of 6.7. It is a naturally phosphorylated protein (Schlievert et al., 1979). A multiplicity of remarkable biological properties are associated with SPEs (Table 12.39). Induction of fever in rabbits is a prominent and constant property of SPEs. The minimal pyrogenic dose at 3 hours is about 0.07 µg/kg by the intravenous route (Watson and Kim, 1970). It is believed to be due to the release of leukocyte endogenous pyrogen (Schuh and Hribalova, 1966). Toxin A is capable of altering cerebral membrane permeability; toxin C is capable of crossing the blood-brain barrier to produce fever by direct stimulation of the hypothalamic fever response control center rather than indirectly by endogenous pyrogen (Okada et al., 1971; Schlievert and Watson, 1978). Perhaps the most significant property of SPEs is the capacity to enhance host susceptibility to other agents such as endotoxin or SLO by inhibiting reticuloendothelial clearance function (Cunningham and Watson, 1978). Prolonged suppression of this function, particularly in the

Table 12.39 Biological Properties of Streptococcal Pyrogenic Exotoxins (SPEs) Pyrogenicity Skin reactivity (erythrogenic effect): erythema in humans and sensitized animals Lethality (rabbit) Cytotoxicity and tissue (cardiac) damage Enhanced susceptibility to endotoxin shock Depression of reticuloendothelial functions Alterations of host antibody response to immunogens in rabbits and mice Mitogenic activity on human and rabbit lymphocytes Source: Compiled from Alouf (1980, 1986).

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liver, could explain the inability of animals to handle other toxic substances. Other biological effects of SPEs include lymphocyte mitogenicity, immunosuppression, cytotoxicity, and tissue (cardiac) damage. Immunopathologically Based Diseases (Rheumatic Fever, Glomerulonephritis) The seemingly unrelated immunopathologically based diseases occur several days to weeks after an acute streptococcal infection has cleared. These types of diseases are almost exclusively associated with infection by S. pyogenes and apparently occur only in humans. They are commonly known as the streptococcal nonsuppurative sequelae and include rheumatic fever and rheumatic heart disease, acute glomerulonephritis, certain forms of psoriasis, and possibly even some forms of obsessive-compulsive disorder (Swedo et al., 1997). The pathogenesis for these sequelae is believed to involve antigenic mimicry. According to this mechanism, the streptococcus expresses epitopes that are molecular mimics of host structures (Gibofsky et al., 1998). As a consequence of the host’s reaction against the streptococcal infection, there is a breakdown of the normal prohibition against responding to self-antigens that results in an autoimmune disorder. The principal molecular evidence in support of this theory is that individuals with acute rheumatic fever often have circulating antibodies that react with numerous human antigens present in heart tissue, including myosin. These same populations of heart-reactive antibodies also recognize streptococcal surface proteins, most notably the M protein, structural mimic of myosin. These antibodies are symptomatic of the expression of molecular mimics by the pathogen that is accompanied by a breakdown of T-cell anergy (Rose, 1998). 12.7.3

Symptoms, Diagnosis, and Treatment

The most common infection due to β-hemolytic streptococci is streptococcal sore throat. Virulent group A streptococci adhere to the pharyngeal epithelium. The infectious dose is probably quite low (<1000 organisms). In infants and small children, the sore throat occurs as a subacute nasopharyngitis with a thin serous discharge and little fever but with a tendency of the infection to extend to the middle ear, the mastoid, and the meninges. The cervical lymph nodes are usually enlarged. The illness may persist for weeks. In older children and adults, the disease is more acute and is characterized by intense nasopharyngitis, tonsillitis, and intense redness and edema of the mucous membranes, with purulent exudates; enlarged cervical lymph nodes; and usually a high fever. Twenty percent of

infections are asymptomatic. Streptococcal infection of the upper respiratory tract does not usually involve the lungs. Pneumonia due to β-hemolytic streptococci is rapidly progressive and severe and is most commonly a sequela to viral infection, e.g., influenza or measles, which seem to enhance susceptibility greatly. As a general rule, elimination of the streptococci terminates the disease process, and the streptococci produce no permanent damage to the infected tissue. This is not the case for infection of deeper layers of tissue. For example, infection of the fascia or underlying muscle results in disease (necrotizing fasciitis and myositis, respectively); it includes numerous streptococci in the tissue, evidence of inflammation, and lateral spread of organisms through the tissue. These infections are life-threatening and characterized by severe irreversible necrotic damage. Treatment must include removal of the damaged tissue. Fulminant, invasive group A streptococcal infections with streptococcal toxic shock syndrome are characterized by shock, bacteremia, respiratory failure, and multiorgan failure. Death occurs in about 30% of patients. Streptococcal toxic shock syndrome and scarlet fever are clinically overlapping diseases. Acute glomerulonephritis sometimes develops 3 weeks after streptococcal infection, particularly with M types 12, 4, 2, and 49. Some strains are particularly nephritogenic. Acute nephritis is characterized by blood and protein in the urine, edema, high blood pressure, and urea nitrogen retention. Serum complement levels are low. The majority of patients recover completely, although in some chronic glomerulonephritis and ultimate kidney failure develop. Rheumatic fever is the most serious sequela of hemolytic streptococcal infection because it results in damage to heart muscle and valves (Bisno, 1990). Certain strains of group A streptococci contain cell membrane antigens that cross-react with human heart tissue antigens. The onset of rheumatic fever is often preceded by a group A streptococcal infection 1–4 weeks earlier, although the infection may be mild and may not be detected. In general, however, patients with more severe streptococcal sore throats have a greater chance of development of rheumatic fever. Typical symptoms and signs of rheumatic fever include fever, malaise, a migratory nonsuppurative polyarthritis, and evidence of inflammation of all parts of the heart (endocardium, myocardium, pericardium). The carditis characteristically leads to thickened and deformed valves and to small perivascular granulomas in the myocardium that are finally replaced by scar tissue. Erythrocyte sedimentation rate, serum transaminase level, electrocardiography, and other tests are used to estimate rheumatic activity.

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Group A streptococcal infections can be diagnosed by culturing of nasal and throat swabs, pus, sputum, blood, suspect food, and environmental samples. Culturing of stool samples, blood, and suspect food identifies group D streptococcal infections. All β-hemolytic group A streptococci are sensitive to penicillin G, and most are sensitive to erythromycin. Some are resistant to tetracyclines. Aminoglycosides often enhance the rate of bactericidal action of penicillin on streptococci, particularly enterococci. Antimicrobial drugs, however, have no effect on established glomerulonephritis and rheumatic fever. However, in acute streptococcal infections, every effort must be made to eradicate streptococci from the patient and eliminate the antigenic stimulus (before day 8) rapidly and thus to prevent poststreptococcal disease. 12.7.4

Sources

Many streptococci (viridans streptococci, enterococci, etc.) are members of the normal flora of the human body. They produce disease only when established in parts of the body where they do not normally occur (e.g., heart valves). The ultimate source of group A streptococci is persons harboring these organisms. Milk and other foods become contaminated when infected individuals create an aerosol by coughing and sneezing. Mastitic cows also may transmit hemolytic streptococci through milk and cause the typical strep throat syndrome and scarlet fever. Raw milk indeed was a major vehicle of food-borne streptococcal infection and cause of scarlet fever before the widespread use of pasteurization. Other food vehicles for group A streptococcal infections include ice cream, eggs, steamed lobster, ground ham, potato salad, egg salad, custard, rice pudding, and shrimp salad (CFSAN, 1998). In almost all cases, the foodstuffs were allowed to stand at ambient temperatures for several hours between preparation and consumption. Entrance into the food is the result of poor hygiene, illness of food handlers, or the use of unpasteurized milk. Group D streptococci sources include sausage, evaporated milk, cheese, meat croquettes, meat pie, pudding, raw milk, and pasteurized milk (CFSAN, 1998). Entrance into the food chain is due to underprocessing and/or poor and unsanitary food preparation. 12.7.5

Outbreaks

Outbreaks of septic sore throat and scarlet fever were numerous before the advent of milk pasteurization. The incidence of food-borne group A streptococcal infection in the United States has decreased considerably since the 1960s.

However, accurate data are difficult to establish because of poor reporting. Between 1969 and 1975, only two foodborne outbreaks involving a total of 575 cases were reported (CDC, 1972, 1973). Recently, salad bars have been suggested as possible sources of infection. Most current outbreaks have involved complex foods, which were infected by a food handler with septic sore throat. The nasal discharges of a person harboring β-hemolytic streptococci are the most dangerous source for spread of these organisms. One ill food handler may infect hundreds of individuals. Food-borne group D streptococcal outbreaks are relatively rare. 12.7.6

Control

Control procedures are directed mainly at the human source. These include detection and early antimicrobial therapy of respiratory and skin infections with group A streptococci. Prompt eradication of streptococci from early infections can effectively prevent the development of poststreptococcal disease. Antistreptococcal chemoprophylaxis in persons who have suffered an attack of rheumatic fever is also recommended. Chemoprophylaxis in such individuals, especially children, must be continued for years. Yet another control measure involves eradication of group A streptococci from carriers. This is especially important when carriers work in areas such as obstetric delivery rooms, operating rooms, classrooms, nurseries, and food service industries. Unfortunately, it is often difficult to eradicate β-hemolytic streptococci from permanent carriers, and individuals may occasionally have to be shifted away from “sensitive” areas for some time.

12.8 YERSINIA ENTEROCOLITICA (YERSINIOSIS) Yersinia spp. are gram-negative bacilli belonging to the Enterobacteriaceae family. Comprising 11 species, the genus Yersinia includes three primary pathogens of humans: Y. pestis, the causative agent of bubonic and pneumonic plague; Y. pseudotuberculosis, an intestinal pathogen of rodents that occasionally infects humans; and Y. enterocolitica, a common intestinal pathogen of humans implicated in diarrheal diseases. The remaining species belonging to this genus may also cause, on rare occasions, opportunistic infections. The three human pathogens share a number of essential virulence mechanisms that enable them to overcome nonspecific immune defenses of the hosts. Similar virulence determinants have also been found in several other important bacterial pathogens, including

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Salmonella and Shigella species, lending credence to the horizontal transfer of the genetic information for virulence determinants between enteric pathogens (Robins-Browne, 1997). In addition, yersiniae may have acquired a number of genetic elements from eukaryotic cells. These factors may enhance bacterial virulence by enabling them to undermine essential aspects of the physiological response to infection. Y. pestis is best known as the “black death” of the Middle Ages that killed millions of people. More recently, there has been an increase in cases of Y. pestis seen in the United States, and isolated outbreaks have also occurred in India (Perry and Fetherston, 1997). It is an obligate parasite. Humans generally become infected with Y. pestis via a bite of a flea that has acquired the organism from a blood meal on an infected animal, such as a rat. In the flea, the infection is limited to the alimentary tract, which becomes so blocked with bacteria that Y. pestis is regurgitated into the next animal the flea feeds on. In humans and other animals, Y. pestis spreads from the site of the flea bite into the regional lymph nodes, and the characteristic buboes (swollen lymph nodes) of bubonic plague form. The bacteria may then spread into the bloodstream to cause septicemia, and to the spleen, liver, and lungs. Pneumonic plague is transmitted by respiratory droplets from the lungs of an infected individual and causes an overwhelming pneumonia. Unlike Y. pestis, Y. enterocolitica and Y. pseudotuberculosis can survive outside animal hosts and are foodborne pathogens. They are responsible for a broad range of gastrointestinal syndromes, ranging from acute gastroenteritis to mesenteric lymphadenitis and, on rare occasions, can even provoke systemic infections such as septicemia and meningitis (Cover and Aber, 1989). First emerging as a human pathogen during the 1930s (Bottone, 1977), Y. enterocolitica causes infection mainly through ingestion of contaminated food or water. Pork meat is a frequent source of this pathogen (Tauxe et al., 1987). The organism has the ability to grow at low temperatures. To date, no food-borne outbreaks caused by Y. pseudotuberculosis have been reported in the United States, but human infections transmitted via contaminated water and foods have been reported in Japan. Y. pseudotuberculosis is genetically very similar to Y. pestis, sharing over 90% DNA homology, and perhaps is a different pathotype of the same species. 12.8.1

Organism

Yersiniae are non-lactose-fermenting gram-negative rods that are urease-positive and oxide-negative. They grow best at 25°C and are motile at 25°C with peritrichous flagella, but nonmotile at 37°C. They are found in the intestinal tract of a variety of animals, in which they may cause

disease, and are transmissible to humans, in whom they can produce a variety of clinical syndromes. However, unlike other members of the Enterobacteriaceae family, they are not part of the normal intestinal microflora of humans. Y. enterocolitica is unusual in its ability to grow at 4°C; most strains grow at temperatures as low as 1°C or even below. Thus, it can readily withstand freezing and can survive in frozen foods for extended periods even after repeated freezing and thawing (Toora et al., 1992). It is, however, destroyed by pasteurization at 71.8°C for 18 seconds (D’Aoust et al., 1988; Toora et al., 1992). The organism is able to grow over a pH range from approximately pH 4 to 10, with an optimal pH of around 7.6 (Schiemann, 1980). It can tolerate NaCl at concentrations of up to 5% (Feng and Weagant, 1993). It is readily inactivated by ionizing and UV irradiation and by addition of sodium nitrate and nitrite to food (de Giusti and de Vito, 1992; Butler et al., 1987; Dion et al., 1994). Y. enterocolitica exists in more than 50 serotypes, which are based on their biochemical activity and LPS O antigens. The biotyping scheme is based on the organism’s ability to metabolize selected organic substrates and provides a convenient means to subdivide this species into subtypes of variable clinical and epidemiological significance. Using this scheme, the species can be subdivided into the following six biovars: 1A, 1B, 2, 3, 4, and 5 (Wauters et al., 1987). With the exception of biovar 1A, the rest contain most primary pathogenic strains of humans and domestic animals. The most frequent Y. enterocolitica biovar obtained from human clinical material worldwide is biovar 4 (Robins-Browne, 1997). Among the serotypes classified on the basis of the O antigen, O3, O8, and O9 are most commonly implicated in human diseases. 12.8.2

Pathogenesis

Y. enterocolitica is an invasive pathogen, which induces an inflammatory response in infected tissues. An inoculum of 108–109 yersiniae must enter the alimentary tract to produce infection. Once ingested, the organism is able to cross the gut epithelium and proliferate locally in the underlying tissue. The bacteria selectively enter via the M cells and reach the intestinal lymphoid follicles known as Peyer’s patches (Hanski et al., 1989; Grutzkau et al., 1990; Autenrieth and Firsching, 1996). The M cells are specialized epithelial cells that carry out transcytosis; they take up small quantities of the intestinal contents and release them at their basolateral side into Peyer’s patches. Entry into Peyer’s patches leads to an enormous recruitment of phagocytic PMNs, formation of microabcesses comprising extracellular Yersinia spp., appearance of dead apoptotic

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cells, and eventually complete destruction of the cytoarchitecture of Peyer’s patches (Hanski et al., 1989; Autenrieth and Firsching, 1996). The hallmark lesion of yersiniosis comprises microcolonies of bacteria surrounded by granulocytic and mononuclear inflammatory cells. Monocytes infiltrate Peyer’s patches and mature into inflammatory macrophages to produce cytokines such as interleukin-12 (IL12), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α). These cytokines aid in the development of the immune response (Autenrieth et al., 1994, 1996; Bohn and Autenrieth, 1996). Phagocytes severely restrict the rate at which Yersinia spp. multiplies in the host tissues, thereby allowing a specific protective immunity to develop in the host. However, the organism can fight back by impairing phagocytosis, inhibiting phagocytic killing, triggering apoptosis, and suppressing the normal release of TNF-α and other cytokines. Once established in Peyer’s patches, the bacteria can disseminate to the mesenteric lymph nodes and eventually to the liver and spleen (Carter, 1975; Robins-Browne, 1997; Boyd and Cornelis, 2001). Because of their innate resistance to killing by macrophages and PMNs, most of the bacteria observed in histological sections are located extracellularly (Hanski et al., 1989). Thus, Y. enterocolitica is predominantly an extracellular pathogen, and its survival strategy strongly relies on its ability to escape phagocytosis. The virulence factors of Y. enterocolitica are chromosomally encoded. These include the invasin (Inv), the adhesive factors Myf/pH6 antigen and Ail, the enterotoxin Yst, and iron acquisition proteins (Table 12.40). In addition, all the three pathogenic species of Yersinia have a common 70- to 75-kb virulence plasmid that encodes an array of tightly regulated and sophisticated antihost factors. The biological characteristics of these virulence factors are briefly described in the sections that follow. Invasin All virulent serovars of Y. enterocolitica produce a chromosomally encoded 91-kDa outer membrane protein called invasin (Inv). Nonpathogenic strains of Y. enterocolitica and Y. pseudotuberculosis often lack a functional Inv (Pierson and Falkow, 1990). The protein in fact was first identified as a 102-kDA product of the chromosomal inv gene of Y. pseudotuberculosis (Isberg and Van Nhieu, 1994; Pierson, 1994). Introduction of this inv gene into other bacterial species, such as E. coli, confers the capacity to invade eukaryotic cells on these bacteria (Pepe and Miller, 1990; Young et al., 1990). Inv promotes both attachment to and invasion into eukaryotic cells, including nonphagocytic cells by Yersinia

Table 12.40

Chromosomal and Plasmid-Mediated Virulence Factors of Yersinia enterocolitica

Protein

Function

Chromosome-mediated Inv

Adherence and invasion

Ail Myf

Adherence and invasion Adherence

Yst FyuA/Psn-Irp Plasmid-mediated YadA YopO/YpkA YscP YopH

Enterotoxin Iron acquisition

YopM YopB LcrV YopD YopT YopN YopP/YopJ

YopE

YopQ/YopK YopR YscM1

Adherence and invasion Effector, serine/threonine kinase Unknown Effector, tyrosine phosphatases of FAK and p130cas; prevents phagocytosis and phagocytic killing Effector? Translocator Translocator effector? Translocator; negative regulator of secretion; effector? Effector, depolymerizes actin: causes cytotoxicity Controls Yop secretion Effector; inhibits NFkB activation; IkB possible target; induces apoptosis; prevents cytokine induction Effector; depolymerizes actin; causes cytotoxicity; prevents phagocytosis Controls size of translocator pore Unknown Negative regulator of secretion

Required for virulence Not absolutely; enhances Peyer’s patches colonization No Found only in pathogenic strains Required for diarrhea Yes Yes Yes Not determined Yes

Yes Yes Yes Yes No Not determined No

Yes

Yes Yes Not determined

Source: Compiled from Robins-Browne (1997) and Boyd and Cornelis (2001).

spp. The amino terminus of this protein is inserted in the bacterial outer membrane; the exposed carboxyl-terminus determines the specificity of binding of Inv to specific ligands, known as β1 integrins, on host cells (Isberg and Van Nhieu, 1994). Inv is not required for virulence of Yersinia spp., but it does favor efficient translocation of the bacteria from the intestinal tract into Peyer’s patches and so to the underlying tissues (Pepe and Miller, 1993; Han and Miller, 1997; Marra and Isberg, 1997). As mentioned earlier, the Inv receptor on eukaryotic cells is β1 integrin. These receptors are able to couple extracellular adhesion events to numerous signaling pathways within the mammalian cell, some of which allow association of the β1-chain to cytoskeletal-associated proteins. Integrins are found on many cell types, including ep-

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ithelial cells, macrophages, and T lymphocytes. Once they are attached, multiple points of contact are established between the cell and bacterium, and the bacterium is taken up into the endocytic vacuole by a “zipper” mechanism (Young et al., 1992). The internalization process mediated by Inv is propagated entirely by the host cell, and the uptake depends on the strong affinity of Inv for integrins. The uptake is accompanied by cytoskeletal rearrangements with accumulation of actin, filamin, talin, and β1 integrins around the incoming bacteria. Invasion can be inhibited by cytochalasin D, which disorganizes actin filaments (Boyd and Cornelis, 2001). Inv mutants of Y. pseudotuberculosis and the virulent serovars of Y. enterocolitica show a significantly reduced ability to invade epithelial cells in vitro (Pierson, 1994; Robins-Browne, 1997).

Ail

Yst Enterotoxin

Ail, a chromosomally encoded, membrane-associated protein, unrelated to Inv, also confers invasive ability to Yersinia spp. (Miller and Falkow, 1988). It is 17-kDa peptide with eight putative membrane-spanning domains and is specified by a chromosomal ail attachment-invasion locus (ail), so called because it mediates bacterial attachment to some cultured epithelial cell lines and invasion of others (Miller, 1992; Pierson, 1994). Like Inv, Ail confers invasion properties on E. coli. Ail is a less powerful adhesin than Inv (Miller et al., 1990), but it does have the additional ability of rendering Yersinia spp. resistant to killing by serum complement (Bliska and Falkow, 1992; Pierson and Falkow, 1993). This protein is preferentially expressed at 37°C under aerobic conditions in the stationary phase (Pierson and Falkow, 1993). Ail is not required for virulence in Y. enterocolitica, neither to establish infection nor to cause systemic infection (Wachtel and Miller, 1995). A number of proteins analogous to Ail have been identified in other pathogenic bacteria. These include the Rck and PagC proteins of Salmonella typhimurium, Lom of bacteriophage lambda in the outer membranes of E. coli lysogens, Tia of enterotoxigenic E. coli (ETEC, see Chapter 13) and OmpX, also of E. coli. However, despite their structural similarities, these proteins are evidently functionally somewhat distinct (Robins-Browne, 1997; Boyd and Cornelis, 2001).

Pathogenic strains of Y. enterocolitica produce a heatstable enterotoxin called Yst. This enterotoxin is responsible for the diarrhea associated with Yersinia spp. infection (Delor et al., 1990; Delor and Cornelis, 1992). Yst is encoded by the chromosomal yst gene and synthesized as a 71-amino-acid polypeptide, the carboxyl terminus of which becomes the mature toxin (Cornelis, 1994). The 18 N-terminal amino acids contain a signal sequence that is cleaved off during secretion. A further 23 central amino acids are then removed during or after secretion, and the mature active peptide contains just 30 amino acids. The toxin is produced during the stationary phase at 30°C, but increased osmolarity and pH at 37°C also induce high expression of this protein (Mikulskis et al., 1994). The carboxyl terminus of Yst is homologous to those of heat-stable enterotoxins from ETEC and Vibrio cholerae non-O1 and to that of guanylin, an intestinal paracrine hormone (Currie et al., 1992). They also share a common mechanism of action, which involves binding to and activation of cell-associated guanylate cyclase. This in turn stimulates cGMP synthesis in the intestinal brush border, leading to an overall effect of fluid loss and lack of fluid absorption.

Myf/pH6 Antigen Originally named the C-antigen, mucoid Yersinia factor (Myf) in Y. enterocolitica and its homolog, the so-called pH 6 antigen in Y. pestis and Y. pseudotuberculosis, are chromosomally encoded proteins that form a fibrillar structure of strands, bundles, and aggregates of 15-kDa subunits surrounding the bacteria (Lindler et al., 1990; Iriarte et al., 1993). Similarly to that of Inv and Ail, the expression of these proteins is restricted to pathogenic strains of the microorganism during the stationary phase of growth. Myf consists of narrow flexible fimbriae, which resemble CS3, an essential colonization factor of many human strains of ETEC. These proteins enhance thermoinducible binding of Yersinia spp. to eukaryotic cells and can bind intestinal luminal mucus. The pH 6 antigen binds specifically to β1-linked galactosyl residues in glycosphingolipids (Payne et al., 1988). It is synthesized inside the macrophages, suggesting it may also play a role in intracellular survival of the organism.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Iron Acquisition Iron is an essential nutrient for Yersinia spp. However, in the host, mammalian proteins such as transferrin, lactoferrin, and hemoglobin chelate iron, thus making it less available to the bacteria. To overcome this problem, Yersinia spp. have a number of iron acquisition systems. The presence of such systems essentially distinguishes low- and high-pathogenicity strains. The highly pathogenic strains, Y. pestis, Y. pseudotuberculosis 0:01, and Y. enterocolitica biotype 1B, share a common iron uptake system called FyuA/Psn-Irp (Boyd and Cornelis, 2001). This system uses yersiniabactin, a novel catechol-containing siderophore that can remove iron from a number of mammalian proteins because of its extremely high affinity for ferric iron. This compound forms a ferrisiderophore complex with iron and then enters the bacteria after binding to a 65-kDa outer membrane protein receptor named FyuA. A number of other iron acquisition systems and iron-regulated proteins have also been identified. The production of these proteins requires the irp2 gene, which is found only in highly virulent yersiniae (De Almeida et al., 1993). Although low-virulence serovars of Y. enterocolitica do not produce these proteins or yersiniabactin, they are capable of acquiring iron from a number of sources, in-

cluding as ferrisiderophore complexes. This has important clinical implications, because siderophores, such as desferrioxamine B, are used therapeutically to reduce iron overload in patients with hemosiderosis and other forms of iron intoxication. Utilization of this siderophore by the organism may explain why septicemia with low-virulence Y. enterocolitica is sometimes observed in these patients. The bacteria may be able to proliferate in tissues where under normal circumstances poor availability of iron would limit their growth. Energy-dependent transport of inorganic iron, analogous to pathways in E. coli, is induced by iron-deficient conditions in all three pathogenic Yersinia species. Under iron-replete conditions, iron transport is repressed and excess iron is stored inside the bacteria. Virulence Plasmid All virulent yersiniae carry a 70- to 75-kb virulence plasmid, referred to as the plasmid for Yerisina virulence (pYV). It is highly conserved among the three species. This plasmid encodes the Yop virulon, an integrated virulence apparatus, as well as an outer membrane protein adhesin called YadA. Nonpathogenic strains lacking this plasmid are unable to cause infection, although they are located intracellularly in vacuoles of PMNs and mononuclear cells. In contrast, virulence plasmid–carrying bacteria are not phagocytosed and remain extracellular, even though they are surrounded by inflammatory cells (Lian et al., 1987). Thus, virulence plasmid–encoded proteins are involved in the resistance of Yersinia spp. to phagocytosis by PMNs and macrophages. These proteins are also involved in inhibition of the PMN oxidative burst, in induction of programmed cell death in macrophages, and in inhibition of the cytokine release that is normally induced by Yersinia spp. infection, thereby limiting the host’s inflammatory response to the infection (Boyd and Cornelis, 2001). Some of the important proteins encoded by this plasmid that are involved in virulence mechanisms of Yersinia spp. are described in the following. YadA YadA, formerly known as YopA, Yop1, or P1, is a 45kDa outer membrane protein, which polymerizes to form fibrillike structures on the bacterial surface. YadA can bind a variety of eukaryotic extracellular and cell surface molecules, including collagens, fibronectin, and laminins, and can mediate internalization of the bacteria into eukaryotic cells, a process in which interaction with β1 integrins plays a role (Boyd and Cornelis, 2001). YadA also contributes to the protection of Y. enterocolitica against killing by the antimicrobial polypeptides from the cytoplasmic granules of

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PMNs that are normally released into the phagolysosome containing the phagocytosed Yersinia spp. In addition, YadA confers resistance to complement-mediated opsonization by binding factor H and reducing deposition of C3b on the bacterial surface (China et al., 1993). In this way, YadA is associated with the ability of yersiniae to resist killing by serum complement, to resist phagocytosis, and to inhibit the respiratory burst of PMNs, all of which require the bacteria to be preopsonized. Structure-function studies have shown a number of functional domains in the YadA protein. The N terminus of the protein contains a typical secretion signal sequence of 25 amino acids. This is followed by amino acids 29–81, which are required for adherence to PMNs (Roggenkamp et al., 1996). Residues 83–101 compose one of the hydrophobic domains of YadA and are required for autoagglutination, and binding to collagen and laminin. Histidines 156 and 159, which are also important for collagen and laminin binding, are needed for binding to fibronectin and to epithelial cells. Finally, the hydrophobic C-terminal amino acids of YadA are involved in surface exposure of the protein and polymer formation (Boyd and Cornelis, 2001). Ysc Secretion System The inner and outer membranes of gram-negative bacteria act as major barriers to protein export. Until recently, only two mechanisms of protein secretion by gramnegative bacteria were known (Robins-Browne, 1997). One of these pathways, termed type I, requires the secreted protein to carry a specific recognition sequence close to the carboxyl terminus and is exemplified by the HlyA hemolysin of E. coli (see Chapter 13). The type II secretion (also known as the general secretory pathway) involves Sec-dependent recognition of a conserved amino-terminal signal sequence, which is removed during secretion. Recently, a novel secretory system has been identified in gram-negative bacteria. This family of type III secretion systems is called contact-dependent because intimate contact between the eukaryotic cell and bacteria triggers secretion and allows delivery of bacterial proteins inside eukaryotic cells. Type III secretion systems have been identified in several animal pathogens, including Salmonella spp., Shigella flexneri, enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, see Chapter 13), Pseudomonas aeruginosa, Chlamydia psittaci, and Bordetella spp.; and plant pathogens that elicit the hypersensitive response and pathogenesis, including Erwinia amylovora, Pseudomonas syringae, Xanthomonas campestris, and Ralstonia solanacearum (Alfano and Collmer, 1997; Hueck, 1998; Yuk et al., 1998; Boyd and

Cornelis, 2001). Some homologs in the type III secretion pathway identified in selected pathogens are listed in Table 12.41. The virulence plasmid encodes a set of proteins that are involved in establishing and building a Yop-specific secretion III apparatus through the bacterial membranes of Yersinia spp. A schematic diagram of the type III or contact-dependent secretion system of Yersinia spp. is shown in Figure 12.7. In Yersinia spp., 29 ysc secretion genes have been identified within four contiguous loci, and, although a mutation in almost any one of these genes causes a lack of secretion, little is known about the exact function of the various encoded components (Boyd and Cornelis, 2001). Four proteins, LcrD, YscD, YscR, and YscU, span the inner membrane (Figure 12.19); YscC is an outer membrane protein that belongs to the family of secretins, a group of outer membrane proteins involved in the transport of various macromolecules and filamentous phages across the outer membrane. The VirG lipoprotein is required for efficient targeting of this YscC complex to the outer membrane, where it forms a ring-shaped pore structure with an external diameter of about 200 Å and an apparent central pore of 50 Å. YscN contains ATP-binding motifs resembling the β-catalytic subunit of FoF1 proton translocase and related ATPases and presumably energizes the secretion of Yops. The type III Ysc secretion proteins are thought to form a structure similar to that of the apparatus involved in flagellar assembly (Lee, 1997; Boyd and Cornelis, 2001).

Table 12.41

Yops The Ysc secretion system is required for the specific secretion of the virulence plasmid–encoded Yop proteins. To date, 14 Yops have been identified (Table 12.40). They appear to be very well conserved among the Yersinia species. Most of the Yop proteins are essential for virulence. The information necessary for Yop secretion is contained in the N-terminus, and the shortest region shown to be sufficient for secretion of a Yop was found to be 15 amino acids for YopE (Sory et al., 1995). The sequence shows no resemblance to classical signal sequences; nor is there any homology between the amino termini of different Yops. Similarly, unlike in proteins secreted by the type II system, there is no cleavage of this secretion signal during export from bacteria. The biochemical properties of individual Yop proteins have been reviewed (RobinsBrowne, 1997; Boyd and Cornelis, 2001). In vitro, all Yops are produced at 37°C, but not at temperatures below 30°C, in the absence or minimal concentration of Ca2+ sufficiently low enough to induce bacteriostasis. The correlation of Yop secretion with growth arrest has long been known as Ca2+ dependency. There seem to be two different regulatory networks. The first permits full expression of all the virulence plasmid– encoded virulence functions when the environment is ideal and the temperature reaches 37°C; the second only prevents Yop production in the presence of 2.5-mM Ca2+ ions. Thus, the presence of Ca2+ ions blocks not only the

Some Homologs in the Type III Secretion Pathway

Yersinia spp.

Shigella spp.

Salmonella spp.

LcrD YopN (= LrcE) YscC YscF YscJ ( = YlpB) YscL YscN YscO YscP YscQ YscR YscS YscT YscU

MxiA MxiC MxiD MxiH MxiJ

InvA InvE InvG

Spa47 Spa13 Spa32 Spa33 Spa24 Spa9 Spa29 Spa40

SpaL SpaM SpaN SpaO SpaP SpaQ SpaR SpaS

Source: From Robins-Browne (1997).

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Pseudomonas solanacearum

Xanthomonas campestris

HrpO

HrpC2

HrpA

HrpA1

Hrp1 HrpF HrpE

HrpB3

HrpN

HrpB6

Figure 12.19 Model of the Ysc secretion apparatus. The Syc chaperones bind to their newly synthesized partner Yops to prevent Yop misfolding, inappropriate protein-protein interaction, and/or degradation. The Yops associate with the secretion apparatus, pass through, and are then released from the bacterium. The Ysc machinery comprises the YscN ATPase; the inner membrane proteins YscR, YscU, and YscD; the lipoproteins VirG and YscJ; the proteins YscS, LcrD, and YscT; and, in the outer membrane, the secretin YscC. ATPase, adenosine triphosphatase.

secretion of Yops but also their synthesis. The former process is a key stimulus for the production of Yops in vivo during infection. The secretion of Yops involves coupling of the Yop to a specific chaperone termed specific Yop chaperone (Syc) that guides it to the export machinery. The genes encoding these chaperones are generally situated beside the genes encoding the Yops they aid. Yop chaperones are analogous to SecB in the general secretory pathway (type

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II system). They differ from SecB, however, in that the latter binds many different proteins, whereas Yop chaperones are highly specific and are named for the individual Yops with which they associate. To date, specific Syc chaperones have been identified for YopE (SycE/year), YopH (SycH), YopN (SycN), YopT (SycT), and the chaperone SycD/LcrH for both YopB and YopD (Boyd and Cornelis, 2001). Although they have no significant homology with each other, all Yop chaperones identified to date are acidic,

low-molecular mass (15- to 18-kDa) proteins that probably occur in the bacterial cytoplasm as dimers (Cornelis, 1994). The secreted Yops include translocators and effectors. The translocators (YopB, YopD, and LcrV) presumably form a pore in the eukaryotic cell membrane through which the effector Yops (YopE, YopH, YopP, YopO, YopT, YopM) enter the cytosol of the host cell. A domain important for translocation of the Yop effectors is found in their N-terminal domain, which immediately follows the secretion domain (Sory et al., 1995). This translocation domain is also the chaperone-binding domain, thereby raising the possibility that the Syc chaperones play a role in the translocation of Yops, as well as in their secretion (Boyd and Cornelis, 2001). Extracellular bacteria adhering to the eukaryotic cell surface carry out the translocation event. Contact between the bacteria and the cell is necessary for the translocation of the Yops inside the eukaryotic cell, as neither Yops produced by nonadherent bacteria nor purified secreted Yops have the ability to enter eukaryotic cells (Sory and Cornelis, 1994). As mentioned earlier, Yersinia spp. only secrete Yops in the absence of Ca2+ and at 37°C. However, contact between the bacterium and the eukaryotic cell is the signal inducing Yop secretion in vivo. The Yop secretion is highly directional in the sense that the majority of the Yop effector molecules produced are directed into the cytosol of the eukaryotic cell and not to the external milieu (Boland et al., 1996). Once inside the host cell, the Yop effectors interfere with the signaling cascades of the host cells. These disruptions of the normal cell processes can be visualized by morphological alterations in the target cell, from cytotoxic rounding up or apoptosis. Two Yop effectors, YopE and YopT, cause disruption of the cytoskeleton, which leads, at least in the case of YopE, to inhibition of phagocytosis (Boyd and Cornelis, 2001). YopH also inhibits phagocytosis, but in this case by dephosphorylating key signaling proteins. In contrast, YopO phosphorylates proteins, but it presumably also interferes with signaling pathways in the cell. YopP disrupts transcriptional signaling in the cell, leading to apoptosis and a lack of induction of cytokine release. This multipronged attack is quite an efficient way of killing a cell. 12.8.3

Symptoms, Diagnosis, and Treatment

An inoculum of 108–109 yersiniae must enter the alimentary tract to produce infection. Gastric acid appears to be a significant barrier to infection. During the incubation period of 24–48 hours (sometimes as long as 5–10 days),

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yersiniae multiply in the gut mucosa, particularly the ileum. This process leads to inflammation and ulceration, and leukocytes appear in feces. The process may extend to mesenteric lymph nodes and, rarely, cause bacteremia. Early symptoms of yersiniosis include fever, abdominal pain, and diarrhea. Diarrhea ranges from watery to bloody and may be due to an enterotoxin or to invasion of the mucosa. At times, the abdominal pain is severe and located in the right lower quadrant, suggesting appendicitis. At 1 to 2 weeks after onset arthralgia, arthritis, and erythema nodosum develop in some patients, suggesting an immunological reaction to the infection. Very rarely, yersiniae infection produces pneumonia, meningitis, or sepsis; in most cases, it is self-limited. Although most episodes of yersiniosis remit spontaneously without long-term sequelae, infections with Y. enterocolitica are noteworthy for the large variety of immunological complications, including reactive arthritis, erythema nodosum, iridocyclitis, glomerulonephritis, carditis, and thyroiditis, after an acute infection (Cover and Aber, 1989). Other autoimmune complications of yersiniosis include Reiter’s syndrome, iridocyclitis, acute proliferative glomerulonephritis, and rheumaticlike carditis. These complications are almost exclusively reported from Scandinavian countries (Larsen, 1980). Yersiniosis has also been linked to various thyroid disorders, including Graves’ disease hyperthyroidism, nontoxic goiter, and Hashimoto’s thyroiditis, although the causative role of yersiniae in these conditions is not proved (Toivanen and Toivanen, 1994). Diagnosis of yersiniosis begins with isolation of the organism from the human host’s feces, blood, or vomit, and sometimes at the time of appendectomy. The number of yersiniae in stool may be small and can be increased by “cold enrichment” at 4°C for 2–4 weeks; many fecal organisms do not survive, but Y. enterocolitica multiply. Confirmation occurs with the isolation, as well as biochemical and serological identification, of Y. enterocolitica from both the human host and the ingested foodstuff. Because of the difficulties in isolating yersiniae from feces, several countries rely on serological evaluation. Acute and convalescent patient sera are titered against the suspect serotype of Y. enterocolitica. In paired serum specimens taken 2 weeks or more apart, a rise in agglutinating antibodies can be shown; however, cross-reactions between yersiniae and other organisms (vibrios, salmonellae, brucellae) may confuse the results. Most yersinia infections with diarrhea are self-limited, and the possible benefits of antimicrobial therapy are unknown. Y. enterocolitica is generally susceptible to aminoglycosides, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, piperacillin, third-generation

cephalosporins, and fluoroquinolones. It is typically resistant to ampicillin and first-generation cephalosporins. Proven yersinia sepsis or meningitis has a high mortality rate; fatalities occur mainly among immunocompromised patients. Yersinia sepsis can be successfully treated with third-generation cephalosporins (possibly in combination with an aminoglycoside) or a fluoroquinolone (possibly in combination with another antimicrobial). In cases in which clinical manifestations strongly point to either appendicitis or mesenteric adenitis, surgical exploration has been the rule unless the occurrence of several simultaneous cases indicates that yersinia infection is likely. 12.8.4

Sources

Y. enterocolitica has been isolated from rodents and domestic animals (e.g., sheep, cattle, swine, dogs, and cats) and waters contaminated by them. It has also been isolated from frogs, fish, crabs, and oysters. Foods that may harbor Y. enterocolitica include pork, beef, lamb, poultry, and dairy products, notably milk, cream, and ice cream (Robins-Browne, 1997). The main source of Y. enterocolitica is undoubtedly pigs, which are known to carry the specific pathogenic strains (especially serovars of O:3) on occasions. De Boer and associates (1986), in a detailed survey of a variety of foods, found the highest isolation rates on raw pork (73%), unpasteurized eggs (43%), raw vegetables (43%), and raw beef (42%), and a surprisingly low recovery rate (10%) of Y. enterocolitica from raw milks, often implicated in outbreaks of yersiniosis. Cattle, however, do not appear to be an important reservoir of these bacteria. These authors stressed that only 4% of the isolates from foods were recognized pathogenic strains; all were obtained from pig samples. In contrast, Anderson (1988) reported that 25% of pig carcasses were contaminated with human pathogenic Y. enterocolitica.

Table 12.42 Year 1976 1976 1980 1981 1981 1982 1982 1983 1989 a

12.8.5

Outbreaks

Considering the widespread occurrence of Y. enterocolitica in nature and its ability to colonize food animals, to persist within the environment, and to proliferate at refrigeration temperatures, yersiniosis does not occur frequently. It is rare unless a breakdown occurs in food processing techniques. The CDC estimates that about 17,000 cases occur annually in the United States (FDA, 1996). Yersiniosis is a far more common disease in northern Europe, Scandinavia, and Japan. Since the 1980s, there has been a marked increase in the number of reported cases of yersiniosis worldwide, but this may be at least partly due to our greater awareness of the pathogenic nature of the organism. Most food-borne outbreaks in which a source has been identified have been traced to milk (Table 12.42). A very large outbreak involving 16,000 people occurred in the United States in 1982 (Dirksen and Flagg, 1988); contamination was traced to a dairy in Tennessee from which outdated milk was transported, in crates, to a pig farm, where the milk was unloaded. On return the crate washing proved inadequate and the crates contaminated milk carton exteriors, on which the yersiniae were shown to survive for up to 21 days. During the mid-1970s, two outbreaks of yersiniosis caused by Y. enterocolitica O:5,27 occurred among 138 Canadian schoolchildren who had consumed raw milk, but the organism was not recovered from the suspected source (Kasatiya, 1976). In 1976, serogroup O:8 Y. enterocolitica was responsible for an outbreak in New York state that affected 217 people, 38 of whom were culture-positive (Black et al., 1978). The source of infection was chocolate-flavored milk, which evidently became contaminated with Y. enterocolitica after pasteurization. In yet another outbreak in 1981 involving the same serotype, the source of infection was traced to reconstituted powdered milk and/or chow mein, which probably became contaminated

Selected Food-Borne Outbreaks of Yersiniosis Location

Canada New York Japan New York Washington Pennsylvania Southern United States Hungary Atlanta

Cases, no. 138 38 1,051 159 50 16 16,000 8 15

Serovar

Source

Reference

O:5,27 O:8 O:3 O:8 O:8 O:8 O:13a, 13b O:3 O:3

a

Kasatiya (1976) Black et al. (1978) Maruyama (1987) Shayegani et al. (1983) Tacket et al. (1985) Cover and Aber (1989) Dirksen and Flagg, 1988 Marjai et al. (1987) Lee et al. (1990)

(?), bacteria were not isolated from the incriminated source.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Raw milk (?) Flavored milk Milk Powdered milk, chow mein Tofu, spring water Bean sprouts, well water Milk (?) Pork cheese (sausage) Pork chitterlings

during preparation by an infected food handler in New York state (Morse et al., 1984). The outbreak affected about 35% of 455 individuals at a diet camp. Seven patients were hospitalized as a result of infection; five of them underwent appendectomy. In yet another major outbreak, in the metropolitan Atlanta area, the source of infection was traced to transmission of bacteria from raw chitterlings (pig intestine) to affected children on the hands of food handlers. It affected 15 infants and children (Lee et al., 1990). Other foods implicated in sporadic outbreaks of yersiniosis include pork cheese (a type of sausage prepared from pork chitterlings), bean sprouts, and tofu washed with contaminated well or spring water. However, in many yersiniosis outbreaks, the source was often not identified. 12.8.6

Prevention and Control

Contact with farm and domestic animals, their feces, or materials contaminated by them probably accounts for most human infections. Meat and dairy products have occasionally been indicated as sources of infection, and group outbreaks have been traced to contaminated food or drink. Conventional sanitary precautions are probably helpful. There are no specific preventive measures.

12.9 AEROMONAS SPECIES Aeromonas spp., particularly A. hydrophila, are universally distributed in aquatic environments, and hence, it has often been assumed that infections are waterborne. However, many foods carry these bacteria. Some strains of A. hydrophila are capable of causing illness in fish and amphibians as well as in humans, who may acquire infections through open wounds or by ingestion of sufficient numbers of the organisms in food and water. Earlier, they were recognized as pathogens for cold-blooded animals (Sanarelli, 1891). The role of Aeromonas spp. as human pathogens became increasingly evident during the 1960s and 1970s (Caselitz, 1966; von Graevenitz and Mensch, 1968; Davis et al., 1978; Trust and Chipman, 1979). A wide variety of clinical manifestations are reported in both healthy and immunologically compromised hosts. 12.9.1

and fermentative metabolism (Blair et al., 2000). They grow over a wide range of environmental conditions, with pH values ranging from 4.0 to 10.0 (optimum around neutrality) and salt concentrations of up to 6.5%. Most members of the genus are mesophiles with an optimal growth temperature of 28°C. Some Aeromonas spp. can grow at temperatures ranging from 4°C to 42°C. Most members of the genus are motile, and flagella, if present, are singular and polar. Their colony morphological characteristics are similar to that of enteric gram-negative rods, and they produce large zones of hemolysis on blood agar. The taxonomy of Aeromonas spp. is complex and still evolving. The genus was previously made up of the species A. hydrophila, A. salmonicida, A. sobria, and A. caviae. Using a DNA-DNA hybridization technique, Popoff (1984) demonstrated that there are at least nine distinct hybridization groups (HGs) among the A. hydrophila strains and that these fall into three main phenotypic groups that can be identified on the basis of the reactions of isolates in 8 to 18 biochemical tests. The 10 genospecies recognized were as follows: A. hydrophila, A. salmonicida, A. caviae, A. media, A. veronii biovar sobria, A. veronii biovar veronii, A. jandaei, A. schubertii, A. trota, and A. allosaccharophila. The three main phenospecies were designated as A. hydrophila, A. sobria, and A. caviae. Most clinical laboratories report Aeromonas spp. isolates as members of 1. 2.

3.

The A. caviae group, which includes A. caviae, A. eucrenophila, and A. media The A. hydrophila group, which includes A. hydrophila and a motile biogroup of A. salmonicida The A. sobria group, which includes A. sobria and A. veronii

Possible new approaches for the identification of aeromonads based on genetic methods include PCR assays with16S rRNA gene-targeted species-specific oligonucleotide primers, determination of rRNA gene restriction patterns, genomic fingerprinting technique that detects DNA polymorphisms by selective amplifications of restriction fragments, MLEE, phage typing, and serotyping (Kirov, 1997; Blair et al., 2000). Currently recognized genospecies and phenospecies of the genus Aeromonas are listed in Table 12.43.

Organism 12.9.2

The genus Aeromonas is a member of the family Vibrionaceae, which includes four other genera: Vibrio, Photobacterium, Plesiomonas, and Enhydrobacter. Aeromonas spp. are chemoorganotrophic, gram-negative, oxidase-positive, facultative anaerobes that demonstrate both respiratory

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Pathogenesis

Most work on the possible virulence factors of aeromonads has been done with strains isolated from patients with gastroenteric disease. Virulence factors of Aeromonas species associated with clinical manifestations of the dis-

Table 12.43 Currently Recognized Genospecies and Phenospecies of the Genus Aeromonas Genospecies

DNA group

2 3

Aeromonas hydrophila A. hydrophila A. salmonicidaa

4 5A 5B 6 7 8 9 10 11 12 13 14

A. caviae A. caviae A. caviae A. eucrenophila A. sobria A. veronii A. jandaei A. veronii A. veronii A. schubertii A. schubertii A. trota

1

Phenospecies A. hydrophila Unnamed A. hydrophila, A. salmonicida A. caviaeb Unnamed A. media A. eucrenophilac A. sobriac A. veronii biovar sobriad A. jandaei A. veronii biovar veroniid Unnamed A. schubertii “Group 501” A. trota

Table 12.44 Species

Potential Virulence Factors of Aeromonas

Extracelluar enzymes (e.g., proteases, lipases, elastase) Siderophores (enterobactin, amonabactin) Exotoxins Cytotoxic (cytolytic enterotoxin, aerolysin, Asao toxin, β-hemolysin)a Cytotonic Heat-stable (56°C, 10–20 min), cholera toxin (CT) crossreacting and non-cross-reacting Heat-labile, non-CT cross-reacting Endotoxins (lipopolysaccharides) Invasins (invasion of HEp-2 and Caco-2 cells) Adhesins Type IV pili Outer membrane proteins (lectinlike, possibly porins) S-layers a

Synonyms. Source: From Kirov (1997).

a

Includes psychrotrophic strains originating from fish. These strains are biochemically distinct (nonmotile and indole-negative) from HG3 isolated recovered from clinical samples. b Some believe that HG4 should be named A. punctate, since the type strain for both species is the same and A. punctate is the older name in the literature. c HG6 and HG7 have not yet been recovered from clinical samples. d HG8 and HG10 are genetically identical, but the type strains are biochemically distinguishable; hence, they are now called biovars of the first described species, A. veronii. Source: Compiled from Joseph and Carnahan (1994) and Kirov (1997).

ease include the expression of extracellular proteins such as exotoxins and exoenzymes, endotoxin (LPS layer), presence of S-layers, presence of fimbriae or adhesins, and production of capsular layers (Table 12.44). S-Layer Proteins The S-layer of A. salmonicida is the only Aeromonas virulence factor linked to overt pathogenicity for salmonids. These are macromolecular arrays of protein subunits (49 to 58 kDa) found on the bacterial cell surface. The biological functions of these proteins are not yet known. However, S-layer increases the capacity of organisms to adhere to the gut mucosa, contributes to protection against the bactericidal activities of both immune and nonimmune serum, influences the outcome of interaction of the organism with macrophages, protects against the action of proteases, and binds immunoglobulins (Trust, 1993). S-layer-producing strains have been particularly associated with a single

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lipopolysaccharides serogrop, O:11, of Aeromonas species (A. hydrophila, A. sobria), which contains some highly virulent strains frequently associated with gastroenteritis (Martinez et al., 1995; Kirov, 1997; Blair et al., 2000). Serogroup O:11 strains, as well as serogroup O:34 strains of A. hydrophila and A. veronii biovar sobria, which reportedly do not possess S-layers but are associated with septicemic disease, produce a capsular LPS during growth in glucose-rich media. Preliminary studies have indicated that possession of this capsule enhances the virulence of these strains (Martinez et al., 1995). Extracellular Enzymes Unlike most other gram-negative organisms, Aeromonas species produce a range of extracellular enzymes and toxins. These include proteases (at least four or five, including a thermolabile serine protease and a thermostable metalloprotease), DNase, ribonuclease (RNase), elastase, lecithinase, amylase, lipases, gelatinase, and chitinase. The roles of the enzymes in virulence have yet to be determined. Proteases may contribute to pathogenicity by causing direct tissue damage or enhancing invasiveness. In addition, they may activate hemolysin. Other enzymes may also constitute virulence factors by interacting with human leukocytes or by affecting several immune system functions by free fatty acids generated during lipolytic activity (Kirov, 1997). Aeromonas spp. that do not express proteases show reduced virulence, indicating that these enzymes contribute to the pathogenic nature of A. hydrophila.

Hemolysins Hemorrhage is a conspicuous feature of Aeromonas spp.–induced infections, which are often referred to as red sore disease and red leg disease in some ectothermic animals. Soft tissue Aeromonas spp. infections in humans may also be indistinguishable from infections caused by Streptococcus pyogenes (Hanson et al., 1977; Rigney et al., 1978; Ljungh and Wadstrom, 1986). These clinical observations, combined with the fact that the vast majority of strains are hemolytic on blood agar, indicate that hemolysin may be an important virulence factor in the pathogenesis of A. hydrophila infections. Aeromonads produce at least two major classes of hemolysins. Wretlind and colleagues (1971, 1973) studied the hemolysin that is released from cells during the late stationary growth phase. It is designated α-hemolysin. The second hemolysin, which is released toward the end of the logarithmic growth phase, is probably identical to the cytolytic toxin called aerolysin (Bernheimer and Avigad, 1974; Bernheimer et al., 1975) and cytotoxic protein (Ljungh et al., 1978). It is now designated β-hemolysin. The α-hemolysin (65 kDa, pI 5.2) causes incomplete lysis of erythrocytes (double-zone lysis on blood agar). It is not expressed at temperatures above 30°C and has not been associated with enterotoxic properties. The hemolytic activity is destroyed by several proteolytic enzymes and is inactivated by DTT and zinc ions (Ljungh et al., 1981). The protein activity is also susceptible to repeated freeze-thaw. α-Hemolysin is dermonecrotic in the rabbit skin and lethal for mice as well as for rabbits (Wretlind et al., 1971). The hemolytic activity varies with erythrocytes from different animal species; rat erythrocytes are most sensitive, sheep the least (Ljungh et al., 1978). It is also cytotoxic to HeLa cells and human embryonic lung fibroblasts. The cytotoxic effects are dose-dependent and temperature-dependent with maximal effect at 37°C. The action of α-hemolysin seems to be restricted to the membrane and may be of enzymatic character. The β-hemolysin (aerolysin) (49–53 kDa, pI ~5.5) causes complete lysis of erythrocytes (clear zones of hemolysis on blood agar). The hemolytic activity is easily destroyed by heat (56°C, 5 minutes). The protein, however, is extremely resistant to destruction by proteolytic enzymes, including pronase, trypsin, subtilisin, and papain. Aerolysin ultimately produces a transmembrane channel that destroys cells by breaking their permeability barriers (van der Goot et al., 1994). There are two inactive forms of the toxin. The first, preproaerolysin, contains a typical 23-amino-acid sequence that directs transport across the inner bacterial membrane and is removed dur-

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ing transit. The resulting protoxin is exported and then activated by proteolysis of about 25 amino acids from the carboxy-terminal end. Aerolysin binds to the eukaryotic receptor glycophorin (Buckley, 1991). After binding, it oligomerizes, in an essential step in channel formation. This step precedes membrane insertion. β-Hemolysin is lethal to mice, rats, and rabbits (Ljungh et al., 1978). It is dermonecrotic in rabbit skin, inducing capillary bleeding, induration, and necrosis. It is also cytotoxic to a wide variety of tissue culture cells, such as HeLa cells, green monkey kidney cells, human diploid lung fibroblasts, adrenal Y1 cells, and CHO cells. Enterotoxins Indian researchers first demonstrated the enteropathogenicity in strains of A. hydrophila by injecting whole cells into ligated rabbit intestinal loops (Sanyal et al., 1975). Subsequently, Wadstrom and coworkers (1976) and Annapurna and Sanyal (1977) independently showed the presence of an extracellular heat-labile enterotoxin. Aeromonas spp. enterotoxin induces fluid accumulation in the rabbit intestinal loop, as well as in rat and mouse loops (Ljungh and Kronevi, 1982). Like cholera toxin, it produces a positive rabbit skin test result, though the dermonecrosis of the hemolysins can occlude the increased capillary permeability and induration caused by enterotoxin. Several cytotonic enterotoxins have been described for Aeromonas spp., including a 15- to 20-kDa heat-stable protein (Ljungh et al., 1982) and a 44-kDa heat-labile protein identified by Chopra and Houston (1989). Aeromonas spp., therefore, may produce different types of cytotonic enterotoxins that are functionally similar. However, only a minority of strains may produce these enterotoxins. Seidler and associates (1980) reported their production in <6% (20 of 330) of strains. Shimada and colleagues (1984) demonstrated the production of a cholera toxin– (CT)-like enterotoxin by Aeromonas spp. in ~4.5% (8 of 179) of strains tested. Most strains of A. veronii biovar sobria (HG8/10), A. hydrophila (HG1, HG2, and HG3), and A. caviae (HG4), whether from clinical or environmental sources, are potentially enterotoxigenic. Endotoxin LPS from Aeromonas spp., like endotoxins from other gram-negative bacteria, has a range of pathogenic effects on animals. The secretion of some exotoxins has been reported to be dependent on the presence of the O-antigen LPS; strains lacking the O-antigen LPS (rough mutants) secrete less toxin than those strains rich in O-antigen LPS

(smooth strains) (Blair et al., 2000). The possession of Oantigen LPS has been reported to be temperature-dependent (smooth at 20°C, rough at 37°C); some strains that possess LPS are more virulent when grown at low temperatures. Fimbriae/Adhesins Aeromonas spp. possess filamentous (fimbriae) and nonfilamentous (outer membrane, S-layer-synonymous) adhesins, which are intimately involved in the adhesion of the bacterium to host cells. Most investigations of Aeromonas spp. adhesion to cultured mammalian cell lines, such as mouse Y1 adrenal, HEp-2, and human intestinal cell lines such as INT407 (embryonic human intestinal cell line) and Caco-2 (colon carcinoma cells that differentiate in culture to express characteristics of small intestinal enterocytes), have shown that strains of A. veronii biovar sobria are the most adhesive (having the highest proportion of adherent strains and the strains with the greatest number of adherent bacteria per cell) (Kirov, 1993a, 1997; Grey and Kirov, 1993). Clinical strains of A. caviae (>30%) were also found to be adherent, whereas A. hydrophila adhesion seems to be less efficient. Thus, there may be species differences in adhesive mechanisms. The mechanisms of Aeromonas spp. cell line adhesion and invasion remain to be elucidated. Siderophores Efficient mechanisms for iron acquisition from the host during an infection are considered essential for virulence. Mesophilic aeromonads produce either of two siderophores, enterobactin or amonabactin. The former is found in many gram-negative bacteria; the latter is found only in Aeromonas spp. Amonabactin is the predominant siderophore in A. hydrophila (HG1, HG2, and HG3), A. caviae (HG4), A. media (HG5), A. schubertii (HG12), and A. trota (HG14); A. veronii biovar sobria (HG8/10) and A. jandaei (HG9) make enterobactin (Kirov, 1997).

Extraintestinal infections with Aeromonas spp. are rare; however, they tend to be severe and often fatal (Kirov, 1997). These include septicemia, peritonitis, endocarditis, pneumonia, conjunctivitis, and urinary tract infections. Those at risk are individuals suffering from leukemia, carcinoma, and cirrhosis and those treated with immunosuppressive drugs or undergoing cancer chemotherapy. Aeromonas spp. are also the causative agents of wound infections, usually linked to water-associated injuries or aquatic recreational activities (Gold and Salit, 1993). There appears to be a species-associated disease spectrum. A. veronii biovar sobria (HG8/10) is more frequently associated with bacteremia than the other species. A. schubertii (HG12) is associated with aquatic wound infections but as yet has not been documented in association with gastroenteritis (Carnahan et al., 1991a). A. hydrophila (HG1 and HG3) also features in wound infections and after the use of medicinal leeches (Snower et al., 1989). A. veronii biovar sobria (HG8/10), A. hydrophila (HG1 and HG3), A. caviae (HG4), and to a lesser extent A. veronii biovar veronii (HG8/10), A. trota (HG14), and A. jandaei (HG9) are the species most commonly associated with gastroenteritis (Carnahan et al., 1991a, 1991b; Janda, 1991; Joseph and Carnahan, 1994; Kirov, 1997). A. veronii biovar sobria has been associated with dysentery more frequently than the other species. A. caviae (HG4) is most common in pediatric diarrhea (Namdari and Bottone, 1991). A. hydrophila can be cultured from stools or from blood by plating the organisms on an agar medium containing sheep blood and the antibiotic ampicillin. Ampicillin prevents the growth of most competing microorganisms. The species identification is confirmed by a series of biochemical tests. The ability of the organism to produce the enterotoxins believed to cause the gastrointestinal symptoms can be confirmed by tissue culture assays. Aeromonas strains are susceptible to tetracyclines, aminoglycosides, and cephalosporins. 12.9.4

12.9.3

Sources

Symptoms, Diagnosis, and Treatment

Aeromonas spp.–associated gastroenteritis occurs most commonly in children, the elderly, and the immunocompromised. There is a distinct seasonal pattern: most cases occur in summer. The most often reported clinical manifestation is a watery diarrhea accompanied by a mild fever. Vomiting may also occur in children under 2 years of age. Diarrhea may last for over 2 weeks. In some patients, dysenterylike symptoms, blood and mucus in stools, may occur. These may be quite severe and may last for several weeks.

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Aeromonas species are primarily aquatic organisms. They are commonly present in drinking water and are found in sinks, drain pipes, and household effluents. However, many foods carry these organisms. A. hydrophila has frequently been found in fish and shellfish. Fricker and Tompsett (1989) reported carriage rates for A. hydrophila and A. veronii biovar sobria of 69% in poultry, 21% in both pork and raw salad, 17% in beef, and 15% in fish. They concluded that both raw and cooked foods were potential sources of human infection. The ability of these organisms to grow and produce toxin at refrigeration

1984). There are relatively few published reports in which Aeromonas species have been associated with food-borne gastroenteritis. Most cases have been sporadic, rather than associated with large outbreaks. In fact, by 1998 there had not been a fully confirmed outbreak in the United States (CFSAN, 1998). Two outbreaks attributed to Aeromonas species have been reported in Japan (Kohbayashi and Ohnaka, 1989) and in Sweden, where 22 of 27 persons became ill 30 to 35 hours after consumption of a smorgasbord containing shrimps, smoked sausage, liver pate, and boiled ham, all of which contained a high number of aeromonads (106 to 107/g of food sample) in virtually pure culture (Krovacek et al., 1995). Fortunately, psychrotrophic strains of Aeromonas spp., which can produce high levels of toxins in many foods, do not appear to be common. Moreover, toxins are inactivated by some foods, such as milk (Kirov, 1997). Thus, disease resulting from intoxication seems an unlikely risk in comparison with the possible risk posed by strains able to colonize the human intestine and express virulence properties in vivo.

temperatures and its recovery from raw milk, beef, lamb, pork, chicken, fish, and shellfish indicate its potential as a food-borne pathogen. Some representative data on the prevalence of Aeromonas species in retail foods are summarized in Table 12.45. Although the organism occurs worldwide, the incidence of different Aeromonas species found in foods varies between and within countries. The most prevalent Aeromonas spp. isolate from food in Europe is A. hydrophila, whereas in Japan, A. caviae is the most prevalent food isolate (Blair et al., 2000). These observations suggest either that there are distinct differences in the geographical incidence of particular species of Aeromonas spp. in foods or that differences among the isolation methods used in different countries result in different isolation rates. Aeromonas species, such as A. hydrophila, are important to the food industry because of their psychrotrophic nature and their ability to express a range of virulence factors under refrigerated storage conditions. Thus, they can be regarded as both spoilage and potentially pathogenic organisms. Aeromonads are not generally considered to be normal inhabitants of the gastrointestinal tract of humans. Fecal carriage rates can, however, approach 3% in asymptomatic persons in temperate climates (Kirov, 1997). In the tropics or developing regions, carriage rates may reach 30% or more. 12.9.5

12.10 PLESIOMONAS SHIGELLOIDES Like Aeromonas spp., Plesiomonas spp. belongs to the family Vibrionaceae. These two genera are very closely related, and differentiation between these two requires biochemical analyses (Table 12.46). Plesiomonas spp. may be more closely related to organisms within the family Enterobacteriaceae (Martinez-Murcia et al., 1992; Joseph and Carnahan, 1994). They are still regarded as controversial

Outbreaks

Increased levels of aeromonads in drinking water have been reported to coincide with increased incidence of Aeromonas spp.–associated gastroenteritis (Burke et al.,

Table 12.45

Prevalence of Aeromonas Species in Retail Foods

Food product Fish (freshwater) Raw milk Pasteurized milk Cold smoked fish Hot smoked fish Cooked/frozen shellfish Minced meat Ham Raw chicken Whipped cream Cream cakes/deserts Mayonnaise/salads Vegetables a

Positive, %

A. hydrophila

A. sobria

A. caviae

Reference

100.0 59.7 3.8 10.9 14.3 8.8 94.1 38.2 84.4 50.0 6.0 10.0 51.0

50.0 39.5 28.6 66.6 100.0 100.0 75.7 47.0 38.7 NDa ND ND 37.0

33.3 9.3 42.9 0.0 0.0 0.0 24.3 11.7 40.8 ND ND ND 0.0

16.7 34.9 28.6 33.3 0.0 0.0 27.0 41.3 20.5 ND ND ND 63.0

Sierra et al. (1995) Kirov et al. (1993) Kirov et al. (1993) Gobat and Jemmi (1993) Gobat and Jemmi (1993) Gobat and Jemmi (1993) Gobat and Jemmi (1993) Gobat and Jemmi (1993) Gobat and Jemmi (1993) Knochel and Jeppesen (1990) Knochel and Jeppesen (1990) Knochel and Jeppesen (1990) Nishikawa and Kishi (1988)

ND, not determined.

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Table 12.46 Differentiation Between Aeromonas and Plesiomonas Species Characteristic

Aeromonasa

Plesiomonas

+b

–

–c

+

dd d d + + – d d +e – 57-63

– – – – – + – – – + 51

Monotrichous flagella (liquid media) Lopotrichous flagella (liquid media) KCN, growth L-Arabinose, utilization Salacin, acid Sucrose, acid D-Mannitol, acid myo–Inositol, breakdown Voges-Proskauer D-Glucose, gas Lipase Sensitivity to O/129 Mol % G + C of DNA a

Except A. salmonicida. +, typically positive. c –, typically negative. d d, differs among species/strains. e Except A. media and A. salmonicida. Source: Compiled from Kirov (1997) and Blair et al. (2000). b

gastrointestinal pathogens, since definitive proof of enteropathogenicity is lacking. Plesiomonas shigelloides is a gram-negative rod with polar flagella. It is most common in tropical and subtropical areas. It has been isolated from freshwater fish and shellfish, and many animals, including cattle, goats, swine, cats, dogs, monkeys, vultures, snakes, and toads. Most isolates from humans have been from stool cultures of patients with diarrhea. Plesiomonas spp. grow on the differential media used to isolate salmonellae and shigellae from stool specimens. More than 100 serovars of P. shigelloides have been described (Kirov, 1997). Some strains share antigens with Shigella sonnei, and cross-reactions with shigella antisera occur. Plesiomonas spp. can be distinguished from shigellae in diarrheal stools by the oxidase test; Plesiomonas spp. is oxidase-positive and shigellae are not. Plesiomonas spp. is also positive for DNase. Most strains of P. shigelloides do not grow below 8°C; their optimal temperature for growth is 38°C to 39°C, with a maximum around 45°C (Farmer et al., 1992; Miller and Kohburger, 1985). The organism grows at pH 5 to 8 but may be particularly susceptible to low pH. Tolerance to salt seems similar to that of Aeromonas species. There are no virulence factors that are widely accepted as being important for Plesiomonas spp.–associ-

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ated infections, and none is tested for routinely. Potential virulence factors that have been described are summarized in Table 12.47. Very few of these have been investigated in any detail. The organism is suspected of being toxigenic and invasive. Endotoxin may play a role in Plesiomonas spp. virulence as it does for other enteric pathogens (Brenden et al., 1988). Several reports, summarized by Kirov (1997), concluded that plesiomonads do not produce enterotoxins. However, more than 90% of 36 Plesiomonas spp. strains tested produced β-hemolysin. The hemolysin was cell-associated and was produced at both 25°C and 35°C (Janda and Abbott, 1993). Large plasmids have been detected in strains from patients with Plesiomonas spp.–associated colitis, and these unstable virulence plasmids may be involved in Plesiomonas spp. pathogenicity (Kirov, 1997). P. shigelloides gastroenteritis is usually a mild selflimiting disease with fever, chills, abdominal pain, nausea, diarrhea, or vomiting. Symptoms may begin 20–24 hours after consumption of contaminated food or water. The infectious dose is presumed to be quite high, at least greater than 106 organisms. Patients who have stools that test positive for P. shigelloides have either a watery diarrhea or diarrhea with blood and mucus. The secretory form is usually reported to last from 1 to 7 days but can be prolonged (~3 weeks), with numerous (up to 30) bowel movements per day at the peak of the disease (Miller and Kohburger, 1985; Brenden et al., 1988). Patients often have severe abdominal cramps, vomiting, and some degree of dehydration. P. shigelloides may also be responsible for extraintestinal infections, including cellulitis, arthritis, endophthalmitis, and cholecystitis (Brenden et al., 1988; Kirov, 1997). Very few systematic studies of the incidence of P. shigelloides in foods have been carried out. The organism is found in fresh and estuarine waters, as well as seawater

Table 12.47 shigelloides

Potential Virulence Factors for Plesiomonas

Extracellular enzymes (e.g., elastase, proteases) Enterotoxins/cytotoxins Suckling mouse assay (SMA), ligated rabbit ileal loop (LRIL), lysis of Y1 cells (after passage in LRIL) CHO elongating factor (when grown in iron-poor medium) β-Hemolysin (cell-associated) Endotoxin Invasins (invasion of HeLa cells) Adhesins (glycocalyx?) Source: From Kirov (1997).

in warm weather (Madema and Schets, 1993). By the early 1990s, P. shigelloides had been isolated predominantly from fish and seafood (Farmer et al., 1992). P. shigelloides has been implicated in two waterborne epidemics involving over 1000 people (Tsukamoto et al., 1978). In one, a single predominant serotype (O17:H2) was identified in patients. This serotype was also found in tap water from different parts of the area where the outbreak occurred. Outbreaks of P. shigelloides–associated gastroenteritis have been attributed to contaminated oysters, chicken, fish (salt mackerel, cuttlefish salad), and shrimp (Brenden et al., 1988; Miller and Kohburger, 1985; Rutala et al., 1982). Oysters have been the major food incriminated in the United States (Kirov, 1997). Consumption of raw seafood and foreign travel (particularly to Mexico) have been identified as major risk factors in the United States.

12.11 BOVINE SPONGIFORM ENCEPHALOPATHY (“MAD COW DISEASE”) Noninflammatory degenerative central nervous system diseases that have similar pathological features are described as subacute transmissible spongiform viral encephalopathies (TSEs) or as transmissible degenerative encephalopathies (TDEs) (Table 12.48). The causative agents do not appear to be conventional viruses; infectivity is associated with proteinaceous material devoid of detectable amounts of nucleic acid. The term prion is used to designate this novel class of agents. There are several distinguishing hallmarks of diseases caused by these unconventional agents. The diseases are confined to the nervous system. The basic lesion is a progressive vacuolation in neurons, an extensive astroligal

Table 12.48

hypertrophy and proliferation, and then a spongiform change in the gray matter. Amyloid plaques may be present. Long incubation periods (months to decades) precede the onset of clinical illness and are followed by chronic progressive pathology (weeks to years). The diseases are always fatal; there are no known cases of remissions or recoveries. The host shows no inflammatory response and no immune response (i.e., the agents do not appear to be antigenic), no production of interferon is elicited, and there is no effect on host B- or T-cell function. Finally, immunosuppression of the host has no effect on pathogenesis of the disease. TSEs are not infectious but can be transmitted between mammals through inoculation with infected tissues or through feeding of affected animals to other animals. CJD and variant CJD (vCJD) have also been experimentally transmitted through inoculation of brain tissue into the brains of mice (Bruce et al., 1997). BSE is a relatively new disease of cattle. It was first recognized and defined in the United Kingdom (UK) in November 1986. Over the next few years the epidemic grew considerably and affected all parts of the country but to different degrees. By June 1990, there had been some 14,000 confirmed cases in a UK cattle population estimated at 11 million. Shortly after BSE was identified in cattle, a cluster of human cases of CJD with atypical characteristics occurred in the United Kingdom. This disease is referred to as variant CJD (vCJD). By 1996, molecular markers were identified in vCJD that are also present in BSE in cattle and in BSE experimentally transmitted to macaque, mice, and domestic cats but that differ from the markers identified in sporadic and iatrogenic CJD (Collinge et al., 1996). This was persuasive but not conclusive evidence that human vCJD is caused by transmission of BSE. Thus, concern that BSE was spreading to humans through the food chain initiated a flurry of research to as-

Transmissible Spongiform Encephalopathies

Disease Scrapie Transmissible mink encephalopathy (TME) Chronic wasting disease (CWD) Bovine spongiform encephalopathy (BSE) Feline spongiform encephalopathy (FSE) Creutzfeldt-Jakob disease (CJD) New variant Creutzfeldt-Jakob disease (vCJD) Iatrogenic Creutzfeldt-Jakob disease Kuru Gertsmann-Straussler-Scheinker (GSS) syndrome Fatal familial insomnia (FFI)

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Affected species Sheep, goats, moufflon Farmed mink Elk, mule-deer Cattle, captive exotic ruminants Domestic cats, captive exotic felids Humans Humans Humans Humans Humans Humans

certain whether, in fact, they were the same disease and, if so, whether they were spreading to humans through the food supply. By October 1997, brain extracts from patients with vCJD injected into brains of inbred mice produced disease with the same incubation period and neuropathological features as extracts from brains of cows with BSE. This disease differed from that provoked by extracts from patients with classical CJD (Bruce et al., 1997). This was compelling evidence that the same agent causes BSE and vCJD. The UK Advisory Committee on Dangerous Pathogens concluded that BSE agent should be classified as a human pathogen. The concern, whose validity is still unconfirmed, is that the BSE agent had spread to humans through ingestion of BSE-infected tissues. In this section, current literature on the nature and cause of BSE and its spread to humans, if any, via the food chain is reviewed. An excellent review on this topic has been published (Brewer, 2001). 12.11.1 Origin BSE is considered a “common source” epidemic; animals contract the disease from some common environmental element with which many have contact. As such the epidemic involves many individual, independent disease outbreaks. BSE appears to have originated from scrapie, an endemic spongiform encephalopathy of sheep and goats that has been recognized in Europe since the mid18th century (Brown and Bradley, 1998). Scrapie is so named because of the pruritus-induced rubbing and scratching (scraping on fences, etc.), which causes loss of wool. Scrapie is the “archetypical” spongiform encephalopathy (Groschup and Haas, 1996). It appears to be the only “naturally occurring” spongiform encephalopathy (SE) that can be transmitted both horizontally (animal-toanimal) and vertically (dam-to-fetus). All other animal SEs appear to be artificially transmitted; the infected animals become “dead-end host” (Schreuder, 1994). Scrapie in sheep now is endemic in most sheepbreeding countries except Australia and New Zealand. One-third of all British flocks have at least one case of scrapie; overall UK scrapie prevalence is between 0.5% and 1.0% (Morgan et al., 1990). In the United States, it is estimated that about 1% of sheep have scrapie (Brewer, 2001). Scrapie is a reportable disease in the United States, which has had a USDA Animal and Plant Health Inspection Service (APHIS) voluntary scrapie-free flock certification program since October 1992. Epidemiological evidence indicates that the primary cause of BSE in British cattle was probably the use of rendered carcasses of livestock (including sheep) as a proteinrich nutritional supplement in animal feed concentrates.

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During rendering, carcasses from which all consumable parts had been removed were milled and then decomposed in large vats by boiling at atmospheric or higher pressures, producing an aqueous slurry of protein under a layer of fat (tallow). After the fat was removed, the slurry was desiccated into a meat and bone meal product that was packaged by the animal feed industry and distributed to owners of livestock and other captive animals (e.g., zoo and laboratory animals, breeding species, pets). Although elements of the ensuing story are still disputed (including its origin from scrapie, rather than from unrecognized endemic BSE), it appears likely that changes in the rendering process that had taken place around 1980 allowed the etiological agent in infected carcasses to survive, contaminate the protein supplement, and infect cattle (Brown et al., 2001). Cattle carcasses and carcass wastes were then recycled through the rendering plants, increasing the levels of the now cattle-adapted pathogen in the protein supplement and eventually causing a full-scale BSE epidemic (Wells et al., 1987; Wilesmith et al., 1992a, 1992b; Collee and Bradley, 1997a, 1997b; Brown, 1997). Before 1970, high-temperature batch rendering was the predominant method of preparing the feed supplements. During the late 1970s and early 1980s, rendering temperature was lowered, and the solvent extraction process for recovering tallow was abandoned. Processing temperatures were lowered to save energy; in addition, solvent removal was no longer necessary. By 1982, nearly all rendering was without solvent at substantially reduced temperatures (AABP, 1996). At around the same time, the proportion of meat and bone meal in animal feeds was also increased from 1% to 12%. This may have allowed the infectious (scrapie and/or BSE) agent to concentrate through recycling of infected animals in the meat and bone meal. Apparently the pre-1970 processing methods eliminated the infective agent, or at least reduced the concentration, in the rendered materials prior to their inclusion as a supplement in cattle rations (Prusiner, 1995). The cessation of solvent extraction methods coincides with the emergence of BSE in 1985 when the extended incubation time for the disease is considered (Wilesmith et al., 1992b). Current data are consistent with 90% of BSE cases’ resulting from infected feed. Recognition of this source of infection has led to a series of countermeasures taken by the United Kingdom and other countries to break the cycle of cattle reinfection, restrict the geographical spread of disease, and eliminate potential sources of new infection (Table 12.49). The feeding of animal protein specifically derived from ruminants was banned in the United Kingdom in July 1988; by 1992 the epidemic began to be controlled. However, by then the loss of nearly 200,000

Table 12.49

Measures Taken to Prevent the Spread of Bovine Spongiform Encephalopathy to Animals

Precautionsa

United Kingdomb

European Unionc

BSE made a notifiable disease BSE surveillance, with histological examination of brains Ban on ruminant protein in ruminant feed Ban on export of UK cattle born before July 1988 feed ban Ban on import of live ruminants and most ruminant products from all BSE countries Ban on export of UK cattle >6 months of age Ban on SBO for use in animal nutrition; ban on export of SBO and feed containing SBO to EU countries High-risk waste to be rendered at 133°C/3 bar/20 min (or other approved procedure) Ban on export of SBO and feed containing SBO to non-EU countries Ban on MBM from SBO in fertilizer After Jan. 1, 1995, rendering methods required to sterilize BSE Ban on mammalian MBM in ruminant feed BSE surveillance including immunohistological features of brains Ban on mammalian protein in ruminant feedc Ban on import of live ruminants and most ruminant products (including meat products) from all countries of Europe Immunological testing for ruminant protein in animal feed Mammalian MBM prohibited from all animal feed/fertilizer Slaughtered cattle >30 months old (except certain beef cattle >42 months old) ruled unfit for animal use (hides for leather excluded) Mammalian MBM and MBM-containing feed recalled All mammalian waste to be rendered at 133°C/3 bar/20 min (or other approved procedure) Cattle tracing system improved Quarantine of 3 sheep flocks imported from Europe with possible exposure to BSE (4 animals dead of atypical TSE) BSE surveillance of fallen stock (downer cows) intensified Proposal to eradicate scrapie rejuvenated Export of deboned beef from cattle >30 months old born after July 1996 allowed Prohibition of use of animal protein, including MBM and blood meal (but excluding milk, or fish meal for nonruminants) in feed for any farmed animal species (effective Jan. 1, 2001) Prohibition of importation of rendered protein and rendering wastes originating or processed in Europe

June 1988 June 1988 July 1988

Apr. 1990 May 1990

a

United States Nov. 1987 May 1990

July 1989 July/Nov. 1989 Mar. 1990 Sept. 1990 Nov 1990 July 1991 Nov. 1991 June 1994 July 1994 Oct. 1993 Nov. 1994

Aug. 1997 Dec. 1997 July 1995

Mar./Apr. 1996 Mar. 1996

June 1996 July 1996 Sept. 1988 Oct. 1998

Oct. 1998 Nov. 1999 Aug. 1999 Dec. 2000

Dec. 2000

BSE, bovine spongiform encephalopathy; EU, European Union; MBM, meat and bone meal (protein residue produced by rendering); SBO, specified bovine offals (brain, spinal cord, thymus, tonsil, spleen, and intestines from cattle >6 months of age); TSE, transmissible spongiform encephalopathy. b In Northern Ireland and Scotland, dates of implementation sometimes differed from those shown for England and Wales; in addition, individual European Union countries often adopted different measures on different dates. c Some exemptions, e.g., milk, blood, and gelatin.

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diseased cattle, followed by preemptive slaughter and destruction of nearly 4.5 million asymptomatic cattle >30 months of age, had crippled the British livestock and many ancillary industries. The estimated number of UK BSE infections during the 1974–1995 period is about 1 million cattle, of which an estimated 730,000 entered the human food chain (Anderson et al., 1996a, 1996b). This followed the introduction of a ban on using ruminant protein in cattle feed and the destruction of animals showing signs of disease (introduced in 1988) and a specified offal ban on the use of brain, spinal cord, tonsils, thymus, spleen, and intestine of cattle origin in the human food chain in 1989. 12.11.2 Outbreaks As mentioned earlier, BSE was first observed in the United Kingdom in April 1985; it was specifically diagnosed in November 1986, when an affected cow was referred to the Central Veterinary Laboratory, Weybridge, England. By June 1990, there had been some 14,000 confirmed cases. When BSE was first observed in 1985, 200–300 infections had probably occurred already. BSE has occurred predominantly in Friesian and Friesian crossbred cattle. Incidence is much higher in dairy herds and mixed herds (90% of cases), probably because cows are fed more protein and remain on feed for a longer period (Anderson et al., 1996a, 1996b). In herds in which supplementary feed is given to calves, feed intake rises with age for the first 2 years. BSE, however, is not restricted to the United Kingdom alone. Cases have occurred in many other countries, especially in the European Union (EU), as a result of imported live animals or livestock food supplements (Table 12.50). In some countries, including those in the United Kingdom, the incidence of new cases is decreasing, but in other countries, for example, France, Portugal, Germany, Spain, and the Republic of Ireland, the incidence appears to be increasing, or initial cases have only recently appeared. The explanation for this phenomenon is most probably improved case ascertainment (supported by active surveillance and immunological methods), but new infections from contaminated feed intended for other species (e.g., pigs and poultry) may also be a contributing factor (Brown et al., 2001). Although in many countries BSE has been identified in native-born cattle, no indigenous index case has been reported outside the United Kingdom: i.e., no case originating de novo or from cow-to-cow transmission. Whatever the origin of these cases, recycling of their contaminated tissues through livestock feed supplements could have occurred in the same way as in the United Kingdom. The international marketing arrangements for trading the meat and bone meal make it impossible to deter-

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Table 12.50 Reported Cases of Bovine Spongiform Encephalopathy in the United Kingdom and Other Countries as of December 2000

Country United Kingdom Republic of Ireland Portugal Switzerlandb Franceb Belgium The Netherlands Liechtenstein Denmark Luxembourg Germany Oman Italy Spainc Canada Falklands (UK) Azores (Portugal)d

Native cases

Imported cases

Total cases

180,376a 487 446 363 150 18 6 2 1 1 3 — — — — — —

— 12 6 — 1 — — — 1 — 6 2 2 2 1 1 1

180,376 499 452 363 151 18 6 2 2 1 9 2 2 2 1 1 1

a

Includes 1287 cases in offshore British Isles. Includes cases detected by active surveillance with immunological methods. c Origin and dates of imported cases are under investigation. d Case imported from Germany. Source: Data from Organization of International Epizootics (Paris) and Ministry of Agriculture, Fisheries, and Food (UK). b

mine the ultimate destination of all such animal feed exported from the United Kingdom. Nevertheless, the strain of the agent responsible for the Swiss BSE epidemic has been found to be identical to the unique strain associated with the British epidemic. Considering that the British rendering industry was particularly successful in exporting animal feeds containing the meat and bone meal to Europe after the 1988 UK ban on feeding ruminantderived proteins to ruminants, it is entirely possible that any European country that imported the animal feed during this period could have received it from the United Kingdom. Other countries thus have had, at the most, a couple of hundred cases in native cattle, which are thought to be related to the use of imported UK cattle feed. These include France, Portugal, Switzerland, Netherlands, and Ireland. Furthermore, there is also the question of the cases that should have been observed from the exportation of adult cattle from the United Kingdom to Europe for breeding purposes between 1985 and 1990 (Taylor and Somerville, 2000). BSE has been reported only in imported cattle in Denmark, Germany, Italy, Falkland Islands, Oman, and Canada. Around 1700 contaminated cattle can be expected

to have been among some 58,000 purebred cattle exported from the United Kingdom to other European countries between 1985 and 1990. However, a much smaller number has been reported from these countries (Schreuder and Straub, 1996). With the ban on animal feeds containing the meat and bone meals, the decline in the BSE outbreaks in the United Kingdom and the EU is expected to continue, and elimination of the disease is expected within the next few years. The pattern of the epidemic also suggests that most transmission occurred through contaminated feed. Vertical transmission from cow to calf through mechanisms not yet known may occur at low levels, but these are probably not enough to maintain the epidemic (Anderson et al., 1996a; Dora, 1999). BSE has not occurred in the United States or other countries that have historically imported little or no live cattle, beef products, or livestock nutritional supplements from the United Kingdom. Even though rendering procedures in other countries underwent changes similar to those in the United Kingdom during the late 1970s, BSE has apparently emerged solely within the United Kingdom. The most plausible explanation is that the proportion of sheep in the mix of rendered animal carcasses and the proportion of scrapie infections in such sheep were probably higher in the United Kingdom than elsewhere (Brown et al., 2001). These proportions were apparently sufficient to raise very low levels of the etiological agent in batches of rendered carcasses over the threshold of transmission in the United Kingdom but not in other countries (Brown, 1997). An alternative explanation proposed in the Report of the BSE Inquiry (2000) is that a pathogenic mutation occurred in cattle in the 1970s. 12.11.3 Symptoms BSE is an afebrile neurological disease. As in other TSEs, it elicits no immune or inflammatory response. The clinical symptoms of BSE are varied. Most cattle with BSE show a gradual development of symptoms over a period of several weeks or even months, although some can deteriorate very rapidly. Only a small proportion of affected cattle show what would be considered typical “mad cow” signs, which are related to abnormal motor nerve control coupled with aggressiveness, which are also symptoms of rabies. Most suspects show several (but not all) of the symptoms listed in Table 12.51 if they are observed closely enough. Stress appears to cause the more rapid development of clinical signs in some animals, particularly when brought in before calving or if transported. Some symptoms may be subtle in the early stage but can usually be recognized by experienced stockmen and veterinarians. BSE usually

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Table 12.51 Some Clinical Manifestations of Bovine Spongiform Encephalopathy in Cattle • • • • • • • • • •

Apprehensiveness Nervousness Reluctance to cross concrete, turn corners, enter yards, go through doorways, or permit milking Occasional aggression directed at other cattle or humans Manic kicking when milked Head shyness, with head held low High stepping gait, particularly hind legs Difficulties in rising Skin tremors Loss of condition, weight, or milk yield

results in death or humane destruction within 4 months. Confirmation of disease is through postmortem examination of brain tissue. Brian tissue sections of infected cattle appear spongy and infiltrated with amyloid (starchlike) plaques when examined microscopically (Davis et al., 1991). 12.11.4 Diagnosis At present there is no fully validated, practical preclinical diagnostic test for BSE or other TSEs. There is no classical immune response or other host reaction to disease, and normal serological tests are therefore not applicable. Initial diagnosis of BSE depends on the observation of clinical signs of disease, which include incoordination, increased fear, increased startle response, and decreased rumination. Postmortem confirmation of diagnosis of TSEs traditionally relies on histopathological examination of the brain, where vacuolation (spongiform change), neuronal loss, and a reactive astrocytosis can be observed to differing degrees (Taylor and Somerville, 2000). In BSE, vacuolation in the mesencephalon, medulla, and pons is particularly prominent. Examination of the medulla has been found to be a reliable means of confirming diagnosis. A significant number of clinically suspect cattle are not confirmed as BSE-positive cases. At the height of the UK epidemic, about 10% of cases were found not to be BSE on neuropathological examination, but this ratio has risen to around 20% as the epidemic has waned. Histopathological evaluation as the primary test for BSE diagnosis continues to be the gold standard against which all other tests have to be validated. Since the association of abnormal, protease-resistant forms of the prion protein, PrPsc with the TSEs was discovered, its detection has been a potentially valuable diagnostic tool. Western blotting and immunocytochemical testing for the detection of PrPsc are used routinely for all suspects born from 1996

onward and for suspects born before 1996 that have negative findings on routine histopathological examination. The Western blot test is used to detect PrPsc protein purified from treated brain material obtained after postmortem by its molecular weight and reaction with specific antibodies. Further developments of this technique may increase the sensitivity and allow its application to different, more accessible tissues; to date only brain material testing has been fully validated with this technique. The immunocytochemical evaluation also relies on the detection of PrPsc using specific antibodies. Postmortem material is once again required and preserved. This test is performed directly on the tissue section and does not involve any protein purification steps. This technique can also work on other tissues, such as the third eyelid or tonsil of sheep. Work is under way both in the United Kingdom and in other countries to determine exactly how useful it would be to take biopsy samples of these tissues while the animal is still alive as a means of screening flocks. These tests, however, are not perfect. The results may be influenced by the genetic makeup of the animal, which determines how soon after the infection one of these tissues yields positive findings of abnormal PrPsc. Newer immunologically based tests that will allow larger-scale testing are being developed. Because high throughput is no longer required for the BSE epidemic, these tests are likely to prove more valuable as screening tools for active surveillance of cattle, e.g., especially for fallen stock and casualty slaughtered animals. There will still be a need to keep the traditional tests to confirm that any positive results of new methodology are correct, and not false-positive. This is because the newer tests have not all been thoroughly validated, especially for preclinical diagnosis. Some of the commercial tests that are currently under evaluation include a Western blot test from Prionics, a Swiss company; an enzyme-linked immunosorbent assay (ELISA) from Enfer Scientific in Ireland; a sandwich immunoassay technique from CEA, a research group based in France, marketed by Bio-Rad Laboratories; and a dissociated enhanced lanthanide fluorescence immunoassay (DELFIA) test from EG&G Wallace (now Perkin Elmer). Bioassays, the standard for detecting the infectivity of tissues, are used in transmission experiments to provide a model for assessing the likelihood that infective tissues could act as agents for transmitting BSE. Bioassays are able to detect infectivity directly, rather than by relying on a correlation with the presence of PrPsc, as detected by alternative methods. The tissue to be tested is either fed to or injected into the experimental animal, which is then observed for signs of the disease. On the basis of a combination of observations of incubation period and brain pathological characteristics, as well as other factors, as-

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sumptions can be made about the infectivity of tissue and type of disease (strain typing). The majority of TSE bioassays are conducted in panels of inbred mice. These are groups of mice in which individuals within a group are genetically similar and so exhibit similar, comparable responses to the disease. Bioassays in mice allow a large amount of data to be generated in a relatively short time and reduce the need to experiment on larger mammals; the latter is slower, more expensive, and ethically less acceptable. The effectiveness of murine bioassay is clearly demonstrated by the success of the assay in detecting BSE infectivity by both intracerebral and oral routes of exposure. The degree to which a tissue can transmit disease is expressed in infective units (IUs); 1 IU is the minimal amount of that tissue required to infect another animal of the same species by intracerebral inoculation. Infectivity is calculated in log10 of infective units/per gram (Dealer, 1993). Using sheep as an example, the infectivity of muscle, kidney (about 2.5) < adrenal < pituitary < nerve, lymphoid < spinal cord, gut < brain (about 7). Infectivities of TSEs for humans appear to be similar (Brown et al., 1994). It is also relatively easy to transmit BSE to mice, but even easier to transmit BSE to calves because of the lack of a species barrier. Wilesmith (1996) reported that experimental intracerebral inoculation of BSE-infected brain tissue from cows into calves has resulted in a detection level 1000-fold more sensitive than that of the mouse bioassay. Tissues not found to be infective by mouse bioassay may prove to be infective via intracerebral inoculation into calves. Experiments to determine whether or not infectivity was present in the tissues of clinically affected cattle began in 1987 with the inoculation of mice with brain from field cases of cattle affected with BSE. By 1988, there was clear evidence that BSE was transmissible to mice via inoculation of brain tissue. The aim of the tissue assays was to identify which, if any, of the tissues that might be consumed by humans contained detectable quantities of infectivity. This would of course be of significance in determining the pathogenesis of BSE, too. A large number of tissues were inoculated into mice, usually by a combination of intracerebral and intraperitoneal routes. The initial assays identified infectivity only in brain, spinal cord, and retina of the clinically affected cattle. Transmission studies based on intracerebral injection into mice of blood from clinical BSE cases have shown no detectable infectivity. The other tissues in which infectivity was not detectable are listed in Table 12.52. In contrast, infectivity in scrapie-infected animals is distributed through many more tissues than BSE in cattle (Table 12.53).

Table 12.52 Tissues from Clinically Affected Cattle with No Detectable Bovine Spongiform Encephalopathy Infectivity by Parenteral Inoculation of Mice Blood Cerebrospinal fluid Fat Gastrointestinal tract

Heart Kidney Liver Lung Lymph nodes Muscle Nerves Pancreas Reproductive

Buffy coat, clotted blood, fetal calf blood, serum Midrum Abomasum, colon (distal, proximal), esophagus, omasum, small intestine (distal, proximal), rectum, reticulum, rumen (esophageal groove, pillar)

Mesenteric, prefemoral, retropharyngeal Musculus (M.): M. semitendinosus, M. diaghragma, M. longissimus, M. masseter Cauda equina, peripheral nerves (N.): N. sciaticus (proximal), N. splanchnic, N. tibialis) Female: milk, ovary, placental cotyledon, placental fluids including amniotic fluid and allantoic fluid, udder, uterine caruncle Male: epididymis, prostate, semen, seminal vesicle, testis

Skin Spleen Trachea Tonsil

12.11.5 Causative Agent There is still considerable scientific uncertainty about the precise causative agents of BSE and other TSEs. A bacterium, virus, or parasite does not cause these diseases; the causative agent contains no nucleic acid. Furthermore, these agents are unusually resistant to standard means of inactivation. They are resistant to treatment with formaldehyde, β-propiolactone, ethanol, proteases, deoxycholate,

and ionizing radiation. However, they are sensitive to phenol (90%), household bleach, acetone, urea (6 M), strong detergents (10% lauryl sulfate), iodine disinfectants, and autoclaving. Guanidine thiocyanate appears to be highly effective in decontaminating medical supplies and instruments and tissues. The prion protein (PrP), a normal membrane-associated protein found most commonly in the central nervous system, appears to be very important in the development

Table 12.53 Relative Scrapie Infectivity Titers in Tissues and Body Fluids from Naturally Infected Sheep and Goats with Clinical Scrapie Category I: High infectivity Category II: Medium infectivity

Category III: Low infectivity Category IV: No detectable infectivity

a

Brain, spinal cord, eyea Ileum, lymph nodes, proximal colon, spleen, tonsil, duramater, pineal gland, placenta, cerebrospinal fluid, pituitary, adrenal Distal colon, nasal mucosa, peripheral nerves, bone marrow, liver, lung, pancreas, thymus Blood clot, feces, heart, kidney, mammary gland, milk, ovary, saliva, salivary gland, seminal vesicle, serum, skeletal muscle, testis, thyroid, uterus, fetal tissue, bile, bone, cartilaginous tissue, connective tissue, hair, skin, urine

The result for the eye is from cattle with bovine spongiform encephalopathy (BSE), and not sheep.

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of TSEs (Prusiner, 1995). PrP is a normal protein that can assume an abnormal (infective) confirmation, which is often referred to as PrPsc, the scrapie isoform of the normal protein. This nomenclature reflects the fact that the moststudied TSE in animals is scrapie in sheep. A PrP is a normal host-encoded glycoprotein found in the brain cell membrane. It has many O- and N-linked glycosylation sites in its precursor form. It is coded for by about 750 base pairs composing about 250 codons, which code for the 231 amino acids of the protein. The gene for PrP is present in most mammals and in some other warm-blooded animal, and is located on chromosome 20 in most. Although the physiological function of this protein is still uncertain, its conservation among mammals implies that it does/did have an important role (Oesch et al., 1991; Roberts and James, 1996). Its normal function has been evaluated by creating knockout mice in which the PrP gene has been inactivated by homologous recombination; these mice are resistant to prion diseases (Collinge et al., 1995). However, as the knockout (PrP null) mice age, symptoms of neuronal disease develop; mice lose cerebellar Purkinje neurons and exhibited weakened gamma-aminobutyric acid– (GABA)-receptor-mediated neuron activity (Sakaguchi et al., 1995). The abnormalities such as altered circadian rhythms, fragmented sleep, and sleep deprivation seen in the knockout mice were similar to those observed in the human FFI (Tobler et al., 1996). Because the amino acid sequences of PrP and PrPsc are identical, some type of posttranslational modification must form the latter. The change appears to be in conformation or shape (Howard, 1996; Horwich and Weissman, 1997). PrP changes from predominantly α-helix to predominantly β-sheet, creating PrPsc. Whereas the former is readily degraded by lysosomal proteases, the latter is protease-resistant. Normal cellular PrP can be easily converted into PrP sc in vitro by simply mixing the two proteins together (Lansbury and Caughey, 1996). Cohen and associates (1994) speculated that the tendency to convert from PrP to the PrPsc form “spontaneously” is due to a genetic mutation in the original PrP. The mutation is usually due to a substitution/insertion of incorrect amino acids at those positions that would destabilize a helix. This increases the likelihood that the affected helix and its neighbors will refold into a β-sheet form. At least 20 such mutations in the PrP gene sequence have been identified thus far; each mutation results in a slightly different conformation and slightly different abnormality. The process is self-perpetuating (Heaphy, 1996). Once formed in the neurons, PrPsc autocatalytically converts normal PrP to PrPsc (Heaphy, 1996). The nerve cell eventually attempts to degrade the prion in the lysosome. However, PrPsc is highly resistant to several endoge-

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nous proteases, and any partial cleavages produce PrPsc fragments that accumulate in the lysosome and then kill the cell. This leaves holes known as spongiform damage. The breakdown products of PrPsc aggregate and precipitate, forming plaques. Even though significant amounts of tissue damage occur, the white blood cells do not infiltrate the brain, indicating that no inflammatory response is generated. The prion hypothesis is currently the most popular, but a number of scientists find difficulties with it, primarily as it contradicts the scientific orthodoxy that the inheritance of a trait, such as disease, must be associated with nucleic acid. The central dogma of biology expects that such a process must involve conversion of the blueprints (DNA, or some viruses that carry their blueprints as an RNA genome) into building blocks via replication of DNA, transcription of the message into RNA, and translation of the RNA’s message to form proteins, the building blocks of cells, tissues, organs, and whole organisms. In contrast, abnormal proteins such as PrP appear to direct the refolding of normal proteins just by direct contact. 12.11.6 Subacute Presenile Dementia and Variant Subacute Presenile Dementia First described in 1920s, subacute presenile dementia, Creutzfeldt-Jakob disease (CJD), is the most common clinicopathological subtype of human transmissible spongiform encephalopathy (TSE). It occurs in inherited, acquired, and sporadic forms. CJD in humans develops gradually, with progressive dementia, ataxia, and somnolence, and leads to death in 8–12 months. The GerstmannStraussler-Schenker (GSS) syndrome and fatal familial insomnia (FFI) are two familial forms of CJD. The disease occurs with a frequency of approximately 1 case per 1 million population per year in the United States and Europe. Most cases occur sporadically and involve patients over 50 years of age. The estimated incidence is less than 1 case per 200 million for persons below 30 years of age. During the 10 years after the first case of BSE was identified in the United Kingdom, cases of CJD did not increase in groups at high risk and continued to occur in the general population with the same spectrum of clinical and neuropathologic features as those before the appearance of BSE (Brown et al., 2001). In contrast, the variant form of CJD (vCJD) that may be linked to BSE has primarily affected people below the age of 30. From May to October 1995, the CJD Surveillance Unit, established in the United Kingdom in 1990, was notified of three cases of CJD in patients 16, 19, and

29 years of age (Britton et al., 1995; Bateman et al., 1995). On neuropathological examination, all three patients had amyloid plaques, which were unexpected in view of their occurrence in only 5%–10% of sporadic cases of CJD. By December 1995, the Surveillance Unit had been informed of 10 additional suspected cases of CJD in persons less than 50 years of age. Neuropathological studies on these and similar cases from other European countries later confirmed that an unrecognized variant of CJD that occurs only in persons less than 45 years of age was probably due to exposure to BSE (Will et al., 1996). This link has now been convincingly established in laboratory studies showing identical distinctive biological and molecular biological features of the pathological agent isolated from BSE-infected cattle and human cases of vCJD (Collinge et al., 1996; Bruce et al., 1997; Scott et al., 1999). The source of contamination appears to have been beef. However, muscle has never been reproducibly shown to contain the infectious agent in any form of TSE, whatever the affected species, and thus infection most probably resulted from beef products contaminated by nervous system tissue. According to Brown and colleagues (2001), contamination could have occurred in any of the following ways: 1.

2. 3. 4.

Cerebral vascular emboli from cranial stunning instruments used to immobilize cattle before killing by exsanguinations Contact of muscle with brain or spinal cord tissue by saws or other tools used during slaughter Inclusion of paraspinal ganglia in cuts of meat containing vertebral tissues (e.g., T-bone steaks) Presence of residual spinal cord and paraspinal ganglia tissue in the past of “mechanically recovered meat” (a carcass compression extract) that could legally be added to cooked meat products such as meat pies, beef sausages, and various canned meat preparations

Although the amount of infectious tissue ingested must be a critical determinant for the transmission of BSE to humans in the form of vCJD, the human genotype at polymorphic codon 129 of the PrP gene appears to play an important role in susceptibility to infection. The encoding alternatives, methionine (Met) and valine (Val), are distributed in the general white population in the approximate proportions of 50% Met/Val, 40% Met/Met, and 10% Val/Val. All 76 vCJD patients tested have been homozygous for methionine, and the apparently single infecting strain of BSE may not be able to replicate in any other human genotype. However, it is also possible that heterozygotes are comparatively resistant to disease and become ill

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after longer incubation periods than those of homozygotes (Brown et al., 2000, 2001). The onset of illness in the first case of vCJD occurred in early 1994, nearly a decade after the first case of BSE was recognized in cattle. Assuming that the earliest appearance of vCJD reflects the earliest exposure to BSE, this incubation period is consistent with those that follow peripheral infections in experimental animals and in cases of iatrogenic CJD in humans. The chronological record of vCJD in the United Kingdom and other European countries is summarized in Table 12.54. Unlike the BSE epidemic, the vCJD outbreak showed only a modest increase during its first 6 years, and the number of cases with onsets in 2000 remained well below the previous year’s total, although additional cases will certainly be identified in the future. The difference between BSE and vCJD may be due to the fact that, in humans, recycling of infected tissue has not occurred, and thus the epidemic will evolve much more slowly than in cattle, or the difference may indicate a limited outbreak in humans due to very small infectious doses that, except in genetically susceptible persons, cannot surmount the combined effects of a species barrier and a comparatively inefficient route of infection (Brown et al., 2001). A major uncertainty about the extent of vCJD outbreak is that the incubation period of vCJD remains unknown. Depending on assumptions about the incubation period and other variables, mathematical modeling predicts that the total extent of the outbreak could range from fewer than one hundred to hundreds of thousands of cases (Cousens et al., 1997; Ghani et al., 1998, 2000).

Table 12.54 Chronological Record of Variant Creutzfeldt-Jakob Disease in the United Kingdom and Other European Countries, as of December 2000

Year of onset

United Kingdom

1994 1995 1996 1997 1998 1999a 2000a

8 10 11 14 17 20 (+4) 1 (+2)

a

France

Ireland

1

1 (+1)

1

Parentheses indicate still-living persons with probable variant Creutzfeldt-Jakob disease (vCJD) or deceased persons whose diagnosis has not yet been confirmed by neuropathological examination. In 2000, additional cases that did not yet meet the minimal clinical criteria for a premortem diagnosis of “probable” vCJD were identified. Dates are for year of onset of illness, not year of death.

The small number of cases of vCJD in the United Kingdom, which as a population of around 50 million, could indicate a low risk to public health associated with exposure to BSE agent from beef products in comparison to other health risks. However, one cannot exclude the possibility that this might be the beginning of a much larger problem, the size of which can only be clarified by continuing surveillance employing standard comparable methods in the countries where it is already implemented and developing that elsewhere. The World Health Organization (WHO) has proposed criteria for identifying vCJD cases in general populations (Table 12.55). 12.11.7 Prevention The emergence of vCJD after the BSE epidemic generated intense public debate about food safety risk assessment

Table 12.55 Clinical Criteria for Variant Creutzfeldt-Jakob Disease Case Definition as Set Forth by the World Health Organization (WHO)a I

II

III

A B C D E A B C D E A

B IV A Definite vCJD Probable vCJD Possible vCJD

Progressive neuropsychiatric disorder Duration of illness >6 months Routine investigations do not suggest an alternative diagnosis No history of potential iatrogenic exposure No evidence of a familial form of TSE Early psychiatric symptomsb Persistent painful sensory symptomsc Ataxia Myoclonus or chorea or dystonia Dementia EEG does not show the typical appearance of sporadic CFJd (or no EEG performed) Bilateral pulvinar high signal on MRI Positive tonsil biopsye I A and neuropathological confirmation of vCJDf I and 4/5 of II and III A and III B or I and IV Ae I and 4/5 of II and III A

a

TSE, transmissible spongiform encephalopathy; EEG, electroencephalogram; MRI, magnetic resonance imaging; vCJD, variant CreutzfeldtJakob disease. b Depression, anxiety, apathy, withdrawal, delusions. c This includes both frank pain and/or dysesthesia. d Generalized triphasic periodic complexes at approximately one per second. e Tonsil biopsy is not recommended routinely, nor in cases with EEG appearances typical of sporadic CJD, but may be useful in suspect cases in which the clinical features are compatible with vCJD and MRI does not show bilateral pulvinar high signal. f Spongiform change and extensive PrP deposition with florid plaques throughout the cerebrum and cerebellum.

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and regulation, agricultural practices, consumer trust, and its effects on food markets. The report of the vCJD caused a collapse of beef markets across Europe, even in countries using little UK beef and before the export ban on UK beef and beef products. Consumption of beef has not yet returned to pre–March 1996 levels. Measures to protect the public from the potential risk of acquiring the animal disease have been issued by both national and international regulatory agencies. Their purposes are threefold: to prevent BSE-contaminated animals and meat products from entering the food chain or being used in the production of medicinal products, to provide monitoring and surveillance of BSE and vCJD, and to identify research needs and support their implementation. These measures are being revised regularly. Some of the measures taken to prevent the spread of BSE to humans are summarized in Table 12.56. The WHO has also issued recommendations to protect public health (WHO, 1996). These include the following: 1.

2.

3. 4.

5. 6.

7.

No part or product of any animal that has shown signs of a TSE or tissues that are likely to contain BSE agent should enter the human or animal food chain. All countries should establish surveillance and compulsory notification of BSE based on recommendation of the Office International des Epizooties (OIE). All countries should ban the use of ruminant tissues in ruminant feed. There is no evidence that milk or milk products transmit BSE. These products are therefore considered safe, even in countries with high BSE incidence. Gelatin and tallow are only considered safe if effective rendering procedures are used. Medical products should be obtained and medical devices containing bovine tissues should be obtained from countries with no or sporadic cases of BSE, and measures recommended to minimize the risk. Persons with CJD should not donate blood.

The United States has had an active BSE surveillance program since 1986. In 1990, over 60 veterinary diagnostic laboratories throughout the country began participating in the BSE Surveillance Program, jointly administered by the USDA Food Safety and Inspection Service (FSIS) and APHIS. These laboratories are responsible for examining brain tissue from cattle over 2 years of age that show signs of neurological disease. BSE is a notifiable disease in the United States (Title 9, CFR, Parts 71 and 161). In addition to international importation restrictions, APHIS has in-

Table 12.56 Chronological Record of Measures Taken to Prevent the Spread of BSE to Humans in the UK, European Union, and United States Precautionsa

United Kingdomb

Compulsory slaughter of BSE-affected cattle Destruction of milk from affected cattle (except milk fed to cows’ own calves) Ban on import of UK cattle born after July 1988 feed ban Ban on SBO for domestic consumption Ban on export to EU of SBO and certain other tissues, including lymph nodes, pituitaries, and serum Ban on export of live UK cattle (except calves <6 months old) Ban on use of head meat after skull opened FDA recommendation of use of BSE-/scrapie-free sources for materials used in dietary supplements; request for safety plans Cell lines used for biologicals to be BSE agent–free FDA request that bovine source materials (except gelatin) used in manufacture of regulated products be restricted to BSE-free countries Bone-in beef only from farms with no BSE for 6 years; if not BSE-free, must be deboned with visible nervous and lymphatic tissue removed FDA request that bovine-derived materials for animal use or for cosmetic and dietary supplements not to be sourced from BSE countries Thymus and intestines from calves <6 months old made of SBO Import of beef only from UK cattle (1) >30 months, or (2) from herds BSE-free for 6 years, or (3) if not BSEfree, deboned with visible nervous tissue and specified lymph nodes removed SBO ban broadened to include whole skull (SBM) MRM from bovine vertebral column banned and export prohibited Removal of lymph nodes and visible nervous tissue from bovine meat >30 months exported to EU Ban on export of all UK cattle and cattle products except milk SBM ban broadened to include entire head (excluding uncontaminated tongue) Slaughtered cattle >30 months (or certain beef cattle >42 months) ruled unfit for animal or human use (hides excepted) FDA urging of manufacturers of FDA-regulated human products to take steps to assure freedom from BSE agent Partial lifting of export ban on tallow and gelatin SBM ban broadened to include certain sheep and goat heads, spleens, and spinal cords (SRM) FDA recommendation withdrawal of plasma and plasma products made from pools to which persons who later died of CJD had contributed

Aug. 1988 Dec. 1988

European Unionb

United States

July 1989 Nov. 1989 Apr. 1990

Apr. 1990

June 1990

June 1990

Mar. 1992 Nov. 1992

May 1993 Dec. 1993

July 1994

Aug. 1994

Nov. 1994 July 1995

Aug. 1995 Dec. 1995 Jan. 1996 Mar. 1996 Mar. 1996 Mar. 1996

May 1996

June 1996 Sept. 1996 Dec. 1996

(table continues)

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Table 12.56

(continued)

Precautionsa CNS tissues excluded from cosmetic products for use in EU BSE cohort cattle in UK ordered slaughtered and destroyed Proposed ban on SRM in cosmetics for use in EU (effective October 2000) SBM controls for cosmetics and medicinal products FDA request to manufacturers that no bovine gelatin from BSE countries be used in injectable, implantable, or ophthalmic products and that special precautions be applied to gelatin for oral and topical use Ban on marketing cosmetic products containing SRM prepared before Apr. 1, 1998 Allow export of beef and beef products from cattle >30 months in certified BSE-free herds from Northern Ireland Importation of all plasma and plasma products for use in UK FDA limit on plasma product withdrawals to pools at risk for contamination by vCJD donors Slaughter and destruction of offspring born to BSEaffected cattle after July 1996 FDA guidance to defer blood donors with >6 months cumulative residence in UK during 1980–1996 Leukodepletion of whole blood donations from UK residents Public FDA discussion about possible risk associated with vaccines produced with bovine-derived materials from BSE countries Withdrawal and destruction of potentially tainted 1989 lot of polio vaccine from one manufacturer SRM ban implemented (effective October 2000) Ban on slaughter techniques that could contaminate cattle carcasses with brain emboli (e.g., pithing or pneumatic stun guns), effective Jan. 2001 All cattle >30 months old to have brain examinations for PrP before entering food chain (effective Jan.–Jun. 2001) a

United Kingdomb

European Unionb

United States

Jan. 1997 Jan. 1997 July 1997 Mar. 1997 Sept./Dec. 1997

Mar. 1998 Mar. 1998

Aug. 1998 Sept. 1998 Jan. 1999 Nov. 1999 July/Nov. 1999 July 2000

Oct. 2000 July 2000 July 2000

Dec. 2000

CNS, central nervous system; EU, European Union; MRM, mechanically recovered meat; SBM, specified bovine materials (SBO plus entire head, including eyes but excluding tongue); SBO, specified bovine offals (brain, spinal cord, thymus, tonsils, spleen, and intestines from cattle >6 months old); SRM, specified risk materials (SBM plus sheep and goat heads and spleens from animals of any age, and spinal cords from animals >1 year old); PrP, proteinase-resistant protein. b In Northern Ireland and Scotland, dates of implementation sometimes differed from those shown for England and Wales; in addition, individual EU countries often adopted different measures on different dates.

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creased surveillance efforts to detect BSE if it is accidentally introduced into the United States. More than 250 APHIS and state veterinarians specially trained to diagnose foreign animal diseases regularly conduct field investigations of suspicious disease conditions. By the end of 1998, over 7000 brains from throughout the United States had been received at the USDA National Veterinary Service Laboratory (NVSL), and no evidence of BSE has been detected. About 15% of these specimens also had immunohistochemical testing for the PrP protein; it also has not been detected in any of these specimens. This coupled with the fact that no products that appear to pose potential risk to either human or animal health have been imported suggests that the U.S. beef and beef products appear to be safe, and there is little, if any, likelihood of emergence of BSE as a public threat.

12.12

PARASITIC PROTOZOA AND WORMS

12.12.1 Giardia lamblia Giardia lamblia, a single-celled flagellate, is the only common pathogenic protozoan found in the duodenum and jejunum of humans. It is also known as Giardia duodenalis in Europe and as Lamblia intestinalis in the former USSR. It is the cause of giardiasis, which is the most frequent cause of nonbacterial diarrhea in North America. G. lamblia is usually only weakly pathogenic for humans. Cysts may be found in large numbers in the stools of entirely asymptomatic persons. Human giardiasis may involve diarrhea within 1 week of ingestion of the cyst. Ingestion of one or more cysts may cause disease; in contrast, in most bacterial illnesses, the infectious dose is quite large. Large numbers of parasites attached to the bowel wall may cause irritation and low-grade inflammation of the duodenal or jejunal mucosa, with consequent acute or chronic diarrhea associated with crypt hypertrophy, villous atrophy, or flattening, and epithelial cell damage. The stools may be watery, semisolid, greasy, bulky, and foul-smelling at various times during the course of the infection. Malaise, weakness, weight loss, abdominal cramps, distention, and flatulence can occur. Normally illness lasts for 1 to 2 weeks, but there are cases of chronic infections lasting months to years. Giardiasis can be treated with metronidazole. Oral quinacrine hydrochloride and furazolidone are alternatives. Only symptomatic patients require treatment. Giardiasis is most frequently associated with the consumption of contaminated water. In the United States, five outbreaks have been traced to food contamination by

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infected or infested food handlers, and the possibility of infections from contaminated vegetables that are eaten raw cannot be excluded (CFSAN, 1998). Cool moist conditions favor the survival of the organism. 12.12.2 Entamoeba histolytica Entamoeba histolytica is a single-celled common parasite that infects the large intestine of humans, certain other primates, and some other animals. Many cases are asymptomatic except in humans or among animals living under stress. E. histolytica infections are commonly known as amebiasis (or amoebiasis). They are either asymptomatic or characterized by vague gastrointestinal distress or dysentery with blood and mucus. Complications include ulcerative and abscess pain and, rarely, intestinal blockage (CFSAN, 1998). Onset time is highly variable. Theoretically, the ingestion of one viable cyst can cause an infection. Asymptomatic (cyst-passing) amebiasis can be treated with iodoquinol or diloxanide furoate. Metronidazole is probably a drug of choice for symptomatic amebiasis. Cysts are usually ingested through contaminated water. In the tropics, contaminated vegetables and food are also important cyst sources. The most dramatic outbreak in the United States occurred at the Chicago World Fair in 1933 (CFSAN, 1998). It was caused by contaminated drinking water; defective plumbing permitted sewage to contaminate the drinking water. There were 1000 cases and 58 deaths. In recent times, food handlers are suspected of causing many scattered infections, but there has been no single large outbreak of amebiasis. 12.12.3 Cryptosporidium parvum The protozoon Cryptosporidium parvum is an obligate intracellular parasite. It has probably been an unrecognized cause of self-limited, mild gastroenteritis and diarrhea in humans. The infections are known as intestinal, tracheal, or pulmonary cryptosporidiosis. The organism inhabits the brush border of mucosal epithelial cells of the gastrointestinal tract, especially the surface of villi of the lower small bowel. The infectious dose is as low as 30 organisms. The prominent clinical feature of cryptosporidiosis is diarrhea, which is mild and self-limited (1–2 weeks) in normal persons but may be severe and prolonged in immunocompromised or very young or old individuals. Treatment is unnecessary for patients with normal immunity. Cryptosporidiosis is acquired from infected animal or human feces or from fecal contamination of food or water. Fertilization of salad vegetables with manure is a possible source of human infection. Mild cases are also

common in farm workers. Occasional outbreaks, such as the one that occurred in Milwaukee in early 1993 and affected 37,000 people, can result from inadequate protection, treatment, or filtration of water supplies for large urban centers (CFSAN, 1998). 12.12.4 Toxoplasma gondii Toxoplasma gondii is a coccidian protozoon of worldwide distribution that infects a wide range of animals and birds but does not appear to cause disease in them. The normal final hosts are strictly the cat and its relatives in the family Felidae, the only hosts in which the oocyst-producing sexual stage of Toxoplasma spp. can develop. The oocyst, when ingested by certain birds or by a rodent or other mammal, including humans, can establish an infection in which it reproduces asexually. The oocyst opens in the human’s duodenum and releases sporozoites, which in turn pass through the gut wall, circulate in the body, and invade various cells, especially macrophages. Subsequently, the infection spreads to lymph nodes and other organs. The organism in humans produces either congenital or postnatal toxoplasmosis. Congenital infection, which develops only when nonimmune mothers are infected during pregnancy, is usually of great severity; postnatal toxoplasmosis is usually much less severe. Congenital infection leads to stillbirths, chorioretinitis, intracerebral calcifications, psychomotor disturbances, and hydrocephaly or microcephaly. Most human infections are asymptomatic. In acute infections, parenchymal cells and those of the reticuloendothelial system are destroyed. A low-grade lymph node infection resembling infectious mononucleosis may occur. Acute toxoplasmosis can be treated with a combination of pyrimethamine and sulfadiazine or trisulfapyrimidines. Domestic cats have been incriminated in the transmission of the parasite to humans. Rodents also play a role in transmission. Avoidance of human contact with cat feces is clearly important in control. Humans can become infected either from oocysts in cat feces or from tissue cysts in raw or undercooked meat.

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HeLa cells results in listeriolysin O-mediated transient activation of the Raf-MEK-MAP kinase pathway. FEMS Microbiol. Lett. 148:189–195. Weinberg, E.D. 1984. Pregnancy-associated depression of cellmediated immunity. Rev. Infect. Dis. 6:814–831. Weiss, M., Roberts, T., and Linstrom, H. 1993. Food safety issues: Modernizing meat inspection. Agricultural Outlook. AO-197. Economic Research Service, U.S. Department of Agriculture, Washington, D.C. Wells, G.A.H., Scott, A.C., Johnson, C.T., Gunning, R.F., Hancock, R.D., and Jeffrey, M. 1987. A novel progressive spongiform encephalopathy in cattle. Vet. Rec. 121: 419–420. Wexler, D.E., Chenoweth, E.E., and Cleary, P.P. 1985. Mechanism of action of the group A streptococcal C5a inactivator. Proc. Natl. Acad. Sci. (U.S.A.) 82:8144–8148. WHO. 1984. The role of food safety in health and development. Technical Report Series No. 705, World Health Organization, Geneva. WHO. 1996. International experts propose measures to limit spread of BSE and reduce possible human risks from disease. World Health Organization. Press release WHO/28, April 3, 1996 (Revised 15 April, 1996). WHO. 1997. Fact sheet no. 149: Typhoid fever. World Health Organization, http://www.who.ch/. WHO. 1998. Typhoid fever. World Health Organization, http://www.who.ch/. Wilesmith, J.W. 1996. Bovine spongiform encephalopathy. BBC TV (Channel 2). “Horizon” programe, Part 2, November 18. Wilesmith, J.W., Ryan, J.B.M., and Hueston, W.D. 1992a. Bovine spongiform encephalopathy: Case-control studies of calf feeding practices and meat and bonemeal inclusion in proprietary concentrates. Res. Vet. Sci. 52:325–331. Wilesmith, J.W., Ryan, J.B.M., and Hueston, W.D. 1992b. Bovine spongiform encephalopathy: Epidemiological features 1985–1990. Vet. Rec. 130:90–94. Will, R.G., Ironside, J.W., Zeidler, M., Cousens, S.N., Estibeiro, K., Alperovitch, A., Poser, S., Pocchiari, M., Hofman, A., and Smith, P.G. 1996. A new variant of Creutzfeldt-Jakob disease in the UK. Lancet 347:921–925. Winans, S.C., Burns, D.L., and Christie, P.J. 1996. Adaptation of a conjugal transfer system for the export of pathogenic molecules. Trends Microbiol. 4:64–68. Wood, R.C., MacDonald, K.L., and Osterholm, M.T. 1992. Campylobacter enteritis outbreaks associated with drinking raw milk during youth activities. A 10-year review of outbreaks in the United States. JAMA 268:3228–3230. Wretlind, B., Heden, L., and Wadstrom, T. 1973. Formation of extracellular hemolysin by Aeromonas hydrophila in relation to protease and staphylolytic enzyme. J. Gen. Microbiol. 78:57–65. Wretlind, B., Mollby, R., and Wadstrom, T. 1971. Separation of two hemolysins from Aeromonas hydrophila by isoelectric focusing. Infect. Immun. 4:503–505.

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Yuki, N., Sato, S., Itoh, T., and Miyatake, T. 1991. HLA-B35 and acute axonal polyneuropathy following Campylobacter infection. Neurology 41:1561–1563. Yuki, N., Taki, T., Inagaki, F., Kasama, T., Takahashi, M., Saito, K., Handa, S., and Miyatake, T. 1993. A bacterium lipopolysaccharides that elicits Guillain-Barre syndrome has a GM1 ganglioside structure. J. Exp. Med. 178: 1771–1775. Yuki, N., Taki, T., Takahashi, M., Saito, K., Tai, T., Miyatake, T., and Handa, S. 1994. Penner’s serogroup 4 of Campylobacter jejuni has a lipopolysaccharides that bears a GM1 ganglioside epitope as well as one that bears a GD1a epitope. Infect. Immun. 62:2101–2103. Yunes, R., Goldhammer, A.R., Garner, W.K., and Cordes, E.H. 1977. Phospholipases: Mellitin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes. Arch. Biochem. Biophys. 183:105–112.

13 Bacterial Toxins

13.1 INTRODUCTION Bacterial toxins are substances that cause damage to host cells and that are often implicated in pathogenesis. Toxigenic bacterial pathogens are a constant feature of our environment. Bacterial toxins were recognized by the 1890s as the potent substances responsible for such infectious diseases as diphtheria (Loeffler, 1884), tetanus (von Behring and Kitasato, 1890), and botulinum (van Ermengen, 1897b). The nomenclature for these toxins was created in the early studies by coupling the term toxin with the disease name, e.g., diphtheria, tetanus, and botulinum toxins. More recently, because some toxins isolated from different organisms were found to cause the same type of cellular disorder, they were also named after their specific type of action. Therefore, toxins that cause enteric disorders, such as cholera, salmonellosis, and Escherichia coli infection, are called enterotoxins; those that cause neurological disorders such as paralysis are called neurotoxins. Bernheimer (1976) has defined bacterial toxins as a collection of bacterial products whose principal common feature is their capacity to produce injury or to kill when administered in relatively small quantities in living entities. It is possible to arrive at a more precise definition if one confines the scope to certain groups of well-characterized toxins. However, the diversity of structure, mode of action, predilection, potency, and immunogenicity of the many known bacterial toxins seems to preclude any less general description. Indeed, the field of bacterial toxinology, which addresses itself only to poisonous substances derived from living organisms (bacteria), appears always to be expanding and becoming more diverse.

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The number of different bacterial toxins described since these first “classical” toxins (diphtheria, tetanus, and botulinum) currently exceeds 150. Unfortunately, the pathophysiological role of the vast majority of bacterial toxins in specific diseases remains obscure. In most cases, bacterial toxins are relatively high-molecular-weight substances in the form of proteins (simple or more complex conjugated forms), peptides, or lipopolysaccharides. However, a few, such as E. coli ST and Legionella spp. toxin, appear to have molecular weights of less than 10,000 Da. The toxins mentioned are all considered exotoxins, which are special proteins excreted by toxigenic bacteria in various foods and are, in general, extremely poisonous and fatal. Botulinum, cholera, and gastroenteritis are wellknown examples of severe food poisoning diseases that result from the ingestion of these toxins. In 1935, Boivin and Mesrobeanu extracted new toxic substances from gramnegative bacteria, the so-called endotoxins. The outer membrane of gram-negative bacteria consists of heteropolysaccharides covalently linked to a lipid region (commonly known as lipopolysaccharides or the LPS complex). Lipopolysaccharides have three main regions: 1.

2.

Repeating oligosaccharide (e.g., man-rha-gal) combinations make up type-specific haptenic determinants (outermost in cell wall). It is commonly referred to as O-specific polysaccharide, which induces specific immunity. Contain N-acetylglucosamine, glucose, galactose, and heptose. It is a core polysaccharide that is common to all gram-negative bacteria.

3.

A backbone of alternating heptose and phosphate groups is linked through 2-keto-3-deoxyoctonic acid (KDO) to lipid. The lipid is linked to peptidoglycan by glycoside bonds. Lipid A with KDO is responsible for primary toxicity.

The representative chemical structure for the LPS complex is shown in Figure 13.1. The base molecule consists of a complex lipid (lipid A) to which is attached a polysaccharide made up of a core and a terminal series of repeat units (Figure 13.1A). Lipid A itself consists of phosphorylated glucosamine disaccharide units to which are attached a number of long-chain fatty acids (Figure 13.1B). β-Hydroxy myristic acid, a C14 fatty acid, is always present (Brooks et al., 1998) and is unique to this lipid; the other fatty acids, along with substituent groups on the phosphates, vary according to the bacterial species. The polysaccharide core, shown in Figure 13.1C, is similar in all gram-negative species that have LPS. Each species, however, contains a unique repeat unit; that of Salmonella newington is shown in Figure 13.1D. The repeat units are usually linear trisaccharides or branched tetra- or pentasaccharides. The negatively charged LPS molecules are noncovalently cross-bridged by divalent cations; this structure stabilizes the membrane and provides a barrier to hydrophobic molecules. Removal of the divalent cations with chelating agents or their displacement by polycationic antibiotics, such as polymyxins and aminoglycosides, renders the outer membrane permeable to large hydrophobic molecules. In contrast to protein toxins, these cell components of gram-negative bacteria elicit a variety of biological effects in the host, some of which are toxic: pyrogenicity, abortion, bone marrow necrosis, leucopenia, leukocytosis, hypotension, Shwartzman phenomenon, and death of the animal. Endotoxins can also produce in the host biological effects of metabolic and immunological consequence. Thus endotoxin is characterized by numerous and diverse biological activities, only a few of which are toxic. Endotoxins are released at the disintegration or death of bacterial cells in the body. Once absorbed into the circulatory system, they produce specific toxic effects, either in specific tissues or in the entire organ system. Endotoxins are weakly toxic, are rarely fatal, and often cause fever. Because they are only liberated from cell bodies by the disruption of cell walls, they do not play a significant role in pathogenesis in their natural state (Hsieh and Gruenwedel, 1990). They manifest their toxicity, however, when there is a massive bacterial infection in the body. The mechanism by which endotoxins induce systemic body reactions is not well understood, but the harmful effects of these toxins are considered to be the result of an

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overreaction of the host’s immune system or an imbalance in the body’s functions (Lai, 1980). Kabir and associates (1978) have comprehensively reviewed the biological activities of various endotoxins. The lipid A moiety has been well established as responsible for the toxicity of these bacterial components. Even then, the exact role of endotoxins in various disease syndromes is not always clear because of possible involvement of other toxins and virulence factors that can mimic or complicate some of the typical activities of the lipid A moiety of the LPS complex. The peptidoglycan of grampositive organisms’ cell walls has also been suggested as a possible factor in development of endotoxinlike systemic effects, as well as having the potential to be a potent enhancer of strain virulence (Sparling, 1983). Some important characteristics of exotoxins and endotoxins are summarized in Table 13.1. Since the microbial products historically classified as toxins have been identified as proteins, the term toxin should be restricted to proteins that possess toxic properties in animal models or tissue cultures (Bonventre, 1970; van Heyningen, 1970). Actively growing organisms usually excrete bacterial toxins (the so-called exotoxins); in some cases the toxin is a product of bacterial cell lysis. In general, protein toxins from different bacterial species have unique and rather specific biological properties and appear to possess distinct mechanisms of action. The prefixes exo- and endo- were used originally to describe the differences in apparent cellular location of the toxins, as well as to indicate the gram reaction of the producing bacterial strain. Experimental evidence now suggests that such terminology is inadequate and misleading. Bonventre (1970) has in fact suggested that an effort should be made to discontinue their usage in scientific publications. A more descriptive term should be sought to replace endotoxin to point up the differences in biological and pharmacological action between pure protein toxins and these cell membrane components. Bonventre (1970) has suggested endobacterial poison. It is also quite common that toxins are either named or classified according to their categorical role in pathogenesis. For example, bacterial toxins that exert some deleterious effect and host response exclusively in the small or large intestine are classified as enterotoxins. Members of this group of toxins, which has grown quite large since the 1970s, elicit alterations in intestinal cell structure or function by a diversity of modes of action, target cell types, receptors, and impacts upon the intestine and host. Another group of toxins, the neurotoxins, act primarily on the host’s nervous system by one means or another. A few other general classes of toxins named for their primary activity are hemolytic, cytolytic, and cytotoxic toxins, as

A

C Glu-GlcNAc Repeat units (up to 25) Gal Glu-Gal Hep Core

Hep-P-P-Eth KDO

P

P

Disaccharide-diphosphate

KDO-KDO-P-Eth.N

Fatty acids

B

D

CORE O CH2

Mannose

CH2

O O

Rhamnose

O

Galactose H2O3P

O

OH O C

O

HN C

CH2

CH2

CH

CH O

C

O

(CH2)12

C CH3

(CH2)10 O

(CH2)10

CH3

(MM)

O

CH3

C

PO3H2

NH

O

O

(CH2)10

O

O

C

O

CH2

CH2

CHOH

CHOH

(CH2)10

(CH2)10

CH3

CH3

(HM)

(HM)

CH3

(LM)

Figure 13.1 The lipopolysaccharides of the gram-negative cell envelope. A, A segment of the polymer, showing the arrangements of the major constituents; B, the structure of lipid A of Salmonella typhimurium; C, the polysaccharide core; D, a typical repeat unit (Salmonella newington). These may be repeated up to 25 times. Serological specificity is determined in part by the type of bond (α or β) between monosaccharide units. LPS, liposaccharide; KDO, keto-deoxy-octulonate; Hep, L-glycero-D-mannoheptose; HM, β-hydroxymyristic acid (C14); LM, lauroxymyristic acid; MM, myristoxymyristic acid; Eth.N, ethanolamine; Glu, glucose; GlcNAc, N-acetylglucosamine; Gal, galactose.

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Table 13.1 Characteristics of Bacterial Exotoxins and Endotoxins (Lipopolysaccharides) Exotoxins Excreted by living cell; high concentrations in liquid medium

Produced by both gram-positive and gram-negative bacteria Polypeptides with a molecular weight of 10,000–900,000 Relatively unstable; toxicity often destroyed rapidly by heating at temperatures above 60°C Highly antigenic; stimulate formation of high-titer antitoxin. Antitoxin neutralizes toxin Converted to antigenic, nontoxic toxoids by formalin, acid, heat, etc.; toxoids used to immunize (e.g., tetanus toxoid) Highly toxic; fatal to animals in microgram quantities or less

Endotoxins Integral part of cell wall of gram-negative bacteria; released on bacterial death and in part during growth; release may not be necessary to biological activity Found only in gram-negative bacteria Lipopolysaccharide complex; lipid A portion probably responsible for toxicity Relatively stable; withstand heating at temperatures above 60°C for hours without loss of toxicity Weakly immunogenic; antibodies antitoxic and protective; relationship between antibody titers and protection from disease less clear than with exotoxins Not converted to toxoids

Frequently controlled by extrachromosomal genes (e.g., plasmids)

Moderately toxic; fatal for animals in tens to hundreds of micrograms Specific receptors not found on cells Usually produce fever in the host by release of interleukin (IL-1) and other mediators Synthesis directed by chromosomal genes

well as toxins that inhibit macromolecular synthesis by some direct mechanism. Once again, the exact mechanism by which one toxin causes, say, cell death, may be quite different from that of another in the same class. Representative organisms known to produce toxins in the categories described are listed in Table 13.2. Some

of these microbial toxins are among the most poisonous chemicals known to humans. The relative potencies of a few classes of chemicals are shown in Table 13.3. It is quite obvious from the data summarized here that the microbial toxins are orders of magnitude more potent than human-made chemical compounds.

Usually bind to specific receptors on cells Usually do not produce fever in the host

Table 13.2 Bacteria That Produce Characterized Toxins Belonging to General Toxin Classes Based on Their Role in Pathogenesis Enterotoxin Vibrio cholerae, Escherichia coli, Bacillus cereus, Clostridium perfringens, Salmonella spp., Staphylococcus aureus, Shigella spp., nonagglutinable (NAG) vibrios, Aeromonas hydrophila Hemolytic Streptococcus spp., Staphylococcus spp., C. perfringens, V. parahaemolyticus, B. cereus, A. hydrophila Endotoxin All gram-negative bacteria Exoenzymes Invasive pathogens Neurotoxin C. botulinum, C. tetani, Shigella dysenteriae Cytotoxic, cytolytic Streptococcus spp., Staphylococcus spp., S. dysenteriae, A. hydrophila, V. parahaemolyticus, C. difficile, Legionella spp. Direct inhibitors of macromolecular synthesis C. diphtheriae, B. thuringiensis, Yersinia pestis, Pseudomonas spp. V. cholerae, E. coli

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Table 13.3 Relative Potency of Some Classes of Chemicals

Class of chemicals

Lethal dose to mice, µg/kg body weight

Bacterial toxins Animal venoms Algal toxins Mushroom toxins Mycotoxins Sodium cyanide Pesticides

0.00003 to 1 10 to 100 10 to 1,000 1,000s 1,000 to 10,000 10,000 10,000s

Source: Compiled from Loomis (1978) and Hsieh and Gruenwedel (1990).

13.2 NOMENCLATURE One of the main problems in bacterial toxin nomenclature is the lack of a uniform set of guidelines for use of symbolic notations to identify toxins, isotoxins, and their component parts. As a result, investigators continue to use arbitrarily capital and Greek letters, Arabic and Roman numerals, and various other symbols. Whatever notation system is finally employed, a few specific rules need to be followed. According to Bonventre (1970), a toxin nomenclature symbol should be used in the following situations: 1. 2.

3.

4.

When two or more independently acting toxins are produced by the same organism For toxic bacterial substances only, i.e., proteins that injure animals or cell culture preparations (other substances not directly toxic should be termed virulence factors) When coupled with the specific generic and species name of the organism (for example, Clostridium perfringens, C. septicum, and C. novyi all produce different α-toxins) Until such time as the biochemical properties of the toxin are revealed, when the name of the toxin should be changed to reflect the mode of action (thus, Staphylococcus aureus β-toxin should be described as S. aureus sphingomyelinase C)

Greek letters have been used for the identification of products associated with Staphylococcus aureus and clostridia of the gas gangrene group. Unfortunately, they also have been assigned to some bacterial products, which should not be classified legitimately as toxins.

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The capital letter system of nomenclature has been employed to signify staphylococcal enterotoxins. These toxins are immunologically distinct proteins of similar molecular size (27,800–34,100) and presumably have the same active site and mechanism of action in vivo. The neurotoxins of C. botulinum also are identified by capital letters, and they are immunologically distinct toxins. The toxin nomenclature system can only become more confusing if both Greek and capital letters are employed to indicate subunit components of toxins (Thorne and Gorbach, 1986). A universal symbolic notation must be sought in order to describe clearly multicomponent toxins. Bonventre (1970) has proposed the term MT as the symbol for a multicomponent toxin. Each individual component could then be designated as MT-1, MT-2, etc. This system, however, has not been accepted in the toxin literature. For the four multicomponent toxins best described to date (diphtheria, cholera, E. coli heat-labile toxin [LT], and Shigella spp. toxins), capital letters have been used to signify the active component(s) (A) and the binding component(s) (B). A subscript number is added to indicate the production of more than one active or binding component. If this nomenclature were adopted universally, it would establish a uniform and consistent system. Whatever the symbol chosen to indicate a toxin initially, a descriptive toxin terminology should be established as soon as possible. Eventually, the name of the toxin should reflect its mode of action.

13.3 PATHOGENESIS Recent advances in biochemistry, molecular biology, genetics, and physiology have allowed the isolation, purification, and structural characterization of many bacterial toxins, as well as elaboration of their modes of action, genetic coding and regulation of expression, structure-activity relationships, and roles in pathogenesis. These advances have been particularly helpful in increasing our understanding of disease syndromes that involve a previously undefined toxin, more than one toxin, or a combination of bacterial invasion and toxin production. As the sophistication of technology continues to increase, the relevant application of new methods should continue to increase our understanding of the role of toxins in pathogenesis. Bacterial toxins of diverse origins with very similar structure, immunological identity, or mode of action can readily be distinguished from one another in terms of their physical or chemical characteristics, the organism of origin, and/or the disease syndrome that results from their ac-

tion upon the susceptible host. The exact role of various toxins in pathogenesis depends to a certain degree upon the complexity of the cause of the disease. The easiest roles to define are those in diseases that stem from simple intoxication. Examples are food-borne diseases such as botulism and staphylococcal food poisoning. In both diseases, it is not uncommon that live bacterial cells are not present at the time of exposure to the disease-causing agent. The toxin is often produced and excreted by the cells into the food environment, after which unfavorable intrinsic or extrinsic conditions (such as heating of foods for serving) result in complete destruction of viable cells. Sickness results solely from ingestion of a heat-stable toxin. It is now a well-recognized fact that the complete disease syndrome can be experimentally induced by simple challenge with the toxin alone. In other disease syndromes, a single toxin may be produced and excreted by bacteria that multiply in the host. Examples are disease due to tetanus toxin, diphtheria toxin, botulism toxin (wound or infant botulism), plague murine toxin, cholera toxin, and E. coli LT or heat-stable toxin (ST). In the case of tetanus, diphtheria, and botulinum toxins, the toxin often spreads throughout the body beyond the location of the multiplying cells, whereas cholera toxin and E. coli LT or ST usually act in the vicinity of the site of cell multiplication. In the case of plague murine toxin, the organism spreads throughout the body, causing widespread distribution of the toxin as well. Of course, the necessary conditions for multiplication and production of toxin in each of these syndromes must also be met. This process can involve, among other factors, certain types of tissue damage, breakdown of host defenses, presence of suitable receptors for attachment of bacteria, or adequate route of inoculation of the organism. Defining the role of toxins in bacterial pathogenesis becomes much more difficult when more than one toxin may be involved and when other processes such as bacterial invasion are also necessary for the development of the disease. For example, more than 20 toxic factors that are produced by Streptococcus species have been described. Similarly, C. perfringens and Bacillus cereus are also capable of producing a broad spectrum of deleterious compounds, including toxins and enzymes that enhance bacterial invasion or survival or host susceptibility to toxin spread or action. However, there are organisms that are capable of elaborating more than one toxin but that produce a disease whose primary symptoms can be attributed to one agent, or in which the importance of one or more toxins is fairly well defined. Such examples include diseases caused by C. difficile, nonagglutinable (NAG) vibrios, Shigella spp., Salmonella spp., and possibly C. diphtheriae.

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Sometimes it is quite difficult to mimic a disease in its entirety in an animal model. For example, pertussis can never be replicated in animal models. One primary clinical feature of this disease, the paroxysmal coughing syndrome, develops and can persist for months after the complete disappearance of the organisms. The symptoms can result from tissue alterations distant from the initial site of infection. Indeed, three toxins from B. pertussis have been reasonably well characterized, but so far it has not been possible to delineate the exact contribution of each or any to the coughing syndrome. Certain biological activities of the toxins described in animal or cell systems could explain aspects of the basic pathological mechanism of the disease in humans, but in the absence of a good model system, precise studies are difficult. Anthrax is another disease whose cause is clearly multifactorial. It can vary from a localized cutaneous infection in humans to a fulminant, systemic infection in cattle, leading to death, which sometimes occurs so quickly that the animals drop dead before overt preliminary symptoms have developed. The exact role of toxin(s) is unclear in this complicated disease syndrome. In some instances, e.g., Salmonella spp. and NAG vibrio species, the role of elaborated toxins varies according to whether the organisms remain localized in the gut or become invasive and spread throughout the host. Generally speaking, as the clinical picture and degree of spread become more complicated, the roles of primary and secondary toxins, enzymes, and other virulence factors become more confused and difficult to resolve experimentally. Needless to say, in diseases due to organisms such as Streptococcus spp. and C. perfringens, the role of each possible active factor is far from clear. Strep throat, for example, can be characterized by the normal clinical picture resulting from respiratory mucosal inflammation, but the role of toxic factors in longer-term and more serious developments, such as rheumatism and damage to heart tissue, is far more difficult to assess. It is quite obvious that bacterial toxins play a major role in the pathogenesis of nearly every disease attributable to bacteria. When one includes endotoxin, peptidoglycan, and exoenzymes, which facilitate bacterial penetration of the host defenses, in the definition of toxin, then it can be said that all bacterial diseases develop either directly or indirectly through these substances.

13.4 TOXIGENICITY The ability of a particular organism to produce toxin is influenced by several factors, including the strain of organism, culture medium, and physiological conditions of

culture. Great variation in the capacity to produce toxin can be found among different toxigenic strains of the same organism, even when grown under similar conditions. On repeated subculture, toxigenic strains may lose their ability to produce toxin. For this reason, it is important to freeze-dry fresh isolates of toxigenic strains. The use of appropriate culture medium for toxin production cannot be overemphasized. van Heyningen (1970) noted culture medium that promotes good growth of a particular toxigenic organism might not necessarily facilitate optimal toxin production. Generally, factors such as pH, organic and inorganic constituents, and the gas phase must be monitored in order to develop the best culture conditions. Toxin production can also be potentiated or adversely affected by production of a proteolytic enzyme by the toxigenic organism. With a toxin-destroying protease, the toxin yield depends on the relative timing of synthesis of toxin and enzyme and the length of time the two are together in the culture medium. Examples of proteolytic enzyme enhancement of toxin activity are also known: C. botulinum type A toxin (Boroff and DasGupta, 1971) and E. coli LT toxin (Rappaport et al., 1976). Toxin production is one of the genetic traits that are concerned with interactions of the bacteria with their host. In the course of history, some toxigenic species have become less prominent and others have become more prevalent. These changes reflect the emergence of new bacterial populations in response to changes in the environment. Examples are the present lower frequency of Vibrio cholerae infections and the rise in toxic shock syndrome–producing staphylococci. From past history, it seems that toxigenic pathogens are a constant feature of our environment. Genetic studies dealing with toxin production can be considered in terms of (a) structure of the genes controlling toxin production; (b) gene expression and its control involving the transfer of information from deoxyribonucleic acid (DNA) to other macromolecules, such as messenger ribonucleic acid (mRNA) and proteins; and (c) location and movement of genes within cells and between cells. Gene expression gives information about the mechanism of toxin biosynthesis; one often uses mutants affecting toxin production to study gene expression and its regulation. The study of location and movement of genes is important mainly for epidemiology and the evolution and spreading of toxin-producing strains. Studies on the genetic characteristics of bacterial toxins are for the most part of recent origin. They have dealt largely with basic cellular processes, first the elucidation of metabolic pathways and their controls and later the analysis of gene replication and recombination and the formation and function of macromolecular structures, such

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as ribosomes and membranes. The study of extrachromosomal elements, plasmids and phages, has been a significant part of these developments. As our knowledge of the genetic machinery of the cell expanded, it became easier to manipulate genes and to isolate mutants, and this development culminated in the sophisticated cloning and sequencing procedures used in the present-day recombinant DNA technology. Genetic methods are now routinely used in the study of almost all cellular activities. One reason for the late arrival of genetic studies of toxins is the difficulty of isolating mutants in the absence of direct selection procedures. With the refinement of mutagenesis procedures, involving site-specific mutagenesis, and the application of restriction enzyme technology, it is now much easier to isolate mutants affecting toxin production than in the past. In addition, some organisms for which genetic analysis has become available have only recently produced many bacterial toxins. Examples can be found among several gram-positive bacteria, such as staphylococci and clostridia. Genetics of individual toxins are described later in this chapter in the appropriate sections. Yet another factor important in understanding the toxigenicity of bacterial toxins is their specific interaction with the corresponding cell surface receptors in the host. The apparent subversion of physiological pathways of uptake of cell surface molecules efficiently delivers the toxic activity to its site of action. The study of strategies adopted by organisms in evolving such potent toxins allows the use of toxins as tools to probe cellular processes, perhaps leading to clinical exploitation in the control of disease. The knowledge of membrane receptors specifically recognized by bacterial toxins is therefore instrumental to the understanding of the mechanism of action of toxins. The interaction of many toxins with target cells can be conceived as a sequence of three main steps (Alouf, 1982): 1.

2.

3.

Interaction of the toxin with the receptor, resulting in the induction of an initial stimulus, as the toxin acts as a first messenger (message reception) Transduction, modulation, and amplification processes transmitting the initial stimulus to the molecular effector system (message translation) Generation of an effect (biological response)

The binding sites or receptors of some well-studied bacterial toxins are summarized in Table 13.4. Bacterial toxins and other exotoxins exploit a variety of mechanisms to affect cell surface components or to penetrate that barrier to interfere with some particular internal process(es) of the cell. A number of the more complex toxins, i.e., those that do not act simply by nonspecific destruction of the cell membrane permeability barrier, sub-

Table 13.4 Chemical Nature of Membrane Constituents Acting as Receptors or Inhibitors of Some Bacterial Toxins Membrane constituent acting as receptor Carbohydrates Triacetylchitotriose Lipids and glycolipids Cholesterol Phospholipids

Ganglioside GM1

Ganglioside GM2 Gangliosides GT1 and GD1b

Ganglioside GM1-GlcNAc Unidentified gangliosides Protein a

Bacterial toxina Shigella toxin Sulfhydryl-activated toxins (e.g., streptolysin O) Staphylococcal leucocidin (F component) Staphylococcal δ-hemolysin Streptolysin S Cholera Escherichia coli and related LT enterotoxins Staphylococcal leucocidin (S component) Clostridium perfringens δ-toxin Tetanus toxin Vibrio parahemolyticus hemolysin Botulinum toxin Staphylococcal α-toxin Aeromonas hydrophila β-hemolysin Vibrio cholerae hemolysin Diphtheria toxin

LT, heat-labile toxin.

vert a variety of mechanisms that the cell normally utilizes to obtain information about its environment and to communicate with other cells. Some toxins such as tetanus toxin and botulinum toxin appear to interfere with a presynaptic neurosecretory membrane component whose function is directly affected at the membrane surface (Le Vine and Cuatrecasas, 1986). Other toxins exploit uptake processes and perhaps pathways to arrive at the internal cellular compartment and then attack various cytosolic processes. The subunit structure and sequence homologies between the β (binding) subunits of thyroid stimulating hormone (TSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), follicle stimulating hormone (FSH), and the α (active) subunits of the same hormones and the corresponding subunits of choleragen may be significant in this regard (Kohn, 1978). Hormonal signal transduction and control of cell sensitivity to hormones may be envisioned as occurring by a variety of mechanisms. Model hormone systems exist for some of these mechanisms, although they are far from being proved. A number of the nonpeptide hormones seem to act at the cell surface and do not appear to be taken up into the cytosolic compartment although signal transduction through the membrane occurs, i.e., ion transport or adenylate cyclase activation. For these substances (catecholamines and other neurotransmitter or neuromodulatory

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materials, prostaglandins, and a variety of other factors too numerous to mention here), internalization does not appear to be important for function. Desensitization phenomena involving loss of response to a hormone occur primarily at the coupling level between receptor and function—however that is accomplished—and are followed on a longer time scale by a decrease in receptor number (Harden et al., 1979). Several systems that appear to require some form of internalization of hormone receptor have been documented, and these bear directly on the mode of action of some of the bacterial toxins. The toxin receptors and their apparent resemblance or partial identity to some hormone receptors, in particular for the glycoprotein hormones, have provided some clues as to hormone receptor function. The binding characteristics of hormones such as glucagon, insulin, prolactin, and ACTH are not modulated by gangliosides. Gangliosides, however, seem to be involved in the binding of many other bacterial toxins as well as the glycoprotein hormones, TSH, and interferon (Lee et al., 1977; Lee et al., 1979; Kohn et al., 1976). The mechanisms by which bacterial toxins interact with their receptors and penetrate the cell membrane and the subtle processes of transmembrane signal transmission between hormone receptors and effectors within the cell are described for the individual toxins later in this chapter.

13.5 FOOD-BORNE BACTERIAL INTOXICATIONS As defined in Chapter 12, bacterial food-borne intoxications are illnesses produced as the result of growth of bacteria in the food prior to consumption. The toxins are not digested or otherwise modified by the enzymes or conditions of the digestive tract. These incidences are strictly poisoning, implying the ingestion of a toxin. Outbreaks of bacterial intoxications may occur with explosive violence, involving from only 1 to more than 1000 individuals within 1 to several hours after consumption of the food. They thus differ from the acute metal poisonings of cadmium and chromium salts, which take effect within minutes after consumption. If bacterial intoxication is not fatal, and with the exception of botulinum intoxication, the duration of illness is relatively brief, and only weakness and malaise persist. Most food-borne bacterial intoxications are expectedly enteric in nature; the initial symptoms take the form of gastrointestinal disturbances, such as nausea, vomiting, abdominal pain or cramps, or diarrhea. In fact, five (staphylococcal enterintoxication, C. perfringens gastroenteritis, enteropathogenic E. coli, B. cereus intoxication, and cholera) of the eight major bacterial intoxications result in diarrheas. The body response to the irritating substance is to flush the system with fluid that is drawn from all body tissues. In extreme instances, sufficient salt is lost from the bloodstream to produce ionic imbalance, leading to coma and death. Recommendations for treatment of such intoxications include administration of lightly salted water to aid the flushing mechanism and to conserve body fluid and maintaining body warmth until medical relief is obtained. Two other bacterial toxins (botulinum and pressor amines) are neurotoxins that induce paralysis. One other metabolic product (paralytic shellfish poison and ciguatoxin produced by dinoflagellate algae in seafood, described in Chapter 14) is not truly a toxin; yet it is a food-borne substance with effects on peripheral nerves. Four toxigenic bacteria produce live cell intoxications. In each instance, intoxication is associated with living cells in the food. The toxic substance is released in the digestive tract. Heating or reheating foods in which these bacteria have grown destroys them, and the process renders the food safe for consumption. The staphylococcal enterotoxins, ciguatera toxin, and the pressor amines are not destroyed or inactivated by heat. The remaining toxins are heat-labile. Some microorganisms produce toxins only, whereas other bacteria, such as E. coli, may also be invasive, that is, penetrate the defense mechanisms to invade and destroy epithelial tissue

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or invade the body and establish residence in various organs. In this chapter, the etiological agents of food-borne bacterial intoxications and symptoms in humans, the conditions or factors leading to the contamination of food products and the growth and proliferation of pathogenic bacteria, means of prevention and detection, and methods of treatment are described.

13.6 STAPHYLOCOCCUS AUREUS ENTEROTOXINS From all historical accounts, staphylococcal enterointoxication was unquestionably the most prevalent noninfectious food-borne illness during the first half of the 20th century. For many years, the illness was termed ptomaine poisoning, but ptomaines are amines with relatively mild effect on humans. Dr. Gail Dack and colleagues finally elucidated the nature of this intoxication during the 1930s (Dack et al., 1930). Of all food-borne intoxications, those caused by staphylococci have the potential for occurring most frequently and for affecting the largest numbers of individuals in single outbreaks. Outbreaks are often associated with large gatherings at which readily and economically prepared foods are served. Surprisingly, in the United States, staphylococcal poisoning accounted for well over 50% of the reported cases for many years (Cliver, 1987), although more recently this figure has dropped to about 10%. Staphylococci are ubiquitous in the environment and are part of the normal flora of the skin and mucous membranes of humans and other warm-blooded animals. S. aureus is carried in the anterior nares (i.e., nose) of 20% to 40% of the general population; carriage is higher in hospital personnel. It escapes through the mouth and the nasal orifices in nasal drainage and as droplets during coughing, sneezing, and wheezing and by finger contact with the lips through smoking, nibbling, and the use of the handkerchief. The average palm has approximately 100 white staphylococci/25 cm2 area, but 5000 can be counted when the palm is used to “catch a sneeze.” Staphylococcus aureus is also found in purulent skin infections as pimples, boils, and infections arising from penetration of the skin by thorns and slivers, cuts, and abrasions. Drainage from such infections through bandages that become wet during food preparation has been responsible for outbreaks of enterointoxication. Humans thus appear to be the single most important source of staphylococci contamination of food products. Although food handlers are usually the main source of food contamination in food poisoning outbreaks (Hobbs,

1987; Concon, 1988; Miller et al., 1998), equipment and environmental surfaces can also be sources of contamination with S. aureus. Human intoxication is caused by ingesting enterotoxins produced in food by some strains of this bacteria, usually because the food has not been kept hot enough (60°C [140°F] or above) or cold enough (7.2°C [45°F] or below). 13.6.1

Organism

Staphylococcus, along with two other closely related species, Micrococcus and Stomatococcus, are genera in the Micrococcaceae family. Biochemical characteristics that differentiate these species are summarized in Table 13.5. There are currently 32 staphylococcal species; approximately half of them are associated with humans (Martin and Iandolo, 2000). The three main species of clinical importance are S. aureus, S. epidermidis, and S. saprophyticus. Some differential characteristics of these three species are summarized in Table 13.6. Staphylococcus aureus is coagulase-positive (i.e., possesses an enzyme coagulating blood plasma), a characteristic that differentiates it from the other species. However, only about 30% of strains are capable of producing the enterotoxins associated with food poisoning (Forsythe and Hayes, 1998). The identification of various species of staphylococci by slide and tube coagulase and latex agglutination tests is schematically shown in Figures 13.2 and 13.3, respectively. Almost every person has some type of S. aureus infection during a lifetime, ranging in severity from food poisoning or minor skin infections to severe life-threatening infections (Brooks et al., 1998). The coagulase-negative staphylococci are normal human flora and sometimes cause infection, often associated with implanted appliances and devices, especially in very young, old, and immunocompromised patients. Staphylococci are gram-positive, aerobic or facultatively anaerobic cocci that tend to form “grapelike” clusters (staphyle is Greek for “bunch of grapes,” and coccus

means “grain” or “berry”). They are nonmotile and do not form spores. They grow readily on many types of media and are active metabolically, fermenting carbohydrates and producing pigments that vary from white to deep yellow. The pathogenic staphylococci often hemolyze blood, coagulate plasma, and produce a variety of extracellular enzymes and toxins. The growth temperature range is 7°C to 48°C (35°C–37°C optimal) and it can grow over a wide pH range, 4 to 10, with a pH of 6 to 7 optimal. The effect of pH on S. aureus growth varies with the strain and is affected by the inoculum level, growth medium, sodium chloride concentration, temperature, and redox potential (Miller et al., 1998). An important characteristic of S. aureus is that it can tolerate high levels of salt; it can grow in media containing 5% to 7% sodium chloride, and some strains are capable of growth in the presence of 20% sodium chloride. The organism can also grow over a much wider water activity range than other food-borne pathogens; growth can occur down to an aw of 0.83 and the optimum is greater than 0.99. The minimal water activity supporting growth by S. aureus depends on a number of factors, including type of humectant, pH, and redox potential (Bergdoll, 1989). The organism is more resistant to heat than are most gram-negative bacteria, requiring exposure to 60°C for 20–30 minutes for destruction. Foods that may harbor staphylococci should be heated to an internal temperature of 74°C to ensure their destruction. Enterotoxigenic strains of staphylococci have been well characterized on the basis of a number of genotypic and phenotypic characteristics. An extensive phage typing system is available for S. aureus. Most strains therefore are typable with strain-specific bacteriophages. Most enterotoxin-producing isolates of S. aureus belong to phage group I or III or are nontypable. Although enterotoxin production by other phage groups is less common, it has been documented (Jablonski and Bohach, 1997). The phages are useful in relating outbreaks of intoxications to the hu-

Table 13.5 Biochemical Characteristics That Differentiate Staphylococcus, Micrococcus, and Stomatococcus Speciesa Test Catalase Modified oxidase Furazolidone disk Bacitracin disk a

Staphylococcus spp.

Micrococcus spp.

Stomatococcus spp.

+ 0 S R

+ + R S

0 0 S R

+, positive; 0, negative; S, susceptible; R, resistant.

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Table 13.6 General Characteristics of Clinically Important Staphylococcus Species Characteristic

S. aureus

S. epidermidis

S. saprophyticus

Coagulase Thermostable nuclease Clumping factor Yellow pigment Hemolytic activity Phosphatase Lysostaphin Hyaluronidase Mannitol fermentation Novobiocin resistance

+ + + + + + Sensitive + + –

– +/– – – +/– +/– Slightly sensitive – +/– –

– – – +/– – – Not determined Not determined +/– +

man source or, in rare instances, to cows with staphylococcal mastitis. Hajek and Marsalek (1973) developed a classification scheme based largely on the animal host of origin. They were able to differentiate S. aureus into at least six biotypes. By far, the enterotoxin production was most prevalent

among human isolates within biotype A. That by other biotypes is reported to be rare except for biotype C bovine and ovine mastitis isolates. Enterotoxins may also be produced by S. intermedius and S. hyicus (formerly S. aureus biotypes E and F, respectively), albeit less frequently.

Figure 13.2 Identification of staphylococci by slide and tube coagulase tests.

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Figure 13.3 Identification of staphylococci by latex agglutination test.

13.6.2

Enterotoxins

Staphylococcus aureus is capable of producing a formidable array of extracellular proteins, many of which are inimical to the tissues of humans and animals. These include enzymes capable of degrading connective tissue (hyaluronidase), membranes and serum components (phospholipases and lipases), nucleic acids (deoxyribonuclease), proteins (proteases), and a variety of esterases (sugar phosphates and cholesteryl esters). In addition, the clotting of plasma is induced by staphylococcal coagulase, and clot dissolution is promoted by staphylokinase. There is yet another group of extracellular proteins, the exotoxins, also produced by this species. These include the membrane-damaging agents α-, β-, γ-, and δ-lysins (also called cytolysins, hemolysins, or toxins); Panton-Valentine leucocidin, active against human and rabbit leucocytes; epidermolytic toxins A and B (exofoliatins A and B), which induce cell separation in the stratum granulosum of the epidermis resulting in a variety of skin lesions; enterotoxins A, B, C1, C2, D, and E, responsible for the symptoms of staphylococcal food poisoning; and the pyrogenic exotoxins A, B, and C, implicated in the generation of scarletiniform rash in cases of staphylococcal scarlet fever. Pyrogenic exotoxin C and enterotoxin F have been implicated in the syndrome known as staphylococcal toxic shock syndrome (TSS). Altemeier and colleagues (1981)

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list 52 infective diseases of various organs caused by S. aureus, including acute gastroenteritis. In this section, only the entertoxins involved in staphylococcal food poisoning are discussed. The enterotoxins of S. aureus form a group of eight serologically (i.e., antigenically) distinct extracellular proteins, A, B, C1, C2, C3, D, E, and H, that are recognized as the causative agents of staphylococcal food poisoning (Harvey and Gilmour, 2000). Analysis of food-borne staphylococcal outbreaks indicates that other unidentified staphylococcal enterotoxins (SEs) exist. In accordance with the current nomenclature for these toxins, they are designated as SEA to SEE; SEA is the abbreviation for staphylococcal enterotoxin A. Strains of S. aureus have been found that produce enterotoxins F (SEF) (Bergdoll et al., 1981) and H (SEH) (Su and Wong, 1995); Betley and coworkers (1992) isolated a strain of S. aureus possessing the gene for enterotoxin G (SEG). So far, SEF and SEG have not been linked with food poisoning (Miller et al., 1998), although the former has been linked to toxic shock syndrome (TSS) (Harvey and Gilmour, 2000; Bergdoll, 1992). There is some evidence that SEH may be involved in food poisoning. This toxin was implicated in a familial outbreak in Brazil caused by cheese (Pereira et al., 1996); both an SEH-producing S. aureus strain and low levels of SEH were detected in the cheese. Additionally, a study by Su and Wong (1996) using an enzyme-linked im-

munosorbent assay (ELISA) method showed that 10 of a total of 21 strains previously shown to produce enterotoxins of unknown type produced SEH. Of the six SEs identified, types SEA and SED appear to be most commonly involved in food poisoning (Halpin-Dohnalek and Marth, 1989; Bergdoll, 1989) because they can be produced over a wider range of microbial growth conditions. About two-thirds of the enterotoxinproducing strains form one toxin only, usually type SEA, and the majority of the remainder form only two. SEA is produced during the late log phase of growth, and SEB is produced during the stationary phase. Growth and Production There is a very extensive literature on the topic of production of SEs in both natural and synthetic media. Bergdoll (1970, 1972, 1983) has comprehensively reviewed all aspects of toxin production under a variety of growth conditions. To some extent, the choice of medium is dictated by the purpose of the study; for instance, a medium suitable for screening strains may not be the best medium for toxin production and purification. In a general assessment of enterotoxin synthesis, Bergdoll and associates (1974) pointed out that the mechanisms of synthesis of SEA may differ from that of SEB and SEC. SEA-producing colonies typically yield uniform small haloes of immunoprecipitate on antiserum-containing agar, but strains producing SEB and SEC give colonies that differ widely in the diameter of the ring of precipitate produced. Also SEA production tends to be stable and to occur at a low level, whereas colonies producing high levels of SEB and SEC can be selected but are unstable on subculture. Czop and Bergdoll (1970), in a study of L forms of SEA-, SEB-, and SEC-producing strains, observed that only L forms of SEA-producing stains sustain enterotoxin production. This finding taken together with the results of Friedman (1968), who reported that detergents and agents that specifically block cell wall synthesis also inhibit SEB formation, suggest that SEB and SEC production may be associated in some way with the cell surface. Up to 67% of cell-associated SEB is surfacebound, located external to the cytoplasmic membrane (Miller and Fung, 1976). The conditions under which SEs are produced are summarized in Table 13.7. The optimal temperature for SE production is higher than that for bacterial growth, ranging from 40°C to 45°C. The pH range for toxin production is from 4 to 9.6; optimal pH is 7 to 8. Both growth and toxin production affected by the pH vary with the strain, inoculum level, growth medium, sodium chloride concentration, temperature, and redox potential. The minimal aw support-

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ing enterotoxin production by S. aureus depends on a number of factors, including type of humectant, pH, and redox potential. S. aureus is capable of growing in conditions of temperature, pH, and water activity that do not support enterotoxin production (Baird-Parker, 1990). Genetic Control The evolutionary history and the genetic control of toxin production by infective strains of S. aureus have been studied extensively. This aspect first attracted attention because of the apparent association between SEB production and methicillin resistance in hospital isolates. Soon after methicillin entered clinical use, there were several hospital outbreaks caused by methicillin-resistant S. aureus (MRSA). These organisms have now achieved global distribution (Grubb, 1990; Ochman, 2001). Methicillin resistance is conferred by the expression of a modified penicillin-binding protein encoded by the mec gene. The multilocus enzyme electrophoresis (MLEE) technique was employed to determine the extent of genetic diversity of MRSA strains and the relationships among strains from temporally and geographically separated outbreaks (Musser and Selander, 1990; Musser and Kapur, 1992). The mec gene is harbored by many divergent lineages of S. aureus and has spread through multiple episodes of horizontal transfer. Many of the MRSAs recovered from Europe and Africa soon after methicillin was introduced into clinical use in the 1960s were identified as the same electrophoretic type, suggesting that most early outbreaks were caused by dissemination of a single clone that had acquired the mec gene. In food-poisoning S. aureus strains, however, the genetic control of SEB appears to be chromosomal (Shafer and Iandolo, 1978). Working with MRSAs, Shafer and Iandolo (1979) later observed that the entB gene occupied either a plasmid or a chromosomal locus but was not physically linked to the mec gene. Shalita and colleagues (1977) earlier strongly implicated the involvement of a small plasmid (pSN2) in SEB production. However, the plasmid does not contain the structural gene for SEB, but it encodes for a regulatory function that can control the synthesis of SEB in both plasmid-bearing and chromosomal producers (Dyer and Iandolo, 1981). In the latter strains, it is proposed that pSN2 plasmid becomes integrated into the chromosome. According to Baird-Parker (1990), the production of SEB and SEC is controlled by plasmids and occurs mainly at the end of the stationary phase of cell growth. The production of SEA, SED, and SEE is under chromosomal control and occurs throughout the logarithmic growth phase. This difference is reflected by differences in the

Table 13.7 Factors Affecting Staphylococcus aureus Enterotoxin Production

Factor Temperature, °C pH Water activity, aw NaCl, % Oxygen, % dissolved

Aerobic, range

Aerobic, optimal

Anaerobic, range or lower limita

10–48 4.0–9.6 0.85–>0.99 0–10 5–50

40–45 7.0–8.0 0.98 0 5–20

10–45 5.3 0.90 ND NA

a ND, not determined; NA, not applicable. Source: Compiled from Baird-Parker (1990), Tranter and Brehm (1990), and Miller et al. (1998).

formation of toxins in foods. As mentioned earlier, SEA and SED are produced under a much wider range of growth conditions than SEB or SEC. SEA, SED, and SEE are produced in relatively small amounts and their production is closely related to the growth of the organism, whereas SEB and SEC can be produced in relatively large amounts and their production is more closely related to the incubation conditions (Bergdoll, 1989). It is because of the relatively large enterotoxin yield that most research on toxin production by S. aureus has been conducted with SEB-producing strains. Biochemical Properties All of the SEs have the same basic structure, a single polypeptide chain containing a single disulfide loop in the

center of the molecule. The significance of the loop is not known; however, it is assumed to stabilize the molecular structure and may contribute to resistance to proteolysis (Bergdoll, 1992). An essentially common sequence of amino acids follows the cystine loop. They contain no carbohydrate, lipid, or nucleotides. The molecular weights are within the range of 27,500–30,000. Their isoelectric points vary from just below neutral to pH 8.6 and are consistent with the number of basic and acidic amino acids (Spero et al., 1988). They are readily soluble in water and salt solutions. Each of the SEs has been obtained in highly purified form, and they have been well characterized (Table 13.8). The amino acid compositions of selected SEs are shown in Table 13.9. The most striking features are that all have a high content of lysine, aspartic acid, and tyrosine and that

Table 13.8 Physicochemical Properties of Staphylococcal Enterotoxins Enterotoxina Property Amino acid residues Mature toxin Enterotoxin gene Molecular weight Mature toxin Enterotoxin gene Isoelectric point Nitrogen content, % C-terminal residue N-terminal residue Emetic dose for monkeyb Maximal absorption, nm Extinction coefficient, 1% Sedimentation coefficient, S20 a

SEA

SEB

SEC1

SEC2

SEC 3

SED

SEE

233 257

239 266

239 266

239 266

238 266

228 258

230 257

27,078 29,700 6.8 16.5 Ser Ala 5 277 14.3 3.03–3.04

28,494 31,400 8.6 16.1 Lys Glu 5 277 14.0 2.78–2.89

27,500 30,511 8.6 16.2 Gly Glu 5 277 12.1 3.00

27,531 30,608 7.0 16.0 Gly Glu 5–10 277 12.1 2.90

27,438 — 8.15 — — — <10c — — —

26,360 — 7.4 — Lys Ser 20 278 10.8 —

26,425 29,358 7.0 — Thr — 10–20 277 12.5 2.60

SE, staphylococcal enterotoxin. ED50, µg/monkey. c Per os; 0.05 µg/kg by intravenous route. Source: Compiled from Freer and Arbuthnott (1986) and Martin and Myers (1994). b

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Table 13.9 Amino Acid Composition of Staphylococcal Enterotoxins Enterotoxin, g/100 g protein Amino acid

SEA

SEB

SEC1c

SEC2c

SEEd

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophan Amide NH3 Total

11.26 3.16 4.02 15.53 5.96 2.99 12.36 1.35 2.96 1.94 0.66 4.93 0.96 4.11 9.78 10.63 4.31 1.46 1.80 98.37

14.85 2.34 2.69 18.13 4.50 4.05 9.45 2.11 1.78 1.32 0.68 5.66 3.52 3.53 6.86 11.50 6.23 0.95 1.66 100.15

14.43 2.91 1.71 17.85 5.31 4.58 8.95 2.16 2.99 1.85 0.79 6.50 3.20 4.09 6.54 9.80 5.35 0.99 1.71 100.00

13.99 2.87 1.75 18.38 5.80 4.81 8.93 2.23 2.90 1.61 0.74 5.87 3.60 4.02 6.13 10.27 5.25 0.84 1.62 99.99

10.83 3.04 4.50 15.10 6.36 4.72 12.15 1.93 4.10 2.38 0.81 4.36 0.45 4.30 10.08 9.79 4.47 1.51 1.66 100.88

a

b

a

Staphylococcal enterotoxin A. (From Schantz et al. [1972].) Staphylococcal enterotoxin B. (From Bergdoll et al. [1965].) c Staphylococcal enterotoxins C1 and C2. (From Huang et al. [1967].) d Staphylococcal enterotoxin E. (From Borja et al. [1972].) b

SEA and SEE are similar in methionine, leucine, and arginine content and differ in this regard from SEB, SEC1, and SEC2. The amino acid sequence of SEB consists of 239 amino acids (Huang and Bergdoll, 1970). Half-cystine residues found at positions 93 and 113 form a disulfide bridge. Bergdoll and coworkers (1974) suggested that the primary structure in this region may be common to all of the SEs. Reduction of the disulfide bridge and subsequent alkylation of the sulfhydryl groups do not affect the physical or emetic properties of the toxin. Acetylation of SEB and SEC with acetylimidazole and nitration with tetranitromethane have no effect on the toxic or immunological properties of toxin. However, modification of the methionine residues in SEB indicates that emetic activity is lost when six of the eight residues react (Chu and Bergdoll, 1969). Similar modification of most of the carboxyl groups in the SEB molecule inactivates the toxin (Chu and Crany, 1969). The free amino groups can be guanidated with little change in conformational or biological properties (Spero et al., 1971). However, removal of the ε-amino groups or neutralization of the positive charge on the molecule inactivates the toxin.

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On the basis of amino acid sequences, the currently known SEs can be divided into three groups (Figure 13.4). Group 1 contains SEB and the SEC subtypes and molecular variants. Toxins in this group are highly related to each other (66% to 99% homology) and to several streptococcal pyrogenic toxins (PTs). Group 2 contains the highly related SEA and SEE (84% identical) and SED, which is more distantly related. SEH alone currently forms group 3. It shares only 38% homology with its closest SE relative, SEE. Secondary structure studies using circular dichroism show that SEB and SEC are similar and that both differ from SEA (Middlebrook et al., 1980). The analysis suggested that the enterotoxin molecules contain little αhelix. For SEB, 29 amino acid residues were in α-helices, 71 in β-pleated sheets, 88 in β-turns, and 55 in aperiodic conformation. The native SEB and SEC1 are highly susceptible to limited proteolytic cleavage by trypsin in the cystine loop, with full retention of their immunological and emetic activities (Spero et al., 1976; Bergdoll, 1992). The enterotoxins are markedly resistant to heat. Most are able to withstand boiling in food for up to 30 minutes; SEB shows

SEC - bovine SEC - ovine SEC1

SEC2 SEC3 - FRI 913

SEC3 - FRI 909

SEB

SEA

SEE

SED

SEH

Figure 13.4 Tree representation demonstrating molecular relatedness of the currently known staphylococcal enterotoxin family.

the greatest stability (Bergdoll, 1970). Typically there is a 60%–70% loss of activity within a few minutes when SEB is heated above 80°C, but the remaining activity is lost much less rapidly; indeed, reactivation can occur during prolonged storage at room temperature or by heating to a higher temperature (Reichart and Fung, 1976). The crude form of toxin shows greater resistance, which is enhanced by other substances. This heat stability explains why the toxin is sometimes detected even when the food product shows no signs of viable S. aureus (Casman et al., 1966; Foster and Bergdoll, 1968). In addition, many food products that are ordinarily eaten almost immediately after being cooked or heated sometimes can cause staphylococcal poisoning. Mode of Action The symptoms of staphylococcal food poisoning are well defined (Bergdoll, 1972). Yet the explanation of the symptoms in cellular and molecular terms remains vague. At least part of the reason is the difficulty of devising appro-

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priate animal models. Some species, such as the cat and the dog, are relatively insensitive to intragastric administration of the enterotoxins, whereas rodents lack a vomiting mechanism. Accordingly, much work has had to be carried out using rhesus monkeys. The emetic response seen in staphylococcal food poisoning may result from a stimulation of nerve centers in the gut, transmitted to the vomiting center via the vagus and sympathetic nerves (Sugiyama and Hayama, 1965; Baird-Parker, 1990; Miller et al., 1998). The basis for the diarrheal response is even more nebulous; whereas the enterotoxins of Vibrio cholerae and E. coli cause diarrhea by stimulating the adenylate cyclase of target cells, staphylococcal enterotoxins show no such stimulation (DeRubertis et al., 1974; Spero et al., 1988). In addition to in vivo gastrointestinal effect, staphylococcal enterotoxins SEA, SEB, and SEC1 are potent mitogens (Peavy et al., 1970; Warren et al., 1975). Greaves and associates (1974) demonstrated that the effect is predominantly on T cells. The enterotoxins also stimulate the

production of interferon from human and murine lymphocytes (Georgiades and Johnson, 1981a,b). 13.6.3

ful when viable staphylococci can be isolated from the incriminated food, from victims, and from suspected carrier such as food handlers.

Symptoms and Diagnosis 13.6.4

The onset of symptoms in staphylococcal food poisoning is usually rapid and in many cases acute, depending on individual susceptibility to the toxin, the amount of contaminated food eaten, the amount of toxin in the food ingested, and the general health of the victim. After ingestion of the contaminated food, the symptoms appear quickly, within 1–6 hours, with an average of about 3 hours. The most predominant and severe symptom is vomiting, preceded by a feeling of nausea. Vomiting can be very frequent and is followed in the later stages by retching, abdominal cramping, and prostration. Blood and mucus may appear in the stools and vomitus. In more severe cases, headache, muscle cramping, and transient changes in blood pressure and pulse rate may occur. There is no fever. The symptoms usually last for 1–2 days, and the mortality rate is extremely low, although fatal cases have been recorded. It is known that large numbers of S. aureus must be present in foods for them to be hazardous, but the precise number necessary to produce enough enterotoxin to induce the symptoms is not certain. Figures in excess of 1 million/g, a number that roughly corresponds to an enterotoxin level in the food consumed of about 1 µg, have been suggested (Halpin-Dohnalek and Marth, 1989). This level is sufficient to cause illness in adult humans, but in schoolchildren the dose may only be about 0.2 µg, as was found by Evenson colleagues (1988) when investigating a large outbreak of staphylococcal food poisoning involving chocolate milk. Studies using human volunteers have shown that ingestion of 0.4 µg enterotoxin (SEA, SEB, and SEC) per kilogram body weight causes illness, with a minimal dosage of 0.05 µg/kg (Bergdoll, 1989). In the diagnosis of staphylococcal food-borne illness, conducting proper interviews with the victims and gathering and analyzing epidemiological data are essential. Incriminated foods should be collected and examined for staphylococci. The presence of relatively large numbers of enterotoxigenic staphylococci is good circumstantial evidence that the food contains the toxin. The most conclusive test is the linking of an illness with a specific food, or in cases in which multiple vehicles exist, the detection of the toxin in the food sample(s). A number of serological methods for determining the enterotoxigenicity of S. aureus isolated from foods as well as methods for the separation and detection of toxins in foods have been developed (discussed later) and used successfully to aid in the diagnosis of the illness. Phage typing may also be use-

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Detection

The detection of enterotoxin in a food implicated in food poisoning is acceptable proof of staphylococcal food poisoning. The methods for detection of the enterotoxin in foods are borderline for the detection of the small amounts (0.5 ng/g) of toxin that may be present; hence, the detection of any amount of enterotoxin is considered adequate proof of food poisoning (Bergdoll, 1992). This point was demonstrated in the food-poisoning outbreak from chocolate milk, which contained as little as 0.4 ng/ml of milk (Evenson et al., 1988). For detecting trace amounts of staphylococcal enterotoxin in foods incriminated in food poisoning, the toxin must be separated from food constituents and concentrated before identification by specific precipitation with antiserum. Two principles are used for this purpose: (a) the selective adsorption of the enterotoxin from an extract of the food onto ion exchange resins and (b) the use of physical and chemical procedures for the selective removal of food constituents from the extract, leaving the enterotoxin(s) in solution. The use of these techniques and maximal concentration of the resulting products have made it possible to detect small amounts of enterotoxin in food. The Association of Official Analytical Chemists (AOAC)-approved standard method for detecting SEs in foods is the microslide gel double diffusion test. This is a qualitative method with a sensitivity of 10 to 100 ng/ml of enterotoxin in culture supernatant and concentrated food extracts (Andrews and Messer, 1990). The sensitivity of this method is barely adequate for detecting the small levels of toxin likely to be present in foods (0.5 to 10 mg/100 g) (Bergdoll, 1989). Several rapid and sensitive methods based on immunological techniques (radioimmunoassay [RIA], enzymelinked immunosorbent assay [ELISA], agglutination tests) are now commercially available for monitoring the enterotoxin levels in a variety of foods, with detection limits as low as 0.1 fg/ml pure toxin and 10 pg/g enterotoxin in artificially contaminated foods (e.g., minced beef, cheese). A comprehensive review of detection methods for SEs has been published (Su and Wong, 1997). Biological assay remains the only method, however, for detecting new serotypes of enterotoxins. The most reliable bioassay is feeding suspected samples to young rhesus monkeys, as described by Bergdoll (1970). Intraperitoneal or intravenous injections of cats or kittens have been used to some extent, but cats are subject to nonspe-

cific reactions to other staphylococcal products. Many other species of animals have been tested, but none is as sensitive and reliable as the monkey. The criterion, then, for a new enterotoxin serotype is that administration of the purified protein produces emesis and diarrhea in monkeys. An assay for the biological activity of SEA based on the proliferation of human and rat lymphocytes (T cells) has been developed. This assay may be applicable to the detection of SEA in food samples (Rasooly et al., 1997). As little as 1 pg/ml SEA stimulated proliferation, and by using this assay system 1 ng/ml SEA was detected in food samples. This is, therefore, a promising alternative to the current test for SEA biological activity that requires monkeys. 13.6.5

Foods Associated with Staphylococcal Poisoning

Foods incriminated in staphylococcal intoxication usually have been heated and are moist and bland or low in acid. Heating destroys competing microflora and denatures proteins for use by the staphylococci. It also provides a favorable temperature, for the staphylococci initiate growth at 45°C on a cooling food, a temperature at which few other bacteria are able to grow. One category of food includes ham, poultry, creamed meats, and baked foods such as custards, meringues, pumpkin pies, and cream pies, which receive no further treatment on emergence from the cooking process and are contaminated at the surface during cooling. The surfaces of such foods must not be retained in the range of 10°C–50°C for more than 4 hours. The other category is that of the formulated foods prepared with cooked or heated ingredients. They include all meat, poultry, and seafood salads; macaroni salad; some vegetable preparations; icings; cake fillers; whipped cream; filled cakes and pastries; and sandwiches. Safe preparation of all such foods dictates the cooling of all ingredients to or below 10°C before formulation. S. aureus is capable of growing and producing enterotoxins in brine (6%–10% salt); hence, salt-preserved foods are sometimes a source of intoxication. Outbreaks of staphylococcal food poisoning from raw or pasteurized milk are rare, but raw milk and raw milk products such as cream and cheese have given rise to outbreaks in many countries. In the latter cases, S. aureus can originate from a cow suffering from bovine mastitis. This contamination route, however, is the exception rather than the rule. In contrast, canned foods have rarely been implicated in staphylococcal food poisoning because the organism is readily destroyed by postcanning heat treatments (Miller et al., 1998). However, postprocessing contamination through faulty seals is possible, with the entry of S. aureus

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cells into an environment both free of microbial competition and conducive to its growth (Hardt-English et al., 1990). In fact, four outbreaks of staphylococcal food poisoning in the United States in 1989, affecting 102 people, were linked to canned mushrooms from China (CDC, 1989). Thermal processing records indicated that the canned mushrooms were subsequently inadequately processed, and sufficient levels of enterotoxin survived the canning process to cause illness (Hardt-English et al., 1990). Further studies have also shown that preformed staphylococcal enterotoxin in mushrooms can survive the typical thermal processes used on canned foods, reinforcing the need to prevent contamination by and growth of S. aureus in foods prior to processing through proper staff and plant hygiene and adequate refrigeration (Anderson et al., 1996). It is not always possible to trace the source of staphylococcal food contamination to a human or animal origin. Regardless of source, numerous studies have demonstrated the common presence of S. aureus in many types of food products (Table 13.10). The levels of staphylococci are often low initially. However, their widespread presence clearly provides a potential source of organisms capable of producing intoxications if conditions appropriate for SE expression are provided. The presence of large numbers of S. aureus in foods does not necessarily mean that enterotoxin has been produced. Scheusner and Harmon (1973) found that whereas all preinoculated samples of various commercial foods that contained enterotoxin also contained high populations of S. aureus, enterotoxin was not detected in preinoculated samples of meat and fish pies containing similar high final numbers of the organism. As described earlier, there are many factors affecting enterotoxin production including type of food, pH (little if any production below pH 5.0), temperature (optimal production at 37°C but wide range tolerated), presence of oxygen (poor enterotoxin production under anaerobic conditions), and presence of other organisms whose main effect is to inhibit the growth of S. aureus rather than affect enterotoxin production per se (Notermans and van Otterdijk, 1985; Halpin-Dohnalek and Marth, 1989). There are, therefore, many interrelated factors determining whether or not S. aureus food poisoning occurs. Bryan (1976) described them as follows: 1.

2. 3.

A source of an enterotoxigenic strain of S. aureus must be in a food production, processing, or preparation environment. The organism must be transferred from the source to a food. The food must be contaminated with thousands of S. aureus per gram, or, more usually, the food

Table 13.10

Prevalence of Staphylococcus aureus in Several Common Food Products

Product Ground beef

Big game Pork sausage Ground turkey Salmon steaks Oysters Blue crabmeat Peeled shrimp Lobster tail Assorted cream pies Tuna pot pies Delicatessen salads a

Samples tested, no.

Positive, %

Organisms/g, no.a

Reference

74 1,830 1,090 112 67 50 75 86 59 896 1,468 1,315 465 1,290 517

57 8 9 46 25 6 80 2 10 52 27 24 1 2 12

≥100 ≥1,000 >100 ≥10 100 >10 >3 >4 >4 ≥3 ≥3 ≥3 ≥25 ≥10 ≥3

Surkiewicz et al. (1975) Carl (1975) Pivnick et al. (1976) Smith et al. (1974) Surkiewicz et al. (1972) Guthertz et al. (1977) Guthertz et al. (1976) Everson et al. (1988) Everson et al. (1988) Wentz et al. (1983) Swartzentruber et al. (1980) Swartzentruber et al. (1980) Todd et al. (1983) Wentz et al. (1984) Pace (1975)

Determined by either direct plate count or most-probable-number (MPN) techniques.

4.

5. 6.

7.

must be heated before it becomes contaminated, or it must contain high levels of salt or sugar. The organism must survive in the food; it must not be outgrown or inhibited by competing organisms or killed by heat, low pH, or other adverse conditions before it can produce enterotoxin. The food, after it becomes contaminated, must support the growth of S. aureus. The contaminated food must stay within the temperature that is suitable for the proliferation of S. aureus long enough for this organism to multiply and produce enterotoxin. A sufficient quantity of enterotoxin-bearing food must be ingested to exceed the enterotoxin susceptibility threshold of persons eating the food.

The involvement of various individual food types in staphylococcal food poisoning outbreaks has been reviewed (Concon, 1988; Bergdoll, 1992; Miller et al., 1998). 13.6.6

Outbreaks of Staphylococcal Food Poisoning

Of all food-borne intoxications, those caused by staphylococci have the potential for occurring most frequently and for affecting the largest numbers of individuals in single outbreaks. Outbreaks are often associated with large gatherings at which readily and economically prepared foods are served. In fact, food poisoning due to staphylococcal

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enterotoxins is one of the four most common types of reported food poisoning worldwide (Genigeorgis, 1989). Staphylococcal food poisoning occurs as either isolated cases or outbreaks affecting a large number of individuals. Since the disease is self-limiting, the incentive to report cases has not been as great as for other food-borne diseases. Although there is a national surveillance system for staphylococcal food poisoning, it is not an officially reportable disease (Jablonski and Bohach, 1997). It has been estimated that only 1% to 5% of all staphylococcal food poisoning cases are reported in the United States, usually at the state health department level. The majority of these are highly publicized outbreaks. Isolated cases occurring in the home are not usually reported. Staphylococci account for an estimated 14% of the total of food-borne disease outbreaks within the United States. Annually about 25 major outbreaks occur within the United States. The highest incidence is typically in the later summer, when temperatures are warm and food is more likely to be stored improperly. A second peak occurs in November and December. Approximately one-third of these outbreaks are associated with leftover holiday food. Bergdoll (1992) has extensively reviewed the literature on staphylococcal food poisoning and its relationship to various locations. The most common locations for intoxications to occur include general public eating places (restaurants, fast-food outlets, vending machines), semiprivate eating places (conferences, hospitals, picnics and outings, wedding receptions, schools, senior citizen centers, military), private eating places (nursing homes, jails), public transportation (foods served on cruise ships, air-

planes and trains), and commercial foods (canned foods and baked goods). S. aureus is widespread in nature, and many of the raw materials arriving at food establishments for processing and manufacture of foods contain this organism. If such materials are not processed and handled properly during food manufacture, there is a risk of resulting staphylococcal food poisoning (Table 13.11). Although staphylococcal food poisoning is decreasing in many nations, the relative incidence in various countries varies substantially, depending on geographical conditions and local eating habits. In the United States, for example, it is one of the most economically important diseases, reported to cost $1.5 billion each year (Harvey and Gilmour, 2000). Large outbreaks, however, have become relatively rare in recent years in the developed countries. Only two large outbreaks have been reported since the late 1980s in the United States, one from chocolate milk involving over 850 schoolchildren (Everson et al., 1988) and the other in butter-blend spreads involving 265 cases (Bergdoll, 1992). In the chocolate milk outbreak, analysis of 12¹⁄₂-pint cartons of the 2% chocolate milk revealed an average of 144 ng of SEA per carton, and a range of 94–187 ng/carton (Table 13.12) In the United States, pork, particularly baked ham, has been the food most frequently involved in outbreaks. Several large outbreaks that occurred in the United States

Table 13.11

involved baked ham sandwiches served at picnics; one of the largest outbreaks involved 1300 individuals. In yet another such outbreaks, 1364 children became ill after consuming chicken salad (Foodborne pathogenic microorganism, 1992). It is believed that the chicken became contaminated after cooking when it was deboned. Most likely, the storage of the warm chicken in the deep aluminum pans used did not permit rapid cooling and provided an environment favorable for staphylococcal growth and SE production. Further growth of the bacteria probably occurred during the period when the food was kept in the warm classrooms. Prevention of the incident would have entailed more rapid cooling of the chicken and refrigeration of the salad preparation. Cream-filled bakery goods, such as éclairs, are infrequently involved in the United States, but in 1990, 484 individuals became ill in Thailand with typical staphylococcal food poisoning symptoms (Bergdoll, 1992). The cream filling contained more than 5 × 106 staphylococci per gram, which produced SEA. In Japan, rice balls are a common item taken on outings and have been a major cause of staphylococcal food poisoning; one outbreak involved 1500 individuals. In Brazil, the two most frequently involved are cream-filled cake and a white cheese frequently produced on the farm or in small establishments. Although staphylococcal food poisoning may indeed be the leading cause of food-borne illness worldwide, re-

Sources, Risks, and Consequences of Staphylococcus aureus in the Food Chain

Sources Natural environment

Food processing environment Raw materials

Processing

Handling

Food preparation environment

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Risks

Consequences of failure to control risks

Animals Humans Air, water, vegetation

Poor animal husbandry and poor human health care

Increase in animal and human S. aureus infections and occurrence in nature

Animal carcasses Animal products Added ingredients Food contact surfaces Air, water Human operatives

High numbers of S. aureus

S. aureus survival after processing; cross-contamination of raw and processed materials S. aureus survival after processing; postprocessing contamination of product with S. aureus

Food product

Animals Humans Food contact surfaces Air, water

Processing failure, insufficient cleaning or disinfection, poor ventilation, insufficient water treatment, poor hygiene Temperature abuse during storage, intrinsic growth control factors incorrectly adjusted Contamination of processed food with enterotoxigenic S. aureus; temperature abuse during holding of prepared food

Multiplication of S. aureus and production of staphylococcal enterotoxin Multiplication of S. aureus and production of staphylococcal enterotoxin

Table 13.12 Enterotoxin Analysis in 2% Chocolate Milk Implicated in Food Poisoning Outbreaks Enterotoxin Sample

ng/ml

ng/half-pint

1 2 3 4 5 6 Average

0.40 0.73 0.48 0.63 0.78 0.65 0.61

94 172 113 149 184 153 144

Source: Everson et al. (1988).

porting in other countries is less complete than in the United States. Some of the representative data are shown in Table 13.13. Outbreaks due to improper manufacturing of canned corned beef have been reported in England, Brazil, Argentina, Malta, northern Europe, and Australia. Cases in England have also been attributed to milk and cheese contaminated through sheep mastitis (Jay, 1986). In some countries, ice cream has been a major cause of staphylococcal food poisoning outbreaks. 13.6.7

Prevention

Staphylococci are ubiquitous in the environment. Thus measures aimed at eliminating S. aureus would be totally impracticable because of its widespread occurrence. Therefore, control measures should aim to limit contamination and the subsequent growth of the organism in foods. At least 50% of humans carry these microorganisms in their nasal passages, throat, or hands. Any foods subjected to human handling during preparation and processing are

Table 13.13 Food-Borne Outbreaks Due to Staphylococcus aureus

Period 1973–82 1976–84 1975–84 1960–68 1976–80 1977–82 1973–85 1972–82

Country

Food poisoning outbreaks, %

Canada England and Wales Finland Hungary Japan Netherlands Scotland United States

26.9 2.2 39.5 56.0 30.8 17.7 1.0 26.6

Source: Data from Kramer and Gilbert (1989) and Miller et al. (1998).

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therefore at risk. Similarly, the very nature of the locale or residence indicates the probability that any or all foods prepared and handled after cooking can be contaminated. S. aureus is not heat-resistant. Thus heating is considered to be the most effective method for inactivating staphylococci in foods. In contrast, freezing and thawing have very little effect on the viability of the microorganism, but storage for prolonged periods does decrease the number of viable organisms. Drying also lowers the number of recoverable S. aureus from several products. S. aureus cells are also easily killed by irradiation (Miller et al., 1998). Most of the food-borne bacteria inhibitory to S. aureus are from the genera Streptococcus, Lactobacillus, and Leuconostoc. The presence and/or growth of other bacteria and their effects on S. aureus growth and enterotoxin production are of most importance in milk and other dairy products and in fermented meats (Genigeorgis, 1989). The heat stability of staphylococcal enterotoxins is well documented. Enterotoxins in food are not easily inactivated by heat, and higher levels of toxin require higher levels of heat for inactivation. Generally, the toxins are more rapidly denatured at higher temperatures, and until recently the temperatures used in conventional canning processes were considered sufficient to inactivate levels of SEs that may be typically present in foods. There is evidence, however, that SEA, SEB, SEC, and SED may survive legal heat processing temperatures and times (Tibana et al., 1987; Bennett and Berry, 1987; Anderson et al., 1996). Factors such as the purity of the toxin, the serological type of toxin, the amount of toxin present, the heating medium, and the pH of the medium influence the thermal inactivation of SEs (Genigeorgis, 1989). SEs are also very resistant to gamma irradiation; more than 2.7 rads and 9.7 rads are required to produce one decimal reduction in SEB in buffer and milk, respectively (Read and Bradshaw, 1967; Genigeorgis, 1989). Rose and coworkers (1988) showed that SEA was more resistant to irradiation than type A botulinum toxin. Again, food was shown to have a protective effect. Up to 26% of SEA remained biologically active after a dosage of 23.7 kGy, which is twice the dose proposed for legal acceptance in the European Union. The only satisfactory control of staphylococcal food poisoning appears to be refrigeration of susceptible foods except when they are being prepared and during serving (Bergdoll, 1992). The lower the temperature, the slower is the growth of the staphylococci, and little or no growth occurs at refrigeration temperatures. In general, the following control measures must be taken to prevent staphylococcal food poisoning outbreaks:

1.

2. 3.

4.

5.

6. 7.

Keep handling of cooked foods to a minimum. Particular care should be taken with warm cooked foods, which should preferably be cooled to below 20°C when subsequent handling is essential. Recognize the susceptible foods in which staphylococci can grow. Personnel with septic lesions should not handle foods; because of the high nasal carriage rate in humans it would be impracticable to prohibit such carriers from handling foods, but all operatives should wear disposable gloves. Adequate heat treatment of the food, followed by prompt cooling to 10°C or below where foods are to be stored, are essential. Minimize cross-contamination from raw to cooked foods and from dirty working surfaces, equipment, and utensils. Minimize the retention of foods to not more than 4 hours in the range of 10°C–50°C. Use (HACCP) principles in both assessing and controlling the risk of S. aureus contamination, growth, and enterotoxin production in food products. To assure food safety with regard to this hazard, the following critical control points should be considered: a. Use raw materials containing the lowest practicable number of S. aureus. b. Use treatments to reduce microbial load and eliminate S. aureus. c. Use additives and/or low temperature to prevent multiplication of S. aureus during handling and storage. d. Use hygienic handling to prevent reintroduction of S. aureus.

Thus, even though staphylococcal food poisoning is mostly nonfatal and of short duration, its symptoms are severe enough to warrant care in the handling of food. Needless suffering can easily be prevented by knowledge of the hazard of staphylococcal poisoning.

13.7 BACILLUS CEREUS POISONING Wide recognition of the pathogenicity of Bacillus cereus has been slow to develop; for years, researchers who encountered B. cereus were only concerned with ensuring that it was not the closely related B. anthracis. Also contributing to this slow recognition was the state of confusion of the taxonomy within the genus Bacillus before Smith and associates (1952) imposed some order on it.

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However, there is ample evidence that, under the guise of a variety of names up to 1952 and, since then, as B. cereus, this organism has been associated from as early as 1898 with abscess formation, bacteremia and septicemia, cellulites, ear and eye infections, endocarditis, gastroenteritis, meningitis, kidney and urinary tract infections, osteomyelitis, puerperal sepsis, pulmonary infections, and wound infections, sometimes gangrenous (Goepfert et al., 1972; Gilbert, 1979; Tuazon et al., 1979; Turnbull et al., 1979; O’Day et al., 1981a, 1981b; Turnbull and Kramer, 1983). B. cereus has also been brought into particular focus since approximately the 1960s in the context of food poisoning. Initially, interest was in what is now described for convenience as the diarrheal-type syndrome characterized by symptoms predominantly of diarrhea and abdominal cramps 8–16 hours after eating the incriminated food; the types of foods involved have ranged widely from meats and vegetables dishes to pastas, desserts, cakes, sauces, and milk (Gilbert, 1979; Granum, 1997; Batt 2000a). The diarrheal-type syndrome was first recognized after a hospital outbreak caused by contaminated vanilla sauce in Oslo, Norway, in 1948 (Hauge, 1950, 1955). More recently, it has become accepted that B. cereus is the etiological agent of a second distinct type of food poisoning, referred to as the emetic-type syndrome, notably characterized by vomiting after a short incubation period of less than 1 to 5 hours. All but a small number of recorded incidents have been associated with rice dishes. It is becoming apparent from studies on the factors of B. cereus responsible for these two forms of food poisoning that the types of food involved are relevant from the standpoint of the growth conditions they provide. In the United States, known B. cereus intoxication is relatively rare and often been confined to starchy foods such as fried and cooked rice, leftover potatoes, bean sprouts, and meat dishes. In contrast, the European literature from the beginning of the 20th century contains numerous accounts of food poisonings caused by B. cereus or B. cereus–like organisms. One of the earliest recorded episodes appeared in 1906, when Lebenau described a hospital outbreak involving 300 patients and staff who experienced symptoms of acute gastroenteritis shortly after eating dinner. The main item on the menu had been meatballs, remnants of which were grossly contaminated with an aerobic spore-forming bacterium. By current epidemiological standards, early reports of food poisoning associated with Bacillus species were somewhat sketchy. Hauge (1950, 1955) finally succeeded in placing B. cereus on the map as a recognized food-poisoning organism with the publication of his meticulous investigations into four large outbreaks in Norway. B. cereus intoxication was ranked third among food-borne illnesses

in Hungary between 1960 and 1968 (Ormay and Novotny, 1970). Outbreaks in that country have been associated with the use of spices in meat dishes, but the less common availability of home refrigeration may also be a contributing factor (Goepfert et al., 1972). 13.7.1

Organism

Members of the genus Bacillus have a ubiquitous environmental distribution and can be isolated from soil, water, and most types of vegetation. It is prevalent on dried foods such as potatoes, milk, flours, and starches. In contrast to other bacterial genera, the genus Bacillus encompasses a great diversity of species and strains. There are more than 50 different species of Bacillus; B. anthracis and B. cereus are the two most important human pathogens. Two other members of this group, B. thuringiensis and B. mycoides, are also closely related. These four species can be differentiated by the criteria defined in Table 13.14. Bacillus species produce spores, which are metabolically inactive and resistant to heat and chemicals. The members of this genus appear as large gram-positive or gram-variable rods. Spores, if present, are not stained by gram-stain reagents and appear as “holes” inside the bacterial cells or are refractile, cell-free structures. Bacillus species are aerobic or facultatively anaerobic, typically growing over a temperature range of 10°C–48°C, with an optimal growth temperature of 35°C–45°C. Of concern in recent years, however, has been the finding of psychrotrophic variants of B. cereus that are capable of growth and toxin production down to 4°C (van Netten et al., 1990). Because of the ubiquity of this bacterium, it is impossible to eliminate it from foods by any process short of sterilization. It is very active both physiologically and biochemically but is highly variable in its properties. No known cultural procedures distinguish toxigenic from nontoxigenic strains of B. cereus. Toxigenic strains are detected by any one of several methods, including Evans blue or the vascular permeability test, the infant mouse test, and tissue culture. A summary of the basic microbiological properties of B. cereus is presented in Table 13.15.

Table 13.14

13.7.2

Toxins

There are at least seven different types of toxins produced by B. cereus (Table 13.16). The principal toxic metabolites of B. cereus are the subject of an extensive and detailed review by Turnbull (1986). Among these various toxins, only emetic (vomit-inducing) and diarrheagenic enterotoxin are involved in food poisoning (Granum, 1994; Batt, 2000a). The emetic toxin is preformed in the food but is quite heatresistant, unaffected by 90-minute heating at 126°C (Melling and Capel, 1978). The diarrheagenic enterotoxin is secreted into the food during logarithmic growth. It is relatively heat-sensitive and is destroyed in 30 minutes at 56×C (Goepfert et al., 1973). Different strains produce either of these two toxins, depending on ability (enterotoxin) or inability (emetic toxin) to hydrolyze starch. Emetic Toxin Little is known about the structure and function of B. cereus emetic toxin. Since the majority of incidents have been associated with rice dishes, most studies used a rice slurry medium for elucidating the pathogenic mechanisms involved in this syndrome (Melling et al., 1976; Melling and Capel, 1978). These studies indicated that the emetic toxin is a distinct heat-stable low-molecular-weight (<10,000 Da) compound, which has been variously speculated to be a peptide (Kramer and Gilbert, 1989) or a lipid (Shinagawa et al., 1992). It can withstand heating at 121°C for 90 minutes. The toxin is resistant to proteolysis and is stable at pH values between 2 and 11 (Kramer and Gilbert, 1989; Granum, 1994). The optimal temperature for the production of emetic toxin has been reported to be 25°C to 30°C in rice culture (Melling and Capel, 1978). These early studies on the nature of emetic toxin were hampered because the only detection method available involved primates (Hughes et al., 1988; Kramer and Gilbert, 1989). Observation that the toxin could be detected in HEp-2 cells (vacuolation activity) led to its isolation and characterization (Agata et al., 1994). Although there has been some uncertainty regarding the relationship between the emetic toxin and the vacuolating factor (Shi-

Criteria for Differentiating Four Closely Related Bacillus Species

Criterion Colony morphological features Hemolysis Motility Susceptibility to penicillin Parasporal crystal inclusion

Copyright 2002 by Marcel Dekker. All Rights Reserved.

B. cereus

B. anthracis

B. thuringiensis

B. mycoides

White + + – –

White – – + –

White/gray + + – +

Rhizoid (+) – – –

Table 13.15 cereus

Basic Microbiological Properties of Bacillus

Property Gram stain result Morphological features

Spore formation Metabolism Growth temperature range, °C (optimal) pH Range for growth Minimal aw for growth Toxin types, number

Characteristic Positive Rod, 1 µm × 3–5 µm long, in chains, motile by peritrichous flagella Yes Aerobic or facultative anaerobe 4–50 (28–35)

liminary epidemiological evidence these studies provide suggests that the production of an emetic toxin may not be restricted to B. cereus among the Bacillus species. On the basis of the documented cases of B. cereus emetic syndrome food poisoning outbreaks, the following conclusions can be drawn: 1.

2. 4.35–9.30 0.83–0.95 7

3. 4.

nagawa, 1993; Shinagawa et al., 1992), they are now confirmed to be the same toxin (Shinagawa et al., 1995; Agata et al., 1995). The emetic toxin, named cereulide, consists of a ring structure of three repeats of four amino and/or oxy acids: [ D -O-Leu- D -Ala- L -O-Val- L -Val] 3 . This ring structure (dodecadepsipeptide) has a molecular mass of 1.2 kDa and is closely related to the potassium ionophore valinomycin (Agata et al., 1994; Granum, 1997). The biosynthetic pathway and the mechanism of action of the emetic toxin are as yet unknown, although the toxin was determined in 1995 to stimulate the vagus afferent through binding to the 5-HT3 receptor (Agata et al., 1995). It is not known whether the toxin is a modified gene product or is enzymatically produced through modification of components in the growth medium. However, considering the structure of cereulide, it is more likely to be an enzymatically synthesized product than a gene product (Granum, 1997). Some physicochemical and biological properties of cereulide are summarized in Table 13.17. There is conflicting evidence as to whether toxin production occurs at the late exponential phase of growth or during sporulation of vegetative B. cereus cells in the stationary phase. Emetic toxin may be formed as a result of the enzymatic modification of the food in which B. cereus is growing, but this has yet to be established (Granum, 1994; Miller et al., 1998). It is not known whether other Bacillus species can produce the emetic toxin. B. subtilis is periodically isolated in high numbers and occasionally in pure growth during investigations of food poisoning; in some of these incidents, histories similar to those of B. cereus emetictype food poisoning are recorded (Mortimer and Meers, 1975; Winton and Sayers, 1975; Turnbull, 1979), but no feeding tests have been done with such isolates. The pre-

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The food vehicles responsible are almost invariably farinaceous, containing either cereal (mainly rice) or cereal-derived ingredients, such as flour. In the vast majority of episodes, that food vehicle has been held for too long at unsatisfactory storage temperatures. As a result, the incriminated food is found to contain substantial levels of B. cereus. Acute-phase fecal specimens from those affected by this form of gastroenteritis carry significant numbers of B. cereus.

Diarrheal Enterotoxin The results of human volunteer feeding studies carried out by Hauge (1955) supported the concept of B. cereus–induced diarrhea. These observations were later reproduced in feeding studies with cats and dogs given experimental diets containing >10 5 B. cereus/g (Nikodemusz, 1964, 1966a, 1966b, 1967). In the test animals severe diarrhea and dehydration developed. Subsequently, the feeding of whole-cell cultures of strains isolated from food-poisoning incidents to rhesus monkeys (Melling et al., 1976; Goepfert, 1978; Logan et al., 1979; Gilbert and Kramer, 1986) and similarly cell-free culture filtrates and ammonium sulfate–precipitated preparations (Goepfert, 1978; Goepfert et al., 1973) have been shown to induce diarrhea in these animals. The first clear indication of the possible existence of an enterotoxin was found in the study carried out by Goepfert and associates (1972), who elicited fluid accumulation after injection of whole-cell B. cereus cultures into ligated rabbit intestinal loops (LRILs). Positive fluid accumulation responses were obtained with 19 of 22 B. cereus strains. On the basis of its loop fluid-inducing ability, Goepfert and colleagues (1972) termed it an enterotoxin, but with circumspect caution based on their inability actually to induce diarrhea experimentally, they called it diarrheagenic factor rather than diarrheal toxin. Spira (1974) and Spira and Goepfert (1972) studied in greater detail the properties of the enterotoxin in both crude and partially purified (by Sephadex G-75 gel filtration) preparations. Evidence was obtained, by the LRIL test, that the effect of the enterotoxin on intestinal permeability was transient and that the molecule bound weakly

Table 13.16

Principal Exotoxins of Bacillus cereus

Exotoxin “Diarrheagenic” toxin

Emetic toxin/factor

Synonyms

Properties

Diarrheagenic factor Fluid accumulation factor Vascular permeability factor Dermonecrotic toxin Mouse lethal factor 1 Intestinonecrotic toxin Vomiting toxin/factor

Thermolabile antigenic protein; MW ca. 38,000–57,000; multicomponent or subunit structure, pI 4.9–5.1 and 5.6 for the two subunits, respectively; susceptible to trypsin and pronase digestion; necrotic, lethal; pathogenic role in food-borne illness and extraintestinal infection

Primary hemolysin

Hemolysin I (H-I) Cereolysin Mouse lethal factor 2

Secondary hemolysin

Hemolysin II (H-II)

Phospholipases C

Lecithinase Egg yolk turbidity factor

Exoenterotoxin (Ezepchuk and Fluer, 1973) Exotoxin (Ezepchuk et al., 1979)

Highly stable; probably a peptide; MW <10,000; not formed above 40°C; survive conditions of 126°C for 1.5 hr, pH extremes, and exposure to trypsin and pepsin; may be associated with sporulation or rice breakdown products Thiol-activated cytolysin; thermolabile antigenic protein; MW ca. 49,000–59,000, pI 6.3–6.7; inactivated by cholesterol and antistreptolysin O in horse serum; necrotic, lethal to mice; pathogenic role in extraintestinal infection Thermolabile antigenic protein; MW ca. 29,000–34,000, pI 4.9–5.3; susceptible to pronase, pepsin, and trypsin; in vitro activity unaffected by thiols, cholesterol, and antistreptolysin O; in vivo toxicity not yet established Relatively stable metalloenzymes Phosphatidylcholine hydrolase, MW 23,000, pI 6.5–8.5; phosphatidylinositol hydrolase, MW 29,000, pI 5.4, phosphatasemic factor; sphingomyelinase, MW 24,000, pI 5.6; hemolytic, depending on sphingomyelin content of erythrocyte Thermolabile antigenic protein; MW 57,000; inactivated by 60°C for 20 min; lethal to mice and rabbits; IV injection induction of emesis in cats; possibly related to diarrheagenic toxin “Previously unknown protein”; MW 100,000; not sensitive to trypsin; lethal to mice; IV administration to cats induction of fever but no emesis; rabbit loop result negative but skin permeability alteration; relationship to exoenterotoxin not known

Source: Compiled from Turnbull (1986) and Kramer and Gilbert (1992).

to receptor sites in the ileum. In contrast to the enterotoxins of V. cholerae and V. perfringens, B. cereus enterotoxin activity was inhibited by injection of antiserum 10 minutes after injection of an enterotoxic culture filtrate into a test loop. Also, LRIL activity was not affected by antisera raised against choleragen, C. perfringens enterotoxin, or the B. cereus lethal factor (Johnson and Bonventre, 1967). On Sephadex G-75, LRIL fluid-inducing, vascular permeability reaction (VPR) in rabbit skin, and lethal activities eluted coincidentally; distinct from phospholipase C but incompletely from hemolysin. Subsequently, on the basis of Sephadex chromatography and electrofocusing supported by tests on temperature, enzyme, and pH stabilities in crude filtrates or purified fractions, Turnbull and coworkers (1979) concluded that the toxin was a protein of molecular weight approximately 50,000 and pI in the order of 4.9. Immunological cross-reaction tests in VPR and gel diffusion tests

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using antisera raised to purified fractions and to purified choleragen and C. perfringens enterotoxin confirmed the findings of Spira and Goepfert (1975) and Katsaras and Hartwigk (1979) that no immunological relationship existed among these three. With reference to the toxin of Ezepchuk and Fluer (1973) and Gorina and associates (1975, 1976), purification was based on lethality in mice and emesis in cats; however, as judged by the molecular size, it is possible their toxin was the same one described by Goepfert and coworkers (1972) and Turnbull and colleagues (1979). Given that the conditions necessary to obtain optimal yields in closed culture systems had been well defined (Carpenter et al., 1975; Glatz and Goepfert, 1976, 1977; Spira and Silverman, 1979), nonetheless, attempts to isolate and purify the enterotoxin were repeatedly hampered by a combination of factors associated with B. cereus toxigenesis, which included the following:

Table 13.17 Physicochemical and Biological Properties of Cereulide, the Emetic Toxin of Bacillus cereus Parameter

Property or activity

Molecular mass Structure Isoelectric point Antigenicity Biological activity on living primates Receptor Ileal loop tests (rabbit, mouse) Cytotoxicity HEp-2 cells Stability to heat Stability to pH Effect of proteolysis (trypsin, pepsin) Toxin produced Production

1.2 kDa Ring-form dodecadepsipeptide Uncharged No(?) Vomiting (emesis) 5-HT3 (stimulation of the vagus afferent)a None No Vacuolation activity 90 Min at 121°C Stable at pH 2–11 None In food: rice and milk at 25°C–32°C Not known, probably enzymatic

a

5-HT3, 5-hydroxytryptamine. Source: Compiled from Agata et al. (1994, 1995), Kramer and Gilbert (1989), Shinagawa (1993), and Shinagawa et al. (1995).

1. 2. 3. 4.

The labile nature of the molecule Its susceptibility to proteolytic enzymes The attenuation of toxigenicity that occurred in producer strains on repeated subculturing Separation methodology problems posed by the coproduction of other metabolites sharing similar molecular properties

Experiments with various novel biochemical approaches to these problems eventually led several research groups independently to report on the purification and characterization of the enterotoxin and the development of immunoassay techniques (Ezepchuk and Fluer, 1973; Gorina et al., 1975, 1976; Kramer et al., 1978; Ezepchuk et al., 1979; Kramer, 1984; Thompson et al., 1984; Shinagawa et al., 1985). The diarrheal enterotoxin produced by B. cereus is a vegetative growth metabolite. It has a molecular weight of approximately 40, 000 Da (Granum et al., 1993) and is a thermolabile protein inactivated by heating at 56°C for 5 minutes. The enterotoxin is unstable below pH 4 and above pH 11 and is degraded by digestive enzymes, including pepsin, trypsin, and chymotrypsin, calling into question the ability of preformed toxin in foods to cause disease (Kramer and Gilbert, 1989; Miller et al., 1998). The optimal temperature for toxin production is 32°C to 37°C, and it is produced near the end of the logarithmic growth phase (Kramer and Gilbert, 1989). Physiologically, the requirement for an apparently ideal nutrient and the lability to even moderate tempera-

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tures and to digestive enzymes may explain why B. cereus is not more frequently involved in food poisoning. Glatz and Goepfert (1976) raise the question as to whether B. cereus food poisoning is due to intake of preformed toxin in the food; with the observation of increased cell numbers in ileal loops (Spira and Goepfert, 1972) in mind, they postulate that, if large numbers of organisms are consumed in mishandled food, they may have just enough time to multiply and produce the toxin in situ before competitive inhibition of the intestinal microflora brings the process to a halt. Granum (1994) took the view that, on balance, the evidence was weighted toward infection rather than intoxication. Preformed B. cereus diarrheal enterotoxin has been detected in foods in a 1997 study (Tan et al., 1997). Lund (1990) states that B. cereus enterotoxin is both preformed in food and formed by the organism in the intestine. Whether or not the preformed toxin is stable enough to cause illness after ingestion remains uncertain (Granum, 1994; Miller et al., 1998). Comparative properties of B. cereus diarrheal and emetic toxins are summarized in Table 13.18. 13.7.3

Symptoms and Diagnosis

It is now widely recognized that distinct clinical forms of gastroenteritis can result from the ingestion of foods heavily contaminated with B. cereus. The signs produced by the enterotoxin (diarrheal syndrome) are similar to those of C. perfringens infections but are much milder. The diarrheal syndrome is usually associated with protein-

Table 13.18

Comparative Properties of Bacillus cereus Diarrheal and Emetic Toxinsa

Property

Diarrheal enterotoxin

Nature/molecular weight

Protein 38,000–46,000 (SDS PAGE) 50,000–57,000 (Sephadex G-100)

Isoelectric point, pI Stabilities Heat pH Proteases Other enzymes, etc.

4.9–5.6

Production In foods Optimal laboratory culture medium Optimal temperature Growth phase Biological activities Monkey challenge Rabbit ileal loop Mouse ileal loop Rabbit VPR Mouse VPR Suckling mouse FAT Mouse lethality (intravenous) Cytotoxicity Antigenicity Action

Disease characteristics Infective dose (cells) Toxin produced Incubation period, hr Duration of illness, hr Symptoms

Foods most frequently implicated

Laboratory detection In vivo assays

Emetic toxin Dodecadepsipeptide, three repeat units of 4 amino and/or oxy acids: [D-OLeu-D-Ala-L-O-Val-L-Val]3; MW 1200 Neutral, uncharged

Inactivated at 56°C for 5 min Unstable <4 and >11 Pronase-, trypsin-, and pepsin-sensitive EDTA-, β-glucuronidase-, and alkylationresistant

90 Min at 121°C Stable at pH 2–11 Stable to proteolysis Not determined

Preformed(?) BHIG, CAM 32°C–37°C Later exponential

Preformed Rice culture slurry 25°C–3 0°C Late exponential-stationary (sporulationrelated?)

Diarrhea 0.5–3.5 hr Positive (150–300 µg); necrotic Positive (12 µg) Positive (0.05–1.25 µg); necrotic Positive (0.2 µg) Positive Positive (12–30 µg) Positive (0.1–0.5 µg) (CHO, INT-407) Positiveb; may be “multicomponent” Fluid (and Na+, Cl–) absorption reversed; strongly necrotic, mucosal damage, other tissues affected; glucose and amino acid malabsorption; may stimulate adenylate cyclase–cAMP system or reduce cell energy through microvilli damage; capillary permeability altered

Emesis 1.0–5.0 hr Negative Negative Negative Negative ND ND Negative No(?) Stimulates vagus afferent through binding to 5-HT3 receptor

105–107 (Total) In small intestine of host 8–16 (Occasionally >24) 12–24 (Occasionally several days) Abdominal pain, watery diarrhea, occasionally nausea

105–108/g Preformed in food 0.5–5 6–24 Nausea, vomiting, malaise (sometimes followed by diarrhea due to abdominal enterotoxin production?) Fried and cooked rice, pasta, pastry, noodles

Meat products, soups, vegetables, puddings and sauces, milk and milk products Ligated ileal loop test Vascular permability reaction Suckling mouse test

Monkey challenge

(table continues)

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Table 13.18

(continued) Diarrheal enterotoxin

Property Laboratory detection (continued) In vitro assays

Tissue culture (Vero, CHO, INT-407) Immunodiffusion Immunoelectrophoresis Reverse passive hemagglutination Reverse passive latex agglutination ELISA

Emetic toxin Vacuolation of HEp-2 cells

a

BHIG, brain heart infusion with glucose; CAM, casamino acids medium; VPR, vascular permeability reaction; FAT, fluid accumulation test; ND, data not available; CHO, Chinese hamster ovary; INT-407, human embryonic intestinal epithelium; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; EDTA, ethylene diamine tetraacetic acid; cAMP, cyclic adenosine monophosphate; 5-HT3 , 5-hydroxytryptamine; ELISA, enzyme-linked immunosorbent assay. b No cross-reactivities with Shigella dysenteriae, Vibrio cholerae, Clostridium perfringens, or Escherichia coli heat-labile toxin (LT) enterotoxins.

aceous foods, vegetables, sauces, and puddings. The onset of watery diarrhea, abdominal cramps, and pain occurs 6–12 hours after consumption of the contaminated food. Nausea may accompany diarrhea, but vomiting (emesis) rarely occurs. Symptoms generally persist for 24 hours in most instances. The enterotoxin has been found to exert its action by disrupting cell membranes, resulting in cell leakage (Granum et al., 1993; Miller et al., 1998). This form of the disease has been reported in several European countries, the United States, and Canada. The emetic syndrome form of B. cereus intoxication is almost exclusively associated with farinaceous foods, particularly cooked rice. The signs produced by the emetic toxin (emetic syndrome) are similar to those of S. aureus intoxications, including sudden onset of nausea and vomiting 30 minutes to 6 hours after consumption of the contaminated foods. Occasionally, abdominal cramps and/or

diarrhea may also occur. The duration is short, as symptoms usually last less than 24 hours (Kramer and Gilbert, 1989; Granum, 1994; Miller et al., 1998). The mode of action of this toxin remains elusive. This form of the disease was first reported in the United Kingdom in 1971; since then, similar incidents have been reported in other parts of the world (Gilbert, 1979). Comparative clinical and epidemiological data on B. cereus–related illnesses and their parallelism with C. perfringens and S. aureus food poisoning are listed in Table 13.19. The confirmation of B. cereus as the etiological agent in a food-borne outbreak requires (a) isolation of strains of the same serotype from the suspect food and feces or vomitus of the patient, (b) isolation of large numbers of B. cereus serotype known to cause food-borne illness from the suspect food or from the feces or vomitus

Table 13.19 Comparative Clinical and Epidemiological Data on Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus Food Poisoning B. cereus Diarrheal syndrome Onset of symptoms, hr Duration of illness, hr Diarrhea, abdominal cramps Nausea, vomiting Pathogenesis Principal food vehicles

Emetic syndrome

8–16 12–24 Predominant

1–5 6–24 Fairly common

Occasional Toxin-mediateda Meat products, soups, vegetables, puddings, sauces

Predominant Toxin-mediatedb Cooked rice and pasta

a

Toxin may be preformed in food or produced in the small intestine. Toxin preformed in food. c Sporulation-associated toxin release in the small intestine. Source: From Kramer and Gilbert (1989, 1992). b

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S. aureus 2–6 6–24

C. perfringens

Common

8–22 12–24 Predominant

Predominant Toxin-mediatedb Cold cooked meat and poultry, dairy products

Rare Toxin-mediatedc Cooked meat and poultry

of the patient, and (c) isolation of B. cereus from suspect foods and determination of their enterotoxigenicity by serological (diarrheal toxin) or biological (diarrheal and emetic) tests. In most cases, large numbers of B. cereus, between 105 and 108 per gram, are isolated from the incriminated foods. The rapid onset time to symptoms in the emetic form of disease, coupled with some food evidence, is often sufficient to diagnose this type of food poisoning. 13.7.4

Detection

A variety of methods have been recommended for the recovery, enumeration, and confirmation of B. cereus in foods. At present, there are no practical tests available for detecting the emetic toxin in foods. Enterotoxin has been detected in foods by using a commercial reversed passive latex agglutination (RPLA) kit (van Netten et al., 1990). The manufacturer claims the sensitivity of this kit to be 4 ng enterotoxin/g food macerate. A commercially available enzyme-linked immunosorbent assay (ELISA) kit has also been used to detect B. cereus enterotoxin in food samples (Tan et al., 1997). The emetic toxin can be detected by animal models (cats, monkeys) or possibly by cell culture. 13.7.5

Foods Associated with Bacillus cereus Intoxications

B. cereus is found commonly in soil and water, and its spores and vegetative cells can be readily isolated from a wide variety of plant foods, including cereal dishes, mashed potatoes, vegetables, and vegetable soups. These foods together with various cooked meat dishes have been responsible for the diarrheal form of food poisoning common in several European countries. It has often been associated with meat dishes that are frequently well seasoned with spices. Spices often contain large numbers of aerobic spore-bearing bacilli including B. cereus. Inadequate cooking of these foods allows the spores to survive so that the warm storage conditions after cooking result in spore germination and subsequent heavy growth of vegetative cells. As mentioned earlier, the emetic type of food poisoning appears to be associated almost exclusively with the consumption of fried rice dishes, particularly from Chinese restaurants and take-out outlets. In these establishments, portions of boiled rice have been allowed to dry off at ambient temperatures for periods of up to 24 hours or longer. In this way, the boiled rice may take many hours to cool, thus creating ideal conditions for the germination of any surviving spores and for the subsequent multiplication of vegetative cells. When required, the rice is fried with beaten egg for about 1 minute and kept warm until

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served. The frying process reduces bacterial numbers substantially, but the final holding period and preserving can again be extensive, and further heavy growth of bacteria may take place. Although the vast majority of cases of B. cereus emetic toxin food poisoning are due to the consumption of Cantonese-style cooked rice, other foods that have been involved include other ethnic rice dishes (curries, risotto, and rice salad), vanilla slices, pasteurized cream, milk pudding, chicken supreme, infant milk formulas, and cooked pasta (Turnbull, 1986; Kramer and Gilbert, 1989; Miller et al., 1998). In most cases, it is the holding of foods for too long at improper temperatures, allowing spores to germinate and vegetative cells to grow and produce toxin, that is the most important factor in the causation of the emetic form of B. cereus food poisoning. 13.7.6

Outbreaks of Intoxication

The number of outbreaks of B. cereus food-borne intoxications is highly underestimated in the literature and official statistics. The main reason for this is the relatively short duration of both types of syndromes (diarrheal and emetic) as well as the mild nature of intoxication. The dominant type of illness caused by B. cereus differs from one country to another. For example, in Japan, the emetic type is reported about 10 times more frequently than the diarrheal type (Shinagawa, 1993), whereas in Europe and North America, the diarrheal type is most frequently reported (Aas et al., 1992; Kramer and Gilbert, 1989; Granum, 1997). This variance is probably due to differences in eating habits, although milk is reported to have caused at least one large outbreak of the emetic type in Japan (Shinagawa, 1993). Several reports indicate that some patients have experienced both types of B. cereus food-borne illness simultaneously (Kramer and Gilbert, 1989). It is also clear that many strains of this organism have the ability to produce both types of toxins. The percentage of reported outbreaks and cases attributed to B. cereus in Japan, North America, and Europe varies from about 1% to 22% of outbreaks and from about 0.7% to 33% of cases (reports from different periods between 1960 and 1992) (Granum, 1997). The greatest number of reported outbreaks and cases were in the Netherlands and Norway. B. cereus food-borne illness has been a focus of research and food control authorities in both of these relatively small countries. Hauge (1950, 1955) provided the first and most comprehensive description of the diarrheal syndrome of B. cereus poisoning. He investigated four outbreaks in Norway involving some 600 cases. The suspect meal, a Sunday dinner, included chocolate pudding with vanilla

sauce for dessert. Both items had been prepared on the previous day and stored in a large container at room temperature. Of the 80 persons who ate the meal, 61 were affected; of the 19 who were not ill, 11 had refused the dessert and 8 had eaten only small quantities. The average incubation period was 10 hours for the patients and 12.5 hours for 20 staff who ate 2–3 hours later. The staff was more severely affected, and their symptoms persisted longer. The symptoms included abdominal pain, profuse watery diarrhea, rectal tenesmus (spasms), and some nausea (seldom accompanied by vomiting); fever was uncommon. In most cases, symptoms resolved within 12 hours. Although a sample of the sauce contained between 2.5 × 107 and 1.1 × 108 B. cereus/mL, there was apparently little change in the odor, taste, or consistency of the product. Numerous spores of B. cereus, up to 104 cfu/g, were found in the cornstarch used to prepare the sauce. Since the 1950s, increasing awareness and recognition of B. cereus–associated illness have resulted in a substantial increase in the number of reports of the diarrheal syndrome of food poisoning, the majority originating in northern and eastern European countries (Kramer and Gilbert, 1989). The first well-documented outbreak in the United States occurred in 1969 (Midura et al., 1970). The food vehicle was a meat loaf contaminated with 7 × 106 B. cereus/g, and in 15 persons a typical diarrheal syndrome illness developed approximately 10 hours later. During the following years up until 1981, 37 outbreaks of food poisoning attributed to B. cereus and involving 698 cases were reported to the CDC (Gilbert and Kramer, 1986; Kramer and Gilbert, 1992). Examples of selected B. cereus food poisoning outbreaks are summarized in Table 13.20. Testing for B. cereus is not a routine practice in a number of state health laboratories within the United States. Food poisoning due to B. cereus emetic toxin was first identified in the early 1970s in the United Kingdom and was associated with consumption of cooked rice from Chinese restaurants and take-out outlets (Lund, 1990; Miller et al., 1998). Most reported cases of B. cereus food

Table 13.20

Year 1969 1975 1980 1985 a

poisoning in the United Kingdom have been attributable to the emetic form of toxin. Two outbreaks in Norway in the 1990s were associated with the consumption of contaminated stew. The infective dose was estimated at 104 to 105 cells. Of the 17 affected people, 3 were hospitalized, 1 for 3 weeks (Granum, 1994). The onset of symptoms for the three hospitalized patients occurred more than 24 hours after the consumption of the stew. The second outbreak, in February 1995, involved 152 cases among competitors of the Norwegian junior ski championship (Granum et al., 1995). The young atheletes (16 to 19 years old) had the most severe symptoms, whereas their coaches and officials were not affected. The time to onset of symptoms for some patients was more than 24 hours, and the duration of illness was from 2 days to several days. Perhaps some strains of B. cereus colonize in the small intestine of some patients and cause more severe symptoms by producing enterotoxin at the site of colonization. In the United States, beyond a general concern about any pathogen in foods, specific attention has been directed to the contamination of infant formula with B. cereus. The U.S. FDA has historically expressed concern “due to levels of B. cereus that exceed 1000 in powdered infant formula” (Batt, 2000a). Moreover, infant formula is of concern because of the ability of the organism to replicate rapidly on rehydration of dried formula. Therefore, current efforts by the FDA are directed to reducing the maximal permissible level of B. cereus in infant formula to 100 colony forming units (cfu) or most probable number (MPN) per gram. 13.7.7

Prevention

Because of the widespread occurrence of B. cereus, it is impossible to prevent contamination of raw foods. The ingestion of low numbers of B. cereus, however, is not harmful (Kramer and Gilbert, 1989). Freshly cooked food eaten hot is safe, but if the food is maintained at temperatures between 10°C and 60°C, spores that have survived cook-

Examples of Selected Bacillus cereus Food Poisoning Outbreaks Reported in the United States

Incriminated food

B. cereus, cfu/g

Ill, number

Symptomsa

Incubation period, hr

Reference

Meat loaf Mashed potatoes Macaroni and cheese Beef stew

7 × 10 2 × 107 108–109 NDb

15 2 8 46

N, (V), D, A V, A, D N, V, A, (D) A, D, (N)

10 3–9 1–3 2–23

Midura et al. (1970) Yrios et al. (1975) Holmes et al. (1981) DeBuono et al. (1988)

6

A, abdominal cramps; D, diarrhea; N, nausea; V, vomiting. ND = not determined.

b

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ing can germinate and the resulting vegetative bacteria can multiply in the food. Effective control measures depend on the prevention of spore germination and the prevention of growth and toxin production of B. cereus cells in cooked, ready-to-eat foods. Gilbert and associates (1974) have suggested the following preventive measures to prevent emetic poisoning syndrome from rice dishes: 1.

2.

3.

4.

Rice should be boiled in smaller quantities on several occasions during the day, thereby reducing the storage time before frying. After boiling, the rice should either be kept hot, at not less than 63°C, or cooled quickly and transferred to a refrigerator within 2 hours of cooking. The cooling of rice, especially large amounts of boiled rice, is hastened by dividing the product into separate portions or by spreading in clean shallow containers. Boiled or fried rice must not be stored under warm conditions and never at a temperature between 15°C and 50°C. Under no circumstances, therefore, should cooked rice be stored at kitchen temperature for more than 2 hours. The beaten egg used in the preparation of fried rice should be freshly prepared.

In general, there has been limited research done on control measures to prevent B. cereus food poisoning. According to Miller and colleagues (1998), this may be due, at least in part, to its relatively recent recognition as a food-borne pathogen and to the short duration of the illness that it causes.

13.8 CLOSTRIDIUM PERFRINGENS GASTROENTERITIS Historically, Clostridium perfringens is best known for its role as a causative agent for gas gangrene (clostridial myonecrosis) especially as a result of wound infections (Labbe, 1992). The advent of antibiotics has reduced the incidence and severity of such infections considerably. Food-borne intoxications caused by this organism were first recognized during the Second World War. Although food poisoning due to C. perfringens was described as early as 1945 (McClung, 1945), it was not until the classic report by Hobbs and coworkers (1953) that the organism began to acquire its deserved attention as one of major public health importance. The basic aspects of the illness, isolation, and identification of the organism, and purification of the toxin involved occurred in the 1960s and early 1970s. Since then, a voluminous literature has accumulated on this subject.

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C. perfringens actually causes two quite different human diseases (Table 13.21) that can be transmitted by food, i.e., C. perfringens type A food poisoning (gastroenteritis) and necrotic enteritis (also known as Darmbrand and Pig-Bel). The C. perfringens enterotoxin is responsible for one of the most common types of food poisoning in the United States (Genigeorgis, 1975; Duncan, 1970; Chakrabarty et al., 1977; Shandera et al., 1983). It is most often associated with meat, poultry, and gravy-containing dishes that are served or stored in large quantities. The organism has become a major agent of food-borne bacterial intoxication in recent years as the result of the shift in meal preparation from the home to the institutional and commercial kitchen. Concomitant with this shift are the prolonged lapse between cooking and serving, the opportunity for abuse in refrigeration, the generally larger volumes or pieces of foods, and the failure to reheat properly foods prepared far in advance of serving. 13.8.1

Organism

Species within the genus Clostridium have both medical and industrial significance. They produce a wide diversity of exoproteins, many of which function as virulence factors. Some of these proteins are antigenic in nature, and some have associated enzyme activity. Table 13.21 lists a representative group of clostridial species that cause various diseases in humans. The most important species with respect to human disease include C. botulinum, C. perfringens, C. tetani, and C. difficile. The role of toxins produced by these species in causing disease has been well characterized. C. perfringens, also known as C. welchii, is a large nonmotile, encapsulated, gram-positive spore-forming rod. The spores, which are rarely seen, are oval and subterminal. Although classified as an obligate anaerobe, C. perfringens can grow in the presence of low levels of oxygen.

Table 13.21 Clostridium Species Involved in Causing Human Diseases Species C. perfringens

C. tetani C. botulinum c. difficile C. novyi C. histolyticum C. septicum

Diseases Food poisoning, gas gangrene, necrotic enteritis, minor wound infection Tetanus Botulism food poisoning Pseudomembraneous enterocolitis Gas gangrene Gas gangrene Gas gangrene

The organism grows between 15°C and 50°C, with an exceedingly rapid growth and a brief generation time of 9.5 minutes at 43°C. In cooling foods, growth is begun by germination of the spore at the upper permissible temperature and continues as the cooling wave moves inward. Compared to S. aureus, C. perfringens is not very tolerant to low water activity. The lowest level supporting growth is between 0.97 and 0.95 (Bartsch and Walker, 1982). It grows at pH 5–9; optimal pH is 5.6, the ultimate pH of red meats. The maximal tolerance to sodium chloride is 5%–8%, a concentration that is far beyond human tolerance in foods. C. perfringens produces at least 12 different soluble toxins that may be involved in pathogenesis (Table 13.22). At least eight of the toxins are believed to be lethal. Four of the lethal toxins, α, β, ε, and ι, are considered to be the major toxins and are used to group the species into five toxigenic types, A, B, C, D, and E (Hauschild, 1971; McDonel, 1986). The toxins produced by the different types are given in Table 13.23. Traditionally, toxin typing of C. perfringens has involved laborious toxin antiserum neutralization tests in mice and thus has been done in only a few laboratories. However, the recent development of polymerase chain reaction– (PCR)-based schemes (Daube et al., 1994) for toxin typing of C. perfringens isolates should now simplify this process considerably. Type A strains produce predominantly alpha (α) toxin, type B beta (β) and epsilon (ε) toxins, type C beta and delta (δ) toxins, type D ε

Table 13.22 Soluble Toxins Produced by Clostridium perfringens and Their Activities Toxin Alpha (lecithinase) Beta Epsilon Iota Delta Theta Kappa (collagenase) Lambda (protease) Mu (hyaluronidase) Nu (deoxyribonuclease) Gamma Eta Enterotoxin Neuraminidase

Activity Lethal, necrotizing, hemolytic Lethal, necrotizing Lethal, necrotizing Lethal, necrotizing Lethal, hemolytic Lethal, hemolytic Lethal, necrotizing, gelatinase Disintegrates azocoll and hide powder, gelatinase Release glucosamine from hyaluronic acid Leucocidic in gas gangrene and postuterine infection Existence doubtful, may be lethal Existence doubtful, may be lethal Diarrheagenic, cytotoxic, causes gut damage, lethal Inhibits cell receptor function

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toxin, and type E iota (ι) toxin. All strains produce the α toxin, also known as lecithinase or phospholipase C. Type A strains are by far the most commonly encountered under noninfectious conditions, and they are the only ones associated with the microflora of both soil and the intestinal tract. The remaining types are invariably restricted to the intestinal tract, primarily of animals and occasionally of humans, under disease conditions. Various disease conditions caused by different types of C. perfringens are listed in Table 13.24. Virtually all food poisoning outbreaks are caused by type A strains; type C causes a more serious condition, called necrotic enteritis. It is the heat-resistant strains of type A, the spores of which can resist boiling for 1 to 5 hours, that are responsible for the majority of outbreaks of food poisoning caused by C. perfringens, although heatsensitive strains may sometimes be implicated. C. perfringens type A has been isolated from a wide variety of foods, most frequently from raw meats and poultry. The extensive contamination in meats led Smart and associates (1979) to suggest that strains of C. perfringens capable of causing food poisoning are present on every commercial carcass of raw meat, even if only in low numbers; similar results have been obtained for poultry (Nakamura and Schulze, 1970). 13.8.2

Toxin

The discovery that an enteropathogenic factor is produced during sporulation led to the isolation and purification of a protein toxin (Duncan and Strong, 1969; Hauschild et al., 1970). This toxin has been shown to be the factor primarily responsible for experimental disease induced in several animal species, including lambs, sheep, calves, chickens, dogs, rats, rabbits, guinea pigs, mice, horses, monkeys, and humans. C. perfringens enterotoxin is apparently synthesized during sporulation. Attempts to demonstrate enterotoxin in vegetative cultures have repeatedly failed, and the degree of sporulation correlates with the quantity of enterotoxin produced by individual strains (Hauschild et al., 1971; Duncan and Strong, 1969). As a result of these observations, attempts to purify the enterotoxin are generally initiated with sonicates of young sporulating cells (Hauschild and Hilscheimer, 1971; Sakaguchi et al., 1973; Skjelkvale and Duncan, 1975). A number of researchers have isolated and purified the enterotoxin by using classical biochemical techniques, such as gel filtration followed by anion-exchange chromatography, ribonuclease (RNase) treatment, and affinity chromatography. The average molecular weight of the protein appears to be approximately 35,000 Da. The protein is a single-

Table 13.23

Toxins Produced by Different Types of Clostridium perfringensa

Toxin Major toxins Alpha Beta Epsiolon Iota Minor toxins Delta Theta Kappa Lambda Mu Nu Eta Gamma Other toxins Enterotoxin Neuraminidase a

Type A

Type B

Type C

Type D

Type E

+ – – –

+ + (+) –

+ + – –

+ – (+) –

+ – – (+)

– + + – + + Θ –

+ + + + + + – Θ

+ + + – + + – Θ

– + + + + + – –

– + + + – + – –

+ +

O +

+ +

+ +

O +

+, Produced by some strains of the type given; quantities of toxin produced by different strains can vary; –, not known to be produced by any strains of the given type; (+), prototoxin, activation requires enzymes; Θ, existence doubtful; O, not studied.

polypeptide chain with an isoelectric point of 4.3. Enders and Duncan (1976) have suggested that the acidic protein may have hydrophobic regions created by clustered hydrophobic amino acids, which constitute about 40% of the residues in the molecule. It is free of fatty acids, nucleic acids, and carbohydrates. Smith and McDonel (1980) suggested it to be a spore coat component. Amino acid sequence analysis of the enterotoxin has yielded a molecular weight of 34,262 consisting of a single polypeptide of 309 amino acids (Richardson and

Table 13.24 Disease Conditions Caused by Different Types of Clostridium perfringens Type A

B C

D E

Disease Gas gangrene in humans and animals, food poisoning, equine grass sickness, necrotizing colitis and enterotoxemia of horses Lamb dysentery, enterotoxemia of foals, sheep, and goats Enterotoxemia of sheep (struck), calves, lambs, piglets; necrotic enteritis of humans (Pig-Bel, Darmbrand) and fowl Enterotoxemia of sheep, lambs (pulpy kidney or overeating disease), goats, cattle, possibly humans Role in pathogenicity unclear; found in sheep and cattle; possibly responsible for colitis in rabbits

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Granum, 1985). The sequence of the toxin has no homology with any other known toxins for which sequence data are available, indicating that the enterotoxin represents a unique polypeptide primary structure (Duffy et al., 1982; Richardson and Granum, 1985). The enterotoxin is heat-labile; heating in saline solution at 60°C for 5 minutes destroys its biological activity. However, 10% of the original serological activity is retained even after 80 minutes at 60°C (Naik and Duncan, 1978). Enterotoxin stored at 37°C for 7 days or at –20°C for 28 days loses biological activity but not serological activity ((McDonel and McClane, 1981). However, neither activity is affected by storage at 4°C for several weeks (Granum et al., 1981). The toxin is also quite sensitive to pH extremes but resistant to some proteolytic treatments (McDonel, 1986). In fact, limited trypsinization or chymotrypsinization actually causes a two- to three-fold activation of the toxin activity (Granum et al., 1981; Granum and Richardson, 1991). These results suggest that intestinal proteases may produce an activated toxin during food poisoning. However, no direct in vivo evidence supporting this hypothesis has been presented. The protein is susceptible to freeze-thaw injury. About 5%–10% of the original activity of the toxin is lost each time it is thawed. The enterotoxin loses its activity during pronase and subtilisin treatment but is insensitive to trypsin, chymotrypsin, bacterial lipase, steapsin, α-amy-

lase, papain, and neuraminidase (Duncan and Strong, 1969; Hauschild and Hilsheimer, 1971). Lyophilization is the preferred method of storing toxin. Attempts to determine the mode of action and the responsible enterotoxin initially sought to reproduce the fluid loss by using animal models. The toxin causes desquamation of intestinal epithelial cells at villus tips (McDonel and Asano, 1975; Labbe, 1992). Brush borders (microvillus membrane) lose their characteristic folded configuration, and large quantities of membrane and cytoplasm are lost to the lumen. The brush border of villus tip epithelial cells is the primary site of action of the enterotoxin (McDonel et al., 1978). In contrast to the effects of cholera toxin and E. coli heat-labile enterotoxin, C. perfringens enterotoxin does not increase the levels of cyclic adenosine monophosphate (cAMP) in intestinal mucosa that is actively secreting fluid (McDonel, 1979). Research since the 1980s directed to the toxin’s action during C. perfringens type A food poisoning has helped to generate a time line depicting the overall sequence of events that are believed to lead to its intestinal effects (Figure 13.5). These events represent a novel mechanism of action for this bacterial enterotoxin. The recent studies have focused primarily on relating this se-

quence of events to the enterotoxin’s mode of action that ultimately leads to the cytotoxic effects seen on mammalian cells and the associated tissue damage. Wieckowski and associates (1994) proposed the current overview model for early steps in the enterotoxin’s action. The enterotoxin action is a multistep process involving at least four early events (Figure 13.6). The initial event is the binding of the enterotoxin to its receptor, which is believed to be a 50-kDa membrane protein. This action results in the formation of a 90-kDa small complex. This is almost immediately followed by some physical change in the enterotoxin protein sequestered in the small complex. This step could correspond either to the insertion of the toxin (or the complex itself) into the membrane bilayer or to a conformational change in the small complex. The third early event involves the formation of a large (160-kDa) complex, whose formation apparently results from an interaction between the enterotoxin-containing small complex and a 70-kDa protein. In the fourth and final early step in the enterotoxin action, the large complex causes the plasma membrane to lose its normal permeability properties. This effect could be the result of the large complex’s serving directly as a pore, or it could be the result of a less direct mechanism (Figure 13.6).

Figure 13.5 Time line of events in Clostridium perfringens enterotoxin action. In this scheme, it is assumed that the enterotoxin has been added at time zero. (Redrawn from McClane and Wnek [1990] and McClane [1997].)

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Figure 13.6 The current model for early events in Clostridium perfringens enterotoxin action. This model reflects the four known early events in toxin’s action: binding of enterotoxin to its receptor to form a small (90-kDa) enterotoxin-containing complex (step 1), a physical change in this enterotoxin sequestered in small complex (an effect that could correspond to either toxin insertion or a conformational change in small complex (step 2), formation of a large enterotoxin-containing complex resulting from an interaction between small complex and a 70-kDa membrane protein (step 3), and development of permeability changes in large-complex-containing plasma membranes of mammalian cells (step 4). All the steps can occur at 37°C; steps 3 and 4 do not occur at low temperature (e.g., 4°C). (From McClane [1997].)

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The model proposed by Wieckowski and colleagues (1994) emphasizes the uniqueness of C. perfringens enterotoxin action at the molecular level by predicting that membrane proteins are intimately involved in every early step of the action. No other membrane-active toxin is known to date to involve eukaryotic proteins so heavily in its action. The initial interaction between the enterotoxin and the mammalian plasma membranes exhibits characteristics typical of a receptor-mediated process. The binding occurs rapidly, is clearly saturable, and is temperature-sensitive (McClane, 1994; Wieckowski et al., 1994). C. perfringens enterotoxin receptors have been found in the small intestines of several mammalian species (McClane, 1994; Sugii and Horiguchi, 1988). To exert its toxic action, the toxin-receptor complex must be inserted into the lipid bilayer of the plasma membrane of mammalian cells. The insertion of the enterotoxin does not represent simply the static binding of the enterotoxin to its receptor to form a protease-resistant complex; instead it represents some postbinding physical change that occurs in the bound enterotoxin, a change such as its insertion into the membranes. This step is considered to be essential in the enterotoxin action (McClane, 1997). Once inserted into the plasma membrane, the enterotoxin becomes sequestered in a large complex (160 kDa) quite rapidly by noncovalent forces. The large complex formation requires intact membranes. The large complex formation does not occur at lower temperatures, e.g., at 4°C, and its formation is a necessary step in enterotoxininduced cytotoxicity. There is an extremely close temporal correlation between large complex formation and the onset of C. perfringens enterotoxin-induced small molecular permeability alterations in the membrane (McClane and Wnek, 1990). This correlation precludes the existence of extensive intermediate steps between these two early events and is consistent with the large complex’s directly causing the toxin’s effects on plasma membrane permeability. The alterations in the membrane permeability are possibly due to the large complex’s functioning as a unique membrane pore. However, this hypothesis is at yet unproved. As an alternative possibility to the large complex’s representing a pore, the complex itself could function indirectly via a nonpore mechanism. For example, perhaps the large complex formation interferes with the regulation of existing membrane pumps in such a manner that these pumps become continuously activated or inactivated (McClane, 1997). The secondary consequences of the C. perfringens enterotoxin action include inhibition of DNA, RNA, and protein synthesis; permeability alterations for large mole-

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cules of up to 3000 to 5000 molecular weight; development of morphological damage, such as blebs; and finally cell lysis. These effects are related to the disruption of the normal colloid-osmotic equilibrium of the mammalian cell that results from the toxin’s initial membrane effects on small molecular permeability (McClane, 1984, 1994, 1997; McClane et al., 1988). 13.8.3

Symptoms and Diagnosis

The symptoms of C. perfringens type A food poisoning first appear after an incubation period of 6 to 22 hours after the consumption of foods containing large numbers of toxin-producing bacteria. The symptoms are characterized by severe abdominal cramps, nausea, and profuse diarrhea; there is normally no vomiting or fever. Recovery is usually rapid, within 12 to 24 hours, although this period may be extended in elderly people for a further 1 or 2 days. A few deaths as a result of dehydration and other complications have been reported. However, the mortality rate is very low. Immunity to future episodes is not conferred. The symptoms are caused by ingestion of large numbers (greater than 108) vegetative cells. Toxin production in the digestive tract is associated with sporulation. A more severe type of illness occurs among natives of the New Guinea Highlands. It is classified as a necrotizing hemorrhagic jejunitis, commonly called Pig-Bel, and is caused by type C strains of C. perfringens that produce β toxin. Deaths of necrotic enteritis are caused by infection and necrosis of the intestines and from resulting septicemia. The illness, which follows traditional pig feasting, is rare in Western countries (Walker, 1985; Lawrence, 1986). C. perfringens poisoning is diagnosed by its symptoms and the typical delayed onset of illness. Diagnosis is confirmed by detecting the toxin in the feces of patients. Bacteriological confirmation can also be done by finding exceptionally large numbers of the causative bacteria in implicated foods or in the feces of patients. Diagnostic criteria suggested by the CDC are (a) detection of 105 C. perfringens/g in a common food source and (b) stools obtained within 48 hours of symptoms showing 106/g of C. perfringens spores (Shandera et al., 1983). 13.8.4

Detection

Several biological and serological assay systems have been developed for the detection and quantitation of enterotoxin. The earliest assay of the enterotoxin’s biological activity was the LRIL test (Duncan and Strong, 1969). This assay is based upon the response of the intestine to the enterotoxin, which is a net secretion of fluid and elec-

trolytes into the lumen. The result is a noticeable expansion of the loops due to the excess fluid. Probably the most generally applicable assay of biological activity reported to date is one based on the observations of Hauschild (1970) and Stark and Duncan (1971) that the enterotoxin, when injected intradermally, causes erythema in guinea pig or rabbit skin. The erythemal unit (EU) has been defined as that amount of enterotoxin that causes a zone of erythema 0.8 cm in diameter on the skin of a dipilitated guinea pig that has received intradermal injections of the enterotoxin (Stark and Duncan, 1971). Results are usually read at 18–24 hours after injection. This assay is more sensitive, less expensive, and easier than the LRIL test. Systemic effects of the enterotoxin using this assay in a number of animal species are listed in Table 13.25. It can detect, with reasonable accuracy, quantities of enterotoxin as low as 0.25–0.5 EU. A linear dose-response curve can be seen over a range of about 0.5–2.0 EU. Mouse lethality assay has also been used to determine the biological activity of the C. perfringens enterotoxin (Hauschild and Hilsheimer, 1971; Stark and Duncan, 1971). A highly sensitive biological assay utilizing inhibition by the enterotoxin of the plating efficiency of Vero (African green monkey kidney) cells grown in tissue culture has been reported by McClane and McDonel (1979). This effect has been used to detect as little as 0.1 ng of protein. Immunological assays have also been used for quantitating this enterotoxin (Duncan and Somers, 1972; Naik and Duncan, 1977; Wimsatt et al., 1986). The only commercially available (Oxoid, Inc., Nepean, Ontario, Canada) serological assay for C. perfringens enterotoxin is the RPLA test. 13.8.5

Foods Associated with Poisoning Outbreaks

The organism’s requirement for many amino acids makes meat, meat products, gravies, and casseroles good candi-

Table 13.25 Systemic Effects of Clostridium perfringens Enterotoxin When Injected Intravenously into Lambs, Rabbits, and Guinea Pigs Parasympathomimetic properties Capillary permeability, vasodilation in intestine, increased intestinal motility Other effects Diarrhea, lacrimation, salivation, nasal discharge, lassitude, dyspnea, hyperemic intestinal mucosa, congestion of liver, lungs, spleen, and kidneys Source: From Niilo (1971) and McDonel (1986).

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dates for this type of food poisoning. According to the CDC statistics, beef alone accounted for nearly 30% of all C. perfringens type A food poisoning in the United States, and turkey and chicken together accounted for another 15% during the period 1973–1987 (Bean and Griffin, 1990). These statistics also indicated that Mexican foods containing meats are emerging as another important vehicle for poisoning outbreaks. It has not been associated with seafoods, with vegetable dishes that do not contain meats, or with fruit dishes. The majority of C. perfringens food poisoning outbreaks result from the ingestion of meat and poultry dishes that have been subjected to incorrect temperature treatment. This type of food poisoning is generally associated with poor catering practices, and the number of individuals affected per outbreak (15–25) is, on average, greater than that for other forms of food poisoning. Illness is most frequently associated with those items held for a long time before cooking or with precooked meat items either eaten cold or reheated. Holding food in the danger zone allows this microorganism to proliferate. The problem of temperature control needs further clarification. Foods can be inadequately heated so that the vegetative cells of the organism are destroyed but the heatresistant spores remain viable. Even during inadequate cooking, oxygen is driven off and conditions in the food become anaerobic, thus enabling spore germination to commence once the meat has cooled to 50°C. Slow cooling between 50°C and 25°C is particularly hazardous with this organism as growth rates are high at these temperatures and large numbers of vegetative cells may therefore develop within a few hours. The problem is exacerbated if the foods, such as cooked meats, stews, and gravies, are then stored at ambient temperature before being eaten, as further growth can occur. Sutton and coworkers (1972) studied the effect of cooking and cooling on C. perfringens in meat. They showed that even vegetative cells might not be killed near the center of larger joints of meat. Cooling at 15°C was ineffective in controlling the growth of the organism and even at 4°C almost 4–5 hours would be necessary to reduce the temperature to 15°C, the minimal growth temperature for C. perfringens. In catering, foods are often prepared on the day prior to consumption and eaten after warming. This practice can be dangerous, and in addition, the final reheating is often only sufficient to stimulate further growth of the organisms rather than destroy them. Food containing the enterotoxin may be different in odor, color, and texture (Al-Obaidy et al., 1985), but consumers may overlook differences that would be detected by taste panelists.

13.8.6

Outbreaks

C. perfringens food poisoning is a relatively common form of food-borne outbreak of disease. UK studies show 30–50 outbreaks involving 1000–2000 persons annually (Mead, 1979; Bartlett, 1988). Data from the CDC indicate that C. perfringens accounts for about 7% of food-borne disease outbreaks in the United States with an average of 24 victims per outbreak. It is quite likely that many outbreaks are overlooked as a result of the relatively mild symptoms and specialized laboratory techniques required for case confirmation. Todd (1989a, 1989b) has estimated 652,000 cases of C. perfringens type A food poisoning in the United States each year, with an average of 7.6 deaths per year and annual costs of approximately $123 million. Identified cases of C. perfringens type A food poisoning usually involve large outbreaks, often in institutionalized settings. This epidemiological pattern results from at least two factors (McClane, 1997). First, large institutions often prepare food in advance and then hold it for later serving, thereby allowing the possible growth of C. perfringens in any temperature-abused foods. Second, given the relatively mild and nondistinguishing symptoms of most cases of C. perfringens type A food poisoning, it is only when a significant number of people become simultaneously sickened with diarrheal symptoms that public health officials are sufficiently motivated to investigate, identify, and report this illness. Outbreaks described in the following sections illustrate the important underlying processes in this type of food poisoning. In 1994, the CDC published a report on an investigation of two outbreaks of C. perfringens type A food poisoning that were associated with St. Patrick’s Day meals (CDC, 1994). The first occurred in Cleveland, Ohio, and involved 156 persons, all of whom acquired the disease from ingesting corned beef that had been prepared at a local delicatessen. During its preparation, the corned beef had been boiled for 3 hours and then allowed to cool slowly at room temperature before refrigeration. Four days later, portions of this corned beef were warmed to 48.8°C and served; some sandwiches prepared with this corned beef were held at ambient temperature from the time of their preparation until they were eaten throughout the afternoon. The second St. Patrick’s Day outbreak described in this CDC report occurred in Virginia and involved 86 persons. The outbreak occurred after the consumption of a dinner that included corned beef contaminated with large numbers of C. perfringens cells. The corned beef involved in this outbreak was a frozen commercially prepared brined product that had been thawed, cooked in large (10lb) pieces, stored in a refrigerator, and held for 90 minutes

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under a heat lamp before being served. Both these outbreaks illustrate the importance of temperature abuse of cooked food as a major contributing factor in this type of food poisoning. A similar type of outbreak that occurred in a British hospital in 1995 sickened 17 patients (Regan et al., 1995). Large numbers of C. perfringens cells were later identified in the vacuum-packed pork served to them. The pork had apparently become contaminated as a result of very slow cooling after cooking at a commercial meat preparation facility. Unlike the U.S. outbreaks that occurred on St. Patrick’s Day, this British outbreak was somewhat unusual in that it involved a commercially prepared food product. 13.8.7

Prevention

Obviously because of the widespread occurrence on foods as well as in the environment generally of C. perfringens, eliminating it would be impractical. Carcass meat and poultry inevitably are contaminated with this bacterium, and survival of the heat-resistant spores after cooking can also be anticipated. The control measures should therefore be aimed at restricting both spore germination and the subsequent proliferation of vegetative cells while the food is cooling and during its storage. It is also important to cook foods thoroughly, especially large roasts and turkeys, since because of their size, generating internal temperatures high enough to kill C. perfringens spores is difficult. This difficulty is undoubtedly a major reason why large roasts and poultry products are such common food vehicles for C. perfringens type A food poisoning outbreaks. To prevent such large outbreaks, the following measures are recommended: 1. 2.

3.

4.

Where possible, the food should be cooked and eaten immediately. Where food is to be held over after cooking it should be cooled as rapidly as possible to below 20°C within 1 hour and refrigerated; placing hot foods in a refrigerator should be avoided as cooling is likely to be too slow; blast coolers should be used. Partial or complete cooking of foods on one day and reheating it the next should preferably be avoided, but when it is necessary, it is important to ensure that the food is thoroughly heated before being consumed. Cross-contamination of cooked foods by raw or by dirty working surfaces, equipment, and utensils should be prevented.

Although C. perfringens spores are usually destroyed when the medium is kept at a temperature of

100°C for 30 minutes, this time-temperature regimen often may alter the aesthetic quality of the food. Whereas the pH of raw meat and poultry normally stabilizes at 5.4–5.7, C. perfringens multiplies at pH 5.0 and above, so this is a suitable medium. It also multiplies at warmer temperatures sufficient to retard the growth of other organisms, thereby allowing it to thrive under some conditions associated with poor food handling. The basis for controlling food-borne outbreaks is keeping hot things hot and cold things cold: i.e., to prevent replication, cooked foods should not be held at temperatures between 4.4°C and 60°C. The occurrence of this troublesome enteric infection is thus highly preventable by simple application of hygienic principles, by recognition and prevention of the conditions that permit multiplication of the causative organism in food, and by avoidance of food that has been improperly prepared and stored.

13.9 CLOSTRIDIUM BOTULINUM NEUROTOXINS The toxins of C. botulinum are some of the most potent biological poisons known because of their lethality and severity of symptoms. Even though its occurrence is relatively rare, botulism caused by poisoning of food contaminated by these toxins is a serious health hazard worldwide. The exotoxins produced by C. botulinum are extremely toxic to humans. A fatal dose for an adult of type A toxin, the most potent, can be as little as 1 × 10–8 g; in other words, 1 g can kill 100 million people. The toxins are classed as neurotoxins since their action is specifically directed to nerves, especially the peripheral nerves of the involuntary muscles of the body. Fortunately, these toxins are not very resistant to heat. Botulism has probably been associated with humans since they first attempted to preserve foods (Eklund, 1982). Its history has been reviewed extensively (Dolman, 1964; Smith and Sugiyama, 1988; Sakaguchi, 1986; Hauschild, 1989). The association of this disease with sausages, especially smoked blood sausages in Germany, led to introduction of the term botulism, which is derived from the Latin word botulus, meaning “sausage” (Dolman, 1964). Acute food poisoning caused by the ingestion of spoiled sausages has been known in Europe for more than 2000 years (Dack, 1956). It was first identified as an illness in 1793. In Russia, the disease was reported in 1818 under the name of ichthyism or fish poisoning; the term dalphinapterism was suggested for the illness, which followed the consumption of white whale and sea meat (Dolman, 1964). The etiological agent of botulism was first isolated in 1896 by Van Ermengem (1897a) from an anaerobic

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spore-forming bacillus from the remains of the incriminated raw ham and the livers of the dead involved in a food poisoning outbreak from the consumption of an improperly salted ham in the Belgian village of Ellezelles in the province of Hainot. Culture filtrates of the isolates, when administered to various experimental animals through different routes, produced paralytic symptoms resembling those of human botulism and led to death. He ascribed the extracellular toxin produced by the isolate to human botulism. The bacterium was first named Bacillus botulinus, later renamed Clostridium botulinum. On the basis of the excellent documentation of Van Ermengem, the isolate from the salted ham would now be classified as nonproteolytic type B strain. In 1904, 11 persons died after eating wax-bean salad in Darmstadt, Germany (Landmann, 1904). This episode proved that botulinum toxin is produced not only in meat and meat products but also in boiled vegetables. The antitoxic serum prepared again the toxin of Elzele strain did not neutralize the toxin of Darmstadt strain; nor did the antitoxic serum against the toxin of Darmstadt strain neutralize the toxin of Elzele strain. Thus, the toxins produced by the two strains were immunologically distinct (Leuchs, 1910). Botulinum toxins were later classified into types A and B (Meyer and Gunnison, 1929); the toxin of Darmstadt strain may have corresponded to type A, as opposed to the type B toxin of Elzele strain. Additional types of C. botulinum strains, A through G, were identified in subsequent years. On the basis of the heat resistance of van Ermengem’s culture, however, a farmer’s bulletin that outlined procedures for home canning of foods was published in the United States in 1909. Because of lack of knowledge of existence of more heat-resistant C. botulinum strains, the heat treatments recommended were inadequate and may have led to a number of botulism outbreaks in the early 1900s caused by consumption of underprocessed home-canned foods (Eklund, 1982; Sofos, 1992). 13.9.1

Types of Botulism

Human botulism is classified into the following four types, which are based on the mode of intoxication (CDC, 1979). There is no transmission from one person to another, although sometimes many people are affected at one time after consumption of a common toxin source (Iida et al., 1964; Terranova et al., 1978; Sakaguchi, 1986; Sugiyama and Sofos, 1988). Food-Borne Botulism Food-borne botulism is the classical intoxication caused by consumption of food contaminated with preformed

toxin of C. botulinum that has proliferated in food. Adult cases are mostly of this type. Type A, B, and E toxins have often been involved in this type of botulism. Laboratory diagnosis of food-borne botulism is dependent upon the detection and identification of the toxin in the blood serum of the patient and the incriminated food. Detection of the toxin in the fecal specimen is also useful for diagnosis. Wound Botulism Wound botulism, a rare form first recognized in 1943, occurs when a wound is infected with type A or B spores. These spores germinate, grow, and produce toxin locally. The toxin reaches other parts of the body via the bloodstream. Foods are not involved in this type of botulism. The incubation period has ranged from 4 to 18 days. A single case per outbreak is a characteristic feature, and young children have most often been affected in the reported cases so far (Sakaguchi, 1986). Infant Botulism Infant botulism is an infection involving infants of less than 1 year of age, first recognized in 1975. Over 200 cases have been documented globally so far for this form of botulism. The number of confirmed infant botulism cases has increased significantly as a result of greater awareness of health officials since its recognition. It is now internationally recognized, and cases are reported in more countries. No toxin source has been found in any case; it is caused by ingestion of spores of C. botulinum, colonization of the intestinal tract, and elaboration of toxin in the lumen of the intestine. Infants do not always possess the normal intestinal flora needed to suppress C. botulinum when its spores are ingested. Contaminated honey has often been incriminated as a probable vector of the spores (Arnon et al., 1978), although there are many other potential vectors. Type A and B cases of infant botulism are common. Detection and identification of the toxin in the fecal materials are the key to diagnosis. Excretion of the toxin and the organisms in the feces continues for a long period, although patients recover spontaneously. The case fatality rate has been less than 3%; however, it may actually have been higher, since at least 5% of cases of sudden infant death syndrome (SIDS) are suspected to be the result of infant botulism (Arnon et al., 1978). Undetermined Category The undetermined category of botulism involves adult cases in which a specific food or wound source cannot be identified. This form appears to be associated with abnor-

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malities in the gastrointestinal system and its microflora, with in vivo production of the toxin. Reports in the medical literature suggest the existence of this form of botulism in patients who had surgical alterations of the gastrointestinal (GI) tract and/or antibiotic therapy (Sonnabend et al., 1981). Type G toxin is believed to be involved in this form of botulism, although both type D organisms and type A toxin were also detected in known cases of this form of botulism. 13.9.2

Organism

C. botulinum is obligately anaerobic and motile organism that contains 6–20 peritrichous flagella; it is a gram-positive, spore-forming rod of the family Bacillaceae. The organisms all form oval, fusiform, or club-shaped subterminal spores. Germination of the spores, outgrowth, and subsequent anaerobic growth are prerequisites to toxin production. Spores are often in a state of dormancy. The organism is a resident of the soil with global distribution, from which the spores are transferred to the surfaces of vegetation. The organism has not been isolated from raw meats, but meat surfaces may become contaminated during handling. C. botulinum is a strict saprophyte. It does not grow in living tissue but does grow in damaged and dead tissue to produce wound botulism. The various strains of C. botulinum are classified into four groups, which are based on their proteolytic activity and other properties such as resistance to heat and type of toxin being produced (Smith and Sugiyama, 1988; Sugiyama and Sofos, 1988; Hauschild, 1989). Some important characteristics of these four groups are listed in Table 13.26. Group I strains include all known cultures producing toxin type A, which are strongly proteolytic and saccharolytic, and the proteolytic cultures that produce toxin types B and F. The spores of the strains of this group are highly heat resistant. Nonproteolytic, but saccharolytic, cultures producing toxin types B, E, or F are group II strains. These strains are psychrotrophic, producing toxin at low temperatures. The spores of this group are relatively heat-labile. Mildly proteolytic or nonproteolytic but saccharolytic cultures producing toxin types C and D are classified as group III strains. The heat resistance of the spores of this group is intermediate. Finally, weakly proteolytic and nonsaccharolytic cultures producing toxin type G are group IV strains (Sakaguchi et al., 1981; Solomon et al., 1985; Hauschild, 1989; Hatheway, 1995). Type G toxin requires tryptic activation for its full toxicity (Smith, 1977). Group I and II strains that produce type A, B, and E exotoxins are almost entirely responsible for human outbreaks of botulism. Type C and D toxins are important as causes of botulism in animals, including birds, although

Table 13.26

Characteristics of Clostridium botulinum Groupsa

Characteristic

Group I

Group II

Group III

Group IV

Neurotoxin type Associated with human outbreaks Growth temperature, °C Minimal Optimal pH Range for growth Inhibitory pH Inhibitory [NaCl], % Minimal aw for growth Proteolytic Heat resistance of spores, °C D100°C of spores, min D121°C of spores, min

A, B, F +

B, E, F +

C, D –

G –

10 35–40 4.6–9.0 4.6 10 0.94 + 112 25 0.1–0.2

3.3 18–25 5.0–9.0 5.0 5 0.97 – 80 <0.1 <0.001

15 40 ND ND ND ND +/– 104 0.1–0.9 ND

ND 37 ND ND ND ND + 104 0.8–1.12 ND

ND, not determined; +, yes; –, no; ±, rare Source: Compiled from Hauschild (1989), Hatheway (1992, 1995), and Miller et al. (1998).

a

type C has been implicated in a few outbreaks of human botulism. Type F and G exotoxins have also caused human botulism on a few occasions. The optimal temperature for growth of the organism varies from 25°C to 37°C, depending upon the strain; the minimal growth temperature for the majority of the types varies between 10°C and 20°C, but psychrotrophic strains of types B, E, and F have minimal growth temperatures of 3°C–5°C. The spores of C. botulinum are markedly heatresistant and those of types A and B can resist boiling for up to 6 hours. Those of types C and D are rather less heatresistant; type E spores are inactivated at 80°C in 15 minutes. Some of the basic microbiological properties of C. botulinum are summarized in Table 13.26. However, it should be noted that the absolute values can vary according to the particular bacterial strain and the growth medium used. Despite their grouping as a single species, strains of C. botulinum have widely varying physiological and biochemical characteristics (Adams and Moss, 1995). Spores of the same strain but of different origin or produced under different conditions can behave differently in the same environment (Blocher et al., 1982; Sofos et al., 1986). The single common factor is the production of potent neurotoxins that are the cause of botulism. The factors or conditions for growth and toxin production by C. botulinum in foods have been established by various studies. These include (a) type and nutrient composition of substrate, (b) temperature of growth, (c) acidity and salt concentration of substrates, (d) water activity, (e) oxygen concentration, (f) presence of other microorganisms, and (g) other inhibitors.

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13.9.3

Neurotoxins

Botulinum toxins are defined as exotoxins that are released from the cells into the medium when the cells lyse. In culture medium, botulinum toxin is produced during the exponential phase of bacterial growth and is released from the cell into the growth medium. As the cells cease to grow and divide, the toxin is also released partly as a result of cell lysis (Siegel and Metzger, 1979). The group II organisms, which are characterized by nonproteolysis and lack of autolysis, do not efficiently release the toxin. Classification C. botulinum toxins are classified by the toxin-antitoxin neutralization test into types A through G in the chronological order of discovery, except types A and B, as described earlier. The type C toxin is classified into two subtypes, C1 and C2. The same classification is used for cultures, since most strains produce only one type of toxin. Known exceptions to this classification are cultures producing C1, C2, and D toxins, and those producing toxin types in pairs, or other clostridia-producing C. botulinum toxins. Cultures producing C1, C2, and D toxins usually form toxin mixtures and are typed on the basis of the toxin with the highest concentration. When C1 is dominant, the cultures are classified as Cα, Cβ when C2 toxin is dominant, and D when D type toxin is dominant in the mixture (Eklund and Poysky, 1972; Sofos, 1992). The susceptibility of animals to botulinum toxin of a certain type differs from one animal species to another and that of animals of

a certain species to botulinum toxins differs from one immunological type to another (Sakaguchi, 1986). Role of Bacteriophages in Toxigenicity All toxigenic strains of C. botulinum appear to be infected by bacteriophages (Dolman and Chang, 1972; Eklund et al., 1969; Iida et al., 1981; Inoue and Iida, 1968). There is evidence that the toxigenicity of C. botulinum is induced by appropriate phages (Iida et al., 1981; Eklund and Poysky, 1981). Thus toxin production is under the control of a viral gene. Nontoxigenic strains may become toxinproducing by means of these phages. Similarly, toxigenic strains may revert to the nontoxigenic state when the inducing phage is lost (Eklund et al., 1974; Iida et al., 1981). Reversion of a phage to a non-toxin-inducing form that is identical in almost all respects with the parent phage also can occur. Bacterial strains infected by toxigenically inactive phage become resistant to subsequent infection by the parent phage (Iida et al., 1981). The precise mechanism by which phages induce toxigenicity in their host has not been shown conclusively. However, these bacterial viruses carry the genetic template for the production of particular toxins in conjunction with the specific components of the host bacteria. The production of C2 toxin, however, appears not to require the intervention of any specific phage, since some nontoxigenic mutants of type C and D strains obtained by curing of their prophages continued to produce the C2 toxin, which was demonstrated by trypsinization of the culture (Eklund et al., 1972; Eklund and Poysky, 1981).

12S or M. Type A crystalline toxin is composed mostly of LL toxin (Sakaguchi et al., 1988). Purified botulinum toxins are single-chain polypeptides of molecular weights ranging from 128,000 for type F to 170,000 for type B. All toxin types are similar proteins, and an average molecular weight of 150,000 is used when they are considered as a single class of toxins (Sugiyama, 1980; Simpson, 1981). Proteolytic cleavage, either endogenous or exogenous, is necessary to generate active toxin. This peptide bond is located about one-third the distance from the amino terminal group of the protein (Sugiyama and Sofos, 1988; Sofos, 1992). Group II toxins of types B, E, and F and other nonproteolytic cultures of types C2 and G are especially susceptible to activation, and their lethality may increase several log–fold in the presence of proteolytic enzymes such as trypsin (Solomon and Kautter, 1979; Solomon et al., 1985). The activation and molecular structure of C. botulinum progenitor and derivative toxins are shown schematically in Figure 13.7. The nicked toxin consists of a heavy (H) (100,000 Da) and a light (L) (50,000 Da) chain connected by a disulfide bridge. The two individual chains are antigenically distinct, and their individual antibodies can neutralize the complete toxin; anti-H serum is generally more effective than anti-L serum (Sugiyama, 1980). The toxin type C2 is the exception because its subunits are separate molecules. Both chains are required for toxin activity (Montecucco and Schiavo, 1995). Detailed information on botulinum toxin structure can be found in several excellent reviews (Sakaguchi, 1986; Sakaguchi et al., 1988; Sofos, 1992; Hatheway, 1990; DasGupta, 1989; Montecucco and Schiavo, 1995).

Structure and Biochemical Properties Botulinum toxins occur in foods in the form of complexes (progenitor toxin) of 12S or larger size (Hauschild, 1989) consisting of a botulinum toxin subunit (7S) and one or more nontoxic subunits (7S or larger) (Hatheway, 1990). The size of the complexes formed depends on the strain of the organism, the type of food, and the growth conditions. The toxic component is released when progenitor toxin is exposed to pH 7.2 or higher. The freed toxic component is referred to as derivative toxin. Three different molecular-sized progenitor toxins have been found; the 12S toxin was named medium-sized or M toxin; the 16S toxin, large-sized or L toxin; the 19S toxin, extra large-sized or LL toxin. The 7S derivative toxin is sometimes called small-sized or S toxin (Kozaki et al., 1974; Sugii and Sakaguchi, 1975). The type A progenitor toxin involves all three forms, 19S, 16S, and 12S (or LL, L, and M); types B, C, and D toxins in two forms, 16S and 12S (or L and M); and types E and F in a single form,

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Stability Botulinum neurotoxins are highly unstable at alkaline pH, losing all activity at pH 11 at 15°C in 3 hours (Spero, 1958). Their activity is destroyed by denaturing agents (Schantz et al., 1960), amino-reacting (Schantz and Spero, 1956; Spero and Schantz, 1956) and sulfhydryl-reacting (Knox et al., 1970) reagents. Destruction of histidine in the protein chain by irradation in the presence of methylene blue (Weil et al., 1957), as with dilute oxidizing agents, destroys the toxin activity. C. botulinum toxins are highly heat-labile. Heating for 10 minutes at 80°C destroys the toxic activity. Heat stabilities of neurotoxins of different types were compared in acid foods and buffer systems (Woodburn et al., 1979; Bradshaw et al., 1981). In these experiments, the toxins used were crude or purified progenitor toxins. Type A crystalline toxin and type E progenitor toxin are more stable than their derivative toxins, and such higher stabilities

Trypsin

S S

S S

(pH 6.0) Progenitor toxin (12S)

Activated progenitor toxin (12S)

1 x 105 LD50/mg N

5 x 107 LD50/mg N

(pH 8.0)

(pH 8.0)

Trypsin S S

Nontoxic component (7S)

Derivative toxin (7S) 2 x 105 LD50/mg N

(pH 6.0)

S S

Activated derivative toxin (7S)

Nontoxic component (7S)

10 x 107 LD50/mg N

Figure 13.7 Activation and molecular structure of Clostridium botulinum type E toxin. Hatched area, activable toxic components; black area, activated toxic component; unshaded area, nontoxic component

are more pronounced at pH lower than 4 and 5, respectively (Kitamura et al., 1969). The stabilities were compared among type A-S, M, and L (a mixture of L and LL) toxins and among type B-S, M, and L toxins in buffers at pH 1–6 at 35°C (Sugii et al., 1977). The larger the molecular size of the toxin, the more stable it is at any pH value. Toxicity The biological activity of botulinum toxin is expressed quantitatively by titration in susceptible animal species, such as the rat, mouse, or guinea pig. Intraperitoneal (i.p.) injection of serially diluted toxin into mice is usually used as the most sensitive procedure to titrate the toxin. It is a general rule to express the toxicity of botulinum toxin in mouse i.p. LD50/mg N (Sakaguchi et al., 1988). The lethal dose to the mouse is not affected by its body weight (Lamanna et al., 1955). Oral Toxicity Botulinum toxin is an oral toxin and is one of the most poisonous substances known. The oral lethal doses for humans are estimated as the most likely amount ingested in untreated cases of fatal or near-fatal botulism (Hauschild, 1989). Studies involving the oral administration of toxin to mice have shown that the lethal dose decreases as the size of the toxin complex increases

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(Sakaguchi et al., 1981, 1984), probably because of the greater stability of large complexes at low pH, which makes them more resistant to the action of digestive enzymes. The oral lethal dose of type A crystalline toxin to experimental animals was estimated at 50,000 to 250,000 times that of the intraperitoneal lethal dose (Lamanna and Meyers, 1960). A similar ratio was obtained with type E activated progenitor toxin (Sakaguchi and Sakaguchi, 1974). Various factors such as the intestinal flora and the masticated food in the intestines may affect the oral lethal dose (Lamanna and Carr, 1967). The higher sensitivity to oral administration of the toxin of the large than of the small mouse is attributed to the larger surface area of the intestines in the large mouse (Lamanna, 1960). The oral toxicity to mice correlates well with the molecular size of the toxins (Table 13.27). The derivative toxin has an oral LD50 that is more than 10 million times the intraperitoneal LD50. It thus seems impossible for the derivative toxin, if ingested, to cause botulism (Sakaguchi and Sakaguchi, 1974). In contrast, the M toxin has an oral LD50 95,000–3,600,000 times and L toxin 1500–12,000 times the intraperitoneal LD50 of the respective toxin. The oral toxicity of the progenitor toxin is, however, much higher than that of the derivative toxin, and that of L toxin is apparently higher than that of M toxin of the same antigenic type (Sakaguchi et al., 1988).

Table 13.27 Oral LD50/Intraperitoneal LD50 in Mice of Botulinum Toxins of Different Molecular Sizes Toxin Type A

B

C D E

F a

Molecular size

Oral LD50/i.p. LD50 (× 10–3)a

LL L M S L M S L M L M M

120 2,200 3,600 43,000 1.5 1,100 24,000 5.3 160 62 370 220

S M S

>750 1,100 >6,000

Reference Ohishi et al. (1977)

Ohishi et al. (1977)

Ohishi and Sakaguchi (1980) Ohishi and Sakaguchi (1980) Sakaguchi and Sakaguchi (1974) Ohishi et al. (1977)

i.p., intraperitoneal.

It is thus quite apparent that if the same toxicities in mouse intraperitoneal LD50 are ingested, the L toxin is more toxic than the M toxin. In other words, if the same levels of toxin are present, foods that support the production of L toxin are likely to be more fatal than those supporting the production of M toxin. Vegetables (string beans and mushrooms) were shown to support the production of L and LL toxins, whereas meat (pork and tuna) supported production of M toxin by type A and B organisms (Sugii and Sakaguchi, 1977). These findings may explain, at least in part, why boiled vegetables have caused fatal human botulism more often than have meat and meat products (CDC, 1979). Intraperitoneal Toxicity The mouse i.p. LD50/mg N of the progenitor and derivative toxins differs from one immunological type to another and from one molecular form to another (Figure 13.8). Within the same immunological type, the smaller the molecular size of the toxin is, the smaller the quantity of nontoxic protein and therefore the higher its specific toxicity. The specific toxicities of M toxins of types A, B, and D are quite similar; those of M toxins of types C and F are about 20%; and that of type E activated progenitor toxin about 10% those of M toxins of types A, B, and D (Miyazaki et al., 1976). The intraperitoneal toxicity is entirely dependent upon the toxic component, and the nontoxic component seems to play little or no role. The latter,

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however, as described previously, plays a critically important role in the oral toxicity of the toxins. Apparently, the nontoxic component of the toxin functions to protect the toxin from inactivation in the digestive tract. The estimated minimal lethal dose (MLD) in mice is 0.3 ng/kg (Middlebrook, 1989). The lethal dose for type A botulinum toxin in humans is estimated to be 0.1 to 1.0 µg (Schantz and Sugiyama, 1974). Lund (1990) calculated that the lethal dose in humans is 0.005 to 0.1 µg toxin for proteolytic strains, and 0.1 to 0.5 µg toxin for nonproteolytic strains. Intestinal Absorption The toxin is absorbed from all areas of the alimentary tract (Lamanna and Carr, 1967). However, the toxin is absorbed mostly from the upper intestines and appears in the lymphatics (May and Whaler, 1958). As mentioned earlier, the larger the molecular size of the toxin, the greater is its stability in the GI tract. However, the rate of absorption of the toxin does not change. Similarly, the toxic and nontoxic components are absorbed at the same rate. The whole toxin molecule, regardless of whether it is 19S, 16S, or 12S, seems to be absorbed without immediate molecular dissociation of the toxin in the lymphatic tissue (Hildebrand et al., 1961; Kitamura et al., 1969). The exact mechanism involved in absorption of the toxin from the intestines is not known. It may be endocytosis, as in absorption of nutritional proteins (Bonventre,

Figure 13.8 Molecular sizes and structures and toxicities of Clostridium botulinum progenitor and derivative toxins. Black area, fully toxic; unshaded area, nontoxic; horizontally hatched area, nonactivated; vertically hatched area, hemagglutinin-active.

1979; Sakaguchi et al., 1988). The mechanism of the subsequent transport of the toxin (derivative toxin) to the target cells is also not known. Mode of Action The mechanism of action of the botulinum toxin can be divided into three steps: binding, internalization, and intracellular action. In addition, membrane translocation may be involved (Montecucco and Schiavo, 1994). The H chains are responsible for selective binding of the neurotoxin to neurons, internalization of the entire neurotoxin, intraneuronal sorting, and translocation of the L chains into the cy-

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tosol. The L chains block exocytosis as soon as they are released into the cytoplasm (Niemann, 1991). The potency of the botulinum neurotoxins results from the specificity of the toxins for neurons. Type A, B, C1, and D neurotoxins all have a small number of high-affinity binding sites and a large number of sites with much lower affinity on the synaptosomes. A double-receptor model for binding of botulinum toxin has been used to explain the high-affinity binding (Montecucco, 1986). In this model, the neurotoxins first bind to the negatively charged surface of the presynaptic membrane, which contains large amounts of acidic lipids. After binding of the C-terminal half of the H chain to the negatively charged lipids, the

neurotoxin may diffuse laterally in the membrane to bind to a protein receptor. Once the neurotoxin is bound to its receptors at the neuromuscular junctions, the entire neurotoxin is internalized by receptor-mediated endocytosis. Once it has been internalized, the neurotoxin can no longer be neutralized by antitoxin. The H chain aggregates and forms channels in the endosomal membrane to allow the L chain to pass into the cytoplasm. Four neurotoxin molecules combine to form channels that cross the vesicle membrane (Schmid et al., 1993). Thus the L chain exits the endosome by passing through channels created by the H chain. The L chain is capable of inhibiting neurotransmitter (acetylcholine) release independently of the H chain (Niemann, 1991). The L chains of the neurotoxins act as zincdependent endopeptidases, whose substrates are components of the synaptic vesicle docking and fusion complex. Types B, F, D, and G cause selective degradation of vesicle-associated membrane protein (VAMP, also called synaptobrevin). Type A and E neurotoxins degrade the synaptosome-asociated protein SNAP-25. Type C1 neurotoxin degrades syntaxin/HPC-1 (Montecucco and Schiavo, 1994). VAMP, syntaxin, and SNAP-25 form the core of a multicomponent complex that mediates fusion of carrier vesicles to target membranes in eukaryotic cells. Proteolysis of these proteins involved in docking and fusion of synaptic vesicle block neuroexocytosis and subsequent neurotransmitter release. The exact mechanism of exocytosis blockages, however, remains elusive. Future research will address selective alteration of neurotoxins by site-directed mutagenesis to determine which amino acid residues are involved in binding, uptake, proteolysis, and antigenicity. A schematic representation of this mechanism is shown in Figure 13.9. The action of the C2 toxin is different from that of the other toxin types. The C2 toxin causes respiratory difficulty through hypotension, which follows extravasation of fluids, especially in the lungs. In general, the effects of C2 toxin are cytotoxic and not neurotoxic, indicating that it may have become cytotoxic as it evolved into a binary toxin of separated subunits (Hauschild, 1989). Genetic Regulation The neurotoxins are arranged as part of a transcriptional unit, which includes the genes encoding the botulinum toxin components as well as the nontoxic components. This transcriptional unit is referred to as the botulinum neurotoxin gene complex (East et al., 1994). Complete gene sequences have been determined for all the neurotoxins produced by C. botulinum. The locations of the genes coding for the toxic and nontoxic components vary with

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the serotype. The genes coding for types A, B, E, and F and the associated nontoxic proteins are located on the bacterial chromosome (Dodds and Austin, 1997). Those coding for C 1 and D are encoded by bacteriophages, whereas the genes coding for type G and the associated nontoxic proteins are located on a plasmid. 13.9.4

Symptoms and Diagnosis

Food-borne botulism varies from a mild illness, which may be ignored or misdiagnosed, to a serious disease that may be fatal within 24 hours. The clinical signs are related to action of the toxin at peripheral sites of cholinergic nerves. Although it is an intoxication, the first symptoms appear some 12–36 hours after ingestion of food containing the toxin, but they may be delayed a further 8 to 14 days. The earlier symptoms appear, the more serious the disease. Considerable variation occurs among individual cases. The first symptoms are generally nausea and vomiting, followed by neurological signs and symptoms, including visual impairments (blurred or double vision, ptosis, fixed and dilated pupils), loss of normal mouth and throat functions (difficulty in speaking and swallowing; dry mouth, throat, and tongue; sore throat), general fatigue and lack of muscle coordination, and respiratory impairment. Other gastrointestinal symptoms may include abdominal pain, diarrhea, or constipation. Nausea and vomiting appear more often in cases associated with types B and E than in those associated with type A. Dysphagia and muscle weakness are more common in outbreaks of types A and B than in outbreaks of type E. Dry mouth, tongue, and throat are observed most frequently in type B cases. Respiratory failure and airway obstruction are the main causes of death. Fatality rates in the first half of the 20th century were about 50% or higher, but with the availability today of antisera and modern respiratory support systems, they have decreased to about 10%. Treatment of patients include trying to remove or inactivate the neurotoxin by (a) neutralization of circulating neurotoxin with antiserum, (b) use of enema to remove residual neurotoxin from the bowel, and (c) gastric lavage or treatment with emetics (Dodds, 1992). Antiserum is most effective in the early stages of the illness. The impact of antiserum is obvious from the Chinese data: before the availability of antisera in 1960, the death rate in China was approximately 50%, whereas it was only 8% in the nearly 4000 patients who received antitoxin since 1960. None of the 139 cases of botulism in the United States in 1994, or the 13 cases in Canada in 1995, resulted in death (Dodds and Austin, 1997). Although botulism can be diagnosed by clinical symptoms alone, differentiation from other diseases may

Figure 13.9 Schematic representation of the peripheral motor nerve terminal and synapse, depicting sites of action of botulinum toxins. Upper diagram, anatomical features of the presynaptic terminal and synapse; boxed area, enlarged beneath, showing details concerning the small synaptic vesicle. Synaptobrevin (also called VAMP) (open ovals), SNAP-25 (dark ovals), and syntaxin (also called HPC-1) (striped rectangle) are proteins involved in the fusion of synaptic vesicles with the presynaptic plasma membrane. Synaptobrevin is embedded in the synaptic vesicle membrane; SNAP-25 and syntaxin are located on the cytoplasmic surface of the neuron plasma membrane. Arrows, protein that is the specific target of the different botulinum toxins. VAMP, vesicle-associated membrane protein; HPC-1, hereditary prostate cancer-1; SNAP, synaptosome-associated protein.

be difficult. The most direct and effective way to confirm the clinical diagnosis of botulism in the laboratory is to demonstrate the presence of the toxin in the serum or feces of the patient or in the food that the patient consumed. Currently, the most sensitive and widely used method for detecting toxin is the mouse neutralization test. This test takes 48–72 hours. It is based on demonstrating the presence of a mouse lethal agent that can be neutralized by one or more of the botulinal antitoxins (Sugiyama and Sofos, 1988). The method is the only one officially accepted by regulatory authorities in the United States and has remained basically unchanged for many years. The test involves injecting mice intraperitoneally with 0.5 ml of food

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extracts and observing for typical symptoms of botulism over a 48 to 72-hour period. Samples that yield positive findings are typed by means of neutralization tests using monovalent antisera for each toxin type. Culturing of specimens takes 5–7 days. Since the mouse bioassay test is ethically undesirable, is time-consuming (taking up to 4 days), and requires expensive and specialized facilities, great efforts have been made to replace it. Several in vitro tests for detection of botulinum toxin have been developed, but their major drawback has been lack of sensitivity and, in many cases, specificity and reliability. The extremely high lethality of botulinum toxins per unit weight makes the sensitivity of

the mouse bioassay difficult to surpass. The most sensitive of the in vitro methods can detect approximately 1 ng/mL of neurotoxin, which is equivalent to 100 mouse LD50 doses. The more sensitive procedures are electroimmunodiffusion, reverse passive hemagglutination radioimmunoassay, countercurrent immunoelectrophoresis, ELISA, and a RPLA test. Although some of these methods have attained sensitivities similar to that of the mouse bioassay, greater numbers of false-positive and false-negative results are frequently obtained. Also, none of these methods is able to detect all types of botulinum toxins. Nonetheless, these methods may be useful when the sensitivity of the mouse bioassay is not necessary. 13.9.5

Foods Implicated in Botulism

A wide variety of foods have been implicated in human botulism, reflecting the world distribution of the predominant types and the eating habits of the populations affected. Most foods are nutritionally adequate for C. botulinum growth and toxin production, as demonstrated by outbreaks that have involved products such as garlic in oil and sautéed onions (Sugiyama and Sofos, 1988; Sofos, 1992). In general, both plant and animal food products provide the nutrients needed for C. botulinum growth and toxin production. Several surveys have been carried out to learn the incidence of C. botulinum spores in foods (Dodds, 1992; Dodds and Austin, 1997). Some of the representative data are summarized in Table 13.28. Food prod-

Table 13.28

ucts involved in botulism outbreaks worldwide are listed in Table 13.29. Most of these are associated with homeprocessed foods. However, considering that the risk of food-borne botulism is also directly related to the contamination of foods, surprisingly there have been fewer surveys of foods than of the environment. Also, most food surveys have largely focused on fish, meats, vegetables, and infant foods, primarily honey (Table 13.30). Some important characteristics are briefly described in the following. Fish and Fish Products Fish and fish products account for the majority of cases of food-borne botulism in Alaska, Japan, Scandinavia, Iran, and the former Soviet Union. In North America, the incidence and level of contamination appear highest in samples from the Pacific coast, followed by those from the Great Lakes and then from the Atlantic seaboard. In Europe, fish shows a lower level of contamination, except fish from Scandinavia and from the Caspian Sea (Dodds and Austin, 1997). In Indonesia, most positive fish samples contain either type C or type D spores. In contrast, most cases are caused by type E toxin, although in Norway and the former Soviet Union, approximately half are due to type B toxin and type A toxin, respectively (Hauschild, 1992). Unprocessed eviscerated fish and fish fillets pose few problems with regard to C. botulinum as spoilage by other bacteria is rapid and occurs before C. botulinum can grow and produce toxin. However, when fish is subjected to preservation procedures to increase its shelf life, such as

Incidence of Clostridium botulinum Spores in Food

Product Eviscerated whitefish chubs Vacuum-packed frozen flounder Dressed rockfish Salmon Vacuum-packed fish Smoked salmon Salted carp Fish and seafood Raw meat Cured meat Raw pork Cooked, vacuum-packed potatoes Mushrooms Random honey samples Honey associated with infant botulinum

Origin Great Lakes Atlantic Ocean California Alaska Viking Bank Denmark Caspian Sea Osaka, Japan North America Canada United Kingdom The Netherlands Canada United States United States

Source: From Dodds (1992) and Dodds and Austin (1997).

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Sample size, g 10 1.5 10 24–36 20 2 30 3 75 30

30 30

Positive samples, % 12 10 100 100 42 2 63 8 <1 2 0–14 0 1 100

Most probable numbers/kg 14 70 2400 190 63 <1 490 3 0.1 0.2 <0.1–5 0.63 2100 0.4 8 × 104

Toxin type E, C E A, E A E B E C, D C A A, B, C B A, B A, B

Table 13.29

Food Products Involved in Botulism Outbreaks Worldwide Food products, %

Country/state Alaska Argentina Belgium Canada Czechoslovakia China France Hungary Iran Japan Poland Spain Sweden United States USSR

Processing, %

Meat

Fish

Plant

Other

Home

Commercial

45 3 60 70 72 10 86 67 3 0 87 42 17 14 17

55 10 20 22 7 0 5 0 97 99 11 0 83 17 67

0 73 20 8 14 86 7 3 0 1 2 58 0 60 16

0 13 0 0 7 4 2 29 0 0 0 0 0 9 0

100 77 60 97 100 — 88 100 — 98 68 92 83 90 97

0 23 40 3 0 — 12 0 — 2 32 8 17 10 3

Source: From Hauschild (1989) and Sofos (1992).

smoking, drying, salting, marinating, or fermenting, the risk of botulism is increased. These processes selectively destroy or inhibit the spoilage bacteria common to raw fishery products but frequently have little or no inhibitory effect on C. botulinum spores (Eklund, 1992; Miller et al., 1998).

Table 13.30 Food type a

Vegetables

Fish products

Meat productsb

The number of spores found in fish products varies from 1 to 170/kg food (Hauschild, 1989). In general, the types of fish products associated with botulism are homeprocessed, uncooked or light cooked (e.g., hot smoked), traditionally stored at ambient temperature and then eaten without further cooking. Also, many of the processed fish

Incidence of Botulism Outbreaks Related to Food Type Country/state

Total outbreaks, %

Period

United States Spain Italy China Alaska Former USSR Japan Norway Iran Germany (West) Poland France Denmark Former Czechoslovakia Hungary Former Yugoslavia Canada

59 60 77 86 52 67 99 84 97 78 83 89 100 72 89 100 72

1971–89 1969–88 1979–87 1958–83 1971–89 1958–64 1951–87 1961–90 1972–74 1983–88 1984–87 1978–89 1984–89 1979–84 1985–89 1984–89 1971–89

a

Especially after home preservation by heat treatment or fermentation. For example, home-cured hams. Source: From Hauschild (1992) and Miller et al. (1998). b

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products are acidic and prepared from fatty fish species that are often ungutted (Huss, 1981). Other products include pickled fish, salted fish, salted, and dried fish (such as kapchunka); salted fish eggs can also cause botulism. Meat and Meat Products The level of contamination of meats with C. botulinum is generally low; estimates range from less than 0.1 to 7 spores/kg (Lucke and Roberts, 1992). Because of this low level of contamination, meat and meat products have been examined less frequently than fish products. This is due to the lower contamination of the farm environment than of the aquatic environment. The incidence is lower in North America than in Europe (Dodds, 1992). Studies have either failed to detect or detected a very low incidence of botulinum spores in cured meats and meat trimmings and in vacuum-packed sliced processed meat such as bologna, smoked beef, turkey and chicken, liver sausage, luncheon loaf, salami, and pastrami. In continental Europe, the single major source of botulism is home-cured smoked ham, particularly in France, Belgium, Germany, and Poland (Hauschild, 1992; Roblot et al., 1994). Salted pork is apparently the predominant vehicle for meat-borne botulism in Belgium, Norway, and Spain (Lucke and Roberts, 1992). Pork products cause botulism more frequently than those from beef or lamb, whereas poultry and poultry products have not to date been associated with botulism in these countries. Type A and B toxins are most commonly responsible for botulism caused by meats. Type E toxin has been associated with botulism caused by the consumption of raw or parboiled meats from marine animals such as seals, whales, and walruses by the native Inuit population in Canada and Alaska (Hauschild, 1989). Diary Products The level of contamination of dairy products with C. botulinum is very low, and their involvement in food-borne botulism is rare, accounting for less than 1% of the total number of outbreaks recorded since 1899 (CollinsThompson and Wood, 1992). Hazelnut yogurt was responsible for the largest recorded outbreak of food-borne botulism (type B) in the United Kingdom during June 1989. Postcanning treatment of the hazelnut preserves used to flavor the yogurt was apparently insufficient to destroy the C. botulinum spores that were present (O’Mahoney et al., 1990). Other dairy products that have caused outbreaks of botulism include cheese spread, cottage cheese, soft cheese, Brie cheese, and Mascarpone cheese (Collins-Thompson and Wood, 1992; Aureli et al., 1996; Simini, 1996).

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Fruits and Vegetables In the United States, Argentina, Spain, Italy, and China, fruits and vegetables are most frequently implicated in outbreaks of food-borne botulism. These are commonly home canned, except in China, where they are usually fermented. Type A and type B toxins have caused virtually all the outbreaks involving fruit and vegetables (Notermans, 1992). Agricultural practices, such as the use of manure for fertilizer, affect the level of contamination. Products in which contamination has often been detected and that have been involved in botulism outbreaks include asparagus, beans, cabbage, carrots, celery, corn, onions, potatoes, turnips, olives, apricots, cherries, peaches, and tomatoes. Frequently, the product responsible was stored for long periods at ambient temperatures under almost anaerobic conditions and was treated inadequately with regard to the inactivation or elimination of spores (Notermans, 1992). A product of particular concern because of the high number of spores found is cultivated mushrooms, in which up to 2.1 × 103 type B spores/kg have been detected (Hauschild et al., 1975). Other products involved in outbreaks include commercially prepared chopped garlic in soybean oil, which caused an outbreak of type B botulism in Canada, heat-treated canned beetroot, potato salad, and stuffed aubergines. The U.S. FDA has designated baked or boiled potatoes as potentially hazardous food products for botulinum toxin production and recommends that they be stored at 7°C or below or above 60°C (Miller et al., 1998). Honey and Infant Foods The potential presence of spores in honey and infant foods is troublesome because in some infants, the spores can colonize the intestine, produce neurotoxin, and cause infant botulism. Honey is the only food that has even been implicated in infant botulism. Surveys show that the botulinum spore level in random samples of honey is between 1 and 10 spores/kg (Dodds, 1992). The level (104 spores/kg) is higher in honey samples associated with infant botulism. Although spores have been detected in other infant foods, such as corn syrup and rice cereal, these foods do not seem to present the same risk as honey because the level of contamination is low and unlikely to increase during production and storage (Dodds and Austin, 1997). Factors Affecting Toxin Formation in Foods Several factors affect toxin formation in foods. Botulinum toxin formation is obviously dependent on the spores’ withstanding the processing treatment or on postprocess contamination. Subsequently, conditions must allow for

spore germination, growth of the vegetative cells, and toxin production. Conditions affecting this chain of events include the composition of the food, the temperature and period of storage, as well as moisture content, pH, oxygen availability and redox potential, and salt content. Although many types of food have been implicated in outbreaks, these foods do have some common features. Most have been subjected to some form of preservation, stored under conditions that permit toxin to be formed, and either eaten without further cooking or when reheating has been insufficient to destroy the toxin. Temperature is an important factor, but toxin formation is possible over the whole growth temperature range. However, other factors such as the degree of acidity and the salt content of the food become more effective growth inhibitors as the temperature is reduced so that the lower storage temperature (15°C as a maximum) are to be preferred. Again, toxin production is possible at all moisture and pH values at which growth occurs, although toxin is unstable at pH values above 7. No growth or toxin production can occur at pH 4.5 or below under normal conditions. In some foods, such as canned vegetables, toxin production is unlikely unless air is excluded. Foods like meats and fish have a strong reducing tendency so that growth and toxin formation are quite possible even in the presence of air. Strains of C. botulinum differ considerably in their sensitivity to salt in foods. Some, mainly strains of types A and B, tolerate levels of up to 10% sodium chloride, whereas type E strains are inhibited at 4.5%–5% levels. The situation is complex because sensitivities to sodium chloride alter at different pH values and at different sodium nitrite levels if the latter is included as a preservative in the food.

Table 13.31

13.9.6

Outbreaks

Food-borne botulism is quite rare in most areas of the world, although the actual incidence is probably higher than reported since mild cases are probably not diagnosed and botulism may be misdiagnosed as another neurological disorder. Unrecognized and misdiagnosed cases of botulism do occur, as shown by a 1985 outbreak in Vancouver, Canada, in which the initial diagnoses for 28 patients included psychiatric illness, viral syndrome, and a variety of other maladies (St. Louis et al., 1988). Nonetheless, botulism is more likely to be detected and reported than other, milder forms of food poisonings. Therefore, the epidemiological data for botulism are probably more complete than for most other food-borne illnesses. The prevalence of food-borne botulism throughout the world is probably associated with the prevalence of spores in the environment. The primary geographical regions of the world that have reported food-borne botulism are East Asia (China, Japan), North America, certain countries in Europe (Poland, Germany, France, Italy, Spain, Denmark, Norway), the Middle East (Iran), Latin America, Russia, and South Africa (Johnson, 2000). Foodborne botulism is very rare in the United Kingdom, although certain outbreaks, such as the Loch Maree incident, the Birmingham outbreak, and the hazelnut yogurt incident, have attracted much attention and publicity. Recent examples of outbreaks of botulism in commercial or restaurant-prepared foods are presented in Table 13.31. Botulism outbreaks at food service establishments are primarily due to temperature abuse of either food ingredients or the final product. The use of leftover baked potatoes at ambient temperature has caused two reported outbreaks, one in

Examples of Outbreaks of Food-Borne Botulism from Commercial Foods or Restaurant-Prepared Foods

Food product

Year

Canned peppers Canned Alaskan salmon Kapchunka (salt-cured, uneviscerated whitefish) Beef pot pie Sauteed onions Karahi-renkon (vacuum-packed, deep-fried, mustardstuffed lotus root) Chopped garlic-in-oil Kapchunka Chopped garlic-in-oil Hazelnut yogurt Faseikh (salted fish) Cheese sauce Skordalia (Greek salad with baked potato) Marscapone cheese

1977 1978 1981 1982 1983 1984 1985 1987 1989 1989 1991 1993 1994 1997

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Location

Toxin type

Cases, no.

Deaths, no.

United States United Kingdom United States United States United States Japan

B E B A A A

58 4 1 1 28 36

0 2 0 0 1 11

Canada United States/Israel United States United Kingdom Egypt United States United States Italy

B E A B E A A A

36 8 3 27 92 5 19 8

0 2 0 1 20 1 0 1

which the potatoes were used for potato salad and the other in which they were used in a Greek food known as skordalia. Data illustrating the incidence of food-borne botulism are presented in Table 13.32. Botulism has a high incidence in the United States: a total of 597 cases between 1971 and 1989 (Hauschild, 1992). Food products involved in botulism outbreaks in the United States are listed in Table 13.33. The former Soviet Union also had a high incidence, mainly linked to preserved fish, meats, and vegetables. In Japan, botulism is mainly associated with preserved fish and other seafoods (Smith and Sugiyama, 1988). The same is true for Iran. Accurate data about the incidence of botulism in Europe are limited; between 1979 and 1988, 148 outbreaks comprising more than 225 cases and 4 deaths were recorded at the Institut Pasteur, France. It is estimated that these represent about half the total number of outbreaks (Lund, 1990). The largest number of outbreaks and cases per capita worldwide has been reported from Poland; most of the implicated foods were canned (Hauschild, 1992). Germany also has a high incidence of food-borne botulism as compared to other European countries. The United Kingdom has a much lower incidence than the United States: only three outbreaks between 1955 and 1989. The largest recorded UK outbreak occurred in 1989, when 27 people were affected (1 death) as a result of the consumption of contaminated hazelnut

Table 13.32

yogurt (O’Mahoney et al., 1990). Tropical countries generally have a very low incidence of food-borne botulism, which is probably due to the limited use of food preservation (Miller et al., 1998). Commercial products generally have had a good safety record since the early days of canning in the 1920s. Yet many countries report relatively frequent outbreaks (Table 13.31). Bottled garlic in oil, which caused an outbreak in Canada and one in the United States, is a recently implicated commercial product. As a result of these two outbreaks, garlic in oil can be sold in North America only if a second barrier, such as acidification, is present in addition to refrigeration (Dodds and Austin, 1997). Two outbreaks of type B botulism in Italy were also associated with commercially prepared sliced roasted eggplant in oil (D’Argenio et al., 1995). In contrast, in many areas, homepreserved vegetables are the foods most often implicated (Hauschild, 1992). This is true for the continental United States and China, where most outbreaks are caused by type A, and for Italy and Spain, where type B is usually implicated. Temperature abuse of home-processed foods continues to be an important cause of the problem. Botulism is rare compared to many other food-borne microbial diseases but has a relatively high fatality rate in humans and animals. Human botulism outbreaks can have a dramatic impact on communities in which they occur and can lead to the demise of food companies, and out-

Food-Borne Botulism Outbreaks

Country Poland China United States Italy France Germany Japan Former USSR Iran Canada Spain Hungary Portugal Norway Former Czechoslovakia Argentina Former Yugoslavia Belgium Denmark and Greenland United Kingdom

Period

Outbreaks, no.

Cases, no.

Deaths

1984–87 1958–83 1971–93 1979–87 1978–89 1983–89 1951–87 1958–64 1972–74 1971–94 1969–88 1985–89 1970–89 1961–90 1979–84 1980–89 1984–89 1982–89 1984–89 1955–89

1301 986 302 Unknown 175 99 97 95 Unknown 99 63 31 24 19 17 16 12 11 11 3

1791 4377 655 310 304 206 479 328 314 231 198 57 80 42 20 36 51 25 16 33

46 548 70 Unknown 7 12 110 95 11 35 6 2 0 7 0 13 Unknown 4 12 3

Source: From Dodds and Austin (1997) and Miller et al. (1998).

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Table 13.33 Food Products Involved in Botulism Outbreaks in the United States Food product Beef Pork Turkey Chicken Fish Dairy Fruits and vegetables Mushrooms Nondairy beverages Mexican-style food Other Unknown

Outbreaks, no. 2 1 1 1 35 1 99 5 5 3 40 38

Source: From Bean and Griffin (1990) and Sofos (1992).

breaks of animal botulism periodically devastate populations of domestic and wild animals. The economic losses involved in botulism outbreaks can be major. For example, an outbreak of type E botulism from Alaskan canned salmon consumed in the United Kingdom in 1978 resulted in direct and indirect costs of approximately $6 million (Sofos, 1992). Two cases of type E botulism from the same product in Belgium in 1982 cost an estimated $148 million. The estimated total cost of an outbreak from consumption of contaminated potato salad eaten at a restaurant was $8.4 million (Sugiyama and Sofos, 1988). To prevent outbreaks and to minimize the devastating economic impact, it is necessary for the food industry to formulate and process foods properly to prevent growth and toxin formation. 13.9.7

Prevention

There is extensive literature on the control of botulinum toxin formation in foods. In most studies, data on the effects of external factors, such as pH and water activity, on the growth of food-borne pathogens have been obtained by varying only one or two factors, with the others remaining near optimal (Adams and Moss, 1995; Miller et al., 1998). However, in foods these factors are usually present at an individually suboptimal level but collectively are able to affect the ability of microorganisms to grow. This is known as the hurdle effect (Leistner, 1978). This multifactorial approach to preserving foods has been applied empirically for many years, for example, for smoked fish, with which a combination of factors (salt, smoke, and drying) all contribute to the overall preservation and microbial

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safety of the food. Clearly, the hurdle effect has to be taken into account when assessing the risk of C. botulinum toxin production in a food. Conditions that permit C. botulinum to grow and produce toxin constitute a greater risk than conditions that permit the germination of its spores (Miller et al., 1998). The problem of botulism food poisoning can be tackled on three different fronts: 1. 2. 3.

Inhibition of growth and toxin production by vegetative cells of C. botulinum Destruction of C. botulinum spores Destruction of the preformed neurotoxin in foods

The control of C. botulinum in most foods is achieved by the inhibition of growth and toxin production; inhibition is the result of a combination of factors rather than a single factor. Each of the three factors mentioned is briefly discussed in the following section. Inhibition of Growth and Toxin Production The growth characteristics of various strains of C. botulinum were described earlier. Temperature, pH, water activity (aw), redox potential (Eh), added preservatives, and the presence of other microorganisms are the main factors controlling the growth of C. botulinum in foods. Historically, studies have established the maximal and/or minimal limits for these parameters that control growth of C. botulinum (Table 13.26). As mentioned earlier, these factors seldom function independently; usually they act in concert, often with synergistic effects. Temperature The optimal growth temperatures are between 35°C and 40°C for group I organisms and between 18°C and 25°C for group II organisms (Table 13.26). The established lower limits are, respectively, 10°C and 3.3°C. Generally, the major concern is with growth of group II nonproteolytic strains of C. botulinum in refrigerated foods. There is particular concern about foods packed under vacuum or modified atmosphere and sous vide foods (Lund and Notermans, 1992), for which temperature may be the major controlling factor preventing growth and toxin production. Nonproteolytic strains may have sufficient time to grow in refrigerated foods if the shelf life is extensive, whereas the group I proteolytic strains only grow in these foods if temperature abuse occurs (Kim and Foegeding, 1992). The safety of canned foods depends on effective sealing and heat treatment. The heat treatment must be sufficient to destroy spores as well as vegetative cells.

Similarly, after heating, the containers must be cooled and container integrity maintained to prevent recontamination of the can through punctures or leaks. Several large outbreaks have occurred after the consumption of canned foods, possibly via a defect in the tin during the cooling process. Canning poses the greatest risk when carried out in the home environment. Home-canned vegetables have often been involved in botulinum poisoning in the United States, China, Argentina, and Spain (Hauschild, 1992; Miller et al., 1998). pH The minimal pH allowing growth of C. botulinum groups I and II is 4.6 and 5.0, respectively (Table 13.26). Thus, many fruits and vegetables are sufficiently acidic to inhibit the growth of the organism by their pH alone, and acidulants are used to preserve other products. However, acid-tolerant microorganisms, such as yeasts and molds, may grow in acidic products and raise the pH in their immediate vicinity to a level that allows the growth of C. botulinum. C. botulinum can also grow in some acidified foods if excessively slow pH equilibrium occurs (Dodds and Austin, 1997). The acid tolerance of C. botulinum depends on the strain, food composition, type of acidulants, presence of other preservatives, and prior heat and/or other treatments. It is generally accepted that the organism cannot grow and produce toxin in foods with a pH of 4.6 or lower, but this is not the case if large amounts of protein are present. Currently, only canned, pasteurized, acidic, or acidified fruits and vegetables are protected by acidity alone because of their relatively low protein content. Salt and Water Activity The growth-limiting salt concentrations are about 10% for group I and 5% for group II organisms under otherwise optimal conditions. These concentrations correspond well to the limiting aw of 0.94 and 0.97 for these two groups, respectively. Salt lowers the water activity of foods. The limiting aw may be raised significantly by other factors, such as increased acidity or use of preservatives. The organism can be effectively controlled in high-moisture foods by maintaining a pH of less than 4.5 (Lund, 1990). Atmosphere and Redox Potential Since C. botulinum is an obligate anaerobe, it is commonly assumed that it cannot grow in foods that are exposed to oxygen or in foods with a high redox potential (Eh). In practice, the Eh of most foods exposed to oxygen is generally low enough to permit growth (Sperber, 1982).

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Once growth is initiated, the Eh declines rapidly. Modified atmosphere packaging (MAP) is particularly a concern, because the CO2 used to inhibit spoilage and pathogenic microorganisms may in fact stimulate C. botulinum growth. Preservatives The use of preservatives to control growth and toxin production by C. botulinum has been comprehensively reviewed (Kim and Foegeding, 1992). Nitrites used in cured meats and fish inhibit the growth of the organism; however, the exact mechanism of inhibition is not known. Their effectiveness is dependent on complex interactions among pH, salt, heat treatment, time and temperature of storage, and composition of food. Sorbates, parabens, nisin, phenolic antioxidants, polyphosphates, ascorbates, EDTA, metabisulfite, nmonoalkyl maleates and fumarates, and lactate salts are also active against C. botulinum (Kim and Foegeding, 1992). Smoking, in combination with appropriate concentrations of sodium chloride, is effective in preventing the growth and toxin production by C. botulinum types A and B. However, smoking alone should not be considered an effective substitute for sodium chloride and refrigeration (Miller et al., 1998). Irradiation C. botulinum spores are probably the most radiationresistant spores of public health concern. Vegetative cells are inactivated by low doses of radiation, but the main disadvantage is that such doses have little or no effect on the spores (Eklund, 1992; Monk et al., 1995). Destruction of Spores Thermal processing remains the method of choice to inactivate the spores of C. botulinum. The amount of heat required to inactivate the spores varies widely, depending primarily on the composition of food (Miller et al., 1998). The pH of the foods is particularly important, and in the United Kingdom “low-acid” foods with a pH higher than 4.5 require a “botulinum cook” (Shapton and Shapton, 1991): the minimal sterilization process required to eliminate C. botulinum spores, also known as the 12 D (decimal) reduction process. This is defined as the heat required to inactivate a theoretical contamination by 1012 C. botulinum spores. For most foods, this requires an F 0 of 3 (where 1 F0 equals 1 minute at 121°C, assuming instantaneous heating and cooling); therefore, an F0 of 3 is 3 minutes at 121°C. Meats usually receive an F0 of 6 (Miller et al., 1998).

Spores of proteolytic types A and B are the most heat-resistant, having D121°C values between 0.1 and 0.2 minute. These spores are of particular concern in the sterilization of canned low-acid foods. Group I organisms are often used as target organisms for most thermal processes. The strains of group II are considerably less heat-resistant (D100°C < 0.1 minute). Their survival, however, is of particular concern in refrigerated, processed foods of extended durability (REPFEDs) because of their ability to grow at refrigeration temperatures (Lund and Notermans, 1992). Various regulations and guidelines for the safe production, distribution, and sale of such REPFED products have been published. The Sous Vide Advisory Committee of the United Kingdom has produced guidelines for sous vide (vacuum-packed) foods specifying that the vacuum packaging and heat treatment ensure destruction of vegetative organisms and significantly reduce the number of psychrotrophic C. botulinum. American recommendations from the National Advisory Committee on Microbiological Criteria for Foods include inoculated pack studies with C. botulinum to determine shelf life (Dodds and Austin, 1997). In general, the following parameters should be observed to prevent botulism outbreaks: 1.

2.

3.

4.

5.

Ensure that the heat treatment for canned and bottled foods (pH > 4.5) is adequate to destroy the most heat-resistant C. botulinum spores. Use the cleanest possible chlorinated water for cooling cans after processing. Where air-cooling is used, ensure that cross-contamination from raw materials or dirty equipment or by handling is prevented during the cooling process. Where milder heat processes are applied to foods, ensure that suitable inhibitory compounds or preservatives are introduced or that the pH is low enough to prevent the growth of C. botulinum. Ensure adequate heat treatment with semiprocessed or processed vacuum-packed foods and store them at a maximal temperature of 3°C. Never taste a food that is suspect unless it is preheated to 100°C.

Destruction of Preformed Toxin Botulinum toxins can be inactivated by heat; however, the stability of the toxin depends on the nature of the heating medium. Heat treatment at 79°C for 20 minutes or 85°C for 5 minutes can be viewed as a general guideline for the inactivation of toxin types A, B, E, and F (Hauschild, 1989; Miller et al., 1998). Nonetheless, developing processes to inactivate botulinum toxins in foods without af-

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fecting nutritional value or palatability has proved quite difficult. Similarly, these toxins cannot be reliably inactivated by radiation doses that do not adversely affect these properties of foods (Siegel, 1992). Irradiation should only be used in conjunction with good manufacturing practice and other control measure and should not be relied upon as a sole means of control (Miller et al., 1998).

13.10 VIBRIO TOXINS Vibrios are among the most common bacteria in surface waters worldwide. They are responsible for several intestinal and extraintestinal diseases. The medically important vibrios are listed in Table 13.34. Many infections are acquired by ingesting contaminated food (especially seafood) or water. Disease may also occur after contact with an aquatic environment (e.g., seawater). Infection with Vibrio cholerae, the causative organism leading to the disease cholera, continues to threaten large portions of the world’s population even today. The disease process is not very complicated: the disease occurs at the mucosal surface, with no invasion by the microbe into deeper tissue, and disease symptoms are primarily due to the action of a single molecule, the cholera toxin. Cholera has earned a frightful reputation throughout history both for its epidemic nature and for the rapidity with which individuals can become gravely ill and even die. Within this simple system of mucosal pathogenesis, extremely interesting mechanisms have been uncovered at practically every level of study. In this section, some of the important toxins produced by Vibrio spp. and many of the mechanisms of pathogenesis are described.

Table 13.34

The Medically Important Vibrios

Organism Vibrio cholerae serogroups O1 and O139 V. cholerae serogroups non-O1/non-O139 (nonagglutinable [NAG] or noncholera [NCV] vibrios) V. parahaemolyticus Others V. mimicus, V. vulnificus, V. hollisae, V. fluvialis, V. damsela, V. anginolyticus, V. metschnikovil

Human disease Epidemic and pandemic cholera Choleralike diarrhea; mild diarrhea; rarely, extraintestinal infection

Gastroenteritis, perhaps extraintestinal infection Ear, wound, soft tissue, and other extraintestinal infections, all uncommon

13.10.1 Cholera Enterotoxin (Choleragen, Classic or Asiatic Cholera) Cholera is a toxin-mediated disease. Reasonable data to support this prediction first became available in 1959 when two Indian researchers, working independently of one another, showed that cell-free preparations from V. cholerae caused relevant symptoms in animal models that they had developed (De, 1959; Dutta et al., 1959). One model was the LRIL (De and Chatterje, 1953), which responded to sterile culture filtrates of an unidentified Ogawa serotype strain of V. cholerae with luminal fluid accumulation, which suggested enterotoxicity (De, 1959). The other model (Dutta and Habbu, 1955) was the infant rabbit, which, when fed multiple doses of sterile lysates of heavy suspensions of the 569B Inaba strain of V. cholerae (Dutta et al., 1959), responded with fatal choleric diarrhea. By 1964 it had been demonstrated that the enterotoxic activity could be produced routinely and optimally by growing strain 569B Inaba with vigorous aeration in synthetic culture medium (Finkelstein and Lankford, 1955) supplemented with casamino acids (Finkelstein et al., 1964). The putative cholera enterotoxin, which was shown not to be the cholera endotoxin, was then named choleragen—now an accepted synonym for cholera toxin (CT) or cholera enterotoxin. In subsequent years, as an outgrowth of basic research on cholera and the resulting increased understanding of its pathophysiological characteristics, a simple, economical, and effective treatment regimen (oral rehydration therapy [ORT]) for diarrheal disease has been developed (WHO, 1983). Regarded as potentially the most important medical advance of the 20th century, ORT saved countless millions of lives in the decade of the 1980s alone (Finkelstein and Dorner, 1986). Cholera, often regarded as the “scourge of Asia,” is a gastrointestinal disease caused by V. cholerae serogroup O1 and is endemic to parts of the Asian continent, primarily India and the Ganges basin. In the past, this catastrophic disease caused thousands of deaths. Cholera has usually infected impoverished people who live in unsanitary conditions. In these areas, drinking water is often taken from sources that are also used for sewage disposal and other human activities. Cholera is considered by some to be an ancient human affliction (Pollitzer, 1959; De, 1961). The distinct symptoms and explosive epidemic onslaught of the disease are depicted in early Arabian, Chinese, Hindu, Greek, and Roman writings (McNicol and Doetsch, 1983; Kaysner, 1992). For over 2500 years, the word cholera was used to describe diarrhea and vomiting of any cause, not only bacterial. It still affects over 60,000 people annually, mostly in

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tropical countries; approximately 75% of these cases occur in African countries. The pandemic cholera that is clinically recognized today emerged in India around 1817 (Longmate, 1966; Kaysner, 1992). Since this time, seven pandemics have been recorded, the latest spreading from Indonesia in 1959 to Asia, Africa, and Eastern Europe by the early 1970s, with a total of 41 countries’ reporting cases. The last one was caused by the E1 Tor biotype, the hemolytic biotype of V. cholerae O1. Organism Vibrio species are straight to slightly curved aerobic, gram-negative rods possessing a polar flagellum. A positive oxidase test result is a key step in the preliminary identification of V. cholerae and other vibrios. Vibrios are capable of mixed acid fermentations. Most species are halotolerant, and salt often stimulates their growth. Some are halophilic, requiring the presence of salt to grow. Although it is not required for growth, V. cholerae grows in the presence of up to 6% NaCl. As with other gram-negative bacteria, the outermost sugars of the lipopolysaccharides in the V. cholerae outer membrane have been used in a classification scheme (DiRita, 2001). Most vibrios share a single heat-labile flagellar H antigen. However, antibodies to the H antigen are probably not involved in the protection of susceptible hosts. V. cholerae has O lipopolysaccharides that confer serological specificity. There are at least 139 O antigen groups. V. cholerae strains of O group 1 and O group 139 cause classic cholera; occasionally non-O1/non-O139 V. cholerae causes choleralike disease (Table 13.34). Antibodies to the O antigens tend to protect laboratory animals against infections with V. cholerae. The V. cholerae serogroup O1 antigen has determinants that make possible further typing; the main serotypes are Ogawa, Inaba, and Hikojima. Two biotypes of epidemic V. cholerae have been defined, classical and E1 Tor. The E1 Tor biotype produces a hemolysin, gives positive results on the Voges-Proskauer test, and is resistant to polymyxin B. El Tor strains are the cause of the seventh cholera pandemic, which began in Indonesia in 1961 and persists even today (Blake, 1994; DiRita, 2001). The seventh pandemic may be the first of the recorded pandemics dating to the early 19th century to be caused by El Tor and not classical strains; strain data on pandemics preceding the fifth do not exist (Karaolis et al., 1995). On the basis of the epidemiological data, the El Tor biotype appears to be associated with less severe disease than the classical biotype and often causes asymptomatic infections (Kaper et al., 1995; Gangarosa and Mosley, 1974).

V. cholerae O139 is very similar to V. cholerae O1 E1 Tor biotype. The former does not produce the O1 lipopolysaccharide and does not have all the genes necessary to make this antigen. V. cholerae O139 makes a polysaccharide capsule like that of other non-O1 V. cholerae strains, whereas V. cholerae O1 does not make a capsule. Cholera Toxin (Choleragen) Finkelstein and LoSpalluto (1969) first purified choleragen to apparent homogeneity. It is a heat-labile oligomeric protein with a molecular weight of about 84,000 Da. Choleragen is composed of two distinct species of subunits, A (~28,000 Da) and B (~56,000 Da), which are held in association by strong noncovalent forces. When they are separated, neither has significant biological activity in animal models or intact cell systems (Finkelstein et al., 1974). However, activity and normal properties of the holotoxin are recovered on reassociation of the subunits. The CT structure, determined after that of E. coli heat-labile LT enterotoxin, is identical to it, as is predicted from the high level of identity between LT and CT primary sequences (Spangler, 1992). The holotoxin contains five identical, noncovalently linked binding (B) subunits, each consisting of 103 amino acid residues with a total formula weight of 11,604 Da. The A subunit consists of an A1 peptide (21,000–22,000 Da) and an A2 peptide (~6000 Da) joined by a single disulfide bond. This is formed as a result of proteolytic cleavage at residue Arg-192 of the A subunit (Mekalanos et al., 1979). The five noncovalently associated B subunits are responsible for specific binding of the holotoxin to receptors on target cell membranes, which contain the oligosaccharides of the GM1 ganglioside. This pentamer of B subunits (which is synonymous with choleragenoid) is also the portion of the cholera toxin molecule, which is the predominant immunogen. The amino acid sequences of both A and B subunits are known (Kurosky et al., 1977a, 1977b; Lai, 1977; Mekalanos et al., 1983). A schematic representation of the molecular structure of the cholera enterotoxin is shown in Figure 13.10 The A subunit of cholera toxin is an enzyme that is responsible for the biological effects of the toxin. Elegant work by Gill and coworkers (Gill, 1982; Gill and King, 1975; Gill and Meren, 1978) using pigeon erythrocytes established that the A1 portion of the A subunit is the biologically active moiety. The A2 peptide is not required for its enzymatic activity; however, it does apparently serve to link the B subunits to the A protomer because reduction of the nicked toxin in the presence of urea leaves an A2 complex with the five B protomers (Sattler et al., 1975; Mekalanos et al., 1979). Although it was possible to reconstitute

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S

9

9

S

9

S

S S

S

B

9

S

9

S

S S

5

A2

S S

A1 Figure 13.10 Schematic representation of the molecular structure of the cholera enterotoxin (CT, choleragen). The holotoxin is depicted as consisting of five identical, noncovalently associated B subunits (MW ~ 11,500), which together constitute the choleragenoid or binding (B) region (MW ~56,000) of the molecule, and a single A subunit (MW ~ 28,000) that consists of two fragments, A1 (MW ~21,000) and A2 (MW ~7000), joined by a disulfide bond initiated at residue 5 from the amino terminus of the A1 peptide. Of these, the A1 peptide is responsible for the biological (i.e., enzymatic) activity of the enterotoxin; the A2 peptide is shown as providing the noncovalent link between the A and B domains. Each B subunit has an internal disulfide loop starting at position 9 and terminating at residue 86. This structure is conserved in the cholera-related enterotoxins, e.g., Escherichia coli heat-labile (LT) toxin.

the holotoxin after separation of the A and B subunits, this could not be accomplished after reduction and separation of the A1 and A2 fragments of the A subunit. The genes for V. cholerae enterotoxin are on the bacterial chromosome. Cholera enterotoxin is antigenically related to LT of E. coli and can stimulate the production of neutralizing antibodies. However, the precise role of antitoxic and antibacterial antibodies in protection against cholera is not clear. Mode of Action Under natural conditions, V. cholerae is pathogenic only to humans. A person may have to ingest 108–1010 organisms to become infected and contract the disease; in contrast, in salmonellosis or shigellosis ingestion of 102–105 organisms can induce infection (see Chapter 12). Cholera is not an invasive infection. The organisms do not reach the bloodstream but remain within the intestinal tract. Virulent V. cholerae organisms attach to the microvilli of the brush border of epithelial cells. There they

multiply and liberate cholera toxin and perhaps mucinases and endotoxin. After its secretion by the cholera vibrios, the cholera enterotoxin must bind to receptors on host target cell membranes to cause its physiological effects. Thus, the initial action is the binding of the B subunit of the toxin to the mucosal receptor, ganglioside GM1, on the cell membrane (Cuatrecasas, 1973; Holmgren et al., 1973; van Heyningen and Seal, 1983). This binding promotes the entry of subunit A into the host cell. The A1 fragment, which catalyzes the NAD-dependent, adenosine diphosphate (ADP) ribosylation of a regulatory component of the host target cell adenylate cyclase system, is required for stimulation of adenylate cyclase, causing increased levels of cAMP. This in turn results in the activation of a cAMP-dependent protein kinase, leading to protein phosphorylation, alteration of ion transport, and ultimately diarrhea with prolonged hypersecretion of water and electrolytes. There is increased sodium-dependent chloride secretion, and absorption of sodium and chloride is inhibited. The ADPribosylation activity of A1 is stimulated in vitro by a family of proteins, termed ADP-ribosylation factors (ARFs) (Di-

Rita, 2001). Detailed description of the mode of action of cholera toxin can be found in several excellent reviews (Gill and Meren, 1978; Finkelstein, 1988; Moss and Vaughan, 1988; Finkelstein and Dorner, 1986; Mintz et al., 1994; Kaysner and Hill, 1994; Oliver and Kaper, 1997; DiRita, 2001). The classical model of choleragen mode of action involving cAMP is shown schematically in Figure 13.11 Persons of the O blood group are at elevated risk for severe outcomes after infection by El Tor V. cholerae (Chaudhuri and De, 1977). This could be due to differential binding of CT to cells from the different blood groups. In order to enter cells, CT binds to a specific surface glycolipid, ganglioside GM1. Cells from O blood group individuals lack an enzyme found in individuals of other blood groups that is involved in glycosylating surface antigens (DiRita, 2001). Decreased glycosylation may allow greater exposure of CT-binding sites and account for the greater severity of disease in this population. The association of certain populations with more severe disease has important ramifications for vaccine development against cholera, since the vaccine efficacy is significantly lower in persons of the O blood group type.

Figure 13.11 Biochemical events leading to secretion caused by CT. The A1 subunit of cholera toxin catalyzes ADP-ribosyl transfer from NAD to Gsα, a regulatory subunit of the adenylate cyclase complex. The ensuing constitutive adenylate cyclase activity leads to elevated cAMP levels, which activate PKA, leading to opening of normally gated channels in the plasma membrane. ARFs, small GTPbinding proteins that stimulate ADP-ribosyltransferase activity by increasing the affinity of CT for both the substrate and the target, have not been shown to be required in vivo yet. Chloride and other ions leave the cell, followed by water, leading to the characteristic profuse water diarrhea. ADP, adenosine diphosphate; CT, cholera toxin; NAD, nicotinamide-adenine dinucleotide; cAMP, cyclic adenosine monophosphate; PKA, protein kinase A; ARFs, ADP-ribosylation factors; ADPR, ADP-ribosyl; GTP, guanosine triphosphate.

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Several other toxins have also been shown to act via mechanisms similar to that of cholera toxin. Of these, the most closely related are the E. coli heat-labile enterotoxins (LTs), which are very similar structurally, functionally, and immunologically to the cholera toxin. Diphtheria toxin and Pseudomonas aeruginosa exotoxin A also act via a similar mechanism.

Without adequate therapy the mortality rate can reach 60% of cases but can be reduced to less than 1% by intravenous replacement of fluid and electrolytes. Symptoms of the disease rarely last more than a few days if adequate treatment is obtained. Cholera is the only food-borne disease required by International Health Regulations to be reported to the World Health Organization (WHO).

Symptoms

Sources

The symptoms of V. cholerae infection are consistent with the fact that the principal mechanism of pathogenesis is a toxin that causes fluid and electrolyte loss due to diarrhea, muscle cramps, dizziness, and low blood pressure. Unlike diarrheas caused by invasive organisms, in which there is evidence of tissue damage such as the presence in stool of inflammatory cells and red blood cells, cholera stools are characterized by a clouded, milky white appearance termed rice water stool because it resembles water in which rice has been mixed. Symptoms of the classical Asiatic cholera may vary from a mild, watery diarrhea to an acute diarrhea, with characteristic rice water stools. The onset of the illness is generally sudden, with incubation periods varying from a few hours to 5 days (typically 2–3 days). Clinical signs include profuse diarrhea with abdominal pain, vomiting, headache, nausea, and fever. The fluid loss may be as great as 20–30 L fluid/day, which results in severe dehydration, shock, acidosis, and death. In addition to shock and fluid loss associated with this secretory diarrhea, another potentially dangerous aspect of cholera is a rise in the acidity of body fluids that can lead to pulmonary edema in severe cases of disease. This condition, known as metabolic acidosis, results partly from excretion of bicarbonate in stool, but also probably as a consequence of lactic academia from poor tissue perfusion and of other factors related to kidney failure and dehydration (Butterton and Calderwood, 1995; Bennish, 1994). Cholera treatment is simple and inexpensive. Fluids and electrolytes are replaced to prevent both dehydration and the shock. Intravenous therapy may be used in cases of severe disease in which the patient is gravely dehydrated. Oral rehydration therapy, however, is otherwise preferable because it is very effective as well as being cheaper and easier to administer. Formulations for oral rehydration therapy vary; that recommended by the World Health Organization (WHO-ORT) is 60 mM sodium chloride, 30 mM sodium bicarbonate, 20 mM potassium chloride, and 111 mM glucose (Butterton and Calderwood, 1995; Bennish, 1994). In order to achieve a maintainable balance of fluids, proper oral rehydration therapy must continue beyond the diarrheal phase of disease.

The bacteria are shed from the body primarily in the feces. In endemic areas, contamination of food as a result of poor hygiene perpetuates the cycle of transmission. Foods associated with cholera include vegetables, fish, and pork products. Sewage is used to fertilize vegetables in some countries, and sewage contamination of drinking water is a major cause of cholera outbreaks. V. cholerae O1 may be found in water of coastal estuaries, and shellfish harvested from these areas are significant sources of infection. Recent evidence indicates that V. cholerae, as well as other pathogenic vibrios associated with seafood, lives in estuarine environments adhering to zooplankton, such as copepods and water hyacinths. Long-term carriage of V. cholerae O1 in humans is extremely rare and is not considered to be significant in the transmission of disease. However, short-term carriage by humans is quite important in the transmission. Persons with acute cholera excrete 107 to 108 V. cholerae/g of stool; for patients who have 5 to 10 L of diarrheal stool, total output of V. cholerae can be in the range of 1011 to 1013 cfu (Oliver and Kaper, 1997). Even after cessation of symptoms, patients who have not been treated with antibiotics may continue to excrete vibrios for 1 to 2 weeks. Furthermore, a high percentage of persons infected with V. cholerae in endemic areas have inapparent illness and can still carry the organism, although excretion generally lasts for less than 1 week. Asymptomatic carriers are most commonly identified among household members of persons with acute illness; in various studies, the rate of asymptomatic carriage in this group has ranged from 4% to almost 22%. V. cholerae O1 can also be sporadically carried by household animals, including cows, dogs, and chickens, but no animal species consistently carries the organism. A dynamic relationship between human and environmental sources of the organism is quite apparent, as carriage and amplification by human populations play a critical role in epidemic spread of virulent strains of V. cholerae.

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Detection V. cholerae subgroup O1 may be recovered from foods by methods similar to those used for recovering the organism from the feces of infected individuals. Pathogenic and

nonpathogenic forms of the organism exist, so all food isolates must be tested for the production of the cholera enterotoxin.

small outbreak of four cases in Maryland was attributed to frozen coconut milk imported from Thailand, which was subsequently used in a topping for a rice pudding.

Outbreaks

Prevention

The critical role of water in transmission of cholera has been recognized for more than a century. In developing countries, ingestion of contaminated water and food is probably the major vehicle for transmission of cholera, whereas in developed countries, food-borne transmission is more important (Glass and Black, 1992). Such distinctions are often difficult to make since contaminated water is frequently used in food preparation. For example, rice prepared with water contaminated with V. cholerae O1 has been implicated in outbreaks in Bangladesh as well as the Gulf Coast of the United States (Oliver and Kaper, 1997). Fruit juices diluted with contaminated water and vegetables irrigated with untreated sewage have been associated with disease in South America. Seafood may acquire the organism from environmental sources and may serve as a vehicle in both endemic and epidemic disease, particularly if it is uncooked or only partially cooked. In modern times, cholera has been mostly a disease of the underdeveloped countries of Asia and Africa. In recent times, of the cases reported to the World Health Organization, over 95% occurred in Asia; Indonesia and India accounted for 61% and 25%, respectively. Outbreaks frequently occur in several Asian countries every year during the monsoon season, when large-scale flooding is quite a common occurrence. In the United States, both domestically acquired and imported cases of cholera occur. For domestic cases, crabs, shrimp, and oysters have been the most frequently implicated vehicles, although the largest single outbreak (16 cases) was due to ingestion of contaminated rice. In this outbreak, which occurred on a Gulf Coast oil rig in 1981, cooked rice was moistened with water contaminated by human feces and then held for 8 hours after cooking (Mintz et al., 1994). Although the majority of domestic cases occur in states bordering the Gulf Coast, seafood shipped from this area has caused disease in both Maryland and Colorado (Mintz et al., 1994). The risk of imported cholera has greatly increased since the establishment of endemic cholera in South America in 1991 (Oliver and Kaper, 1997). The largest such outbreak (75 cases) involved crab salad served on an airplane flying from Peru to California. A smaller outbreak of eight cases occurred in New Jersey; it was due to crabs purchased in Ecuador and carried to the United States in an individual’s luggage. Importation from Asia can also occur, even in commercially imported food. A

The provision of safe drinking water has been synonymous worldwide with cholera control. Where it has been combined with adequate sewage treatment, epidemic cholera has disappeared. Careful food handling is needed to prevent contamination by fecal carriers or contaminated water. Water from coastal seafood-harvesting sites should be tested for contamination.

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13.10.2 Nonagglutinable Vibrio Toxin The vibrios that have biochemical reactions similar to those of V. cholerae and are not agglutinable by V. cholerae subgroup O1 antiserum have been designated choleralike vibrios (Gardner and Venkatraman, 1935), noncholera vibrios (NCVs) (McIntyre et al., 1965), and nonagglutinable (NAG) vibrios (Felsenfeld, 1967). These organisms are now reported as V. cholerae serogroup nonO1. The NAG vibrios are widely distributed in the environment. They have been isolated from sewage, surface and estuarine waters, seafoods, animals, poultry, cockroaches, and tadpoles (Sanyal, 1986). Although doubts have been expressed about the capability of these free-living strains to cause disease in humans (WHO, 1980), Back and associates (1974) reported cases of diarrheal and extraintestinal infections due to NAG vibrios in which connection with the brackish water was a significant common feature. Yagnik and Prasad (1954) reported the first outbreak caused by NAG vibrios in northern India. Pollitzer (1959), while reviewing the various reports of isolation of the NAG vibrios, concluded that they are frequently implicated in sporadic cases and outbreaks of diarrhea in different geographical areas of the world. It is quite likely that in many countries infections by these vibrios are unrecognized because of inaccurate bacteriological techniques, which are aimed at detecting V. cholerae serotype O1. However, no large epidemic or pandemic like that caused by V. cholerae serotype O1 has yet been reported. Contaminated food and water is probably the exclusive mode of transmission of NAG vibrios. Shellfish harvested from U.S. coastal waters frequently contain these vibrios. Consumption of raw, improperly cooked or cooked, recontaminated shellfish may lead to infection. Water, ice, eating utensils, soft drinks, and fruits and vegetables washed with polluted water have all been implicated as vehicles of transmission (Hoover, 1985). No large out-

breaks attributed to these organisms have as yet occurred in the United States. Sporadic cases, however, continue to occur all year, increasing in frequency during the warmer months. The incubation period is much shorter, usually 20–36 hours. In India and Bangladesh, the classical home of cholera, the NAG vibrios have been associated with the full-blown cholera syndrome (Lindenbaum et al., 1965; Chatterjee et al., 1972; Tiwari et al., 1975). The clinical features of NAG diarrhea range from a choleralike disease with rice water stools to mild gastroenteritis. Approximately 25% of infected individuals have blood and mucus in their stools, which are not found in the classical cholera cases. However, the duration of diarrhea and the volume of fluid excreted and/or required for treatment are usually less than those of patients with severe forms of classical cholera. Septicemia (bacterial entry into the bloodstream and multiplication therein) can occur in individuals who have cirrhosis of the liver or are immunocompromised, but this is relatively rare. The U.S. FDA has warned individuals with liver disease to refrain from consuming raw or improperly cooked shellfish. The methods used to isolate this organism from foods are similar to those used with diarrheic stools. Because many food isolates are nonpathogenic, pathogenicity of all food isolates must be demonstrated. Because the virulence mechanisms of this group are not yet fully elucidated, pathogenicity testing must be performed in suitable animal models. NAG vibrio toxin contains both enterotoxic and vascular permeability factors, as does cholera toxin (Ohashi et al., 1972). The toxin is heat-labile; heating at 56°C for 30 minutes causes appreciable loss of activity of the culture filtrates and complete inactivation of enterotoxicity and the permeability factor at 60°C for 10 minutes (Shanker et al., 1982). The optimal pH for its activity is between 6 and 8. Ghosh and colleagues (1970) suggested NAG vibrio enterotoxin to be a combination of protein and the presence of an exopolysaccharide moiety. Using immunodiffusion and immunoelectrophoresis techniques, these researchers showed its antigenic similarity to that produced by V. cholerae 569B isolate. Its biological activity as well as mode of action also appear similar to those of the classic cholera toxin. At present, good markers of pathogenicity for this group of bacteria are lacking. Animal assays and biochemical markers do exist, but they have not been completely evaluated as to their role in human illness. 13.10.3 Vibrio parahaemolyticus Hemolysin V. parahaemolyticus was first isolated by Fujino and coworkers (1953) from a case of food poisoning in Isaka Pre-

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fecture, Japan, when 20 deaths occurred among 272 stricken consumers of a partially dried raw sardine. It is a common marine isolate found in unpolluted coastal waters throughout the world, especially those adjacent to warm countries where the temperature exceeds 15°C. It does not flourish in estuary waters. First recognized as a pathogen in the early 1950s, it is a significant source of food-borne illness in Japan, perhaps responsible for 50% of the reported cases of such illness. Among the bacteria causing food poisoning in Japan, V. parahaemolyticus is the most frequently isolated, followed in decreasing order by Staphylococcus spp., Salmonella spp., E. coli, and C. botulinum (Miwatani and Takeda, 1976; Takeda, 1986). Several outbreaks of food poisoning associated with V. parahaemolyticus and sporadic cases of V. parahaemolyticus infection have also been reported from the United States, Europe, and Asia (Chatterjee et al., 1972; Fujino et al., 1974; Barker, 1974; Miwatani and Takeda, 1976; Blake et al., 1980). It was not until 1969 that this organism was thought to be a problem in the United States. Since that time a few outbreaks have been attributed to it, principally cases around the Gulf and southeastern Atlantic coasts and in the Pacific Northwest (Liston, 1990). In the United States, it has been associated with cross-contamination of cooked with raw foods, such as the flow of melting ice from raw clams over steamed clams, and possibly undercooking of shrimp. However, most public health authorities around the world acknowledge it as a pathogen of concern even though accurate figures on the disease incidence are not available. At least two factors complicate accurate figures on disease incidence: One is that a special culture medium is necessary to identify it; the other is that not all environmental isolates are capable of causing the disease, a situation similar to that of NAG or non-O1 vibrios. Organism V. parahaemolyticus is a short, slender, curved gram-negative motile rod with a single polar flagellum. The organism is facultatively anaerobic. It is an obligate halophile (i.e., requires NaCl for growth), growing best in the presence of 2%–4% NaCl but tolerating salt concentrations of up to 8%. The temperature range for growth is 10°C–44°C, and optimal growth occurs at 37°C, at which the doubling time is about 10–12 minutes. V. parahaemolyticus is very heatsensitive and is readily killed at temperatures above 50°C. Not all strains are pathogenic to humans, but those that are almost invariably give a positive Kanagawa reaction (i.e., lyse red blood cells). These pathogenic strains appear to possess a “toxic factor” that is released on lysis of the bacterial cells (Brown et al., 1977).

V. parahaemolyticus has many serotypes, which are distinguished by their major antigens: somatic (O), capsular (K), and flagellar (H). Since the H antigen is common to all vibrios, the O and K antigens are the basis for the serological typing of V. parahaemolyticus. Hemolytic Toxin Obara (1971) first isolated a thermostable hemolysin from culture filtrates of Kanagawa phenomenon–positive strains and suggested that this hemolysin might be responsible for the Kanagawa phenomenon. Zen-Yoji and associates (1971), who also purified the toxin, proposed the name enteropathogenic toxin for it. In contrast, Sakurai and colleagues (1973) found that the hemolytic activity of the thermostable hemolysis produced by Kanagawa phenomenon–positive strains was not activated by adding lecithin and thus proposed the name thermostable direct hemolysin. The purified thermostable direct hemolysin does not contain any phospholipid and carbohydrates. It has a pI of 4.2. The hemolytic activity of the toxin is destroyed by pepsin and α-chymotrypsin, but not by trypsin (Zen-Yoji et al., 1976). The protein is composed of two subunits of approximately 21,000 Da each. The amino acid sequence of the toxin is known (Tsunasawa et al., 1983; Nishibuchi and Kaper, 1985). The purified thermostable direct hemolysin is not inactivated by heating at 100°C for 10 minutes, hence its name. The hemolytic activity of the crude hemolysin, however, is partially inactivated by heating at around 60°C for 10 minutes (Miwatani et al., 1972). Mode of Action The purified thermostable direct hemolysin shows various biological activities, such as hemolytic activity, cytotoxic activity on various cultured cells, and lethal toxicity in mice and rats. It shows high hemolytic activity on erythrocytes of various species; its activity decreases in the order rat, dog, mouse, monkey, human, rabbit, and guinea pig (Table 13.35). Sakurai and coworkers (1975) studied the mechanism of hemolysis induced by this toxin and found that hemolysis varied with the temperature and did not occur at low temperatures; however, binding to human erythrocytes did occur at low temperatures (0°C–4°C). The binding was followed by lysis of the cells and was stimulated by divalent cations such as calcium, magnesium, and manganese. The hemolysis appeared to be a two-step process involving the binding of the toxin and a subsequent step(s). It is still uncertain whether the hemolysis involves some enzymic action on the cell membranes.

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Table 13.35 Hemolytic Activities of the Thermostable Direct Hemolysin of Vibrio parahaemolyticus on Erythrocytes from Various Species

Source of erythrocytes

Reciprocal of titer of minimum hemolytic dose

Rat Dog Mouse Monkey Human Rabbit Guinea pig Chicken Sheep Horse

10,240 5,120 2,560 2,560 1,280 640 640 160 30 0

Source: From Zen-Yoji et al. (1971) and Takeda (1986).

Symptoms and Diagnosis The symptoms of V. parahaemolyticus food poisoning usually appear 10–18 hours after ingestion of the contaminated food, although incubation periods varying from 2 to 48 hours have been reported. The main symptoms are diarrhea, abdominal pain, headache, vomiting, fever, general malaise, chill, tenesmus, and nausea. Of these, diarrhea and abdominal pain are the main symptoms. The frequency of diarrhea is usually less than 10 times a day, but some cases show a frequency of more than 21 times a day (Saito, 1967). The diarrhea is usually watery, but mucous and bloody discharges are sometimes observed. The diarrhea or soft stools persist for 4–7 days or more. The enteritis tends to subside spontaneously with no treatment other than restoration of water and electrolyte balance. The mortality rate is very low. Although there is no direct evidence that the thermostable direct hemolysin is a cause of diarrhea due to V. parahaemolyticus infection, several findings suggest that it is at least one of the major causes of diarrhea. The minimal infective dose for this organism in humans is unknown, but a total dose of greater than 1 million organisms may cause disease. Culturing the organism from the diarrheic stools of the patients makes the diagnosis of gastroenteritis caused by V. parahaemolyticus. The organism is usually identified by its oxidase-positive growth on blood agar. Foods Incriminated in the Poisoning V. parahaemolyticus is principally associated with seafoods, and it is these foods that have been responsible for

most of the outbreaks of this type of food poisoning. In Japan, where raw fish is commonly eaten, V. parahaemolyticus accounts for approximately 50%–60% of all food poisoning cases. In European countries and the United Sates, unlike in Japan, most seafood is cooked before consumption and therefore the organism is invariably killed. However, oysters are an exception, but they are rarely implicated in food poisoning outbreaks. This is because the numbers of V. parahaemolyticus present in this mollusk are too low to initiate the illness. Foods that have been involved in food poisoning outbreaks in the United Kingdom and the United States include dressed crabs, lobster, prawns, and shrimps, usually imports from the Far East. Outbreaks appear to have been caused in two ways, either by inadequate cooking or by recontamination after cooking, both of which left viable organisms in the food, which were capable of developing into vast numbers during subsequent storage at room temperature. Improper refrigeration of seafoods contaminated with this organism also allows its proliferation, which increases the possibility of infection. The largest United States outbreak occurred during the summer of 1978 and affected 1133 of 1700 persons attending a dinner in Port Allen, Louisiana (Oliver and Kaper, 1997). The food implicated was boiled shrimp, which yielded positive culture findings of V. parahaemolyticus. The raw shrimp had been purchased and shipped in standard wooden seafood boxes. They were boiled the morning of the dinner but returned to the same boxes in which they had been shipped. The warm shrimp were then transported 40 miles in an unrefrigerated truck to the site of the dinner and held an additional 7 to 8 hours until served that night. In their survey of four Gulf Coast states, Levine and associates (1993) found V. parahaemolyticus to be the most common cause of gastroenteritis (37% of 71 cases) in that area. Similarly, Desenclos and colleagues (1991) found V. parahaemolyticus to be the second leading cause (over 26%) of gastroenteritis cases in those persons who had consumed raw oysters in Florida. In a 15-year survey of vibrio infections reported by a hospital adjacent to Chesapeake Bay, Hoge and coworkers (1989) found 9 (>69%) of 13 vibrio-positive stool specimens to contain V. parahaemolyticus as the sole pathogen. Prevention Since the food poisoning incidents are primarily due to a storage and cross-contamination problem, the control measures should be aimed at the following: 1.

Ensure adequate heat treatment of cooked seafoods.

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2.

3.

4.

Ensure rapid cooling and refrigeration of cooked seafoods, if they are to be stored and ensure raw seafoods are refrigerated at all times. Prevent cross-contamination of cooked by raw seafoods or by surfaces that have had contact with the raw food. Where possible, attempt to import seafoods in the raw state so that final processing can be monitored.

Oysters present a particular challenge as they are frequently eaten raw, and thus for raw product, there is no critical control point that eliminates the hazard. Control includes health education and warnings for high-risk consumers, and pathogen reduction measures, neither of which offers absolute control. Mild heat treatments (e.g. 50°C for 10 minutes) and cold shock are proposed as alternative control measures (Desmarchelier, 2000). Given the potential high mortality rate and the impact of such an event on the industry, the risks are high, although the likelihood of occurrence is low. The usual methods of food preservation such as brining, pickling, acidifying, drying, and smoking should render seafood free of or safe from V. parahaemolyticus. Again, adequate heating during the smoking process must be maintained. Many food preservatives, if used properly, are also valuable in rendering seafood safe. Nonetheless, as one of the major bacteria causing seafood-borne poisoning, V. parahaemolyticus will continue to be a threat to both consumer safety and the seafood industry. 13.10.4 Vibrio vulnificus V. vulnificus is perhaps the most virulent of all the vibrios found in estuarine environments. Of all the pathogenic vibrios, V. vulnificus is the most serious in the United States, alone responsible for 95% of all seafood-related deaths in this country (Oliver and Kaper, 1997). It is also the leading cause of reported deaths of food-borne illness in Florida (Hlady et al., 1993). Among the portion of population that is at risk of infection by this bacterium, primary septicemia cases resulting from raw oyster consumption typically carry fatality rates of 60%. This is the highest death rate of any food-borne disease agent in the United States (Todd, 1989a; Oliver and Kaper, 1997). According to CDC and FDA estimates there are at least 50 food-borne cases per year of V. vulnificus serious enough to be recognized by hospital personnel, although estimates of 17,500 to over 41,000 total cases have been calculated (Todd, 1989b). This halophilic bacterium infects only humans and other primates and can cause severe wound infections,

bacteremia, and probably gastroenteritis. It is a free-living estuarine bacterium found in the United States on the Atlantic, Gulf, and Pacific coasts as well as in Mexico. Cases of illness have also been associated with brackish lakes in New Mexico and Oklahoma. It has been isolated from a wide range of environmental sources, including water, sediment, plankton, and shellfish (oysters, clams, and crabs), especially in warm months. Consumption of these products, raw or recontaminated, may result in illness. Infections have also been reported from Korea, and the organism may be distributed worldwide. Bacteremia with no focus of infection occurs in persons who have eaten infected oysters and who have alcoholism or liver disease. In healthy individuals, gastroenteritis usually occurs within 16 hours of ingesting the organism. The infective dose for gastrointestinal symptoms in healthy individuals is unknown, but for predisposed persons, septicemia can presumably occur with doses of less than 100 total organisms. Wounds may become infected in normal or immunocompromised persons who are in contact with water where the organism is present. Infection often proceeds rapidly, with development of severe disease. About 50% of the patients who have bacteremia die. Wound infections may be mild but often proceed rapidly (over a few hours), with development of bullous skin lesions, cellulites, and myositis with necrosis. Because of the rapid progression of the infection, it is often necessary to treat with appropriate antibiotics before culture confirmation of the cause can be obtained. There are no reported cases of development of V. vulnificus infection in more than one person after consumption of the same lot of oysters. Occasionally reports document the consumption of raw oysters and subsequent disease in one family member, while others exhibiting no symptoms (Oliver and Kaper, 1997). Further, because raw oysters are usually eaten whole, there rarely exist any remains of the implicated oyster to sample. Hence, it is difficult to track the source of the V. vulnificus strains isolated from the patient. It is possible that newly developed fingerprinting methods can enable molecular epidemiology to be used in tracking infections. Acceptable levels of V. vulnificus in raw seafood are difficult to determine. Healthy consumers are able to tolerate the small numbers present in naturally contaminated seafood, whereas the same dose apparently causes infection in high-risk consumers (Desmarchelier, 2000). Because of the severity of the infection and the existing impaired health of the high-risk individuals, human volunteer studies cannot be performed. Small numbers are therefore considered hazardous for at-risk individuals. Oysters are the main product implicated, and groups at risk should avoid eating this food. Some food authorities

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require restaurateurs and retailers of raw oysters from regions known to be naturally contaminated with V. vulnificus to have a health warning on the product notifying the consumer of the risk to certain individuals. Additional measures to health education are aimed at limiting the concentrations of V. vulnificus and controlling its growth in oysters after harvest. The U.S. Interstate Shellfish and Sanitation Conference has recommended that any state whose oyster-growing waters have been confirmed as the original source of oysters associated with two or more V. vulnificus cases be expected to require that the oysters be refrigerated within a specified time after harvest (Desmarchelier, 2000). The time is dependent on the average monthly temperature of the oyster-growing waters. For example, the interval between harvest and refrigeration is 14 hours when the water temperature is 18°C–23°C, 12 hours at 23–28°C, and 6 hr at >28°C. Another reduction strategy is to relay oysters from waters of low salinity to those of higher salinity. It should be noted that the depuration of oysters is effective in the reduction of introduced bacterial species but is ineffective in the removal of natural contamination with vibrios. 13.10.5 Other Vibrios V. mimicus is similar to V. cholerae. This organism is chiefly isolated from cases of gastroenteritis, but occasionally strains have been isolated from ear infections as well. This species is found in Bangladesh waters throughout the year, whereas in Okayama, Japan, it is found only when the water temperature is above 10°C (Chowdhury et al., 1987). Food poisoning by this organism peaks in the summer months. Documented vehicles include raw oysters and boiled crayfish. Symptoms include diarrhea, which may be bloody and may last 1–6 days. Nausea, vomiting, and abdominal pain occur in two thirds of the cases (Hackney and Dicharry, 1988). V. mimicus appears to produce no unique enterotoxins, although many strains produce toxins that were first described in other vibrios. The species V. hollisae is different from other vibrios in that it grows on different media. It has been the documented cause of 36 cases of food poisoning associated with raw oysters, clams, and shrimp. Diarrhea is the major symptom. Vomiting and fever may occur. The median duration of diarrhea is about 1 day (range of 4 hours to 13 days). The organism produces hemolysin that is related to the V. parahaemolyticus thermostable direct hemolysin. V. furnissii has also been documented as a source of food poisoning characterized by diarrhea and cramping, sometimes with nausea and vomiting (Hackney and Dicharry, 1988). There is no fever. The symptoms appear be-

tween 5 and 20 hours after ingestion of contaminated food, and the patients recover within 24–48 hours. V. alginolyticus, a halophilic vibrio found in estuarine environments, is frequently isolated from seafood, particularly shellfish, in markets around the world. It is considered a fish spoilage organism and is usually associated with wound infections in people and marine mammals. Only a few human cases of enteric disease have been reported to date.

13.11 ESCHERICHIA COLI INTOXICATIONS Escherichia coli is the most extensively studied microorganism. It has been a model system for the study of bacterial metabolism, the cell division process, cell wall biosynthesis, chemotaxis, bacterial genetics, and the physiological role of enteric bacteria as part of the normal fecal flora (Neidhardt, 1996). Despite the vast knowledge that has been accumulated over the years, the 1997 release of its full genomic composition has made it obvious that there are still many factors to learn about this microorganism (Blattner et al., 1997). Analysis of the E. coli K-12 genome sequence also shows that about 2% of its DNA consists of mobile genetic elements, including phages, plasmids, and transposons. These elements are responsible for the continuous evolution of the bacterial genomic repertoire, providing significant diversity in E. coli strains. In this regard, pathogenic E. coli appears to have evolved from nonpathogenic strains by acquiring new virulence factors by the horizontal transfer of accessory DNA, which is often organized in clusters (pathogenicity islands) in the chromosome or on plasmids (Hacker et al., 1997). The high genetic diversity of the E. coli genome is also reflected by the large variation in DNA content among different strains (Rode et al., 1999; Bergthorsson and Ochman, 1995, 1998) and by the distribution or genomic location (insertion site) of different virulence determinants (Wieler et al., 1997; Boyd and Hartl, 1998). In this context, it seems that most pathogenic E. coli strains do not have a single evolutionary origin but instead have emerged as a result of different events of DNA transfer, and that even strains capable of causing the same disease do not constitute a monophyletic group (Pupo et al., 1997). E. coli pathogenic variants are represented by strains of specific serogroups possessing a particular set of virulence factors, which are responsible for the different clinical manifestations that characterize E. coli infection. Pathogenic E. coli cause various diseases in humans, including several types of diarrhea, urinary tract infections, sepsis, and meningitis (Table 13.36). E. coli strains that cause human diarrhea of varying severity have been di-

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vided into six major categories: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), and diffuse adhering E. coli (DAEC). E. coli strains causing urinary tract infections are known as uropathogenic E. coli (UPEC); E. coli K1 are often responsible for cases of meningitis or sepsis (SEC) (Table 13.36). Different bacterial virulence attributes appear to dictate the types of interactions that occur between the pathogenic organism and its host cells and the area of the body where these interactions occur. Tissue tropism plays an important role in disease: for example, UPEC infects the urinary tract and kidneys, EPEC the small bowel, and EHEC the large bowel. Once they are localized to a particular tissue, the molecular interactions that occur between pathogenic E. coli and their host cells follow specific steps and are quite different among different pathogenic types. Some strains adhere to mucosal surfaces and secrete specific toxins that either intoxicate localized epithelial cells or spread systemically to affect distant host cells. Others interact more intimately with host cell surfaces, and this intimate interaction results in disease. Finally, some strains actually enter host cells and live as intracellular pathogens or penetrate host barriers and live systemically within the human host, resulting in septic disease (Finlay and Falkow, 1997; Nataro and Kaper, 1998). The wide diversity of virulence factors identified and characterized in different pathogenic E. coli resemble many of the virulence mechanisms found in other pathogens (Finlay and Falkow, 1997; Finlay and Cossart, 1997). ETEC utilizes a choleralike toxin to cause choleralike disease (Sears and Kaper, 1996). EIEC behaves as Shigella spp., in that it contains the same virulence factors (e.g., type III secretion system, invasions, and intracellular spread mechanism) that are responsible for producing a dysenterylike disease (Menard et al., 1996). EHEC produces a Shiga-like toxin (similar to that found in Shigella dysenteriae) that seems to be involved in causing the hemolytic uremic syndrome in a proportion of cases (Paton and Paton, 1998). EHEC and EPEC utilize a type III secretion system, similar to those seen in Salmonella, Shigella, and Yersinia spp., and other gram-negative pathogens, to inject E. coli–specific factors into the host cell. These factors induce actin rearrangements and activation of particular signal transduction pathways that result in disease (Hueck, 1998). As with other pathogens that cause systemic disease and meningitis, E. coli K1 produces a polysaccharide capsule that prevents clearance by phagocytic cells (Moxon and Kroll, 1990). In this section, the literature on the various pathogenic E. coli strains and their importance in the human

Table 13.36

Escherichia coli That Are Pathogenic to Humans

Type

Disease

Virulence factors

Enterotoxigenic (ETEC)

Watery to choleralike diarrhea

Enteroinvasive (EIEC)

Watery diarrhea to dysentery

Enteropathogenic (EPEC)

Watery diarrhea

Enterohemorrhagic (EHEC) Enteroaggregative (EAEC)

Hemorrhagic colitis, hemolytic uremic syndrome (HUS) Watery to mucoid diarrhea

Heat-labile toxin (LT), heat-stable toxin (ST), colonization factors (CFs) Ipas, type III secretion (Mxi and Spa), VirG/IcsA Esps, type III secretion (Sep and Esc), intimin, Tir, and BFP EPEC factors and Shiga toxin, hemolysin

Diffusely adhering (DAEC) Uropathogenic (UPEC)

Watery diarrhea Urinary tract infections

Septic (SEC)

Neonatal sepsis, meningitis

food chain is described. Major attention is focused on E. coli O157:H7, since it has gained recognition in recent years as an important food-borne pathogen. An excellent comprehensive review that examines the epidemiological characteristics, clinical symptoms, detection, diagnosis, and virulence of the diarrheagenic E. coli was published in 1998 (Nataro and Kaper, 1998). 13.11.1 Organism E. coli belongs to the Enterobacteriaceae family, which comprises a large heterogeneous group of gram-negative rods whose natural habitat is the intestinal tract of humans and animals. Other genera belonging to this family include Shigella, Salmonella, Citrobacter, Enterobacter, Klebsiella, and Proteus. The Enterobacteriaceae are facultative anaerobes or aerobes, ferment a wide range of carbohydrates, possess a complex antigenic structure, and produce a variety of toxins and other virulence factors. E. coli is oxidase-negative and grows by using simple carbon sources, including glucose and acetate. The hexose is fermented to a mixture of acids (lactate, acetate, and formate) as well as carbon dioxide. E. coli are citratenegative but methyl red–positive and Voges-Proskauer– negative. It is classified as a coliform, a general term used to describe gram-negative asporogenous rods that ferment lactose within 48 hours and whose colonies are dark and exhibit a green sheen on agar such as eosin methylene blue. E. coli is a normal inhabitant of the gut of warmblooded animals. As such, it is often used as an indicator of fecal contamination. Not all strains of E. coli cause disease, however, and as a consequence the detection of

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AAF adhesins, EAST-1, Pet, Pic, hemolysin F1845 and AIDS-I fimbriae Type I pili, P pili, afimbrial adhesins (Afa), hemolysin, CNF-1 Capsule, type I pili, S-fimbrial adhesin, IbeA and IbeB (invasion proteins)

E. coli in a food, although implying a potential hazard, does not a priori indicate that the food will cause illness if consumed. Of note among the E. coli strains is the serotype O157:H7. This serotype includes highly virulent strains, and has been the focus of much attention during the past 10 years or so, not only because of its association with a number of highly publicized food-borne outbreaks, but also because of its ability to survive acidic conditions that were previously believed to be lethal to E. coli. 13.11.2 Serological Characteristics The Enterobacteriaceae have a complex antigenic structure. E. coli isolates are serologically differentiated on the basis of three major surface antigens, which allow serotyping: the O (somatic), H (flagella), and K (capsule) antigens. At present, a total of 174 O antigens, 56 H antigens, and 80 K antigens have been identified (Doyle et al., 1997; Brooks et al., 1998; Batt, 2000b). The O antigens are the most external part of the cell wall lipopolysaccharides and consist of repeating units of polysaccharides. The oligosaccharide is covalently linked to the lipid A-core polysaccharide and the repeating units define the diversity of the O antigen group. Some O-specific polysaccharides contain unique sugars. As a result of the extreme heterogeneity in the five or more sugars the O group comprises 174 different O groups. O antigens are resistant to heat and alcohol and usually are detected by bacterial agglutination. Antibodies to O antigens are predominantly immunoglobulin M (IgM). Although each genus of Enterobacteriaceae is associated with specific O groups, a single organism may carry

several O antigens, and as a result there is cross-reactivity. Thus, most shigellae share one or more O antigens with E. coli. E. coli may cross-react with some Providencia, Klebsiella, and Salmonella species. Occasionally, O antigens may be associated with specific human diseases; e.g., specific O types of E. coli are found in diarrhea and in urinary tract infections. The consequence of cross-reactivity is that many antibody-based tests that broadly detect E. coli frequently generate false-positive results that are due to cross-reactivity with O antigens of other microorganisms. The K antigens are external to O antigens on some but not all Enterobacteriaceae and are part of the cell capsule. Some are polysaccharides, including the K antigens of E. coli; others are proteins. The polysaccharide is mainly acidic and heat-labile to varying degrees. K antigens may interfere with agglutination by O antisera. Unlike the O and H antigens, the K antigen generally is not used in most serotyping schemes. However, some K antigens may be associated with virulence; e.g., E. coli strains producing K1 antigen are prominent in neonatal meningitis, and K antigens of E. coli cause attachment of the bacteria to epithelial cells prior to gastrointestinal or urinary tract invasion. The H antigens are located on flagella and, hence, found only in motile strains of organisms. They are easily denatured or removed by heat or alcohol. Most E. coli are nonmotile or partially motile on initial isolation from the environment. As a consequence, H antigen typing is not reliable unless efforts are taken to select for the restoration of motility. The antigenic classification of Enterobacteriaceae often indicates the presence of each specific antigen. Thus, the antigenic formula of an E. coli may be O55:K5:H21. The K antigen descriptor, however, has been dropped as a descriptor of serotypes, and only the H and O are commonly employed. The H antigen coupled to the O antigen therefore represents a robust and highly discriminatory typing method for distinguishing various strains of E. coli. The O:H serotypes can be sorted into various virulence groups and also categorized with respect to the host animal. For example, O157:H7 is associated with enterohemorrhagic (EHEC) forms of disease in humans, whereas the O55:H7 is associated with the enteropathogenic (EPEC) forms of the disease, also in humans. However, this is not absolute, as serotype 111, for example, is found among EAEC, EHEC, and EPEC E. coli virulent strains. The distribution of O serotypes among the different virulence groups of E. coli strains is shown in Table 13.37. E. coli that cause diarrhea are extremely common worldwide. These E. coli are classified by the characteristics of their virulence properties, and each group causes disease by a different mechanism. Genes on plamids en-

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Table 13.37 Distribution of O Serotypes among the Different Virulence Groups of Escherichia coli EAECa

EHEC

EIEC

EPEC

ETEC

3 4 6 7 17 44 51 68 73 75 77 78 85 111 127 142 162

2 4 5 6 22 36 38 45 46 82 84 88 91 103 104 111 113 116 118 145 153 156 157 163

28ac 29 112a 124 135 136 143 144 152 164 167

18ab 19ac 55 86 111 114 119 125 126 127 128ab 142 158

6 15 20 25 27 63 78 80 85 101 115 128ac 139 141 147 148 149 153 159 167

a

Refer to Table 13.36 for the abbreviations used.

code the small or large bowel epithelial cell adherence properties. Similarly, the toxins often are plasmid- or phagemediated. The major characteristics of various pathogenic E. coli implicated in diarrheal diseases are described in the following sections. 13.11.3 Enterotoxigenic E. coli ETEC is a common cause of “traveler’s diarrhea” and a very important cause of diarrhea in infants in developing countries. A variety of names have been associated with this disease, including gypsy tummy, Delhi belly, Hong Kong dog, and Aden gut. Humans are the principal reservoir of ETEC that causes human illness. The disease is characterized by a watery diarrhea, ranging in severity from mild and self-limiting to a severe choleralike profuse diarrhea. It is usually without mucus, blood, pus, fever, or vomiting, as is consistent with its being an intoxication (i.e., toxin-mediated), rather than a systemic infection. ETEC is responsible for more than 650 million cases of diarrhea and between 700,000 and 800,000 deaths in children below the age of 5 years. Antibiotics decrease the

severity and duration of diarrhea, but antibiotic-resistant strains of ETEC are increasingly common. Like that for cholera, therapy is mainly rehydration (usually oral). With proper hydration, the disease is usually self-limiting (Nataro and Kaper, 1998). Virulence Factors The pathogenesis of ETEC is mediated by at least two virulence factors. The colonization factors or adhesins (Jones, 1977) (pili or fimbriae e.g., colonization factor/antigen I [CFA/I] and CFA/II) specific for humans promote adherence of ETEC to epithelial cells of the small bowel. Recognized colonization factors occur with particular frequency in some serotypes. Certain serotypes of ETEC occur worldwide; others have a limited recognized distribution. The second virulence factor is the production of two kinds of enterotoxins. Some strains of ETEC produce an LT or an ST toxin that elicits fluid accumulation and a diarrheal response. Approximately 30% of the ETEC strains express LT, 35% ST; the rest express both. Genes for both enterotoxins and colonization factors are present on plasmids (Smith and Linggood, 1971a, 1971b; Nataro and Kaper, 1998; Brooks et al., 1998). Colonization Factors (Adhesins) Colonization factor antigens (CFAs), coli surface antigens (CSs), and putative colonization factors (PCFs) mediate ETEC attachment to the intestinal surface. These are generally referred to as colonization factors (CFs) or adhesins (Cassels and Wolf, 1995; Gaastra and Svennerholm, 1996). These structures are essential for ETEC to colonize the small intestine, a central step in ETEC’s virulence. At least 20 different and antigenically distinct CFs have been described in human ETEC and are found in varying combinations along with LT, ST, or both (Gaastra and Svennerholm, 1996). The main CFs associated with human ETEC strains include CFA/I, constituted by a single fimbrial structure, and CFA/II and CFA/IV, which can be a combination of a particular set of CSs. CFs dictate host and tissue specificity, since animal ETEC CFs are not found in human ETEC isolates. Morphologically, CFs can be subdivided into four major groups: rigid rods (e.g., CFA/I), bundle-forming (longus), fibrillar, and nonfimbrial adhesins (Cassels and Wolf, 1995; Gaastra and Svennerholm, 1996). The intestinal receptors that CFs bind include sialoglycolipids such as GM2, sialic acid containing glycoconjugates, and several other glycoconjugates (glycolipids and glycoproteins) found on the cell surface. The oligosaccharides expressed on mammalian cell surfaces vary

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widely, providing an extensive range of options that might contribute to host and tissue specificity for the ETEC CFs. Heat-Labile Toxin There are two forms of LT: LT-1 and LT-II (Robertson et al., 1986; O’Brien and Homes, 1996). LT-1, the predominant form, is quite similar to CT (choleragen) at the sequence level (about 80% homology) and is thought to act mechanistically in an identical fashion (Spangler, 1992). LT-1 (80,000 molecular weight) is oligomeric in structure with one enzymatic A subunit and five identical B subunits (Gill et al., 1981). The five B subunits are arranged symmetrically in a ringlike structure that binds the ganglioside GM1 and binds weakly to ganglioside GD1b (Fukuta et al., 1988). The A subunit is proteolytically cleaved into two domains, A1 and A2, that remain linked by a disulfide bond and span the center of the ring (Sixma et al., 1991, 1993). The toxin is endocytosed, and the A subunit reaches the basolateral surface of the epithelial cell after escaping the endocytic vesicle. The A1 peptide transfers an ADP-ribosyl group from NAD to the α-subunit of the GTP binding protein GS. This modification of the GSα inhibits its intrinsic guanosine triphosphatase (GTPase) activity, which results in permanent activation of adenylate cyclase, leading to accumulation of intracellular levels of cAMP. As cAMP accumulates inside intestinal cells, it activates a cAMP-dependent kinase (A kinase), which then results in phosphorylation of apically located chloride channel proteins such as cystic fibrosis transmembrane conductance regulator (CFTR), the channel affected in cystic fibrosis patients. This causes channel opening and chloride ion efflux out of cells along with a block in ion and fluid absorption into cells, resulting in a net osmotic imbalance (Figure 13.12). The result is watery diarrhea. Although the preceding mechanism appears likely, several other more complex mechanisms of action have also been implicated in LT- and CT-mediated disease. These mechanisms, reviewed by Nataro and Kaper (1998), include increased production of prostaglandins, stimulation of a mild inflammatory response, and activation of the enteric nervous system. It is likely that the watery diarrhea resulting from ETEC infection is a combination of the classical mechanism described along with one ore more of these other events. The LT-II form shows less homology to LT-1 and CT (approximately 57%) and basically no identity to the B subunit (Pickett et al., 1987, 1989). These differences are also reflected in their specificity for binding to gangliosides, since LT-II binds best to ganglioside GD1b or GD1a (Fukuta et al., 1988) and is associated mainly with animal,

Figure 13.12 ETEC interactions with intestinal epithelial cells. ETEC adherence to intestinal cells is mediated by different CFs. Once established in the proximal small intestine, ETEC strains produce LT and/or ST. The LT holotoxin, consisting of an A subunit and a pentamer of B subunit (a structure similar to cholera toxin), is internalized by endocytosis on binding to its ganglioside GM1 receptor. The A subunit is potentially cleaved into the A1 and A2 subunits, which remain linked by a disulfide bond. The A1 subunit ADP-ribosylates the alpha subunit of the GTP-binding protein, Gs, inhibiting its intrinsic GTPase activity, resulting in constitutive activation of adenylate cyclase at the basolateral membrane. This activation leads to increased levels of intracellular cAMP, activation of a cAMP-dependent A kinase, and supranormal phosphorylation of intestinal epithelial cell chloride channels, such as CFTR. These events result in inhibition of NaCl absorption and stimulation of chloride secretion. ST acts by binding to GC-C, localized in the brush-border membrane of intestinal epithelial cells. Activation of GC-C results in increased levels of intracellular cGMP that stimulate chloride secretion and/or inhibit NaCl absorption, resulting in net intestinal fluid secretion. In vivo chloride secretion may occur through activation of a cGMP-dependent protein kinase (G-kinase), which ultimately activates the chloride channel CFTR. ETEC, enterotoxigenic E. coli; CF, colonization factor; ST, heat-stable toxin; LT, heat-labile toxin; ADP, adenosine diphosphate; GTP, guanosine triphosphate; GTPase, guanosine triphosphatase; cAMP, cyclic adenosine monophosphate; GC-C, guanylate cyclase type C; cGMP, cyclic guanosine monophosphate; CFTR-P, cystic fibrosis transmembrane conductance regulator protein; ADPr, ADP ribosyl.

but not human, disease (Seriwatana et al., 1988). Like ST and the CFs, LTs are usually encoded on a plasmid. LT is antigenic and cross-reacts with the cholera enterotoxin. LT stimulates the production of neutralizing antibodies in the serum (and perhaps on the gut surface) of persons previously infected with ETEC. Persons residing in areas where such organisms are highly prevalent (e.g., in some developing countries) are likely to possess antibodies and are less prone to have diarrhea on reexposure to the LT-producing E. coli.

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Heat-Stable Toxin As mentioned earlier, about one-third of ETEC strains express ST, and another third express ST and LT. Thus, ST alone is capable of causing diarrhea, without the presence of LT. However, the strains with both toxins generally produce a more severe diarrhea. In contrast to LT, ST is heat stable, a property provided by its intramolecular disulfide bonds. There are two major STs: STa and STb (Sears and Kaper, 1996; Giannella, 1995). STa has been studied in greater detail and provides an excellent bacterial

example of a hormonelike peptide that affects normal host cell function (Nair and Takeda, 1998). STa enterotoxins are small, cysteine-rich molecules (18–19 amino acids) that can form three intramolecular disulfide bonds (Shimonishi et al., 1987). These toxins are made from larger precursors (72 amino acids) that are cleaved as the molecule transits out of the bacterium. The eukaryotic membrane receptor for STa is guanylate cyclase C (GCC), which is located in the apical membrane of intestinal cells. Because of the apical location of GCC, STa-mediated cell activation is quite rapid. It has been suggested that STa receptors are more abundant on enterocytes of infants or young animals but that their numbers decrease in older individuals. This variation in receptor abundance may determine the severity of the secretory response and give a plausible explanation for the high susceptibility of human infants and newborn animals to STamediated diarrhea (Al-Majali et al., 1999a, 1999b; Cohen et al., 1988). The GCC activation triggers a cascade of events (Figure 13.12), including the accumulation of intracellular cyclic guanidine monophosphate (cGMP) levels, the cGMP-dependent activation of protein kinase A (PKA), and the PKA-dependent phosphorylation and activation of the CFTR. This process finally leads to increased chloride secretion and blockage of sodium chloride uptake, resulting in diarrhea (Goldstein et al., 1994; Chao et al., 1994). GCC-null mice were protected against an infection with ETEC, further evidencing the importance of this receptor in STa-mediated diarrhea (Schulz et al., 1997). Several other intracellular signals are activated in response to STa, but the contribution of these signals to diarrhea has not been fully defined. STa behaves in the same way as guanylin, the endogenous intestinal peptide hormone that binds to guanylate cyclase (Carpick and Gariepy, 1993; Ieda et al., 1999). Guanylin regulates ion and fluid levels by modulating cGMP levels, thereby mediating intestinal homeostasis. It is a 15-amino-acid peptide that contains four cysteines (two disulfide bonds) and, ironically, is less efficient than STa in activating guanylate cyclase. The discovery of guanylin was one of the first demonstrations that bacteria have evolved the ability to mimic eukaryotic endogenous functions to take advantage of host cells and, also, an indication of how the study of microbial pathogens is teaching us important aspects of the cell biology of eukaryotes. Although STb is primarily found in animal pathogens, it has also been isolated from human ETEC isolates. STb bears no sequence similarity to STa, although it does have four cysteine residues that form disulfide bridges (Arriaga et al., 1995). It is also initially synthesized as a larger precursor of 71 amino acids, which is then pro-

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teolytically processed to a 48-amino-acid mature protein (Dreyfus et al., 1992). Sulfatide has been suggested as a functional receptor for STb (Rousset et al., 1998a, 1998b). STb neither affects cAMP or cGMP levels nor stimulates chloride secretion (Peterson and Whipp, 1995); instead, it seems to elicit an intestinal response characterized by the secretion of bicarbonate, to which human cell lines seem to be insensitive (Weikel et al., 1986). 13.11.4 Enteropathogenic Escherichia coli Certain serotypes of E. coli termed EPEC by Neter and associate (1955) were recognized as a cause of epidemic and sporadic infantile diarrhea in the 1940s and 1950s (Bray, 1945; Taylor et al., 1949; Kauffman and DuPont, 1950). Most EPEC strains isolated in the 1970s and a few isolated in the 1950s that were available for testing did not produce LT or ST or cause Shigella spp.–like enteroinvasion (Goldschmidt and DuPont, 1976; Gross et al., 1976; Gurwith et al., 1977). Some believed that EPEC strains were not pathogenic because they lacked these virulence determinants. They hypothesized that these strains may have originally contained plasmids coding for enterotoxin when initially isolated but had lost them upon storage (Sack, 1976; Gangarosa and Merson, 1977). Hence, they were earlier referred to as “diarrheagenic E. coli belonging to serogroups epidemiologically incriminated as pathogens but whose pathogenic mechanisms have not been proven to be related to either heat-labile enterotoxin, heat-stable enterotoxin, or Shigella-like invasiveness.” Since the mid1970s, considerable progress has been made in defining the pathogenesis of EPEC. EPEC is the predominant cause of infant diarrhea worldwide and represents a major endemic health threat to children less than 6 months of age living in developing countries (Levine and Edelman, 1984). In addition, isolated outbreaks in day care centers, nurseries, and pediatric wards, as well as among adults in developed countries, have been reported. It has been estimated that EPEC kills several hundred thousand children each year worldwide. EPEC disease is characterized by prolonged watery diarrhea of varying severity, with vomiting and low fever often accompanying fluid loss. The actual molecular mechanisms that cause diarrhea remain undefined. Unlike other diarrheas caused by pathogenic strains of E. coli strains such as ETEC (discussed previously), no toxin has been implicated in EPEC-mediated diarrhea. Instead, EPEC binds to intestinal surfaces of the small bowel, causing a characteristic histological lesion called the attaching and effacing (A/E) lesion (Moon, 1997). These lesions are marked by dissolution of the intestinal brush-border surface and loss of

epithelial microvilli (effacement) at the sites of bacterial attachment. Once bound, EPEC resides on cuplike projections or pedestals formed by cytoskeletal rearrangements in host actin. EPEC-associated diarrhea has been attributed to the loss of absorptive surface area due to effacement of epithelial cell microvilli (Kaper, 1998a, 1998b). Although this mechanism remains to be proved, a series of recent in vitro studies have indicated that a more complex, yet not well-defined multifactorial mechanism might be involved. The result of EPEC infection is watery diarrhea, which is usually self-limited but can be chronic. The duration of EPEC diarrhea can be shortened and the chronic diarrhea cured by antibiotic treatment. 13.11.5 Enteroinvasive Escherichia coli EIEC causes a disease very similar to shigellosis. It is characterized by a watery diarrhea that often resembles that caused by ETEC. However, some patients do experience a dysenterylike disease, with mucus, blood, and pus in the stool, along with fever. The virulence factors in EIEC are virtually identical to those in Shigella species. However, the infectious dose of EIEC is much larger than that of Shigella spp. Unlike S. dysenteriae, EIEC does not contain a Shiga toxin and so does not cause hemolytic uremic syndrome (Parsot and Sansonetti, 1996; Menard et al., 1996). EIEC disease occurs most commonly in children in developing countries and in travelers to these countries. The incidence is low in developed countries, but foodborne outbreaks have been reported (Gordillo et al., 1992). Both EIEC and Shigella species invade the colonic epithelium. To achieve this, they follow a series of steps as they interact with the intestinal mucosa: invasion of colonic epithelial cells, lysis of the endocytic vacuole, bacterial multiplication, spread to adjacent cells, and host cell killing by apoptosis if the host cell is a macrophage (Menard et al., 1996; Parsot and Sansonetti, 1996). The virulence factors required for these multiple steps are encoded on a 140-MDa plasmid (pInv), although some other factors, such as regulators, are encoded on the chromosome (Harris et al., 1982; Menard et al., 1996). Nearly all we know about EIEC virulence mechanisms is based on extrapolation from the Shigella spp. systems. The overall process is schematically shown in Figure 13.13. Like Shigella spp., EIEC secretes invasion plasmid antigens (Ipas) A–D into the host cells. Ipas mediate invasion by triggering several events in host cells, resulting in membrane ruffling, macropinocytosis, actin rearrangements, and bacterial engulfment. Once those processes are

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accomplished, internalized bacteria are surrounded by a membrane-bound inclusion in the cytoplasm of host cells. Unlike phagocytosis, this process can occur in nonphagocytic (e.g., epithelial) cells. Like most gram-negative pathogens, EIEC and Shigella spp. utilize a specialized secretion system, designated the type III system, to export the invasion plasmid antigens (Ipa proteins) out of the bacteria and into their host cells (Hueck, 1998). This system is encoded by more than 20 mxi and spa genes located on the large plasmid (Menard et al., 1996). Once free in the cytoplasm, EIEC and Shigella spp. trigger an event, analogous to that in Listeria monocytogenes, that is responsible for their cell-to-cell spreading. These bacteria trigger nucleation of actin at one pole of the bacterial cell, which propels the bacterium through the cytoplasm at the head of this “comet tail” (Theriot et al., 1992). The VirG protein (also called IcsA) is critical and sufficient for triggering of this actin-mediated motion, probably by binding cytoskeletal components such as neural Wiskott-Aldrich syndrome protein (N-WASP) (Kocks et al., 1995; Suzuki et al., 1998). When the moving bacteria encounter the cytoplasmic face of the host cell membrane, they propel outward, pushing into adjacent cells, thereby avoiding extracellular exposure. Most epidemics of EIEC that have been described were food-borne. This is presumably because a large inoculum (>108 cells) is required to cause disease. Epidemic outbreaks have been reported in the United States after eating contaminated French Camembert or Brie cheeses (Schnurrenberger et al., 1971; Tulloch et al., 1973; Barnard and Callahan, 1972), China (Echeverria et al., 1992), Hungary (Lanyai et al., 1959), and Thailand (Taylor et al., 1986). 13.11.6 Enteroaggregative Escherichia coli EAEC is associated with persistent pediatric diarrhea in developed countries and is characterized by its aggregative adherence pattern (Wanke et al., 1991; Law and Chart, 1998; Nataro et al., 1998). EAEC causes a watery secretory diarrhea, often mucoid in nature. There is often lowgrade fever, but no vomiting. Grossly bloody stools can occur, although the majority of cases do not involve bloody diarrhea. Formation of a thick mucus gel on the intestinal mucosa and mucosal damage probably mediated by a mucosa-damaging toxin are pathogenic EAEC histopathological features (Hicks et al., 1996; Nataro et al., 1996). EAEC strains are a heterogeneous collection of pathogenic E. coli that share certain chromosomal and plasmid-borne genes. Their defining feature is that they adhere to cultured HEp-2 cells in small clumps or aggregates (aggregative

Figure 13.13 EIEC interactions with intestinal epithelial cells. EIEC strains secrete Ipa proteins via a type III secretion system onto and into host cells, causing actin rearrangements and membrane ruffling, resulting in bacterial internalization. Once inside a vacuole in the host cell, the IpaB protein degrades the vaucole, releasing the bacterium into the cytosol, where IcsA polymerizes actin. This action propels the organism through the cell and into neighboring cells. EIEC, enteroinvasive E. coli.

adherence [AA]), resembling a stacked-brick configuration (Nataro et al., 1987). A schematic representation of EAEC interactions with intestinal cells is shown in Figure 13.14. Adherence to host cells and neighboring bacteria is mediated by fimbrial adhesins encoded on a large plasmid. EAEC can increase mucus secretion, leading to a blanket of adherent bacteria trapped in a layer of mucus. Some EAEC can cause tissue damage, resulting in villus atrophy and other cytotoxic effects that are probably mediated by toxins, although the contribution of each toxin to disease has not been defined. The virulence factors involved in the pathogenesis of EAEC include adherence factors, enteroaggregative E. coli heat-stable toxin 1 (EAST-1) and Pet and Pic. EAEC produce at least two fimbrial adhesins: aggregative adherence fimbriae I and II (AAF/I and AAF/II, respectively). AAF/I mediates adherence to tissue culture cells and human erythrocytes and is encoded by two regions on the 60MDa plasmid (Nataro et al., 1992). One region encodes the fimbrial structure gene and assembly genes, such as chaperones, while the other is a regulatory region encod-

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ing an AraC-like regulator named AggR. AAF/I is a member of the Dr family of adhesins, which adhere to the Dr blood group antigen. AAF/II is a second fimbria produced by some strains of EAEC (Czeczulin et al., 1997). As in AAF/I, two regions encode them, although, unlike in AAF/I, the structural subunit and the assembly genes are encoded in separate regions (a characteristic of the Dr family of adhesins). Many EAEC strains also produce a plasmid-encoded heat-stable toxin designated EAST-1 (Savarino et al., 1996). This toxin shares identity with STa and probably works in a similar manner by activating guanylate cyclase, leading to secretory diarrhea. It is composed of 38 amino acids (4.1 kDa) and contains four cysteine residues (like guanylin, but differing from STa, which has six) that form disulfide bonds to stabilize the toxin. However, the contribution to disease by EAST-1 has not been established. Many E. coli strains produce EAST-1, including O157, several ETEC, and EPEC strains; however, clinical isolates of EAEC that do not produce EAST-1 are common, and even nonpathogenic E. coli produce EAST-1, indicating that it alone is not suffi-

Figure 13.14 EAEC interactions with intestinal cells. AAF fimbriae mediate the initial adherence of EAEC strains to the intestinal mucosa in a stacked-brick configuration (aggregative adherence). Colonization enhances mucus production, leading to accumulation of a thick mucus layer where bacterial cells remain embedded. During the process, EAEC delivers different toxins that damage the mucosa and promote intestinal secretion. The heat-stable enterotoxin (EAST1) is related to ETEC ST and may act similarly. Pet and Pic belong to the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily. EAEC, enteroaggregative E. coli; AAF, aggregative adherence fimbriae; SPATE, serine protease autotransporter of the Enterobacteriaceae; Pet, plasmid-encoded enterotoxin; Pic, protein involved in intestinal colonization.

cient to cause disease (Savarino et al., 1996). Much as for ETEC, it is likely that EAEC possess multiple adhesins and toxins that, in various combinations, contribute to disease. EAEC also produce a 108-kDa plasmid-encoded enterotoxin (Pet) that may contribute to disease (Eslava et al., 1998). Purified Pet is capable of causing rises in short-circuit current (Isc) and falls in tissue resistance, characteristic of toxins that cause secretory diarrhea. Some EAEC strains also produce and secrete a chromosomally encoded 110-kDa protein designated protein involved in intestinal colonization (Pic). Pic is presumably involved in mucinase activity, serum resistance, and hemagglutination (Henderson et al., 1999).

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13.11.7 Diffusely Adhering Escherichia coli With the characterization of EAEC, DAEC is now recognized as a separate class of E. coli, although little is known about their virulence mechanism, and their association with diarrheal disease remains controversial. DAEC strains are also considered a heterogeneous group that comprises strains with different pathogenic potential that is due to the presence of variable virulence factors. DAEC have been associated with persistent watery diarrhea without blood or fecal leucocytes in children between the ages of 2 and 5 years, particularly in Mexico, Bangladesh, and Chile (Doyle et al., 1997; Baqui et al., 1992; Levine et al., 1993).

DAEC are identified by a characteristic diffuse-adherent pattern of adherence to HEp-2 or HeLa cell lines. These bacteria cover the cell surface uniformly. DAEC generally do not elaborate heat-labile or heat-stable toxins or elevated levels of Shiga toxins; nor do they possess EPEC adherence factor plasmids or invade epithelial cells (Doyle et al., 1997). 13.11.8 Enterohemorrhagic Escherichia coli (O157:H7) Since the 1980s, EHEC strains have emerged as the cause of a major health problem, particularly in developed countries (Kaper, 1998b; Besser et al., 1999; Paton and Paton, 1998). EHEC were first identified as human pathogens in 1982, when E. coli of serotype O157:H7 was associated with two outbreaks of hemorrhagic colitis. Despite the broad variety of EHEC serotypes found in the gastrointestinal tract, only a limited number (particularly O157, O111, O26, O103, O104) have been associated with the serious clinical manifestations seen during human EHEC infections (CDC, 1995a, 1995b; Karmali, 1989). All EHEC produce factors cytotoxic to African green monkey kidney (Vero) cells, which have been described as verotoxins (VTs) or Shiga-like toxins (SLTs), because VT-1 is similar to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1. Serotype O157:H7 is the predominant cause of EHEC-associated disease in the United States and many other countries. Johnson and associates (1983) first reported the ability of this serotype to produce VTs. It has thus been variously described as verotoxigenic E. coli (VTEC), Shiga-like toxin producing E. coli (SLTEC), and, currently, Shiga-toxin producing E. coli (STEC), reflecting the evolution of the nomenclature of the toxins it produces. E. coli O157:H7 differs metabolically from other strains of E. coli in several ways. Most isolates of this serotype are slow fermenters or nonfermenters of sorbitol and lack the enzyme β-glucuronidase, two important differentiating characteristics for identification of the strain on selective media. This serotype also cannot grow at the high-end of the temperature range of growth (44°C–45°C) typical of E. coli but can tolerate more acidic conditions (pH 2.5). In general, EHEC are acid-resistant, and as in Shigella species, acid resistance accounts for their low infectious dose (10–100 organisms). Virulence Factors The precise mechanism of pathogenicity of E. coli O157:H7 has not been fully elucidated. The disease is currently associated with three major virulence attributes:

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1.

2. 3.

The capacity to cause formation of A/E lesions, mediated by genes encoded within the locus of enterocyte effacement (LEE) The expression of Shiga toxin The presence of a 60-Mda plasmid that encodes a hemolysin

However, other pathogenic serotypes (such as O26, O103, and O111) have a distinctive pattern of virulence factors, with respect to that of E. coli O157:H7 (Schmidt et al., 1999). In addition, there are “atypical EHEC” strains that express Stx but do not produce A/E lesions or possess the plasmid. It is believed that the capacity to attach tightly to enterocytes and elicit the formation of A/E lesions contributes to the nonwatery diarrhea; this process appears necessary for intestinal colonization. Once established, EHEC secrete the Stx, which has systemic effects, causing the bloody diarrhea and hemolytic uremic syndrome (HUS). Locus of Enterocyte Effacement EHEC possesses a genetic LEE that is functionally and structurally similar to the LEE found in EPEC (McDaniel et al., 1995). A 1998 characterization of the EHEC O157:H7 revealed that the molecules that are thought to be on the bacterial surface or interact with host cells (i.e., those that would be exposed to the host immune system), such as Esps, Tir, and intimin, are much more diverse than the others when compared to the EPEC products (Perna et al., 1998). In addition, the EHEC LEE also encodes a cryptic prophage of the P4 family. Like EPEC, EHEC O26:H– sorbitol-fermenting serotype produces a Tir (also called EspE) protein that becomes tyrosine phosphorylated on translocation into host cells, serves as the intimin receptor, and focuses cytoskeletal components beneath the adherence site (Deibel et al., 1998). In contrast, EHEC O157:H7 Tir is not tyrosine phosphorylated and acts as the primary determinant of bacterial adherence to epithelial cells (DeVinney et al., 1999a, 1999b; Ismaili et al., 1995). This Tir binds intimin and focus cytoskeletal rearrangements, suggesting that tyrosine phosphorylation is not needed for pedestal formation. Despite the remarkable heterogeneity found in the amino acid sequence of Tir proteins from different EHEC serotypes (Paton et al., 1998), as well as between the Cterminal domains of different intimins (Frankel et al., 1995), the EHEC and EPEC intimins are functionally interchangeable, although EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin (DeVinney et al., 1999b). In vivo studies have shown that E. coli O157:H7 requires intimin to colonize the intestinal

tract efficiently, to cause diarrhea and A/E lesions in neonatal calves and piglets. The LEE region of EHEC also encodes proteins homologous to EspA, EspB, and EspD secreted through the type III secretion apparatus (Perna et al., 1998). EspA is essential for bacterial attachment and is a part of filamentous appendages that appear during the early stages of the attachment process and are necessary for protein translocation of other effector proteins. Similarly, EspD is required to obtain efficient bacterial attachment to target cells and to establish a direct link between bacteria and eukaryotic cells via EspA-containing surface appendages. Shiga Toxin Also known as Shiga-like toxins (SLTs) or verotoxins (VTs), the Shiga toxins produced by EHEC strains possess high similarity to the cytotoxins produced by S. dysenteriae (O’Brien et al., 1992). The production of Stx constitutes a key element of EHEC pathogenesis and a distinctive characteristic that distinguishes EHEC strains from EPEC. Because of this, EHEC is also called Stx-producing E. coli (STEC) or verotoxigenic E. coli (VTEC). Two subgroups of serologically distinguishable toxins, Stx1 and Stx2, have been recognized in EHEC. Whereas Stx1 is identical to S. dysenteriae Stx, Stx2 is 56% homologous to the other toxins and presents a number of variant forms, such as Stx2c, Stx2d, and Stx2e (Jackson et al., 1987; Gyles et al., 1988; Weinstein et al., 1988; Schmitt et al., 1991). The sequence variability, which is mainly observed in the B subunit, is reflected not only antigenically, but also in receptor binding and toxicity for tissue culture cells or in animal models. Stx1 and Stx2 toxins (except Stx2e) are encoded by bacteriophages (i.e., toxin-converting bacteriophages), which are able to spread stx genes among enteric E. coli strains (Schmidt et al., 1999). Like LT, CT, and several other toxins, Stx is an AB5 toxin, with one 32-kDa catalytic subunit (A) and five 7.7kDa binding subunits (B). The A subunit is nicked into two products (A1 and A2) that are linked by a disulfide bond. Toxin binding to its specific glycolipid receptor, globotriaosylceramide, or GB3, on cell surfaces, occurs by the B subunits. After binding, toxin uptake occurs by endocytosis, followed by retrograde transport to the Golgi apparatus and endoplasmic reticulum. The A subunit enters the cytoplasm, where the A1 peptide acts as a specific N-glycosidase that cleaves an adenine from the 28S ribosomal RNA. This elimination of a single adenine nucleotide inhibits the elongation factor 1– (EF-1)-dependent binding to ribosomes of aminoacyl-bound tRNA molecules. This action blocks protein synthesis by truncating peptide chain elongation, resulting in death of the intoxicated cell (Fig-

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ure 13.15). Intestinal cell death may result in hemorrhagic colitis (bloody diarrhea) due to a breach in the intestinal barrier; the pathogenesis of HUS is characterized by Stxmediated destruction of endothelial cells in venules and arterioles. It has been suggested that Stx2-expressing strains are more likely to be associated with the development of HUS (Pickering et al., 1994). The nomenclature of Stxs and their important biological characteristics are listed in Table 13.38. Role of 60-Megadalton Plasmid (pO157) EHEC also possess a 60-MDa plasmid termed pO157 that is not found in EPEC. The contribution of this plasmid to EHEC adherence or colonization has not been clearly established. Despite the lack of consistent experimental evidence about the role of pO157 in disease, the determination of its full nucleotide sequence confirmed that it encodes several potential virulence factors, including a hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP), and a type II secretion system, as well as a protein containing a putative active site shared with the large clostridial toxin (LCT) (Burland et al., 1998; Makino et al., 1998). The hemolysin encoded within the plasmid is highly conserved among many EHEC strains of different serotypes isolated from outbreaks (Doyle et al., 1997). Although its role in pathogenesis has not been clearly established, it may stimulate bacterial growth in the gut by releasing hemoglobin from red blood cells, thus providing a source of iron (Law and Kelly, 1995). The serine protease EspP is a 104-kDa extracellular protein that shares significant similarity with a group of surface-associated or -secreted bacterial proteins that are also known as autotransporters (Brunder et al., 1997). Although present in EHEC O157:H7 and O26:H– serotypes, it was not detected in a significant number of EHEC isolates of different serotypes (e.g., O157:H–) (Brunder et al., 1999). This serine protease cleaves the coagulation factor V and is cytotoxic for Vero cells. These features have led to the suggestion that it might have a synergistic effect during the development of the hemorrhagic disease (Brunder et al., 1997), a role that is supported by the presence of EspP antibodies in patients with EHEC infections. Symptoms Clinical symptoms of disease appear 3 to 4 days after the ingestion of EHEC-contaminated food or water and transit to the large bowel. The onset of disease is characterized by nonbloody watery diarrhea, abdominal pain, and fever. Vomiting may also occur at this stage. As the disease progresses, abdominal pain increases and bloody diarrhea

Figure 13.15 Structure and mode of action of Stx. The Stx holoenzyme is composed of a single catalytic subunit (A) that is associated with a pentameric ring formed by B subunits. The B subunit binds the toxin to a specific glycolipid receptor, globotriaosylceramide (Gb3). Once bound to the cell membrane, the toxin molecules seem to be internalized by receptor-mediated endocytosis, through clathrincoated pits. Vesicles containing toxin-receptor complexes are transported to the Golgi apparatus and then to the endoplasmic reticulum, before being translocated to the cytosol (retrograde transport). The A subunit is proteolytically nicked during this process, generating the catalytically active A1 amino-terminal portion, which remains linked to the C terminus by a disulfide bond. The A1 subunit has an Nglycosidase activity that depurinates the 28S rRNA of 60S, resulting in inhibition of protein synthesis and cell death. rRNA, ribosomal ribonucleic acid.

commences. In the majority of cases, the bloody diarrhea subsides and symptoms resolve within 5–10 days, with an average duration of about 8 days. However, in 10%–20% of cases (especially in the pediatric and geriatric populations), EHEC infections can lead to the development of serious life-threatening complications such as HUS (hemolytic anemia), thrombotic thrombocytopenic purpurea (TTP) (decreased platelets in the blood), and renal failure (Karmali, 1989; Doyle et al., 1997; Tortorello, 2000). Blood transfusions and kidney dialysis are often necessary for treatment of HUS patients. The disease has a

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fatality rate of 5%; about 25% of patients have permanent kidney damage. Outbreaks EHEC outbreaks have been caused by contamination of various foodstuffs, including beef, radishes, lettuce, sprouts, apple juice, salami, yogurt, and even chlorinated water (Morgan et al., 1993; Samadpour et al., 1994; Besser et al., 1993; Swinbanks, 1996; Keene et al., 1994; Doyle et al., 1997; Tortorello, 2000). Cattle can be asymptomatic

Table 13.38

Nomenclature and Biological Characteristics of Shiga Family Toxins Nomenclaturea

Biological characteristics Proposed

Genetic locus

Cross-neutralized by antiserum to

Receptor

Stx VT1 VT2 VT2c (VT2)

Stx Stx1 Stx2 Stx2c

Chromosome Phage Phage Phage

Stx1 Stx Stx2c, Stx2e Stx2, Stx2e

GB3 GB3 GB3 GB3

Human diarrhea, HC, HUS Human diarrhea, HC, HUS Human diarrhea, HC, HUS Human diarrhea, HC, HUS

VT2e (VT2v, VTe)

Stx2e

Chromosome

Stx2, Stx2c

GB4

Pig edema disease

Shiga-like

Verotoxin

Stx SLT1 SLT2 SLT2c (SLT2 vha, SLT2vhb) SLT2e (SLT2v, SLT2vp)

Diseaseb

a

Previous designations are given in parentheses. HC, hemorrhagic colitis; HUS, hemolytic uremic syndrome. Source: Compiled from Calderwood et al. (1996) and Doyle et al. (1997). b

EHEC carriers, and contamination with beef products or manure can often be traced as the source of EHEC (Bell et al., 1994; Heuvelink et al., 1998; Wieler et al., 1998; Dean-Nystrom et al., 1997). Asymptomatic humans can also be transient vehicles of the pathogen and have been epidemiologically implicated in person-to-person transmission cases. The first documented outbreak of E. coli O157:H7 infection occurred in Oregon in 1982, with 26 cases and 19 persons hospitalized (Riley et al., 1983). All patients had bloody diarrhea and severe abdominal pain. The duration of illness ranged from 2 to 9 days, with a median of 4 days. This outbreak was associated with eating undercooked hamburgers from fast-food restaurants of a specific chain. E. coli O157:H7 was recovered from the stools of patients. A second outbreak followed 3 months later and was associated with the same fast-food restaurant chain in Michigan, with 21 cases and 14 hospitalized cases. Contaminated hamburgers again were implicated as the vehicle, and E. coli O157:H7 was isolated both from patients and a frozen ground beef patty. The largest documented outbreak of EHEC infection in the United States occurred in Washington, Idaho, California, and Nevada in early 1993 (Bell et al., 1994; CDC, 1995a; Griffin et al., 1994). Approximately 90% of primary cases were associated with eating at a single fastfood restaurant chain, from which E. coli O157:H7 was isolated from hamburger patties. Transmission was amplified by a secondary spread (48 patients in Washington state alone) via person-to-person transmission. In total, 731 cases were identified: 629 in Washington, 13 in Idaho, 57 in Las Vegas, Nevada, and 34 in southern California. Of these, 176 were hospitalized, HUS developed in 56, and 4 children died. The outbreak was unrecognized because

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neither specific laboratory testing nor surveillance for E. coli O157:H7 was carried out for earlier cases in these states. Its involvement was recognized only when a sharp increase in cases of HUS was identified and investigated in Washington. Other outbreaks of E. coli O157:H7 in the United States were associated with drinking water in Cabool, Missouri, in 1989–1990; with a swimming water outbreak near Portland, Oregon, in the summer of 1991; with an apple cider outbreak in southeastern Massachusetts in the fall of 1991; and with an unusual outbreak associated with the consumption of dry fermented salami in 1994 in Washington and California (Doyle et al., 1997). A partial listing of outbreaks caused by foods contaminated with E. coli O157:H7 is given in Table 13.39. The list, which only includes outbreaks that affected more than 10 persons, was compiled primarily to illustrate the global nature of the events and the types of foods that have been implicated or confirmed as vehicles of transmission and is not intended to be comprehensive. The largest outbreak involved almost 10,000 people and 11 deaths in Japan in the summer of 1996. The majority of cases occurred in schoolchildren, and the organism was isolated from a vegetable salad with a topping of dried bonito shavings served for school lunch. The most frequent cause of illness has been consumption of undercooked ground beef, although a number of other foods have also been implicated. Numbers of reported outbreaks and clusters and numbers of cases of infection in the United States between 1982 and 1994 are presented in Table 13.40. Of outbreaks and clusters reported in the United States, 83.4% (57 of 68) occurred from May to October. The possible reasons for this are threefold (Doyle et al., 1997):

Table 13.39 Selected Worldwide Food-Borne Illness Outbreaks Caused by Escherichia coli O157:H7 Year

Location

Food vehicle

Persons affected, no.

1982 1982 1984 1985 1986 1986 1987 1987 1988 1990 1990 1990 1990 1991 1991 1991 1993 1993 1993 1993 1993 1993 1993 1994 1994 1995 1995 1995 1996 1996 1996 1996 1997 1997 1997 1997

United States Canada United States Canada Canada United States United States United States United States United States United States Scotland Japan United States United Kingdom United States United States United States United States United States United States United States United States United States United States United States Canada United States United States/Canada Scotland United Kingdom Japan Japan Japan United States United States

Hamburger Hamburger Hamburger Sandwich Raw milk Hamburger Hamburger Turkey Hamburger Conference meal Roast beed Restaurant food Tap water Tap water Yogurt Apple cider Hamburger Ground beef Ground beef Ground beef Cantaloupe Salad bar Mayonnaise Supermarket food Salami Venison jerky Lettuce Lettuce Apple cider Meat pies Precooked meats Meat, sprouts Sprouts Hospital food Sprouts Hamburger

47 31 34 73 46 37 51 26 32 70 65 16 174 243 16 23 629 13 58 32 27 53 47 21 23 11 21 74 66 496 14 9500 96 58 60 16

Source: Compiled from CDC (1995a) and Tortorello (2000).

1.

2.

3.

An increased prevalence of the pathogen in cattle or other livestock or vehicles of transmission during the summer Greater human exposure to ground beef or other E. coli O157:H7–contaminated foods during the “cookout” months Greater improper handling (temperature abuse) or incomplete cooking of products such as ground beef during warm months than any other time of the year

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Control Sanitary food handling practices are essential to prevent fecal contamination, particularly excluding anyone with diarrhea from food preparation. Particular care is needed in handling and cooking ground meat. Meat trimmings from external portions of a carcass more likely to be contaminated are often used in preparing ground meat. Meat should be heated to an internal temperature of at least 63°C because excessive contamination is not unlikely. As with all raw meat products, it is crucial to prevent cross-

Table 13.40 Reported Outbreaks and Clusters of Escherichia coli O157:H7 Infection in the United States, 1982–1994 Year

Outbreaks, no.

Cases, no.

1982 1984 1986 1987 1988 1989 1990 1991 1992 1993 1994 Total

2 2 2 1 3 2 2 4 4 17 30 68

47 70 52 51 153 246 75 54 75 1006 511 2334

Source: Compiled from CDC (1995a) and Doyle et al. (1997).

contamination from cutting boards and equipment. Milk must be pasteurized because raw milk is not sterile and fecal contamination with E. coli O157:H7 from a healthy carrier cow is possible. A variety of food processing technologies have been proposed for the control of E. coli O157:H7 in foods. Heating, e.g., pasteurization, is an effective method of killing the pathogen. Steam-vacuuming and organic acid sprays are capable of reducing populations of the pathogen on the surfaces of animal carcasses. New technologies such as the use of high hydrostatic pressure, pulsed power electricity, ohmic heating, bright light pulsing, and ozonation have also shown promise (Tortorello, 2000). HACCP programs for food production facilities may also be effective in improving food safety.

13.12 BONGKREK TOXINS Bongkrek is a traditional Indonesian food product made by the fermentation of coconut pressed cake with Rhizopus oryzae. Many Indonesians eat the food with no apparent harm. In 1932, a bacterium was isolated from this fermented food implicated in an outbreak of food poisoning. The organism was classified as Bacillus cocovenenans (cocos meaning “coconut,” and veneno meaning “to poison”). The organism is a pleomorphic gram-negative bacterium that is usually rod-shaped, but also appears as coccoid, vibrioid, or filamentous, depending on the cultural conditions (Concon, 1988; Cox et al., 1997). It is motile by one to four polar flagella, is catalase-positive, and exhibits

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a very weak oxidase activity. It grows at 30°C, but not at 4°C, 10°C, or 45°C. As the organism conforms to the general description of the family Pseudomonadaceae, it was relocated from the genus Bacillus to the genus Pseudomonas. Subdivision of Pseudomonas into groups and ultimately into several genera on the basis of rRNA analysis created the genus Burkholderia. Early biochemical test data suggested that species B. cocovenenans should remain in the genus Pseudomonas. However, comprehensive biochemical and nucleic acid analysis of P. cocovenenans in 1995 led to reclassification of the species to the new genus (Cox et al., 2000). The organism produces two toxins: bongkrek acid and toxoflavin (Figure 13.16). Bongkrek acid is the major toxin. It is a substituted glutaconic acid derivative of aconitic acid with a molecular weight of 486 (Figure 13.16A). In crude form, and in oil or solvent solutions, bongkrek acid is very heat-stable. The production of bongkrek acid appears to be specific to coconut pressed cake, since the same bacterium does not produce the toxin in soy meal and peanut pressed cake (van Veen, 1967). It is produced only in the presence of fatty acids. The simple lowering of pH during fermentation to below pH 5.5 can prevent toxin production. Below this pH, the toxigenic bacteria do not grow; however, the growth of the mold is in fact stimulated at these lower pH values. Flavotoxin A is the major toxin produced by B. cocovenenans bv. farinofermentans. The compound has not been chemically defined. However, similarities in mass spectra and absorption maxima between this compound and bongkrek acid suggest the compounds are related (Cox et al., 1997). If bongkrek acid is hydrolytically cleaved at the points indicated by dashed lines in Figure 13.16A, compounds with a chemical composition similar to that reported for flavotoxin A are generated. B. cocovenenans and biovar. B. farinofermentans produce a second toxin, known as toxoflavin (Figure 13.16B) because of its physicochemical resemblance (yellow color, green fluorescence, stability against oxidation, absorption spectrum) to riboflavin (Daves et al., 1962). Bongkrek acid is the more toxic of the two (Van Veen, 1950, 1973). The main symptoms of poisoning include an initial hyperglycemia quickly followed by a marked hypoglycemia, which exhausts the glycogen reserves in many tissues, particularly the liver and heart. The acid interferes with mitochondrial oxidative phosphorylation, as well as with the citric acid cycle in heart muscle (Welling et al., 1960). It reacts with ADP and ATP in the mitochondria and inhibits the enzyme adenine nucle-

A. Bongkrek Acid H CH3 H HOOC

C C

H

C

C

C H

H

H2 C

H C

C C

C

C H2

C

C H2

H

H

C

OCH3

C C H2 H

H

COOH C

C CH3

C

C CH3

H

H

H CH2

COOH

CH3 N

O

N N

B. Toxoflavin N H 3C

N O

Figure 13.16 Structures of (A) bongkrek acid and (B) toxoflavin. Hydrolytic cleavage of bongkrek acid, indicated by dashed lines in A, yields flavotoxin.

otide translocase (Henderson and Lardy, 1970; Henderson et al., 1970). Toxoflavin functions under aerobic conditions as an active electron carrier between reduced nicotinamide-adenine dinucleotide (NADH) and oxygen, leading to production of hydrogen peroxide and bypass of the cytochrome system. These characteristics, respectively, probably confer the strong antibiotic and poisoning properties associated with the toxin (Latuasan and Berends, 1961; Cox et al., 1997). It is inactive under anaerobic conditions. Although toxoflavin is lethal in small doses (5 to 10 µg) when administered intravenously to rats, little morbidity or mortality is observed in rats or monkeys when it is given orally, suggesting this toxin plays only a minor role, if any, in the symptoms observed during bongkrek food poisoning. The symptoms of bongkrek poisoning appear 4–6 hours after ingestion of contaminated food. Victims experience a range of symptoms, including malaise, abdominal pains, dizziness, extensive sweating, and extreme tiredness, before lapsing into coma. Death usually occurs 1–20 hours after the onset of the initial symptoms. After death, there is little evidence of cause, as no histopathological changes can be demonstrated on autopsy, no bacterial growth can be obtained from various organs, and laboratory animals do not succumb when fed organ tissue (Cox et al., 2000).

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Although there are no precise figures, the mortality rate is very high compared to that of many food-borne illnesses. About 1 mg of bongkrek acid is required to cause death. About 1–3 mg of bongkrek acid/g food can be produced within 48 hours when high numbers of the causative organism develop, and only a few grams of contaminated food, even after cooking, is sufficient to kill humans. The first deaths were reported in 1895, and since 1951, consumption of contaminated bongkrek has resulted in approximately 10,000 cases of intoxication, including at least 1000 deaths (Cox et al., 1997, 2000). Preventative measures include the use of a sufficiently high quantity of active fungal inoculum; addition of up to 1.5% NaCl or acidification of the substrate to pH 4.5 serves to suppress the synthesis of bongkrek acid during the fermentation process. Crude extracts of various spices, such as garlic, onion, capsicum, and turmeric, may also inhibit toxin production. However, efficacy is inversely proportional to the population of the pathogen. In order to curb the morbidity and mortality associated with the consumption of contaminated product, the Indonesian government banned the production of bongkrek in 1988. Such a legislative approach, however, is unlikely to prove effective in the long run, as consumers see such products as a vital source of nutrition in their daily diet.

13.13 CONCLUSIONS Food spoilage and food poisoning caused by microorganisms were problems that must have beset humans since antiquity. Judging by the earliest documented historical accounts, humans have recognized that diseases could be spread by foods. For example, prohibitions on eating pork in the Jewish and Muslim religions had their origins in medical doctrine. Although most of these edicts carried the weight of religious sanction, they were fundamentally laws of simple hygiene and reflected similar practices derived in several countries. However, it was not until the mid- to late 19th century with the classic works of Louis Pasteur and Robert Koch that food microbiology came of age. Just around the dawn of the 20th century, bacterial toxins were recognized as the potent substances responsible for such infectious diseases as diphtheria, tetanus, and botulism. Since the discovery of these first three toxins, many more have been identified, and the process continues even to this day. The dynamic nature of this field is quite evident, since in just the past 20 years or so, four relatively unknown bacteria (Campylobacter jejuni [previously known as Vibrio fetus], E. coli O157:H7, Vibrio vulnificus, and Listeria monocytogenes) have become associated with widespread food-borne illnesses. Most food-borne bacterial intoxications described here can be attributed to the production of exotoxins, frequently called enterotoxins because of their association with diarrheal diseases. Nausea, vomiting, abdominal pain, diarrhea, and fever are often the major clinical findings in gastrointestinal infections. The predominant symptoms are dependent upon the etiological agent and whether it is toxigenic or invasive or both. When preformed toxins are in food, they often are associated with nausea and vomiting. For example, S. aureus and B. cereus produce enterotoxins in food; nausea and vomiting, and to a much lesser extent diarrhea, occur a few hours after ingestion of the contaminated food. Organisms that produce enterotoxins affect the proximal small bowel and tend to cause watery diarrhea (e.g., enterotoxigenic E. coli, V. cholerae). Maintaining adequate hydration and electrolyte balance is the most important feature of the treatment in most bacterial intoxications, especially in infants and children. The agents that commonly cause toxin-induced gastroenteritis and gastrointestinal infections are listed in Table 13.41. It should be noted that the table includes many more causative agents for toxin-induced diarrheal diseases than are discussed in this chapter, primarily because the aim here is to indicate the most important and common food-borne illnesses occurring worldwide. A few causative organisms were described earlier in Chapter 12.

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Gastrointestinal infections are very common, especially in developing countries, where the associated mortality rate is high in infants and young children. Public health prevention through fostering good hygiene and providing sanitary water and food supplies is of the utmost importance. Even with good sanitary practices and a welldeveloped food processing industry, it is not uncommon in the developed countries for occasional and sporadic outbreaks of food-borne bacterial intoxications to occur. Also, with the exception of few illnesses, such as those due to botulinum toxins and E. coli O157:H7, smaller outbreaks, especially those at individual household levels, often are underreported to the health authorities. In fact, many of these intoxications cause billions of dollars of losses annually in the United States alone because of the accompanying morbidity of such illnesses. The ability of a particular causative organism to produce toxin is influenced by a variety of factors, including the strain of organism, culture medium, and physiological conditions of culture. Great variation in the capacity to produce toxin can be found among different toxigenic strains of the same organism, even when grown under similar conditions. In the food environment, the toxin production is influenced by the nature of the food itself (e.g., pH or acidity of the food, salt content), its processing, and subsequent storage and handling. The number of food-borne intoxications due to toxin formation in foods by a variety of bacteria described in this chapter illustrates the complexity of this subject. Even with the great advances made in food science and technology, these intoxications remain a problem worldwide. The increasing globalization of the food supply and the ease of travel are potentially leading to an even greater risk of these intoxications being spread outside their normal endemic geographical boundaries. Most intoxications can easily be prevented by strictly following good hygienic practices, especially during food handling and preparation; preventing temperature abuse after the food is cooked; avoiding consumption of raw seafood; and educating both food processers and consumers about the importance of appropriate storage conditions for foods at all points in the chain of processing, distribution, and consumption. The use of HACCP during food manufacture and processing will go a long way in improving the safety of our food supply. The efficacy of a HACCP system, however, depends on the rigor and consistency with which it is designed and implemented and the use of (a) critical control point(s) that will control pathogens. It should be noted that most consumers live in urban areas, and the ingredients they obtain for the foods they prepare at home are produced elsewhere, whereas rural and suburban residents may produce some of their own

Table 13.41

Microorganisms That Commonly Cause Toxin-Induced Gastroenteritis and Gastrointestinal Infections in Humans

Organism

Typical incubation period

Signs and symptoms

Epidemiological characteristics

Pathogenesisa

Staphylococcus aureus

1–8 hr (Rarely up to 18)

Nausea and vomiting

Grow in meats, dairy, and other foods and produce enterotoxin

Enterotoxin action on receptors in the gut that transmit impulse to medullary centers that control vomiting

Bacillus cereus

2–16 hr

Vomiting or diarrhea

Reheated fried rice common vehicle

Enterotoxin formed in food or in gut from growth of B. cereus

Clostridium perfringens

8–16 hr

Watery diarrhea

Grow in rewarmed meat dishes; huge numbers ingested

Enterotoxin produced during sporulation in gut; cause of hypersecretion

Clostridium botulinum

18–24 hr

Paralysis

Grows in anaerobic food and produces toxin

Toxin absorbed from gut; blocks acetylcholine at neuromuscular junction

Escherichia coli (enterotoxigenic, ETEC)

24–72 hr

Watery diarrhea

Most common cause of “traveler’s diarrhea”

Escherichia coli (enteroinvasive, EIEC)

48–72 hr

Dysentery

Escherichia coli (enterohemorrhagic, EHEC)

24–72 hr

Watery, bloody diarrhea

Occasional outbreaks of dysentery; infrequent cause of sporadic infection Bloody diarrhea associated with undercooked meats

ETEC in gut produces heat-labile (LT) or heat-stable (ST) enterotoxins; toxinsb cause of hypersecretion in small intestine Inflammatory invasion of colonic mucosa, similar to shigellosis; EIECs closely related to Shigella spp. EHEC production of Shiga-like toxin (vero toxin, Stx); often serotype O157:H7

Escherichia coli (enteropathogenic, EPEC)

Slow in onset

Watery diarrhea

Common cause of diarrhea in neonates in developing countries; classically, cause of epidemic diarrhea in newborn nurseries with high

EPEC attachment to mucosal epithelial cells; produces cytoskeletal changes; may invade cells; different from other E. coli that are diffusely enteroadherent (DAEC) or enteroaggregative (EAEC) and cause diarrhea

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Clinical featuresa Very common, abrupt onset, intense vomiting for up to 24 hr, regular recovery in 24–48 hr; occurs in persons eating the same food; no treatment usually necessary except to restore fluids and electrolytes With incubation period of 2–8 hr, mainly vomiting; with incubation period of 8–16 hr, mainly diarrhea Abrupt onset of profuse diarrhea; vomiting occasionally; recovery usual without treatment in 1–4 days; many clostridia in cultures of food and feces of patients Diploplia, dysphagia, dysphonia, difficult breathing; treatment requirement for ventilatory support and antitoxin; diagnosis confirmed by finding toxin in blood or stool Usually abrupt onset of diarrhea; vomiting rare; serious infection in newborns; adults, usually self-limiting in 1–3 days Acute bloody diarrhea with malaise, headache, high fever, abdominal pain; severe disease in poorly nourished children; WBC present in stool Causes bloody diarrhea, hemorrhagic colitis, and most cases of hemolyticuremic syndrome; culture stool for sorbitol-negative E. coli and serotype isolates with antisera for O157:H7 Insidious onset over 3–6 days, poor feeding, and diarrhea; usually lasts 5–15 days; dehydration, electrolytic imbalance, and other complications that may cause death; antimicrobial therapy important

mortality rates; less common now in developed countries Organisms grow in seafood and in gut and produce toxin, or invade

Vibrio parahaemolyticus

6–96 hr

Watery diarrhea

Toxin cause of hypersecretion; vibrio invasion of epithelium; may be bloody stools

Vibrio cholerae

24–72 hr

Watery diarrhea

Organisms grow in gut and produce toxin

Toxinb cause of hypersecretion in small intestine; infective dose >105 organisms

Shigella spp. (mild cases)

24–72 hr

Dysentery

Organisms grow in superficial gut epithelium

Organism invasion of epithelial cells; blood, mucus, and PMNs in stools; infective dose <103 organisms

Shigella dysenteriae type 1 (Shiga’s bacillus)

24–72 hr

Causes outbreaks in developing countries

Production of cytotoxin and neurotoxin

Salmonella spp.

8–48 hr

Dysentery, bloody diarrhea Dysentery

Organism growth in gut; do not produce toxin

Superficial infection of gut, little invasion; infective dose >105 organisms

Clostridium difficile

Dysentery

Antibiotic-associated pseudomembranous colitis

Makes enterotoxin and cytotoxin, which cause diarrhea and epithelial cell necrosis

Campylobacter jejuni

Days to weeks after antibiotic therapy 2–10 days

Dysentery

Infection via oral route from food, pets; organisms growth in small intestine

Invasion of mucous membrane; toxin production uncertain

Rotavirus

48–96 hr

Watery diarrhea

Virus major cause of diarrheal disease in infants and young children worldwide

Induces histopathological changes in intestinal mucosal cells

Abrupt onset of diarrhea in groups consuming same food, especially crabs, other seafood; recovery usually complete in 1–3 days; food and stool culture results positive Abrupt onset of liquid diarrhea in endemic area; prompt IV or oral replacement of fluids and electrolytes; stool culture results positive Abrupt onset of diarrhea; can be blood and pus in stools, cramps, tenesmus, and lethargy; WBC in stool; stool culture results positive; often mild and self-limiting; fluid restoration required Severe bloody diarrhea in children in developing countries; high fatality rate; rare in United States Gradual or abrupt onset of diarrhea and low-grade fever; WBC in stool; stool culture result positive; no antimicrobials unless systemic dissemination suspected; prolonged carriage frequent Abrupt onset of bloody diarrhea and fever; toxin in stool; patients typically received antibiotics in previous days to weeks Fever, diarrhea; PMNs, and fresh blood in stool, especially in children; usually self-limited; special media needed for culture at 42°C; usually recovery in 5–8 days Fever and vomiting usually before abdominal distress and diarrhea; death in infants in developing countries after dehydration and electrolyte imbalance; typical course 3–9 days; diagnosis by immunoassay detection of rotavirus antigen in stool (table continues)

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Table 13.41

(continued)

Organism

Typical incubation period

Giardia lamblia

Signs and symptoms

Epidemiological characteristics

1–2 weeks

Watery diarrhea

Entamoeba histolytica

Gradual onset 1–3 weeks

Salmonella typhi (S. paratyphi A and B; S. choleraesuis)

Yersinia enterocolitica

a

Pathogenesisa

Clinical featuresa

Most commonly identified intestinal parasite; frequent pathogen in outbreaks of water borne diarrhea

Complex and poorly understood interaction of parasite with mucosal cells and patient’s immune response

Dysentery

Highest prevalence in developing countries; may infect 10% of world’s population

Invades colonic mucosa and lyses cells, including leukocytes

10–14 days

Enteric fever

Humans only S. typhi reservoir

Invade intestinal mucosa and multiply in macrophages in intestinal lymph glands to blood and dissemination

4–7 days

Enteric fever

Fecal-oral transmission; food-borne; animals infected

Gastroenteritis or mesenteric adenitis; occasional bacteremia; toxin produced occasionally

Diarrhea self-limited in 1–3 weeks; chronic symptoms of intermittent diarrhea, malabsorption, and weight loss that may last 6 months; diagnosis by finding trophozoites or cysts in stool or duodenal contents, or by immunoassay detection of Giardia spp. antigen in stool Diarrhea, abdominal pain, weight loss, and fever common; can cause many complications, including fulminant colitis, perforation, and liver abscess; diagnosis by finding trophozoites or cysts in stool Insidious onset of malaise, anorexia, myalgias, and headache; high remittent fever; may be constipation or diarrhea; hepatosplenomegaly in about 50% of patients; diagnosis by culture of S. typhi from blood, stool, or other site; antibiotic therapy important. Severe abdominal pain, diarrhea, fever; PMNs and blood in stool; polyarthritis, erythema nodosum, especially in children; stool specimen kept at 4°C before culture

WBC, white blood cell; PMN, polymorphonuclear leukocyte; IV, intravenous. Cholera toxin and E. coli heat-labile (LT) toxin stimulate adenylyl cyclase activity, increasing cyclic adenosine monophosphate (cAMP) concentration in gut, yielding secretion of chloride and water and reduced reabsorption of sodium. E. coli heat-stable toxin (ST) activates intestinal guanylyl cyclase and results in hypersecretion.

b

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food. The consumers need to be able to handle (i.e., store and prepare) food safely in the kitchen. Those who prepare food at home for persons with lowered resistance to disease must take special care to avoid food-borne hazards. Similarly, people who produce their own eggs, meat, or milk should be prepared to take a safe and sanitary product from the field into the kitchen. On the basis of epidemiological evidence, some foods of animal origin are considered to have a high potential to be vehicles of food-borne pathogens. The reasons for this evaluation are as follows: 1. 2. 3. 4.

High susceptibility to microbial contamination Considerable opportunity for survival of the contaminants High likelihood that growth of the contaminants will occur at some point before serving Reasonable likelihood that the food may be subjected to mistreatment just before serving

Examples of foods processed for safety include pasteurized milk and irradiated poultry. Foods purchased cold should be selected last and promptly taken home to the refrigerator. Fresh meat and poultry should not be stored refrigerated for more than 7 days (1–2 days for raw ground meat), whereas eggs may be kept as long as 3 weeks. All parts of cooked food must reach at least 70°C (184°F). Frozen food should be thawed before cooking in a microwave (and then cooked immediately) or in a refrigerator, but never at room temperature. Do not hold food at room temperature for more than 2 hours. If food must be cooked earlier or leftovers are to be stored, they should be kept either cold or hot (<4°C to >60°C). Foods for infants or others with lowered resistance should never be stored. In warm climates where refrigeration may be limited or nonexistent, many people customarily prepare no more than enough food to be eaten during the meal, thereby avoiding the storage problems altogether. Thoroughly reheat food to at least 70°C. Home-canned foods (especially meats and low-acid vegetables) should be heated before tasting since botulinum toxin in the juice is tasteless and potentially lethal. Cross-contamination between raw foods containing pathogens and cooked foods can reintroduce pathogens into cooked foods. Raw foods of animal origin, e.g., fish, meat, or poultry, can contaminate cutting boards and other surfaces where food is handled. These surfaces should be washed thoroughly after contact with raw product before cooked products are placed on them. Washing hands and utensils, as well as surfaces, with clean cloths or towels after each contact with raw food is also important. Cooked foods should be stored in clean, sealed containers to protect them from contamination. Raw fish, meats, or poultry

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should be stored on a plate or in a container that will prevent contamination of other foods with their juices. Water is an essential component of food preparation and is often overlooked as a source of food-borne pathogens. Ice made from contaminated water has been the culprit on occasion. In rural areas, fecal contamination of well water is not uncommon. If in doubt, to make it safe for drinking and food preparation, raise water to a rolling boil for at least 1 minute to kill vegetative enteric pathogens. The health of the person preparing the food is also critical to the success of safe food handling. Good habits such as regular hand washing are essential. Individuals who have diarrhea, who have pustules on their hands, or who are coughing should not be preparing food. The following rules go a long way in preventing incidences of food-borne poisoning and infections: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Choose foods processed for safety. Cook food thoroughly. Eat cooked foods immediately. Stored cooked foods carefully. Reheat cooked foods thoroughly. Prevent contact between raw foods and cooked foods. Wash hands repeatedly. Keep all kitchen surfaces meticulously clean. Protect foods from insects, rodents, and other animals. Use pure water.

Infectious diseases thus continue to be the leading cause of morbidity and mortality worldwide. Some of these infections and intoxications are caused by eukaryotic parasites; bacterial pathogens, however, continue to present a threat to the well-being of humans and animals in both the developing and developed worlds. The use of vaccines and antibiotics, together with changes in sanitary practices, contributed to an important increase in the life span of humans in the 20th century. However, these significant improvements are now challenged by the appearance of microbes resistant to multiple antibiotics, the emergence of new bacterial pathogens as a result of horizontal and vertical gene transfer, and the use of health care treatments that, while prolonging life, render individuals susceptible to opportunistic pathogens. Novel strategies are currently being tested to prevent and/or treat bacterial infections and intoxications. An increasing number of these strategies are based on our understanding of the mechanisms by which pathogenic microorganisms cause disease. This increase is possibly due to exciting developments in the field of bacterial pathogenesis, which started in the 1980s with the use of molecular genetics to investigate the microorganisms re-

sponsible for causing disease and is now complemented with cell biological and biochemical approaches aimed at unraveling the consequences of infection by such microorganisms for their hosts. We now have a basic understanding not only of the varied nature of virulence determinants but also of their origin and acquisition by pathogenic microbes. We appreciate that expression of virulence determinants is most often regulated in response to host signals and that microbes use different devices to deliver toxic products to host cells. These studies have revealed a set of principles that govern bacterial pathogenesis. Novel aspects of pathogenic microorganisms will continue to be discovered in the years ahead; a basic understanding of the principles governing bacterial pathogenesis not only allows us to appreciate the sophisticated mechanisms used by microbes in their pathogenic life cycle but also is essential to beginning to understand the plethora of information emerging from genomics and to developing new rational approaches to the treatment and prevention of this age-old problem facing humankind. Finally, it is appropriate to conclude the information presented in this chapter and in Chapter 12 with the findings of the task force created by the Council for Agricultural Science and Technology (CAST). The task force was created in 1989 to determine the state of knowledge about U.S. food-borne disease risks, and their report was made available in 1994. They acknowledged that zero risk of food-borne illness is neither possible nor practical and offered the following recommendations for reducing foodborne illness: 1.

2.

3.

4.

5.

6.

The food safety policy should be based on risk assessment using all available data for acute and chronic food-borne diseases. The food safety information database should be expanded to provide more complete information on the incidence of food-borne disease by pathogen and by food. Vigorous fundamental and applied research efforts related to food safety should be encouraged and supported. New rapid, reliable, sensitive, and economical methods continue to be developed to allow fast and accurate detection of hazardous organisms and their toxins. Rigorous epidemiological studies should be conducted to assist in establishing the cause of illness and effect of the occurrence of a particular pathogen or toxin. Although both dose-response and minimal infective or intoxicating dose are difficult types

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7.

8.

9.

10.

11.

12.

13.

14.

15.

of data to accumulate, these data and doses need to be determined or estimated. The estimates of numbers of acute illnesses, chronic illnesses, and deaths; costs of foodborne diseases; severity of illnesses; and duration of chronic illnesses should be improved. Research should be conducted on the mechanisms of chronic illnesses with which foodborne pathogens are associated, so that appropriately targeted detection and control strategies can be developed and implemented. Research should be conducted to identify foods likely to be associated with specific pathogens or toxins and to establish risk minimization controls. Whether new processing methods create an environmental niche for pathogens should be determined. Populations at high risk for opportunistic pathogens that cause acute or chronic illnesses need to be identified and special control programs tailored to inform these populations of their high-risk status so that they can protect themselves. The consumers should be allowed choices in the types of food available to them yet be made aware of their relative risk status, including their risks of acute as well as chronic illnesses. The federal food safety regulations should be modified to reflect that zero risk of food-borne illness is not possible. The food safety goals and priorities should be set so that resources may be allocated and targeted appropriately. Control practices should be applied from food source to consumption, including the incorporation of HACCP principles. New scientific advances should be incorporated into such control practices. The public should be well educated regarding safe food handling and the relative and changing risk status of individuals.

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14 Seafood Toxins and Poisoning

14.1 INTRODUCTION In many areas of the world, intoxication by marine biotoxins or phycotoxins (“fish poisoning”) is a major public health problem affecting some thousands of people each year. Poisonings caused by the ingestion of toxin-containing seafood, in fact, have been a part of human existence and concern for centuries. In years past, poisoning through eating seafoods such as shellfish was considered a public health problem only in local seacoast areas where people consumed considerable amounts of these products. In recent years, reports of food poisoning of marine origin are increasing in frequency of outbreaks. Part of this increase is undoubtedly due to increased consumption of foods of marine origin, although heightened awareness, the result of greater travel to areas of the world where marine toxicity is endemic, and thus a greater opportunity for exposure to oral fish intoxicants, have also contributed. Furthermore, modern methods of freezing and shipping seafood globally have broadened the scope of this problem and have necessitated controls on shipments to make certain that harmful amounts of poisons are not present. There also has been an increase in the importation of toxic marine food products into North America, Europe, Russia, Taiwan, and Japan (Halstead, 1994a). As worldwide distribution of seafood products becomes even more common, the number of cases of seafood poisoning in inland areas will naturally increase, unless proper precautions are taken. However, travel and awareness are only two epidemiological facets of the marine food biotoxication problem. Environmental pollution may be an added factor that also needs to be taken into consideration.

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Many of the phycotoxins involved in cases of food poisoning are toxic components produced by unicellular microalgae. These algae are usually photosynthetic and may be planktonic (free floating or swimming) or benthic, i.e., live on surfaces of, for example, plants and corals, or in or on marine sediments. Seafood poisoning is often associated with the occurrence of algal “blooms,” in which the microscopic algae may reach sufficient density to produce a visible discoloration of the water. These blooms, not necessarily toxic, are a natural phenomenon and occur when there is a particular combination of physical and chemical conditions that allows rapid growth of the organisms (Boney, 1989). Blooms of harmful algae are often confusingly referred to as “red tides,” although the color of the water may actually be red, brown, or green (Anderson, 1994). The incidence of harmful algal blooms appears to be increasing all over the world (Hallegraeff, 1995), but it remains to be established whether this apparent increase is due to increased vigilance by the many countries that have monitoring programs, is a genuine phenomenon caused by as yet unknown factors, or is a combination of both. Phycotoxins enter fishery products through food chains in the aquatic environment. They are difficult to control from the standpoint of public health safety because of the unpredictable and sporadic occurrence of the organisms producing them. Also, they may cause any one of a number of species of fish consuming the organism to become poisonous. Furthermore, most phycotoxins are usually quite stable to heat processing or cooking and are refractory to the action of the digestive enzymes in humans. Phycotoxins generally demonstrate various toxic manifestations, such as paralytic, diarrheic, amnesic, or

neurotoxic effects. Human intoxications caused by phycotoxins occur worldwide through consumption of marine fishery products in which the toxins have accumulated. Toxins of freshwater algae have induced intoxications in farm animals. However, human intoxications related to freshwater toxins have not been identified so far (van Egmond and Speijers, 1999). Because toxin production and accumulation occur only under certain conditions, marine fishery products become incidentally contaminated with relatively high concentrations. Information about the occurrence of phycotoxins in fishery products and about human exposure to these toxins is very limited because of the limited availability of reliable methods of analysis and reference standards of phycotoxins. In contrast, some species of fish, for instance, the puffers, are intrinsically poisonous. Thus these poisons are more controllable in commercial fisheries, because they can be identified with the species of marine animal. Some other important problems are the bacteria that are particularly adapted to contamination and the production of toxins in seafoods. However, these toxins are usually proteins and thus are readily destroyed by the heat but not sufficiently destroyed by digestive enzymes to prevent poisoning in a person consuming them. Venomous marine animals generally do not cause food poisoning problems. Venoms are localized in special organs where they do not contaminate the edible portions of marine animals and, because they are proteinaceous, are readily destroyed by heat and by the digestive enzymes. In this chapter, current information on some of poisonous marine organisms used for food purposes is summarized. The more important and well-studied phycotoxins, including various types of shellfish and ciguatera fish toxins, are described in greater detail in a separate section.

14.2 TOXIC MARINE ORGANISMS 14.2.1

Dinoflagellates

The single-celled dinoflagellates belong to the phylum Protista (or Protozoa). Along with other phytoplankton, dinoflagellates are important as producers of the primary food supply of the sea. They exhibit both animal and plant characteristics, i.e., motility and presence of chlorophyll. Hence, they sometimes are referred to as plant-animals. The nutrition of these organisms is holophytic, sprozoic, holozoic, or mixotrophic. Since the fundamental function of nutrition in dinoflagellates overlaps that of both animals and plants, zoologists designate these organisms as protozoa and botanists classify them as unicellular algae.

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Dinoflagellates abound in neritic waters and in the high seas, ranging from tropics to polar oceans. During their periodic maxima, they may cause yellow, brown, green, black, red, or milky local discolorations of the sea. The “blooming” of these toxic plankton in excessive numbers frequently causes mass mortality of the fish and other animals living in the region. Such blooms are often associated with weather disturbances or weather alterations that may bring about changes in water masses or upwellings. Conditions most favorable for the growth of dinoflagellates are found more often in coastal waters than far offshore areas. Dinoflagellate blooms may cause a serious economic loss to a region because of their toxicity and the mass deaths of fish (Halstead, 1994a). Many species of shellfish become poisonous through the consumption of toxic dinoflagellates. Thus far, over 20 structurally related toxins produced mainly by dinoflagellates have been identified and implicated in paralytic shellfish poisoning. Various dinoflagellates that are known to be poisonous and their observed locations are summarized in Table 14.1. The two main species that produce the paralytic poisons are Gonyaulax (Protogonyaulax) catenella and G. tamarensis. The former occurs most commonly along the north Pacific coast of North America from central California northward to Oregon, Washington, British Columbia, Alaska, and on to Japan. The latter occurs along the east coast of the New England states, the Maritime Provinces of Canada, and the coasts of countries bordering the North Sea. When conditions such as temperature, pH, salinity, and food requirements become favorable for the growth of these particular organisms, they multiply and produce blooms containing anywhere from a few hundred cells (40 to 50 µm in diameter) to several thousand per milliliter. Blooms that contain 20,000 or more cells per milliliter produce what is called a red tide. Another dinoflagellate, G. acatenella, found in some areas along the coast of British Columbia produces several poisons. These dinoflagellates bloom to a maximum within 10 to 15 days, remain for 1 or 2 weeks, and gradually disappear as other organisms bloom in their place. Toxic dinoflagellates play a major role as transvectors of poisons that are ingested by a variety of mollusks, causing paralytic, brevitoxic (neurotoxins), and diarrhetic shellfish poisoning. The dinoflagellate Gambierdiscus toxicus serves as a transvector of the ciguatoxin complex in ciguatera fish poisoning. In addition, several other species of dinoflagellates also are suspected causative agents in ciguatera fish poisoning. They include Amphidinium carterae Hulbert, Ostreopsis ovata Fukuyo, Prorocentrum concavum Fukuyo, P. lima Ehrenberg, and P. mexicanum Tafall (Steidinger and Baden, 1984).

Table 14.1 Geographical Distribution of Various Marine Dinoflagellates Occurrence

Species Gonyaulax (= Protogonyaulax) (= Alexandrium) G. catenella G. tamarensis ( = G. excavata) (= G. tamarensis, var. excavata) G. acatenella Pyrodinium bahamense var. compressa Pyrimidium phoenus Aphanizomenon flos-aquae Jania spp. Gymnodinium breve Exuviaella mariae lebouriae Gonyaulax polyedraa Gonyaulax monilataa Gymnodium veneficuma

Worldwide

North Pacific coasts from central California to Japan North American Atlantic coast and North Sea

North America, West Coast South Pacific North Sea Fresh water Tropical, subtropical red algae Gulf Coast of the United States Japan California coast Gulf Coast of the United States English Channel

a Not involved in known cases of shellfish or fish poisoning in humans. Source: Compiled from Schantz (1973) and Shimizu (1988).

Pelecypods, a class of mollusks consisting of bivalves that includes clams, oysters, scallops, and mussels, reported as transvectors of paralytic dinoflagellate poisons are listed in Table 14.2. Shellfish feed on these dinoflagellates as other organisms do but store the poison in the body organs such as the hepatopancreas in the case of mussels and some clams and in the siphon in the case of the Alaska butter clam. Poison stored in the hepatopancreas is usually excreted or destroyed within a week or two after the bloom of poisonous dinoflagellates has disappeared, but that stored in the siphon is usually retained for many months after the dinoflagellate has disappeared (Schantz, 1973). Mussels and soft shell clams are usually safe to eat again a week or two after the poisonous bloom has receded. 14.2.2

Poisonous Cnidarians (Coelenterata)

Cnidarians, or coelenterates, are simple metazoans having primary radial, biradial, or radiobilateral symmetry. The phylum Cnidaria (Coelenterata) includes three classes: the Hydrozoa, hydroids; Scyphozoa, jellyfish; and Anthozoa, sea anemones, corals, and alcyonarians. Apparently, hydroids are not used as food. Jellyfish commonly are eaten in Japan and elsewhere without any reported cases of poisonings. The nematocysts of hydroids and jellyfish contain proteinaceous toxins that are inactivated by heating and gastric juices. However, oral biotoxications have resulted from the ingestion of sea anemones in the Philippines,

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New Guinea, and Samoa (Halstead, 1994a). The nature of the poison is unknown. Rhodactis howesi (Matalelei) and Physobrachia douglasi (Lumane) generally are considered to be poisonous when raw, but safe to eat when cooked. Radianthus paumotensis (Matamala Samasama) and some of the other members of this genus are considered to be poisonous whether eaten raw or cooked. Small children are frequent victims of sea anemone poisoning in the IndoPacific region. The initial symptoms of jellyfish poisoning consist of acute gastritis with nausea, vomiting, abdominal pain, cyanosis, and prostration. Shortly after ingestion of the sea anemones, the victim may become comatose and may remain so for a period of 36 hours or more. During this period, the superficial reflexes may be absent. The blood pressure and pulse remain normal. Pulmonary edema has been reported (Halstead, 1994a). The patient may go into profound shock, and death usually ensues. There are no specific antidotes for sea anemone poisoning. 14.2.3

Poisonous Echinoderms (Sea Cucumbers, Sea Urchins)

Echinoderms include the starfishes, sea urchins, and sea cucumbers, all members of the phylum Echinodermata. Some species of starfish have been reported to be toxic, but little is known about the nature of the poisons or their effects on humans. Poisonings have resulted from eating

Table 14.2 Pelecypods Acting as Transvectors of Paralytic Dinoflagellate Poisons Common name

Species Arca noae Cardium edule Clinocardium nuttalli

Noah’s ark Heart cockle Basket cockle

Donax denticulatus Donax serra Spisula solidissima Schizothaerus nuttalli

Donax Wedgeshell, bean clam, white mussel Atlantic surf clam Gaper, horse clam

Mya aremaria

Softshell clam

Mytilus californianus Mytilus edulis Mytilus edulis pellucidus Mytilus edulis striatus Mytilus galloprovincialis Mytilus planulatus Modiolus areolatus Modiolus demissus Modiolus modiolus

California mussels Bay mussel Bay mussel Mussel Peocio, miesmuschel Mussel Mussel Ribbed mussel Northern horse mussel

Crassostrea gigas Ostrea edulis Placopecten magellanicus Penitella penita Tugellus californianus Ensis directus Siliqua patula Spondylus americanus Spondylus ducalis Macoma nasuta Macoma secta Anodonta oregonensis

Edible oyster, huitre Atlantic deep-sea scallop Flap-tipped piddock Razor clam Atlantic jack-knife clam Northern razor clam Atlantic thorny oyster Rock scallop, northern thorny oyster Bent-nosed clam, mud clam White sand clam Freshwater mussel

Protothaca staminea Saxidomus giganteus Saxidomus nuttalli Tivela stultorum

Rock cockle, rock clam, quahaug Smooth Washington clam, butter clam Common clam, butter clam Pismoi Washington clam

Distribution Mediterranean Sea European seas From Nunivak, Pribilof, and Commander Islands; Bering Sea; and all coastal waters south to Hakodate, Japan, and to Baja, California West Indies South Africa Labrador to North Carolina Northern Japan; Prince William Sound, Alaska; south to Scammons Lagoon, Baja California Atlantic coast, California and northwestern Pacific coast, English North Sea coast Aleutian Islands, eastward and southward to Socorro Island Worldwide, in temperate water, including Arctic Ocean Greenland to North Carolina Europe Mediterranean Sea Victoria, Tasmania, Australia New South Wales, Australia Virginia to Florida, San Francisco Bay, California Pacific Coast of North America, from Arctic Ocean to San Ignacio Lagoon, Baja California, circumboreal Japan, Pacific Northwest Coast of Europe Labrador to North Carolina Chirikof Island, Alaska to Turtle Bau, Baja California Humboldt Bay, California to Panama Gulf of St. Lawrence to Florida Alaska to Pismo Beach, California North Carolina to West Indies, Texas Philippines, Indonesia, Micronesia, New Guinea Kodiak Island, Alaska to Cape San Lucas, Baja California British Columbia to Cape San Lucas, Baja California Freshwater streams, Kodiak Island, Alaska, southward to northern California and eastward to Great Salt Lake, Utah Aleutian Islands to Cape San Lucas, Baja California Sitka, Alaska, to San Francisco Bay, California Humboldt Bay, California, to San Quentin Bay, Baja California Halfmoon Bay, California, to Magdalena Bay, Baja California

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a, 1994b).

sea cucumbers and toxic ovaries of sea urchins. Sea cucumbers are not important as foods eaten by fish but are used as food by many of the Pacific Islanders and Asians. The causative toxins in sea cucumbers have been termed holothurins, which involve a complex of saponins (Yamanouchi, 1955; Nigrelli and Jakowska, 1960). Little is known concerning the symptoms resulting from the ingestion of poisonous sea cucumbers, but fatalities have been

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reported (Cleland, 1913; Castellani and Chalmers, 1919; Frankel and Jellinek, 1927). Sea urchins are used as commercial food sources in some European and Indo-Pacific regions. Only the gonads are eaten, either raw or cooked. During the reproductive season of the year, generally spring and summer, the ovaries of certain species of sea urchins are reported to develop toxic products injurious to humans (Halstead, 1988;

Hashimoto, 1979). The chemical nature of the poison is unknown. The symptoms of poisoning include epigastric distress, nausea, diarrhea, vomiting, severe migrainelike headaches, and swelling of the lips and mouth. There is believed to be an allergic type of reaction in some cases. 14.2.4

Poisonous Mollusks

Mollusks are unsegmented invertebrates having a soft body and usually secreting a calcareous shell. They have been incriminated in a number of types of oral food intoxications aside from shellfish poisoning. The phylum Mollusca generally is divided into five classes: Amphineura, the chitins; Scaphopoda, the tooth shells; Gastropoda, the snails and slugs with single valves; Pelecypoda, the bivalves (scallops, oysters, and clams); and Cephalopoda, the octopuses, squids, and cuttlefishes. Meglitsch (1967) described a sixth class, Monoplacophora, comprising sea mollusks having limpetlike shells. Most of the toxic species of mollusks are gastropods, pelecypods, and, rarely, cephalopods. The gastropods that have been reported to be poisonous include the following species: abalones belonging to the family Haliotidae (Haliotis discus, H. sieboldi); turban shells belonging to the family Turbinidae (Turbo argyrostomus, T. marmoratus); and whelks belonging to the family Buccinidae (Babylonia japonica, Neptunea Antigua, N. intersculpta) (Asano and Ito, 1959, 1960; Fange, 1960; Halstead and Courville, 1965; Hashimoto and Tsutsumi, 1961; Halstead, 1994a). These species have worldwide distribution; some predominate in specific geographical zones. The abalones are gastropod mollusks that have the outer shell covered by a rough, horny coating, which quite frequently is hidden by a thick cover of algae and other growth. In Asian countries, the entire tissues of the mollusks, including the viscera, are consumed. The toxic principle found in the viscera of abalone is believed to originate through their food web, viz., certain species of seaweeds belonging to the genus Desmarestia (Hashimoto et al., 1960). The muscular foot is generally safe. The toxic compound is a pigment that closely chemically resembles pyropheophorbide A. It is chemically similar to chlorophyll. The symptoms of abalone viscera poisoning have been described as a sudden onset of a burning and stinging sensation over the entire body, followed by an urticarial rash, itching, erythema, pain in the face and extremities, and subsequent development of skin ulceration. The skin lesions are limited to those parts of the body exposed to sunlight, and there is a distinct boundary between covered and exposed parts of the body (Hashimoto et al., 1960).

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Some of the turban shell mollusks have been found to be toxic to humans. Human outbreaks have been reported from eating turban shells taken at Marcus Island, Marianas Islands, and the Western Pacific (Hashimoto et al., 1968; Hashimoto, 1979; Halstead, 1988). Several toxic substances have been isolated from T. argyrostomus, but the chemical nature of these compounds has not been fully elucidated. A poison has been isolated from T. marmoratus that is believed to be identical to ciguatoxin (Yasumoto and Kotaki, 1977). Saxitoxin also has been found in the gut of this same turban shell mollusk. The chemical and toxicological characteristics of these phycotoxins are described later in this chapter. The toxic substances apparently are found in the midgut gland and gut of the turban shell and apparently are obtained by feeding on toxic algae. Ingestion of the whole mollusks may cause poisonings in humans. The symptoms include gastrointestinal disturbances, nausea, vomiting, diarrhea, fatigue, temperature-reversal sensation, and pruritus. In general, the symptoms resemble those of ciguatera fish poisoning. The turban shells are believed to be safe to eat if the viscera are removed. However, since ciguatoxin and saxitoxin may be present, to prevent poisoning, one should be extremely cautious in eating these shellfish. The whelks are carnivorous marine snails that range from tropical to polar seas. They have a vertical distribution that ranges from littoral zone to great depths. Whelks are widely eaten in Europe and in Asia. Several outbreaks of poisonings from the ingestion of the Japanese ivory shell, Babylonia japonica, have been reported in Niigata, Japan (Hashimoto, 1979; Halstead, 1988; Ji-Sheng, 1993). Whelk poison is believed to be restricted to the salivary glands of the mollusk. The toxic substance present in poisonous whelks is tetramine (tetramethylammonium ion) (Figure 14.1A), and its content may range from 3 to 9 mg/g of gland (Asano and Ito, 1960). The amount of poison in the salivary gland of the whelk varies, depending on the season; it is lowest in September and highest in midJuly. Tetramine is considerably less toxic than most marine toxins with an estimated oral lethal dose for an adult human of 250–1000 mg toxin. Tetramine is an autonomic ganglionic blocking agent. Symptoms of tetramine poisoning appear 30 min after ingestion, and recovery is generally complete within a few hours (Anthoni et al., 1989). Intoxication from tetramine may result in nausea, vomiting, anorexia, weakness, fatigue, faintness, dizziness, thirst, dysuria, mydriasis, aphasia, numbness, photophobia, impaired visual accommodation, and dryness of the mouth (Halstead, 1994a). Intoxications result when these salivary glands are ingested in whole shellfish in the raw, cooked, or canned state. Poisonous whelks are said to be safe to eat if the sal-

O CH3 H3C

N

CH

CHCOCH2CH2N(CH3)3OH

CH3 OH

CH3

N

B. Murexine

NH

A. Tetramine CH3

O (CH3)2C

Br

CHCOCH2CH2N(CH3)3Cl

C. Senecioylcholine chloride

D. Aplysin H3C

O CH3

CH3

CH3

CH3 Br

F. Laurene H3C

O CH2OH

CH3

CH3

CH2

CH3

E. Aplysinol Figure 14.1 Toxic substances found in some marine gastropods.

ivary glands are removed prior to eating. There are no documented cases of whelk-derived tetramine’s causing human fatalities. Like whelks, the murices or rock shells are also carnivorous and are found in tropical and temperate waters in shallow tidal zones. They feed on oysters by boring into the oyster shells. The Murex sp. produces a colorless or yellowish fluid from the hypobranchial or the purple gland. This fluid changes to violet or purple on exposure to sunlight. The purple gland also produces murexine (urocanylcholine) (Figure 14.1B). Murexine is a paralytic agent of the skeletal muscles in both invertebrates and vertebrates. It is characterized by marked neuromuscular blocking and nicotinic activity but almost without muscarinic effects. The toxin appears to produce depolarization similar to the effect of suxamethonium (Erspamer and Glasser, 1957). Other active compounds, such as senecioylcholine (isovalerylcholine, Figure 14.1C) and serotonin, also have been isolated from this gland of the murex Thais floridana (Erspamer, 1956; Whitaker, 1960). Cases of human poisoning that followed consumption of Murex species are extremely rare, even though several species are commonly consumed in the European countries. Only one serious outbreak with five fatalities was reported at the beginning of the 19th century in which

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43 persons in the area around the Gulf of Trieste were poisoned after consumption of Murex brandaris (Plumert, 1902). The omnivorous sea hares, found in coastal waters from shallow tidal zones to deeper zones, have also been implicated in human poisoning at various times, although their harmful effects have not been confirmed in modern times (Halstead, 1994a). Several toxins such as aplysin, aplysinol, and laurene have been isolated from the digestive glands of Aplysia californica, A. kurodai, and A. vaccaria (Figure 14.1D–F). These toxins have also been isolated from the dinoflagellates Laurencia okamurai and L. glandulifera on which these mollusks feed. There are no available records of the extent of human consumption of sea hares. Their long-standing notoriety as poisonous mollusks may discourage widespread consumption. This fact may account, in part, for the rarity of reported cases of human poisoning due to sea hares. Furthermore, it is also possible that the sea hares are transvectors of poisonous compounds from dinoflagellates such as Laurencia spp. (Irie et al., 1965). Thus the toxicity of these mollusks may be variable and dependent on the type of algae on which they feed. The bivalve pelecypods probably are the most popular among the mollusks in terms of worldwide consump-

tion. As such, they also rank first among various mollusks as agents of human poisonings. In addition to paralytic shellfish poisoning, amnesic, brevitoxic (neurotoxic), and diarrhetic shellfish poisonings, which are described in greater detail in Section 14.3, there are other forms of bivalve shellfish intoxication, including callistin, venerupin, and giant tridacna clam poisonings. The members of the genus Callista have been incriminated in human intoxications. The first report of an outbreak of poisoning was caused by eating C. brevisiphonata caught in the vicinity of Mori, Hokkaido, Japan (Asano et al., 1950). There was a second report of an occurrence in the same region in 1953. The Mori Health Center subsequently banned the sale of this shellfish, which contains a high concentration of choline and is toxic only during the spawning season of the year from May through September. Callistin poisoning is a classic example of a biologically essential compound choline, that is toxic when ingested in excess. Choline is an essential component of acetylcholine and phospholipids, important in neurotransmission, and of lecithin, involved in lipid function and transport. It shows three distinct actions: muscarinic, nicotinic, and epinephrine mobilization (Sollmann, 1949). The first is shown by parasympathetic stimulation, resulting in the lowering of blood pressure in nonatropinized cats. The second is manifested by the effects of autonomic ganglic stimulation, affecting neuromuscular functions. Thus, the blood pressure of atropinized cats is increased; the denervated gastronemius of these animals can be contracted by choline. The third action is manifested by the dilatation of denervated and atropinized iris. The symptoms of callistin poisoning generally occur within 1 hr after ingestion of the toxic shellfish. They include itching, flushing of the face, urticaria, a sensation of constriction in the chest; epigastric and abdominal pain; nausea; vomiting; dyspnea; cough; asthmatic manifestations; hoarseness, paralysis, or numbness of the throat, mouth, and tongue; thirst; hypersalivation; drop in blood pressure; increase in pulse rate; leukocytosis; sweating; chills; and fever (Asano, 1954; Asano et al., 1953; Halstead, 1994a). In general, this biotoxication resembles a severe allergic reaction. No fatalities have been reported thus far. The venerupin shellfish poisoning was named after the bivalves Dosinia japonica and Tapes semidecussata, which are members of the pelecypod family Veneridae (Halstead, 1994a). In addition, the Japanese oyster Crassostrea gigas, a bivalve of the family Ostreidae, has been incriminated as a causative shellfish species. The toxicity of these bivalves is attributed to the transvectoring of several toxic species of dinoflagellates of the genus Procentrum.

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Venerupin appears to be concentrated in the digestive organs of the mollusks. It is a very stable toxin, resistant to boiling for 3 hr at pH 3 to 8 or heating at 109°C for 1 hr (Akiba and Hattori, 1949). The toxin in these shellfish remains lethal to humans and dogs even after boiling for 1 hr. Venerupin is soluble in water, methanol, acetone, and acetic acid, but insoluble in nonpolar solvents. Like saxitoxin, it appears to be an alkaloid, although its exact structure has not been elucidated. Outbreaks of venerupin shellfish poisoning were reported first in 1957 near Niigata, Japan, and later in the Kanagawa and Shizuoka Prefectures, Japan, during the months of January through April (Akiba, 1961, Hashimoto, 1979). There is no record that this type of shellfish poisoning has occurred elsewhere, despite the fact that the species of shellfish involved are found in other parts of Japan and have been introduced into the United States. The symptoms of venerupin, or asari, shellfish poisoning usually develop within a period of 48 hr after ingestion of the shellfish. The initial symptoms are nausea, gastric pain, vomiting, constipation, headache, and malaise. The body temperature remains normal. Within 36 hr, such additional symptoms as nervousness, hematemesis, and bleeding from the mucous membranes of the nose, mouth, and gums develop (Akiba and Hattori, 1949; Hattori and Akiba, 1952; Meyer, 1953; Halstead, 1994a). In fatal cases, the victim becomes extremely excitable, delirious, and comatose. There is no evidence of paralysis or other neurotoxic effects such as are seen in paralytic shellfish poisoning. The low mortality rates have been credited to early diagnosis and prompt medical care. In severe cases, the victim usually dies within 1 week. Recovery is extremely slow, and the victim remains in a weakened condition for an extended period. Two species of the massive shelled tridacna clams, Tridacna gigas and T. maxima, have been incriminated in French Polynesia in human intoxications. Clinically, tridacna poisoning resembles ciguatera fish poisoning. The symptoms consist of gastrointestinal, vasomotor, and various neurological disturbances, including a loss of motor coordination. Bagnis (1967) has given the most complete account of tridacna shellfish poisoning, which involved 33 persons and a number of domestic animals that had eaten Tridacna maxima at Bora-Bora, Society Islands, French Polynesia. The poison involved was believed to be of the ciguatoxin complex. 14.2.5

Poisonous Arthropods (Crabs and Lobsters)

The phylum Arthropoda is the largest single group within the animal kingdom. It is divided into a large number of

classes. However, only two, Merostomata (horseshoe or king crabs) and Crustacea (lobsters, crayfish, and crabs), are pertinent to toxicologists. Human intoxications caused by eating Asiatic horseshoe crabs (Carcinoscorpius rotundicauda, Tachypleus gigas) have been reported by several researchers (Smith, 1933; Soegiri, 1936; Waterman, 1953; Banner and Stephens, 1966). Most of the reported outbreaks have occurred in Thailand, but outbreaks probably occur elsewhere in Southeast Asia wherever these crabs are endemic. Horseshoe crab poisoning is referred to as mimi poisoning in Thailand. Eating the unlaid green pigs, flesh, or viscera during the reproductive season of the year causes Asiatic horseshoe crab poisoning. The poison is believed to be chemically identical to saxitoxin (Fusetani et al., 1983). Despite their periodic toxicity, the large masses of green unlaid eggs are highly prized by Asiatic people (Waterman, 1953). The onset of symptoms of poisoning is usually within a period of 30 min after ingestion of the poison. The initial symptoms consist of nausea, vomiting, abdominal cramps, headache, dizziness, slow pulse rate, decreased body temperature, aphonia, cardiac palpitation, numbness of the lips, paresthesias of the lower extremities, and generalized weakness. More severe symptoms may occur in rapid succession: aphonia; sensation of heat in the mouth, throat, and stomach; inability to lift arms and legs; generalized muscular paralysis; trismus; hypersalivation; drowsiness; and loss of consciousness. The mortality rate is believed to be quite high. Reports of human biotoxications from tropical reef crabs (lobsters, crayfish, and crabs) have appeared at infrequent intervals from the tropical Indo-Pacific region (Hashimoto, 1979; Cooper, 1964; Guinot, 1967; Inoue et al., 1968; Mote et al., 1970; Hashimoto et al., 1970; Halstead and Cox, 1973). The poison appears to be chemically identical to saxitoxin. Saxitoxin, neosaxitoxin, and gonyautoxins have been reported in various other species of tropical reef crabs of the genera Neoxanthias impressus, Actaeodes tomentosus, Eriphia scabricula, Pilumnus vespertillo, Schizophrys aspera, Thalamita spp., and Percnon planissimum (Yasumoto et al., 1981, 1983, 1986). Yasumura and associates (1986) have reported the presence of tetrodotoxin in the reef crab Zozymus aeneus. The symptoms of tropical reef crab poisoning consist of paresthesias, muscular paralysis, aphasia, nausea, vomiting, and collapse. Death may occur within 2 hr to several days (Hashimoto et al., 1967). The symptoms may resemble those of paralytic shellfish poisoning or tetradotoxism. Coconut or robber crabs (Birgus latro) are often considered a great delicacy. However, under certain conditions, they may be toxic and may cause fatalities. Human

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biotoxications from coconut crabs have occurred from time to time in the Tuamotu and Ryukyu Islands in the Indo-Pacific region (Hashimoto et al., 1967; Bagnis et al., 1970). These crabs become toxic by feeding on the roots of certain toxic terrestrial plants. The poisoning symptoms include violent gastrointestinal upset, headache, chills, joint aches, extreme exhaustion, and muscular weakness. Fatalities have also been reported. In contrast to poisoning by crabs, lobster poisoning generally is considered to be rare, and apparently little is known concerning the nature of the poisons involved or the clinical characteristics of the intoxications they produce. The poisons involved may originate through the food chain of the lobster similarly to that of the toxic tropical reef crabs since they inhabit the same reef environment and have similar eating habits (Halstead, 1994a). 14.2.6

Poisonous Marine Turtles

Marine turtles are reptiles of the order Chelonia (Testudinata). Five species of marine turtles have been reported as poisonous to humans: Caretta caretta gigas (tropical Pacific and Indian Oceans), Chelonia mydas (all tropical and subtropical oceans), Eretmochelys imbricata (all tropical and subtropical oceans), Dermochelys coriacea (largely circumtropical), and Pelochelys bibroni (rivers and coastal areas of Southeast Asia). Very little specific information on human poisoning is available. The origin of turtle poison (chelonitoxin) is unknown, although it may originate in the food chain of the turtle. The symptoms of chelonitoxication appear to vary with the amount of the turtle ingested and the individual. Symptoms develop within a few hours to several days after ingestion and include nausea, vomiting, diarrhea, epigastric pain, tightness of the chest, pallor, tachycardia, sweating, coldness of the extremities, and vertigo (Halstead, 1988, 1994a; Deraniyagala, 1939; Pillai et al., 1962). Acute stomatitis may also develop. 14.2.7

Poisonous Marine Mammals

Several orders of marine mammals have been found to be toxic when eaten: the order Cetacea, which includes the whales, dolphins, and porpoises; Pinnipedia, which includes the walruses and seals; and Carnivora, which includes the polar bear. Toxic species of cetaceans include the liver of sei whale (Balaenoptera borealis), viscera and meat of the white whale (Delphinapterus leucas), the oil and meat of sperm whale (Physeter catodon), and the liver, other viscera, and muscle of the Southeast Asiatic porpoise (Neophocaena phocaenoides).

The symptoms of sei whale poisoning appear within 24 hr after the ingestion of the liver and consist of severe occipital headaches, neck pain, flushing of the face, nausea, vomiting, abdominal pain, diarrhea, fever, chills, photophobia, tearing, and erratic blood pressure. After several days, the victim’s lips become dry and desquamation develops around the mouth, gradually spreading to the cheeks, forehead, and neck. It does not involve the whole body. Acute symptoms generally subside within 2 days, but the desquamation may continue for a longer period (Halstead, 1994a). The eating of the white whale has produced fatalities (Stefansson, 1924, 1944). However, little appears to be known concerning the clinical characteristics. Similarly, no information is available on the clinical characteristics of sperm whale poisoning (Sahashi, 1933). The symptoms of Asiatic porpoise poisoning consist of abdominal pain, nausea, vomiting, bloating, swelling and numbness of the tongue, loss of vision, cyanosis, numbness of scattered areas of the body, hypersalivation, greenish tinge of the saliva, and muscular paralysis. Death may be rapid, and the case fatality rate appears to be very high (Read, 1939). 14.2.8

Poisonous Walruses and Seals

Walruses and seals are members of the mammalian order Pinnipedia. The livers of walruses and certain species of seals at times may be poisonous when eaten. Intoxications are believed to be due to an excessive intake of vitamin A, which is present in the liver. 14.2.9

Poisonous Polar Bears

Polar bears are marine carnivores of the class Mammalia. The only marine carnivore toxic to humans is the polar bear Thalarctos maritimus. Eating the liver or kidneys, which apparently concentrate large quantities of vitamin A, causes polar bear poisoning. The symptoms usually begin about 2 to 5 hours after ingestion of either the kidneys or the liver. The predominant symptoms are intense throbbing or dull frontal headaches, nausea, vomiting, abdominal pain, dizziness, drowsiness, irritability, weakness, muscle cramps, visual disturbances, and collapse. Tonic and clonic convulsions may occur (Jeghers and Marraro, 1958; Sutton, 1942; Rodahl, 1949). 14.2.10 Toxic Fish There are about 25,000 species of fish, yet fewer than 13 are commercially valuable on a large-scale basis worldwide. A few, such as the herring, anchovie, cod, haddock,

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hake, and mackerel, are exploited heavily. Many other edible fish are caught only on a limited scale. There are two major kinds of toxic fish, distinguished on the basis of the origin or source of toxicity: endogenously and adventitiously toxic fish. Many endogenously toxic species are among the most violently poisonous (e.g., pufferfish) and yet are valued as a delicacy in certain parts of the world. The latter type is distributed in a large number of families, many of which are commercially important. The poisonous substances in fish may be concentrated in specific tissues or organs. Accordingly, fish are classified as being ichthyosarcotoxic, ichthyootoxic, and ichthyohemotoxic (Halstead and Courville, 1965). In the first group of fish, the toxin is concentrated in the muscles, skin, liver, intestines, and other tissues in addition to the gonads. Ichthyootoxic fish contain toxins mainly in the gonads, i.e., ovary or testes, and the roe (eggs). The final group concentrates their poison in the blood. Toxic species from these three groups used as human food are briefly described in the following. Ichthyosarcotoxic Fish Ichthyosarcotoxic fish contain a poison within the flesh in its broadest sense (i.e., the musculature, viscera, skin, or slime) that, when ingested, causes biotoxication (Halstead, 1994b). The toxins could be endogenous, derived toxic substances, or poisonous biological contaminants. The endogenous toxins include tetrodotoxin, gempylotoxins, certain elasmobranch toxins, chimaerotoxins, and cyclostomotoxins. Ciguatoxins, clupeotoxins, and some elasmobranch toxins are naturally occurring biological contaminants in fish. Scrombotoxin is a derived substance produced by bacterial action. Of these, tetrodotoxins and ciguatoxins are described in greater detail in Section 14.3. The other types of poisonings are briefly described in the following. Cyclostomotoxic Fish Members of the class Agnatha, the cyclostomes include the lampreys and hagfish. The latter are rarely eaten as food. The species of lampreys reported to be toxic include the Caspian lamprey (Caspiomyzon wagneri), the river lampreys (Lampetra fluviatilis and L. planeri), and the sea lamprey (Petromyzon marinus). Most cyclostome poisonings have been reported to be due to the failure to remove the slime before cooking. The symptoms, including nausea, vomiting, dysenteric diarrhea, tenesmus, abdominal pain, and weakness, may develop within a few hours after eating (Halstead, 1994b; Concon, 1988). The victim generally recovers after several days.

Elasmobranch Fish The elasmobranch fish include the sharks, skates, rays, and chimaeras. The poisoning is referred to as elasmobranch poisoning. Poisonous sharks implicated in human poisonings include mackerel sharks (Carchardon carcharias), found in tropical, subtropical, and warm-temperate seas worldwide; requiem sharks (Carcharhinus melanopterus), found in the tropical Indo-Pacific region; cow sharks (Heptranchias perlo), found in the Atlantic Ocean and Mediterranean Sea, South Africa, and Japan; sleeper sharks (Somniosus microcephalus), distributed from the Arctic, Atlantic, and North Sea east to the White Sea, and west to the Gulf of St. Lawrence and Greenland; and hammerhead sharks (Sphyrna zygaena,) found in the tropical to warm temperate belt of the Atlantic and Pacific Oceans. Eating shark livers and the flesh of some tropical sharks most commonly causes the elasmobranch poisoning. The symptoms resulting from the ingestion of shark livers may be very severe, developing within 30 min after eating. The symptoms consist of nausea, vomiting, diarrhea, abdominal pain, headache, joint aches, tingling about the mouth, and a burning sensation of the tongue, throat, and esophagus. As time progresses, the symptoms involving the nervous system may worsen, resulting in muscular paralysis, followed by coma and finally death (Halstead, 1994b). The nature of the elasmobranch poison is unknown. Gempylotoxic Fish The gempylids, or snake mackerels, are a small group of predatory oceanic or pelagic fish. They resemble the mackerels except for the absence of a lateral keel on the caudal peduncle. Three toxic species are known: the escolar (Lepidocybium flavobrunneum), the castor oil fish (Ruvettus pretiosus), and the snoek or barracouta (Thyrsites atun). The first species is rather rare; the other two have worldwide distribution in the Indo-Pacific, South American Pacific, and tropical Atlantic Oceans and along the South African coastline. The gempylid poisoning is mainly due to ingestion of the flesh or sucking of the rich, oily bones. The oil has a pronounced purgative effect and is similar to castor oil but with different pharmacodynamic properties (Mori, 1966). Diarrhea after gempylid ingestion occurs rapidly, generally without pain or cramps. It is a relatively mild form of intoxication. Clupeotoxic Fish The clupeotoxic group, which includes some of the herrings, anchovies, and related species, apparently become poisonous after eating certain planktonic organisms,

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such as toxic dinoflagellates. This intoxication is rare and resembles ciguatera or paralytic shellfish poisoning but acts very rapidly and produces a very high mortality rate. There are a few scattered reports of clupeotoxicism. Two outbreaks were reported in the Philippines in 1953 and 1955; 87 cases occurred, with 14 to 17 deaths (Halstead and Courville, 1967). The common Philippine sardine tamban (Sardinella longiceps) was implicated. Three cases of sardine (Harengula spp.) poisoning were also reported in Tarawa in the Gilbert Islands in the South Pacific. All three people died (Cooper, 1964). An uncounted number of poisonings resulting in five deaths caused by another sardine (Clupea venenosa [= Harengula ovalis]) were also reported in the Fiji Islands in the South Pacific (Banner and Helfrich, 1964). Clupeotoxic fish are listed in Table 14.3. Most thrive in tropical waters in the Asian, Indo-Pacific, Mediterranean, Arabian, and Red seas; the Indian Ocean; and the South American and other tropical parts of the Pacific Ocean. Clupeotoxicism can be a serious public health problem in certain parts of the world, because of not only the unpredictability of its occurrence, but also the violent and often lethal nature of the poisoning. It has been associated with toxic dinoflagellate blooms (Halstead, 1994b). The viscera are regarded as the most toxic part of the fish. Tropical clupeotoxic fish are most likely to be toxic during the warm summer months of the year. The actual source and nature of the poison never have been identified. The toxin may be derived from the food chain, particularly from certain types of dinoflagellates. The red tide dinoflagellate, Pyrodinium bahamense, is suspected as a possible source of the toxin (MacLean, 1979). The symptoms and signs of clupeotoxicism are distinct and usually violent. The first indication of intoxication is a sharp metallic taste that may be present immediately after ingestion of the fish. This is soon followed by nausea, dryness of the mouth, vomiting, malaise, abdominal pain, and diarrhea. A feeble pulse, tachycardia, chills, and cold may accompany the gastrointestinal upset and clammy skin, vertigo, a drop in blood pressure, cyanosis, and other evidences of vascular collapse. Within a very short period or concurrently, a variety of neurological disturbances rapidly ensure, such as nervousness, dilated pupils, violent headaches, numbness, tingling hypersalivation, muscular cramps, respiratory distress, progressive muscular paralysis, convulsions, coma, and death. Death may occur in less than 15 min. There are no accurate statistics available regarding the mortality rate of clupeotoxicism. However, judging from the documented case reports, the fatality rate appears to be very high, and the victims generally die within min-

Table 14.3 Fish Reported as Clupeotoxic Common name

Species Family Clupeidae Anondostoma chacunda Clupanodon thrissa Clupea sprattus Dussmieria acuta Harengula humeralis H. ovalis, H. zunasi, Sardinella fimbriata, S. longiceps, S. perforata, S. sindensis Ilisha africana Macrura ilisha Nematoilosa nasus Opisthonema oglinum Family Elopidae Megalops cyprinoids Family Engraulidae Engraulis enchrasiculus, E. japonicus, E. ringeus, Thrissina baelama

Shirt-finned gizzard shad Sprat or thread herring Sprat Round herring Red-ear sardine Sardine Herring Sablefish Gizzard shad or long-finned gizzard Atlantic thread herring Tarpon Anchovy

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a).

utes to hours. It is believed that clupeotoxicity in some instances may be related to ciguatera poisoning. This has not been documented, however. Scombrotoxic Fish (Histamine Poisoning) Scombrotoxism is a syndrome that results from ingesting spoiled fish of the families Scombridae and Scombresocidae. The most often affected fish are tuna, bonito, skipjack, saury, and mackerel. Nonscombroid fish can also be involved in outbreaks of histamine poisoning. These include mahi-mahi, yellowtail, bluefish, amberjack, herring, sardines, and anchovies (Pugno et al., 1983; Etkind et al., 1987; Halstead, 1994b). Fish species implicated in histamine poisoning are listed in Table 14.4. Scombroid poisoning is caused by improper preservation of scombroid fish that causes certain bacteria, mainly species of the family Enterobacteriaceae (Clostridium, Lactobacillus, and Vibrio spp.), to act on histidine in the muscle of the fish, converting it to histamine. Pure histamine by itself lacks toxicity. The toxicity of histamine in scombrotoxic fish is enhanced by the presence of certain potentiators (e.g., cadaverine and putrescine) that act by inhibiting intestinal histamine-metabolizing enzymes (Halstead, 1994b). The chemical structures of the potentiators of histamine toxicity are shown in Figure 14.2. The enzyme inhibition increases the intestinal uptake of unmetabolized histamine. Histamine ingested by itself generally is much less toxic.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Histamine poisoning is an intoxication, so the incubation period is rather short, ranging from several minutes to a few hours after ingestion of the contaminated fish. The duration of the illness is usually short also; symptoms subside within a few hours in most cases. The intoxication is characterized by a wide variety of possible symptoms of cutaneous (rash, urticaria, edema, localized inflammation), gastrointestinal (nausea, vomiting, diarrhea, cramping), hemodynamic (hypotension), and neurological (headache, palpitations, flushing, tingling, burning, itching) nature. The acute symptoms generally are transient, lasting only 8 to 12 hours. Histamine poisoning is often confused diagnostically with food allergies, because of the similar symptoms and the equivalent effectiveness of antihistamines. However, histamine poisoning can be easily distinguished from food allergy on the basis of (1) the lack of a previous history of allergic reactions to the incriminated food, (2) the high attack rate in group outbreaks, and (3) the detection of high levels of histamine in the incriminated food (Taylor, 1986). Antihistamine therapy is the optimal mode of therapy for histamine poisoning. Symptoms usually subside rapidly after treatment with H 1 antagonists, such as diphenhydramine or chlorpheniramine, or H2 antagonists, such as cimetidine. Since the disease is self-limited, pharmacological intervention is generally not required in mild cases.

Table 14.4 Scombroid Fish Species Implicated in Histamine Poisoning Common name

Species Family Scombridae Thunnus albacares T. atlanticus T. maccoyii (=T. thynnus maccoyii) T. obesus (Parathunnus mebachi) T. thynnus thynnus T. thynnus orientalis T. tonggol T. alalunga Euthynnus pelamis (Katsuwonas pelamis) E. affinis E. alletteratus E. lineatus Allthunnus fallai Auxis rochei A. thazard Sarda sarda S. orientalis S. chiliensis S. australis Scomber scombrus S. japonicus Scomberomorus cavalla S. maculatus S. concolor S. regalis S. sierra Family Scomberesocidae Scomberesox saurus Cololabis saira Family Pomatomidae Pomatomus saltatrix Family Coryphaenidae Coryphaena hippurus Family Carangidae Trachurus trachurus, T. japonicus T. symmetricus Seriola colburni S. dorsalis, S. grandis S. dumerili Family Clupeidae Clupea harengus harengus C. harengus pallasi C. sprattus Sardinops sagax Sardina pilchardus Sardinella aurita S. anchovia Family Engraulidae Engraulis encrasicolus E. mordax Cetengraulis mysticetus

Yellowfish tuna Blackfin tuna Southern bluefin tuna Big-eye tuna Atlantic bluefin tuna Pacific bluefin tuna Longtail tuna Albacore tuna Skipjack tuna Kawakawa Little tunny Black skipjack tuna Slender tuna Bullet tuna, bullet mackerel Frigate tuna, frigate mackerel, plain bonito Atlantic bonito Indo-Pacific or striped bonito Eastern Pacific bonito Australian bonito Atlantic mackerel Chub or Pacific mackerel King mackerel Spanish mackerel Monterey Spanish mackerel Cero Sierra Atlantic saury Pacific saury, mackerel pike Bluefish Dolphin fish, dorado, mahi-mahi Horse mackerel Jack mackerel Pacific amberjack Yellowtail Greater amberjack Atlantic herring Pacific herring Sprat, brisling Pacific sardine, pilchard Pilchard, sardine Golden sardine Spanish sardine European anchovy Pacific or northern anchovy Anchoveta

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a).

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CH3 H2N

C

N(CH2)4NH2

NH

H3C

N CH3

Agmatine

Choline

CH3

CH3 H3C

N

CH2CH2OH

O

CH3

H3C

N CH3

Trimethylamine oxide

Trimethylamine

H2N(CH2)5NH2

H2N(CH2)4NH2

Cadaverine

Putrescine

Figure 14.2 Potentiators of histamine toxicity in scombroid fish.

Scombroid fish and other related species incriminated in this form of poisoning should be refrigerated promptly or eaten soon after capture to aid in prevention of poisoning. The histamine content in some of the scombroid fish increases from 0.09 mg/100 g tissue to about 95 mg/100 g of tissue when kept at ambient temperatures for about 10 hours (Halstead, 1994b). Toxic scombroid fish cannot always be detected by appearance or odor. The histamine content in the flesh may be very high with little or no evidence of putrefaction. Scombroid or any other fish having a sharp or peppery taste or having histamine levels greater than 20 mg/100 g fish should be discarded. Ichthyootoxic Fish Ichthyootoxic poisoning is one of the lesser-known forms of fish poisoning. These fish produce a poison generally restricted to the gonads and, of course, the roe or eggs. The toxicity is thus related to the reproductive or gonadal activity of the fish. The flesh of these fish is usually harmless. Most of the intoxications resulting from the ingestion of ichthyootoxic fish therefore occur during the reproductive season, during which the gonadal activity of the fish is at its peak. The various fish species reported to contain toxic gonads or roe are summarized in Table 14.5. They include both freshwater and saltwater species, many of which are

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of significant economic value and are highly esteemed as food. The most poisonous in this group are the barbells (Barbus spp.), carps (Schizothorax spp.), tenches (Tinca spp.), and pricklebacks (Stichaeus spp.) (Halstead and Courville, 1965). Fish roe poisoning is quite common in Europe, Asia, and North America, and to a lesser extent, the tropics. The tetrodotoxic fish (see Section 14.3) also qualify as ichthyootoxic, since their gonads are more toxic than any other organ in the fish and any other type of toxic fish roe. The nature of the toxins from all known ichthyootoxic fish has not been identified. The exception is the toxin from the Japanese prickleback (Stichaeus grigorjewi); the toxins appear to be lipoproteins. Four lipoprotein fractions have been isolated from the roe. One fraction, δlipostichaerin, seems to be responsible for human poisonings (Asano and Ito, 1966; Hatano, 1971a, 1971b). This toxin appears to be a phospholipopeptide containing glycerol, choline, phosphorus, fatty acids, and amino acids. Lipostichaerin consists of a nontoxic protein moiety, stichaerin, and a toxic phospholipid, dinogunellin. The intraperitoneal LD50 in mice of dinogunellin is 25 mg/kg. Death occurs in 72 hr (Hatano and Hashimoto, 1974). The intraperitoneal LD50 of lipostichaerin in mice is 180 mg/kg. Roe poisoning has a rapid onset. Symptoms develop soon after ingestion of the roe and consist of abdominal pain, nausea, vomiting, diarrhea, headache, fever, bitter taste in the mouth, dryness of the mouth, intense thirst, a sensation of constriction of the chest, cold sweats, irregular pulse, low blood pressure, cyanosis, papillary dilatation, syncope, chills, dysphagia, and tinnitus (Concon, 1988; Halstead, 1994b). In severe cases, there may be muscular cramps, paralysis, coma, and death. Barbus spp. roe usually do not cause death, but fatalities have resulted from eating Schizothorax spp. roe (Knox, 1888). There are no known antidotes for fish roe poisoning. Although cooking is said to destroy most ichthyootoxins, it cannot be relied upon as a completely safe procedure since the poison in some fish appears to be resistant to heat. It is better to avoid eating the roe of any fish during the reproductive season of the year. This preventative advice is particularly pertinent to the freshwater and brackish water fish of Europe and Asia and all tropical marine species. Although caviar, which is processed salted roe from certain sturgeons, is eaten widely, the roe of some sturgeons have been found to be toxic. Ichthyohemotoxic Fish Ichthyohemotoxic fish largely consist of a variety of different species of eels. These eels are regarded as generally edible, except for their toxic blood. This is a rare form of

Table 14.5 Ichthyootoxic Fish Containing Toxic Gonads or Roe Genus

Family Acipenseridae Agenoiosidae

Cottidae Cyprinidae

Cyprinodontidae Esocidae Gadidae Ictaluridae Lepisosteidae Percidae Plotosidae Salmonidae Serranidae Siluridae Stichaeidae

Acipenser, Huso Ageneiosus Bagre, Pseudobagrus, Sciadeichthys, Selenaspis Scorpaenichthys Abramis Barbus Cyprinus, Schizothorax Diptychus Tinca Aphanius Fundulus Esox Gaidropsarus Lota Ictalurus Lepisosteus Perca Plotosus Salmo Stenodus Paranthias Parosilurus Stichaeus

Group or common name Sturgeon Sheatfish Catfish Cabezon Bream Barbel Carp Osman Tench Killfish or dogpike Mummichog Pike Rockling Barbot White catfish Gar River perch Oriental catfish Salmon Whitefish Creolefish Mudfish, catfish, sheatfish Japanese prickleback or blenny

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a).

marine food poisoning. Hemotoxins are largely parenteral poisons and seldom toxic when taken by mouth. Very little is known concerning the chemical nature of these poisons. However, the toxin from the eels Anguilla vulgaris, Muraena Helena, and Conger vulgaris appears to be proteinaceous, since it is destroyed by trypsin and papain (Ghiretti and Rocca, 1963; Rocca and Ghiretti, 1964; Russell, 1965). Very little is known concerning the symptoms of ichthyohemotoxism in humans. Fish serum intoxications may be of two types: systemic, a form that results from drinking fresh, uncooked fish blood, and topical (Halstead, 1994b). The symptoms of the systemic form consist of diarrhea, bloody stools, nausea, vomiting, hypersalivation, skin eruptions, cyanosis, apathy, irregular pulse, weakness, paresthesias, paralysis, respiratory distress, and possibly death. For the topical form of poisoning, there is a severe inflammatory response when raw eel serum accidentally has contact with the eye or the tongue. Oral symptoms consist of burning, redness of the mucosa, and hypersalivation (Halstead, 1988). Ocular contact invokes a severe burning sensation and redness of the conjunctivae, lacrimi-

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nation, and swelling of the eyelids. Eye irritation may persist for several days. Recovery is usually spontaneous. For prevention, care should be taken in the handling of eel blood. Raw eel blood should not be ingested. Cooking is said to destroy the toxic properties. Ichthyohepatotoxic Fish The livers of certain edible species of fish sometimes are found to be toxic when eaten. These fish include the Japanese mackerel (Scomberomorus niphonius), Japanese sandfish (Arctoscopus japonicus), sea bass (Stereolepis ischinagi), porgy (Petrus aupestris), and halibut (Hippoglossus spp.). These fish also may be involved in other types of poisoning, such as ciguatera poisoning. Ciguatoxicity may also involve the liver of the fish, but in ichthyohepatotoxism, only this organ of the fish is toxic. The tetrodotoxic fish may also be classified as ichthyohepatotoxic fish. Most of the outbreaks of ichthyohepatotoxism have occurred in Japan. Little is known about the chemical nature of the poisons involved in this type of fish poisoning. In some instances, the intoxications were attributed to hypervita-

minosis A (Abe and Kinumai, 1957). In contrast, Mizuta and colleagues (1957) asserted, on the basis of the symptoms, that the toxic substance is histaminelike. Because the liver of the fish may store a variety of compounds, fish liver poisoning is quite likely to be the result of a combined action of different compounds. Symptoms of ichthyohepatotoxism appear within 30 minutes to 12 hours after ingestion of the fish liver. The initial symptoms consist of nausea, vomiting, fever, and headache. The headache may be very severe and is intensely aggravated by the slightest movement of the body, head, or eyes. A mild diarrhea may be present, but abdominal pain generally is absent. The face of the victim usually becomes flushed and edematous (Nater and Doeglas, 1970). A macular rash of large patchy erythematous areas develops shortly. Within 3 to 6 days, desquamation appears. Large areas of skin may peel off around the nose, mouth, head, neck, and upper extremities and gradually extend over the entire body. Desquamation may continue for about 30 days. Most of the more acute symptoms usually disappear in about 3 to 4 days. Residual symptoms consist of chapping of the lips, stomatitis, and mild hepatic dysfunction. Recovery usually is uneventful. No fatalities have been reported (Smith, 1961; Halstead, 1994b). The liver may be enlarged, but no jaundice has been observed. There are no specific antidotes for this type of poisoning. Preventatively, care should be taken in eating fish livers. In general, the liver is one of the most dangerous parts of a fish to eat. If a fish is poisonous, a greater concentration of the poison is likely to be found in the liver than in almost any other part of the fish. Cooking does not destroy the poison. Most outbreaks of ichthyohepatotoxism have resulted from eating fish livers that have been sautéed or as part of soup. Ichthyoallyeinotoxic (Hallucinogenic) Fish Ingesting certain types of reef fish known to occur in the tropical Pacific and Indian Oceans causes ichthyoallyeinotoxic fish poisoning. The biotoxication may result from eating either the head or the flesh of the fish. Ichthyoallyeinotoxic fish species are listed in Table 14.6. The source and chemical nature of the poison are unknown, and the usual cooking processes do not destroy it. The poison is believed to be concentrated in the head of the fish. This type of intoxication is sporadic, uncommon, and completely unpredictable. The poison primarily affects the central nervous system. The symptoms may develop within minutes to 2 hr after ingestion of the fish, persist for about 24 hr, and then gradually subside (Russell, 1965). Symptoms consist of dizziness, loss of equilibrium, lack of motor coordination,

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Table 14.6 Ichthyoallyeinotoxic (Hallucinogenic) Fish Species Species

Common name Sea chubs Convict surgeonfish, tang Mullets Surmullets, goatfish Damselfish, sergeant major Grouper Rabbitfish

Kyphosus cinerascens, K. vaigiensis Acanthurus triostegus sandvicensis Mugil cephalus, Neomyxus chaptalli Mulloidichthys samoensis, Upeneus arge Abudefduf septemfaciatus Epinephelus corallicola Sigamus oranin

Source: Compiled from Concon (1988) and Halstead (1994a).

hallucinations, and mental depression. There is also a sensation of a tight constriction around the chest (Halstead, 1964). This form of poisoning is generally mild; the victim usually recovers completely in 2 to 24 hr. Miscellaneous Toxic Fish The mucous secretions of some fish may contain toxic substances. Among these are the Hawaiian trunkfish or pahu (Ostracion spp.), Pacific bass (Grammistes sexlineatus), soapfish (Rypticus saponaceus), and Pogonoperca punctate (Boylan and Scheuer, 1967; Liquori, 1963; Maretzki and Del Castillo, 1967; Hashimoto, 1968). The toxin from the Hawaiian trunkfish has been called pahutoxin (Figure 14.3). The toxin is hemolytic to vertebrate red blood cells at concentration of 1 ppm (Thomson, 1964). In mice, the crude toxin injected intraperitoneally produces ataxia, labored breathing, coma, and death. The minimal lethal dose in mice is 0.2 mg/g, and there is complete recovery at sublethal doses. The toxin of the soapfish has not been fully characterized. It is believed to be a large peptide, since it is nondialyzable (Maretzki and Del Castillo, 1967). It appears to be an unusual protein, being soluble in water and watersaturated butanol, moderately heat-stable at 65°C for 2 hr, and insoluble in solutions with low ionic strength except in

O CH3(CH2)12CHCH2

C

O

(CH2)2

N(CH3)3Cl

OCOCH3

Figure 14.3 Pahutoxin, a hemolytic toxin found in the Hawaiian trunkfish.

the presence of acid. Injected intraperitoneally into mice, extracts of the soapfish toxin produce motor unrest and death. The toxin from the Pacific bass also appears to be a peptide and is toxic to the killfish at 70 ppm (Liquori, 1963). That from Pogonoperca punctate is probably a smaller peptide than the related soapfish toxin, since it is slowly dialyzable. It has a bitter taste and produces ciguateralike symptoms in cats when administered orally or subcutaneously; cats are killed when fed 10 g raw skin/kg. (Hashimoto, 1968).

14.3 PHYCOTOXINS 14.3.1

Paralytic Shellfish Poisons

As mentioned earlier, many species of shellfish (Table 14.1) become poisonous through the consumption of toxic marine algae or dinoflagellates. The most common cause of paralytic shellfish intoxication occurs when humans consume shellfish (mussels, clams, oysters, scallops, and univalve mollusks) contaminated with toxins stored in the hepatopancreas. Starfish and sand crabs may also contain these toxins (Halstead and Courville, 1965). Paralytic shellfish poison (PSP) is one of the most severe forms of food poisoning caused by the ingestion of seafood. It is acute and often fatal, and there is no effective way to detoxify the toxins or to treat patients. It poses serious health problems, which in turn deter shellfish consumption, causing economic problems. History and Discovery The history of PSP goes back to prehistoric days, and incidents due to the consumption of toxic shellfish are well documented (Halstead, 1978; Hashimoto, 1979). In North America, the best known is Captain George Vancouver’s account of shellfish poisoning of his crews on the West Coast in the 1700s (Vancouver, 1798). In 1927, a series of outbreaks and deaths in the San Francisco Bay area stimulated modern research in PSP (Meyer et al., 1928). The problem chronically exists along both the East and West Coasts, often leading to a total ban on shellfishing in a wide area with enormous economic losses. Until 1937 the shellfish poison problem was indeed puzzling. Shellfish that might have been edible for generations suddenly, and for no reason apparent at the time, became extremely poisonous. The shellfish would remain so for 1–3 weeks and then again become safe for human consumption. Sommer and Meyer at the University of California finally explained this sporadic occurrence of poisonous shellfish in 1937. They observed the presence of a particu-

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lar microscopic plankton in the waters around the mussel beds during several outbreaks that occurred between 1920 and 1937 along the central California coast. They identified this organism as the dinoflagellate Gonyaulax catenella and found that it contained a poison that produced effects in mice similar to those of extracts of the poisonous mussels. Their work showed that the mussels acquired the poisonous properties through the food chain and that they possessed a mechanism in the dark gland or hepatopancreas that binds the poisons. Mussels gradually destroy or excrete the bound poison, so that within 1–3 weeks after the bloom of the poisonous dinoflagellate subsides, they are free of poison. The poison that is bound in the shellfish is readily released when humans consume them. The discovery of the relationship of G. catenella to poisonous shellfish by Sommer and Meyer (1937) soon led to the discovery that G. tamarensis (var. excavata) caused clams and scallops along the northeast coast of North America and the northeast coast of England to become poisonous. In the early years there was much interest in the amount of poison necessary to cause sickness and death of humans. From accidental cases of poisoning along the coast of California, where G. catenella commonly blooms, Meyer (1953) estimated that sickness may result from about 1000 to 20,000 mouse units (MUs) and estimated the minimal amount to cause death at 20,000 MU of poison. A mouse unit of poison is defined as the amount that will kill a 20-g white mouse in 15 min. However, along the St. Lawrence estuary and Bay of Fundy in Canada, Bond and Medcof (1958) found that sickness occurred with about 600 MU and death at 3000–5000 MU. The lower figures were explained on the basis that these persons had not consumed shellfish regularly and had not acquired any tolerance to the poison by consuming small doses; this is most likely the case with many people along the California coast. Transvectors of Paralytic Shellfish Poisons The dinoflagellates generally recognized to be the source of the PSPs in temperate waters were originally assigned to the genus Gonyaulax, within which they form a distinct subgroup. These were referred to collectively as the tamarensis complex (Hall and Reichardt, 1984). Subsequently, they were separated into a new genus, Protogonyaulax. Although several researchers have used Gonyaulax as the genus name, according to the rules of biological nomenclature, the correct name for this genus may be Alexandrium. Despite the profusion of genus names, the group itself seems relatively well defined. Most of the organisms are similar morphologically, to the extent that it is very difficult to make morphological distinctions among species.

Many of the species may, in fact, have been established through historical accident. In contrast, the dinoflagellate Pyrodinium bahamense from the tropical Pacific has been shown to contain PSPs and is clearly distinct from the organisms of the tamerensis group. The taxonomic and biogeographical aspects of PSP-producing dinoflagellates are briefly discussed in the following section. Protogonyaulax Species Three species of this genus are linked to natural PSP events: P. tamarensis (which includes excavata, attempts to distinguish between them by morphological or other means have proved difficult), P. acatenella, and P. catenella. In addition, there are five other species that have not been shown to produce toxins so far. All these were formerly classified under the genus Gonyaulax, but they differ in several respects, including epithecal plate pattern, hypothecal pattern, apical pore, degree of girdle displacement, and cyst type. The morphological distinction between species of Protogonyaulax is quite difficult, since they all share the same basic plate pattern. In this regard, P. catenella and P. tamarensis have been distinguished for the longest period. These two species obtained from a restricted geographical area show a high degree of electrophoretic polymorphism even for relatively conservative enzyme systems. There is also strong circumstantial evidence that Protogonyaulax spp. populations are multiclonal, even when morphologically indistinguishable. The distribution of the tamarensis and catenella morphotypes is also quite interesting, on both global and local scales. P. catenella occurs on the West Coast of North America from southern California to southeastern Alaska. Its type locality is San Francisco Bay. It is absent south of 32°N but reappears in southern Chile. P. catenella has not been recorded throughout most of the Atlantic Ocean but has produced four PSP outbreaks off the west coast of South Africa (Taylor, 1984). The only other region where P. catenella has been recorded is the east coast of Japan. Blooms generally occur when the temperature is close to 20°C, and the form occurs in both estuarine and open coast localities. The P. tamarensis morphotype, in contrast, is found over a much wider area, including both arctic and tropical localities, the latter only in the western tropical Atlantic Ocean. In the North Atlantic it is found from estuarine localities or shallow embayments from Long Island to the Arctic and is particularly common in the Gulf of Maine, Bay of Fundy, and Gulf of St. Lawrence in the west, and from Portugal north to Norway, and the Arctic near Spitsbergen. In the South Atlantic, it is known from the central

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coast of Argentina to southwest Africa. In the Pacific Ocean, it occurs in British Columbia, some coastal locations in the Gulf of Alaska, and bays in northeastern Japan. Gonyaulax Species Although several species of Gonyaulax (other than those transferred to Protogonyaulax and Gessenerium spp.) have produced red tide blooms, only G. grindleyi (= Protoceratium reticulatum) and G. polyedra have been suspected of producing PSPs. The former was associated with PSP and the death of bivalves on several occasions off the west coast of South Africa, although no water-soluble toxin could be extracted from it (Taylor, 1984). Gessnerium and Alexandrium Species Several researchers include all those species considered to be Protogonyaulax under this nomenclature. The species, which may be referred to Gessnerium in the narrow sense, are Gs. monilatum, Gs. balechii, and A. minutum, if the latter genus is conserved. All three have been associated with red water tides, but only Gs. monilatum produces toxins that are lethal to fish, annelids, mollusks, and crustaceans. Pyrodinium Species Pyrodinium compressa is the only variety known to produce toxins, which have produced PSP in the IndoWest Pacific. It has been found in various warm, high-salinity regions, such as the Red Sea and the Persian Gulf, but has not been observed in the Atlantic Ocean. The variety P. compressa bahamense, in contrast, is almost exclusively found in the tropical Atlantic, from the Bahamas to Venezuela, as well as sporadically from the west coast of Central America. Although the latter is not known to produce toxins, there are records of PSP and clupeotoxicity from localities within its range, and it is quite possible that some strains are toxigenic. Peridinium Species The only toxic freshwater dinoflagellate known is that identified as Peridinium polonicum (initially referred to as Glenodinium spp.). The species is cosmopolitan in temperate lakes. Because of the direct relationship between the occurrence of red tides and incidences of PSP, several nations have monitoring programs for harmful algae. By itself, phytoplankton monitoring does not provide sufficient protection to public health. However, it is often used as an early warning system on which to base intensive sampling programs of toxicity testing of shellfish tissue.

Examples of countries that monitor potentially toxic species and the concentrations of these that elicit further action are given in Table 14.7. Occurrence PSP is a worldwide problem in temperate coastal areas. On a global scale, PSP is the most common and widespread of the phycotoxin syndromes, as is the occurrence of PSP toxins in seafood. The actual number of cases per year is unknown, but some suggest an estimated incidence of 1600 cases with possibly 300 deaths per year (Sims and

Ostman, 1986; Haddad et al., 1987; Smith, 1992). The consequences of PSP can be very severe. For example, in 1987 in a significant epidemic along the coast of Guatemala, of 187 people who became intoxicated with PSP toxins, 26 subsequently died (Kao, 1993). The endemic areas of the United States include Alaska, California, Maine, and Oregon. Worldwide, the endemic areas include Canada, England, Scotland, Ireland, Norway, Germany, Belgium, France, Japan, Mexico, Malaysia, South Africa, Taiwan, and Venezuela. The paralytic shellfish poisons are a group of more than 20 structurally related water-soluble toxins produced

Table 14.7 Countries with Toxic Algal Monitoring Schemes and Cell Concentrations at Which Action (Intensified Monitoring to Closure of Fisheries) is Taken Country

Algal species monitored

Cell concentration for action

Australia

Alexandrium catenella Gymnodinium catenatum Alexandrium spp. Pseudonitzschia multiseries Pseudonitzschia pseudodelicatissima Dinophysis spp. Alexandrium spp. Dinophysis spp. Prorocentrum lima Pseudonitzschia seriata Pseudonitzschia delicatissima Nodularia spumigena Dinophysis spp. Alexandrium spp. Dinophysis spp. Pseudonitzschia spp. Alexandrium spp. Dinophysis spp. Pyrodinium bahamense var. compressum

>5 × 104 Cells/liter based on level of toxin >5 × 104 Cells/liter based on level of toxin Presence 5 × 104 1 × 105 ?? 500 500 500 2 × 105 2 × 105 1 × 105 to 2 × 105 Colonies/liter 103 and DSPa in mussels 103–104 100 104–105 Presence in net hauls 500–1.2 × 103 200 Cells/liter

Alexandrium catenella Alexandrium minutum Alexandrium spp. Dinophysis acuminata Dinophysis acuminata Gymnodinium catenatum Prorocentrum lima

2 × 107 to 5 × 107 103 103 103 2 × 107 to 5 × 107 >500 ??

Alexandrium tamarense Dinophysis spp. Prorocentrum lima

Presence >100 Presence

Gymnodinium breve (Ptychodiscus brevis)

>5 × 103

Canada

Denmark

Italy The Netherlands

Norway Philippines Spain Valencia Balearic Islands Balearic Islands Balearic Islands Valencia Andalusia Andalusia United Kingdom Northern Islands Scotland United States Florida a

DSP, diarrhetic shellfish poison(ing); see the section Phycotoxins for detailed description. Source: Compiled from Shumway (1995), Andersen (1996), and Leftley and Hannah (1998).

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by certain species of dinoflagellates. Shellfish grazing on these dinoflagellates accumulate the toxins, but they are rather resistant to the harmful effects of these compounds. A concentration of as little as 200 cells/mL in seawater results in toxicity in shellfish. Shellfish can filter up to 20 L of water/day and, in continuous contact with the red tide (algal blooms containing 20,000 to 50,000 cells/mL), can ingest 3.0 g dry weight of the dinoflagellates in 24 hr and can contain as much as 180 mg/g toxin. Levels greater than 8 µg toxin/100 g of shellfish meat are considered unsafe for consumption (Shantz, 1975). Apart from algal blooms, other routes may also lead to contamination of shellfish with PSP. One of the PSPproducing organisms, Alexandrium tamarensis (formerly Gonyaulax tamarensis), which occurs particularly in European waters, may produce toxin-containing cysts. As a result of their high toxin concentration, these cysts may be a source of contamination of shellfish with PSP. Chemical Characteristics The best known PSP, saxitoxin (STX), was first isolated from the Alaska butter clam, Saxidomonus giganteus, collected in certain areas in Alaska where the shellfish remain toxic year-round (Schantz et al., 1957). The same toxin was later isolated from mussels exposed to G. catenella blooms and from the cultured cells of G. catenella (Schantz et al., 1966). For the next 20 years, saxitoxin was believed to be the sole toxin involved in PSP, although Schantz (1960) observed that the toxin in Canadian east coast scallops could not be purified by the method applicable to the purification of saxitoxin. In 1975, Shimizu and associates isolated four new toxins, named gonyautoxins, from soft-shell clams exposed to a red tide caused by Gonyaulax tamarensis. Buckley and colleagues (1976) also reported the presence of two new toxins in a similar sample. These were the first demonstration that the PSP toxin is actually heterogeneous and has diverse sources. The PSP toxins are now known to be produced by a completely different dinoflagellate, Pyrodinium bahamense var. compressa (Kamiya and Hashimoto, 1978; Harada et al., 1982a, 1982b). It is responsible for PSP in South Pacific waters. There was an earlier report that the toxins found in the blue-green alga Aphanizomenon flosaquae resemble saxitoxin (Jackim and Gentile, 1968). The toxins, called aphantoxins, isolated from a Kezar lake strain were later identified as saxitoxin analogs (Shimizu et al., 1977; Alam et al., 1978; Ikawa et al., 1982). The major toxins found in a different strain of alga from New Hampshire were identified as neosaxitoxin and saxitoxin (Ikawa et al., 1982; Shimizu et al., 1984a). The toxins,

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both saxitoxin and gonyautoxin analogs, were also found in the calcerous macro red algae belonging to Jania spp. (Kotaki et al., 1983). The random occurrences of these rather specific toxins among distantly related organisms have been a puzzling question to many researchers. Consequently, most interest is now focused on finding a possible common denominator that is transferable among those PSP-producing organisms. It is quite possible that an endosymbiotic organism is toxigenic (Shimizu, 1988). A systematic study of the distribution of toxic strains of Gonyaulax spp. along the northeast coast of the United States seems to imply that toxigenicity of Gonyaulax spp. is an intrinsic property of certain strains or subspecies (Maranda et al., 1985). In any event, it is quite remarkable that such a structurally unique class of compounds is distributed in taxonomically unrelated organisms, both eukaryotic and prokaryotic. The chemical structures of several PSPs identified and characterized thus far are shown in Figure 14.4. They consist of the parent compound saxitoxin (STX) and the 11 derivatives formed by the addition of N-1-hydroxyl, 11hydroxysulfate, and 11-sulfo groups. Saxitoxin and its hydroxy derivative, neosaxitoxin, are water-soluble neurotoxic alkaloids. The basic molecule is a tetrahydropurine substituted at various positions. The STX molecule has several interesting features. First, its perhydropurine skeleton with an additional five-membered ring fused at the angular position is unprecedented. It has a ketone hydrate stabilized by two neighboring electron-withdrawing guanidinium groups. The ketone is also easily enolized to effect the rapid exchange of protons at the C11 position. STX is very stable in acidic solutions and can be kept in dilute hydrochloric acid solutions for years without loss of its activity or potency (Shimizu, 1988). Neosaxitonin (NeoSTX) was first isolated as a minor component in an Alaska butter clam sample (Oshima et al., 1977). Later it was proved to be a major component in most toxic shellfish, dinoflagellates, blue-green algae, and crab samples. Its chemical structure is 1-N-hydroxysaxitoxin. The reduction of neoSTX with zinc-acetic acid results in the reductive cleavage of the N-hydroxyl group to give STX. Gonyautoxin-1 (GTX-1) was similarly isolated from soft-shell clams exposed to G. tamarensis blooms (Shimizu et al., 1975). It is now known to be present as a major component in many PSP samples and causative organisms. Its structure was established on the basis of spectroscopic data and chemical correlation with GTX-2, neoSTX, and STX. Thus, the reduction of GTX-1 gives a mixture of neoSTX and GTX-2, which can be further reduced to

STX. GTX-1 and its stereoisomer, GTX-4, are probably the most unstable among the PSP toxins. GTX-2 is a sulfate ester of STX, whereas GTX-3 is the 11-epimer of GTX-2. These two compounds are always found concurrently and form a 7:3 equilibrium mixture in a solution of neutral or higher pH. GTX-4 is the 11-epimer of GTX-1. The relationship of these two compounds is parallel to that of GTX-2 and GTX-3 (Shimizu, 1988).

H2N 14

O

GTX-5 is the carbamoyl-N-sulfate of neoSTX and identical with B1 toxin. It was first isolated from Japanese and Alaskan PSP samples (Oshima et al., 1976; Hall et al., 1980). It is the major toxin in the tropical dinoflagellate P. bahamense var. compressa (Harada et al., 1982a, 1982b). This almost nontoxic substance can be hydrolyzed with a weak acid to give rise to very toxic neoSTX. GTX-6 is the carbamoyl-N-sulfate of STX and identical with the B2 toxin described by Koehn and coworkers

H2N H

13

O

H

H

H N

6

HN 1 2

5

7

4

9

3

8

12

10 11

H

NH2 N H

OH

OH

OH

OH

H

H

H

B. Neosaxitoxin (neoSTX)

O

H2N H

O H

H

H N

O HON

H N

N

H2N

A. Saxitoxin (STX)

H2N

H

O HON 1

NH2

N H

N

H2N

O

H

H N

O HN NH2

H2N

NH2

N H

N

N H

N

H2N OH

OH 11

OH O3SO

OH

H

O3SO

C. Gonyautoxin-1 (GTX-1)

H2N

D. Gonyautoxin-2 (GTX-2)

H 2N

O H O HN

H

N H

N

O H

H N NH2

H2N

H

O HON 1 H2N

H

H N NH2

N

N H OH

OH 11

OH

OH H

OSO3

E. Gonyautoxin-3 (GTX-3)

Copyright 2002 by Marcel Dekker. All Rights Reserved.

H

OSO3

F. Gonyautoxin-4 (GTX-4)

(1982). On acid hydrolysis, the N-sulfate bond is cleaved to yield STX. GTX-8 is the carbamoyl-N-sulfonyl derivative of GTX-3. It is easily converted to GTX-3 with brief acid treatment. GTX-7, first found in a toxin mixture from the sea scallop, Placopecten magellanicus (Hsu et al., 1979), is decarbamoyl neoSTX. Similarly, the C3 and C4 toxins were speculated to be sulfonyl derivatives of GTX1 and GTX-4, respectively. Both toxins easily release free GTX-1 and GTX-4 on treatment with dilute acids.

O3SHN

O3SHN

O H O HN

H

The biotransformation pathways leading to the conversion of GTX family toxins to STX in various shellfish are shown in Figure 14.5; (A1) and (A2) are postulated precursors of GTX-1 and GTX-4, respectively. It is quite possible that these biotransformation processes also occur in the dinoflagellate itself, since trace quantities of the GTXs and neoSTX have been detected in the dinoflagellate extract (Hall et al., 1980). Furthermore, Shimizu and associates (1975) detected the same toxins in both the di-

O

HO H

H N

O HON

H

H H N

H2N

N

N H

H2N

N

N H

H2N

OH OH H

G. Gonyautoxin-5 (GTX-5, B1 toxin)

O3SHN H

O H

H

H N

O HN

H

H N

O HN

NH2

NH2 N H

N

H2N

H2N

N H

N

OH

OH 11

11

OH

OH O3SO

OSO3

H

J. Gonyautoxin-8 (GTX-8)

O

H

K. Epigonyautoxin-8 (epiGTX-8)

O3SHN H

O H

H

H N

O HON

O HON

H

H N

NH2 N H

N

NH2 H2N

N

N H

OH

OH

OH O3SO

H

I. Gonyautoxin-7 (GTX-7)

H. Gonyautoxin-6 (GTX-6, B2 toxin)

O

H2N

N H

N

OH

OH

O3SHN

NH2

OH

OH

O3SHN

H N

HON 1

NH2

NH2

H

H

L. C3 toxin

OH H

OSO3

M. C4 toxin

Figure 14.4 Chemical structures of saxitoxin, the parent compound implicated in paralytic shellfish poisoning (PSP), and its analogs.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

Figure 14.5 Biotransformation pathways leading to the conversion of gonyautoxin (GTX) family toxins to saxitoxin (STX) in various shellfish.

noflagellate (G. tamarensis) and the clams exposed to it. Also, it is rather unusual that G. catenella has been shown to contain only STX (Schantz et al., 1957, 1966), whereas some clams in the area (Mytilus spp.) where the dinoflagellate was found contained the GTX family of toxins in addition to either STX or neoSTX (Shimizu et al., 1978). Certain clams in this area have very low or negligible toxicity (Schantz and Magnusson, 1964), whereas others, like the Alaskan butter clam (Saxidomus giganteus), may retain a significant amount of STX for prolonged or indefinite periods (Schantz and Magnusson, 1964). All these observations may be explained in part if it is assumed that G. catenella as well as the Alaskan butter clam biotransform the GTX family of toxins to STXs in a manner similar to that shown in Figure 14.5. Other shellfish, such as Mytilus spp., are not as efficient as the butter clam or do not have the capability for being so. It is quite possible that these biotransformation processes may also occur in the dinoflagellate itself. The biotransformation pathway from GTX-1 or GTX-4 to STX involves a reductive elimination of N-OH, a relatively common biochemical reaction, and a desulfation reaction. The latter is the reverse of the usual biochemical process, i.e., sulfate conjugation. Such biotransformation reactions at least partly explain the anomalous variability of the presence and proportions of these PSPs in shellfish in neighboring areas. The synonyms and abbreviations of various PSPs isolated and characterized and used in the literature by various researchers thus far are summarized in Table 14.8.

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Biosynthesis Some information is known on the biosynthesis of the PSPs. The guanidine moiety of arginine was found to be incorporated into the carbamoyl moiety and presumably the two guanidine groups in GTX-2 (Shimizu, 1982; Shimizu et al., 1984a). Subsequently, 13C-labeled glycine was found to enrich C11 and C12 of the GTX molecule (Shimizu, 1982; Shimizu et al., 1984b). The result was interpreted to support the arginine precursor hypothesis proposed earlier. Feeding experiments using 13C-labeled acetic acid showed differential incorporation of two acetate units into C5–C6 and C10–C11 positions (Shimizu et al., 1984c). These results indicated that the ring skeleton of the toxin is formed by Claisen-type condensation of an acetate unit on the nitrogen-bearing α-carbon of arginine, followed by decarboxylation, addition of a guanidine group, and cyclization. This synthetic scheme was later confirmed by a feeding experiment using 15N, 13C double-labeled ornithine to the culture of A. flos-aquae (Shimizu et al., 1984c). The origin of the side chain carbon C13 has been attributed to the methyl group of methionine with concomitant migration of a hydride ion and elimination of two of the hydrogens on the methyl group (Shimizu et al., 1985; Shimizu, 1986). However, it remains to be seen why only certain strains of dinoflagellates or blue-green algae acquire toxigenicity. Mode of Action The primary mode of action of PSPs in mammals is their binding readily to sodium channels in nerve cell mem-

Table 14.8 Synonyms and Abbreviations of Paralytic Shellfish Poisons Appearing in the Literature Paralytic shellfish poison

Synonyms used 11-Hydroxyneosaxitoxin-11-O-sulfate

Abbreviations

Saxitoxin Neosaxitoxin Gonyautoxin-1 Gonyautoxin-2 Gonyautoxin-3

11-Hydroxysaxitoxin 11-Hydroxysaxitoxin

STX NeoSTX GTX-1, GTX GTX-2, GTX2 GTX-3, GTX3

Gonyautoxin-4 Gonyautoxin-5

11-Hydroxyneosaxitoxin-11-O-sulfate B1, carbamoyl-N-sulfosaxitoxin

GTX-4, GTX4 GTX-5, GTX5

Gonyautoxin-6

B2, carbamoyl-N-sulfoneosaxitoxin

GTX-6, GTX6

Gonyautoxin-7 Gonyautoxin-8

Decarbamoylneosaxitoxin C2, carbamoyl-N-sulfogonyautoxin-4

GTX-7, GTX7 GTX-8, GTX8

Epigonyautoxin-8

C1, carbamoyl-N-sulfogonyautoxin-2

EpiGTX-8

C3 C4 Decarbamcylsaxitoxin

Carbamoyl-N-sulfogonyautoxin-1 Carbamoyl-N-sulfogonyautoxin-4

branes at nanomolar concentrations (Hille, 1975). This leads to inhibition of impulses along the nerves, resulting in paralysis, respiratory depression, and circulatory failure. The profound paralytic effects of PSPs and the observed decrease in toxicity in the presence of sodium ions are consistent with the effects of these toxins on the nervous system. The action on the central nervous system is shown by the effects on the respiratory and vasomotor centers (Kao, 1966; Murtha, 1960). The action on the peripheral nervous system is due to the effects on the neuromuscular junctions, cutaneous tactile endings, and muscle spindles (Sapeika, 1953). In addition to peripheral transmission, reflex transmission is also completely depressed by these toxins. After PSP, respiration may immediately cease totally or be depressed slowly, depending on the dose. The depression may be characterized by dyspnea with gasping action or short rapid exhalations. These effects appear to be the result of the paralysis of diaphragm muscles. Several researchers have shown that the principal target of the PSPs is the sodium channel in the most excitable membranes, i.e., nerves and muscles (Evans, 1964, 1965; Kao, 1972; Kao and Nishiyama, 1965; Hille, 1975; Narahashi, 1972). The toxins have high specificity and binding capacity (Ka = 10–9 for STX) to the sodium channels. The toxin molecules block the channels to the early entry of sodium ions. Thus, the initial increase in sodium

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Decarbamoyl-1-STX

Reference Schantz et al. (1957) Shimizu et al. (1975) Oshima et al. (1977) Shimizu et al. (1975) Shimizu et al. (1975) Buckley et al. (1976) Shimizu et al. (1975) Oshima et al. (1977) Hall et al. (1980) Oshima et al. (1976) Hall et al. (1980) Hsu et al. (1979) Shimizu (1980) Hall et al. (1980) Kobayashi and Shimizu (1981) Hall et al. (1980) Hall et al. (1980) Hall et al. (1980) Sullivan et al. (1983) Harada et al. (1983)

permeability usually associated with excitation is interfered with, so that the propagation of the impulses in nerve and muscles is blocked without depolarization. The resulting membrane conductances due to potassium and chloride ions are not affected. The action of STX and other PSPs is similar to that of the puffer fish toxin, tetrodotoxin (described in Section 14.3.6) (Ritchie, 1980). Although most studies on the mechanism of action have been carried out with STX, neurophysiological studies with other PSPs indicate that all act by the same mechanism; only their potency differs. Analytical Methods In addition to the use of PSPs as research tools, analytical methods are necessary to support the toxin-monitoring programs in several areas of the world impacted by PSP. To ensure a safe year-round supply of shellfish, it is necessary to monitor the total toxicity of bivalve shellfish continually, especially during the summer months. Generally, when values reach a quarantine level, the area is closed to both recreational and commercial shellfish harvesting and not reopened until levels drop again. Shellfish harvesting is prohibited when assay results reach 80 µg toxin/100 g shellfish meat. These monitoring programs generally rely on intensive sampling protocols that require a rapid, accurate analytical method for the toxins. The monitoring programs have generally been very successful in controlling

PSP, and the majority of illnesses are caused by recreational activities. The toxicity of extracts and purified toxins has generally been determined by using the mouse bioassay first developed by Sommer and Meyer (1937). The relationship between toxin concentration and time of death in mice after intraperitoneal injection is logarithmic. The weight of the mice affects time of death, as lighter mice exhibit significantly faster time of death. Nevertheless, a semiquantitative assay procedure using the MU has been developed. The toxicity of STX is 5494 MU/mg of toxin; therefore, 1 MU equals 0.18 µg STX dihydrochloride. The pH of the injected solution must be below 4.5. The solution is diluted as appropriate to give a median death time close to 6 minutes. Used in this manner, the test does not rely heavily on the shape of the dose-response curve, as it is based on the titration to a 6-min time of death and is therefore internally consistent despite variations in sample toxin composition. MU is determined from Sommer’s table, and concentration of poison (µg/100 g) is calculated from MU. The overall toxicity of a given sample of shellfish is the sum of the products of the separate toxicities multiplied by the quantities of each toxin present in the sample. Any sample with >80µg/100 g (equivalent to 400 MU/100 g) is considered hazardous and unsafe for human consumption. The original bioassay developed by Sommer and Meyer (1937) was later modified by Medcof and associates (1947), and when pure toxin became available (Schantz et al., 1958), it was subjected to a collaborative study (McFarren, 1959). This bioassay was subsequently adopted in 1958 by the Association of Official Analytical Chemists as an Official Procedure (Williams, 1984) and is

in use today as the primary analytical technique to support the majority of toxin-monitoring programs in shellfish. The toxicities of the various PSPs expressed in mouse units are summarized in Table 14.9. Among these, STX and GTX-3 are the most toxic. Thus the degree of toxicity of a shellfish sample depends on the proportions of the different PSPs. Evidently, those containing greater quantities of STX or GTX-3 are more toxic. As mentioned earlier, because of the biotransformation processes, most of the GTX and neoSTX are ultimately converted to STX, so that the level of the latter in shellfish increases with time. Also, cooking may hasten the conversion of the protoxins to more toxic forms. Therefore, the degree of toxicity of the shellfish at the table may depend on whether significant leaching of the toxin during cooking occurs. The LD50 values for STX are summarized in Table 14.10. The human lethal dose is estimated to be about 1 mg. The FDA has established the limit of maximal human tolerance for PSPs at 1200 MU/100 g shellfish or 2500 MU/meal. PSP toxins lack native fluorescence, useful ultraviolet (UV) absorption, and adequate volatility, making traditional analytical procedures such as gas chromatography or spectrometry ineffective in assaying for them. A sensitive fluorometric assay based on alkaline oxidation of PSP toxins to fluorescent derivatives is available (Bates and Rapoport, 1975). This assay appears to be 100 times more sensitive than the traditional mouse bioassay. Immunoassays have also been described for the detection of PSPs (Johnson and Mulberry, 1966; Chu and Fan, 1985). However, since the toxicity of a shellfish extract is due to the collective effect of a number of different toxins present,

Table 14.9 Toxicities of Paralytic Shellfish Poisons Paralytic shellfish poison STX Neo-STX GTX-1 GTX-2 GTX-3 GTX-4 GTX-5

Mouse Unitsa (MU)/µmole

Specific toxicity MU/mg

Relative toxicity

2045 ± 126 1038 ± 44 1638 ± 128 793 ± 83 2234 ± 137 673 ± 38 354 ± 19

5492 ± 339 2363 ± 101c 3976 ± 312d 2003 ± 211d 5641 ± 346d 1634 ± 92d NDe

100 43 72 36 103 30 NDe

a

b

See text for the definition of MU (mouse unit). Weighed as dihydrochloride. c Weighed as diacetate. d Weighed as monoacetate. e Not determined Source: Compiled from Genenah and Shimizu (1981) and Shimizu (1988). b

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Table 14.10 LD50 Values for Various Paralytic Shellfish Poisons Species Human (oral) Pigeons Guinea pigs Dogs and rabbits Rats Cats Mice Monkeys Rabbits Humans

LD50, µg/kg body weight 1–10 91 135 181 192 254 382 364–727 3–4a 3000–5000b

a

Intravenous lethal dose. Lethal oral dose from accidental causes. Source: Compiled from Kao (1966), McFarren et al. (1960), and Schantz (1973). b

the application of immunoassays for accurate detection of “total toxicity” of the PSPs is very difficult. Other analytical approaches for the measurement and quantitation of PSPs include electrophoretic and high-performance liquid chromatography (HPLC) techniques. The pharmacological activity of the PSPs at the molecular level has also been exploited in developing assay techniques. The toxins bind to sodium channels in nerve cell membranes, preventing the influx of sodium and subsequent depolarization of the membrane. A number of electrophysiological systems have been utilized for this purpose, including measurement of the compound action potential of the frog sciatic nerve (Strichartz, 1984), voltage clamp of single nerve cells (Frace et al., 1986), and blockage of sodium conductance through single sodium channels isolated in lipid bilayers (Moczydlowski et al., 1984). These techniques are useful for determining the pharmacological properties of the toxins. However, because of the specialized techniques and instruments required, they are seldom used as routine assay techniques.

5–30 min of consumption, sometimes followed by headache, thirst, nausea, and vomiting. The tingling and burning sensation and numbness then spread to the neck, arms, and legs, and general muscular incoordination ensues. Incoherent speech and aphonia along with paresthesia are seen in all of the cases reported and are pathognomonic. The paresthesia progresses to the neck and extremities in most patients. This is accompanied by dizziness, ataxia, dysmetria, and a floating sensation (cerebellar syndrome). Paresis or paralysis follows. With the onset of respiratory paralysis, cyanosis ensues. Abdominal distention and urinary retention are seen on physical examination. In patients who survive intoxication, asthenia and moderate memory loss occur for up to 3 weeks. Other signs of PSP include weakness, malaise, prostration, headache, hypersalivation, intense thirst, perspiration, dysphagia, myalgia, tachycardia, abdominal cramps, diarrhea, and anuria. Most intoxicated patients remain calm and conscious. Death occurs within 2–25 hr or the patient recovers. Survival for 12–18 hr is a good prognostic sign (McCollum et al., 1968; CDC, 1978, 1983; Roy, 1977; Concon, 1988; Smith, 1992; Leftley and Hannah, 1998). The levels at which PSP intoxications have been reported to occur in humans vary from 144 to 1660 µg PSP/person, expressed as saxitoxin-equivalents, whereas fatal intoxications were reported after a calculated consumption of 456–12,4000 µg PSP/person (van Egmond et al., 1993; van Egmond and Speijers, 1999). There is no antidote for PSPs. Symptomatic treatment to promote emesis, use of absorbents to bind the poison (e.g., charcoal, Lloyd’s reagent), diuresis with 5% ammonium chloride, and anticurare drugs have all been recommended, some with varying degrees of success. Artificial respiration is an important adjunct to treatment (Halstead and Courville, 1965; Concon, 1988). It appears to yield good results experimentally and in mild cases but is of no avail in severe intoxications. Variation in Toxicity

Symptoms The symptoms of PSP poisoning in humans are very characteristic and range from a tingling sensation or numbness as a result of mild intoxication to death through respiratory paralysis in extreme cases. The onset of symptoms usually occurs within 3 hr of ingestion of the toxic shellfish, although this can vary from 15 min to 10 hr, depending on the quantity of ingested toxin and other factors such as age and weight of the patient, alcohol consumption, and source of contamination (e.g., broth, meat). The tingling sensation around the lips, gums, and tongue develops within

Copyright 2002 by Marcel Dekker. All Rights Reserved.

The specific toxicity of these compounds varies significantly. For example, the most potent are the carbamate toxins (STX, neoSTX, and GTX-1 to GTX-4), which are 10 to 100 times more toxic than the N-sulfocarbamoyl derivatives, the B and C toxins (Cembella et al., 1993; Wright, 1995). PSP toxins may form up to 0.2% of the wet weight of PSP-toxic dinoflagellates, and the most predominant toxins are usually the sulfated derivatives (Laycock et al., 1994). However, some of these compounds may undergo degradation or biotransformation after being assimilated by shellfish and other organisms (Cembella et al.,

1994; Oshima, 1995; Wright 1995; Bricelj et al., 1996; Leftley and Hannah, 1998), and novel derivatives are occasionally reported (Gago-Martinez et al., 1996). Intoxicated shellfish that grow in cold or temperate waters most commonly contain the sulfated C toxins GTX-2 and GTX-3, and STX (Wright, 1995). That sulfamate STXs have very low potencies relative to their carbamate hydrolysis products has been shown in every assay system tried, including the standard mouse bioassay, squid giant axon, frog sciatic nerve, mammalian brain, and single rat sarcolemma sodium channels incorporated into lipid bilayers. It seems unlikely that human oral potencies (HOPs) are an exception to this trend. Although the sulfamates themselves probably have low HOP, they can hydrolyze at low rates to the more toxic carbamates under conditions of food storage, preparation, or digestion. It is also likely that the latent sulfamate toxicity can also be potentiated through enzymatic conversion in some shellfish to the corresponding decarbamoyl toxins. It is therefore advisable to use the potential HOP, the HOP that product might attain under a worst-case scenario of conversions, for describing the PSPs for public health protection. Control Because PSPs do not affect the physiological characteristics of the shellfish, there are no characteristics that distinguish between poisonous and nonpoisonous specimens to serve as a guide to a person collecting them. The most practical means for the detection of STX or other PSPs in shellfish products is the bioassay with mice. Control of PSP poisonings is also very difficult, principally because of the wide geographical occurrence. Toxins are accumulated rapidly in shellfish, and in some species, depuration is also very slow, if it occurs at all (Pugsley, 1939; Prakash et al., 1971). The rapid kinetics of accumulation and apparent equilibrium in favor of toxin retention when coupled with the high oral potency of some of the PSPs are sufficient to translate PSP accumulation in potential human food sources to a real public health hazard. The potency of the suspect food source is largely dependent upon bloom characteristics and accumulator species. The wide multiplicity of toxins present in the toxigenic organisms and the propensity of accumulator for altering the toxin complement assimilated make precise measurement of public health significance quite difficult. PSPs can also be assimilated from resting cysts of the toxigenic organisms. The absence of dinoflagellates in the water may also not totally guarantee the safety of shellfish in the area, particularly if the part of the sea has just experienced a red tide. Natural detoxification processes may take more than 1 month to rid shellfish of the toxins. Har-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

vesting shellfish during the winter months might minimize poisoning, since in most cases, toxicity is much lower during this period. Steaming or cooking does not destroy STX, but because it is water-soluble, it concentrates in the cooking broth. Since STX and other PSPs are very water-soluble, the broth in which the shellfish is cooked therefore should contain a significant amount of the toxins and should be discarded. PSP toxins are heat-stable in acid but can be destroyed in alkali. Because they are unstable in alkaline medium, adding sodium bicarbonate to the cooking medium and heating for 20 to 30 minutes are often recommended (Sommer and Meyer, 1941). However, although this method destroys over 85% of the toxins, it produces an undesirable flavor. In spite of this reduction in toxicity, the shellfish may retain enough toxins to cause serious poisoning. During the last three decades, there has seemed to be an increase in PSP poisoning in both tropical and moderate climatic zones. As yet it is not clear whether the increase is real; whether it could be a consequence of improved identification, detection, and medical registration; or whether it is due to expanded shellfish culture and consumption (van Egmond and Speijers, 1999). Currently a toxicological risk evaluation for PSPs can only be based on the acute toxicity data in humans. Because the acute toxic effects of PSPs include fatality and show great variability, the lowest published lethal dose level should be used for safety evaluation. Currently, a provisional dose level that would not cause mortality could be calculated in principle. However, the present data concern observations in adults but not in children, who are claimed to be more vulnerable. Moreover, toxic effects other than mortality would not be considered in such a risk evaluation. If the present lowest published toxic dose level were used to establish a tolerance level, the limit for PSPs in mussels would be lower than the limits currently used. The PSP tolerance levels and official assay and/or analytical methods used in various countries are listed in Table 14.11. When shellfish resources are well developed, intensive monitoring has proved cost-effective and has permitted flexible management so as to optimize the areas of shellfish open to harvesting. This strategy has been used in France (Berthome and Lassus, 1986), Japan (Yamamoto and Yamasaki, 1996), New Zealand (Trusewich et al., 1996), and Maine in the United States (Shumway et al., 1988). In contrast, when shellfish resources are limited or underexploited, it may only be cost-effective to impose widespread closures in a region, with little or no monitoring. Such is the case in California, where the harvesting of mussels is prohibited every year between April and October regardless of the level of toxicity (ICES, 1992; Hungerford and Wekell, 1993).

Table 14.11

Paralytic Shellfish Poison Tolerance Levels and Official Assay and/or Analysis Methods in Various Countries

Country Australia Canada European Union (EU)c Guatemala Hong Kong Japan Korea New Zealand Norway Panama Singapore Sweden United States

Product

Toxin

Shellfish Mollusks Bivalve mollusks Mollusks Shellfish Bivalves Bivalves Shellfish All types of mussels Bivalves Bivalves Mollusks Bivalves

Saxitoxin PSPa PSP Saxitoxin PSP PSP Gonyautoxins PSP PSP PSP Saxitoxin PSP PSP

Tolerable level (per 100 g soft tissue)

Method of assay or analysis

80 µg <80 µgb 80 µg 400 MUe 400 MU 400 MU 400 MU 80 µg 200 MU 400 MU 80 µg 80 µg 80 µg

Mouse bioassay Mouse bioassay Mouse bioassayd Mouse bioassay Mouse bioassay Mouse bioassay + HPLC Mouse bioassay + HPLC Mouse bioassay Mouse bioassay Mouse bioassay Mouse bioassay Mouse bioassay Mouse bioassay

a

PSP, paralytic shellfish poisoning. In Canada products having levels between 80 and 160 µg/100 g before closure of fisheries may be canned. c European Union (EU) countries include Belgium, France, Germany, Greece, Ireland, Italy, Luxembourg, the Netherlands, Portugal, Spain, and the United Kingdom. d In the majority of countries within the EU, the mouse bioassay is used. However, the Netherlands uses high-performance liquid chromatography (HPLC) alone and Denmark and the United Kingdom supplement the bioassay with HPLC. e 1 Mouse unit (MU) = approximately 0.18 µg saxitoxin. Source: Compiled from Shumway (1995), Andersen (1996), and Leftley and Hannah (1998). b

14.3.2

Diarrhetic Shellfish Poisoning

Diarrhetic shellfish poisoning (DSP) is distinctly different from PSP in both symptoms and causes. Unlike in PSP, the predominant human symptoms of DSP are gastrointestinal disturbances, and no fatal cases have been reported. Nevertheless, the high morbidity rate and global distribution of DSP make it a serious threat to both public health and the shellfish industry. Even though diarrhea is the most characteristic symptom of intoxication, several other effects may be of relevance. Also, some of the toxins in the DSP group do not cause diarrhea at all. Consequently, the term diarrhetic shellfish poisons is a misnomer for the whole group of structurally related toxins (van Egmond et al., 1993; van Egmond and Speijers, 1999). It is actually the absence of symptoms, especially those of neurotoxicity (e.g., paralysis) that implicates DSP rather than other marine intoxications. DSP is also easily distinguished from bacterial infection by its rapid onset time and heat stability. DSP can result from consumption of filter-feeding shellfish, especially mussels and clams. In Japan, it was first reported in 1976 as a consequence of eating mussels and scallops harvested from the Tohuku District (Yasumoto et al., 1978, 1979). The illness, a predominantly selflimiting but severe gastroenteritis, is now well recognized as a worldwide threat to human health, affecting many

Copyright 2002 by Marcel Dekker. All Rights Reserved.

thousands of consumers of molluscan shellfish and posing great economic hardships on the shellfish industry. Epidemiological records indicate 1300 persons were affected between 1976 and 1982 in Japan (Yasumoto et al., 1984), 5000 cases in Spain in 1981 alone (Campos et al., 1982), and an ever-increasing number of reported cases in the United States each year. Infrequent intoxication is also observed in the Netherlands (Kat, 1985) and Chile (Gervais and MacLean, 1985). DSP is now recognized as the second most widespread intoxication of humans and shellfish caused by algae. The DSPs are a group of toxins produced mainly by dinoflagellates belonging to the genera Dinophysis and Prorocentrum. These occur in several marine environments around the world. The causative organisms include D. acuminata, D. acuta, D. fortii, D. norvegica, D. mitra, D. rotundata, D. tripos, and P. lima. Difficulties in culturing Dinophysis spp. under laboratory conditions and their low population densities in the sea make the assignment of a species to a particular DSP outbreak challenging and somewhat controversial. Cell densities of D. fortii as low as 200 cells/L have been associated with shellfish toxic to humans (Yasumoto et al., 1978). The DSP complex consisting of at least 12 toxins can be separated into three different groups of polyether compounds exerting toxicity. One group consists of oka-

daic acid (OA) and dinophysis toxin (DTX). The most important of this group of acidic toxins as regards shellfish contamination are OA, DTX-1, and DTX-2 (Figure 14.6), which may occur singly or together. The polyether backbone of all three toxins may be acylated in the digestive glands of shellfish to produce a mixture of compounds with diarrhetic activity, collectively known as DTX-3, which have fatty acid side chains (Quilliam and Wright, 1995; Wright, 1995). Tachibana and coworkers (1981) first isolated OA from the Pacific sponge Halichondria okadai and the Caribbean sponge Halichondria melanodocia. Later this toxin was found in the free-living microalga Prorocentrum lima (Murakami et al., 1982). Polyether toxins related to OA were also isolated from other sponges (Yasumoto et al., 1985). A number of other OA derivatives are also known to occur, including diol esters that do not inhibit protein phosphatases. These esters are labile and might undergo chemical or biochemical transformation to toxic derivatives (Quilliam and Wright, 1995). Two sulfated derivatives of DTX, DTX-4 and DTX-5, which are unusual in being water-soluble, have also been isolated from the benthic dinoflagellate Prorocentrum lima (Hu et al., 1995a, 1995b; Quilliam et al., 1996). Their significance in shellfish poisoning, however, is presently unknown. The second group of DSP toxins comprises neutral macrolide polyether lactones. These are called pectenotoxins (PTXs). Seven PTXx have been isolated from algae or shellfish, and four, PTX-1, PTX-2, PTX-3, and PTX-6, have so far been chemically characterized (Figure 14.7). PTX-2 has only been found in the dinoflagellate Dinophy-

sis fortii and never in shellfish. All other PTXs appear to be derived from it by metabolic reactions in the hepatopancreas of shellfish (Yasumoto et al., 1989; Draisci et al., 1996). Pectenotoxins are hepatotoxic to mice and lead to necrosis after intraperitoneal injections. The final group of DSP toxins are yessotoxins (YTXs), so called because they were originally isolated from the scallop Patinopecten yessoensis. They resemble the brevetoxins and ciguatoxins (described in Sections 14.3.4 and 14.3.5) in that they have contiguously fused ether rings (Figure 14.8) but differ in that they have a longer backbone of 47 carbon atoms, a terminal side chain of 9 carbons, and 2 sulfate esters and lack carbonyl groups (Murata et al., 1987; Takahashi et al., 1996). Two derivatives of YTX have been described, 45-hydroxyYTX and 45,46,47 trinorYTX (Yasumoto et al., 1989; Satake et al., 1996). Which type of toxin(s) is (are) produced by the algae is in part geographically determined. DTX-1 is the most common DSP toxin in mussels in Japan, whereas OA is reported to be the major DSP toxin in Europe. Studies of Norwegian mussels show a more complex picture: DTX-1 predominates in the Sognefjord, whereas OA is the major DSP toxin at other locations along the Norwegian coast (van Egmond and Speijers, 1999). The DSP toxin profile is thus diverse and dynamic, resulting in both spatial and temporal variations. Ratios of OA to DTX-1 in Dinophysis spp. cells show significant intraspecies variation (Yasumoto, 1990). Toxin profiles also vary by region, by season, and even within a given area, from year to year (Yasumoto and Murata, 1990). In fact, levels of toxins (e.g., OA) can vary in adjacent shellfish

CH3 OH

O

2

O

O

HO H3C

12 7

OH

R1

O

16

O OR3

H

31

CH3

O

O

H OH

DSP Toxin Okadaic acid (OA) Dinophysistoxin-1 (DTX-1) DTX-2 DTX-3

R1 CH3 CH3 H

R2 H CH3 CH3 H or CH3

O

26

CH3

35

R2

R3 H H H Acyl

Figure 14.6 Chemical structure of okadaic acid (OA) and some related compounds implicated in diarrhetic shellfish poisoning (DSP).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O O

O O

O

O O

OH

HO OH

O O

R

O

O

Pectenotoxin (PTX)

R CH2OH CH3 CHO COOH

PTX-1 PTX-2 PTX-3 PTX-6

Figure 14.7 Chemical structures of pectenotoxins (PTXs) implicated in diarrhetic shellfish poisoning (DSP).

R HO O

O O

O O NaO3SO

O

O

O

O NaO3SO

O

O

Figure 14.8 Chemical structures of yessotoxin (YTX) (R = H) and 45-hydroxyyessotoxin (45-OHYTX) (R = OH).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

samples, such as mussels, at a given sampling site; shellfish at the same site but at different depths in the water column also show differences in total DSP levels (Edebo et al., 1988). The highest toxicity was obtained in mussels from the upper level (3–6 m) whereas toxicity was reduced to half that level at 6–8 and 8–12 meters. The dynamics of the toxin profile are mediated by toxin removal and possibly by toxin transformation. Since only OA, DTX-1, and PTX-2 were found in Dinophysis spp. cultures and other analogs found in scallops were absent in the algae (Lee et al., 1989), Yasumoto (1990) suggested that acylation of DTX-1 to DTX-3, and a series of oxidations of the 43-CH3 of PTX-2 to PTX-1 (CH20H), PTX-3 (CHO), and finally to PTX-6 (COOH) occur in the scallop hepatopancreas. The organism that produces YTX remains unknown. The rate of removal of toxin from shellfish (depuration rate) most likely depends upon the species and may be affected by such interrelated factors as the feeding or pumping rate of the shellfish, temperature, salinity, and level of nontoxic algae and particulates. In Japan with the use of intact mouse bioassays, DSP toxins decreased from 4.4 to 2.5 MU in 1 week, and then to 0.5 MU/g by the next week (Yasumoto et al., 1978). In the Netherlands, toxicity in mussels (Mytilus edulis) was no longer detectable by rat bioassay after 4 weeks at water temperatures of 14°C– 15°C (Kat, 1983). In the North Sea off the coast of Sweden (water temperatures 1°C–3°C), mussels in 1 week became toxic quite rapidly (0.4–5.4 µg OA/kg hepatopancreas). After the bloom had subsided, OA levels in mussels decreased in 1 week from 7.2 to 1.8 µg/kg as measured by HPLC fluorescence detection (Edebo et al., 1988). These data show that in endemic areas, weekly sampling may not be sufficient for maximal protection of public health. Most of the methods developed for the detection and/or quantification of DSP toxins identify the presence or the physiological action of OA toxins. Methods developed to date include animal bioassays, suckling mouse assay, intestinal loop assay, cytotoxic assay, immunological assays (radioimmunoassay [RIA], enzyme-linked immunosorbent assay [ELISA]), gas chromatography, and HPLC (fluorescence, UV detection). Hungerford and Wekell (1992) have reviewed the various analytical methods used for the quantitation of DSP toxins. Gastrointestinal disturbance is the main effect of DSP. The symptoms usually disappear within 3 days. In a typical DSP incident, the first appearance of symptoms occurs after 30 minutes to a few hours after ingestion of the contaminated shellfish. Symptoms of individuals affected with DSP as reported by Yasumoto and associates (1978) include diarrhea (92%), nausea (80%), vomiting (79%),

Copyright 2002 by Marcel Dekker. All Rights Reserved.

and abdominal pain (53%). Victims recover after 3 days regardless of medical treatment. Pure DSP symptoms develop over a period of about a day; they include abdominal cramps (1 hr elapsed time), nausea progressing to diarrhea (2–7 hr), and a raw, burning feeling in the stomach (Gervais and MacLean, 1985; Yasumoto et al., 1984). There is an additional delayed onset syndrome attributed to a Prorocentrum minimum–derived component of DSP, progressing from vomiting (35 hr) through dizziness, diarrhea, cramps, and headaches (35–83 hr) to recovery (3 days to 2 weeks) (Freudenthal and Jijina, 1985). DSP toxins are stable to heat, for cooked shellfish have been responsible for many causes of illness (Yasumoto et al., 1978; Kat, 1983), although prolonged boiling of hepatopancreas homogenates can decrease the concentration of one of the toxins (OA) to half of its original concentration in 163 min (Edebo et al., 1988). The OA and DTX groups of toxins exert their effects mainly on the small intestine, leading to diarrhea and degenerative changes of the absorptive epithelium. At a biochemical level, OA and DTXs are powerful inhibitors of protein phosphatases (PPs) PP1 and PP2 and 2A (Leftley and Hannah, 1998). These enzymes are involved in regulating many important metabolic processes in eukaryotic cells. This biological action has been utilized in developing assays for the detection of these toxins. OA and DTX-1 are also powerful tumor promoters (Aune and Yndestad, 1993; van Egmond et al., 1993). In the two-stage carcinogenesis experiments on mouse skin, they have potency comparable with that of phorbol esters. The possible effects of chronic exposure to these toxins in humans remain to be established. Haystead and colleagues (1989) suggested that tumor promotion might stem from increased phosphorylation of one or more proteins that are substrates for protein kinase C and dephosphorylated by protein phosphatases 1 and 2A (which are inhibited by OA). The DSP toxins have varying toxicities and toxicological effects. Only OA, DTX-1, and DTX-3 are believed to be responsible for diarrhea. These toxins cause diarrhea by a mechanism similar to that caused by cholera toxin, by stimulating the phosphorylation of a protein that controls sodium secretion by intestinal cells (Cohen et al., 1990). Only these three toxins of those tested caused fluid accumulation in ileal loop assays (Hamano et al., 1985). Intraperitoneal and oral doses of OA and DTXs also cause marked changes in the small intestine, such as fluid accumulation and distention in suckling and adult mice and rats. Ultrastructural changes include degeneration of the intestinal absorptive epithelium. Terao and coworkers (1990, 1993) also observed liver damage in rats and mice given intraperitoneal and oral doses of OA and DTX-1.

PTX-1 had no effect on the intestines of suckling mice but caused extensive liver damage. Similar results were also obtained with adult mice, and extensive necrosis of hepatocytes was observed after 24 hr. PTX-1 appears to be nondiarrheagenic but does damage the liver in a similar way to that caused by fungal toxins such as cyclochorotine (from Penicillium islandicum) and phalloiodine (from Amanita phalloides) (Terao et al., 1986). YTX also appears to be nondiarrheagenic and has no effect on the intestines of mice. However, it can cause severe cardiac damage including swelling of cardiac muscle cells and of cells of the endothelial lining of capillaries (Terao et al., 1990). Desulfated YTX caused minimal damage to heart muscle but liver damage was observed. The toxicities (LD99) of OA, DTX-1, DTX-3, and YTX given intraperitoneally to mice are 200, 160, 500, and 100 µg/kg body weight, respectively (Yasumoto and Murata, 1990; Yasumoto et al., 1988). That of PTX toxins to mice ranges from 160 to 770 µg/kg. DSP tolerance levels in shellfish in countries with monitoring programs are summarized in Table 14.12. 14.3.3

Amnesic Shellfish Poisons

Amnesic shellfish poisoning (ASP) is the most recently discovered toxic shellfish syndrome. It was first recog-

nized in 1987 on Prince Edward Island, on the east coast of Canada, when a serious incident occurred in which there were four human fatalities and over 100 cases of acute poisoning after the consumption of blue mussels (Mytilus edulis) (Quilliam and Wright, 1989). Several of the severely affected cases during the initial incident still suffered memory loss 5 years later (Todd, 1993). A less serious outbreak affecting humans occurred near Monterey Bay, California, in 1991. It was traced to razor clams. Prior to this incident the deaths of cormorants and pelicans in this area had been shown to be due to the consumption of anchovies that had accumulated domoic acid, the toxic principle responsible for ASP, after feeding on a bloom of diatoms composed of Nitzschia australis. ASP gets its name from the fact that one of the recognized symptoms of this type of poisoning is loss of memory. The class of ASP poisons at present includes only domoic acid (Figure 14.9), a toxic amino acid mainly produced by the diatoms Nitzschia pungens f. multiseries (=Pseudonitzschia multiseries), N. australis, N. actydrophila, N. seriata, and N. pseudodelicatissima, occurring in the coastal waters of the United States, Canada, and New Zealand, and by the red macro alga Chondria armata, found in Japanese coastal waters. Domoic acid–producing species were also identified in Dutch coastal waters in the summer of 1994 (van Egmond and Speijers, 1999). Although the toxin was not detected in shellfish, this finding

Table 14.12 Diarrhetic Shellfish Poison Tolerance Levels in Shellfish and Official Assay and/or Analysis Methods in Countries with Monitoring Programsa Country Canada Denmark France Italy Ireland Japan Korea The Netherlands Norway Portugal Spain Sweden United Kingdom (including Northern Ireland) Uruguay

Tolerance level (per 100 g tissue)

Method of assay or analysis

20 µg Presence (2 out of 3 die within 24 hr) Presence (2 out of 3 die within 5 hr) 5-hr Mouse test Positive bioassay result 5 MU 5 MU 20–40 µg (Digestive gland) 5–7 MU Presence (20 µg) Presence 40–60 µg 200 µg

Mouse bioassay, HPLC, ELISA Mouse bioassay Mouse bioassay Mouse bioassay Mouse bioassay + LC-MS Mouse bioassay Mouse bioassay Rat bioassay Mouse bioassay Mouse bioassay Mouse bioassay HPLC, mouse bioassay for confirmation Rat bioassay

Mortality in 24 hr

Mouse bioassay

a

HPLC, high-performance liquid chromatography; ELISA, enzyme-linked immunosorbent assay; LC-MS, liquid chromatography–mass spectrometry; MU, mouse unit. b One mouse unit = approximately 5 µg okadaic acid. Source: Compiled from Shumway (1995), Andersen (1996), and Leftley and Hannah (1998).

Copyright 2002 by Marcel Dekker. All Rights Reserved.

CH3

1'

5 4

2'

HN 1 3

3'

2 6

4'

HOOC 5'

HOOC HOOC

CH3

Figure 14.9 Domoic acid implicated in amnesic shellfish poisoning (ASP).

demonstrated the potential for widespread occurrence. ASP is the first recorded diatom species implicated in shellfish intoxication. Domoic acid is a water-soluble, acidic, non-proteinexcitatory amino acid closely related to kainic acid, a neurotoxin isolated from seaweed. The presence of two conjugated double bonds in the molecule results in a strong absorbance at 242 nm, which allows for detection by monitoring absorbance at this wavelength. A number of isomers of domoic acid have been isolated from the diatom Nitzschia pungens f. multiseries and from mussel tissues, but these are less toxic than domoic acid (Wright, 1995; Wright and Quilliam, 1995). Domoic acid is neurotoxic and acts as a glutamate antagonist on the kainite receptors of the central nervous system. This action causes depolarization, influx of calcium, and eventual cell death (Teitelbaum et al., 1990). Extracts of toxins may be more lethal because of synergism between domoate and other excitatory amino acids such as glutamate. Symptoms of ASP poisoning appear within 3 to 5 hr of contaminated shellfish ingestion; they include headache, loss of balance, disorientation, nausea, vomiting, diarrhea, and the usual gastrointestinal symptoms typical of most food-poisoning episodes. Extreme cases of intoxication are characterized by decreased reaction to deep pain, dizziness, hallucination, confusion, short-term memory loss that sometimes becomes permanent, seizures, damage to the hippocampus in brain, and coma. Death may occur in extreme cases of ASP poisoning. The average fatality rate appears to be around 3%. There is no treatment at

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present for ASP poisoning other than life support systems if required. Examination of epidemiological data and laboratory studies of the mammalian toxicity of domoic acid—LD50 (intraperitoneal) of 3.6 mg domoic acid/kg in mice, and lethality at 4 mg/kg in the monkey M. fascicularis—have led to the establishment of an action level of 20 ppm. The mouse bioassay for domoic acid is performed by using essentially the same procedure as in the official bioassay for PSP (Helrich, 1990), except that observation times are extended. Since the mouse bioassay is not sufficiently sensitive to monitor domoic acid at the 20-ppm action level, instrumental methods such as HPLC were developed to provide the required sensitivity. After the initial outbreaks in Canada and the United States, the regulatory authorities in those countries introduced monitoring and control measures, and since then no serious cases of ASP have been reported. ASP tolerance levels in countries with monitoring programs are listed in Table 14.13. 14.3.4

Neurotoxic Shellfish Poisoning

Neurotoxic shellfish poisoning (NSP), sometimes also referred to as neurological shellfish poisoning, is caused by the ingestion of a group of lipophilic polyether toxins, the brevetoxins (PbTXs), produced by the naked dinoflagellate Gymnodium breve (=Ptychodiscus brevis). Because the dinoflagellate is “naked” (has no polysaccharide shell), the toxins are readily released in sea surf. Until recently, the incidence of NSP was only noticed in the Gulf of Mexico and the east coast of Florida, where red tides due to blooms of P. brevis are a common occurrence. The predominant season for outbreaks of red tide and, thus, epidemics of NSP, as well as irritative aerosolized dinoflagellate respiratory disease, is between January and April (Baden, 1983). In 1987, however, a bloom of P. brevis originating in Florida was spread by the Gulf Stream to North Carolina, where incidents of poisoning were reported (Morris et al., 1991), and it has since continued to be present there (Hallegraeff, 1995). An NSP outbreak in New Zealand also indicated the toxigenic species was more abundant than initially thought (Jasperse, 1993; Ledoux and Fremy, 1994). Species similar or closely related to P. brevis have been reported in Spain and Japan (Chou et al., 1985), but there have been no reports of poisonings in these regions. Oysters, clams, and coquina are the most common shellfish to filter the dinoflagellate and store the toxin in the hepatopancreas. The feeding shellfish can become toxic after being exposed during a red tide bloom of <5000 cells/mL seawater. Blooms of P. brevis also cause fish kills (Steidinger et al., 1973; Steidinger, 1979); fish deaths oc-

Table 14.13 Neurotoxic Shellfish Poison and Amnesic Shellfish Poison (Domoic Acid) Tolerance Levels and Official Assay and/or Analysis Methods in Countries with Monitoring Programsa

Country

Product

Canada Denmark Italy

Shellfish Bivalve shellfish Shellfish

New Zealand Portugal Spain United States

Shellfish Shellfish Shellfish Bivalve shellfish Bivalve shellfish Cooked crab viscera

Toxin

Tolerable level (per 100 mg soft tissue)

Domoic acid Domoic acid NSP Domoic acid NSP Domoic acid Domoic acid NSP Domoic acid Domoic acid

2 mg 2 mg n.d.b n.d. 2 mg n.d. 2 mg 3 mg

Method of analysis HPLC HPLC Mouse bioassay Mouse bioassay Mouse bioassay Mouse bioassay and HPLC Mouse bioassay HPLC HPLC

a

HPLC, high-performance liquid chromatography; NSP, neurotoxic shellfish poison. n.d., must not be detectable. Source: Compiled from Shumway (1995), Andersen (1996), and Leftley and Hannah (1998). b

cur when P. brevis cell densities approach 5 × 105 cells/L. There is no evidence to suggest that fish exposed to P. brevis are toxic to humans. Because the dinoflagellate is taken in through the gills, it is toxic to fish (LD50 0.2–0.5 mg/kg), but not to shellfish. Notorious are the massive fish kills that occur every 3–4 years on the west coast of Florida, associated with blooms of P. brevis. Sea birds may also be the victims of these red tides. The compounds responsible for the toxicity of P. brevis are a group of polycyclic polyether lactones commonly known as brevetoxins (PbTXs). So far nine have been isolated. Six of the brevetoxins (type 1) are based on a single structural polyether backbone (Figure 14.10), and an additional three (type 2) are based on a different polyether backbone (Figure 14.11). Brevetoxins are structurally related to yessotoxin (YTX, a DSP component described earlier) and ciguatoxin. Various investigators have used several names for the brevetoxins. Most are based on an earlier classification of the plankton as Gymnodinium breve and have not been applicable since it was changed to P. brevis. Poli and associates (1986) proposed a new notation system correlating names of toxins isolated from all laboratories. In this notation system, the numbering system proposed by Shimizu (1982), preceded by the letters PbTX denoting P. brevis toxin, is used. As well as PbTXs, P. breve has also been found to produce two phosphorus-containing ichthytoxins (Premazzi and Volterra, 1993). Brevetoxins have also been discovered in algae other than dinoflagellates, the chloromonads Fibrocapsa japonica and Hetrosigma akashiwo, both of the class Raphidophyceae (Khan et al., 1996, 1997), but these have not been implicated in human poisonings.

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Most brevetoxins are lipid-soluble, acid-stable, and base-labile compounds. In the dry state, they are stable up to 300°C. The brevetoxins are affected by autoclaving for 30 min at 122°C (18 psi), but this is not sufficient to detoxify them completely; however, heating at 500°C is effective (Poli, 1988). They decompose at extremes of pH (<2 and >10). The composition of the brevetoxins produced by P. brevis can vary according to growth phase (Roszell et al., 1990) and clone type (Baden and Thomas, 1988). In the log phase, cultures have greater levels of the aldehyde PbTX-2. As cultures progress into the stationary and declining phases, the levels of the alcohol PbTX-3 increase with a concurrent decline in PbTX-2, and other toxins such as PbTX-7 and PbTX-9 also begin to appear. The levels of PbTX-1 fluctuate during all culture phases. The findings suggest that in any new culture the aldehyde is the natural form of the toxin found and that changes in the profile can be explained by detoxification mechanisms of the dinoflagellate (Roszell et al., 1990). In other experiments using laboratory-grown cultures with growth phase controlled (log phase) and six different clone types, P. brevis isolates showed wide clonal variabilities in levels of PbTX-1, -2, and -3 (Baden and Thomas, 1988). Several different methods for the detection and quantitation of brevetoxins are available, including animal bioassays (mouse, fish), molecular pharmacological assays using voltage-dependent sodium channel preparations, immunoassays such as RIA and ELISA, and HPLC. The mouse bioassay has been routinely applied to the detection of the brevetoxins in shellfish. Some of the symptoms of NSP are similar to those seen in mild cases of PSP and usually resolve within a

R2O

R1O O

R3

O H

O

O O

O

A O

H

O H

H

H

H O

O

O

O H

H

H

R2O

R1O O

R3

O H

O

O O

O O H

H

O H

H

H

R1

R2

R3

H H COCH3 H H

CH2 CH2 CH2 CH2 O

CHO CH2OH CHO CHO (27,28-β-epoxide) CH2Cl

H

CH3

CH2OH

Brevetoxin (PbTX) Polyether backbone A Brevetoxin-2 Brevetoxin-3 Brevetoxin-5 Brevetoxin-6 Brevetoxin-8 Polyether backbone B Brevetoxin-9

H

O

H

H

B O

O

O

Figure 14.10 Chemical structures of brevetoxins implicated in neurotoxic shellfish poisoning (NSP). Compounds shown here share the same polyether backbone. The R groups for compounds belonging to A and B groups are shown in the table.

few days. They include tingling and numbness in the mouth and digits, ataxia, hot-cold reversal of temperature sensation, mydriasis, reduced pulse rate, diarrhea, and vomiting (Baden, 1983; Steidinger et al., 1973; Baden and Mende, 1982). In extreme cases, paresthesia, altered perception of hot and cold, difficulty in breathing, double vision, and difficulties in talking and swallowing have also been reported. No human fatalities have been documented thus far.

Copyright 2002 by Marcel Dekker. All Rights Reserved.

NSP toxins are the only algal toxins that cause problems to humans through inhalation. Sailing around in a red tide and breakers on the seashore may lead to inhalation of enough toxin to cause symptoms such as conjunctival irritation, copious nose bleeding, and nonproductive cough. The physiological action of brevetoxins is well understood and is similar to that of ciguatoxins (discussed later). They act specifically on site-5 voltage-dependent sodium channels and postganglionic cholinergic nerve fi-

R2 R1O O

R3

O H

O O H

O

O

O

O

H

H

H

H

O O

H

H

H O

Brevetoxin (PbTX)

R1

R2

R3

Brevetoxin-1 Brevetoxin-7

H H

CH2 CH2

CHO CH2OH

Figure 14.11

Brevetoxins having a polyether backbone different than those in Figure 14.10.

bers and induce influx of sodium ions into affected cells (Baden and Trainer, 1993; Premazzi and Volterra, 1993). Brevotoxin causes the channel to remain open longer and stabilizes preopen and open states, thereby allowing sodium inflow and hence neuronal membrane depolarization. The action of brevetoxins on the sodium channel is thus similar to that of PSP toxins, but at a site distinct from that affected by the saxitoxins. Some of the symptoms of NSP are also similar to those of ciguatera. Especially noteworthy is paresthesia in which sensations of hot and cold are reversed, a symptom often thought to be unique to ciguatera poisoning. The duration of NSP is, however, considerably shorter than that of ciguatera. NSP episodes last from 1 to 72 hr (17 hr is most typical); ciguatera symptoms are far more persistent. In contrast, in PSP, symptoms and physical findings are similar to those in NSP but are more severe and accompanied by respiratory paralysis. The latter is not seen in brevetoxin intoxication. In the United States, Florida has had the greatest impact from brevetoxins since most blooms of P. brevis occur there. The Florida Department of Natural Resources (FDNR), in accordance with their procedures (FDNR, 1985) and the recommendations of the National Shellfish Sanitation Program Manual of Operations (1990), closes waters to shellfish harvesting when P. brevis cells number 5000/L. Waters are reopened when cell densities remain

Copyright 2002 by Marcel Dekker. All Rights Reserved.

below 5000/L for 2 weeks. Commercial shellfish beds are frequently reopened within 1–2 months after bloom termination. Beds are reopened when the mouse bioassay detects less than 20 MU/100 g shellfish tissue (about 80 µg). The depuration rate is bloom concentration–dependent and has been shown to be a simple function of feeding and elimination rates. It is unlikely that the toxins are sequestered in shellfish, although few quantitative data are available. NSP tolerance levels in countries with monitoring programs are summarized in Table 14.13. 14.3.5

Ciguatera Fish Toxins (Ciguatoxin)

Ciguatera is a serious human intoxication that results from eating certain tropical and subtropical fish associated with coral reefs and adjacent coastal waters. The term ciguatera is derived from a name used in the 18th century in the Spanish Antilles for intoxication brought about by ingestion of the cigua or turban shell Cittarium (Livona or Turbo) pica (Yasumoto and Kanno, 1976). Historically ciguatera poisoning has been defined more by symptoms and by the epidemiological aspects of the illness than by the chemical properties or characterization of toxin(s). Ciguatera has been one of the most difficult of the marine toxins to study because of the unpredictable and variable nature of fish toxicity, the scarcity of toxic

fish to study and absence of any toxin standards, the logistics problems encountered in working in some of the endemic areas, the diversity of fish species implicated, the tedious isolation procedures required by the extremely low toxin concentration in fish (parts per billion levels), and the complex nature and multiplicity of the toxin(s). Epidemiological Characteristics Reports of possible ciguatera intoxication date back to Egypt between 2700 and 2400 B.C. In 1492, Columbus reported a clinical case while exploring the Caribbean. In 1789, Captain Bligh almost succumbed to fish poisoning in Tahiti after the mutiny aboard his H.M.S. Bounty (Halstead, 1978). In 1978, ciguatera intoxication in the United States accounted for 12.3% of food poisoning cases reported to the Centers for Disease Control (CDC), and it is presently the most frequent reported fish-borne intoxication worldwide. From 1983 to 1987, 87 outbreaks and 332 cases were reported to the CDC with no deaths (CDC, 1990). The predominant intoxications were from semitropical areas such as Hawaii, the Virgin Islands, Puerto Rico, and Florida. Significant outbreaks have also occurred in nontropical states as a result of the shipment of fish from endemic areas (CDC, 1986). Globally, ciguatera poisoning is mainly concentrated in tropical and subtropical areas, especially islands, in a band between 35°N and 35°S during the spring and/or fall. It is a serious public health problem in those countries where fish constitute the main source of dietary protein. With the advent of rapid air transport, ciguatera poisoning may occasionally occur in countries outside those latitudes where specialist seafood restaurants import the fish or tourists may experience symptoms after they have returned home. It is also not uncommon for patients living outside the high endemic areas to have ciguatera intoxication diagnosed as another disease, such as multiple sclerosis, which has signs easily confused with the acute intoxication by ciguatera. A psychosomatic/conversion disorder may also be hard to differentiate from chronic intoxication (Schatz, 1989). Ciguatera has been estimated to cause annually more than 50,000 illnesses worldwide and accounts for more than one-third of all finfish-borne illness outbreaks (Leftley and Hannah, 1998; van Egmond and Speijers, 1999). It is, however, rarely fatal. Levels as low as 1 ppb can cause intoxication in adults. Toxic Fish Ciguatera toxin outbreaks are most often associated with transitory human or natural disturbances (e.g., hurricanes) of the coral reef ecosystem. Three types of fish tend to be-

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come ciguateric: herbivorous reef fish, detritus-feeding reef fish, and large carnivores that feed on these two types of reef fish. Over 400 species distributed among at least 57 families of fish harbor the toxin, including many that are highly prized for food (Table 14.14). Their distribution is worldwide, although most are found mainly in tropical waters. Ciguatoxic fish are generally bottom dwellers or shore fish, found usually at depths less than 300 feet. These fish accumulate toxins via the marine food web. The toxin enters the food chain when the dinoflagellate responsible attaches itself to marine algae, which are consumed by small, herbivorous fish. Larger carnivorous fish then consume these. All susceptible species in a certain area usually become toxic. The fish exhibits no odor or visible difference in appearance; therefore, the presence of poison is difficult to detect. The internal organs of the fish—liver, intestine, roe, and gonads—are more toxic than the more commonly consumed muscle tissues. Unlike the toxins described earlier in this chapter, which accumulate mainly in shellfish, the group of ciguatera-causing toxins are harbored in finfish. These fish include the barracuda (Sphynaenidae spp.), red snapper (Lutjanus bokar), grouper (Cephalopholis spp.), amberjack (Seriola spp.), surgeon fish (Acanthunidae), sea bass (Serradinae), and even moray eel (Muraedinae). Barracuda and red snapper are the species most commonly implicated in the United States. There is a good correlation between fish size and toxicity: the heavier and larger fish are generally more toxic. Also, unlike in other types of fish or shellfish toxicity, in ciguatoxicity there is no seasonal pattern. Transvectors The dinoflagellate Gambierdiscus toxicus is responsible for the toxins found in ciguatoxic fish. Wild specimens of G. toxicus collected from the surface of dead corals in the Gambier Islands yielded upon extraction significant amounts of ether-soluble toxin, ciguatoxin (Yasumoto et al., 1979a). The identity of ciguatoxin from wild G. toxicus cells was confirmed by comparison of its chromatographic and pharmacological properties with those of moray eel toxin (Yasumoto et al., 1979b). The dinoflagellate is a photosynthetic species that has a relatively slow growth of approximately one division every 3 days (Withers, 1984). In its coral reef habitat, cells of G. toxicus are biflagellate and swim if disturbed but usually are motionless and attached to certain macroalgae, especially rhodophytes (e.g., Spyridea filamentosa and Acanthophora spicifera) and phaeophytes (Sargassum polyphyllum, Turbinaria ornate, and Dictyota acutiloba) (Taylor, 1980; Withers, 1982, 1984). Thus far, no specific

Table 14.14

Ciguatoxic Fish Species

Family Acanthuridae

Albulidae Aluteridae Antennariidae Apogonidae Arripidae Aulostomidae Balistidae Batrachoididae Belonidae Blenniidae Bothidae Carangidae

Chaetondontidae

Chanidae Cirrhitidae Clupeidae

Congridae Coryphaenidae Elopidae Engraulidae Exocoetidae Gempylidae Gerridae Gobiidae Hemiramphidae Holocentridae Istiophoridae Kuhliidae Kyphosidae

Group common name Surgeon fish Unicorn fish Tang Bonefish Filefish Sargassum fish Cardinal fish Tommy rough Australian salmon Trumpetfish Triggerfish

Genus

Toadfish Garfish Needlefish Blenny Flounder Jack Rainbow runner or Hawaiian salmon Leatherjacket Lookdown Amberjack Rudderfish Pompano, permit, palmometa Horse mackerel Atlantic moonfish Surgefish Butterflyfish

Acanthurus, Ctenochaetus, Prionurus Naso Zebrasoma Albula Alutera, Anacanthus, Pseudaluteres Histrio Apogon, Cheilodipterus, Paramia Arripis Arripis Aulostomus Abalistes, Balistapus, Balistes, Balistoides, Canthidermis, Melichthys, Odonus, Pseudobalistes, Rhinecanthus Coryzichthys, Opsanus, Thalassophryne Belone Belone, Strongylura Entomacrodus, Ophblennius Bothus, Scophthalmus Carangoodes, Caranx. Selar, Seriola Elegatis Oligophites, Scomberoides Selene Seriola Seriola Trachinotus Trachurus Vomer Zalocys Chaetodon

Bannerfish Angelfish Milkfish Hawkfish Shad Sprats Herring Sardines Sablefish Conger eel Dolphin Tarpon Anchovy Flying fish Oilfish Silverfish Goby Halfbeak Squirrelfish Soldierfish Sailfish Mountain bass Rudderfish

Heniochus Holocanthus, Pomacanthus, Pygophites Chanos Paracirrhites Anodontostoma, Nematolosa Clupanodon, Clupea Dussumieria, Ilishia, Opisthonema Harengula, Sardinella Macrura Conger Coryphaena Megalops Engraulis, Thrissina Cypselurus Ruvettus Gerres Acentrogobius, Ctenogobius, Oligolipes, Zenogobius Hemiramphus, Hyporhamphus Holocentrus Myripristis Istiophorus Kuhlia Doydixodon, Kyphosus (table continues)

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Table 14.14

(continued)

Family

Group common name

Labridae

Hogfish Wrasse

Lophiidae Lutjanidae

Goosefish Snapper

Monacanthidae

Filefish

Mugilidae Mullidae Muraenidae

Mullet Surmullet, goatfish Mottled or spotted eel Moray eel Batfish Snake eel Worm eel Cowfish Trunkfish

Ogcocephalidae Ophichthyidae Ostraciontidae

Phempheridae Pomacentridae Pomadasyidae Priacanthidae Scaridae Scatophagidae Sciaenidae Scombridae

Scorpaenidae

Serranidae

Siganidae Sparidae Sphyraenidae Syngnathidae Synodontidae Xiphiidae Zanclidae

Sweeperfish Damselfish Grunt Bigeye (glasseye) snapper Parrotfish Spadefish Croaker (drum) Wahoo Skipjack Bonito Mackerel Zebrafish Barbfish, lionfish Scorpionfish Oceanfish, redfish Grouper Creolefish Seabass Soapfish Rabbitfish Porgy Scup Barracuda Seahorse Lizardfish Swoardfish Moorish idol

Genus Bodianus, Lachnolaimus Cheilinus, Coris, Ctenolabrus, Epibullus, Halichoeres, Thalassoma Lophiomus, Lophius Apareus, Aprion, Gnathodentrix, Gymnocranius, Lethrinus, Lutjanus, Lythrulon, Monotaxis, Ocyurus, Plectorhinchus Amanses, Monacanthus, Navodon, Oxymonocanthus, Paramonocanthus, Pervagor, Pseudomonocanthus, Stephanolepsis Chelon, Crenimugil, Mugil Mulloidichthys, Parupeneus, Openeus Echidna Gymnothorax, Muraena Ogcephalus Callechelys, Leiuranus, Myrichthys, Ophichthus, Oxystomus Echelus Acanthosthracion Kentiocapros, Lactophrys, Lactoria, Ostracion, Rhinesomus, Rhynchostracion Phempheris Abudefduf, Dascyllus, Pomacentrus Anisotremus, Orthopristis Priacanthus Chlorurus, Euscarrus, Scarops, Scarus Scatophagus Johnius, Nibea, Odontoscion Acanthocybium Euthynnus Sarda Scomberomorus Pterois Scorpaena Scorpaena, Scorpaenopsis Sebates Anyperodon, Cephalopholis, Permatolepsis, Epinephelus, Mycteroperca, Paralabrax, Plectropomus, Variola Paranthias Promicrops, Epinephelus Rypticus Siganus Calamus, Erynnis, Pagellus, Pagrus, Sparus Stenotomus Sphyraena Hippocampus Synodus Xiphias Zanclus

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a).

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nutrient or environmental factor that stimulates the benthic blooms of G. toxicus in nature has been found. The specific nature of the ciguatoxigenesis in a localized area remains obscure (Baden, 1983). Several hypotheses have been developed to explain the coincidence of ciguatera outbreaks after the appearances of denuded surfaces in a coral reef area. These include the possibility (1) that the surfaces may provide sites for algal hosts of the ciguatoxigenic dinoflagellates; (2) that disruption in an area may cause resuspension of hypnozygotes of toxic benthic dinoflagellates, similar to the known initiation of red tides (like P. brevis and Gonyaulax spp.); (3) that resuspension of detritus populations may cause “reseeding” of algal hosts by toxic dinoflagellates; (4) that “rafting” of dinoflagellate populations on a macroalgal surface may contribute to the initiation of ciguatera in new locales; and (5) that there may be a “ciguatoxin-inducing factor” that acts on in situ populations to stimulate their ciguatoxin production. Other dinoflagellates implicated in ciguatera poisoning include Prorocentrum mexicanum, Coolia monotix, Ostreopsis ovata, P. concavum, and Amphidinium spp. Thus, there are potentially different progenitors of the ciguatera toxins, with different ecological factors that affect each. These additional species must also be considered as likely sources of toxins found in ciguateric fish. Chemical Characteristics The principal active component of ciguatera poisoning was isolated from the red snapper, Lutjanus bohar; the shark Carcharchinus menisorrha; and the moray eel, L. javanicus in 1967 (Scheuer et al., 1967). This lipophilic compound, named ciguatoxin, was purified to a toxicity level of 500 µg/kg minimal lethal dose by intraperitoneal injection in mice. Later, Yasumoto and Scheuer (1969) found that the L. javanicus viscera, especially the liver, were an excellent source of ciguatoxin because of its high concentration and the relatively small amounts of minor toxins in that tissue. The L. javanicus liver has since been the major source of ciguatoxin for chemical and pharmacological studies (Tachibana, 1980; Nukina et al., 1984). Since these beginnings, the ciguatera toxins were later found to be a group of three different toxins. The ciguatoxins (CTXs) are lipophilic polyethers (Figure 14.12). They are some of the most potent known marine toxic substances. Gambiertoxin-4, isolated from the dinoflagellate G. toxicus, is probably the parent compound (Lewis and Holmes, 1993). Ciguatoxins have been isolated and purified from relatively few organisms so it is likely that the CTX molecules that occur in carnivorous fish are modified during their passage through the food chain and many more variants exist. This could explain the wide

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range of clinical symptoms that has been observed (Bagnis, 1993; Wright, 1995). At present eight ciguatoxins from the Pacific area have been fully characterized. There are at least 11 oxidized ciguatoxins (Lewis and Jones, 1997). At least four new ciguatoxins have been identified from the Caribbean area but have yet to be structurally defined (Venoux and Lewis, 1997). The characteristic feature of CTX is the presence of 13 contiguously transfused ether rings of five through nine members resembling the brevetoxins and YTX (Yasumoto, 1990). The 59-carbon polymer contains four olefins, five hydroxyl, and five methyl groups. Ciguatoxin is stable in water, pyridine, acetic acid, and 1 N NaOH at 100°C for at least 10 min; under continuous airstream overnight, under sunlight exposure in methanol for 1 hr, and in metal salt solution for at least 36 hr. Toxicity was lost by treatment with 1 N HCl at 100°C for 10 min and treatment with acetic anhydride in pyridine (Nukina et al., 1984). Maitotoxin (MTX), a water-soluble compound that is often associated with CTXx in the viscera of herbivorous fish, was originally isolated from the viscera (mainly gut) of the surgeonfish, or maito (Tahitian name), Ctenochaetus striatus. This fish has caused much of the ciguatera intoxication in Tahiti (Yasumoto, 1971). Subsequently, Murata and coworkers (1994) isolated MTX from cultures of the dinoflagellate G. toxicus and determined its structure and partial stereochemical characteristics. It is a large and complex bisulfated molecule (molecular weight = 3422) containing 32 ether rings, not all of which are contiguous. The complete structure and stereochemical characteristics of MTX have been determined by Nonomura and associates (1996) and Zheng and colleagues (1996). Relatives of MTX, MTX-2 and MTX-3, have been identified by mass spectrometry (Lewis et al., 1994). MTX is thought to play only a minor role in ciguatera fish poisoning in humans, since it has not been detected in significant quantities in the muscle tissue of either herbivorous or carnivorous fish (Bagnis, 1993; Lewis and Holmes, 1993). Little is known about the other toxins associated with these two principal ciguatera toxins. Scaritoxin, originally isolated from the flesh of the parrotfish, Scarops gibbus (Chungue et al., 1977), may be present in a wide variety of fish (Hashimoto, 1979). Scaritoxin is lipophilic and causes symptoms in mice similar to those of ciguatoxin, but the two can be distinguished by silicic acid chromatography. OA and its analogs (DSP toxins) have also been implicated as contributory agents in ciguatera poisoning (Gamboa et al., 1990; Dickey et al., 1990). Several other dinoflagellate species common to endemic areas are known to produce toxins; therefore, the full spectrum of toxins responsible for ciguatera may still be unknown.

H

HO

H H

H

O

H

O

H OH

A

H H

H

H

O

H

H

H

H

O

H

H

H

H

H

O

O

H

O

O

O

O O

O HO

H

O

H

H

H

H

H

OH

OH

OH

H

HO

H H

H

O

H

O

H OH

H

B

H

H

H

O

H O

H

H

H

O

H H

H

O

O O

O O

H

H

H

H

H H

O

O H

O

H

H

OH

Figure 14.12 Ciguatoxins of known structure. Form A is the parent compound ciguatoxin (CTX), isolated from moray eels; form B is a congener isolated from the dinoflagellate Gambierdiscus toxicus.

Mode of Action The major toxins of ciguatera fish poisoning, CTX and MTX, apparently have no effect on the fish that harbor them. However, when injected intraperitoneally into mice, they are lethal and produce characteristic symptoms. Early reports on the pharmacological characteristics of ciguatoxin are conflicting as a result of both the variability in purity of the samples (and thus the dose) and the presence of secondary toxins. Nonetheless, these early studies showed that the primary action of ciguatoxin is to increase the permeability of the excitable membranes to sodium, causing depolarization. CTX-induced depolarization is blocked by tetrodotoxin (discussed later), and both this depolarization and the consequent changes in membrane excitability are antagonized by increased extracellular calcium ion concentrations (Rayner et al., 1969). Subsequent research has further defined the action of CTX. Bidard and associates (1984) showed that CTX belongs to a new class of toxins acting on sodium chan-

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nels. In rat brain synaptosomes, CTX caused stimulation of neurotransmitter (γ-aminobutyric acid [GABA] and dopamine) release. This action was completely blocked by tetrodotoxin. Electrophysiological studies on neuroblastoma cells showed that CTX induces a partial membrane depolarization by an action that enhances sodium permeability and is prevented by tetrodotoxin. The depolarization in turn opens calcium channels, provoking calcium entry and neurotransmitter release. CTX does not affect the sodium-potassium adenosine triphosphate (ATP) pump, and so membrane depolarization and transmission are not due to the inhibition of the sodium-potassium adenosine triphosphatase (ATPase). The transmitter release is not caused by an effect of CTX on slow calcium channels, as indicated by the finding that calcium channel antagonists had no effect. CTX also had no effect on the electrical properties of neuroblastoma cells in the presence of excess calcium. It can create spontaneous oscillations in the membrane polarization level and repeated action potentials.

When used synergistically with other sodium channel toxin classes II, III, and V (Table 14.15), viz., veratridine, batrachotoxin, pyrethroids, and sea anemone or scorpion toxins, CTX stimulates sodium entry through the voltage-dependent sodium channels. This stimulation is abolished by tetrodotoxin. Therefore, CTX does not associate with the receptors of three of the five known classes of sodium channel toxins. CTX also has no effect on binding of 125I-labeled Asv and AaII (class III) or 125I-labeled Tityus toxin (class IV) to receptor sites in synaptosomes. Tetrodotoxin (class I) is a noncompetitive inhibitor of CTX action; the concentration dependence of tetrodotoxin inhibition of CTX action is independent of CTX. So tetrodotoxin and CTX occupy different binding sites. Therefore, CTX acts selectively on voltage-dependent sodium channels in nerve and muscle cells and on synaptic terminals and thus represents a new sodium channel toxin (Bidard et al., 1984). Legrand and colleagues (1982) investigated the effects of CTX, STX, and MTX in pentobarbital-anesthetized cats and found both central and peripheral effects and both cholinergic and adrenergic actions by CTX. Lewis and Endean (1984) also found CTX is approximately as active on cholinergic nerves as it is on adrenergic nerves. Although the symptoms of ciguatera are primarily neurological and gastrointestinal, the cardiovascular system is also implicated frequently. CTX has been reported to cause either hypotension with bradycardia or hypertension with tachycardia (Rayner et al., 1969). Ohshika (1971) found a dual effect of CTX that was inhibited by a variety of adrenergic and ganglionic blocking agents. This

Table 14.15 Class

Classes of Neurotoxins Acting on Sodium Ion Channels Property

I

Water-soluble

II

Lipid-soluble

III

Polypeptide

IV

V

VI

effect was explained as an indirect action via the neural release of catecholamines. Lewis (1985) found that CTX has a direct effect on the sodium channel of the myocardium in addition to an indirect effect on nerves in the myocardium. He also noted that three types of arrythmias were induced by CTX. Maitotoxin produces calcium-dependent excitatory effects on various cells and tissues, increasing contractile force of cardiac cells and inducing a rise in diastolic tension followed by atrial arrest in experimental animals. At very low concentrations (10–8 g/mL), MTX induces a profound increase in calcium influx and the calciumdependent release of norepinephrine from the pheochromocytoma cells (Takahashi et al., 1982). The effects of MTX are not influenced by treatment with tetrodotoxin or the removal of external sodium but were inhibited by verapamil, manganese, and tetracaine. Thus the action of MTX differs from that of calcium ionophores (which are blocked by manganese but not affected by verapamil and tetracaine). Its effect is possibly due to an increase in calcium permeability through voltage-sensitive calcium channels. MTX also causes excitatory actions on cardiac tissue, probably as a result of an increase in calcium permeability to cardiac and smooth muscle membranes that is antagonized by calcium antagonists or divalent cations (Ohizumi et al., 1985). Scaritoxin appears to have pharmacological properties similar to those of CTX (Yasumoto and Murata, 1990). It depresses the oxidative metabolic process in the rat brain and has a depolarizing action on excitable mem-

Examples Tetrodotoxin Saxitoxin Grayanotoxin Veratridine Batrachatoxin α-Scorpion toxins (Leiurus, Androctonus spp.) Sea anemone toxin β-Scorpion toxins (Centuroides, Tityus spp.) Pyrethroids Brevetoxin

Lipid-soluble

Ciguatoxin

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Mode of action +

Na channel antagonist; inhibition of transport Persistent activation of Na+ channels

Slow Na+ current inactivation; enhancement of persistent activation Shift in voltage dependence of activation; no persistent membrane activation; repetitive firing Transformation of fast Na+ channels into slower ones; persistent activation; repetitive firing Activation of voltage-dependent Na+ channels

branes. It may be speculated that CTX and scaritoxin are related compounds, the latter resulting from CTX metabolic transformation in some fish species. Toxicity and Analysis Ciguatera toxins are some of the most potent marine toxicants known to date. The purified CTX has an LD50 of 0.45 µg/kg in mice. The LD50 of MTX is 0.17 µg/kg, making it the most toxic of the dinoflagellate toxins (Baden and Trainer, 1993). MTX is also 80 times more potent than saponin in hemolytic ability and can induce severe pathomorphological changes in the stomach, heart, and lymphoid tissues in mice and rats by intraperitoneal injection of as little as 200–400 ng/kg (Terao et al., 1989). The ciguatoxins appear to be quite stable to a variety of treatments. Cooking, freezing, drying, or salting of fish does not alter the potency of these toxins. MTX is also extremely stable to heat and acid. However, because it is water-soluble, some of its toxicity will apparently leach with soaking fish in water. Bioassays for ciguatera toxicity have involved many animal species including fish, crayfish, frogs, mosquitoes, dogs, pigs, hamsters, guinea pigs, turtles, mongoose, rabbits, chicks, cats, rats, and mice. Rakotoniaina and Miller (1990) have thoroughly reviewed the subject of ciguatera bioassays as well as other in vitro assays used for their detection and quantification. At present, the most widely used bioassay utilizes the intact mouse. In general, mouse bioassays involve an intraperitoneal injection into mice (15–21 g) of a toxic fraction (solvent extracted from algal cultures or from flesh or viscera of fish) diluted in either saline solution or phosphate-buffered saline solution with or without Tween-60 (0.2%). The mice are observed for symptoms, which can include all or some of the following: prolonged suppression of body temperature, reduced activity, lumbar muscle contractions, unsteadiness or difficulties in walking, breathing difficulties, diarrhea, cyanosis, pilorection, reduced reflexes, and death. Toxin levels have been estimated by using rating scales such as that used by Kimura and coworkers (1982) or by regression analysis of the relationship of the injected dose to the survival time (Tachibana, 1980). Mouse lethality (intraperitoneal) estimated by Legrand and associates (1989) for both crude and pure CTX was LD 8900 µg/kg and LD 0.4 µg/kg, respectively, where LD is the extrapolated lethal dose that would kill mice at infinite time. Symptoms The symptoms produced by ciguatera toxins are complex, and the effects may be long-lasting. The symptom complex is polymorphous, and the diagnosis is based only on

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clinical presentation with no confirmatory laboratory tests available. Paresthesia, especially in the South Pacific, is considered a clinical hallmark of ciguatera poisoning and differentiates this form of intoxication from other forms of food poisoning or mild gastroenteritis (Dawson, 1977; Lewis, 1981). These abnormal burning or tingling sensations occur typically in the extremities and circumoral region. Temperature reversal in which cold seems hot, referred to as dry ice phenomenon, is also characteristic of ciguatera poisoning. Clinical manifestations of ciguatera intoxication usually begin within 1–6 hours after ingestion of contaminated fish; however, signs can occur within several minutes if a large quantity of toxin is consumed. Initial signs are nausea, vomiting, watery nonbloody diarrhea, abdominal pain, and cramping. Circumoral or facial paresthesia and numbness, which proceed to the extremities, may follow. In certain ethnic groups, the signs of paresthesia, which do not follow dermatomal patterns, can precede the gastrointestinal signs (Sims, 1987). Cold-to-hot sensory reversal dysesthesia that develops 2–5 days after intoxication is considered by some to be pathognomonic. The median duration of the paresthesia and dysesthesia is 17 days. Weakness, dizziness, dry mouth, blurred vision, photophobia, transient blindness, myalgia, and arthralgia are common. Either generalized pruritus or localized itching may be present; fatigue and malaise that can last for weeks usually follow. In severe cases, fatigue may persist for years. In severe intoxication or reintoxication, circulatory collapse (shock), acute respiratory failure, and coma may occur (Lewis, 1986; Withers, 1982; Hamberger, 1986; Smith, 1992). In acute cases, mortality rate has been reported to be as high as 7%–20% (Craig, 1980). These figures, however, are derived from severe, hospitalized cases. In the Pacific, mortality rate is 0.1% of reported cases (Lewis, 1981). Gastrointestinal symptoms of ciguatera poisoning usually abate within 48 hours. In French Polynesia, these symptoms are more common in victims who have eaten herbivorous fish, such as the surgeonfish or maito. This fish contains MTX, mostly in the gut, as well as CTX. Cardiovascular symptoms (hypotension and bradycardia), if present, abate in 7 days; neurosensitivity lasts for 1–2 weeks. Cardiovascular and other disorders prevail in cases caused by ingestion of toxic carnivores, such as snapper and grouper (Bagnis, 1968). Pruritis and paresthesia are often reactivated within the first several weeks of poisoning by consumption of any kind of fish or alcohol. In the Gambier Islands, the parrotfish causes most of the ciguatera intoxications. Symptoms from ingestion of this fish begin like those of the typical ciguatera syndrome, but after 5–10 days, a second phase, characterized by a staggering walk, dysmetria, loss of equilibrium, and ki-

netic muscle tremor, which appears to be caused by an effect on the cerebellum, begins and lasts for about a week, requiring a month for complete recovery. These symptoms are primarily due to the presence of scaritoxin in parrotfish. Since no specific antidote for ciguatera poisoning has been found, treatment is symptomatic and supportive only. It includes gastric lavage, calcium gluconate, magnesium sulfate, opiates for pain, oxygen and ventilation assistance, atropine sulfate, oximes, procainamide, intravenous fluid for severe dehydration, aspirin, cold showers for pruritus, antidiarrheal and antiemetic agents, B and C vitamins, cortisone, and dexamethasone (Halstead, 1978; Lawrence et al., 1980; Smith, 1992). There is really no curative treatment presently known, and immunity through previous exposure to ciguatoxin does not develop. On the contrary, evidence suggests that individuals who have been previously exposed are more susceptible and react to lower levels of toxins. The treatment thus remains nonspecific, symptomatic, and supportive despite major advances in the understanding of the pharmacological characteristics of ciguatera toxins. Since the ciguatoxic fish cannot be distinguished from the wholesome species by their appearance and taste, there are no practical means for the consumer to determine accurately whether or not the fish is toxic. Nevertheless, certain precautions may be observed to minimize the possibility of poisoning. Halstead and Courville (1967) offer the following helpful suggestions: 1. 2.

3. 4.

5.

6.

7.

Avoid eating large predatory reef fish such as groupers, barracudas, snappers, and jacks. In particular, avoid eating the viscera and roe of all tropical marine fish, especially during the reproductive season. Eat only a small quantity of any unknown variety of tropical fish. Avoid eating any tropical moray eels; these species may be highly poisonous and may produce instant death. If at all possible, select for the table only fish captured in the open sea away from reefs or the entrance of a lagoon. If it is a matter of survival, the suspect fish could be cut in small strips and soaked with several changes of water; however, this procedure does not guarantee the safety of the fish. It is wise to ask the advice of the inhabitants of an island regarding the safety of the local fish.

Management of chronic ciguatera fish poisoning remains a difficult problem. Psychotherapy plays an important role in helping the patient live with the disability. The prevention of reintoxication is an important consideration

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because a small amount of toxin can cause exacerbation of the disease. A diet free of fish, shellfish, alcohol, and nuts has been advocated for at least 3–6 months after acute ciguatera poisoning and for life for patients suffering from the chronic form of the disease (Smith, 1992). 14.3.6

Tetrodotoxins

Tetrodotoxin (TTX) is a naturally occurring toxic alkaloid found in a variety of animals, the most notable of which is the pufferfish. Accounts of pufferfish intoxication date back to the Egyptian experience in the years 2700 to 2400 B.C. (Mills and Passmore, 1988). Captain James Cook provided one of the first accounts of what some believe to be tetrodotoxin from the pufferfish (Beaglehole, 1961). The more recent Japanese clinical and management experience reports over 6000 cases over 82 years (Fuhrman, 1986). The U.S. Food and Drug Administration (FDA) has permitted the importation of Japanese puffers for sale in fugu restaurants in the United States (Ahmed, 1991; Halstead, 1994b). This, however, does not guarantee the food safety of puffer products. All puffers are potentially toxic unless they have been cultivated artificially. Tetrodotoxic Fish The tetrodotoxic fish are grouped into five families (Table 14.16). These are the pufferfish (blowfish, globefish, and fugu) found worldwide in both tropical and semitropical waters. The most poisonous pufferfish are in the family Tetraodontidae, but not all fish in this family produce the poison. The smaller species of pufferfish are found between 47°N and 47°S latitude; the larger species are found in open waters as far as 63°N latitude. Not all puffers thrive in salt water; some species have been found in fresh water. The pufferfish can be easily recognized by their peculiar and unique behavior. When frightened, injured, or taken out of the water, these fish engulf air or water and inflate like balloons. This defense mechanism is not seen in any other group of fish. The toxin is secreted by the pufferfish exocrine glands, acting as a natural defense mechanism to repel predators (Kodama et al., 1985; Saito et al., 1985). Although not commonly consumed by humans, the eggs of the California newt (Taricha), porcupine fish, ocean sunfish, triggerfish, and boxfish are also sources of tetrodotoxin. The toxin is also reported to occur in the ivory shell Babylonia japonica (Yasumoto et al., 1981), the frog shell Tutufa lissostoma (Noguchi et al., 1984), the trumpet shell Charonia sauliae (Narita et al., 1981), and the lined moon shell Natica lineata (Hwang et al., 1990). Tetrodotoxin is also found in certain tropical and subtropi-

Table 14.16

Examples of Tetrodotoxic Fish Species

Family (common name) Canthigasteridae (sharp-nose puffer) Diodontidae (porcupine fish)

Molidae (ocean sunfish) Tetraodontidae (puffer, rabbit fish)

Triodontidae (puffer)

Canthigaster bennetti, C. cinctus, C. compressus, C. jictator, C. margaritatus, C. rivulatus Chilomycterus affinis, C. antenatus, C. atinga, C. orbicularis, C. schoepfi, C. spinosus, C. tigrinus Diodon holocanthus, D. hystrix, D. jaculiferus Mola mola Ranzania laeuis Amblyrhynchotes biocellatus, A. glaber, A. honckeni, A. hypselogeneion, A. loreti, A. richei Arothron aerostaticus, A. alboreticulatus, A hispidus, A. immaculatus, A. mappa, A. meleagris, A. nigropunctatus, A. reticularis, A. setosus, A. stellatus Boesemanichthys firmentum Chelonodon fluviatilis, C. laticeps, C. patoca Colomesus ephippion guttifer Fugu basilevskianus, F. chrysops, F. exascurus, F. niphobles, F. oblongus, F. ocellatus obscurus, F. ocellatus ocellatus, F. pardalis, F. poecilonotus, F. pseudommys, F. rubripes chinensis, F. rubripes _rubripes, F. stictonotus, F. vermicularis, F. porphyreus, F. vermicularis radiatus, F. vermicularis vermicularis, F. xanthopterus Lagocephalus cheesemani, L. laevigatus inermes, L. laevigatus, L. lagocephalus, L. lunaris, L. oceanicus, L. scleratus Liosaccus cutaneous Monotreta cutcutea, M. palembangensis Sphaeroides annulatus, S. armilla, S. greeleyi, S. maculatus, S. nephelus, S. sechurae, S. spengleri, S. testudineus Tetraodon lineatus Torquigener hamiltoni Xenopterus naritus Triodon bursarius

Source: Compiled from Halstead and Courville (1965) and Halstead (1994a).

cal crabs (Noguchi et al., 1986) and in the eggs of the blueringed octopus (Hapalochlaena maculosa) (Sheumack et al., 1984). Pufferfish contain tetrodotoxin in the ovaries, liver, intestine, skin, and spawn. In female pufferfish, the ovaries are the principal site of toxin concentration, and female fish contain more toxin than males. The level of toxin in the female also appears to be related to the spawning season. In Japan, pufferfish poisoning presents the highest risk of severe intoxication from March through June during the spawning season, when the toxin level is at its highest. The flesh of pufferfish caught in temperate waters is believed to have low or nonexistent toxicity, but improper handling can easily result in release of the toxin from highly toxic tissues. As with other toxic marine species, certain tissues of the pufferfish appear to accumulate the poison more than any other. The most toxic parts are the ovaries and liver;

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the intestines in some species (e.g., Fugu niphobles) are also quite toxic. The skin in several species can be moderately toxic. The muscles and testes are generally nontoxic, except in the very toxic species, in which these tissues are weakly or moderately toxic. The ovaries, however, can be mistaken frequently for the testes and thus pose a significant risk of poisoning. Heating the fish does not inactivate the toxin enough to ensure safety. The toxin loses some toxicity during commercial canning (116°C, 75 minute), but not enough to allow safe human consumption of toxic fish. Proper evisceration of the fish is the best way to prevent poisoning if these species are consumed. The meats of the most poisonous pufferfish, Fugu poecilonotus, F. pardalis, F. vermicularis, F. chrusops, and F. rubripes, are esteemed delicacies in Japan, where the government licenses professional pufferfish cooks. These cooks must take special training and pass a government examination in the art and science of puffer preparation

(Tsunenari et al., 1980). The Japanese government licenses trained persons who can correctly identify the species and properly eviscerate the poisonous organs without contaminating the white meat. The Japanese know well the violently toxic nature of some parts of the tissues of the pufferfish. However, the tradition or the enjoyment of eating puffer meat overcomes whatever fear or reservations the fuguphagists may have in the consumption of puffer meat, even at the risk of death. Even with experts preparing the puffer meat, the risk of serious poisoning is not eliminated; it is only minimized. Even the finest cooks have been known to succumb to their own cooking (Fukuda, 1951). The penchant for puffer or fugu meat among the Japanese is reflected by the high rates of mortality from fugu poisoning in that country. For example, prior to World War II, puffer poisoning accounted for 44.6% of all food poisonings in Japan. Since the war, education, rigid government control on pufferfish harvesting and preparation, and other factors have reduced this figure to approximately 20% (Fukuda, 1951). Between 1967 and 1976, puffer poisoning accounted for 4.8% of all outbreaks of food poisoning and 0.3% in terms of patients of food poisoning. However, puffer poisoning still accounts for over 60% of all food poisoning fatalities and over 80% of all seafood poisoning deaths in Japan. Chemistry Tetrodotoxin (TTX) is the most toxic form of a series of structurally related compounds (Figure 14.13). TTX is much more toxic than the other forms, and most of the toxicity of finfish and shellfish is due to TTX. Many other members of the TTX family compounds have been detected as minor components in species of Fugu pufferfish: F. niphobles (Endo et al., 1988; Yasumoto et al., 1989), F. pardalis (Nakamura and Yasumoto, 1985; Yasumoto et al., 1989), and F. poecilonotus (Nakamura and Yasumoto, 1985). These include 4-epi-TTX, 6-epi-TTX, 4,9-anhydroTTX, 11-deoxyTTX, and 11-norTTX-6(R)-ol. Tetrodonic acid has also been reported. These compounds and a new TTX-related compound, 11-oxoTTX, have been found in the puffer Arothron nigropunctatus (Khora and Yasumoto, 1989). TTX has been released from a nontoxic, high molecular fraction of livers of F. poecilonotus by treatment with ribonuclease (RNAse) (Kodama et al., 1983). Many species of bacteria are also capable of producing TTX and/or anhydroTTX (Simidu et al., 1987). It is now well established that these toxins accumulate in marine sediments (Kogure et al., 1989). Do and colleagues (1990) have identified TTX-producing bacteria in deepsea sediment and postulated that TTXs found in marine

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sediments are synthesized only by bacteria and are concentrated and deposited by benthic organisms in the food web. It is likely that toxin-producing bacteria may in fact be responsible for much of the TTX toxicity of several marine finfish and shellfish. For example, TTX-producing bacteria have been isolated from the intestines of the trumpet shell, Charonia sauliae (Narita et al., 1989). This fish has been implicated in poisoning incidents (Narita et al., 1981). The bacteria were also isolated from the intestines of the xanthid crab, Atergatis floridus (Noguchi et al., 1983, 1986). One study suggests that TTX found in pufferfish is a result of their exposure to TTX-producing marine bacteria (Matsui et al., 1990). It would seem that the production of TTXs by a wide variety of bacteria, many of which are prevalent in the marine environment, could lead to TTX contamination of finfish other than pufferfish. TTX is an alkaloid and a derivative of aminoperhydroquinazoline with a molecular weight of 319. It is sparingly soluble in water, soluble in acid solutions, and unstable above pH 7 and below pH 3 (Kao, 1966). It is also partly inactivated by heating at 116°C under 12.5 psi for 75 minutes (Halstead and Bunker, 1953). The monoacidic base has a pKa of 8.5. The terminal amino group tends to form a zwitterion with one of the hydroxyl groups (Camougis et al., 1967). Mode of Action The mechanism of tetrodotoxin poisoning is similar to that of saxitoxin (STX), but more intense. Like STX, it interferes with nerve conduction by blocking the sodium channel. Thus, the initial increase in the permeability of nerve membranes to sodium ions is curtailed. The evidence in support of the notion that TTX selectively blocks the sodium channel when using frog skeletal muscle fibers was first obtained by Narahashi and coworkers (1960). TTX was found to block the action potential without affecting the resting potential, resting membrane resistance, or delayed rectification, as is indicative of delayed potassium conductance increase. These researchers concluded that TTX blocked the action potential through its selective inhibition of the sodium conductance mechanism. This notion was later demonstrated more clearly by voltage clamp experiments with lobster giant axons (Narahashi et al., 1964). TTX has a specific affinity for the sodium channel. Although it is highly potent in blocking the sodium current when applied outside the membrane, it has no effect when perfused internally. It does not permeate the nerve membrane; therefore, the internal membrane surface is much less sensitive to TTX than the external surface with more than a 1000-fold difference (Cuervo and Adelman, 1970).

O

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3 1

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(A) Tetrodotoxin O O H H2N

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(B) Anhydrotetrodotoxin Tetrodotoxin (TTX) Tetrodotoxin (A) TTX 4-epiTTX 6-epiTTX 11-deoxyTTX 11-norTTX-6(R)-ol 11-deoxy-4-epiTTX 11-oxoTTX Anhydrotetrodotoxin (B) 4,9-anhydroTTX 4,9-anhydro-6-epiTTX 4,9-anhydro-11-deoxyTTX

Figure 14.13

R1

R2

R3

R4

H OH H H H OH H

OH H OH OH OH H OH

OH OH CH2OH OH H OH OH

CH2OH CH2OH OH CH3 OH CH3 CH(OH)2

OH CH2OH OH

CH2OH OH CH3

(A) Tetrodotoxin (TTX) and (B) anhydrotetrodotoxin (anhydroTTX) derivatives occurring in nature.

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The stoichiometrical characteristics of TTX binding to the sodium channel have been studied extensively, and most studies give a value of one toxin molecule binding to one channel (Almers and Levinson, 1975; Bay and Strichartz, 1980; Keynes and Ritchie, 1984). Electrophysiological experiments have also indicated 1:1 stoichiometry. This stoichiometry provides support to the hypothesis that one TTX molecule plugs a sodium channel at its external mouth, the model originally proposed by Kao and Nishiyama (1965) and later elaborated by Hille (1975). Later on, Kao and associates proposed a model in which the TTX binding site is located adjacent to the external orifice of the sodium channel, and not the selectivity filter inside the orifice of the channel (Kao, 1983; Kao and Walker, 1982; Kao et al., 1983). This revised model of binding site has further been extended to include two cooperative sites (Benoit and Dubois, 1985). On the basis of the markedly delayed onset of TTX block of the sodium channel in the node of Ranvier and of the high Hill coefficient of 1.74, it was proposed that two TTX molecules bind to separate sites in the immediate vicinity of the external orifice of the sodium channel in a sequence with dissociation constants of K1 = 3 nM and K2 = 0.8 nM. Only after two TTX molecules have occupied both sites does the channel become nonconductive. For TTX, the 1, 2, 3guanidinium group and the C-9 and C-10 hydroxy groups are important, and the C-4 and C-8 groups also contribute to the binding. Apparently, other special types of sodium channels outside the nerve membrane, such as in the fibroblast, are also blocked by TTX (Pouyssegur et al., 1980). However, not all sodium channels in these cells are blocked; thus, those channels essential to DNA synthesis and cell proliferation are not blocked. Similarly to STX, TTX also causes hypotension in addition to the classic muscular paralysis; in this case, hypotension is more pronounced and predictable. This follows from a direct relaxation of the smooth muscles and a blockage of the vasomotor nerves, resulting in a release of the vasomotor tone (Kao, 1972). TTX has no effect on the release of neurotransmitters from the presynaptic nerve terminals at the synaptic and neuromuscular junctions (Halstead, 1981). It has little or no effect on smooth muscles such as cardiac tissue. This toxin also has little, if any, influence on active sodium transport. TTX has a profound effect on respiration, is a powerful emetic, and produces hypothermia that also indicates its action on central nervous system. Very low doses, 4 to 5 µg/kg, can cause immediate respiratory arrest in animals (Evans, 1969).

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Toxicity The lethality of pufferfish is determined by the mouse bioassay. Pufferfish is considered to be very toxic if less than 10 g of tissue is lethal, moderately toxic if 10 to 100 g is lethal, weakly toxic if 100 to 1000 g is lethal, or in terms of mouse units, 20,000, 2000, and 200 MU or more, respectively. A mouse unit in this case is the amount of toxin per gram of mouse required to kill a 15- to 21-g mouse. Thus, 1000 MU is sufficient to kill 50 20-g mice. The minimal lethal dose (MLD) in mouse units is 0.4 µg tetrodotoxin/20 g mouse. On the basis of the MLD for mice, the calculated MLD for humans is 4 µg/kg (approximately 280 µg for an average human) (Halstead, 1981). This would amount to 100 g of tissue containing 2.8 µg/g. By means of capillary isotachophoresis assay, the amount of TTX in the ovary of the more commonly consumed pufferfish species (F. vermicularis vermicularis, F. rubripes rubripes, and F. poecilonotus) ranges from 0.146 to 0.404 mg/g; that of the liver, from 0.0018 to 0.113 mg/g; and of skin, from 0 to 0.301 mg/g (Shimada et al., 1983). Thus, it is obvious that consumption of as little as 10 g of tissue may be fatal to humans. However, in these species of pufferfish, the muscles are essentially free of toxin. Several analytical methods, including bioassays, TLC, HPLC, pharmacological assays, and immunoassays, are available for the qualitative and quantitative analysis of TTX. The mouse bioassay is similar to that for the PSP toxins since both groups cause blockage of the sodium channel with death by respiratory paralysis. The bioassay method for determining TTX requires that the ground tissue be extracted with slightly acidic hot water, cooled, and then clarified by filtration or centrifugation. One-milliliter aliquots of the extract are then injected into the mice. One mouse unit is defined as the amount of TTX required in a ddY strain of mouse weighing 18–21 g in 30 min after an intraperitoneal injection. Because of their common mechanism of intoxication, many of the same pharmacological assays developed for STXs have been applied to TTX (Frace et al., 1986; Kogure et al., 1989; Daigo et al., 1989). TLC, cellulose acetate membrane electrophoresis, and infrared and nuclear magnetic resonance (NMR) spectra can do qualitative analyses of TTX. Nunez amd coworkers (1976) treated TTX with strong alkali to produce a fluorescent compound with a maximal excitation at 370 nm and maximal emission at 495 nm. Shimada and associates (1983) described a method for quantification of TTX in crude extracts of pufferfish by capillary isotachophoresis. The lower limit of detection for this method is about 10 µg toxin/g of tissue.

Symptoms The symptoms of pufferfish poisoning in humans are consistent with the observed effects of TTX. The initial signs of tetrodotoxism usually begin with circumoral paresthesia. Nonspecific signs such as nausea, vomiting, and diarrhea commonly precede the sign of paresthesia. The onset of signs usually begins within 3 hours of ingesting the toxin-containing organ, although this could vary from 10 minutes to 15 hours, depending on the quantity of toxin consumed. Other common complaints include headache, diaphoresis, unusual taste sensation with hypersalivation, generalized muscle weakness, myalgia, lethargy, floating sensation, and progressive numbness of the entire body. If the signs of paresthesia progress from the lip, tongue, and oropharynx to the distal extremities, this diagnosis should be confirmed. Clinical signs associated with tetrodotoxin intoxication include dysarthria, dysphagia, hyperemesis, progressive weakness, ataxia, hypersalivation, progressive ascending paralysis, loss of deep tendon reflex, cranial nerve dysfunction, muscle fasciculations, cardiac arrhythmia (particularly sinus bradycardia), hypotension (shock), and acute respiratory failure with associated cyanosis, pallor, and dyspnea (Goldfrank et al., 1981; Lyn, 1985; Kodama et al., 1985; Sims, 1987; Concon, 1988; Lang, 1990; Smith, 1992). Death may occur as early as 17 min after ingestion of puffer meat, but usually between 6 and 24 hr. Survival after 24 hr may indicate a good prognosis for the patient. In summary, the appearance of the symptoms of puffer poisoning may be divided into four stages: 1. 2. 3.

4.

Oral paresthesias and gastrointestinal symptoms Paresthesias spreading to other areas and motor paralysis of the extremities, with reflexes intact More severe symptoms of gross muscular incoordination, e.g., aphonia, respiratory distress, hypotension, with no loss of consciousness Agonal symptoms of severe hypotension, respiratory paralysis, and impairment of mental acuity (cardiac beat is maintained for a short period in this stage)

Differential diagnosis for tetrodotoxism includes paralytic shellfish poisoning and ciguatera fish poisoning, as well as organic phosphate intoxication. In PSP, hypotension may be present but is not a hallmark of the intoxication as with TTX. The latter directly affects the vascular smooth muscle and the neurovascular innervation of blood vessels. In ciguatera poisoning, nausea, vomiting, diarrhea, and ab-

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dominal pain followed by paresthesia are among the first presenting signs (Smith, 1992). The suggested treatment for puffer fish poisoning is symptomatic. If the toxin has not already induced emesis or hyperemesis, then an emetic should be administered. Residual toxin in the stomach can be removed by gastric lavage with particular care taken to prevent aspiration. More aggressive therapy includes endoscopy to remove ingested toxin from the proximal small bowel (Goldfrank et al., 1981). Activated charcoal is effective in binding the toxin, thus preventing further gastrointestinal absorption. General supportive care depends on the signs and symptoms and may include early intubation with ventilatory assistance and oxygen supplementation, use of intravenous normal saline solution and dopamine, and intravenous atropine. The best safeguard against puffer poisoning obviously is to avoid these species altogether. By all means, the pufferfish must be prepared by an expert and bought from a reputable and licensed restaurant. Nevertheless, even under these conditions, the risk of serious poisoning remains. 14.3.7

Palytoxin

Palytoxin (PTX) is widely distributed and found in triggerfish, filefish, parrotfish, and xanthid crabs from the Philippines and Singapore. Two palytoxin analogs were isolated from a red alga, Chondria armata, a species that also produces domoic acid. Human illnesses and death have occurred after consumption of xanthid crabs (Alcala et al., 1988), mackerel (Kodama et al., 1989), and parrotfish (Yasumoto and Murata, 1990). Originally isolated from various corals of the genus Palythoa, palytoxin (Figure 14.14) has one of the most complicated structures of a natural product (Moore and Bartolini, 1981; Cha et al., 1982). Like OA, it is a potent tumor promoter (Fujiki et al., 1986). Palytoxin increases cell permeability for sodium and potassium but not calcium and at concentrations over 100 nM inhibits sodium/potassium adenosine triphosphatase (ATPase). Because the enzyme is a transmembranal protein, palytoxin can trigger a pore as soon as it has recognized the target enzyme (Habermann, 1989). The acute toxicity of PTX in several animal species has been examined (Wiles et al., 1974; Vick and Wiles, 1975). The lethal doses (LD50) of the toxin in rats, mice, guinea pigs, rabbits, dogs, and monkeys range between 33 and 450 mg/kg when injected intravenously. However, when administered by the intragastric or intrarectal route, PTX is relatively nontoxic. The toxic symptoms are species-dependent. Early signs in monkeys included ataxia,

OH O

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O

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OH OH

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103

OH

HO

OH

OH

80 90

O

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97 93 Me HO

OH OH

OH OH OH

71

OH O

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OH

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f

d

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a

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35

62 58

OH

O

20

26

HO

OH

Me OH

O HO

OH OH

HO

OH

50

43 47

OH

Figure 14.14 Palytoxin, the most potent marine toxin isolated from various corals of the genus Palythoa.

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OH OH

11 15 HO

67

OH

drowsiness, and weakness of the limbs, followed by collapse and death. Those in dogs are defecation and vomiting followed by ataxia, weakness, collapse, and death. Rats, guinea pigs, rabbits, and mice became drowsy and inactive after injection of PTX. It caused marked irritation and tissue injury when topically applied to the skin or eyes, as well as having a general necrotizing action on cells when injected. Death caused by PTX is generally attributed to multiple failure of the cardiovascular system (Vick and Wiles, 1975). 14.3.8

Seafood Allergy

Reactions to seafood, particularly fish and crustacea, are among the most commonly encountered food allergies in adults (Saavedra-Delgado and Metcalfe, 1984; Taylor and Bush, 1988). The prevalence of particular food sensitivities in various populations often depends on the eating habits of the region. For example, allergic reactions to the ingestion of fish are particularly prominent in Scandinavian countries, possibly because fish consumption is more frequent in that area. In one study fish provoked asthma in 50 (6.9%) and urticaria in 76 (10.3%) of the 825 pediatric patients of an asthma and allergy clinic (Aas, 1966). Many, if not all, seafood species may be allergenic. However, most seafood-allergic patients are not sensitive to all species. The more commonly consumed species are likely to be among the most frequently allergenic seafoods (Taylor and Bush, 1988). Cross-reactions can occur when individuals are sensitive to genetically related fish species. However, the taxonomic diversity among edible seafoods suggests that complete cross-reactivity is unlikely; few, if any, individuals have allergic reactions to all seafoods. Food allergies represent an immediate hypersensitivity reaction. The immunogenic potential of the seafood, i.e., the likelihood that some protein antigen in the seafood will elicit an immunoglobulin E (IgE) response, is an important factor in determining the frequency of allergic reactions. Allergenic reactions are due to the interaction of food antigens with antigen-specific IgE bound to the surface of mast cells and basophils. The binding triggers the release of the preformed chemical mediators contained in the cell granules. Other chemical mediators are generated from the phospholipids of cell membranes. Together these mediators exert their influence by pharmacological action on target organs, such as blood vessels and airway smooth muscle. They increase vascular permeability, constrict bronchial smooth muscles, stimulate mucus production in the airways, and recruit inflammatory cells into the reaction site. The reaction may be localized in the skin, producing urticaria and angioedema, or may be a more

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generalized process known as systemic anaphylaxis, a generalized life-threatening allergic reaction that involves multiple organ systems (Table 14.17) Allergic reactions develop rapidly, usually within minutes to an hour after the food antigen is ingested. The avoidance of offending seafoods is the most common approach to the treatment of seafood-induced allergies. Care should be taken to avoid all foods that induce reactions. The avoidance diet should be as specific as possible, thereby allowing the patient to have access to the widest possible variety of foods. Appropriate diagnostic procedures can lead to the identification of reactions to the various classes of seafoods and the development of more specific advice on avoidance diets. 14.3.9

Miscellaneous Phycotoxins

There are many other toxic compounds that are known to occur in foods of marine origin. Examples include cyanobacterial toxins and the macrocyclic lactones. Various cyanobacteria (blue-green algae) are known to produce a number of potent toxins. The major ones are the microcystins and nodularin, which are hepatotoxic peptides; anatoxin-a, an alkaloid neurotoxin; and anatoxin-a(s), a guanidinium phosphate ester that inhibits the enzyme cholinesterase (Carmichael and Falconer, 1993; Negri and Jones, 1995). The effects of cyanobacterial toxins on human health through potable water supplies and recreational use of affected fresh water are well documented (Carmichael and Falconer, 1993; Codd, 1994; Ressom et al., 1994). These toxins have also been found in crustaceans and shellfish harvested from waters with high cyanobacterial concentrations (Falconer, 1993).

Table 14.17 Signs and Symptoms of Anaphylactic Reactions Due to Seafood Allergy Respiratory tract Nasal congestion, dyspnea, stridor, cough, wheezing, tachypnea Cardiovascular system Hypotension, syncope, faintness, arrhythmia (sinus bradycardia, nodal rhythm, atrial fibrillation, ventricular fibrillation, asystole) Skin Urticaria, pruritus, flushing, angioedema Gastrointestinal tract Abdominal cramping, nausea, vomiting, diarrhea Eye Ocular pruritus, periorbital edema, conjunctival inflammation, lacrimation

The discovery that chronic exposure to some cyanobacterial toxins may promote tumor production (Falconer, 1991; Falconer and Humpage, 1996; Yu, 1994) will likely increase the awareness of these toxins, although the main risk is posed by fresh water rather than food. Examples of toxic macrocyclic lactones are prorocentrolide (PC), prorocentrolide-B, and gymnodinine (Torigoe et al., 1988; Hu et al., 1996). PC cooccurs with OA and DTX-1 in the dinoflagellate Prorocentrum lima but has an entirely different skeleton, which includes an imine group. PC was found to be lethal to mice at a dose of 400 µg/kg injected intraperitoneally. Gymnodinine sp. has been isolated from oysters in New Zealand and the dinoflagellate Gymnodinium spp. (Seki et al., 1995, 1996; Mackenzie et al., 1996). The molecule is composed of a 16-membered carbocyclic ring, a butenolide ring, and a cyclic imine. Its lethality to mice is 450 µg/kg intraperitoneally, but it is not thought to constitute a hazard to human health. Examples of other phycotoxins include cooliatoxin (Holmes et al., 1995) and amphidinol-2 (Shimizu, 1996). Although these toxins have not yet been directly implicated in human poisonings, they may be present in the food chain. Some of these compounds are ichthyotoxic, and algal blooms have been responsible for the large-scale mortality rate of both wild and cultured fish (Andersen, 1996). As a ready reference, the characteristics of most important phycotoxins are summarized in Table 14.18. It is quite obvious from this table that bivalve shellfish are the most common vectors of phycotoxins other than in ciguatera fish poisoning. This is because they are filter feeders and naturally ingest any phytoplankton or particles to which they may be attached and thereby concentrate any toxins in their tissues. The shellfish that have been most studied with regard to accumulation of algal toxins are those consumed directly by humans, such as clams and mussels. Nonetheless, the importance of carnivorous gastropods and crustaceans as vectors should not be ignored.

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Similarly, data on the site of action and primary physiological effects of phycotoxins and the comparative potency of these toxins as compared to other toxins from natural sources are summarized in Tables 14.19 and 14.20. The effects of processing on the phycotoxins content of various shellfish are presented in Table 14.21. It appears that with the possible exception of the cooking method employed, there have been no useful methods devised for effectively reducing phycotoxins in contaminated shellfish. Even though cooking may reduce levels of toxins, it does not completely eliminate the danger of intoxication. Physical and chemical means of possible detoxification studied include temperature and salinity stress, electrical shock treatments, reduction of pH, chlorination, and ozone treatment. Most methods tested to date have been unsafe or economically unfeasible or have yielded products unacceptable in appearance and taste. It is quite evident here that many species of marine life that are actual or potential food sources for humans present a complex food toxicological and public health dilemma. The phycotoxins represent a particularly problematical group of compounds as regards detection and quantitation in food. Fortunately, only a few seafood toxicants, such as ciguatera and shellfish, present significant public health problems worldwide. The shellfish poisoning can at least be minimized by finding ways to predict and control algal blooms and by eliminating dinoflagellates in clam beds. This is unfortunately not the case with ciguatera poisoning. Although not as serious as paralytic shellfish poisoning, ciguatera toxicity occurs in many normally edible fish. The fact that marine phycotoxins pose a threat to human health led a number of countries to establish regulations to control the presence of these toxins in seafood. Currently, only a few countries have established regulations for shellfish toxins other than PSP and DSP. Four have proposed regulations for domoic acid; a few regulations exist for NSP, specifically for brevetoxins, and for ciguatera toxins in finfish.

Table 14.18

Phycotoxin Poisoning Causative and Vector Organisms, Clinical Symptoms, and Treatment

Paralytic shellfish poisoning (PSP) Causative organisms Dinoflagellates (e.g., Alexandrium catenella, A. minutum, A. tamarense; Gymnodinium catenatum; Pyrodinium bahamense) Vector organisms Shellfish and crustaceans (e.g., mussels, clams, oysters and lobsters)

Effects and symptoms Neurotoxic Mild case: within 30 min: tingling sensation or numbness around lips, gradually spreading to face and neck; prickly sensation in fingertips and toes; headache, dizziness, nausea, vomiting, and diarrhea

Diarrhetic shellfish poisoning (DSP)

Amnesic shellfish poisoning (ASP)

Neurotoxic shellfish poisoning (NSP)

Dinoflagellates (e.g., Dinophysis acuminata, D. acuta, D. fortii, D. norvegica; Prorocentrum lima)

Diatoms (e.g., Nitzschia actydrophila, Pseudo-nitzschia australis, P. pseudodelicatissima, P. pungens f. multiseries, P. seriata)

Dinoflagellates (e.g., Gymnodium breve [= Ptychodiscus brevis]) Raphidophytes (e.g., Fibrocapsa japonica, Heterosigma akashiwo)a

Dinoflagellates (e.g., Gambierdiscus toxicus, Prorocentrum lima [?])

Bacteria (e.g., toxin-producing bacteria in deep-sea sediments, concentrated in vectors by benthic organisms in food web)

Shellfish (e.g., mussels)

Shellfish (e.g., blue mussels and crabs)

Shellfish (e.g., mussels and clams); may also affect humans by direct contact with toxic algae via sea spray or swimming

Carnivorous fish, mainly barracudas, groupers, jacks, sea bass, snappers, and surgeon fish, that feed on herbivorous fish that inhabit coral reefs

Pufferfish (e.g., blowfish, globefish, fugu), certain tropical and subtropical crabs, eggs of blue-ringed octopus

Gastrointestinal disturbance After 30 min to a few hours (seldom more than 12 hr): diarrhea, nausea, vomiting, abdominal pain

Neurotoxic

Neurotoxic

Neurotoxic

After 3 to 5 hr: nausea, vomiting, diarrhea, abdominal cramps

After 3 to 6 hr: chills, headache, diarrhea, muscle weakness, muscle and joint pain, nausea and vomiting, irritation to eyes and nasal membranes where in direct contact with algae

Complex, primarily neurotoxic Symptom development 12 to 24 hr after eating fish, gastrointestinal symptoms, diarrhea, abdominal pain, nausea, vomiting

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Ciguatera fish poisoning

Tetrodotoxin fish poisoning

Within 15 min to 3 hr: cirumoral paresthesia, headache, diaphoresis, hypersalivation, muscle weakness, numbness of entire body

Extreme case: muscular paralysis, pronounced respiratory difficulty, choking sensation, death through respiratory paralysis possible 2 to 24 hr after ingestion

Average fatality rate 1%–14%

Treatments Patient stomach pumping and artificial respiration, no lasting effects

a

Chronic exposure potential promoter of tumor formation in the digestive system

Decreased reaction to deep pain, dizziness, hallucinations, confusion, short-term memory loss (sometimes permanent), damage to hippocampus in brain, coma, death in some extreme cases

Paresthesia, altered perception of hot and cold, difficulty in breathing, double vision, trouble in talking and swallowing

Neurological symptoms, numbness and tingling of hands and feet, cold objects perceived as hot to touch, difficulty in balance, low heart rate and blood pressure, rashes, death in extreme cases through respiratory failure

Acute respiratory failure with associated cyanosis, pallor, and dyspnea; cardiac arrhythmia; death within 15 min to 6 to 24 hr, depending on quantity ingested

0%

3%

0%

<1%

Up to 60% in severe cases, generally 10%–20% accounting for over 80% of all seafood poisoning deaths in Japan

Intravenous infusion of electrolytes, recovery after 3 days irrespective of medical treatment

None at present other than life support systems if required

Support as required

No antidote or specific treatment available, neurological symptoms that may last months and years, potential symptom relief with calcium and mannitol

Symptomatic, gastric lavage, emetics, artificial respiration, recovery expected if patient survives beyond 24 hr

Brevetoxins have been detected in these algae but have not yet been implicated in NSP.

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Table 14.19

Sites of Action and Primary Physiological Effects of Phycotoxinsa

Phycotoxin PSP toxins

DSP toxins Okadaic acid and derivatives

Pectenotoxins Yessotoxin NSP toxins

Domoic acid (ASP)

Ciguatera toxins Ciguatoxin

Blocks sodium ion influx in neuronal and muscular sodium channel; prevents propagation of action potential essential for conduction of nerve impulses and contraction of muscles; peripheral nervous system particularly affected in vertebrates

Catalytic subunit of protein phosphorylase, phosphatases type 1 and 2A Unknown Unknown Site 5 on voltage-dependent sodium channels

Inhibits protein phosphorylase phosphatases; powerful tumor promoters in two-stage carcinogenic experiments Has hepatotoxic effect Damages heart muscle Induces channel-mediated sodium ion influx; depolarizes isolated muscle and nerve cells Binds to glutamate receptors, i.e., competes with glutamate; causes receptorinduced depolarization and excitation

Glutamate receptors in central nervous system

Site 5 on voltage-dependent sodium channels

Calcium channels

Scaritoxin

Site 5 on voltage-dependent sodium channels

Palytoxin

Cyanobacterial toxins (microcystins, nodularin)

Primary physiological effects

Site 1 on voltage-dependent sodium channels

Maitotoxin

Tetrodotoxins

a

Biochemical site of action

Site 1 on voltage-dependent sodium channels Inhibits sodium/potassium ATPase

Inhibition of cholinesterase

Opens sodium channels at resting potential and causes inability of open channels to be inactivated during subsequent depolarization; Produces calcium ion influx, which may lead to cell death Depresses oxidative metabolic processes in brain; causes depolarization of excitable membranes Has same effects as PSP, but more intense and severe Increases cell permeability for sodium and potassium but not calcium ions; acts as potent tumor promoter Has hepatotoxic effects; affects neurotransmission; acts as powerful tumor promoter

PSP, paralytic shellfish poison; DSP, diarrhetic shellfish poison; NSP, neurotoxic shellfish poison; ASP, amnesic shellfish poison; ATPase, adenosine triphosphatase.

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Table 14.20

Comparative Potency of Some Phycotoxins Compared to Other Natural Toxins

Toxin

LD50 (µg/kg)a

Ciguatoxin Maitotoxin Saxitoxin Tetrodotoxin Palytoxin Botulinum A Okadaic acid 35S-methyl okadaic acid d-Tubocurare Brevetoxin a Brevetoxin b

0.45 0.13 3.0 8.0 0.15 2.6 × 10–5 192.0

a

200.0 95.0 500.0

1,111 145,000 309 319 3,300 150,000 786 696 900 885

Source Gymnothorax javanicus Gambierdiscus toxicus Spondylus butreli Fugu vermicularis porphyrreus Palythoa spp. Clostridium botulinum Prorocentrum lima Dinophysis fortii Chondodendrum tomentosum Ptychodiscus brevis Ptychodiscus brevis

Intraperitoneal in 20-g mice.

Table 14.21 Toxin PSP

Effects of Processing on the Phycotoxin Content of Some Shellfish Shellfish American lobster (Homarus americanus) Butter clams (Saxidomus giganteus) Mediterranean cockle (Acanthocardia tuberosum) Mussel (Mytilus edulis) Soft clam (Mya arenaria) Scallop (Patinopecten yessoensis)

DSP Domoic acid

a

Molecular weight

Mussel (Mytilus sp.) Crab (Cancer magister)

Process

Reference

Cooking

Reduction

Cooking

Reduction

Lawrence et al. (1994) Watson-Wright et al. (1991) Quayle (1969)

Freezing Cooking

Minimal effect Reduction

Quayle (1969) Berenguer et al. (1993)

Cooking Cooking Cooking

Reduction Reduction Reduction

Freezing

Migration to other tissues No reduction Reduction Migration to other tissues

Prakash et al. (1971) Prakash et al. (1971) Noguchi et al. (1980a, 1980b) Ohta et al. (1992) Noguchi et al. (1984)

Cooking Cooking Chilling and freezing

PSP, paralytic shellfish poison; DSP, diarrhetic shellfish poison.

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Effect

Vernoux et al. (1994) Hatfield et al. (1995) Hatfield et al. (1995)

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Oshima, and Y. Fukuyo, pp. 19–22, Intergovernmental Oceanographic Commission of UNESCO, Paris. Yamanouchi, T. 1955. On the poisonous substances contained in holothurians. Publ. Seto Mar. Biol. Lab. 4(2–3):183–203. Yasumoto, T. 1971. Toxicity of the surgeon fishes. Bull. Jpn. Soc. Sci. Fish. 37:724–734. Yasumoto, T. 1990. Marine microorganisms toxins, an overview. In Toxic Marine Phytoplankton, Proceedings of the Fourth International Conference on Toxic Marine Phytoplankton, 1989, ed. E. Graneli, B. Sundstrom, L. Edler, and D. M. Anderson, pp. 3–8, Elsevier, New York. Yasumoto, T. and Kanno, K. 1976. Occurrence of toxins resembling ciguatoxin, scaritoxin, and maitotoxin in a turban shell. Bull. Jpn. Soc. Sci. Fish. 42:1399–1404. Yasumoto, T. and Kotaki, Y. 1977. Occurrence of saxitoxin in a green turban shell. Bull. Jpn. Soc. Sci. Fish. 43:207–211. Yasumoto, T. and Murata, M. 1990. Polyether toxins involved in seafood poisoning. In Marine Toxins: Origin, Structure and Molecular Pharmacology, ed. S. Hall and G. Strichartz, pp. 120–132. American Chemical Society, Washington, D.C. Yasumoto, T. and Murata, M. 1990. Polyether toxins involved in seafood poisoning. In Marine Toxins: Origin, Structure, and Molecular Pharmacology, ed. S. Hall and G. Strichartz, pp. 120–132. American Chemical Society, Washington, D.C. Yasumoto, T. and Scheuer, P. J. 1969. Marine toxins of the Pacific. VIII. Ciguatoxin from moray eel livers. Toxicon 7:273–276. Yasumoto, T., Inoue, A., Bagnis, R. A., and Garcon, M. 1979b. Ecological survey on a dinoflagellate responsible for the induction of ciguatera. Bull. Jpn. Soc. Sci. Fish. 45:395–399. Yasumoto, T., Murata, M., Lee, J. -S., and Torigae, K. 1988. Polyether toxins produced by dinoflagellates. In Mycotoxins and Phycotoxins, ed. S. Natori, K. Hashimoto, and Y. Ueno, pp. 375–382. Elsevier, Amsterdam. Yasumoto, T., Murata, M., Lee, J. -S., and Torigoe, K. 1989. Polyether toxins produced by dinoflagellates. In Mycotoxins and Phycotoxins ’88, ed. S. Natori, K. Hashimoto, and T. Ueno, pp. 375–383. Elsevier, New York. Yasumoto, T., Murata, M., Oshima, Y., Matsumoto, G. K., and Clardy, J. 1984. Diarrhetic shellfish poisoning. In Seafood Toxins, ed. E. Ragelis, pp. 207–214. American Chemical Society, Washington, D.C. Yasumoto, T., Murata, M., Oshima, Y., Sano, M., Matsumoto, G. K., and Clardy, J. 1985. Diarrhetic shellfish toxins. Tetrahedron Lett. 41:1019–1025. Yasumoto, T., Nakajima, I., Oshima, Y., and Bagnis, R. A. 1979a. A new toxic dinoflagellate found in association with ciguatera. In Toxic Dinoflagellate Blooms, ed. D. L. Taylor and H. H. Seliger, pp. 65–70. Elsevier, Amsterdam. Yasumoto, T., Oshima, Y., and Konata, T. 1981. Analysis of paralytic shellfish toxins of xanthid crabs in Okinawa. Bull. Jpn. Soc. Sci. Fish. 47:957–959.

Yasumoto, T., Oshima, Y., Tajiri, M., and Kotari, Y. 1983. Paralytic shellfish toxins in previously unrecorded species of coral reef crabs. Bull. Jpn. Soc. Sci. Fish. 49:633–636. Yasumoto, T., Oshima, Y., and Yamaguchi, M. 1978. Occurrence of a new type of shellfish poisoning in the Tohoku District. Bull. Jpn. Soc. Sci. Fish. 44:1249–1255. Yasumoto, T., Yasumura, D., Ohizumi, K., Takahashi, M., Alcala, A. C., and Alcala, L. C. 1986. Polytoxin in two species of xanthid crabs from the Philippines. Agric. Biol. Chem. 50:163–167.

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Yasumura, D., Oshima, Y., Yasumoto, T., Alcala, A. C., and Alcala, L. C. 1986. Tetrodotoxin and paralytic shellfish toxins in Philippine crabs. Agric. Biol. Chem. 50:593–598. Yu, S. -Z. 1994. Blue-green algae and liver cancer. In Toxic Cyanobacteria, Current Status of Research and Management, ed. D. A. Steffenson and B. C. Nicholson, pp. 75–85. Australian Center for Water Quality Research, Salisbury. Zheng, W. J., Demattei, J. A., Wu, J. P., Duan, J.J.W., Cook, L. R., Oinuma, H., and Kishi, Y. 1996. Complete relative stereochemistry of maitotoxin. J. Am. Chem. Soc. 118: 7946–7968.

15 Mushroom Toxins

15.1 INTRODUCTION Mushrooms, from either commercial cultivation or natural habitats, are highly valued for their characteristic and often delicate flavor. Whereas the commercial sources may be limited to a few varieties, the wild sources provide thousands of varieties. The sudden demand for new mushroom tastes and a trend toward the ingestion of uncooked vegetables in previously mycophobic North America during the past two decades have further increased the popularity of mushrooms in the human diet and the world trade in mushrooms. For many centuries, mushroom gathering in the wilds has been a common practice in many parts of the world. However, this practice is replete with danger from serious and often fatal poisonings, not only for the unfamiliar, but also for the experienced mushroom gatherer. Concon (1988) has cited several reasons for this: 1.

2.

3.

There are thousands of mushroom species, some 3000 in the United States alone (Smith, 1958). Not all have been documented for their possible poisonous and intoxicating effects. Several mushroom species exhibit very similar morphological characteristics. Sometimes the differentiation between a poisonous and a nonpoisonous species can only be made with microscopic examination of tissues and cellular structures. Thus human and animal poisoning can result from mistaken identity. An edible or poisonous species may change its morphological characteristics as a result of en-

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4.

5. 6.

vironmental or nutritional growth conditions. For example, the poisonous mushroom, Amanita muscaria may exist in three shades: dark red, yellowish orange, and white. These shades also vary in intensity with age or exposure to the sun and rain. The orange-capped variety may be confused with the edible A. cesarea mushroom. Sometimes a characteristic identifying feature of a mushroom species may be modified by mechanical damage. Such changes may cause confusion and mistaken identity. For example, such essential identifying features of the amanitas as the remnants of the universal and partial veils, the volva, and the annulus can be removed mechanically. This can happen by the force of heavy rains or the pressure exerted by the growing mushroom on the soil; the volva may also remain in the ground when the mushroom is pulled up during gathering. Different individuals react with varying susceptibilities to different poisonous mushrooms. The quantity of mushrooms consumed may determine the cause and nature of poisoning. This factor varies with the age and health status of the individual as well as the amount of toxin present in the mushrooms. The latter varies with the conditions of growth. For example, the toxic isoxazole content of A. muscaria harvested in Switzerland varied markedly from 0.03% of the wet weight in specimens collected in 1962 to a very high concentration of 0.1% of wet weight in specimens collected in the summer of 1966

7.

8.

(Eugster, 1968). Concentrations of the psychoactive ibotenic acid and muscimol from various psychoactive amanitas vary markedly according to geographical locations, season, and year of growth (Good et al., 1965; Takemoto et al., 1964a, 1964b). The edibility or toxicity depends on the way the mushroom is prepared or cooked. For example, insufficiently cooked Paxillus involutus killed three of four people so poisoned (Bschor and Mallach, 1963). Yet this mushroom seems harmless after boiling. Lactarius porninsis, L. plumbeus, and Pleurotus olearius (Clitocybe olearia) are also poisonous only when eaten raw or insufficiently cooked (Pilat, 1961). Although several species belonging to specific genera are proved edible, one or more species in those genera may be toxic. Because members of a genus are often very similar in morphological characteristics, the risk of mistaken identity is quite high.

Many toxic mushrooms have well-defined characteristics that may allow for easy identification. However, there are variations from the norm and intergradations within the species. Thus, the task of determining what is toxic and what is not within a genus, and especially within a species, can be a dangerous one. Indeed, even accomplished mycologists have erred (Smith, 1958; Litten, 1975). Identification of species may require both chemical and microscopic histological examination, especially of the spores. It should be noted that among the thousands of species of mushrooms known, fewer than 100 are poisonous or toxic to humans and animals (Buck 1961; Dalvi, 1991). Although the toxicity of most of the poisonous mushroom species has long been known, that of others has been recognized only in recent decades. Several mushroom species poisonous to humans and grazing animals with their potential toxic effects are listed in Table 15.1. Detailed descriptions can be found in several excellent articles and books (Hesler, 1960; Krieger, 1967; Pilat, 1961; Singer, 1961; Smith, 1958; Gray, 1973;

Table 15.1 Commonly Encountered Poisonous Mushrooms and Their Toxic Effects Mushroom species (common names) Agaricus. albolutescence A. arvensis var. palustris (meadow mushroom) A. hordensis A. placomyces (flat-capped mushroom) A. sylvicola A. xanthodermus Amanita bisporigera A. brunnescens A. capensis (cape death cap) A. chlorinosma A. citrina A. cothurnata A. flavoconiaa A. flavivolva A. gemmata A. mappa (false death cap) A. muscaria (fly agaric) A. pantherina (panther mushroom, panthercap) A. parcivolvata A. phalloides (death cap) A. porphyria A. regalis A. rubescence A. solitaria A. spreta A. strobiliformis

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Poisonous effects Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Cytotoxic (lethal) Cytotoxic (lethal) Cytotoxic (lethal) Gastrointestinal Neurological, hallucinogenic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic (very toxic) Neurological, muscarinic Cytotoxic (very lethal) Hallucinogenic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic

Table 15.1 (continued) Mushroom species (common names) Amanita bisporigera (continued) A. tenuifolia A. vernab (Spring amanita) A. virosab (destroying angel) Amanitopsis volvata Boleltus eastwoodiae B. erythropus B. luridus (lurid boletus) B. purpureus B. satanas (Satan’s boletus) Calvatia gigantea Cantharellus floccosus (Gomphus floccosus) (shaggy cantharelle) Chlorophyllum molybditis (Lepiota morgani, L. molybditis) Clavaria Formosa (coral fungi) C. gelatinosa Clitocybe cerussata C. claviceps C. dealbata C. illudens (jack-o-lantern, copper trumpet) C. olearia C. rivulosa C. subilludens C. sudorifica C. toxica C. truncicola Coprinus atramentarius Cortinarius orellanus C. traganusc (Inoloma traganus) Entoloma lividumb (leaden entoloma, livid entoloma) E. salmoneum (salmon-colored entoloma) E. sinuatuma E. strictusa E. vernum Galerina autumnalis (gill mushroom) G. marginata G. sulcicepsd G. venenata Gomphus kauffmanii Gymnopilus decurrens G. spectabilis Helvella esculenta (Gyromitra esculenta) (false morel) H. gigas (Gyromitra gigas) H. infula (hooded helvella) H. underwoodii Hygrophorus conicus (conic hygrophorus) Inocybe godego I. infelix (unfortunate inocybe) I. infida (untrustworthy inocybe) I. lacera I. napipes

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Poisonous effects Cytotoxic (lethal) Cytotoxic (lethal) Cytotoxic (lethal) Gastrointestinal

Probably gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal

Gastrointestinal Gastrointestinal Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Cytotoxic Neurological, muscarinic Disulfiramlike effect Cytotoxic Gastrointestinal Gastrointestinal

Cytotoxic Cytotoxic Cytotoxic (very lethal) Cytotoxic Neurological, cytotoxic Hallucinogenic Hallucinogenic Cytotoxic, hemolytic Cytotoxic Cytotoxic Cytotoxic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic Neurological, muscarinic (table continues)

Table 15.1 (continued) Mushroom species (common names) Inocybe godego (continued) I. nigrescence I. patouillardiib (red-staining inocybe) I. picrosma Lactarius chrysorheus L. glaucescens L. helvus L. lignyotus L. plumbeus L. porninsis L. rufus L. torminosus L. trivialise L. uvidus L. vellereus Lampteromyces japonicus Lepiota brunneoincarnata L. fuscovinacea L. helvella L. schulzeri Lycoperdon subincarnatum Morchella augusticeps Mycena purae Naematoloma fasciculare Panaeolus acuminatus P. retirugis P. semioviatus P. subbalteatus (P. venenosus) Panus stypticus Paxillus involutus Phallus ravenelii Psilocybe baeocystis P. cubensis P. mexicana P. semperviva Rhodophyllus sinuatus Rumaria flavobrunescence Russula densifolia R. emetica R. foetens (fetid russula) R. fragilisa R. nondorbingi Scleroderma aurantium S. cepa Stropharia aeruginosa S. coronillaa Tricholoma album T. flavobrunneum T. pardinumb T. pessundatum T. pessundatum var. montanum T. saponaceum

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Poisonous effects Neurological, muscarinic Neurological, muscarinic (lethal) Neurological, muscarinic Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Cytotoxic Cytotoxic Cytotoxic Cytotoxic Cytotoxic Gastrointestinal

Gastrointestinal, cytotoxic Hallucinogenic Hallucinogenic Hallucinogenic Hallucinogenic Gastrointestinal Gastrointestinal, cytotoxic Hallucinogenic Hallucinogenic Hallucinogenic Hallucinogenic Gastrointestinal Cytotoxic Gastrointestinal Gastrointestinal Neurological, cerebral Neurological Neurological

Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal Gastrointestinal, hemolytic

Table 15.1 (continued) Poisonous effects

Mushroom species (common names) Tricholoma album (continued) T. sejunctum Verpa bohemica Volvaria speciosaa V. gloiocephalaa

Neurological, gastrointestinal Neurological

a

Probably poisonous, conflicting reports. Very poisonous, high fatality rate. c Mildly poisonous. d Probably the most poisonous mushroom reported so far. e Suspected as poisonous. Source: From Concon (1988). b

Concon, 1988; Faulstich et al., 1980; Flammer and Horak, 1983; Flammer, 1985a, 1985b).

15.2 SYMPTOMS OF MUSHROOM POISONING Virtually all systemic mushroom intoxications can be divided into two main groups, depending on the duration of the latent period between the consumption of the mushroom and the appearance of symptoms. Usually the longer the latent period before the symptoms appear after ingestion the more dangerous the intoxication may be. This relationship may cause the diagnosis to be delayed, thus making the treatment difficult. Mushrooms that produce signs of toxicity within 2 hr of ingestion, or immediately after the consumption of alcohol, are rarely considered to be of serious consequence and require only conservative, symptomatic management. Intoxications characterized by a later onset, usually 6 hr or more after ingestion, are associated with serious intoxications with a grave prognosis. Each of the two groups can be further subdivided by its characteristic symptoms (Lampe, 1983). The rapid onset group may include the following: 1.

2. 3.

Mushrooms whose response is primarily gastroenteric irritation, which results in nausea, abdominal discomfort, diarrhea, and sometimes vomiting Mushrooms evoking sweating and other signs of parasympathetic hyperactivity The psilocybin-containing species, which produce delirium or hallucinations, not associated with sleep, or the deliriant mushrooms associated with sleep or coma (the pantherine syndrome)

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4.

Mushrooms not producing any adverse effects unless alcohol also is consumed

The delayed onset of poisoning category includes Gyromitra esculenta, which produces headache, nausea, and fatigue about 6 to 8 hr after ingestion; the Amanita phalloides group, which produce severe emesis and diarrhea approximately 12 hr after ingestion; and those producing a marked increase in fluid consumption and urination 3 days or more after ingestion, belonging to the Cortinarius spp. group. Unspecific intoxications may also occur when edible mushrooms or those that are conditionally edible are consumed uncooked in the form of raw salads. Many species, e.g., Boletus luridus, Armillaria mellea (syn. Clitocybe mellea, honey fungus or boot-lace fungus), Clitocybe nebularis (clouded agaric), and the very tasty Tricholoma nudum (syn. Lepista nuda), for which parboiling is recommended, cause intoxications, usually gastric upsets with symptoms of nausea, vomiting, and stomachache, when consumed uncooked. Usually, the intoxication by mushroom ingestion may be properly recognized on the basis of different symptoms, a mycological inquiry, and the recognition of the consumed mushroom, mainly via identification of the spores in the stomach contents. In the case of the most dangerous poisoning by death cap, Amanita phalloides (as well as by some Cortinarius species), biochemical tests are also very important since they make it possible to confirm the diagnosis, to correct the treatment, and to follow the dynamism of the pathological changes in the intoxicated organism. Naturally, all attempts should be made to obtain a specimen of the offending mushroom and to have it identified. Since it is not always possible to accomplish this rapidly, if at all, then the details concerning the preparation of

the mushroom may provide additional aid in the differential diagnosis and may even eliminate mushrooms as the cause of an illness. Von Clarmann (1964) gives a useful checklist for the adult patient. 1. 2.

3. 4.

5. 6. 7. 8. 9. 10.

Was more than one kind of mushroom consumed? How were the mushrooms stored between collection and preparation, and what was their condition at the time of preparation? Were the mushrooms eaten raw, sautéed, or prepared in boiling water as in a soup or stew? If they were prepared by simply boiling in water, was the cooking water discarded or ingested? At what time were the mushrooms eaten? Were they eaten again later and, if so, were they reheated? When and what were the first sign of illness? Are all the persons who ate the mushrooms ill? Are persons in the group who ate none of the mushrooms ill? Was alcohol drunk with the meal or within a day after the meal?

General procedures recommended in the treatment of various mushroom intoxications have been reviewed (Koppel, 1993; Pore, 1993; Spoerke and Rumack, 1992; Lampe, 1986; Gosselin et al., 1984; Henneberg et al., 1984).

15.3 INCIDENCES OF MUSHROOM POISONING Incidences of mushroom poisoning or mycetism, even epidemic ones with fatal consequences, are well documented in the medical literature. However, as compared to food poisoning incidences due to microbial contaminants, mushroom poisoning is sporadic in nature and often produces far fewer fatalities. Such incidences have been quite common in the European countries, where mycophagy is widely practiced. In contrast, in North America, where gathering of wild mushrooms is not so popular, relatively few incidences have occurred. More than 100 severe intoxications, 11 of which resulted in death, occurred in a small area near Konin, Poland, during a few months in 1952 after the ingestion of Cortinarius orellanus (Grzymala, 1957, 1959). In Switzerland in 1943 and 1944, 356 persons suffered from mushroom poisoning, and 4 died (Pilat and Usak, 1950). There were 1980 recorded cases of mycetism in Switzerland between 1918 and 1958, of which 5% were fatal (Alder, 1961). In the United States,

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675 cases of mycetism were reported to the U.S. Poison Control Centers, with no fatalities (Concon, 1988). In the period between 1931 and 1968, there were 64 deaths of mushroom poisoning reported in the United States (Benedict, 1972; Buck, 1961, 1964). In general, mushrooms raised and processed commercially may be considered safe for consumption. However, poisoning from commercial products still can occur, such as the poisoning from canned mushrooms from Taiwan (Rose and Reiders, 1966). That imported mushroom soup poisoned 55 of 86 women within 15 to 30 min after consumption. It should be emphasized that toxic species, such as Clitocybe dealbata and toxic Panaeolus species, may also grow in the same bed as the cultivated mushrooms (Krieger, 1967) and be carelessly harvested together with the edible species. Thus, for safety, one must discard any piece of mushroom that is different from the majority in the group. According to Bielski and Sikorski (1992), the intoxications from mushroom ingestion still compose an essential part of food poisonings causing a serious toxicological problem. Although lower fungi are known to be capable of causing serious health problems both to humans and animals, as in the case of ergotism or intoxications by aflatoxins and other mycotoxicoses (see Chapter 11), mushrooms can be especially dangerous to humans because of their use for culinary purposes. It appears from the statistical data from several European countries that especially in the time of hunger that follows wars and bad crops, the intoxications caused by mushrooms increase as they serve as a natural source of substitute food. Additionally, the intoxication by poisonous mushrooms very often occurs because they are confused with similar edible species and sometimes because they are believed to be edible.

15.4 TYPES OF POISONING Toxic mushrooms from one or more genera can be grouped together according to the toxic syndrome or principal symptoms produced after consumption. As a rule, a specific group of symptoms predominates. In many cases, the clinical manifestations may not immediately indicate the tissue or cellular damage that has occurred (Concon, 1988). Frequently, this is seen only during an autopsy or by closer biochemical and histological examination. Many cases of mycetism cannot be definitely assigned to a specific type of poisoning in spite of the clinical manifestations. On the basis of clinical manifestations, mushroom intoxications can be grouped into the following six categories: (a) cytotoxic and choleriform, including mushroom producing potent hepato- and nephrotoxins as

secondary metabolites; (b) neurotoxic; (c) hallucinogenic; (d) hemolytic; (e) gastrointestinal; and (f) those with obscure toxicity patterns. Mushroom intoxications can also be classified, as described before, as those with early or rapid onset and those having delayed onset reactions. The former category includes mushrooms that are gastrointestinal irritants, sweat-inducing mushrooms, those that induce delirium or are hallucinogenic, and those that induce sensitivity to alcohol. The delayed onset group includes the amatoxins (Amanita phalloides group), Gyromitra esculenta, and the Cortinarius spp. mushrooms. The following classification and discussion on mushroom toxins use the mechanism of toxic action as a general guideline. Some of the important mushroom toxins are described.

15.5 CYTOTOXIC HEPATO- AND NEPHROTOXINS Cytotoxic mushrooms cause most fatalities due to mycetism. Depending on the species or group, such mushroom poisoning may reveal different syndromes and durations of latency. Important members of this class are described in the following sections. 15.5.1

Amatoxins, Phallotoxins, and Virotoxins

Excluding those in Eastern Europe, by far the largest numbers of cases of severe mushroom poisoning are caused by the ingestion of the green death cap Amanita phalloides and by A. virosa (known as destroying angel or deadly agaric) and related species. The characteristic toxins of these mushrooms, cyclopeptide alkaloids, are also produced by A. verna, A. bisporigera, A. tenuifolia, A. ocreata, A. suballiacea, Lepiota brunneoincarnata, L. helveola, Galerina marginata, G. autumnalis, and G. venenata. Given the high content of toxins these mushrooms produce and their abundance all over Europe, it is not surprising that they account for more than 95% of all fatal cases on the continent. One medium-sized mushroom of A. phalloides contains 10 to 12 mg of amatoxins, a dose highly deleterious to an adult. Estimates of amatoxin poisonings in Europe vary between 50 and 500 cases per year. Considering that 64 children were treated for amatoxin poisoning in 1988 at the Children’s Health Center in Warsaw, Poland, alone and that 16 of these died, the higher number is probably realistic (Faulstich and Wieland, 1992). Increasingly, cases of A. phalloides poisoning are being reported in North America.

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The composition of toxins occurring in these mushrooms growing in North America is found to be similar to that of the corresponding European species (Benedict et al., 1970). Among the white amanitas, the white subspecies A. phalloides var. verna is very rare and only occasionally causes poisoning (Hazani et al., 1983). In contrast, the destroying angel, A. virosa, occurs frequently on the East Coast of the United States as well as in some places in Europe, e.g., southern Sweden and central France. Although its content of amatoxins is somewhat lower than that of the green species, it still represents one of the most dangerous mushroom species (Piering and Bratanow, 1990). Although visually not very attractive to mushroom gatherers, some amatoxins-containing species of the genera Lepiota and Galerina also appear to be increasingly involved in casualties (Piqueras, 1984; Sanz et al., 1989; Haines et al., 1985; Schulz-Weddingen, 1986; Langer et al., 1990). Chemical Characteristics On the basis of their toxicological properties and chemical features, the orally active thermostable amanita cyclopeptidic alkaloids can be divided into three groups: amatoxins (the amanitin family), phallotoxins (phalloidins), and virotoxins. The latter two groups have too low mammalian toxicity to be considered of clinical significance, whereas amatoxins are actually solely responsible for the fatal intoxications in humans. The basic differences between amatoxins and both phallotoxins and virotoxins lie in the mode and rapidity of their action. Phallotoxins and virotoxins act quickly, within 2–5 hr, whereas the action of amatoxins is delayed and the animals succumb to death within 2–8 days after toxin administration. Amatoxins are also 10–20 times more toxic than phalloidins. Chemically, amatoxins are bicyclic octapeptides (Figure 15.1). Although all the amino acids have the Lconfiguration, three of them differ from amino acids in proteins in the hydroxylation of their side chains: OH-proline, (OH)2-isoleucine, and (6′-OH)-tryptophan. Another unusual feature of these toxins is an R-configured sulfoxide bridge between the side chains of cysteine and (6′OH)-tryptophan, which results in the rigid, pretzellike shape of the molecules (Wieland and Faulstich, 1978; Faulstich and Wieland, 1992). Similar to phallotoxins and virotoxins, amatoxins contain the indole nucleus of a tryptophan building block substituted in 2-position by a sulfur atom in various oxidation states. The family of amatoxins contains nine members (Figure 15.1); most of them, however, are found in very small or even trace amounts. Hence, they do not contribute significantly to poisoning. In A. phalloides mushrooms,

R2 H CH3 C HN

HC

H

C R1 CO

HN

R5

NH

CH2

CO

HC OS

N

CH3

NH

CH N H

R4

CO

CH2

NH

CH C2H5

CH2 OC

α-Amanitin β-Amanitin γ-Amanitin ε-Amanitin Amanin Amanin amide Amanullin Amanullinic acid Proamanullin

CO

H 2C

OC H

CH

CO

H 2C

HN

H2 C

COR3

CH

R1

R2

R3

R4

R5

CH2OH CH2OH CH3 CH3 CH2OH CH2OH CH3 CH3 CH3

OH OH OH OH OH OH H H H

NH2 OH NH2 OH OH NH2 NH2 OH NH2

OH OH OH OH H H OH OH OH

OH OH OH OH OH OH OH OH H

NH

CO

Figure 15.1 Chemical structures of natural amatoxins.

amatoxins are made up of nearly equal amounts of αamanitin, the acidic β-amanitin, and some γ-amanitin (<10%). On average, these mushrooms contain amatoxins in amounts of approximately 3.5 mg/g dry weight. The phallotoxins consist principally of seven distinct toxins (Figure 15.2). These bicyclic heptapeptides contain cysteine; threonine; hydroxyproline; tryptophan; mono-, di-, or trihydroxyleucine; and two alanine residues. One of the alanine residues may be substituted with L-valine, and D-erythrohydroxyaspartic acid is substituted for threonine in the case of phallacidine. Phallotoxins are produced by several Amanita species, including A. phalloides, A. phalloides var. verna, A. virosa, and A. bisporigera. The bicyclic structure of phallotoxins gives the molecules their relative stability. Chemical changes on the side chains do not alter the geometric characteristics of the basic peptide ring nor diminish the toxicity in most cases (Wieland and Wieland, 1972). However, cleavage of the ring by acid hydrolysis or heating with Raney nickel produces nontoxic products.

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Virotoxins (Figure 15.3) are monocyclic heptapeptides with phalloidinlike activity found only in A. virosa (Faulstich et al., 1980). The amino acid sequence of virotoxins appears to be very similar to those of phallotoxins; the difference is that they contain D-serine instead of Lcysteine and two amino acids not generally found, 2,3trans-3,4-dihydroxy-L-proline and 2′-(methylsulfonyl)-Ltryptophan. Because they are monocyclic peptides, the conformation of virotoxin molecules is not rigid. The cyclopeptide toxins may actually be fragments of larger molecules (Concon, 1988). These high-molecularweight substances appear to consist of a polysaccharide core surrounded by several cyclopeptide molecules attached to it by means of oxygen bridges. The high-molecularweight toxins have been designated as myriamanins. Those found in A. phalloides are called myriaphalloisins and those from A. virosa, myriavirosins. The former appear to be less toxic than the latter. The presence of a polysaccharide core may make it possible to develop a vaccine against these toxins.

H H3C

H CO

C

HN

CH

CO

NH

H2C

NH

R5

C

CH2

C

R6

OH

CO NH

CO S

R1

N H

C H

CH2 N

CO

R4

C

H

H

NH

CO

C

CO

NH

HC R2

R3

Phalloin Phalloidin Phallisin Prophalloin Phallacin Phallacidin Phallisacin

R1

R2

R3

R4

R5

R6

CH3 CH3 CH3 CH3 CH(CH3)2 CH(CH3)2 CH(CH3)2

CH3 CH3 CH3 CH3 OH OH OH

OH OH OH OH COOH COOH COOH

OH OH OH H OH OH OH

CH3 CH2OH CH2OH CH3 CH3 CH2OH CH2OH

CH3 CH3 CH2OH CH3 CH3 CH3 CH2OH

Figure 15.2 Chemical structures of natural phallotoxins.

Analysis Several methods for the quantitation of these toxic cyclopeptides have been developed (Wieland, 1986; Faulstich and Wieland, 1992). The target proteins, immunoglobulin antibodies, or specific enzyme reactions affected by the poisons can be used for determining very small amounts of these toxic peptides. In addition, several chromatographic methods for the detection of the deadly amanita toxins have been developed (Enjalbert et al., 1989). These include a reversed-phase high-performance liquid chromatography (HPLC), the sensitive and rapid high-performance thinlayer chromatography (HPTLC), mass spectrometry, and fluorometry. Symptoms of Poisoning Ingestion of mushrooms containing the cyclopeptide toxins causes the phalloides syndrome, usually after a delay of 6 to 24 hr after ingestion. It is characterized by severe abdominal pain accompanied by nausea, violent bloody vomiting, and profuse diarrhea. The first period of acute gastritis symptoms gradually resolves and the patient enters a symptom-free period. This interphase is of unpre-

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dictable duration but has been as much as 3 to 5 days. This situation is often misleading, since in the meantime, progressive liver and kidney injury continues to occur. Heavy vomiting, severe abdominal cramps, nausea, water diarrhea causing severe dehydration and oliguria, together with loss of electrolytes and acid-base disorders, characterize the second phase. Persistence of diarrhea during this period is a strong indication of amatoxin poisoning (Zilker, 1987). During phase II, which lasts 24–36 hr, disturbances of blood coagulation begin, in particular a decrease of prothrombin time. Phase III, lasting 12–24 hr, is characterized by an improvement of clinical symptoms, while elevated levels of transaminases reveal the beginning of liver necrosis. The final phase IV is determined by hepatic failure, encephalopathy, and very often acute renal failure. In addition, internal bleeding is observed and may cause complications or even death. Patients usually die within days 5–8, but deaths may occur as late as 20 to 25 days after the mushroom meal (Faulstich and Wieland, 1992). The mortality rate of A. phalloides intoxication in children below 10 years of age is much higher (51%) than that in adults (17%) (Floersheim and Bianchi, 1984).

H H3C

H HN

CO

C

H

C

CO

H 2C

NH

N H

C

R2

OH

R1 C

CH2OH CO

C NH

H H

H CO

OH

C

NH

CO

HC HO

Viroidin Deoxoviroidin Ala1-viroidin Ala1-deoxoviroidin Viroisin Deoxoviroisin

CH2

CO

CH3X

OH

H

C

NH

CO H

N

NH

CH2OH

X

R1

R2

SO2 SO SO2 SO SO2 SO

CH(CH3)2 CH(CH3)2 CH3 CH3 CH(CH3)2 CH(CH3)2

CH3 CH3 CH3 CH3 CH2OH CH2OH

CH3

Figure 15.3 Chemical structures of virotoxins found in Amanita virosa.

Faulstich (1979) suggested the following therapeutic procedures after diagnosis of amatoxin poisoning by analysis of urine and serum: 1.

2.

3. 4.

Obligatory washing of stomach and intestines during the first 36 hr after the toxic meal, use of activated charcoal to bind amatoxins in the gastrointestinal (GI) tract, restoration of water and electrolyte balance During the first 48 hr after the meal, obligatory removal of amatoxins from blood by any of several means, such as forced diuresis, hemofiltration, hemoperfusion using polymeric adsorbents, or plasmapheresis Where hypoglycemia is imminent, glucose infusion Chemotherapy with thioctic acid (100 to 300 mg/day) or high doses of silymarine or penicillin

Mechanism of Action The biological activity of amatoxins and phallotoxins is strictly dependent on the three-dimensional structures of the peptide molecules. The toxicity can be completely lost as a result of even a minor change of structure, which would alter the conformation and, consequently, the ability

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to bind to the protein receptor. The phallotoxins and amatoxins differ in their mechanisms of action with regard to their cytopathologenic effects as well as their primary target tissues. The phallotoxins are primarily hepatotoxic, whereas the amatoxins are both strongly hepatotoxic and nephrotoxic. Biochemically, amatoxins (particularly α-amanitin) bind to ribonucleic acid (RNA) polymerase II and block the catalytic site of the enzyme, thereby inhibiting the deoxyribonucleic acid (DNA) transcription, formation of messenger RNA (mRNA), and consequently, protein synthesis. The inhibition of RNA polymerase II explains some of the biochemical defects noted after α-amanitin poisoning, viz., a decrease in blood protein level, a decrease in glycogen synthetase level in the liver and to a lesser extent in muscles, and consequent glycogen content of these tissues. The decrease in liver adenosine triphosphate (ATP) and nicotinamide-adenine dinucleotide (NAD) levels and the leakage of potassium from liver cells also follow from the inhibition of protein synthesis (Faulstich and Wieland, 1992). Phallotoxins and virotoxins interact very specifically with the equilibrium system G ↔ F actin that plays a fundamental role in the existence and functions of a living cell. By tightly binding to F-actin, they stabilize the struc-

ture of the filamentous assembly, preventing its dissociation and thus shifting the equilibrium almost entirely to the F-actin state. The liver is the organ almost exclusively affected by these toxins. Phalloidins also cause inhibition of glycogen synthesis, acceleration of autolytic reactions on liver nucleotides and proteins, and release of lysosomal enzymes. That both phallotoxins and virotoxins may be poorly absorbed in the GI tract at least partially explains their low mammalian toxicity. The LD50 values of several amatoxins and phallotoxins are listed in Table 15.2. 15.5.2

Orellanine

The orellanus syndrome, characterized by an exceptionally long latent period and renal tubular necrosis, is caused by Cortinarius species, especially C. orellanus, C. speciosissimus, and C. splendens. The mushrooms may contain up to 1% toxins on a dry weight basis (Grzymala, 1961). Cortinarius is the largest genus of fungi in Europe. The toxicity of C. orellanus first became apparent in 1952 when 102 inhabitants of Bydgosz, Poland, fell ill after ingestion of this mushroom (Grzymala, 1962). Eleven of them died 4–16 days after the mushroom meal with acute renal failure. Similarly, the closely related C. speciosissimus, found more frequently in northern Europe, was proved similarly toxic (Mottonen et al., 1975). Intoxications by

Table 15.2 LD50 Values for Amatoxins and Phallotoxins Toxin

LD50 (mg/kg, white mouse)

Amatoxins α-Amanitin β-Amanitin γ-Amanitin ε-Amanitin Amanin Amanin amide Amanullin Amanullinic acid Proamanullin Phallotoxins Phalloin Phalloidin Phallisin Prophalloin Phallacin Phallacidin Phallisacin

0.3–0.6 0.5 0.2–0.5 0.3–0.6 0.5 0.5 >20 >20 >20

Chemical Characteristics The main toxic substance, orellanine, was first isolated from C. orellanus by Grzymala (1962). Antkowiak and Gessner (1975) identified two homogeneous toxic substances. Orellanine, the main component, was determined to be 3,3′-4,4′-tetrahydroxy-2,2′-bipyridyl-N,N′-dioxide (Figure 15.4a); the second component, orellinine, was suggested to be the monodeoxo derivative (Figure 15.4b). Chemically, the pyridine-N-oxide structures of orellanine are unstable. On heating or on ultraviolet (UV) irradiation on cellulose thin layer plates orellanine is decomposed into its bisdeoxo form (Figure 15.4c), orelline, a yellow and blue fluorescent compound that is nontoxic to animals (Antkowiak and Gessner, 1985). Symptoms of Poisoning The toxic symptoms that follow orellanine ingestion do not appear until 3 to 14 days later. In the first stage of intoxication, apart from general weakness, the symptoms may be a typical form of acute gastritis. There is a marked increase in liquid intake, during which the patient may drink several liters a day. Nausea, emesis, constipation, headaches, muscular pains, and chills follow. Initially there is a corresponding increase in urinary output, but this is followed by renal failure, which is ultimately responsible for the death of the patient. Signs of renal failure include oliguria and sometimes anuria, albuminuria, uremia, and electrolyte imbalance. Hepatic damage is indicated by pain in the abdomen, vomiting of bile, and subicteric state. Death may be preceded by drowsiness, coma, and convulsions. Fatalities have been shown to vary between 10% and 20% (Flammer, 1982; Concon, 1988). The treatment for orellanine poisoning resembles that for acute renal failure with the hemodialysis and hemoperfusion to eliminate the toxins from the blood circulation. Mechanism of Action

1.5 2 2 >20 1.5 1.5 4.5

Source: Compiled from Concon (1988) and Antkowiak (1996).

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C. splendens were reported from France (Gerault, 1981). Schumacher and Hoiland (1983) have extensively reviewed several cases of Cortinarius spp. poisoning from various countries.

Unlike the well-documented clinical symptoms and histopathological changes caused by the ingestion of Cortinarius spp. mushrooms, the mode of action of their toxins is still not fully elucidated. There is a relationship between the structure and physiological activities of orellanine and two biologically active cationic bipyridines with wellknown herbicidal properties, diquat (N,N′-dimethyl-2,2′bipyridine) and its 4,4′-isomer, paraquat. Schumacher and

HO

HO

HO

O

HO

OH

OH N

N

N

OH

OH

OH (a) Orellanine

HO

O

OH

O

N

N

N HO

(b) Orellinine

(c) Orelline

Figure 15.4 Toxic principles of Cortinarius orellanus mushroom.

Hoiland (1983) suggested a similar pathogenic mechanism of action for all these compounds, assuming their ability to inhibit the intracellular metabolic systems dependent on reduced oxidized nicotinamide-adenine dinucleotide (NADPH) plus H+, by participation in the electron transport process (Figure 15.5). Additionally, the concomitantly produced superoxide also has cytotoxic properties, either direct or by generation of other reactive forms of oxygen such as singlet oxygen. This proposed intracellular toxic mechanism with NADPH plus H+ depletion would need a long time to impoverish the cells to the degree of inevitable necrosis. This is in accordance with the delayed toxic effects observed in this type of mushroom poisoning. This hypothesis, although convincing, has not been con-

firmed by the studies of the electrochemical behavior of orellanine, which was shown to be different from diquat and paraquat (Richard et al., 1988). Rapior (1988) suggested that the Cortinarius spp. toxicity is caused by metabolites with the isoxazolium core derived from the photochemical rearrangement of orellanine. These intermediates can bind covalently with numerous proteins in the body, leading to organ damage. Rapior (1988) also found that orellanine purified in the dark and administered to laboratory animals showed low toxicity, whereas that extracted in the light induced a toxic response. These observations indicating the consequence of photoexcitation supported the proposed phototoxicity mechanism of orellanine (Figure 15.6).

OH N Enzyme reduced form

O

OH

HO

O22–

O N

HO

OH N O Enzyme oxidized form

HO

O N

HO

Figure 15.5 Intracellular toxic mechanism proposed for orellanine.

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OH O2

Orellanine

Orellinine

HO

Orelline

HO

HO N

N HO

O

O

N

HO

HO OH

O

OH

OH

N

N

N

OH

OH

hν

OH hν

hν

HO

hν

HO N

HO

N O

O OH

O N OH

Intermediates capable of binding with proteins

N OH

Figure 15.6 Photodecomposition of orellanine.

The LD50 values of orellanine are 4.9, 8.3, and 8.0 mg/ kg in the cat (per os), mouse, and guinea pig (parenteral), respectively. These values and the symptoms observed in victims indicate the extreme toxicity of this compound (Concon, 1988). 15.5.3

Gyromitrin

Gyromitrin poisoning is associated predominantly with the consumption of Gyromitra esculenta, the false morel. This mushroom was considered a delicious edible mushroom for centuries until it turned out to contain, in the raw state, toxic substances that can cause death. Franke and associates (1967) presented a compilation of 513 cases of poisoning including 14.5% fatalities. The severe cases occurred after ingestion of raw or insufficiently cooked false morels, whereas after extensive cooking and discarding of the water or after drying of the mushrooms, poisoning occurred only rarely. Intoxications resulting from the ingestion of this mushroom are seen most frequently in Eastern Europe and only occasionally elsewhere. In Poland, it is the third most common poisoning after those caused by A. phalloides and Paxillus involutus (Antkowiak, 1996).

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Chemical Characteristics The toxic properties of G. esculenta and other related species result from the presence of harmful hydrazine derivatives in the mushrooms. The main component, gyromitrin (2-ethylidene-1-formyl-1-methylhydrazine, Figure 15.7a) is a volatile compound. It is an unstable compound, prone to hydrolysis and oxidation. On acid hydrolysis in the GI tract, gyromitrin is converted first to 1-formyl-1-methylhydrazine (Figure 15.7b), which after the loss of the formyl residue yields the highly toxic N-methylhydrazine (Figure 15.7c). Additional homologs of gyromitrin, in which the acetaldehyde is replaced by other aldehydes, now also are known to exist. These include the derivatives of pentanal, 3-methylbutanal, and hexanal. All of these compounds readily hydrolyze to form the active toxin, monomethylhydrazine. The toxin is volatile and water-soluble and thus can be eliminated from the mushroom by air-drying or extraction with boiling water. The content of gyromitrin in dried mushrooms is about 3 mg/kg, compared to 1.2 to 1.6 g/kg in fresh mushrooms. The instability of gyromitrin suggests that in the mushroom it is probably bound to high-molecular-weight components, possibly as glycoside (Stijve, 1978).

CH3 H3C

CH

N

CH3 H2N

N CHO

(a) Gyromitrine

CH3 H2N

N CHO

N H

(b) N,N-Formylmethylhydrazine

(c) N-Methylhydrazine

Figure 15.7 Chemical structures of Gyromitra esculenta toxins.

Symptoms of Poisoning The symptoms of gyromitrin poisoning are similar to those caused by cyclopeptide poisoning. They appear suddenly, usually 6 to 12 hr after ingestion or inhalation of the vapor from cooking mushrooms. They are characterized by nausea, vomiting, abdominal pain, dizziness, headache, abnormal thirst, general weakness, and painful liver enlargement, but only exceptionally accompanied by diarrhea. In most cases this phase ends with recovery within 2–6 days. However, after ingestion of large amounts of weakly cooked false morels, a hepatorenal phase can follow with symptoms of liver injury, hemoglobinuria, or anuria. Predominant symptoms of this phase are nervous disturbances such as difficulties in moving, cramps, deliria, and even unconsciousness. Death can occur through collapse of circulation (Faulstich and Wieland, 1992). The treatment for gyromitrin poisoning is the same as for isoniazid overdose and consists of administering pyridoxine and correcting the systemic acidosis. In the case of jaundice and coma, it is the same as for phalloides poisoning. The monomethyl derivative of gyromitrin appears to be neurotoxic and carcinogenic, causing liver and intestinal tumors in animals. It is also shown to be embryotoxic and teratogenic (Slanina et al., 1993).

15.6 NEUROTOXIC MUSHROOMS The symptoms caused by neurotoxic mushrooms may occur almost immediately from 15 to 180 min after ingestion. The composite symptoms as observed in various cases of poisoning of this type include increased salivation, lacrimation, perspiration, and severe gastrointestinal disturbance with vomiting and copious water diarrhea. The pulse becomes slow and irregular (Concon, 1988). There may be labored or asthmatic breathing, vomiting, hallucinations, and confusion. Well-known mushroom toxins belonging to this group are described in the following sections.

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15.6.1

Muscarine

Muscarine certainly is the most famous mushroom poison. In Europe, muscarine intoxication is most often caused by Inocybe patouillardii, commonly known as red-staining inocybe. This mushroom is often confused with the edible Tricholoma georgii or Agaricus campestris. Muscarine was long considered to be the toxin produced by all poisonous mushrooms (Schmiedeberg and Koppe, 1869). The toxin was first isolated from Amanita muscaria, from which the name of the toxin originated. This mushroom, however, contains muscarine in only traces, whereas muscarine is found in large amounts in mushrooms of the genera Clitocybe and Inocybe. Almost all Inocybe species contain the toxin and hence should be avoided. The most popular, I. patouillardii, is definitely known to cause death. It contains 370 mg/kg muscarine, relative to the fresh weight of the fruit body, as compared to 2–3 mg/kg in A. muscaria. Additionally, low concentrations (≤ 2 mg/100 g dry weight) of muscarine and its stereoisomers have been found in the fruiting bodies of the following species of agaricales: A. pantherina, A. phalloides, Boletus calopus, B. luridus, Clitocybe dealbata, C. hydrogramma, C. gibba, C. vermicularis, Clitopilus intermedius, Collybia peronata, Hygrocybe nigrescens, Hypholoma fasciculare, Lactarius rufus, L. trivialis, Mycena pelianthia, M. pura, Paxillus involutus, Rhodophyllus rhodopolius, R. sinuatus, Russula emetica, Tricholoma sulphureum, and Tylopilus felleus (Antkowiak, 1996). Chemical Characteristics Muscarine is 2-methyl-3-hydroxy-5-trimethylammoniummethyl-tetrahydrofuran. Eugster (1956a, 1956b) and simultaneously Kogl and colleagues (1957) revealed its complete chemical structure by using x-ray diffraction techniques. The compound has three chirality centers, and hence, four pairs of enantiomers are possible. All diastereomers: (+)-(2S,3R,5R)-muscarine, (–)-(2S,3R,5R)-allomuscarine, (+)-(2S,3S,5S)-epimuscarine, and (+)(2S,3S,5R)-epiallomuscarine (Figure 15.8a–d), can occur

HO

HO 3

3

4

2

5

H3C

2

N(CH3)3 H3C

1 O

(a) L-(+)-Muscarine (2S, 3R, 5S)

5

N(CH3)3

1 O

(b) L-(+)-Epimuscarine (2S, 3S, 5S)

HO

HO 3

4

2 H3C

4

5

N(CH3)3

N(CH3)3

H3C

1 O

O

(c) L-(-)-Allomuscarine (2S, 3R, 5R)

(d) L-(+)-Epiallomuscarine (2S, 3S, 5R) O N(CH3)3

H3C

O (e) Acetylcholine

Figure 15.8 Diastereomeric muscarines and their structural similarity with acetylcholine.

in nature. The physiological activity of the stereoisomers differs; for natural muscarine it is 100-fold stronger than that of the others, whereas D-(–)muscarine is only 10–3 as active as the naturally occurring enantiomers. Mechanism of Action Through its structural similarity to acetylcholine (Figure 15.8e), muscarine binds to the acetylcholine receptor on synapses of the nerve endings of smooth muscles and endocrine glands, causing the well-known parasympaticomimetic effects. However, it is more selective. Muscarine binds strongly with the muscarine type of acetylcholine receptors in smooth muscles of the GI tract, eye, exocrine glands, and heart; acetylcholine exerts an additional “nicotinic action” by affecting ganglia and voluntary muscles. Because muscarine is not an ester like acetylcholine and hence resists esterase activity, it is not degraded but can cause continuous stimulation of the affected neurons (Faulstich and Wieland, 1992). The binding affinity of muscarine with the receptors is so high that the muscarine stereoisomers are routinely used to study cholinergic pharmacological characteristics. The cholinomimetic effects of muscarine are successfully antagonized by atropine. The involvement of muscarine in

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the phospholipid metabolism in ganglia has also been shown (Horwitz et al., 1986). The selective binding affinity of stereoisomers of muscarine strongly depends on even small changes in the structure, conformation, and configuration of the rigid muscarine molecule. The hydroxyl group trans-arranged to the other two substituents plays a key role (Antkowiak, 1996). The change of the CH-OH group into carbonyl yields muscarone, which is more potent than muscarine but less selective. As does acetylcholine, it exhibits strong stimulating and blocking effects on ganglionic synapses and neuromuscular junctions. Symptoms of Poisoning The symptoms of parasympathetic stimulation evoked by muscarine are usually evident within 15 to 30 min after ingestion. The toxic response is not affected by cooking. There appears to be a dose-response relationship as to the appearance of any particular effect, the most sensitive of which is profuse sweating. As the quantity of muscarine increases, one also may observe nausea, vomiting, and abdominal pain. More severe intoxications produce blurred vision, salivation, lacrimation, rhinorrhea, and diarrhea. Tremors, dizziness, and bradycardia are only rarely seen.

Death may occur in a few hours unless the poisoning is diagnosed and promptly treated with atropine. Atropine competes with muscarine for binding to the receptor sites but does not evoke a signal. In mice, muscarine has an LD50 of approximately 0.25 mg/kg body weight when administered intraperitoneally, and higher if administered orally (Faulstich and Wieland, 1992). Even if humans were several times more sensitive to muscarine than mice, severe intoxication of humans would require a large amount of A. muscaria. 15.6.2

Muscimol and Ibotenic Acid

The toxins muscimol and ibotenic acid cause the so-called pantherine syndrome, which is attributed to the consumption of Amanita muscaria (fly agaric), A. pantherina (panther cap), and A. strobiliformis (grows only in Japan) mushrooms. The symptoms of illness occurring after the ingestion of these mushrooms cannot be attributed to muscarine, which is present only in trace amounts. Numerous reports of poisoning after the consumption of these mushrooms for food are documented in the older literature. Two

Chemical Characteristics The toxicity of these mushrooms is attributed to two isoxazoline compounds, ibotenic acid (also known as pramuscimol, α-toxin) and muscimol (initially also called pantherine, pyroibotenic acid, β-toxin, or agarin). In addition, an oxazolone, muscazone, has been isolated from these mushrooms. Both ibotenic acid and muscimol appear to be limited to these three mushroom species only. Ibotenic acid is α-amino-(4-hydroxy-isoxazole-2)-yl-acetic acid, a structure related to and possibly derived from βoxo-L-glutamine (Figure 15.9a). Muscimol (Figure 15.9b) is the decarboxylation product of ibotenic acid. Muscazone (Figure 15.9c), found only in A. muscaria collected in summertime, appears to be formed from ibotenic acid by a photoinduced rearrangement (Goth et al., 1967). Tricholomic acid (Figure 15.9d) is a related isoxazole found only in Tricholoma muscarium. This mushroom is found only in Japan.

(a)

HO

Ibotenic acid

medium-sized mushrooms of A. muscaria and only one of A. pantherina are sufficient for intoxication of an adult.

(b) O Muscimol

N

NH3

N

O

NH3 O

COO

(d)

(c) O

HN

Tricholomic acid

H

Muscazone

NH3 O

H HN

NH2

O O

COO

COOH (e)

(f)

HO

O NH3

O

NH3 O

COO Glutamic acid

γ-Aminobutyric acid (GABA)

Figure 15.9 Ibotenic acid and muscimol as analogs of glutamic acid and GABA along with related toxins.

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The amounts of the isoxazoles present in these mushrooms are approximately 0.2% for dried A. muscaria and 0.4% for A. pantherina.

a source of muscimol that is generated in the body by decarboxylation.

Symptoms of Poisoning

15.7 HALLUCINOGENIC MUSHROOMS

These mushroom toxins exhibit a powerful action on the central nervous system, causing atropine-type symptoms. When they are eaten in large amounts, the poisoning symptoms may appear as early as 15 to 30 min; however, usually they occur 1 to 3 hr after ingestion. The initial weakness, balance disorder, sweating, and gastrointestinal discomfort resembling advanced alcoholic intoxication are followed by anxiety, dizziness, elation, increased motor activity, tremors, illusions, and even maniac delirium. This may alternate with periods of drowsiness or sleep, after which the awakened patient has no memory of his or her experience (Antkowiak, 1996). Increased pulse rate and body temperature up to 40°C (104°F) may be observed. In severe cases, unresponsive, maximally dilated pupils may also be observed. Finally, coma accompanied by cardiovascular collapse and respiration depression may occur. However, in the majority of cases, symptoms abate rapidly, usually after approximately 24 hr. In the past, these mushrooms were eaten deliberately in several European countries in order to produce hallucinogenic effects. Incidences of poisoning have been increasing in the United States, Europe, and South Africa, especially in children. Mechanism of Action The neurotransmitting properties of ibotenic acid and muscimol are linked to the structures of these alkaloids, which resemble those of some amino acids of the nervous system. They reveal the powerful properties of glutamate (Figure 15.9e) and γ-aminobutyric acid (GABA) (Figure 15.9f) agonists. In fact, ibotenic acid and muscimol may be considered as conformationally restricted analogs of these amino acids, binding alternatively to their respective receptors (Gore and Jordan, 1982). Baldelli and coworkers (1994) have shown that GABAA receptors on rat cellular granule cells are even more potently activated by muscimol than by GABA itself in inducing Cl– currents. Both ibotenic acid and muscimol have been suggested as possible models for the development of potent drugs in the treatment of specific nervous diseases. In rats the LD50 of muscimol was determined to be 45 mg/kg by oral and about 4.5 mg/kg by intravenous administration; the corresponding values for ibotenic acid are 129 and 42 mg/kg, respectively (Theobald et al., 1968). Perhaps ibotenic acid exerts its toxic effects only as

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Besides Amanita muscaria and A. pantherina, which cause sedative-hypnotic actions due to the presence of the toxic isoxazole alkaloids, a few other mushrooms with such hallucinogenic properties are known. The physiologically active substances responsible for such symptoms are 4- or 5substituted tryptamines that may be represented by psilocybin and bufotenine, respectively. Striking features of the symptoms produced by hallucinogenic mushrooms are the hallucinatory or psychedelic effects and, depending on the species, prolonged euphoria and excitation. The effects may be accompanied by other serious symptoms such as muscular incoordination and weakness of the arms and legs, sometimes with complete but temporary paralysis. The unpleasant effects may last for several hours. Mushrooms containing hallucinogenic indole derivatives are found in the genera Psilocybe, Panaeolus, Pholiotina (Conocybe), Gymnopilus, and possibly Panaeolina. Psilocybe semilanceata is widely distributed in Europe and North America. 15.7.1

Psilocybin and Psilocin

Psilocybin and its accompanying congener psilocin were first isolated from Psilocybe mexicana and identified as active compounds of this hallucinogenic mushroom by Hofmann and associates (1958a, 1958b; Hofman, 1959). Like the mycotoxin ergot alkaloids (Chapter 11), psilocybin and related alkaloids have a 3,4-distributed indole ring system, though the structure is less complicated. Psilocybin is a derivative of tryptophan; its chemical structure is 4-phosphoryloxy-dimethyltryptamine or 4-phosphoryloxy-3-[2-(dimethylamino)-ethyl]-indole (Figure 15.10a). The dephosphorylated compound, psilocin (Figure 15.10b), is not hallucinogenic and, unlike the phosphoric ester, easily decomposed by oxidation. In addition to these two compounds, baeocystine (Figure 15.10c) and norbaeocystin (Figure 15.10d) are the monomethylated and nonmethylated derivatives of psilocybin, respectively. These are found in Psilocybe baeocystis (Leung and Paul, 1968). The highest concentration (>0.5% of dry weight) of psilocybin is generally found in Psilocybe semilanceata (liberty cap mushroom), Panaeolus subbalteatus, Conocybe cyanopus, and Pluteus salicinus (Ohenoja et al., 1987). The first two are the only psilocybin containing mushrooms that can be gathered in middle and northern

(d)

O

(a)

O

P

O

O O

P

O

OH

OH CH2CH2

NH

CH2CH2NH3

CH3

CH3 N H

Norbaeocystin

N H

Psilocybin

(e) HO

(b)

CH2CH2

N

OH

CH3

CH3 CH2CH2

N

N H

CH3

CH3 N H

Bufotenine

(f) CH

Psilocin

CH

O

O

Yangonin H3CO

O

(c) O

P

OCH3

O

(g)

OH CH2CH2

NH2 CH

CH

O

O

CH3

bis-Noryangonin

N H

Baeocystin

HO OH

Figure 15.10

Structures of some hallucinogenic toxicants found in various species of mushrooms.

Europe in sufficient quantities for intoxication to occur (Stijve and Kuyper, 1985). The dried fruiting bodies of Psilocybe mexicana contain 0.2%–0.4% psilocybin and only trace amounts of psilocin. Symptoms after ingestion of about 20 g of fresh mushrooms, or 4–8 mg psilocybin, start within the first half hour. Reactions to the drug vary between individuals from a feeling of relaxation to one of tension, anxiety, or dizziness. Sometimes nausea and abdominal discomfort including vomiting and diarrhea may occur. During the second half hour the occurrence of visual effects is generally seen, including the perception of brilliant colors and objects with closed eyes. Panic reactions, such as fear of death or insanity, have also been reported. During the following hours the visual effects increase and then disappear. Systemic effects are mostly due to the stimulation of the central or sympathetic nervous system and may comprise papillary dilatation, rapid heartbeat, and high blood

Copyright 2002 by Marcel Dekker. All Rights Reserved.

pressure, combined with low blood sugar level and decreased body temperature (Vergeer, 1983; Faulstich and Wieland, 1992). Therapeutic intervention is rarely sought or required for adults. The clinical effects may be terminated, however, by the administration of diazepam or a phenothiazine. The LD50 values of psilocybin in mice are 275 mg/ kg intravenously and 420 mg/kg intraperitoneally (Antkowiak, 1996). 15.7.2

Bufotenine and Related Compounds

Bufotenine, 5-hydroxy-N,N-dimethyltryptamine (Figure 15.10e), was first identified and isolated from false death cap mushroom, Amanita mappa (identical with A. citrina), by Wieland and colleagues (1953). It is also found in other Amanita species, such as A. muscaria, A. pantherina, A. porphyrea, and A. tormentella. Stijve (1979) demonstrated

the presence of 0.03%–0.06% of another related alkaloid, bufotenine N-oxide, in A. citrina mushrooms collected in various parts of Europe. Bufotenine has always been considered a psychomimetic agent. Fabing and Hawkins (1956) gave an intravenous injection of a dose up to 16 mg of this compound to a healthy young man, which was feasible without jeopardizing his life. It resulted primarily in a burning sensation in the face, which turned purple and returned to its normal color at the end of an hour; visual disturbances (red spots passing before the eyes) accompanied by nystagmus and mydriasis, as well as a change in the perception of time and space. These effects resembled those of lysergic acid diethylamide (LSD25) and mescaline but developed and disappeared more quickly. Subsequently, Bhattacharya and Sanyal (1971) demonstrated the anticholinesterase activity of bufotenine in rats at the dose of 2.5 mg/kg given intraperitoneally. Bufotenine is capable of increasing the basal neurotransmitter efflux by blocking the neural membrane transport carrier. Its action on 5-hydroxytryptamine (5-HT) neurons is comparable to that of p-chloroamphetamine (Monroe et al., 1994). The concentrations of bufotenine and related compounds are generally too small to cause any toxicity of these mushroom species. 15.7.3

Psychoactive Effects of Gymnopilus spectabilis

In Japan, the mushroom Gymnopilus spectabilis is believed to cause hallucinations and abnormal behavior. However, this mushroom is frequently consumed without any psychoactive effects after the bitter taste is removed by boiling water. Intoxications generally occur as a result of ingestion of inadequately cooked mushrooms (Kusano et al., 1986). The hallucinogenic effects of this mushroom are attributed to yangonine (Figure 15.10f) and bis-noryangonin (Figure 15.10g). Unlike the other hallucinogenic principles in mushrooms, which are all tryptamine derivatives, the yangonines are styrylpyrones.

15.8 MUSHROOM TOXINS AFFECTING THE HEMATOLOGICAL SYSTEM 15.8.1

Mushrooms with Disulfiramlike Activity (Coprinus Syndrome)

Several mushroom species are capable of inducing a marked sensitivity to alcohol. This effect is encountered most commonly with Coprinus atramentarius (common inky cap mushroom), which is a particularly desirable edi-

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ble mushroom. However, if alcohol is consumed up to approximately 72 hr after the ingestion of this species, the individual may experience flushing, hypotension, tachycardia and palpitations, paresthesias, severe nausea and vomiting, and an intense headache (Faulstich and Wieland, 1992). The effects normally disappear after 3–4 hr, but occasionally may last up to 50 hr. These effects are similar to those caused by disulfiram (Antabuse), employed in the treatment of alcoholism. Therapeutic intervention usually is not required. The agent causing these symptoms has been identified as coprine, N5-(1-hydroxycyclopropyl)-L-glutamine (Figure 15.11). This phototoxin was isolated almost simultaneously in the United States (Hatfield and Schaumberg, 1975) and in Sweden (Lindberg et al., 1975). This substance is converted in the body into 1-aminocyclopropanol (Figure 15.11) and thence to the active toxin, 1-hydroxycyclopropylammonium ion (Figure 15.11). The active toxin reversibly inhibits the low-Km acetaldehyde dehydrogenase of the liver by blocking an essential sulfhydryl group of the enzyme (Wisemann and Abeles, 1979). This results in the accumulation of acetaldehyde in the blood during alcohol metabolism. The elevated acetaldehyde levels are primarily responsible for the intoxicating symptoms described. The coprine content of fresh C. atramentarius mushroom is about 160 mg/kg (Lindberg et al., 1977). Coprine is also found in C. quadrifidus, C. variegates, and C. insignis. Other mushrooms producing similar alcohol-sensitizing activity include Boletus luridus, Clitocybe clavipes, and Verpa bohemica; however, none of them has been found to contain coprine.

H2C

OH C

H2C

NH

NH3

O C

CH2

CH2

CH

COO

Coprine

H2C

OH

H2C

NH2

H2C

C

C H2C

1-Aminocyclopropanol

O

Cyclopropanone

Figure 15.11 Chemical structure of coprine and its conversion to the toxic metabolite cyclopropanone.

15.8.2

Mushrooms Causing Hemolysis

Hemolysis-causing agents have been found in several mushroom species, including the quite popular Agaricales spp. and rarely in Boletaceae and Russula spp. These toxins have been also detected in the species from the families Hygrophoraceae and Strophariaceae, and from genera Mycena, Oudemansiella (Tricholomataceae), Hebeloma, Gymnopilus (Cortinariaceae), and Amanita. The latter genus includes species such as A. muscaria (causes agglutination of red blood cells), A. gemmata, A. verna, A. citrina, A. porphyria, A. spissa, A. echinocephala, and, invariably, A. rubesscens and A. phalloides, which contain high activity (Seeger and Widemann, 1972; Seeger et al., 1973; Antkowiak, 1996). Although in most cases, the chemical identity of the toxin(s) has not yet been determined, the principal hemolytic activity in A. phalloides is attributed to phallolysin. It appears to be a mixture of two to three cytolytic proteins. The hemolytic activity of phallolysin results in the release of hemoglobin from erythrocytes into the blood plasma, and its toxicity exceeds even that of α-amanitin severalfold (Faulstich et al., 1983). Although the sensitivity of mammals to phallolysin increases with body size (LD50 intravenously in mice is 120 µg/kg, in rats is 50 µg/kg, and in rabbits is even less than 25 µg/kg) and death occurs by acute hemolysis only a few minutes after intravenous or intraperitoneal application, it still cannot contribute to human intoxications. This is because phallolysin, as a protein, is unstable when heated over 65°C or when treated with acid, which should cause the inactivation of the toxin on contact with the gastric juice (Wieland, 1986). 15.8.3

The Paxillus Syndrome

The paxillus syndrome is associated with the consumption of Paxillus involutus, commonly known as brown roll-rim mushroom, and Suillus luteus. Both are abundant and sometimes consumed in large quantities. If not thoroughly cooked before eating, these mushrooms very often have been the cause of intoxications, sometimes leading to severe consequences. The symptoms of poisoning occur within 1–2 hours after ingestion and include intense abdominal pain, nausea, vomiting, diarrhea, and acute circulatory collapse, followed by oliguria and hemoglobinuria. Schmidt and coworkers (1971) reported on a hemolytic anemia that developed after eating of P. involutus followed by a massive hemolysis with subsequent shock and acute renal failure that occurred after renewed consumption of this mushroom. According to Winkelmann and associates (1986), fatal poisoning with P. involutus is caused by an immune reaction in which immune complexes at-

Copyright 2002 by Marcel Dekker. All Rights Reserved.

O OH

HO HO

HO

OH

Figure 15.12 Chemical structure of involutin, the toxic secondary metabolite of Paxillus involutus mushroom.

tached to the erythrocyte surface initiate hemolysis. Therefore, in addition to adequate shock treatment, elimination of these immune complexes by plasma separation seems to be the therapy of choice. Edwards and colleagues (1967) isolated the only secondary metabolite of P. involutus not found in other mushrooms, called involutin (Figure 15.12). However, there is no evidence that this nitrogen-free compound may be the toxin. 15.8.4

Mushrooms with Hemagglutinating Activity

Lectins or hemagglutinins are present in the aqueous extracts of many species of various families including Hygrophoraceae, Tricholomataceae, Agaricaceae, Strophariaceae, Cortinariaceae, and Russulaceae, provided they are collected no earlier than September (Seeger and Wiedemann, 1972). These carbohydrate-binding proteins, which cause agglutination of red blood cells, have been purified from a variety of mushrooms, such as Agaricus bisporus, A. campestris, Clitocybe nebularis, Marasmius oreades, Fomes fomentarius, Flammulina velutipes, Psathyrella velutina, Grifola frondosa, and Aleuria aurantia (Antkowiak, 1996).

15.9 MUSHROOMS CAUSING GASTROINTESTINAL DISTURBANCES Although poisoning that produces gastrointestinal symptoms has been estimated to amount to up to 40% of all cases of mushroom poisoning (Alder, 1960), reports are not numerous because fatalities are quite rare, and the patients normally recover within a few days. In the few casualties reported so far, only children were involved (Chapuis, 1984). A large number of widely distributed mushrooms are capable of causing gastrointestinal irritations (Table 15.1). Usually symptoms occur 15 min to 3 hr after the mushroom meal. Depending on the mushroom, the intoxi-

33H16C

HOOC

29H14C

COOH

CH

C

CH2

HOOC

COOH

COOH C

CH

CH2

COOH

OH

OH Norcaperatic acid

Agaricic acid

O

N

H N

N

COO N H

N H

O

NH3

Agaricone

HO

OCH3

OH

OH

Xanthodermin OH CH3

N

OHC

CH3

CH3

N Agaricin

Figure 15.13

COOH

N

N

SO3Na

CH3

HO

O

O

N Psalliotin

Illudinine

Illudalic acid

Chemical structures of some gastrointestinal irritants found in various mushrooms.

cation may produce persistent nausea, vomiting, diarrhea, and abdominal pain, which may lead to dehydration and hypovolemic shock, especially in children. The importance of fluid and electrolyte replacement in children must be emphasized. Mushrooms eaten by adults almost invariably have been cooked, by a process that markedly reduces or even inactivates some gastroenteric irritants. Many childhood fatalities that follow the ingestion of normally “nontoxic” mushrooms are secondary to uncompensated fluid and electrolyte losses resulting from profound emesis and diarrhea in response to gastroenteric irritants in the raw mushrooms. Otherwise, the management is entirely symptomatic as for gastritis of any other cause. Unlike in the cases of well-known fatal consequences of ingestion, most of the mushrooms have not been thoroughly investigated yet with regard to their poisonous principles and pathogenic effects. The chemical structures of some of the gastrointestinal irritants identified thus far are shown in Figure 15.13.

15.10 MUSHROOMS WITH OBSCURE TOXICITY PATTERN Several species of mushrooms can only be listed as “suspected” because there are insufficient clinical case re-

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ports to have attracted the attention of investigators. In addition to the possibility of obtaining a specimen of the offending mushroom for identification, there is the problem of the great variability in toxicity. Thus a species growing in one part of a country may produce sickness, whereas in another section of the same country it may be eaten with relish. Part of this is due to variability in growing conditions. Certain mushrooms are also capable of forming hybrids. Hypholoma fasciculare (Naematoloma fasciculare), for example, seems to be edible, although not particularly desirable, in some locations and dangerously toxic elsewhere. In a few cases of fatal intoxications by this mushroom studied by Herbich and colleagues (1966), about 9 hr after the mushroom meal gastrointestinal symptoms occurred. These were, however, preceded by extensive liver damage that later resulted in death. Ikeda and coworkers (1977a, 1977b, 1977c) isolated two groups of active compounds from these mushrooms, fasciculols (Figure 15.14) and naematolins (Figure 15.15). Fasciculols are hydroxylated tetracyclic triterpenes of a common lanostane skeleton with one tetrasubstituted double bond. They are rare natural compounds containing two α-glycol systems at the positions C-2,C-3, and C-24,C-25. Naematolin is a sesquiterpenoid with a caryophyllane bicycloundecane skeleton.

R4 H

OH

2HC

R3 OH

R1O

R2O

O

OH

H

O

O

OH

OCH3

OH

N H

(Y)

O

(X)

O

O

OH

O OH N H O

(Z)

Fasciculol A Fasciculol B Fasciculol C Fasciculol D Fasciculol E Fasciculol F Fasciculic acid A Fasciculic acid B Fasciculic acid C

Figure 15.14 mushroom.

R1

R2

R3

R4

H H X H H X Y Y H

H H H H X H H H Z

H OH OH OH OH OH H OH OH

H H H OH OH OH H H OH

Active hepatotoxic compounds of fasciculol type isolated from Naematoloma fasciculare (Hypholoma fasciculare)

Yet another example is the ugly milk cap mushroom, Lactarius necator (synonyms L. plumbeus, L. turpis), which is widely distributed in Europe, Asia, and North America. Studies on the chemical composition of this mushroom by Suortti (1984) and Suortti and von Wright (1983) led to the identification of necatorin, the activity of which appeared to be comparable with that of aflatoxin B1 in the microbiological mutagenicity tests. Several other alkaloids were also found in this mushroom (Figure 15.16). The commercially cultivated Agaricus bisporus mushroom was found to contain several phenylhydrazine

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and aniline derivatives of potential toxicity. Both have been implicated in carcinogenesis. The well-characterized compound of this class is agaritine. Agaritine is hydrolyzed in the mushroom by γ-glutamyl transferase to the active agent 4-hydroxymethylphenylhydrazine (Figure 15.17), which is a well-known antipyridoxine factor. The hydrolysis of agaritine is accelerated if the cells of the mushrooms are disrupted. The mechanism underlying the antipyridoxine activity is believed to be condensation of the hydrazines with the carbonyl compounds pyridoxal and pyridoxal phosphate, resulting in the formation of inactive hydrazones.

HO

OH

H HO

Naematolin B OAc

H O

HO

O

H

Naematolone OAc H O

Figure 15.15

Chemical structures of naematolin B and naematolone found in Naematoloma fasciculare (Hypholoma fasciculare) mushroom.

HO

HO

N

Necatorin

O N

Necatorone

O

O

N

N OH OH

OH

N

N

N

O

O

O N

N

N

HO

HO

HO

HO

HO

HO

N

N

O

O N

N

N O N

OH

4,4'-Binecatorone Figure 15.16

10-Deoxy-4,4'-binecatorone

10,10'-Dideoxy-4,4'-binecatorone

Alkaloids isolated from Lactarius necator (ugly milk cap mushroom) having mutagenic activity similar to that of aflatoxin B1.

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O HO2HC

NHNH C

(CH2)2

CH

COOH

NH2 Agaritine γ-Glutamyl transferase

O HO2HC

NHNH2

HO

C

(CH2)2

CH

COOH

NH2 4-(Hydroxymethyl)-phenylhydrazine Figure 15.17

Glutamic acid

Hydrolysis of agaritine, the antipyridoxine factor found in the commercially cultivated edible Agaricus bisporus mushroom.

15.11 CONCLUSIONS Although mushrooms do not compose a significant portion of the human diet, they are a major toxic hazard in terms of the rate of poisoning and the number of fatalities caused. There are, as yet, no infallible methods of distinguishing poisonous mushrooms from edible ones. Furthermore, their chemical composition appears to be quite dependent on several factors. They often seem to have variable effects on individuals. As a general rule, one should consume only personally known edible wild species. Eating unfamiliar species may be quite hazardous. With advances in food production technologies, a vast variety of edible mushroom species is now available year round for one’s enjoyment. Thus, there appears to be no need for mushroom gathering in the wild. However, despite the culinary enjoyment that mushrooms offer, the risks accompanying wild mushroom consumption are not commensurate with the benefits.

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Antkowiak, W. Z. 1996. The chemistry and toxicology of mushroom alkaloids. In Chemistry and Toxicology of Diverse Classes of Alkaloids, ed. M. S. Blum, pp 185–300. Alaken, Inc., Fort Collins, CO. Antkowiak, W. Z. and Gessner, W. P. 1975. Isolation and characteristics of toxic components of Cortinarius orellanus Fries. Bull. Acad. Polon. Sci. Ser. Sci. Chim. 23:729–733. Antkowiak, W. Z. and Gessner, W. P. 1985. Photodecomposition of orellanine and orellinine, the fungal toxins of Cortinarius orellanus Fried and Cortinarius speciossimus. Experimentia 41:769–771. Baldelli, P., Cupuello, A., and Robello, M. 1994. GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam. Amino Acids 6:155–163. Benedict, R. G. 1972. Mushroom toxins other than amanita. In Microbial Toxins: A Comprehensive Treatise, Vol. VIII, Fungal Toxins, eds. S. Kadis, A. Ciegler, and S. J. Ajl, pp. 281–320. Academic Press, New York. Benedict, R. G., Brady, L. R., Stuntz, D. E., and Spurr, J. 1970. Occurrence of the deadly Amanita verna in the Pacific Northwest. Mycologia 62:597–599. Bhattacharya, S. K. and Sanyal, A. K. 1971. Anticholinesterase activity of bufotenine. Indian J. Physiol. Pharmacol. 15:133–134. Bielski, J. and Sikorski, M. 1992. Epidemiology of mushroom poisonings in Poland. In Recent Advances in Toxicology Research, Vol. 3, eds. P. Gopalakrishnakone and C. K. Tan, pp. 135–148. The Venom and Toxic Research Group, National University of Singapore, Singapore.

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Gerault, A. 1981. Intoxication collective de type orellanine provoquee par Cortinarius spendens. R. Hry. Bull. Soc. Mycol. Fr. 97:67–72. Good, R., Muller, G.F.R., and Eugster, C. H. 1965. Isolation and characterization of premuscimol and muscazone from Amanita muscaria (L. ex Fr.). Helv. Chim. Acta 48:927–930. Gore, M. G. and Jordan, P. M. 1982. Microbe single-column analysis of pharmacologically active alkaloids from the Fly Agaric mushroom Amanita muscaria. J. Chromatogr. 243:323–328. Gosselin, R. E., Smith, R. P., and Hodge, H. C. 1984. Mushroom toxins. In Clinical Toxicology of Commercial Products: Acute Poisoning, 5th ed., ed. R. E. Gosselin, pp. 289–309. Williams and Wilkins, Baltimore, MD. Goth, H., Gagneux, A. R., Eugster, C. H., and Schmid, H. 1967. 2(3H)-Oxazolone durch Photoumlagerung von 3-Hydroxyisoxazolen. Synthese von Muscazon. Helv. Chim. Acta 50:137–142. Gray, W. D. 1973. The Use of Fungi as Food and in Food Processing. Part II. CRC Press, Boca Raton, FL. Grzymala, S. 1957. Massenvergiftungen durch den orangefuchsigen Hautkopf. Z. Pilzk. 23:139–142. Grzymala, S. 1959. Zur toxischen Wirkung des orangefuchsigen Hautkopfes (Dermocybe orellana Fr.). Dtsch. Z. Gerichtl. Med. 49:91–99. Grzymala, S. 1961. Fatal poisoning by a pseudoedible species. III. Isolation of the toxic substance orellanine. Roczniki Panstwowego Zakladu Hig. 12:491–498. Grzymala, S. 1962. L’isolement de l’orellanine, poison du Cortinarius orellanus Fries et l’etude de ses effects anatomopathologiques. Bull. Soc. Mycol. France 78:394–404. Haines, J. H., Lichstein, E., and Glickerman, D. 1985. A fatal poisoning from an amatoxin-containing Lepiota. Mycopathologia 93:15–17. Hatfield, G. M. and Schaumberg, J. P. 1975. Isolation and structural studies of coprine, the disulfiramlike constituent of Coprine atramentarius. Lloydia 38:489–496. Hazani, E., Taitelman, U., and Shasha, S. M. 1983. Amanita verna poisoning in Israel. Report of a rare case and of time and place. Arch. Toxicol. Suppl. 6:186–189. Henneberg, M., Klawitter, M., Kozlowski, J., Marciniak, J., and Skrzydlewska, E. 1984. Zatrucia roslinami wyzszymi I grzybami (Poisoning by Higher Plants and Mushrooms). Pantswowy Zaklad Wydawnictw Lekarskich, Warszawa, Poland. Herbich, J., Lohwag, K., and Rotter, R. 1966. Todliche vergiftung mit dem grunblattrigen schwefelkopf. Arch. Toxikol. 21:310–320. Hesler, C. R. 1960. Mushrooms of the Great Smokies. University of Tennessee Press, Knoxville, TN. Hofman, A. 1959. Psychotomimetic drugs, chemical and pharmacological aspects. Acta Physiol. Pharmacol. Neerl. 8:240–258. Hofman, A., Frey, A., Ott, H., Petrzilka, T., and Troxler, F. 1958b. The structure and synthesis of psilocybin. Experientia 14:397–399.

Hofman, A., Heim, R., and Kobel, H. 1958a. Psilocybin, a hallucinogen from Psilocybe mexicana. Experientia 14:107–109. Horwitz, J., Anderson, C. H., and Perlman, R. L. 1986. Comparison of the effects of muscarine and vasopressin on inositol phospholipid metabolism in the superior cervical ganglion of the rat. J. Pharmacol. Exp. Ther. 237:312–317. Ikeda, M., Niwa, G., Tohyama, K., Sassa, T., and Miura, Y. 1977c. Structures of fasciculol C and its depsipeptides, new biologically active substances from Neamatoloma fasciculare. Agric. Biol. Chem. 41:1803–1805. Ikeda, M., Sato, Y., Izawa, M., Sassa, T., and Miura, Y. 1977a. Isolation and structure of fasciculol A, a new plant growth inhibitor from Neamatoloma fasciculare. Agric. Biol. Chem. 41:1539–1541. Ikeda, M., Watanabe, H., Hayakawa, A., Sato, K., Sassa, T., and Miura, T. 1977b. Structures of fasciculol B and its depsipeptide, new biologically active substance from Neamatoloma fasciculare. Agric. Biol. Chem. 41:1543–1545. Kogl, F., Salemink, C. A., Schouten, H., and Jellinek, F. 1957. Uber Muscarin III. Rec. Trav. Chim. Pays-Bas 76:109–127. Koppel, C. 1993. Clinical symptomatology and management of mushroom poisoning. Toxicon 31:1513–1540. Krieger, L.C.C. 1967. The Mushroom Handbook. Dover, New York. Kusano, G., Koike, Y., Inoue, H., and Nozoe, S. 1986. The constituents of Gymnopilus spectabilis. Chem. Pharm. Bull. (Tokyo) 34:3465–3470. Lampe, K. F. 1983. Mushroom poisoning. In Handbook of Naturally Occurring Food Toxicants, ed. M. Rechcigl, pp. 193–212. CRC Press, Boca Raton, FL. Lampe, K. F. 1986. Toxic effects of plant toxins. In Casarett and Doull’s Toxicology, The Basic Science of Poisons, 3rd ed., eds. C. D. Klaassen, M. O. Amdur, and J. Doull, pp. 757–767. Macmillan, New York. Langer, M., Vesconi, S., and Costantino, D. 1990. Update in Intensive Care and Emergency Medicine. Springer, Berlin. Leung, A. Y. and Paul, A. G. 1968. Baeocystin, a monomethyl analog of psilocybin from Psilocybe baeocystis saprophytic culture. J. Pharm. Sci. 57:1667–1671. Lindberg, P., Bergman, R., and Wickberg, B. 1975. Isolation and structure of coprine, a novel physiologically active cyclopropane derivative from Coprinus atramentarius and its synthesis via 1-aminocyclopropanol. J. Chem. Soc. Chem. Commun. 946–947. Lindberg, P., Bergman, R., and Wickberg, B. 1977. Isolation and structure of coprine, the in vivo aldehyde dehydrogenase inhibitor in Coprinus atramentarius; syntheses of coprine and related cyclopropanone derivatives. J. Chem. Soc. Perkin 1:684–691. Litten, W. 1975. The most poisonous mushroom. Sci. Am. 232:90–101. Monroe, P. J., Smith, D. L., Williams, G. M., and Smith, D. J. 1994. Bufotenine has a parachloroamphetamine-like action on the storage and release of serotonin in rat spinal cord synaptosomes. Biog. Amines 10:273–284.

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Mottonen, M., Nieminen, L., and Heikkilae, H. 1975. Damage caused by two finnish mushrooms Cortinarius speciosissimus and C. gentiles on the rat kidney. Z. Naturforsch. 30c:668–679. Ohenoja, E., Jokiranta, J., Makinen, T., Kaikkonen, A., and Airaksinen, M. M. 1987. The occurrence of psilocybin and psilocin in Finnish fungi. J. Nat. Prod. 50:741–744. Piering, W. F. and Bratanow, N. 1990. Role of the clinical laboratory in guiding treatment of Amanita virosa mushroom poisoning: Report of two cases. Clin. Chem. 36:571–574. Pilat, A. 1961. Mushrooms and Other Fungi. Nevill, London. Pilat, A. and Usak, O. 1950. Mushrooms. Spring Books, London. Piqueras, J. 1984. Intoxicacion de tipo ciclopeptidico (Faloidina) producida por pequenas Lepiotas. Bull. Soc. Catalana Micol. 8:33–37. Pore, R. S. 1993. Mushroom poisoning. In Fungal Infections and Immune Responses, eds. J. W. Murphy, H. Friedman, and M. Bendinelli, pp. 493–519. Plenum Press, New York. Rapior, S. 1988. Contribution a l’etude de Cortinarius orellanus (Fr.): Chimiotaxinomic, culture in vitro, toxicite. Ph.D. Thesis, University of Montpellier, France. Richard, J. M., Louis, J., and Cantin, D. 1988. Nephrotoxicity of orellanine, a toxin from the mushroom Cortinarius orellanus. Arch. Toxicol. 62:242–245. Rose, E. K. and Reiders, P. 1966. An episode of food poisoning attributed to imported mushroom. Ann. Intern. Med. 64:372–377. Sanz, P., Reig, R., Piqueras, J., Marti, G., and Corbella, J. 1989. Fatal mushroom poisoning in Barcelona, 1986–1988. Mycopathologia 108:207–209. Schmidt, J., Hartmann, W., Wurstlin, A., and Deicher, H. 1971. Akutes nierenversagen durch immunhamolytische anamie nach genuss des kahlen kremplings (Paxillus involutus). Dtsch. Med. Wochenschr. 96:1188–1191. Schmiedeberg, O. and Koppe, R. 1869. In Das Muscarin, das Giftige Alkaloid des Fliegenpilzes. F.C.W. Vogel, Leipzig. Schulz-Weddingen, I. 1986. Beitrage zur Kenntnis der Gattung Lepiota. I. Eine Intoxikation mit Lepiota brunneo-incarnata in Nordwestdeutschland. Z. F. Mykol. 52:91–110. Schumacher, T. and Hoiland, K. 1983. Mushroom poisoning caused by species of the genus Cortinarius Fries. Arch. Toxicol. 53:87–106. Seeger, R., Kraus, H., and Wiedmann, R. 1973. Zum Vorkommen von Hamolysinen in Pilzen der attung Amanita. Arch. Toxicol. 30:215–226. Seeger, R. and Wiedmann, R. 1972. Zum Vorkommenn von Hamolysinen and Agglutininen in hoheren Pilzen (Basidiomyceten): Untersuchungen an 293 Arten. Arch. Toxicol. 29:189–217. Singer, R. 1961. Mushrooms and Truffles. Hill, London. Slanina, P., Cekan, E., Halen, B., Bergman, K., and Samuelsson, R. 1993. Toxicological studies of the false morel (Gyromitra esculenta): embryotoxicity of monomethylhydrazine in the rat. Food Addit. Contam. 10:391–398. Smith, A. H. 1958. The Mushroom Hunter’s Field Guide. University of Michigan Press, Ann Arbor, MI.

Spoerke, D. G. and Rumack, B. H. 1992. General approach to mushroom poisoning. Recent Adv. Toxicol. Res. 3: 120–134. Stijve, T. 1978. Ethylidene gyromitrin and N-methyl-N-formylhydrazine in commercially available dried false morels, Gyromitra esculenta Fr. Mitt. Geb. Lebensm. Hyg. 69: 492–504. Stijve, T. 1979. Bufotenine concentrations in carpophores of Amanita citrina (Schff) S. F. Gray. Mitt. Geb. Lebensm. Hyg. 70:246–253. Stijve, T. and Kuyper, T. W. 1985. Occurrence of psilocybin in various higher fungi from several European countries. Planta Med. 385–387. Suortti, T. 1984. Improved analytical and preparative methods for necatorin from Lactarius necator (Fr.) Karst mushroom. J. Chromatogr. 301:303–307. Suortti, T. and von Wright, A. 1983. Isolation of a mutagenic fraction from aqueous extracts of the wild edible mushroom Lactarius necator. J. Chromatogr. 255:529–532. Takemoto, T., Nakajima, T., and Sakuma, R. 1964a. Isolation of an insecticidal constituent (ibotenic acid) from Amanita muscaria and A. pantherine. Yakugaku Zasshi 84: 1233–1234. Takemoto, T., Nakajima, T., and Yokobe, T. 1964b. Structure of ibotenic acid. Yakugaku Zasshi 84:1232–1233. Theobald, W., Buch, O., Kunz, H. A., Krupp, P., Stenger, E. G., and Heimann, H. Z. 1968. Pharmakologische und experimental-psychologische Untersuchungen mit 2 Inhaltsstoffen des Fliegenpilzes (Amanita muscaria). Arzneim. Forsch. 18:311–315.

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Vergeer, P. P. 1983. Poisonous fungi: Mushrooms. In Fungi Pathogenic for Humans and Animals. Part B, eds. H.D.H. Howard and L. F. Howard, pp. 374–412. Marcel Dekker, New York. Von Clarmann, M. 1964. Pilzvergiftung. Fortschr. Med. 82: 508–529. Wieland, T. 1986. Peptides of poisonous Amanita mushrooms. In Springer Series in Molecular Biology, ed. A. Rich, pp. 256–271. Springer Verlag, New York. Wieland, T. and Faulstich, H. 1978. Amatoxins, phallotoxins, phallolysin, and antamanide: The biologically active components of poisonous Amanita mushrooms. CRC Crit. Rev. Biochem. 5:185–260. Wieland, T., Motzel, W., and Merz, H. 1953. Uber das Vorkommen von Bufotenin imgelben Knollenblatterpilz. Liebigs Ann. Chem. 577:10–16. Wieland, T. and Wieland, O. 1972. The toxic peptides of Amanita species. In Microbial Toxins, Vol. VIII, Fungal Toxins, eds. S. Kadis, A. Ciegler, and S. J. Ajl, pp. 212–239. Academic Press, New York. Winkelmann, M., Stangel, W., Schedel, I., and Grabensee, B. 1986. Severe hemolysis caused by antibodies against the mushroom Paxillus involutus and its therapy. Klin. Wochenschr. 64:935–938. Wisemann, J. S. and Abeles, R. H. 1979. Mechanism of inhibition of aldehyde dehydrogenase by cyclopropanone hydrate and the mushroom toxin coprine. Biochemistry 18:427–435. Zilker, T. 1987. Diagnose und Therapie der Pilzvergiftungen (Teil II). Leber, Magen, Darm 1987:173–197.

16 Toxic Metals, Radionuclides, and Food Packaging Contaminants

16.1 INTRODUCTION Incidental or unintentional food additives are substances present in our food that can alter its properties and have not been added deliberately. This group of food chemicals can be broadly classified under the general heading of contaminants and residues. These substances end up in our food supply by indirectly entering the food chain. Some of these chemicals are extremely hazardous to human health, whereas others are responsible for inducing plasmid-mediated drug resistance in several human pathogens. Still others may interact with natural food constituents, particularly the lipids, to generate free radicals with carcinogenic potency. Generally, most of these chemicals are broken down naturally or washed away. Nevertheless, residual amounts do remain in our food supply. Such unintentional addition is unfortunate but unavoidable. Incidental food additives have several sources: 1.

2.

3.

Contamination of soil and water supplies with heavy metals, radioisotopes, pesticides, and other toxic industrial chemicals Manufacturing processes, which may contribute packaging contaminants (monomers, polymer stabilizers, plasticizers, etc.), particles of the equipment used, and the remains of errant animals Chemicals applied to crops (plant hormones and pesticides, such as insecticides, fungicides, herbicides, and antisprouting agents) and administered to livestock and poultry to maintain or

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improve their health (veterinary medicines, hormones, and feed additives) Any discussion of the hazards presented by the incidental contaminants must be cognizant of those features they may have in common. For example, although differing widely in chemical structures, they possess certain common physical properties that tend to increase their potential hazard to humans. Contaminants that are persistent in the environment resist degradation and are extremely stable. Second, they tend to accumulate in the human food chain, especially in fish, and it is this accumulation that renders them a potential hazard to humans. The characteristic that makes them an actual hazard is their slow rate of elimination and/or metabolism, which results in their accumulation in tissues. Finally, their toxicity is usually greater in higher-order mammals than in species of lower phylogenetic orders. For example, fish, seals, and crustaceans can tolerate much higher tissue levels of mercury and arsenic than can humans. Chemicals released into the environment that have one or more of these properties can be predicted to have a potential to cause human harm. Although not every environmental contaminant possesses all of these properties, there is a surprising similarity among these substances, to the extent that any aromatic halogenated substance or organometallic substance detected in the environment with these properties should be viewed with suspicion. In this chapter, the toxicological aspects of heavy metals, radioisotopes, and food packaging contaminants are described. Various toxicological aspects of pesticide

residues and industrial contaminants as well as drug residue contamination in our food chain are described in the two subsequent chapters.

16.2 TOXIC METALS Metals are considered the oldest toxins known to humans since the Stone Age. They are redistributed in the environment by natural geological and biological cycles. The biological cycles bioconcentrate elements through both plants and animals, through biomagnification in food cycles. Because of the elemental nature of metals and their diverse affinities for organic ligands in biological cycles, their toxicities arise as multiple organ effects more often than do those of any class of toxicants. In view of this multiplicity of effects, the concepts of critical organ and critical dose have evolved to delineate the most sensitive ones. Thus, the critical organ is the one showing adverse effects at the lowest dose; other organs and systems may be much more severely affected but only at higher doses. This concept is important with respect to toxicants for which a tolerance greater than zero has been assigned for technical or economic reasons (Nordberg, 1976; Ahmed, 1999). Factors that influence the toxicity of metals at a certain level of exposure are important, particularly in susceptible populations. These include metabolic interactions of essential metals, formation of metal-protein complexes, age and state of development of exposed individuals, life-style factors, chemical form or speciation, and immune status of the host (Burns et al., 1995; Fowler, 1991; Oehme, 1978). Toxicity of a metal is determined by the dose at the cellular level and such factors as chemical form (or speciation) and ligand binding (Ahmed, 1999). For example, alkyl compounds are lipid-soluble and pass readily across biological membranes, causing them to accumulate inside the cell, with the result that their toxicity differs from that of the inorganic form (e.g., alkylmercury is primarily neurotoxic, whereas mercuric chloride, HgCl2, is nephrotoxic). The strong attraction between metal ions and organic ligands influences not only their availability for absorption, but also the deposition of a metal in the body and its excretory route. Most biologically important metals bind strongly to tissues and therefore are slowly excreted, leading them to accumulate in vivo. Blood, urine, and hair are most frequently used to assess the exposure to metals and are thus often referred to as indicator tissues. Blood and urine exposure usually represent recent effects and correlate best with acute toxicity. Quantitation of metals in organs can be carried out by techniques such as atomic absorption, atomic fluorescence, flame emission, electron

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probe, ion probe, neutron activation analysis, spark source mass spectroscopy, proton-induced x-ray emissions, x-ray fluorescence spectroscopy, and scanning x-ray analysis (Meloan, 1978; Jacobs, 1996; Ahmed, 1999). It is difficult to draw a clear distinction between essential—especially the trace elements in human nutrition—and toxic metals. Nearly all metals are toxic to humans if ingested in abnormal amounts. Similarly, the interaction of a toxic metal with an essential one occurs when they have similar metabolism. Moreover, absorption of a toxic metal from the gastrointestinal (GI) tract or lung is often influenced by an essential dietary element if they share—or if the toxic metal influences—a homeostatic mechanism, as for lead, cadmium, and iron. In addition, the physiological effects of some metals, such as cadmium, are closely related to the amount of other essential nutrients in the human diet (Reilly, 1991; Deshpande and Salunkhe, 1995). All metals are capable of interacting in the body with other cellular constituents. Nevertheless, it is possible to differentiate among elements that are known with certainty to be essential and those that display severe toxicological symptoms at extremely low levels and have no known beneficial physiological functions. The heavy metals, most noticeably mercury, lead, and cadmium, probably constitute the single largest group of elements that contaminate agricultural soils, water supplies, and the environment and eventually find their way into the human food chain. Other toxic metals include arsenic, beryllium, boron, selenium, and other metals and metalloids. The possible sources of contamination of our food and the physiologically adverse and toxic effects of these metals are described in the following section. 16.2.1

Sources of Contamination

Soil Soil is the primary source of toxic metals found in food crops. Although most nutrients are absorbed from the top 10–30 cm of soil, plants that are capable of developing extensive deep root systems can effectively penetrate the soils to a depth of more than 6–10 m. Hence, the toxic metal content of agricultural soils needs to be considered from the viewpoint of both surface contamination as well as the nature of the underlying soil and the surrounding area (Hall et al., 1953; Deshpande and Salunkhe, 1995). Although most toxic metal contamination of soils occurs because of environmental pollution, natural factors also play an important role in this regard. For example, the volcanic soils that are intensively cultivated in Java and Sumatra contain inherently high levels of mercury and other toxic metals. Reilly and associates (1989) reported the mercury and arsenic contents in soil, water, and foods

grown in the Dieng Plateau area of Java, where they found a significant accumulation of mercury in locally grown vegetables. It was estimated that the consumption of as little as 100 g potato each day would contain almost twice the World Health Organization’s (WHO) tolerable level of mercury intake. Accumulation by Pasture Plants and Crops Plants are capable of absorbing toxic metals from contaminated soils and accumulating them in various tissues. Selenium toxicity was first noticed in livestock grazing on pastures and herbage grown in selenium-rich soils (Knott and McCray, 1959; Gardiner et al., 1962). Cadmium poisoning of humans was reported in Japan when rice paddy fields were irrigated with water contaminated by effluent from a local zinc-cadmium-lead mine (Reilly, 1991). Similar incidences of cadmium, copper, and zinc toxicity were also reported in England and Zambia (Reilly and Reilly, 1971; Morgan, 1988). Some plants are also capable of absorbing and accumulating certain toxic metals in large amounts. The shrub Camellia sinesis absorbs large quantities of both aluminum and manganese from the soil and concentrates them in its leaves (Pennington, 1987). The dried leaves of this shrub are used to make tea. In the United Kingdom, tea drinking alone appears to contribute a significant dietary intake of manganese in the elderly population (Wenlock et al., 1979). Metal accumulation in foods thus is a natural occurrence, with benefits as well as possible disadvantages for those who consume the foods. Sewage Sludge The application of sewage sludge to agricultural lands constitutes a significant source of food contamination by toxic metals. Although it contains more than 40% organic matter and is a rich source of both nitrogen and phosphorus, sewage sludge, especially from heavily populated urban and industrial areas, can contain relatively high levels of several toxic metals. Incidences of crop failures from soils where such sludge is applied are not uncommon (Mackenzie and Purves, 1975). Normal concentrations of several metals in typical sludge samples are presented in Table 16.1. The metals are usually industrial in origin, although domestic waste also makes a substantial contribution. Mercury, zinc, lead, and cadmium have been reported to occur at high levels in household dust as well as in domestic garbage (Harrison, 1978; Price, 1988). The levels of toxic metals found in sewage sludge are considerably higher than those found in typical agricultural land, with more than 300 times as much zinc and 100 times as much boron and copper as

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Table 16.1 Normal Ranges of Metals in Dry Matter of Sewage Sludge Metal Boron Cadmium Chromium Cobalt Copper Iron Lead Manganese Mercury Molybdenum Nickel Scandium Silver Titanium Vanadium Zinc

Content, mg/kg 15–1,000 60–1,500 40–8,800 2–260 200–8,000 6,000–62,000 120–3,000 150–2,500 3–77 2–30 20–5,300 2–15 5–150 1,000–4,500 20–400 700–49,000

Source: Compiled from Pike et al. (1975) and Capon (1981).

would occur in the normal arable rural soils (Berrow and Webber, 1972). Significant accumulation of these metals in food crops may result in potential health problems for the consumers. Of the metals found in sewage sludge, lead, cadmium, and mercury pose more serious problems than copper and zinc (Strenstrom and Vahter, 1974). Agricultural Chemicals and Fertilizers Some of the widely used commercial fertilizers are capable of introducing significant levels of cadmium to the soil. Such incidences have been reported from Sweden and Australia (Williams and David, 1973; Strenstrom and Vahter, 1974). The acid rain phenomenon observed in both Europe and North America further enhances the mobilization of toxic metals in agricultural soils, thereby facilitating a greater uptake by the food crops (Reilly, 1991). Certain toxic metals, e.g., mercury and arsenic, have also been used in both inorganic and organic forms in fungicides and other agrochemicals. The use of organomercurial compounds has been the cause of far more serious and better-documented cases of food poisoning in Iraq, Pakistan, and Guatemala (Bakir et al., 1973; Reilly, 1991). Mercurial compounds are no longer common in agricultural practices and have been largely replaced by less persistent fungicides. The use of arsenic pesticides in horticulture has similarly declined appreciably (MAFF, 1982).

Metal-Containing Water Contamination of both surface water and groundwater by industrial wastes is a prime source of heavy metal toxicity in human and animal nutrition. The contamination and subsequent consumption of seafood are particularly serious in this regard. The water used for food production and drinking, however, is often treated to remove excessive levels of toxic metals. Besides that by cadmium and mercury, large-scale pollution of water by other metals is quite common in several industrial countries (Prater, 1975; Reilly, 1991; Deshpande and Salunkhe, 1995). Food Processing Metal contamination may occur at several stages during food processing. Contamination sources include the following: 1. 2. 3. 4. 5.

The factory door Plant and equipment Catering operations Ceramic and enameled utensils Metal containers

Generally, only high-quality stainless steel, plastics, and other structural materials approved for contact with foods are used in food-processing plants. The use of ceramics and enameled utensils is a significant source of metal poisoning, particularly those made of aluminum, copper, lead, and cadmium, in the less-developed countries. Wrapping paper, cardboard containers, as well as the print and color applied to plastic containers are also capable of contaminating food (Klein et al., 1970; Watanabe, 1974; Heichel et al., 1974; Gramiccioni, 1984). 16.2.2

Occurrence and Toxicity

The occurrence and toxicity of some of the more common metal pollutants in the human food chain are briefly described in the following. Lead Of all the heavy metals, lead has probably the longest history of environmental contamination and toxicity to humans. Its presence in the human food chain continues to be a major health problem worldwide. Lead is the ubiquitous toxic metal and is detectable in practically all phases of the inert environment and in all biological systems. There is evidence that lead in the environment has increased during the past 200 years (Shukla and Leland, 1973). It is used on a very wide and increasing scale in the modern world, with production in the Western world alone totaling 4.8 million

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tons per year (Deshpande and Salunkhe, 1995). For this reason, lead poisoning, or plumbism, has been intensely studied. Because lead is toxic to most living things at high exposure and there is no demonstrated biological need for it, a major issue is to determine the dose at which it becomes toxic. Specific toxicities vary with age and circumstances of the host, but the major risk is toxicity to the nervous system. The most susceptible populations are children, particularly toddlers, infants in the neonatal period, and fetuses (CDC, 1991; Ahmed, 1999). Lead is naturally present in the soil. Environmental lead is a product of storage batteries, ammunition, solder, pigment colors and dyes, galvanizing and plating processes, pipes, and insecticides. It is also used in alloys with antimony, tin, and copper. It is a constituent or a contaminant of houseware materials such as crystal and pewter. Tetraethyl lead is an antiknock additive in gasoline, introduced into the environment through exhaust fumes from leaded gasoline used in vehicles; in recent years, this use has been drastically curtailed worldwide. Because the diet, including drinking water, is considered the principal source of the total body burden of lead, lead contamination of foodstuffs and the possibility of chronic lead poisoning from this source are indeed important food toxicological problems. Estimates place the total contribution of dietary sources at 90% of the total body burden of lead (Concon, 1988). The daily intake of lead via food in human nutrition was estimated to be 100–300 µg, with considerably higher levels the result of increasing environmental pollution (Jelinek, 1992). At the international level, the United Nations (UN) Joint Food Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) expresses the tolerable intake of cumulative environmental contaminants having toxicological effects when taken in the diet on a weekly basis to allow for variations in intake levels. These values are expressed as provisional tolerable weekly intake (PTWI) (UNEP/FAO/ WHO, 1988). This committee recommended a PTWI for lead from all sources of 50 µg/kg body weight for adults. This value was based on the data collected from 26 countries in 1972 covering a wide variety of foods (WHO, 1972). Because of increased sensitivity of infants and children, JECFA lowered the PTWI for this group of the population to 25 µg/kg body weight in 1986. Lead content of some representative food groups is shown in Table 16.2. Generally, shellfish and finfish have higher lead content than milk, fruits, vegetables, and meat, and levels in mollusks and crustaceans are higher than in fish. Lead levels in kidney and liver are substantially higher than those in meat muscle.

Table 16.2 Lead Content of Selected Foods Food

Range, µg/100 g

Cereal grains Cereal grain products Seafood Raw Canned Meats Gelatin Eggs, whole Vegetables, leafy Legumes Raw, dried, or frozen Canned Apples Pear Milk Whole, fresh Skim, dried, and packaged Skim, bulk package Evaporated Tea, leaves Cocoa, dry Sugar, white Molasses Baking powder Yeast, dry Black pepper Cinnamon Nutmeg Allspice Chili powder Bay leaves Cider, apple Vinegar, cider Cola, 2 samples Ginger ale Beer, canned Wine, red Drinking water Alcoholic beverages

0–62 0–749 17–250 6–30 7–37 0–15 0–126 0–16 3–11

4–5

Mean 22 10.5 62 16 19 57 7 37 7 7 38 3 0 2 2 4.5 1.37 0.10

0–7

16–85 µg/L

1–50 µg/L 50–100 µg/L

53 150 117 40 11 41 64 v18 55 90 µg/L 100 µg/L 10 µg/L 40 µg/L 50 µg/L 5 µg/L

Source: Compiled from WHO (1976), Reilly (1991), Deshpande and Salunkhe (1995), and Janssen (1997).

The lead levels in vegetables are somewhat higher than in fruits, probably because of lead translocation from soil to the edible portion of vegetables compared with that of fruits grown on trees and bushes (Ahmed, 1999). Of all vegetables studied, spinach, with a large surface area compared to weight, usually contains high lead levels. Another factor affecting lead content of vegetation worthy of discussion is the growing location with respect

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to major highways. Perhaps not surprisingly, there is a good correlation between average traffic counts and average soil and plant lead content at sites close to the roadside (Concon, 1988; Ahmed, 1999). As a corollary, an inverse relationship between distance from the road and lead content has been observed in various plants and vegetables. Processing and packaging also can significantly increase the total lead content in foodstuffs. In this regard, canning may markedly increase lead content. For example, in a study of 256 cans of food, the contents of 62% contained a lead level of 100 ppb or more, 37% contained 200 ppb or more, and 12% contained 400 ppb or more (Mitchell and Aldons, 1974). Of products in glass and aluminum containers, only 1% had lead levels in excess of 200 ppb. This is indicative of leaching of lead from the soldered seam of the can and is an especially serious problem with canned acidic foods, such as tomato paste. The consistently high lead content of canned foods unequivocally shows that canning and the attendant handling and processing operations significantly increase the degree of lead contamination of foodstuffs. Lead solder used in cans is a major controllable source of lead in food. Food processors in many countries have switched to nonsoldered cans. This is particularly important in the case of infant foods. The use of nonsoldered cans can cut lead concentrations on average to between one-fifth and one-tenth of previous levels. In contrast, attempts to decrease lead content by improving processing operations for food packed in lead-soldered cans have achieved only a 50% reduction (UNEP, 1992). However, nonsoldered cans are more expensive than lead-soldered cans and hence are not widely used. Drinking water consumed directly and used in food processing also contributes lead to the dietary intake. In addition to the efforts of FDA to control lead levels in foods, the EPA has also moved to limit lead intake from water and air. In 1993, WHO proposed a guideline value of 10 mg/L for lead in drinking water. The widespread use of lead piping and soldering of water tanks in some countries contributes to incidences of lead poisoning much higher than those attributable to leaded gasoline (Grobler et al., 1996). No organic forms of lead have been reported to occur in food. Lead in foodstuffs exists exclusively as salts, oxides, or sulfhydryl complexes. Most lead salts and oxides are insoluble in water, and, hence, lead absorption is low. The absorption of lead from food is estimated to be 10% in adults and 40% in children (Reilly, 1991). Several dietary factors influence the level of absorption. A low body-calcium status, iron deficiency, and diets rich in carbohydrates but lacking protein and those containing high levels of vitamin D result in increased absorption of lead.

In the normal adult, about 90% of the ingested lead is generally excreted in the urine and feces. The absorbed lead may be distributed into three compartments (Rabinowitz et al., 1973): (a) the freely diffusible lead, which probably includes blood lead and free exchangeable lead of soft tissues; (b) the more firmly bound but exchangeable soft tissue lead; and (c) the hard tissue lead, such as in bones, teeth, hair, and nails. Lead is present in practically every organ and tissue of the human body, with amounts ranging from 100 to 400 mg or about 1.7 µg/g tissue (Barry, 1975). Over 90% of the lead in the human body occurs in the bone. The retention of lead in soft tissues is greatest in liver, followed by kidneys, aorta, muscle, and brain in decreasing order (Whanger, 1982). Lead also passes the placental barrier readily. Blood and soft tissue lead are likely to be responsible for the symptoms of poisoning. Lead in the hard tissues is more tightly bound. However, an equilibrium state may exist among bone, blood, and soft tissue lead, so that hard tissue lead may be an important source of blood and soft tissue lead (Goyer and Chisolm, 1972). Lead accumulated in the hard tissues, therefore, must be viewed as potentially toxic. The levels of lead in the bones, teeth, and hair continue to increase with age, suggesting a gradual accumulation of lead in the body. Therefore, contamination of food with lead and the possibility of chronic lead intoxication through the diet necessitate constant monitoring. The halflife of lead in the hard tissues has been estimated to be greater than 20 years; that of blood lead is 27 days (Rabinowitz et al., 1973). The toxic effects of lead form a continuum from clinical or overt effects to subtle or biochemical effects, which involve several organ systems and biochemical activities. The symptoms of acute lead poisoning in humans are well documented. The major effects are related to hemopoietic, nervous, gastrointestinal, and renal functions (Reilly, 1991). Generally, anorexia, dyspepsia, and constipation are followed by an attack of colic with intense paroxysmal abdominal pain. Lead encephalopathy is also observed in young children (NAS, 1972; NRC, 1993). However, little is known about chronic lead poisoning over a long period. Mild anemia, mental deterioration, hyperkinetic or aggressive behavior, peripheral neuropathy, lead palsy, and kidney damage are some of the clinical symptoms of chronic lead poisoning (WHO, 1976). Kehoe (1966a) has listed four principles that govern the conditions under which lead poisoning may be induced: 1.

The time of appearance of symptoms of lead poisoning depends on the magnitude of the

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2.

3. 4.

daily oral dose: i.e., large intakes cause symptoms to appear sooner. The dose, which is absorbed at a constant rate, is large enough that progressive accumulation of lead in critical quantities can occur within the lifetime of the individual. A critical concentration of lead must be attained in the blood. The likelihood of poisoning increases markedly if the concentration of lead in the tissues increases rapidly in response to relatively high dosage and there is a failure in some mechanism that inhibits the toxic effects of lead.

In Kehoe’s experiments with human volunteers, a dose of 0.62 mg lead daily was sufficient to bring about a slight accumulation of lead in the body; by extrapolation, it was estimated that daily dosages of 3.3, 2.3, and 1.3 mg produce a critical blood level of 80 mg/100 g in 8 months and 4 and 8 years, respectively. An oral intake of 10 to 15 mg of lead per day results in gastroenteric plumbism in about 30 days (Kehoe, 1966b). The toxic effects of lead and the minimal blood lead levels at which the effects are most likely observed are summarized in Table 16.3. Lead’s toxicological mode of action depends on its molecular configuration; inorganic lead, which is the form most available environmentally, contaminates foods, producing clinical signs different from those of organic forms (e.g., tetraethyl and tetramethyl lead) and is distributed differently in the body. Organic lead compounds may be absorbed in large quantities through the skin, but in these forms, their toxicities are primarily a problem of the petroleum industry (Ahmed, 1999). The organic forms are rapidly dealkylated by the liver to the trialkyl metabolites, which are responsible for toxicity. These metabolites in turn are slowly converted to inorganic lead (Hammond and Beliles, 1980). The most effective way to treat lead toxicity is removal of individuals from the source(s) of exposure. Chelation has a role in the treatment of symptomatic workers and children and is warranted in adults with blood lead levels above 60 µg/100 mL after assessment of biological and clinical parameters of exposure (Burns and Currie, 1995; Trachtenbarg, 1996). The Centers for Disease Control (CDC) in Atlanta has established guidelines to assist in evaluating exposure factors for lead toxicity in children (CDC, 1991). For children with severe lead poisoning, chelation is the standard procedure, even though the mortality rate may be 25%–35% when ethylene diaminetetraacetic acid (EDTA) or British AntiLewsite (BAL) is used individually; when

Table 16.3 Lowest Observed Effect Levels from Lead-Related Health Effects Blood lead concentration, µg/dL Effects Neurological Encephalopathy (overt) Hearing deficit Intelligence deficits In utero effects Peripheral neuropathy Hematological Anemia U-ALAa B-Eppb ALA inhibition Py-5-Nc inhibition Renal Neuropathy Vitamin D metabolism Blood pressure (males) Reproduction

Children

Adults

80–100 20 10–15 10–15 40

100–112

80–100 40 15 10 10

80–100 40 15 10

40 <30 30 40

a

Aminolevulinic acid in urine. Concentration of erythrocyte protoporphyrin. c Enzyme pyridimidine-5-nucleosidase inhibition results in accumulation of nucleotides in red blood cells, altering their energy metabolism and affecting their membrane stability and survival. Source: Compiled from Goyer (1995) and Ahmed (1999). b

both agents were combined, mortality was reduced. The oral chelating agent, meso-2,3-dimercaptosuccinic acid (DMS [Succimer]), has been used at lead blood levels of 45 µg or greater. Although it lowers the blood lead levels, its effectiveness in removal of lead from the brain and in reversing cognitive functions and behavioral development has not been demonstrated (Porru and Alessio, 1996). Cadmium Cadmium is widely distributed in the environment. Its extensive technological uses have resulted in widespread contamination of soil, air, water, vegetation, and food supplies. Cadmium and its compounds are widely used in electroplating metals and alloys. They are also used in many industrial, household, and office products and machines and in pigments in paints, enamels, glazes, textiles, and plastics (NRC, 1969). Cadmium is commonly found in its metallic form and as sulfides and sulfates. In foods, only inorganic cadmium salts are present. Organic cadmium compounds are

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very unstable. Sewage sludge, which is used as fertilizer and soil conditioner, is an important source of soil pollution with cadmium. In contrast to lead and mercury ions, cadmium ions are readily absorbed by plants and are equally distributed throughout them. Cadmium exposure arises from the ambient air, drinking water, tobacco, the working environment, soil, dust, and food; food is the main source of exposure to cadmium in nonoccupational settings (IARC, 1976). Unless contamination has occurred, the levels of cadmium in most foods are normally very low (Table 16.4). The overall range appears to be 0.095–0.987 mg/kg, with a mean of 0.469 mg/kg. Generally, meat and seafoods tend to contain higher levels of cadmium than any other food group. In 1988, the JEFCA established a PTWI of 7 µg/kg body weight for adults and infants over an accumulative period of 50 years at an exposure rate equivalent to 1.0 µg/kg/day for adults. Intakes above this level may be tolerated provided they are not sustained over long periods (WHO, 1989a). The WHO standard for cadmium levels in the drinking water was established at 10 µg/L (WHO, 1963). The United States has set a safety limit for drinking water and bottled water at 5 µg/L. The intake of cadmium in the United States from 1982 to 1991 ranged from 3.7 to 14.4 µg/day (Janssen, 1997). The dietary intake of cadmium in several countries was estimated to be 10–80 µg/day (Dabeka et al., 1987). Most well documented cases of cadmium contamination of foods and subsequent human poisoning are reported from Japan and Australia (Asami, 1984; Rayment et al., 1989).

Table 16.4 Cadmium Content of Selected Foods Food Bread Potatoes Cabbage Apples Poultry Minced beef Kidney (sheep) Dairy products Grains and cereals Prawns Seafoods Drinking water

Cadmium, µg/kg <2–43 <2–51 <2–26 <2–19 <2–69 <2–28 13–2000 <1–6 <2–28 17–913 50–3660 <1–21 µg/L

Source: Compiled from WHO (1963), Dabeka et al. (1987), Reilly (1991), and Deshpande and Salunkhe (1995).

The absorption of cadmium from food varies, depending on genetic factors, age, and nutritional factors. Infants absorb and accumulate more cadmium than adults. Under normal dietary conditions, about 6% of the cadmium ingested in food and beverages is absorbed by the human body (Reilly, 1991). Higher dietary levels of calcium and protein tend to increase cadmium absorption. Calcium or iron deficiency can increase, whereas a pyridoxine deficiency appears to decrease the absorption of cadmium. Most of the absorbed cadmium is retained in the kidneys bound to a metal-binding, high sulfhydryl protein, metallothionein. The half-life of cadmium in human kidneys may be as long as 30 years (Ahmed, 1999). Because of cadmium’s high solubility in organic acids, cadmium contamination of the human food chain is quite common. Being highly toxic, it is recognized as one of the most dangerous trace elements in food and the environment (Vos et al., 1987). Therefore, similarly to those of lead and mercury, cadmium levels are often monitored in the food supply. The principal long-term effects of low-level exposure to cadmium are chronic obstructive pulmonary disease and emphysema and chronic renal tubular disease. The kidney, particularly the cortex, was identified as the target organ relative to low levels of exposure to cadmium. Thus, long-term chronic ingestion of cadmium often results in serious renal damage, as well as bone disease leading to brittleness and even collapse of the skeleton (Frieberg et al., 1974). Cadmium toxicity is the prime cause of itai-itai disease observed in certain population segments of Japan (Asami, 1984). It is attributed to the consumption of rice heavily contaminated with cadmium by environmental pollution; 0.1 to 1 mg/day was ingested for a period of possibly more than 12 years. The painful disease that developed was characterized by skeletal deformation, reduced body weight, and multiple fractures. Vitamin D deficiency appeared to be a predisposing factor in this case. Cadmium has been considered as a category 1 (human) carcinogen primarily on the basis of its induction of pulmonary tumors (IARC, 1994). Abnormally high levels of cadmium in the diet also enhance the rates of several cancers in humans (Browning, 1969). In human and animal nutrition, cadmium toxicity is counteracted by the presence of cobalt, selenium, and zinc. Their protective effects are attributed to the induction of metallothionein. Chelating agent therapy is detrimental to the host as it increases cadmium uptake by the kidney, which in turn leads to nephrotoxicity in spite of increased urinary excretion of cadmium. The only effective treatment for cadmium-induced toxicity is to eliminate the

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source of exposure to this element (Nogawa and Kido, 1996). Exposure to dietary cadmium remains a health risk. Thus, more countries should carry out diet intake studies and appropriate measures should be taken to minimize its occurrence in the diet, especially in animal organs, shellfish, vegetables, fruits, and grains from areas of known cadmium contamination. Well-designed dietary studies should be conducted in locations of potential cadmium contaminations, such as those near mining and metal extraction operations, phosphate fertilizer plants, high-cadmium-bearing strata in the soil, municipal sludgedeposition areas, and shellfish growing areas affected by improperly treated industrial or municipal discharges (UNEP, 1992). Mercury Its persistent presence in the environment, bioaccumulation and transport in the aquatic chain, and levels in a variety of foods make mercury among the most dangerous of all metals in the human food chain. The widespread use of mercury and its derivatives in industry and agriculture (now banned in most countries) has resulted in serious environmental pollution. Today, mercury is still commonly used in thermometers, batteries, and fluorescent lights and in industrial processes such as production of paints and fungicides (Goyer, 1995). Mercury occurs in three different forms: elemental mercury, inorganic salts (monovalent and divalent, mercuric mercury), and organic mercury compounds such as phenylmercuric salts and alkylmercuric compounds. The latter have been used as fungicides and herbicides. Each has its own toxicity and health effects. Its organic form is easily absorbed after ingestion and has a half-life varying from 60 to 120 days in humans, but up to 20 years in fish, in which it is the predominant form (Al-Shahristani and Shihab, 1974). Organic mercury compounds easily pass across biomembranes and are lipophilic. Normal human diets generally contain less than 50 µg mercury/kg food (Bouquiaux, 1974). In the absence of gross contamination of soil or irrigation water, some of the commonly found mercury values for various foods and food products are summarized in Table 16.5. Seafood is a prime source of mercury in the human diet (Table 16.6). The reason for the greater toxicological significance of mercury in seafood is that, as a rule, it is in the methyl form. Together with ethyl mercury, this is the most toxic form of the metal. Some species of fish are able to concentrate methyl mercury by a factor of 2000 in the muscles and 9000 in the kidneys (Hannerz, 1968).

Table 16.5 Levels of Mercury Residues in Food in Several Countries Foods Cereal (grains) Bread and flour Meatsa Fishb Dairy products Milk Cheese Butter Fruits Vegetables Fresh Canned Eggs White Yolk Beer

United States, µg/kg

United Kingdom, µg/kg

Japan, µg/kg

2–25 20 1–150 0–60

5

12–48

10–40 70–80

310–360 35–540

8 80 140 4–30

10 170 10 10–40

3–7 — — 18

0–20 2–7

10–25 20c

30–60 0

10 62 4

NDd

80–125 330–670

a

Includes beef, pork, beef liver, canned meats, and sausages. Includes canned salmon, shellfish, and whitefish. c Canned peas. d Not detectable. Source: Compiled from Concon (1988) and Janssen (1997). b

JECFA established a PTWI of 300 µg for the general population, of which no more than 200 µg should be present at methyl mercury; these amounts are equivalent to 5 and 3 µg/kg body weight, respectively. The United States has set limit values for seafood only, since nearly all of the mercury intake in the American diet is from seafoods. Mercury is a cumulative poison and is stored mainly in the liver and kidney. The level of accumulation depends on the type of organism and the chemical form of mercury. Mercury in its pure metallic form is poorly absorbed, readily excreted from the body, and thus unlikely to cause poisoning. In contrast, the inorganic and organic compounds of mercury are highly toxic to humans. Methyl mercury has been listed as one of the six most dangerous chemicals in the environment (Bennet, 1984). It is efficiently absorbed from the food in the intestine, rapidly enters the bloodstream, and is bound to plasma proteins. Methyl mercury also accumulates in the human brain. It is thus neurotoxic to both the adult and the fetus (Berlin et al. 1963). Human response data are available from epidemics of methyl mercury poisoning in Japan and Iraq. The first epidemic (Minamata disease) was caused by consumption of fish from water that was heavily contaminated by industrial wastewater (Frieberg and Vostal, 1972). In Iraq, the

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poisoning appeared to result from the ingestion of wheat treated with a mercurial fungicide (Clarkson et al., 1976). The clinical signs of methyl mercury poisoning generally manifest in sensory disturbances in the limbs, the tongue, and around the lips; irreversible damage to the central nervous system resulting in ataxia, tremor, slurred speech; tunnel vision blindness; loss of hearing; and finally death (Reilly, 1991; Deshpande and Salunkhe, 1995). Selenium appears to counteract both inorganic and organic mercury poisoning in several animal species by readily complexing with methyl mercury (Stoewsand et al., 1974; NRC, 1989). The treatment of mercury poisoning should aim at lowering its concentration in the critical organ(s) or site of injury. In severe cases, hemodialysis is the first step, followed by infusion of chelating agents, such as cysteine or penicillamine (Ahmed, 1999). In less severe cases of inorganic mercury poisoning, chelation with BAL may be effective (Goyer, 1995). However, chelation therapy is not very useful for alkylmercury exposure (Berlin, 1986). Arsenic Arsenic has been traditionally associated with homicide and the forensic scientist. Arsenic intoxication has been widely associated with occupational, chemical, and che-

Table 16.6 Mercury Content of Selected Species of Fish Species Catfish Cod Dogfish Garfish Haddock Halibut Mackerel Meka Milkfish Perch Pollock Purbeagle Shark Snapper Swordfish Tuna Whiting

Range, µg/kg

Typical value

50–230 20–2700 160–720 130–685 70–370 140–520 890–1540

130 190 450 550 170 340 1200 1600 640 500 230 1800 1000 380 1000 300 140

50–870 100–450 300–2100 100–2500 400–1500 10–1500 40–210

Source: Compiled from Concon (1988), UNEP/FAO/ WHO (1988), and Ahmed (1999).

motherapeutic exposures. It has no known vital functions in humans and is ubiquitous in the biosphere. The toxicity of arsenic is related to the chemical form of the element. Its inorganic compounds are the most toxic, followed by the organic arsenicals and finally arsine gas (Buck, 1978). In the past, arsenic-based herbicides, fungicides, wood preservatives of several kinds, insecticides, rodenticides, and sheep dips were in common usage. Because of their toxicity and the persistent nature of arsenic poison, their use at present has been severely restricted in many countries. Because of its wide distribution in the environment and its past uses in agriculture, arsenic is present in most human foods. With the exception of that in seafood, it is generally present in very low levels of less than 0.5 mg/kg. A Canadian survey (Dabeka et al., 1987) has reported the following levels (micrograms per kilogram [µg/kg] with ranges in the parentheses) in different food groups: cereals 8.6 (0.71–61), dairy products 2.58 (0.6–11.3), starchy vegetables 13.69 (4–81.9), other vegetables 2.60 (0.6–8.3), and meat and fish 60.1 (4–625). Cereals, starchy vegetables, and meat and seafood thus together accounted for over 65% of the daily intake of 2.6–101 µg of arsenic in the Canadian diet. The average daily intake of arsenic was reported to be 62 µg in the United States (Gartrell et al., 1985), 55 µg in New Zealand (Dick et al., 1978), 89 µg in the United

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Kingdom (FACC, 1984), 15–45 µg in Sweden (Slorach et al., 1983), and 12 µg in Belgium (Buchet et al., 1983). The FAO/WHO maximum allowable daily intake of arsenic in the human diet is restricted to 2 µg/kg body weight (CAC, 1984). Arsenic is also present in almost all potable waters with levels ranging from 0 to 0.2 mg/L. The U.S. Federal Regulations for drinking water set a maximal limit of 0.01 mg arsenic/L (Drinking Water Standards, 1962). Both tri- and pentavalent arsenic are easily absorbed from food in the GI tract. It is then rapidly transported to all organs and tissues. Arsenic is primarily accumulated in skin, nails, and hair and, to some extent, in bone and muscle. Total body levels of arsenic in humans have been estimated at 14–20 mg (Schroeder and Balassa, 1966). Arsenic is a general protoplasmic poison; its pentavalent form is less toxic than the trivalent. It binds to organic sulfhydryl groups and thus inhibits the action of several enzymes, especially those involved in cellular metabolism and respiration (Reilly, 1991). Its clinical symptoms are manifested in the dilatation and increased permeability of capillaries, especially in the intestine. Chronic arsenic poisoning generally results in loss of appetite, leading to weight loss, gastrointestinal disturbances, peripheral neuritis, conjunctivitis, hyperkeratosis, and skin melanosis. Arsenic is also a suspected carcinogen (IARC, 1987). Arsenic is believed to counteract the toxicity of an excessive intake of selenium in animal feeds (Rhian and Moxon, 1943). Dietary arsenic does not seem at the moment to pose a public health risk. However, potential problems may occur in the future with continued increased use of arsenical herbicides and defoliants. This is especially true for foods associated with cottonseed by-products; in areas where coal is used as a fuel source, and the resultant fly-ash contaminates the soil and water; with arsenic emissions into runoff waters from geothermal power plants, contaminating rivers that receive these discharges; and near gold mines. Monitoring of these sources of environmental pollution should be increased as a precaution against the potential toxic effect of this element. Selenium Selenium at a low concentration is an essential element and is an antioxidant present in essential enzymes such as glutathione peroxidase and heme oxidase and with the cofactor ubiquinone. However, its excessive intake often results in the manifestation of toxic syndromes. Selenosis of livestock has been widely reported from several parts of the world, including China, the United States, Australia, Mexico, Canada, Colombia, Israel, and Ireland (Reilly,

1991). Its toxic manifestations include embryotoxicity, teratogenicity, and mutagenicity (Harr, 1978; NRC, 1989; Schamberger, 1985). Its levels in agricultural soils primarily influence the presence of selenium in the human food chain. Its dietary intake, therefore, varies greatly with geographical region. Selenium consumption (micrograms/day) in different parts of the world was estimated to be as follows: New Zealand, 28; the United States, 132; Canada, 98–224; Japan, 88; Venezuela, 326; the United Kingdom, 60; Italy, 13; China, 4.99 mg in a selenosis region, 750 µg in a high-selenium but nonselenosis region, and 11 µg in a selenium-deficient region (Thomson and Robinson, 1980; Reilly, 1991). The Food and Nutrition Board of the U.S. National Research Council recommends a range of 50–200 µg/day, and 10–40 for infants and 20–120 for children under the age of 6. The FAO/WHO has no set PTWI for selenium. Excessive intake of selenium in the human diet results in dermatitis, dizziness, brittle nails, gastric disturbances, hair loss, and a garlic odor on the breath. The margin of safety between essential trace levels of selenium in the human diet and manifestation of its toxic symptoms appears to be quite small. Antimony Antimony is toxic and occurs widely in many foods. Its levels are restricted by food regulations in several countries. High levels of antimony in food are generally attributed to contamination from containers glazed with antimony-containing enamel in which the food is cooked or stored, and from rubber or soldered tinfoil used for packaging. Very little is known about the dietary intake of antimony. The daily intake is believed to be 0.25–1.25 mg for children in the United States (Murthy et al., 1971). The U.S. Environmental Protection Agency (EPA) has recommended a limit normally not exceeding 0.1 mg/L for antimony in drinking water. Prolonged exposure to antimony results in dermatitis, conjunctivitis, and nasal septum ulceration. Ingested antimony apparently has low inherent toxicity (Nielsen, 1986). It is primarily stored in the liver, kidney, and skin. The human adult body contains about 7–9 mg antimony, with about 25% in the bones and 25% in blood. Soluble antimony salts are more toxic than similar lead or arsenic compounds, and trivalent antimony salts are 10 times more toxic than pentavalent salts. Symptoms of antimony poisoning include colic, nausea, weakness, and collapse with slow or irregular respiration and a lowered body temperature (Reilly, 1991).

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Aluminum Aluminum is widely used in several industrial applications. Its compounds are used in the food industry as food additives; in baking powder, processed cheese, manufactured meats, and other products; in cooking and storage utensils; and in foil and takeout food containers. Aluminum is also widely used in the pharmaceutical and cosmetic industries, toothpastes, antiperspirants, and a variety of therapeutic agents and related products. Aluminum sulfate and other compounds are also used for particle sedimentation in water treatment (ALCOA, 1969). In recent years, high aluminum levels associated with short-term memory loss, dementia, parkinsonism, motor neuron disease, amyotrophic lateral sclerosis (ALS), and the brain tissue of Alzheimer’s disease patients have generated a great deal of interest in its presence in the human food chain. Intakes of aluminum in the U.S. diet were estimated to be 9 mg/day for teenage and adult females and 12–14 mg/day for teenage and adult males (Pennington, 1987). Pennington and Jones (1988) have reported the following aluminum levels (milligrams per kilogram [mg/kg]) in 234 foods sampled in FDA’s Total Diet Survey in 1984: Cow’s milk, 0.6; cheddar cheese, 0.19, American (processed) cheese, 0.411; infant formula (milk-based), 0.05 Meat: beef, 0.28; meat loaf, 1.26; bacon, 3.63; sausage, 1.82; roast chicken, 0.22 Fish: cod, 0.47; fish sticks, 51.4; canned tuna, 0.67 Fruits: apples, 0.14; cherries, 0.19; grapes, 1.81 Vegetables: broccoli, 0.98; canned beets, 0.26; carrots, 0.19 Cereals: corn grits, 0.2; oatmeal, 0.68; rice, 1.42 Cereal products: biscuits, 16.3; white bread, 2.33; rye bread, 4.07; pancakes from mix, 69.0; tortilla flour, 129.0; chocolate cake, 86.0 Others: milk chocolate, 6.84; chocolate chip cookies, 5.61 Tea from bag, 4.46 Domestic water (eastern United States), 0.12 With the exception of those in certain spices and tea leaves, natural levels of aluminum in foods tend to be quite low. In contrast, most dietary aluminum primarily is derived from the use of food additives such as sodium aluminum phosphates and aluminum silicates. These compounds are widely used as acidifying agents, emulsifiers, binders, anticaking agents, stabilizers, thickeners, bleaching agents, and texturizers. Contamination may also result from the use of aluminum utensils and cans in the food industry. Water generally is not an important source of dietary aluminum.

The chemical form of the element is an important factor controlling the absorption of aluminum in human nutrition. In one study, about 7% of ingested aluminum was absorbed by healthy young men consuming a diet containing 10–33 mg of the element over a 28-day period (Gormican and Catli, 1971). As compared to its phosphate salts, aluminum citrates appear to be readily absorbed from the gut. Vitamin D, parathyroid hormone, and iron levels appear to influence the absorption of aluminum (Reilly, 1991). In the human body, aluminum is primarily stored in liver, kidney, spleen, bone, and brain and heart tissue. Under certain conditions, aluminum is known to be toxic to plants and fish. Accumulation of aluminum in the human brain has been associated with Alzheimer’s disease (Crapper et al., 1973; Martyn et al., 1989). The use of aluminum-containing water in kidney dialysis and the ingestion of aluminum-containing phosphate binding gels by renal patients were reported to cause dialysis dementia (Platts et al., 1977). Excessive intakes of aluminum in human diets are also associated with osteomalacia and bone fractures (Boyce et al., 1982). Aluminum has also been implicated in metabolic alkalosis, parkinsonian dementia, and bowel obstruction (Pennington and Jones, 1988). Tin Tin is widely distributed in small amounts in most soils. It occurs at <1.0-mg/kg levels in all major food groups except canned vegetables (9–80 mg/kg) and fruit products (12–129 mg/kg) (Sherlock and Smart, 1984). A primary source of tin contamination is the use of lacquered cans in the canning industry. Tin in foods appears to be poorly absorbed and excreted mainly in feces (WHO, 1973). Small amounts of absorbed tin may be retained in kidney, liver, and bone. High levels of tin in food can cause acute poisoning; the fatal toxic dose for humans is 5–7 mg/kg body weight. Chronic tin poisoning is manifested in growth retardation, anemia, and histopathological changes in the liver. Tin also influences iron absorption and hemoglobin formation (Reilly, 1991). Nitrates Nitrate (NO3–), nitrite (NO2–), and nitrosamines (R2-NN=O) are chemically and toxicologically related and, therefore, are generally considered as a group with respect to their toxicological significance. However, the latter two, which are mostly formed in situ in food products, are discussed elsewhere in this book. The principal natural source of nitrates in the biosphere is microbial nitrification. Thus, this process is re-

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sponsible for the nitrate conversion of ammonia in fertilizers, used either as such or in the form of urea, derived from the decomposition of human and animal waste matter. The level of nitrate in any given situation depends on the balance between nitrification and denitrification reactions. Levels of nitrates in various foods are summarized in Table 16.7. However, what is more important is the relationship of these nitrate-containing foodstuffs to their level of consumption. Thus, potatoes would probably contribute a greater quantity of total nitrate intake than foods such as sugar beets. Heavy nitrogen fertilizer applications can cause accumulation of potentially hazardous concentrations of nitrates, thereby adversely affecting the nutritional quality of vegetables. Other factors that affect the nitrate

Table 16.7 Average Nitrate Content of Common Foodstuffs Food Total vegetables Asparagus Beets Beans, dry Beans, lima Beans, snap Broccoli Cabbage Carrots Celery Corn Cucumbers Eggplant Lettuce Melons Onions Peas Peppers, sweet Pickles Potatoes Potatoes, sweet Pumpkin/squash Spinach Sauerkraut Tomatoes Breads All fruits Juices Cured meats Milk and milk products Water

Nitrate, mg/100 g 1.3–27.6 2.1 276 1.3 5.4 25.3 78.3 63.5 11.9 234 4.5 2.4 30.2 85 43.3 13.4 2.8 12.5 5.9 11.9 5.3 41.3 186 19.1 6.2 2.2 1.0 0.2 20.8 0.05 0.071

Source: Compiled from White (1975), Concon (1988), and Deshpande and Salunkhe (1995).

content of plants include species, variety, plant part, stage of maturity, drought, high temperature, shading or cloudiness, time of day, deficiencies of certain nutrients, excessive soil nitrogen level from manure, legume residues, and plant damage from insect and weed control chemicals (Keeney, 1970; Deshpande and Salunkhe, 1995). Herbicides such as alachlor, linuron, lenacil, prometryne, and chloropropham also tend to increase the nitrate content of vegetables. The amount of nitrates in drinking water is equally important. The U.S. Public Health Service Standard is 10 mg nitrate/L. In this regard, deep-water wells pose serious health hazards. Mayo (1895) first reported the toxic effects of excessive amounts of naturally occurring nitrates in foods and feeds. In three cases of fatal poisoning in cattle, the animals showed tremors, diuresis, collapse, and cyanosis after feeding of cornstalks that were later shown to contain 25% by dry weight potassium nitrate. The corn had apparently absorbed large amounts of nitrate from the accumulated soil manure. The symptoms were also experimentally reproduced by oral dosing of cattle with doses of potassium nitrate at about 1.3 g/kg body weight. The occurrence of inorganic nitrate in vegetables has, at times, given rise to serious toxic effects, especially those resulting from methemoglobin formation. Holscher and Natzschka (1964) reported two cases of methemoglobinemia in infants who had eaten spinach puree. The spinach contained large amounts of nitrite (about 218 mg/100 g wet weight) and only small amounts of nitrate. Heavy metal toxicity generally is less of a problem in the well-developed Western countries. This is primarily due to the stricter regulatory guidelines and monitoring of industrial discharges. In developing countries, in contrast, the situation is grimmer. The widespread and frequent contamination of foods indicates that this problem will persist, unless a serious effort is made by the governments in these countries to establish regulatory actions and to monitor compliance with sufficient resources (Gzyl, 1997; Jung and Thornton, 1997; Menkes and Fawcett, 1997; Romieu et al., 1997; Ahmed, 1999).

nonradioactive counterparts with one major difference: they decompose with the liberation of ionizing radiation. Although it is a well-known fact that large amounts of radioactivity deposited in the human body can produce harm, no evidence exists of deleterious effects from the amounts of radionuclides that occur naturally in the diet and in the body. This does not mean that no such effects occur, but they have not been, and possibly cannot be, observed. It is also quite evident that there is no interference from them with the ability of the human population to develop and progress throughout history. Because of radionuclides’ very low levels in foodstuffs, contamination by them probably has no significance in terms of chemical toxicity. However, their levels in foods may be important when viewed from different toxicological perspectives for the following reasons (Concon, 1988; Gofman, 1981; Lambert and Mondon, 1999; Jones, 1992): 1.

2.

3. 4.

5.

These considerations underscore the toxicological importance of radionuclides in the human food chain. In particular, the following parameters must be established: 1.

16.3 RADIONUCLIDES 2. Radioactivity occurs naturally and results from the use of radiation in nuclear power, nuclear weapons, and medicine. Radionuclides have existed in foodstuffs from the beginning of the universe. Their existence in foods is readily accounted for by the many elements in food that have radioactive counterparts that can replace them. The radioactive elements have the same chemical properties as their

Copyright 2002 by Marcel Dekker. All Rights Reserved.

In terms of the no threshold concept, radionuclides may present carcinogenic, mutagenic, and teratogenic hazards. Several radionuclides have special, strong affinities for a specific organ or tissue, so that the relative dose to that organ or section of an organ may be several times higher than the ingested or absorbed dose. These affinities may cause radionuclides to accumulate to even higher doses with time. Except for the excretion mechanism and radioactive decay, the body possesses no detoxification mechanism for radionuclides, although other body processes or substances may afford some mitigation of their effects. Several radionuclides have long half-lives, and thus their radioactive effect may persist throughout a person’s lifetime.

3.

4. 5.

The presence, persistence, and levels of radionuclides in food and water The manner in which radionuclides enter the human food chain The factors and conditions leading to an increase in the concentration (biomagnification) of radionuclides in food The means of preventing these increases The geographical and ecological distributions of radionuclides and factors affecting them

6. 7.

The biological effects and factors mitigating or exacerbating these effects The toxicological relationships of radionuclides with other substances

Some of these factors are briefly described in the following section. 16.3.1

Basic Concepts

Radionuclides, or radioactive isotopes, are elements with unstable nuclei, which decay or disintegrate at predictable rates. The radioactive decay, or radioactivity, is manifested by the emission of radiation consisting of two general types: electromagnetic radiation, such as gamma (γ) and xrays, and particulate radiation. The latter includes alpha(α-) and beta (β–-) particles, electrons (e–), positrons (β+), and neutrons. The most common forms of the radioactive emissions are the α–, β–, e–, and γ-rays. Since the discovery of polonium and radium in the late 19th century, over 40 naturally occurring radionuclides have been identified and characterized. Most are elements of high atomic weight (atomic number >81). They fall into three distinct series, each of which begins with a very long-lived radionuclide and ends with a stable isotope of lead. These are known as the uranium series, the thorium series, and the actinium series (Table 16.8). In addition to these, there are a few naturally occurring radionuclides that are not members of these series (Table 16.9). Some of them have half-lives sufficiently long to have enabled them to exist from the time of formation of the Earth’s crust, whereas others must be produced continuously. The latter are formed by nuclear reactions between components of cosmic radiation and stable nuclei. Carbon-14 and tritium (3H) are examples of this type. The classical unit of radioactivity is the curie (Ci), which is equivalent to 3.7 × 1010 nuclear disintegrations per second. Environmental radioactivity is generally expressed in terms of the millicurie (mCi), microcurie (µCi), nanocurie (nCi), or picocurie (pCi), which are equal to 10–3, 10–6, 10–9, and 10–12 curie, respectively. In some literature, radioactivity is also expressed in the Système international (SI) unit the becquerel (Bq), where 1 Bq is one radioactive disintegration per second. As radiation interacts with matter, it deposits energy, called the absorbed dose, measured in grays (Gy), where 1 Gy is equal to 1 joule/kg. In the older literature, it is often expressed as rad (radiationabsorbed dose), equivalent to the deposition of 0.01 joule/kg of tissue. Dose for dose, the α-particles are more damaging to living tissues than β-particles or γ-rays, and, hence, for the purposes of comparing doses from different radiations, the absorbed dose is multiplied by the factor

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that takes account of how a particular radiation distributes energy in tissues. It is then called equivalent dose, measured as the sievert (Sv), where 1 Sv = 1 Gy × Wr where Wr is the radiation weighting factor for the radiation under consideration. For γ-rays and β-particles, Wr is taken as 1, and for α-particles as 20. The equivalent to the sieverts is the rem, which stands for “radiation equivalent man.” Organs in the body have different susceptibilities to radiation. To sum the effects of radiation on the overall body, the effects on different organs are weighted by a factor Wt, and the resultant dose is known as the effective dose, measured in sieverts. Finally, the kinetics of the turnover of a radionuclide taken into the body depends on the physical half-life (T1/2), the time taken for the radioactivity to halve by decay, and the biological half-life (Tbiol). The latter is governed by the behavior of the radionuclide once in the body. It usually follows the same pattern as for stable chemical analogs (Lambert and Mondon, 1999). The overall residence time in the body is described by the effective half-life (Teff), where 1/Teff = [1/T1/2 + 1/Tbiol] The biological effects of radiation are regularly reviewed by expert groups, in particular the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR, 1993) and the Biological Effects of Ionizing Radiation Committee (BEIR, 1989) in the United States. Recommendations on radiation protection standards for both workers and the public are made by the International Commission on Radiological Protection (ICRP), and most countries use ICRP publications as the basis for their own national guidelines. In its 1991 publication on dose limits, ICRP recommended an annual dose limit to the public of 1 mSv excluding medical uses and doses from natural sources (ICRP, 1991). Current annual average doses to adults from all sources are given in Table 16.10. 16.3.2

Sources of Exposure

Humans are exposed to radiation through a number of pathways: direct exposure, inhalation, and ingestion. A breakdown of some background radiation levels is shown in Table 16.11. The largest natural portion is from inhaled radon decay products. Only about 400 mSv per year is found in the body, and this level must be derived from food and water. Thus, food constitutes roughly 10% of the average annual dose (Moeller, 1988; Silini, 1988; Stroube et al., 1985; Jones, 1992). Under normal conditions, contem-

Table 16.8 Naturally Occurring Radionuclides of the Uranium, Thorium, and Actinium Series

Element Uranium series Uranium Thorium Protactinium Uranium Thorium Radium Radon Polonium Lead Bismuth Polonium Thallium Lead Bismuth Polonium Lead Thorium series Thorium Radium Actinium Thorium Radium Radon Polonium Lead Bismuth Polonium Thallium Lead Actinium series Uranium Thorium Protactinium Actinium Thorium Francium Radium Radon Polonium Lead Astatine Bismuth Polonium Thallium

Symbol

Common name

Major type of radiationa

238

U Th 234 Pa 234 U 230 Th 226 Ra 222 Rn 218 Po 214 Pb 214 Bi 214 Po 210 TI 210 Pb 210 Bi 210 Po 206 Pb

Uranium I Uranium X1 Uranium X2 Uranium II Ionium Radium Radon Radium A Radium B Radium C Radium C’ Radium C” Radium D Radium E Radium F Radium G

α, γ, e– β–, γ, e– β–, γ α, γ, e– α, γ, e– α, γ, e– α, γ α β–, γ, e– α, β–, γ α, γ β–, γ, e– α, β–, γ, e– α, β–, γ α, γ None

4.5 × 109 yr 24.1 days 1.17 min 2.5 × 105 yr 8.0 × 104 yr 1.6 × 103 yr 3.82 days 3.05 min 26.8 min 19.7 min 1.6 × 10–, sec 1.32 min 20.4 yr 5.0 days 138 days Stable

232Th 228Ra 228Ac 228Th 224Ra 220Rn 216Po 212Pb 212Bi 212Po 208TI 209Pb

Thorium Mesothorium I Mesothorium II Radiothorium Thorium X Thoron Thorium A Thorium B Thorium C Thorium C Thorium C Thorium D

α, γ, e– β–, e– β–, γ, e– α, γ, e– α, γ, e– α, γ α β–, γ, e– α, β–, γ, e– α β–, γ, e– None

1.41 × 1010 yr 6.7 yr 6.13 yr 1.90 yr 3.64 days 55.3 sec 0.145 sec 10.6 hr 60.6 min 3 × 10–7 sec 3.1 min Stable

235U 231Th 231Pa 227Ac 227Th 223Fr 223Ra 219Rn 215Po 211Pb 219At 211Bi 211Po 207TI

Actinouranium Uranium Y Protactinium Actinium Radioactinium Actinium K Actinium X Actinon Actinium A Actinium B Astatine Actinium C Actinium C’ Actinium C”

α, γ β–, γ, e– α, γ, e– α, β–, γ, e– α, γ, e– β–, γ, e– α, γ, e– α, γ, e– α β–, γ α α, γ, e– α, γ β–, γ

7.1 × 108 yr 255 hr 3.25 × 104 yr 21.6 yr 18.2 days 22 min 11.44 days 4.0 sec 1.78 × 10–3 sec 36.1 min 0.9 min 2.16 min 0.5 sec 4.79 min

234

Half-life

a α, alpha-particle emission, β–, beta-particle emission; γ, gamma-ray emission; e–, electron emission. Source: Compiled from Lederer et al. (1967) and Comar and Rust (1973).

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Table 16.9 Some Naturally Occurring Nonseries Radioactive Nuclides Element Formed with Earth’s crust Potassium Vanadium Rubidium Indium Lanthanum Samarium Lutecium Produced continuously Tritium Beryllium Beryllium Carbon Sodium Silicon Phosphorus Phosphorus Sulfur

Major type of radiationa

Half-life

K V 87 Rb 115 In 138 La 147 Sm 176 Lu

β+, β–, γ β–, γ, x β– β– β–, γ, x α β–, e–, γ

1.3 × 109 yr 6 × 1014 yr 4.8 × 1010 yr 6 × 1014 yr 1.1 × 1011 yr 1.05 × 1011 yr 2.2 × 1011 yr

3

β– γ β– β– β+, γ β– β– β– β–

12.3 yr 54 days 2.5 × 106 yr 5730 yr 2.6 yr 650 yr 14.3 days 24.4 days 88 days

Symbol 40 50

H Be 10 Be 14 C 22 Na 32 Si 32 P 33 P 35 S 7

α, alpha-particle emission; β–, beta-particle (negatron) emission; β+, positron; γ, gamma ray emission; e–, electron emission; x, x-rays. Source: Compiled from Lederer et al. (1967) and Comar and Rust (1973).

a

porary waste discharges to the atmosphere and to rivers and the sea from the nuclear power industry do not now represent a serious threat in terms of the contamination of food (Lambert and Mondon, 1999). This has not, however, always been so, and, in addition, accidents have added substantially to both local and continental pollution. Together with radioactive fallout from atmospheric nuclear weapons tests (now banned), these sources have resulted in significant food contamination in the past. Consequently, the need for control of the contamination of food has been recognized and standards have been developed internationally. Natural radionuclides enter the food chain from the soil. The level in the plant depends first on the amount in the soil and second on how well a particular plant takes up the elements through the root system. The chemical properties and speciation of the radionuclide as well as the nature of the soil, notably its pH and organic content, usually determine the uptake of a radionuclide by a plant. Thus, the soil is a prime source of geographic variability. In general, values are low for milk products, fruits, and vegetables but high for cereals and nuts (Comar and Rust, 1973). The natural radioactivity in various types of water is highly variable, depending upon origin and treatment. Certain natural springs in areas of high soil levels of uranium

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and thorium have high levels of radioactivity originating from these elements. The levels of natural radioactivity in seawater in terms of picocuries per liter (pCi/L) are approximately as follows: 40K, 300; 87Rb, 3; uranium series, 3; and 3H, 0.6–3 (Comar and Rust, 1973). The movement of radionuclides through the food chain is a complex subject, which has been reviewed elsewhere (Carter, 1988; Eisenbud and Gesell, 1997; Simmonds et al., 1995). Since many radionuclides are isotopes of biologically essential stable elements, they behave according to biochemical expectations. Tritium (3H) and 14C, for example, follow the same metabolic pathways as their stable analogs. Others behave similarly to essential elements that are in the same chemical group, radiostrontium like calcium and radiocessium potassium. Some radionuclides, including uranium, technetium, and the actinides, that occur in plants and animals largely because of pollution from human activity have no obvious chemical analogs. Data on their environmental behavior have, therefore, been derived empirically. Radionuclides found naturally in food include 226Ra, 228 Ra, 14C, 87Rb, 210Po, 228Th, and 40K. 40K is the most prevalent, contributing about 0.01% of total body potassium and providing 25% of the background radiation received by cells (Miller and Miller, 1986; Jones, 1992).

Table 16.10 Current Annual Average Millisievert Doses to Adults

Table 16.11

Involuntary Sources of Radiation

Source Dose, mSv/yr

Source

(a) Doses from all sources Natural sources Cosmic rays 380 Cosmogenic radionuclides 10 Terrestrial radiation External exposure 460 229 Internal exposurea Radon + daughters 1,270 Total 2,349 Artificial sources Medical uses 1,000–2,000 Weapons testing 5 1–200 Nuclear powerb Occupational doses 1,000–10,000 (b) Doses from ingestion of naturally occurring radionuclides Radionuclide 14 C 12 Uranium isotopes 0.28 Thorium isotopes 0.79 Radium isotopes 7.7 210 Pb/210Po 43 40 K 165 Total (rounded) 229 a

Excludes radon; data for individual isotopes presented in part (b). b Most highly exposed people living near nuclear installations. Source: From UNSCEAR (1993) and Lambert and Mondon (1999).

Most foods contain 1–10 µCi/g 40K. For example, rice contains a very small amount, less than 1 µCi/g; coffee and tea contain 35 µCi/g. 16.3.3

Physiological Effects

The body in two ways may absorb radiation. In the first, the radiation source is outside the body, as in an x-ray. This is called external radiation. The second, internal radiation, occurs when the radiation occurs within the body, as when radionuclides are either inhaled or ingested. From a food toxicology viewpoint, the latter internal emitters are of concern. The first effects are on the gut. Fortunately, most of the radionuclides are insoluble; they simply pass through the GI tract, causing minor irradiation to the stomach and the intestine during their short transit. Obviously, the shorter the transit time before excretion, the less the time available in which to inflict any cell damage.

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Radon Natural radiation Inside body Cosmic radiation Rocks and soil (other than radon) Medical x-rays Nuclear medicine Consumer products Nuclear power production Miscellaneous

Total, % 54 11 8 8 11 4 3 0.1 <1

Source: Compiled from Living with Radiation (1989) and ACSH (1988).

Low doses of ionizing radiation have often been linked to changes in reproductive physiological characteristics and to carcinogenesis (Concon, 1988). However, sporadic low doses of radiation can produce only transient effects. Of the various types of radiation, the high-energy α-emitter can cause great damage because of its high ionizing capability. Fortunately, its large size prevents deep penetration into tissues. Radionuclides that are the most destructive are those that can penetrate the soft tissue and become part of active metabolism. 137Cs is particularly notorious, since its chemical similarity to potassium means that it is rapidly adsorbed by the bloodstream and can be distributed to all cells of the body. Its half-life is 27 years. 14 C can also irradiate the whole body since all organic molecules contain carbon. It is a soft β-emitter with a halflife of 5600 years. Other radionuclides that can cause physiological damage include 90St and 131I. The toxicity of 90St has been studied extensively in mice, rats, dogs, and pigs. Since it is an analog of calcium, it is readily absorbed either from the GI tract or from the lung and is deposited in the bone. The actual amount absorbed by the gut is dependent on the body’s trace mineral status. Adequate calcium and phosphate intakes substantially decrease its absorption (Concon, 1988, Jones, 1992). A single brief oral intake of 90St results in a high incidence of cancers of bone and leukemias. Age at exposure to the ionizing radiation determines the susceptibility to leukemias. Children below age 10 are at greatest risk. The Federal Radiation Council has stated that 90St should not exceed 1500 mrem above background levels. The same recommendation applies to 131I (Jones, 1992). Radioiodine is produced in abundance in nuclear reactor operation and during nuclear weapon fallout. High levels of this radionuclide result in the almost complete

destruction of the thyroid, with an attendant decrease in thyroid hormone production. Levels of 131I that damage the thyroid but leave the tissue capable of proliferation lead to hyperplastic cancers. Children appear to be twice as susceptible as adults are. As a preventive measure, where exposures are greater than 10 rad, potassium iodide should be administered to minimize the dose to the thyroid (Upton and Linsalata, 1988). Since internal irradiation by radionuclides ingested in food and water is only a very small part of the average total dose received by most tissues, the carcinogenic effect attributed to such irradiation is indeed small. It is estimated that, on average, 0.3% of fatal cancers can be attributed to this cause (Upton and Linsalata, 1988; Jones, 1992). However, in areas where the natural background level of radiation is high, as in certain parts of India, China, and Brazil, internal radiation doses are higher. Since a dose relationship appears to exist between level of radiation and cancer risk, in areas where the background radiation is high the cancer rates attributable to internal radiation are also likely to be relatively high. Overall, the radioactive contamination of foods needs to be monitored along lines similar to those for other chemical pollutants.

16.4 FOOD PACKAGING CONTAMINANTS Protecting foods against infestation and general contamination as well as the ingress of light, moisture, and oxygen, which can induce chemical degradation of food constituents, is a major challenge to the food industry. Food packaging provides a means of responding to this challenge, and its role in the preservation, distribution, and sale of foods is of paramount importance. Food packaging encompasses a broad spectrum of food contact articles— from the bottles, jars, tubs, cans, trays, cartons, bags, boxes, tubes, closures, and film wraps used at the retail level to the drums, barrels, pails, crates, tote boxes, baskets, bags, holding tanks, and transport vehicles used for the commercial handling of bulk foods. The materials used to fabricate these articles range from wood, plant fibers, and glass, which have been used for centuries, to those of more modern origin such as paperboard, steel, tinplate, aluminum, and the myriad plastics that have been developed (Sacharow and Griffin, 1980; Kirkpatrick et al., 1989). Packaging materials are generally in intimate contact with the foods they protect, often for extended periods and in some instances at elevated temperatures. Since such conditions are conducive to the migration of constituents of the packaging materials to foods, many countries have

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enacted regulations or established codes of practice to control food-packaging materials to ensure that migrating constituents that enter the food supply do not pose a health risk to consumers (Briston and Katan, 1974; Concon, 1988). Although none of the materials used in food packaging is truly inert, the move away from glass and metal toward less inert materials such as plastics has increased the need for regulatory control. The degree of regulatory control over food packaging materials varies around the world. In the United States, the Code of Federal Regulations (21 CFR, parts 175–178) lists eight different categories of indirect food additives: (a) adhesive components, (b) coating components (e.g., paraffin, polyvinyl fluoride), (c) paper and paperboard components, (d) polymeric compounds for use as basic components of materials for single or repeated contacts to food (e.g., copolymers of vinyl chloride with other monomers), (e) components of articles intended for repeated use (e.g., rubber articles and filters), (f) antimicrobials (e.g., iodine and sodium lauryl sulfate), (g) antioxidants and stabilizers for use in packaging and other contact surfaces, and (h) various adjuvants and production aids (e.g., lubricants and fatty alcohols) (FDA, 1983). In the following section, properties and toxicological aspects of some of the more commonly used food packaging materials are described. 16.4.1

Types of Packaging Materials

Glass Glass has been used for several years and has raised very few toxicological concerns. The only major concern is the leaching of lead from high-quality crystal glass, which may contain up to 30% lead oxide (Barlow, 1999). The lead content of wines and spirits stored in lead crystal decanters increases over time and has been shown to reach levels of 1–2 mg/L after 3–4 months storage or, exceptionally, up to 21 mg/L after several years of storage (Graziano and Blum, 1991; Falcone, 1991). Ceramics Like glass, ceramic materials have really only caused problems in the leaching of heavy metals from enamel and pottery glazes, particularly when in contact with acidic beverages such as fruit juices and cider (Lead poisoning, 1988). Human lead toxicity has resulted from just a few months’ exposure to lead glazed ceramic ware (Zuckerman, 1991). Although cadmium from ceramic glazes has not been identified as a definite cause of toxicity, damage to the kidney from ingestion over a period of years could

occur (WHO, 1989b). In the European Union (EU), the amounts of lead and cadmium permitted to transfer from ceramic articles are controlled by legislation (EEC, 1984).

chemicals, solvents, and fuel oils (Franz et al., 1994; Feron et al., 1994). Plastics

Tin Cans Food packed in tin cans with lead-soldered seams are a source of a number of metals, including lead, chromium, tin, and cadmium (Jorhem and Slorach, 1987; Meah et al., 1991; Sherlock and Smart, 1984; Barlow, 1999). Technological advances such as the introduction of cans without side seams (two-piece cans) and cans with electrowelded side seams have been very beneficial in reducing the intake of lead from this source. Lacquering of the inside of cans has similarly greatly reduced contamination of foods by tin; in the past canned foods were the major contributor to tin intake from the diet. Textiles Few direct problems have been encountered with the use of textiles, such as jute sacks for the packing of rice, beans, sugar, and flour. Mineral hydrocarbons may be used to soften sacking fibers and can leave residues on the food, and these are under toxicological scrutiny at present (Barlow, 1999). Of more concern with the use of textiles is the occasional contamination of the food when carried in bulk by other chemicals leaking from adjacent surfaces. Aluminum The leaching of aluminum from cooking vessels used to prepare high-acid foods such as rhubarb has been known for a long time (Poe and Leberman, 1949). When substantial leaching into acid foods occurs, it may form the highest contribution to the daily intake of aluminum apart from drinking water (Liukkonen-Lilja and Piepponen, 1992). In general, aluminum intakes should not regularly exceed the FAO/WHO provisional tolerable weekly Intake of 7 mg/kg body weight (WHO, 1989b). However, a study showing an association between Alzheimer’s disease and aluminum levels in drinking water (Martyn et al., 1989) raised concerns about all sources of aluminum. Recycled Materials The recycling of relatively inert materials, such as glass and cans, has presented few problems compared with potential difficulties with the recycling of paper, board, and plastics for food use. Nevertheless, these issues are now being addressed with research aimed at assessing worstcase situations in which plastic materials may have been contaminated, prior to recycling, by consumers’ using them to store other liquids, such as pesticides, household

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Unlike the other packaging materials described, plastics, in contrast, although offering many advantages as a new range of packaging materials, involve contact between food and a whole new range of chemical components not previously used in the food industry and for which no previous experience was available. Migration of additives used in their manufacture causes the greatest concern relating to food safety issues. Since their share of the food packaging market worldwide continues to increase, various concerns associated with their use are described at greater length. The principal base polymers, primarily derived from the petroleum industry, used in food packaging applications are listed in Table 16.12. Well-known chemicals used in the production of polymers are vinyl chloride and styrene. The former is the monomer of polyvinyl chloride; styrene is used in the manufacturing of a number of plastics. In addition to the base polymer, two other major

Table 16.12 Materials

Examples of Polymers Used in Food-Packaging

Polyethylene (C2-8 1-alkene comonomers) Polypropylene (ethylene comonomer) Ethylene-vinyl acetate copolymers Ethylene-vinyl alcohol copolymers Ethylene-methylacrylate copolymers Ionomers (ethylene, vinyl acetate, isobutylacrylate, methacrylic acid salt copolymers) Acrlyonitrile copolymers (styrene, butadiene, methylmethacrylate comonomers) Polystyrene (butadiene, maleic anhydride comonomers) Poly(vinyl acetate) Poly(vinyl chloride) (ethylene, propylene, vinyl acetate comonomers) Poly(vinylidene chloride) (vinyl chloride, acrylonitrile, methacrylate comonomers) Polyisobutylene Poly(p-methyl styrene) Polyamides (Nylon 6:6, 6:10; Nylons 6, 11, and 12) Poly(ethylene terephthalate) Poly(ether sulfone) Polysulfone Polycarbonate Polyurethanes Polyesters (unsaturated) Polyacetal Source: Compiled from FDA (1987) and British Plastics Federation (1980).

sources of migrants from plastics are processing aids and end-service additives. Some examples and levels of use in food packaging plastics are presented in Table 16.13. These are added either deliberately during manufacturing and processing or unavoidably as residues from polymerization reactions. 16.4.2

Source of Contamination

Packaging contaminants from plastics primarily arise from two sources: 1.

2.

Polymerization residues, including monomers, oligomers (with a molecular weight of up to 200), catalysts (mainly metallic salts and organic peroxides), solvents, emulsifiers and wetting agents, raw material impurities, plant contaminants, inhibitors, decomposition and side reaction products Processing and end-service aids listed in Table 16.13, including antioxidants, antiblocking

Table 16.13

agents, antistatic agents, heat and light stabilizers, plasticizers, lubricants and slip agents, pigments, fillers, mold release agents, and fungicides The more volatile gaseous monomers, e.g., ethylene, propylene, and vinyl chloride, usually decrease in concentration with time, but very low levels may persist in the finished product almost indefinitely (Crompton, 1979; Crosby, 1981; Deshpande and Salunkhe, 1995). Styrene and acrylonitrile residues are generally the most difficult to remove. Since compounds of the first group are present inadvertently, not much can be done to remove them. However, the efforts made by the industry to reduce vinyl chloride monomer levels in particular illustrate the effects of optimal manufacturing processes on the purity of the final product. In contrast, chemicals added deliberately during formulation to alter the processing, mechanical, or other properties of the polymer are likely to be present in greater amounts than polymerization residues and should be sub-

Examples of Processing and Service Aids Used in Food-Packaging Materials

Technical function Antioxidant Stabilizer

Plasticizer

Lubricant Processing agent Melt fracture eliminator Slip agent Antistatic agent Blowing agent Antiblock agent Impact modifier Clarifying agent Light stabilizer

Coupling agent Filler, extender Reinforcing agents Colorant

Example Tetrakis[methylene (3,5-di-tert-butyl-4-hydroxyhydrocinnamate)]methane Tris(2,4-di- tert-butylphenyl) phosphite Di(n-octyl)tin S,S′-bis(isooctylmercaptoacetate) Epoxidized soybean oil Stearoylbenzoylmethane Cuprous iodide Di(2-ethylhexyl) phthalate Di(2-ethylhexyl) adipate Acetyltributyl citrate N,N′-Ethylenebisstearamide Pentaerythritol adipate-stearate Styrene/butadiene/methacrylate copolymer Vinylidene fluoride-hexafluoropropylene copolymer Fatty acid amides (erucamide, oleamide) N,N′-Bis (2-hydroxyethyl)alkyl-C14–18-amine Azodicarbonamide Silica, talc Butadiene/styrene/methacrylate copolymers Dibenzylidene sorbitol 2-Hydroxy-4-n-octoxybenzophenone Dimethylsuccinate-(4-hydroxy-2,2,6,6-tetra-methyl-1-piperidyl)-ethanol polycondensate 3-(triethoxylsilyl)propylamine Calcium carbonate, clay, talc Glass, fiber, mica, calcium silicate Titanium dioxide, ferric oxide, carbon black, ultramarine blue, phthalocyanine blue

Source: Compiled from FDA (1987) and British Plastics Federation (1980).

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Use level, wt%, polymer 0.25 (Polystyrene) 0.2 (Polyolefins) 1.5 (PVC) 6 (PVC) 0.5 (PVC) 0.01 (Nylon 6,6) 40 (PVC) 20 (PVC) 5 (PVDC) 1 (PVC) 1 (PVC) 2 (PVC) 0.1 (Polyethylene) 0.2 (Polyolefins) 0.15 (Polyolefins) 0.15 (Polyethylene) 0.2 (Polyethylene) 10 (PVC) 0.25 (Polyolefins) 0.5 (Polyolefins) 0.25 (Polyolefins) 0.5 (Nylon 6,6) >5 (Various polymers) >5 (Various polymers) 0.1–5 (Various polymers)

ject to strict quality control. Additives in the second group listed in Table 16.13 are normally restricted to compounds appearing on an approved list for food contact use. Most legislative authorities now require extraction and migration tests on the finished product for the protection of the consumer. 16.4.3

Migration and Assessment of Dietary Exposure

Risk assessment in the food packaging area involves two key pieces of knowledge: the hazard or inherent toxicity of the substance and the known or predicted level of human exposure. In common with that in other types of chemicals found in food, the exposure side of the equation in food packaging risk assessment contains a number of uncertainties. Although the initial composition of any packaging material may be known, the behavior of its constituent substances once in contact with food will vary. The extent of migration of a substance depends on several factors (Figge, 1988; Barlow, 1999), including the following: 1. 2. 3. 4.

5. 6.

The concentration of the residue or contaminant in the material The degree to which it is bound or mobile within the matrix of the material The thickness of the packaging material The nature of the food with which the material is in contact (dry, aqueous, fatty, acidic, alcoholic, etc.) The solubility of the substance in the food The duration and temperature of the contact

Migration of chemicals into foods can have two adverse consequences: they can impart undesirable flavors and/or odors to the food, and they may be potentially toxic. The former situation is usually detected by the manufacturer and corrective action taken. The plastic monomer styrene, for example, can be detected organoleptically by a substantial proportion of the population at levels lower than its current regulatory specific migration limit (Barlow, 1999). However, almost all national and international regulatory efforts are concentrated on its potential toxicity. Approaches for estimating dietary exposure of consumers to chemical substances have been described in a number of publications (WHO, 1985; Bunyan, 1985; Gunner and Kirkpatrick, 1979). Data on migrant levels in foods can be obtained either directly by analysis of foods or indirectly by use of food stimulants. In this regard, it is necessary to ascertain (a) the soundness of the analytical methodology so that chemical identity of the migrant is

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confirmed, interferences and artifacts are excluded, etc., and (b) the adequacy of detection limits and sensitivity of the method to allow a meaningful estimate of migrant intake to be determined. The latter consideration is important to the determination of actual residues in food of toxicologically undesirable substances such as acrylonitrile and vinyl chloride. The use of food simulants to determine levels of potential migrants from food packaging materials has found widespread application, particularly in the context of regulatory preclearance assessment of such materials (Kirkpatrick et al., 1989; Barlow, 1999). This situation has arisen largely because of practical problems in analyzing actual foods for trace migrant levels, including analytical sensitivity limitations, interfering food constituents, and food instability problems. This indirect approach involves exposing test packaging material substrates containing the potential migrant to certain prescribed food simulants under stipulated surface-to-volume exposure ratios and under time/temperature test conditions chosen to reflect the intended end use and then analyzing the simulants for migrant levels. The food simulants that are usually accepted by regulatory agencies as surrogates for aqueous, acidic, alcoholic, and fatty foods are distilled water, acetic acid solutions, ethanol solutions, and rectified olive oil, respectively (FDA, 1976; EEC 1982). Correction factors are applied to the results in the case of fatty food simulants since it is recognized that they have a greater extraction capacity than fatty foods themselves. In both the United States and the European Union, other test conditions, such as duration of contact test time and temperature, are also specified, depending on the likely contact time and contact temperature in actual use. The information obtained from migration tests may then be used to estimate dietary exposure. In the United States, the FDA estimates the fraction of the daily diet that is in contact with the different packaging materials to arrive at consumption factors. Estimated daily intakes (EDIs) are then calculated by multiplying migration figures from relevant simulants with the consumption factors to give a concentration in the total diet, with a figure of 3000 g/day used for total daily food and beverage intake (Schwartz, 1988). It has been questioned whether it is necessary for food packaging substances to be tested to this degree. However, consideration of potential intakes of some substances illustrates the necessity for toxicological testing. In general terms, a considerable number of substances migrate into food in amounts in excess of 5 mg/kg food. This is particularly true of fat-soluble additives in plastics. Data from UK surveys carried out in 1986 and 1988 showed

that migration of some plasticizers into wrapped fatty foods such as cheese, meats, cakes, sandwiches, and confectionery readily occurs at levels in excess of 5 mg/kg food (MAFF, 1987, 1990). The plasticizers involved and the EDIs are shown in Table 16.14.

ity, teratogenicity, and allergenicity. These effects are more relevant in terms of the levels in which these compounds may be present in foodstuffs. The toxicological aspects of several of these chemicals are briefly described in the following sections.

16.4.4

Vinyl Chloride

Toxicological Aspects

The safety of substances used in packaging materials usually must be judged from animal toxicology data, because human data are rarely available. For the majority of industrial chemicals in manufacture, acute toxicity, irritation, and sensitization data are available, but these are not useful for risk assessment of orally ingested chemicals. Given the very large numbers of substances already in use, regulatory systems have evolved in the European Union (for plastics) and in the United States in which the amount of toxicology testing required is related to exposure. The greater the likely exposure, the more testing is required (Heckman, 1994; EEC, 1977, 1992). In the United States, it is related to the EDI; in the European Union, the Scientific Committee for Food (SCF) has set testing requirements that depend on the level of migration in the relevant simulants. These requirements are summarized in Table 16.15. The core set of tests, which should generally be sufficient to identify any main targets of toxicity, are listed in Table 16.16. Aside from those additives that are also used for other purposes (e.g., pesticides, food preservatives, and solvents for paints and varnishes), little is known of the toxicological characteristics of the majority of these products, especially in relation to carcinogenicity, mutagenic-

Table 16.14 Estimated Maximal Daily Intakes of Plasticizers from United Kingdom Surveys

Vinyl chloride polymers (PVCs) in various forms have been used worldwide for several years in the manufacture of food-contact articles. Plasticized flexible PVC is used for fabricating food-wrap film and beverage bottle cap liners. Semirigid PVC sheet is used to fabricate processed meat blister packs and portion packs for jams and jellies, and rigid PVC bottles are used for packaging liquor, wine, vinegar, vegetable oils, and mineral water (Codex Committee on Food Additives, 1984; Kirkpatrick et al., 1989; Deshpande and Salunkhe, 1995). IARC (1973) has reviewed the relevant biological data and epidemiological studies on humans on the safety of vinyl chloride. Rats, mice, and hamsters have all been exposed to varying levels of vinyl chloride monomer (VCM), either by inhalation or by oral administration. Maltoni and colleagues (1974) showed that inhalation of levels of VCM in the range of 50–10,000 ppm in air for 4 hours a day 5 days a week for 12 months resulted in the

Table 16.15 Toxicity Testing Requirements in the European Union and the United States Level of exposure, mg/kg food European Union None detectable LODa–0.05 ≤ 0.05–5

Extreme intake, mg/kg body weight/day Substance

1986

1988

Di-2-ethylhexyl adipate (DEHA) Dibutyl phthalate Dicyclohexyl phthalate

0.26 0.03 0.02 — 0.001 0.0003 0.0003 0.0003 —

0.14 — — 0.02 0.025 — — — 0.007

Epoxidized soybean oil acetyl tributyl citrate (ATBC) Di-isodecyl phthalate Di-2-ethylhexyl phthalate Di-isooctyl phthalate

Source: Compiled from MAFF (1987, 1990).

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>5

United States None detectable LOD–0.05 0.05–1 >1 a

Requirements Three mutagenicity tests Three mutagenicity tests Three mutagenicity tests 90-Day oral study Bioaccumulation ADMEb Reproduction Teratogenicity Lifetime toxicity/carcinogenicity None Acute toxicity test Two 90-day oral studies (one in utero exposure rodent and one nonrodent) Lifetime toxicity/carcinogenicity studies Others to be decided

LOD, limit of detection. ADME, absorption, distribution, metabolism, and excretion. Source: From Barlow (1999). b

Table 16.16 Core Tests Required by the Scientific Committee for Food, European Union, on FoodPackaging Materials 90-Day oral study Three mutagenicity tests Gene mutations in bacteria (Ames test) Chromosomal aberrations in cultured mammalian cells Gene mutations in cultured mammalian cells Absorption, distribution, metabolism, excretion (ADME) Reproduction Teratogenicity Long-term toxicity/carcinogenicity Source: From Barlow (1999).

70% acrylonitrile content based on styrene, butradiene, and methacrylate comonomers and with exceptional gasbarrier characteristics led to their use in the manufacturing of bottles for carbonated beverages and other oxygen-sensitive foods, such as vegetable oils. Acrylonitrile is considerably more toxic than the chlorinated monomers and has LD50 values of 80–90 mg/kg body weight in rats and 27 mg/kg in mice (Crosby, 1981). It converts to a mutagen after metabolic activation by liver enzymes. In animals, acrylonitrile is metabolized to cyanide, which is subsequently converted to thiocyanate and excreted in the urine. There is also some evidence of carcinogenicity in animals and possibly humans (IARC, 1973). Vinylidene Chloride

production of a number of tumors at different sites, including angiosarcomas of the liver. Oral administration of doses as low as 3.33 mg/kg body weight were also shown to be carcinogenic. VCM has also been shown to be mutagenic by the Ames test, and metabolic studies provide some evidence of alkylation of nucleic acids (Crosby, 1981). The principal products formed during metabolism are chloroethylene oxide and chloroacetaldehyde. In the IARC (1973) study, the principal toxic effects of VCM in humans included lesions of the bones in the terminal joints of the fingers and toes as well as histopathological changes in the liver and spleen. Long-term exposure gave rise to a rare form of liver cancer (angiosarcoma). VCM also has a long latent period for tumor development, requiring up to 20 years for the symptoms to appear. The IARC (1973) study concluded that VCM causes angiosarcomas of the liver as well as tumors of the brain, lung, and hematolymphopoietic system in humans. Dietary exposure to vinyl chloride is now controlled in several countries by limiting either monomer content of food-contact materials (typically <1 ppm) or monomer migration to foods to nondetectable levels (typically <0.01 ppm) or both (EEC, 1980). It appears that the overall thrust of such controls is to maintain migration of vinyl chloride to foods from packaging materials at the lowest level technologically achievable, in concert with the FAO/WHO Joint Expert Committee on Food Additives recommendation (JECFA, 1984) for this migrant.

Little toxicological information regarding the safety of vinylidene chloride (VDC) is available. The LD50 values for rats and mice are around 1500 and 200 mg/kg body weight, respectively (Crosby, 1981). VDC affects the activity of several rat liver enzymes and decreases the store of glutathione. Some tumors have been observed after prolonged exposure, but no teratogenic effects have been seen in rats or rabbits. The main pathway of excretion is via the lungs; other metabolites are discharged by the kidneys. Styrene Liebman (1975) has extensively reviewed the biological properties of styrene. The LD50 value for rats is 5 g/kg body weight. It is metabolized to styrene oxide, which is a potent mutagen in a number of test systems. Both styrene and its oxide have been shown to produce chromosomal aberrations under certain conditions. Antioxidants Little is known of plastic antioxidants except the usual ones used as food additives (e.g., butylated hydroxyanisole [BHA], butylated hydroxytoluene [BHT]). These groups consist of alkyl or alkylidenyl phenols and polyphenols, phenolic condensation products, esters, amines, hydroquinones, and organophosphorus compounds; many are well-guarded proprietary compounds or mixtures.

Acrylonitrile

Antistastic Agents

Acrylonitrile copolymers of low acrylonitrile content (<30%), such as acrylonitrile/butadiene/styrene (ABS), have been used for many years to fabricate such diverse food-contact materials as refrigerator liners, cake packaging inserts, and tubs for margarine, dairy products, and salads. Development in the 1970s of copolymers with up to

The antistatic agents consist of amino and quarternary ammonium compounds and anionics, and little is known about their toxicological properties. The polar nature of these compounds makes them readily leachable by waterbased products. They are also chemically stable even at high temperatures.

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Flame Retardants Flame retardants consist of organophosphate esters, halogenated hydrocarbons, and other halogenated organics and inorganics. The experience with halogenated compounds regarding possible carcinogenic and teratogenic hazards, and organophosphorus compounds regarding neurological and mutagenic hazards should be sufficient cause for a toxicological reevaluation of these materials. Serious reservations regarding the toxicological hazards relating to flame retardants have been expressed (Liepins and Pearce, 1976; Concon, 1988). Plasticizers The plasticizer group consists of esters of various organic acids, such as phthalates, which are the most widely employed plastic materials used in food packaging (Woggon and Koehler, 1967). Di(2-ethylhexyl) phthalate (DEHP) has been used extensively worldwide as a plasticizer in flexible PVC compounds used in a variety of industrial and consumer products. The film wrap used in packaging may have a DEHP content of up to 40% by weight. It is also used for beverage bottle-cap liners and jar-lid sealants and as tubing, conveyor belting, and liners for bulk liquid holding tanks in food plants. DEHP has been shown to be a hepatocarcinogen in rats and mice (National Toxicology Program, 1982). Toxicological data on DEHP, including results of studies of its carcinogenic potential, were reviewed by the JECFA (1984). This committee recommended that the level of DEHP in food-contact material and the extent of its migration into food should be kept at the lowest levels that are technologically possible. Di(2-ethylhexyl) adipate (DEHA) is yet another plasticizer used commonly in PVC and PVDC food-packaging materials. Its principal use is in PVC food-wrap film sold to consumers as household wrap or to supermarkets for wrapping such foods as fresh meats, poultry, fish, and cheese. PVC film wraps typically contain 15wt%–25 wt% DEHA. DEHA has been shown to be hepatocarcinogenic in the mouse but not in the rat (National Toxicology Program, 1981). Although little is known about the toxicity and carcinogenicity of other monomers and processing and end-service aids used in the production of food-grade plastics, there is clear evidence of the need for concern in the case of VCM and acrylonitrile. Further studies are required to elucidate the interactions of various monomers with body components, their biotransformation, and their metabolism in humans.

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16.4.5

Regulatory Aspects

Although the majority of packaging contaminants are nontoxic in the small amounts in which they are present, food/package and package/process compatibility are food safety issues from FDA’s viewpoint. The packaging materials are thus legally considered food additives, requiring premarket safety evaluation by the FDA. As mentioned earlier, FDA requirements include conducting extraction tests to measure the amount of migration and providing appropriate toxicological data for the migrants by the manufacturers and processors (FDA, 1976). Adjuvant use, level of migration (or the lack thereof) under various conditions of use for foods, and consumption of these foods in the diet must be evaluated with regard to safety. The potential toxicological hazards of foods contained in polymeric packages have received a great deal of attention since the passage of the 1958 amendment to the FFDCA. The determination of whether substances in food-contact materials may migrate into food and, if so, which substances and at what levels, is usually made on the basis of extraction experiments. Prior to the 1958 amendment, this determination was government’s responsibility. Since then, it has been industry’s responsibility. A petition for use of an unregulated material should include the following information (FDA, 1976): 1. 2. 3. 4. 5. 6. 7. 8. 9.

Identity, composition, properties, and specifications of the additive Amount used and the type of food contact proposed Intended physical or technical effect and efficacy data Description of practicable extraction methods for estimating migration of the additive to food Details of analytical methodology applicable to the additive and to extractive determination Safety of the food additive Proposed tolerance Amendments for later use Environmental impact analysis report

Although no specific harm has been verified for any specific packaged food, at least three cases involving the additive polychlorinated biphenyl (PCB) and the monomers acrylonitrile and vinyl chloride (VCM) have been of major economic consequence. The PCB situation was primarily the result of contamination in recycled paper; the acrylonitrile case resulted in multimillion-dollar losses in the beverage container market (FDA, 1977). The VCM problem threatened to ban the usage of PVC for food packaging, but the ability of the industry to reduce the res-

idues of VCM to the low-parts-per-billion (ppb) range has delayed the proposed ban. A more practical concern is the presence of compounds in toxicologically insignificant amounts but at levels affecting quality by taste and/or odor changes in the packaged food. These residues can arise from a variety of origins, including acrylonitrile and VCM monomers; reaction aids, such as catalysts or solvents; manufacturingrelated decompositions, such as from thermal oxidation in extrusion; coating resin components and solvents from inks and adhesives; and complex interactions, such as transesterification and hydrolysis to produce volatile or transferable products. The formation of stilbene from the antioxidant BHT is another example of a detrimental chemical reaction (Gilbert, 1985; Deshpande and Salunkhe, 1995). The development of aseptic processing and packaging systems, especially those employing chemical sterilization of the container and the use of ionizing radiation, raised a number of safety issues. With regard to the use of a chemical sterilant, FDA’s concerns were twofold: (a) to assure that the residue levels of the sterilant in food were kept within safe limits and (b) to assure that sterilization did not substantially alter the quality and nature of the migrants from the package. Consequently, the regulation for the use of hydrogen peroxide to sterilize food-contact surfaces, 21 CFR 178.1005, contains both a limit on residual hydrogen peroxide (0.1 ppm in food measured immediately after packaging) and a list of permitted materials. The effects of ionizing radiation on packaging materials are of concern to FDA whether the irradiation is done as part of the manufacturing process for the food-contact material or the food is irradiated during manufacturing for various purposes. These include cross-linking a polymer curing an adhesive or promoting adhesion prior to lamination and controlling microorganisms. Only three regulations for food-contact polymers in 21 CFR 177 specifically address irradiation: 21 CFR 177.1350 covers ethylene-vinyl acetate polymers; 21 CFR 177.1520, olefin polymers; and 21 CFR 177.1550, perfluorocarbon resins. In each case, the regulation is specific as to the technical effect to be achieved, as well as the maximal permitted intensity of the source and/or maximal allowed absorbed dose. Since the 1990s, the development of more sensitive and accurate analytical methods has greatly facilitated the control of packaging residues as well as identification of their origins. These methods also have aided in developing the theory of migration in very low concentration gradients to elucidate the mechanisms involved. Thus, a better scientific data base has been developed to allow a more

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rational approach to packaging design for optimal retention of food quality in packaged foods. Considerable improvements in design effectiveness have also arisen from the introduction of new materials and processes for food packaging. National and international efforts to tailor the amount of regulatory control to the size of the potential risks to human health issues, however, must continue in order to ensure that the use of valuable industry testing and regulatory resources is commensurate with the likely problems.

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Romieu, I., Lacasana, M., and McConnell, R. 1997. Lead exposure in Latin America and the Carribean: Lead Research Group of the Pan-American Health Organization. Environ. Health Perspect. 105:398–405. Sacharow, S. and Griffin, R. C. 1980. Principles of Food Packaging, 2nd ed., AVI, Westport, CT. Schamberger, R. J. 1985. The genotoxicity of selenium. Mutat. Res. 154:29–48. Schroeder, H. A. and Balassa, J. J. 1966. Abnormal trace metals in man: arsenic. J. Chron. Dis. 19:85–106. Schwartz, P. S. 1988. Food packaging regulation in the United States. Food Addit. Contam. 5(suppl. 1):537–541. Sherlock, J. C. and Smart, G. A. 1984. Tin in foods and the diet. Food Addit. Contam. 1:277–285. Shukla, S. S. and Leland, H. V. 1973. Heavy metals: a review of lead. J. Water Pollut. Control Fed. 45:1319–1331. Silini, G. 1988. Biological effects of ionizing radiation. In Radionuclides in the Food Chain, eds. J. H. Harley, G. D. Schmidt, and G. Silini, pp. 35–44. ILSI Monographs. Springer-Verlag, New York. Simmonds J. R., Lawson, G., and Mayall, A. 1995. Methodology for Assessing the Radiological Consequences of Routine Releases of Radionuclides to the Environment. European Commission (Radiation Protection), EUR 15760 EN, Luxembourg. Slorach, S., Gustafsson, I. B., Jorhem, L., and Mattsson, P. 1983. Intake of lead, cadmium and certain other metals via a typical Swedish weekly diet. Var Foda 35:1–16. Stoewsand G. S., Bache, C. A., and Lisk, D. J. 1974. Dietary selenium protection of methylmercury intoxication of Japanese quail. Bull. Environ. Contam. Toxicol. 11:152–156. Strenstrom, T. and Vahter, M. 1974. Heavy metals in sewage sludge for use on agricultural soil. Ambio 3:91–92. Stroube, W. B., Jelinek, C. F., and Baratta, E. J. 1985. Survey of radionuclides in foods, 1978–1982. Health Phys. 49:731–735. Thomson, C. D. and Robinson, M. F. 1980. Selenium in human health and disease with emphasis on those aspects peculiar to New Zealand. Am. J. Clin. Nutr. 33:303–323. Trachtenbarg, D. E. 1996. Getting the lead out: when is treatment necessary? Postgrad. Med. 99:201–202, 207–218. UNEP. 1992. The Contaminants of Food. United Nations Environment Program, Nairobi. UNEP/FAO/WHO. 1988. Assessment of Chemical Contaminants in Food. Report of the Results of UNEP, FAO and WHO Program on health-related environmental monitoring. World Health Organization, Geneva. UNSCEAR. 1993. Sources and Effects of Ionizing Radiation, United Nations Scientific Committee on the Effects of Atomic Radiation. United Nations, New York. Upton, A. C. and Linsalata, P. 1988. Long-term health effects of radionuclides in food and water supplies. In Radionuclides in the Food Chain, eds. J. H. Harley, G. D. Schmidt, and G. Silini, pp. 218–234. ILSI Monographs, Springer-Verlag, New York.

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17 Pesticides and Industrial Contaminants

17.1 INTRODUCTION No other types of food contaminants are as pervasive as synthetic chemicals. Not only have they contaminated most of the Earth’s surface, but some also accumulated in the vast majority of human and animal populations in only a few decades after 1940. Synthetic organic chemicals constitute a significant part of modern-day commerce. Many industrial chemicals, although not manufactured to be used in direct contact with food or feed, can become general environmental contaminants by diverse routes and eventually contaminate the food supply during agricultural production, food processing, packaging, and storage. Water is also a major vehicle of contamination, especially contaminated water used in agriculture and food processing and manufacture. Similarly, food from marine sources is contaminated by the unwarranted intentional or unintentional pollution of the rivers, lakes, and seas (Concon, 1988). From sheer numbers of synthetic organic chemicals, their universal distribution and persistence in the environment, and their toxicological properties, these contaminants constitute a major potential hazard to public health throughout the world. A full understanding of their toxicological characteristics must involve knowledge of the benefits arising from their use. Only a few compounds are of such basic interest that their toxicological study is merited in the absence of any real or prospective use; only the economic value and use of most industrial chemicals justify such study. Their important contributions to our health and economy guarantee their continued use as a class and require the most complete knowledge of their toxicological

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properties we can achieve and the avoidance of their hazards. As societies become more industrialized, constant surveillance is required to evaluate their residual levels in the world food supply. In this chapter, food toxicological aspects of pesticides and some major persistent environmental contaminants, such as polyhalogenated hydrocarbons (PHHs) and polycyclic aromatic hydrocarbons (PAHs), are reviewed.

17.2 PESTICIDES Pesticides, in general, are a group of chemicals used worldwide in agricultural production to control, destroy, or inhibit weeds, insects, fungi, and other pests. They are defined under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) as including “(1) any substance or mixture of substances intended for preventing, destroying, repelling, or mitigating any pest [insect, rodent, nematode, fungus, weed, other forms of terrestrial or aquatic plant or animal life or viruses, bacteria, or other microorganisms on or in living man or other animals, which the Administrator declares to be a pest] and (2) any substance or mixture of substances intended for use as a plant regulator, defoliant or desiccant.” The internationally adopted definition of the Food and Agriculture Organization (FAO) of the United Nations is as follows: “Pesticide means any substance or mixture of substances intended for preventing, destroying, attracting, repelling or controlling any pest including unwanted species of plants or animals during the production, storage, transport, distribution and processing of food, agricultural commodities, or animal feeds or

which may be administered to animals for the control of ectoparasites” (Edwards, 1986). The term includes substances intended for use as plant growth regulators, defoliants, desiccants, fruit thinning agents, or sprouting inhibitors and substances applied to crops either before or after transport. The term normally excludes fertilizers, plant and animal nutrients, food additives, and animal drugs. Approximately 320 active pesticide ingredients are available in a few thousand different registered formulations (Hotchkiss, 1992; Deshpande and Salunkhe, 1995). However, not all compounds or formulations are used. Some formulations may have only limited use, some none at all; others are used in large quantities. The contributions of pesticides to health and economy are closely interrelated. They contribute directly to our health through control of certain vector-borne diseases (e.g., malaria); directly to the economy through increased production of food and fiber, and through the protection of many materials during storage. Improved health has sometimes permitted more prosperous and stable economies around the world. In fact in some countries (e.g., India), greater and more dependable production of food has virtually eliminated famine and thus contributed as much to health as to the economy. It is now a widely accepted fact that pesticides return to producers economic benefits well in excess of costs. Benefit estimates for developed countries range from $3.50 to $5.00 for every dollar spent on pesticides (Headley, 1968; Archibald and Winter, 1990); returns run as high as $14.00 in developing countries (FAO, 1972). Less than 1% of all 500,000 estimated species of plants, animals, and microorganisms are known pests. The remaining greatly benefit agriculture and other sectors of the world economy by degrading organic wastes; removing pollutants from water and soil; recycling vital chemicals within the ecosystem; buffering air pollutants; moderating climatic changes; conserving soil and water; providing medicines, pigments, and spices; preserving genetic diversity; and supplying food via the harvest of fish and other animal life (Pimentel, 1991). Nevertheless, the 1% pest component is extremely costly. Insects, plant pathogens, and weeds destroy about 37% of U.S. agricultural production, and losses are 50% to 60% in the developing world. According to Pimentel (1991), approximately 500 million kg pesticides is applied annually in an effort to control pests in the United States alone at a cost of about $4 billion. This figure, however, does not consider the indirect costs related to the destruction of beneficial organisms, disturbance of ecological systems, and human

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poisoning and illness. Such indirect costs are estimated to total at least $1 billion and may be twice that amount (Pimentel, 1991). Of the estimated total pesticide usage in the United States, 60% are herbicides, 24% insecticides, and 16% fungicides applied to about 61% of the U.S. cropland (Pimentel, 1981). The application of agricultural pesticides, however, is not evenly distributed. Pimentel (1981) estimates that 93% of all food crops are treated with some type of pesticide as compared to less than 10% of forage crops. Nearly 75% of the total herbicides used are applied to corn and soybeans; corn alone accounts for about 52% of the total usage. Of the total insecticides used, nearly 40% are applied to cotton and another 20% to corn. In contrast, fungicides and soil fumigants are used primarily on fruit and vegetable crops, which account for a relatively small percentage of agricultural land. These compounds are, however, important residues in the human diet. 17.2.1

History and Development of Pesticides

The idea of combating the ravages wrought by pests and crop diseases by the use of chemicals is not new. Homer wrote of the fumigant value of burning sulfur, and Pliny wrote about the insecticidal use of arsenic (Jones, 1992; Cremlyn, 1978). Historical Chinese writings also show the use of arsenic salts as well as tobacco extracts as pesticides. Evolution of chemical insecticides, however, essentially began in the 19th century with readily available material such as arsenicals, petroleum oils, and botanical insecticides (e.g., nicotine, pyrethrin, rotenone). French grape growers began using Paris green (impure copper arsenite) as an insecticide. This agent was later used successfully in the United States to check the spread of the Colorado beetle. Bordeaux mixture (copper sulfate, lime, and water) was discovered in 1896 in France for the control of pathogenic fungi such as potato blight or vine mildew (Table 17.1). In the early part of the 20th century, seed stock in Germany was first treated with organomercury to prevent sprouting before planting. The 1930s can be considered as the real beginning of the modern era of synthetic organic pesticides (Holland, 1996; Hajslova, 1999). Important examples of compounds introduced include dinitro-ortho-cresol (1932), controlling weeds in cereals; thiram (1934), the first of the dithiocarbamate fungicides; pentachlorophenol (1936), a wood preservative against fungi and termites; and tetraethylpyrophosphate (TEPP), the first organophosphorus insecticide. In 1939, the Swiss chemist Paul Muller discovered the pesticide properties of dichlorodiphenyltrichloroethane

Table 17.1 History of Development and Use of Insecticides Year

Chemicals and locationa

Era of natural products 900b Arsenites in China 1690 Tobacco used in Europe 1787 Soaps used in Europe Pyrethroids in Caucasus 1800b 1845 Phosphorus (inorganic) in Germany 1848 Derris root powder in Malaya Era of Fumigants, inorganics, and petroleum products 1854 CS2 for fumigation (France) 1867 Paris green (United States) 1868 Petroleum products (United States) 1874 DDT synthesized (Zeidler) 1877 HCN as fumigating agent 1880 Lime-sulfur (United States) 1883 Bordeaux mixture (France) 1886 Pine resin for scales 1892 Lead arsenate (United States) 1918 Chloropicrin (France) 1932 Methyl bromide (France) Modern synthetic insecticides 1925 Dinitro compounds 1932 Thiocyanates 1939 DDT insecticidal properties discovered (Muller) 1941 2,4-D synthesized (United States) 1941 BHC (France) 1942 BHC (United Kingdom) 1944 Parathion (Germany, Schrader) 1940–50 Aldrin, dieldrin, endrin, other chlorinated hydrocarbons (United States) 1945 Chlordane (Julius Hyman, United States; Riemschneider, Germany) 1947 Development of insecticidal carbamates, Geigy, Switzerland (Dimetan, Pyrolan, and Isolan) 1950 EPN (DuPont) 1952 Malathion 1953 Lidov: aldrin, dieldrin, Shell patent 1958 Sevin (carbaryl) (Union Carbide, United States) 1962 Rachel Carson’s Silent Spring published (United States) Hormones and pheromones 1967 First hormone mimic (juvenile) insecticides (United States) 1970 Beginning of DDT trial (Sweden, United States) 1970 Development of Bacillus thuringiensis (United States) 1970s Development of modern synthetic pyrethroids (Rothamstead, United Kingdom; Sumitomo Chemical Co., Japan) 1980 Discovery of avermectins (Merck, United States) a

DDT, dichlorodiphenyltrichloroethane; HCN, hydrocyanic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; BHC, benzene hexachloride; EPN, o-ethyl-o-[p-nitrophenyl]-phenylphosphorothiolate. b The years are approximations. Source: From Matsumura (1985), Coats (1987), and Jones (1992).

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(DDT), which soon became the most widely used single insecticide in the world. This was soon followed by the introduction of several other organochlorine compounds as contact insecticides. Next was the discovery of the first hormone herbicides, the phenoxyacetic acids 2-methyl4-chlorophenoxyacetic acid (MCPA) and 2,4-dichlorophenoxyacetic acid (2,4-D). In 1945 soil-acting carbamate herbicides were discovered, and the invention of insecticidal carbamates followed shortly thereafter. In the 1950s, several groups of pesticides that are still in use were commercialized. Because of their unheard degree of pest control on farms, these new chemicals were viewed as miracle chemicals. The early euphoria was soon replaced by the first round of postpesticide blues when the Senate Select Committee on Chemicals in Food and Cosmetics stated that there was insufficient evidence that pesticides were safe and that more regulation was needed. These proceedings resulted in the passage of the Pesticide Chemical Act of 1954. The specter of a birdless environment painted by the biologist Rachel Carson’s 1962 book Silent Spring created dire concern bordering on damnation. This book shocked the public, generated legitimate fears for the environment, and caused a flurry of government and scientific activity that resulted in environmental protection laws. Analytical work on pesticide residues in the diet began in the 1960s in reaction in alarming new findings on environmental hazards resulting from the use of pesticide. Fears about human and animal health also rose with the knowledge that DDT could degrade into dichlorodiphenyldichloroethane (DDD) and dichlorodiphenyldichloroethylene (DDE). The finding that these metabolites were stored in human and animal fat tissues ultimately led to the banning of DDT and some other persistent organochlorine insecticides (aldrin, dieldrin) from use all over the world at the beginning of the 1970s. The discovery of some subtle long-term effects of some other pesticides in the environment on nontarget organisms including humans resulted in the cancellation of registration of several other pesticides in many countries (e.g., toxaphene and dinoseb). Attempts to maximize benefits and minimize risks to public health, agriculture, and the environment ultimately resulted in an ecologically sound system for pest control. The integrated pest management (IPM) approach uses many of the techniques that farmers and manufacturers judiciously applied before the advent of pesticides. Many new and alternative pest control techniques are constantly being sought. Improved knowledge of host-pest interactions is reflected by new strategies employed in the design of modern pesticides and their formulations. At the same time, new methods of pesticide application are being developed around the world, aiming to reduce the risk of pesticide poisoning.

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17.2.2

Classification of Pesticides

The word pesticide is often used as a generic term covering all classes of compounds used in plant protection and the control of plant, insect, and animal populations. Pesticides may be divided into three main classes: insecticides, fungicides, and herbicides. However, the term also covers acaricides, molluscicides, rodenticides, fumigants, plantgrowth regulators, defoliants, desiccants, repellants, attractants, and sterilizing agents. The range of chemicals covered by this classification is very wide, and it is impossible to classify them into a few discrete groups. The World Health Organization (WHO, 1990) prefers to classify pesticides by using two main criteria, viz., the target pest and the chemical structure. A general classification of pesticides based on this approach is shown in Table 17.2. The characteristics of various chemical classes are briefly described in the following sections. Chlorinated Hydrocarbon Insecticides A large group of insecticides developed in the late 1940s and early 1950s were called chlorinated hydrocarbons or organochlorines. These compounds are characterized by (a) the presence of carbon, chlorine, hydrogen, and sometimes oxygen atoms, including a number of CCl bonds; (b) the presence of cyclic carbon chains (including benzene rings); (c) lack of any particular active intramolecular sites; (d) apolarity and lipophilicity; and (e) chemical unreactivity (i.e., they are stable in the environment). In addition to being persistent, these chemicals become concentrated in the food chain. Once deposited in the fat of mammals including humans, chlorinated hydrocarbons remain there indefinitely. Their mode of action has still not been elucidated, although they appear to be nervous system poisons (Coats, 1987). They also inhibit γ-aminobutyric acid– (GABA)-activated chloride uptake in nerves, producing destabilized, more easily stimulated nerve membranes. Major categories of these chemicals include the chlorinated bicyclodienes (or cyclodienes), hexachlorocyclohexanes (or BHCs, a term generated by the incorrect assumption that they were benzene hexachlorides), toxaphene (from the chlorination of the natural terpene camphene), and the DDT group. The cyclodienes include aldrin, dieldrin, chlordane, heptachlor, endrin, and endosulfan. The one important hexachlorocyclohexane is lindane (γ-BHC). Toxaphene is a complex mixture of chlorination isomers that also possess the bicyclic cageline form of the cyclodienes.

Table 17.2 Classification of Pesticides by Main Groups Pesticide group according to target pest, application and action, and chemical class Insecticides Chlorinated hydrocarbons Nonsystemic Organophosphates Carbamates Systemic Organophosphates Carbamates Synthetic pyrethroids Specific acaricides Nonfungicidal Organochlorine Organotin Fungicidal Dinitro compounds Molluscicides Aquatic Botanical Other chemical Terrestrial Carbamates Others Protectant fungicides Nonsystemic Dithiocarbamates Phthalimides Dinitro compounds Organomercurials Organotin compounds Chlorine-substituted aromatics Cationic detergents Others Eradicant fungicides Systemic Antibiotics Benimidazoles Morpholines Pyrmidines Nonsystemic Piperazines Others Herbicides Foliar application, systemic, or translocated Phosphonoaminoacids Benzoic acids

Common names within groupa Aldrin, dieldrin, DDT, dicofol, chlordane, endrin, HCH, heptachlor, methoxychlor, toxaphene Azinphos methyl, diazinon, dichlorvos, fenitrothion, malathion, parathion ethyl, parathion methyl, pirimiphos-methyl, tetrachlorvinphos Carbaryl, methomyl, propoxur Dimethoate, disulfoton, phorate, phosphamidon, trichlorfon, vamidothion Aldicarb, carbofuran, pirimicarb, methomyl, oxamyl Allethrin, bifenthrin, bioresmethrin, cyfluthrin, cyhalothrin, cypermethrin, deltamethrin, fluvalinate

Chlorobenzilate, tetradifon Cyhexatin Binapacryl, dinocap

Endod Niclosamide, sodium pentachlorophenate, triphenmorphe, tributyltin, trifenyltin Aminocarb, mexacarbate, methiocarb Mataldehyde

Mancozeb, maneb, metiram, propineb, thiram, zineb Captan, folpet, captafol, dichlofluanid Binapacryl Phenylmercury Fentin (acetate and hydroxide) Chlorothalonil, dichlone, dicloran, PCNB, TCNB Dodine acetates, glyodin acetate Iprodiione, procymidone

Blasticidin, cyclohexamide, kasugamycin, streptomycin Benomyl, thiophanate-methyl, thiabendazole Dodemorph, tridemorph Ethirimol, bupirimate Triforine Benomyl, carboxin, thiabendazole, thiophanate-methyl

Glyphosate, glufosinate Chlorfenprop-methyl, dicamba, 2,3,6-TBA (table continues)

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Table 17.2 (continued) Pesticide group according to target pest, application and action, and chemical class

Common names within groupa

Herbicides (continued) Foliar application, systemic, or translocated (continued) Chlorinated aliphatic acids Dalapon, TCA Oxyphenoxy acid esters Cycloxydim, diclofop-methyl, fenoxaprop, fluazifop-butyl, haloxyfopmethyl, quizalofop-methyl Phenoxyalkanoic acids 2,4-D, 2,4-DB, dichlorprop, mecoprop, MCPA, MCPB, Silvex, 2,4,5-T Quaternary ammonium compounds (bipyridiliums) Diquat, paraqut Foliar application, contact Benzonitriles Bromoxynil, dichlobenil, ioxynil Benzothiadiazoles Bentazon Carbanilates Phenmedipham Cyclohexenones Clethodim, Cycloxydim, sethoxydim Dinitrophenols Dinoseb Diphenyl ethers Acifluorfen, lactofen, nitrofen, oxyfluorfen Soil application Acetanilides Alachlor, butachlor, metolachlor, propachlor Amides and anilides Benzoylprop-ethyl, diphenamid, naptalam, pronamide, propanil Carbanilates and carbamates soil Asulam, barban, bendiocarb, carbetamide, chlorpropham, propham, triallate Dinitroanilines Benefin, phendimethalin, trifluralin Pyridazinones and pyridinones soil Amitrole, dimethazone, fluridone, norflurazon, oxadiazon, pyrazon Pyridinoxy and picolinic acids Clopyralid, fluroxypyr, picloram, triclopyr Phenylureas or substituted ureas Diuron, fenuron, fluometuron, linuron, monolinuron, siduron Sulphonylureas Chlorimuron-ethyl, chlorsulfuron, metsulfuron-methyl, sulfometuronmethyl, thiameturon-methyl Thiocarbamates Butylate, cycloate, EPTC, molinate, pebulate, thiobencarb, triallate Triazines Ametryn, atrazine, cyanazine, desmetryne, hexazinone, methoprotryne, metribuzin, prometon, propazine, terbutrizine, terbutryne Uracils or substituted uracils Bromacil, lenacil, terbacil Dessicants, defoliants Organophosphates Merphos, DEF Phenol derivatives Dinoseb, PCP Quaternary ammonium compounds (bipyridiliums) Diquat, paraquat Plant growth regulators Growth promoters Auxins 2,4-D, MCPB, NAD Cytokinins BA, PBA, adenine, kinetin Gibberellins Gibane, GA3 Ethephon Ethylene generators Inhibitors and retardants Quaternary ammonium compounds Chlormequat, mepiquat Hydrazides Maleic hydrazide, daminozide Triazoles Paclobutrazol, uniconazole Rodenticides Aluminum phosphide, calcium cyanide, chloropicrin, methyl bromide Fumigants Anticoagulants Hydroxycoumarins Brodifacoum, difenacoum, coumafuryl, caumatetralyl, warfarin Indandiones Chlorophacinone, diphacinone, pindone Noncoagulants Arsenicals Arsenious oxide, sodium arsenite Benzenamine Bromethalin Botanicals Red squill, strychnine

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Table 17.2 (continued) Pesticide group according to target pest, application and action, and chemical class Rodenticides (continued) Noncoagulants Thioureas Others

Common names within groupa

Antus promurit Fluoroacetamide, fluoroacetate, norbormide sodium, zinc phosphate

a

Most chemical pesticides have common names agreed on by the International Organization for Standardization through its Technical Committee 81 (ISO/TC 81). The principles for coining these common names are explained in ISO 257:1988. DDT, dichlorodiphenyltrichloroethane; PBA, 6-benzylamino-9-(tetrahydropyran-2-yl)-9H-purine; HCH, hexachlorocyclohexane; GA3, gibberellins; PCNB, quintozene; TCNB, thenazene; 2,3,6-TBA, 2,3,6trichlorobenzoic acid; TCA, trichloroacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; MCPA, 2-methyl-4-chlorophenoxyacetic acid; 2,4,5-T, 2,4,5trichlorophenoxyacetic acid; PCP, pentachlorophenol; 2,4-DB, 4-(2,4-dichlorophenoxy)-butanoic acid; MCPB, 4-(4-chloro-2-methylphenoxy)-butyric acid; EPTC, S-ethyl-N,N-dipropylthiolcarbamate; NAD, naphthylacetamide; DEF, SSS-tributylphosphorotrithiolate; BA, benzylaminopurine. Source: From Hajslova (1999), based on WHO (1996) recommendations for classification.

These chemicals are often considered to belong to the group of organochlorine pesticides. However, their group characteristics make them very different from other organochlorine pesticides, such as fumigants, chlorinated organophosphates, and chlorinated aliphatic and aromatic acids. Organophosphates The organophosphate group accounts for approximately 40% of the registered synthetic insecticides and acaricides in the United States. They are often referred to as organophosphates, because most of the member chemicals are some form of phosphate. The most commonly used kinds today are esters of phosphorothionates and phosphorodithioates. These chemicals possess the common characteristic that they are, or can become, excellent inhibitors of cholinesterases. The organophosphate group of insecticides includes such generally toxic compounds as parathion and TEPP and such selective compounds as malathion and ronnel. They are used as stomach and contact poisons, as fumigants, and as systemic insecticides for nearly every type of insect control. Unlike the chlorinated hydrocarbons, organophosphates are not persistent, so they are not concentrated in the food chain. Because they are volatile, more frequent application is required to get the same degree of pest control throughout the crop cycle. As with some other pesticides, various pests are evolving to be resistant to these chemicals.

principal alkaloid of the plant Physostigma venenosum (calabar bean). The carbamic esters are structurally more similar to the neurotransmitter acetylcholine. Carbaryl, a naphthylcarbamate that was introduced in 1956 under the trade name Sevin, is the most widely used carbamate today. It is a wide-spectrum insecticide that controls 100–150 species of insects but is virtually ineffective against houseflies, certain aphids, and spider mites. It also has low mammalian toxicity. The most important difference between organophosphates and carbamates lies in the rates at which the phosphoryl and carbamoyl groups are released from the active site of cholinesterase. The release step, which occurs very quickly (milliseconds) with the natural substrate acetylcholine, proceeds at a moderate rate for carbamate inhibitors (seconds or minutes), but only at extremely low rates for organophosphate inhibitors (hours or days). Carbamate poisoning is caused by a reversible inhibitor, whereas organophosphate poisoning is the result of an essentially irreversible inhibition. Bipyridilium Herbicides The bipyridilium class of herbicides includes paraquat and diquat, which, unlike most herbicides, are toxic to mammals. The mode of action is through their potent oxidative capability. Paraquat, for example, generates peroxides, which in mammals cause a slow but irreversible fibrosis of lung tissue (McEwen and Stephenson, 1979).

Carbamates

Thiocyanate Insecticides

The carbamates are the latest arrival in the field of anticholinesterase insecticides and are synthetic derivatives of physostigmine (commonly called eserine), which is the

The organic thiocyanates were important insecticides before DDT and the chlorinated cyclodiens came into widespread use. Methyl- and ethylthiocyanates have been used

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as fumigants. However, they are also toxic to plants, a property that limits their use somewhat. Many of the thiocyanates are known commercially as Lethanes. Phenols The dinitrophenols were early insecticides and acaricides that were first used in the 1890s. They still have some usefulness today as dormant sprays and herbicides. Examples include dinitro-o-cresol (DNOC) and pentachlorophenol. They act by uncoupling oxidative phosphorylation. The fungicides hexachlorobenzene and pentachloronitrobenzene are considered to act via the same action after metabolic conversion to a phenolic derivative. Fluoroacetate Derivatives The fluoroacetate deriviative compounds are characterized by their rigid structural requirement that only the compounds that can give fluoroacetic acid on activation in the animal or plant tissues are active. Fluoroacetate is further converted in vivo into fluorocitric acid, which inhibits cisaconitase of the tricarboxylic acid (TCA) cycle. They are effective as systemic insecticides and acaricides against aphids and mites. Acaricidal Chemicals The acaricidal compounds normally contain two chlorinated benzene rings and are sulfones, sulfonates, or sulfides (but never sulfates). Examples include Ovotran, Sulphenone, and Fundal. Fumigants Methyl bromide (CH3Br) is still the most widely used fumigant. It is extremely toxic and flammable and is most widely used for insect pests in grain elevators and warehouses. Other examples are dibromochloropropane (DBCP), ethylene dibromide, ethylene dichloride, and chloropicrin. These fumigants act primarily by inhibiting the conversion of succinate to fumarate in the TCA cycle through binding with sulfhydryl groups in succinic dehydrogenase and other enzymes. Inorganic Insecticides Inorganic insecticides are relatively nonspecific, and since they are not very toxic to insects, large quantities are required to control insect pests. Because of these limitations, they have been gradually replaced by organic, particularly synthetic, chemicals. Nevertheless, two groups of inorganic chemicals are still used today as insecticides: arseni-

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cals and fluorides. The former include lead arsenate, calcium arsenate, and sodium arsenite; sodium fluoride, cryolite (sodium fluoroaluminate), and sodium fluorosilicate are the commonly used fluoride insecticides. Trisubstituted tin hydroxides (trialkyl, tricycloalkyl, triaryl) are used as acaricides. They act as inhibitors of oxidative phosphorylation. Botanical Insecticides The botanical insecticide group mainly consists of heterocyclic nitrogen-containing compounds obtained from certain species of plants. Well-known examples are nicotinoids from Nicotiana tabacum and N. rustica, nornicotine from N. sylvestris, and anabasine from Anabasis aphylla; rotenoids; and pyrethroids. Nicotine has neurotoxic effects. It acts at the synapses but not as an inhibitor of acetylcholinesterase. Rather, it is an agonist of acetylcholine, resulting in overstimulation of all mammalian cholinergic junctions. Plants containing rotenoids have been used as fish poisons for many centuries. Rotenone and allied substances are found in a large number of plants, all in the family Leguminosae. Economically important sources of rotenone are the plants Derris elliptica and D. malaccensis from Malaya and the East Indies and Lonchocarpus utilis and L. urucu from South America. Rotenone is used in the form of ground roots or resins or as a crystalline material that is extracted by organic solvents. They act as either a contact or a stomach poison. Rotenone is extremely toxic to fish. Rotenoids ostensibly act on the electron transport system by blocking the reduction of flavoprotein and/or ubiquinone. Pyrethrum is found in the flowers of plants belonging to the family Compositae and the genus Chrysanthemum. Commercially, it is manufactured from C. cinerariaefolium and C. coccineum. Synthetic pyrethroids were first developed in the 1950s. Pyrethrum is essentially nontoxic to mammals and is very fast-acting to insects. It is a contact insecticide that acts as a nerve poison. Synergists The toxicity of certain insecticides, notably pyrethrin, can be enormously increased by the addition of compounds that may not be insecticidal at all. These compounds are called synergists. The majority of synergists contain an active moiety, a methylenedioxyphenyl group. Examples are sesamin and sesamolin (both from sesame oil) and piperonylbutoxide, which is perhaps the most widely used synthetic pyrethrin syngergist.

Microbial Insecticides Bacillus thuringiensis, a bacterium species related to Bacillus cereus, produces substances that are extremely toxic mostly to lepidopterous larvae. The most important of all is δ-endotoxin. The commercially available preparations are a mixture of spores and crystals. To be toxic the material must be ingested by the larvae: i.e., it is a stomach poison. Avermectin B1a (AVM) is a macrocyclic lactone derived from the mycelia of Streptomyces avermilitis. It has been shown to be anthelmintic, insecticidal, and acaricidal. The chemical structures of some of the pesticides most widely used since the 1950s are shown in Figure 17.1. 17.2.3

Movement of Residues in the Environment

The major source of environmental contamination by pesticides are the deposits resulting from application of these chemicals to control agricultural pests and pests causing public health problems. A major proportion of the applied pesticide does not immediately enter any organism, remaining in soil, water, and air, where it is subjected to further transformation and transport to different locations as well as uptake by organisms at that site (Fuhr, 1982). Pesticides are dispersed mainly by water and air movements and are picked up by various biological systems. At the same time, they are chemically or biochemically transformed to other nontoxic or toxic compounds in the environment. A simplified scheme illustrating the various processes to which pesticides applied for plant protection are subjected is shown in Figure 17.2. Major physical factors involved in pesticide distribution in the environment include mobility into soil, surface runoff from soil, leaching downward through soil, binding to soil and subsequent transport by wind or water, spray drift, and volatilization. These processes result in residues’ being deposited in “reservoirs” such as the soil, surface water, groundwater, or air, in addition to the originally intended location. Biological movement can include uptake and translocation of some pesticides by plants as well as bioaccumulation through food chains. The movement of pesticide residues invariably depends on specific molecular structure and associated physical properties. For example, the more water-soluble pesticides tend to dissolve in water and move very easily in the water. The less soluble types tend to bind to soil or vegetation, thus being physically less mobile (Elgar, 1983; Coats, 1987). Hydrophobicity also provides some potential for a chemical to accumulate in lipid reservoirs such as adipose tissue in animals, or oils and oily products of

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plants. A lipophilic compound accumulates to a high level only if it also is resistant to degradation. Long exposure times in biological systems subject even a lipophilic chemical to repeated enzymatic challenges, and only a relatively nonbiodegradable substance can resist biotransformation effectively enough to be sequestered in large quantities in plant or animal lipids. Thus, a pesticide can bioaccumulate significantly only if it is of high lipid solubility, of low water solubility (not easy to excrete), persistent (not easily degraded in vivo), and stable enough in the environment to allow adequate uptake into biological components (McEwen and Stephenson, 1979). The most notorious examples include pesticides belonging to the chlorinated hydrocarbon group, such as DDT, dieldrin, chlordane, BHC, aldrin, endrin, heptachlor, and lindane. 17.2.4

Mechanisms of Toxicity

Although designed to control pests, pesticides can also be toxic to nontarget organisms, including humans. This is primarily because the same basic enzyme, hormone, and other biochemical systems are present in species from insects to humans. When pesticides are applied improperly, resulting residues in foods can pose significant health risks to consumers. The use of pesticides in agricultural production represents three related but distinct risks defined as the quantifiable probability that harm or injury will occur (Hotchkiss, 1992): 1.

2.

3.

The environmental risks associated with adverse effects on nontarget organisms and groundwater contamination Occupational risks to agricultural workers and pesticide factory workers, which are considerably higher than those of other sources of human exposure and pose the foremost human health concern related to pesticides The occurrence of pesticides as residues on or in edible foods

Within each type of exposure, pesticides may enter the body dermally, inhalationally, or orally. Accidental encounters often cause acute poisoning, which has been studied by medical and veterinary toxicologists. Many of the pesticides are potent poisons. Like those of most poisons, the acute and subacute symptoms associated with pesticide poisoning are dose-related and disappear at very low doses such as those generally found in foods. Several examples of incidences of human poisonings are shown in Table 17.3. In spite of these rare occurrences, the acute toxicity of these compounds is only marginally relevant to food toxicological effects. This is because most humans

Figure 17.1 Chemical structures of some commonly used pesticides. Not all are currently allowed for use in agriculture.

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Figure 17.1 (Continued)

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Figure 17.1 (Continued)

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Figure 17.1 (Continued)

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Figure 17.2 Pesticide movement in the environment.

are exposed continuously to low levels of pesticides. Even though we are chronically exposed to these substances and accumulate some of them in our tissues, acute symptoms from such exposures or accumulations are seldom encountered. In contrast, occupational exposure usually results in chronic poisoning, which provides rough estimates of toxicity to humans for some of the pesticides. The occupational risks can be reduced by strict controls and use of appropriate protective technology.

As mentioned earlier, environmental effects are associated with some pesticides, especially chlorinated hydrocarbons. These are quite toxic and are resistant to biodegradation. They can last for decades in the soil and, being fat-soluble, can be stored in human and animal fat tissues, liver, and the central nervous system to produce suspected chronic and delayed effects. In lactating animals, residues are removed more quickly and enter the animal’s milk. The use of fat-soluble pesticide or disposal of

Table 17.3 Recorded Acute Mass Pesticide Poisonings in Various Countriesa Pesticide Dieldrin Diazinon Endrin Endrin Endrin Hexachlorobenzene Mevinphos Nicotine Organomercurials Organomercurials Organomercurials Parathion Parathion Parathion Parathion Parathion Toxaphene

Contaminated foodstuffs b

Mixed diet Doughnut mixb Wheat flourb Wheat flourb Wheat flourb Seed grainc Vegetablesb Mustardc Seed graind Seed graind Seed graind Wheatb Flourb Flourb Barleyb Sugarb Collards and chardsc

a

Country

Cases, number

Mortality

On ship United States Wales Saudi Arabia Qatar Turkey United States United States Guatemala Iraq Pakistan India Egypt Columbia Malaya Mexico United States

20 20 159 183 691 3000+ 6 11 45 321 34 360 200 600 38 300 7

0 0 0 2 24 90–330 0 0 20 35 4 102 8 88 9 17 0

Other pesticide poisonings unrelated to food use were also reported. Food became contaminated by spillage during transport or storage. c Food was contaminated by improper application of pesticides. d Poisonings were caused by eating pesticide-treated seed grains. Source: From Concon (1988). b

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their wastes where they might volatilize or be washed away from soil or foliar surfaces into water can also lead to the accumulation of residues in plants and animals. It is now a well-known fact that residual levels of DDT, a pesticide widely used in the 1950s and 1960s, occur in every living form on the planet, and heptachlor, a toxic metabolite of chlordane, is now found in over 90% of the population (Deshpande and Salunkhe, 1995). Chlorinated hydrocarbons have become most hazardous to human health through a process called biomagnification. Spray drifts and drainage from treated fields contaminate water bodies. Small plankton and other organisms living in the water absorb the pesticides and store them in their tissues. The next animal feeding on the plankton in the food chain takes in a diet enriched with the pesticides and their metabolites. After a period, the concentration of these residues rises in the animal. There is thus a stepwise increase in pesticide concentration along the food chain. Eventually, they all accumulate in human adipose tissue with increasing age. This property is often expressed as a bioconcentration factor (BCF) (Kenaga, 1980; Hajslova, 1999) and is reported for hydrophobic chemicals having a tendency to partition from the water column and bioconcentrate in aquatic animals. BCF is given by the concentration of the chemical in the organism at equilibrium divided by the concentration of the chemical in water. It correlates very well with the octanol-water partition coefficient (Kow). The BCFs for selected pesticides are summarized in Table 17.4. The route of entry of pesticides bears considerably on the type, timing, and severity of effects. The inhalation route usually is the most rapid mechanism of uptake and most direct route to the bloodstream. Immediate and dire

effects can result when certain types of vapors are inhaled. However, the general population seldom is exposed to dangerous airborne concentrations of pesticidal compounds for any extended time; also, an olfactory warning frequently alerts us to such a hazard before we suffer deleterious consequences. The dermal route of absorption of pesticides is important primarily through occupational types of exposure, for example, in manufacturing and applying the pesticides. Many organic insecticides of relatively high water solubility penetrate the skin readily and can be toxic by this route, including some organophosphorus and carbamate pesticides. Chemicals with relatively low water solubility are not easily absorbed through the skin. The resultant hazard of a dermal exposure is always a composite of penetration rate plus the distribution and potency of the toxicant once inside the body. The oral route of entry is one of greatest concern to a large segment of the population. Most pesticides are more acutely toxic via the oral route than the dermal route. In addition, low-level residues that occur in foods and beverages are virtually unavoidable components of our diet. It, therefore, is critical that we perceive and understand the potential toxic manifestations of ingested pesticides. The modes of action for many of the earlier, inorganic pesticides have been determined through decades of research. Among the organic chemicals, both synthetic and natural, some mechanisms of action are well known and others have not been elucidated (Kuiper, 1996). For example, the mechanism for organophosphorus and carbamate insecticides results in inhibition of cholinesterase (among the commonly used compounds of this group, parathion and aldicarb are especially toxic), and nitrophenols

Table 17.4 Bioconcentration Factor for Some Persistent Chlorinated Hydrocarbons and Other Priority Pollutants

Pesticide p,p′-DDTa p,p′-DDEa HCBa Toxaphenea Aldrina Dieldrina Lindane Pentachlorophenol Atrazine

Chemical abstracts reference no.

n-octanol-water partition coefficient, Kow

log biocentration Factor, fish

50-29-3 72-55-9 118-74-1 8001-35-2 309-00-2 60-57-1 58-89-9 87-86-5 1912-24-9

1.00 × 106 5.80 × 105 2.00 × 105 6.60 × 104 3.20 × 106 2.10 × 104 4.00 × 103 1.30 × 105 5.60 × 102

4.468 NAb 4.342 4.522 4.209 4.100 2.505 2.892 1.000

a

Not registered for use in agriculture. p,p′-DDT, p,p′-dichlorodiphenyltrichloroethane; p,p′-DDE, p,p′dichlorodiphenyldichloroethylene; HCB, hexachloro-2-butanone. b Data not available. Source: Compiled from Kenaga (1980) and Hajslova (1999).

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and higher chlorinated phenols are inhibitors or uncouplers of oxidative phosphorylation (Hassall, 1990). Fatsoluble substances are accumulated in the body and, when stored in fatty tissues, are not readily metabolized (WHO, 1990). However, in times of poor nutrition or relative starvation, the deposits of these compounds are mobilized and are released into the bloodstream, with possible toxic effects if concentrations reach a high enough level. The toxic effects of pesticides on major body organs are briefly described in the following sections. General symptoms of pesticide poisoning in humans are listed in Table 17.5. The discussion that follows is not intended to serve as a comprehensive list of all pesticidal chemicals that can cause such effects. Because of the chronically low levels of exposures to pesticide residues in food, the toxicological characteristics of such residues focus primarily on carcinogenicity, mutagenicity, and teratogenicity. This emphasis is based again on the assumption that humans may be susceptible to these effects even at these low doses because of long lifespan and the influences of other carcinogens, toxicants, and endogenous and exogenous factors. Liver The liver plays a central role in the ability of an animal to deal effectively with both acute and chronic exposure to toxic chemicals. Indeed, the ability of animals to survive chronic low levels of environmental toxicants may depend in large measure on the adaptive potential of the liver. In

general, the liver reacts to toxicant stress by certain patterned responses, including increases in enzymes, in cellular organelles, and in cell and organ size. One of the most consistent responses in vertebrates to pesticide exposure is an increase in liver mass. The response is dependent on dose, age, and sex. The enlargement represents “real” growth in that there are appropriate increases in protein and lipid levels per unit liver. In many instances, the increase in liver mass appears to be unrelated to toxicity. It is rather a compensatory response in which the liver is responding to increased functional demand (Schulte-Hermann, 1979; Yarbrough et al., 1982). Initial observations of liver enlargement resulting from chronic insecticide exposure involved DDT (Fitzhugh and Nelson, 1947; Laug and Fitzhugh, 1946). Since these initial studies, a consistent pattern of liver enlargement by a wide range of insecticides, including pyrethrum, aldrin, dieldrin, chlordane, endrin, mirex, and chlordecone, has been reported. Liver enlargement also occurs in most vertebrate species; mice, rats, dogs, rabbits, and birds demonstrate liver enlargement in response to pesticide exposure. Liver weight induction by chronic exposures to insecticides is most often associated with proliferation of smooth endoplasmic reticulum (SER) and mixed-function oxidase induction. The physiological and biochemical effects of chronic exposure of vertebrates to pesticides have included increases in protein and lipid synthesis, changes in carbohydrate metabolism, hepatic glycogen depletion, changes

Table 17.5 Symptoms of Pesticide Poisoning in Humans System Central nervous system and somatomotor

Examination Body movements Muscular tone Clinical signs

Autonomic Respiratory

Ocular

Gastrointestinal General side effects

Pupil size Secretion Nostrils Character of breathing Clinical signs Eyelids and eyeball Clinical signs Clinical signs Acute poisoning Chronic poisoning

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Signs of toxicity Twitch, tremor, ataxia, convulsion, paralysis, fasciculation Rigidity, flaccidity Headache, disturbed dreams and poor sleep, perspiration, nervousness, dizziness, test on reflex Myosis, mydriasis Salivation, lacrimation Unusual discharge or movements, rhinorrhea Bradypnea, dyspnea, yawning Constriction of chest, cough, and wheezing Ptosis, exophthalmos Pain on accommodation, dimness, lacrimation, conjunctival injection Anorexia, nausea, vomiting, diarrhea Temperature, skin texture and color, cardiovascular effects, cyanosis, jaundice Food intake, body weight, tumor, disease, sleep time

in hepatic soluble enzymes (specifically the gluconeogenic enzymes), and induction of the microsomal mixed-function oxidases (MFOs). The mechanisms by which these effects are brought about are not well understood. However, many of the initial biochemical alterations observed in the liver relate to the stress response mediated principally by steroid hormones (Copeland and Cranmer, 1974; Foster, 1968; Szot and Murphy, 1970). Increases in protein and lipid synthesis generally accompany the increase in liver mass, keeping pace with the liver mass increase. Alterations in basic metabolism involve both anabolic and catabolic processes and occur in both acute and chronic treatments with pesticides (Darsie et al., 1976; Robinson and Yarbrough, 1980). In contrast, the increase in gluconeogenic enzyme activity may represent a carbohydrate sparing effect, i.e., a switch from utilizing carbohydrate as a primary energy source to utilizing protein and lipid (Yarbrough et al., 1982). One of the most common effects reported for either acute or chronic pesticide exposure in animals is the induction of mixed-function oxidases (MFOs). Indeed, this is probably the most widely reported and studied of all the xenobiotic effects. There is an apparent relationship between induction of liver enlargement and induction of hepatic microsomal drug metabolism. Schulte-Hermann (1974), in surveying a wide range of xenobiotics, indicated that with few exceptions, xenobiotics that induce liver growth also induce hepatic drug metabolism. Of the pesticides, DDT, pyrethrum, and dieldrin appear to be moderate inducers and mirex appears to be a highly potent inducer. Although the acute induction of MFO has been extensively investigated, studies of MFO induction during chronic exposures to pesticides have been limited. The alterations in hepatocyte morphological characteristics resulting from chronic pesticide exposure seem to follow a general pattern that includes, but is not necessarily limited to, the listing in Table 17.6. This basic group of histological alterations has been reported to occur for a wide range of doses (5 to 500 ppm), duration of exposures (30 days to 2.6 years), and pesticides, primarily chlorinated hydrocarbons, used. Perhaps the three most consistent histological alterations are cellular enlargement (hypertrophy), increased SER, and cytoplasmic vacuolization, which is most often interpreted as fatty degeneration. The changes often appear to be dose- and time-related. It should be noted that the changes observed in the liver do not necessarily represent cytotoxicity but are indicative of a compensatory response in which the liver (as the main organ of xenobiotic processing) is responding to xenobiotic challenge. However, there are a dose level and a length of exposure time at which the animal apparently

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Table 17.6 Common Pathological Findings in Livers Related to Chronic Pesticide Exposures Cellular hypertrophy Fatty degeneration Cellular margination Increased basophila Increased fat granules Cytoplasmic vacuolization Necrosis Increase in smooth endoplasmic reticulum (SER) Atypical mitochondria Scattered swollen degenerated hepatocytes (celluar edema) Enlargement of nuclei Hypertrophy of nucleoli

can no longer adapt. At this point, the compound begins to disrupt essential cellular processes. Kidney The mammalian kidney is a dynamic and complex organ. Excretion of wastes is a primary function, but the kidney also plays a significant role in the regulation of total body homeostasis. Regulation of extracellular volume and control of electrolyte and acid-base balance are important renal functions. Extensive descriptive data concerning the effect of pesticides on the kidneys are available. Only the salient features are described here. Because of its normal function, the kidney is susceptible to the noxious effects of many chemicals in the blood. Renal blood flow is quite high; the two kidneys together receive about 25% of the cardiac output (Hook, 1980). Approximately one-third of the plasma water reaching the kidney is filtered. From this material, approximately 98% to 99% of the salt and water is reabsorbed. Maintenance of normal function requires delivery of large amounts of metabolic substrates and oxygen to the kidney. Because of the high blood flow, any chemical in circulation is delivered in relatively high amounts to this organ. As salt and water are reabsorbed from the glomerular filtrate, the materials remaining (including the potential toxicant) in the urine may be concentrated in the tubular lumen. Thus, a nontoxic concentration of a chemical in plasma may become toxic in the kidney subsequent to concentration within the urine. Furthermore, a chemical reaching the kidney may be concentrated in the cells by other mechanisms. If the material is actively secreted into the tubular urine, it is first accumulated within the cells of the proximal tubule, thus exposing these cells to very high concentrations of the agent, which may produce toxicity. Similarly, a material that is reabsorbed even by passive means from the urine into the

blood passes through the cells of the nephron in a relatively high concentration. In addition, the kidney is exposed to chemicals in another unique fashion. Binding of chemicals to plasma proteins may protect most cells of the body from the potential toxic action of these compounds. However, if a compound is actively secreted by the nephron, it can be removed from plasma binding sites and transported into renal tubular cells and into the tubular urine. Thus, although other cells of the body may be protected by protein binding, the renal cells may be exposed to these potentially toxic molecules or ions. The renal medulla is unique in relation to the nephrotoxicity of chemicals. Since only about 10% of total renal blood flow enters the renal medulla, relatively less toxicant may reach this region via the blood than would enter the cortex. However, any chemical in the tubular urine passes through the loop of Henle and the medullary collection duct, exposing the cells of the medulla to high concentrations. In addition, the countercurrent mechanism within the medulla and the relatively low blood flow may trap compounds, further leading to the development of relatively high concentrations (Duggin and Mudge, 1976; Valtin, 1973). Nervous System Neurotoxicology is a relatively new branch of toxicology that relies on a number of disciplines, including psychology, neurology, physiology, pharmacology, biochemistry, and pathology. Each discipline relies on a range of methods of varying complexity, specificity, and applicability to investigations into neurotoxic effects of chemicals. Potential damage to the nervous system is difficult to assess because of wide variation in the normal function of the nervous system as well as its plasticity, residual capacity, and compensatory mechanisms (NRC, 1992). Both organophosphorus and carbamate pesticides inhibit acetylcholinesterase, an enzyme that regulates neurotransmission by hydrolyzing acetylcholine that accumulates at the synaptic junction during propagation of nerve impulses. However, there is a critical difference in the mode of action of carbamates and organophosphates. For carbamates, the esterase inhibition is readily reversible; for organophosphates, it is more permanent. However, many organophosphorus pesticides exhibit a special kind of neurotoxicity, which appears unrelated to acetylcholinesterase inhibition. In this case, the neurotoxic signs appear several hours or days after exposure or ingestion, and thus, the condition has been termed delayed neurotoxicity. Delayed neurotoxicity is characterized by irreversible demyelination of nerves in the central and peripheral

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nervous system in many species of mammals, including humans. It has been described as a polyneuritis with a flaccid paralysis of distal muscles of the upper and lower extremities, accompanied by degeneration of the axons and associated myelin sheaths of the spinal cord, sciatic nerve, and medulla (Davies, 1963). The onset of paralysis in humans is marked by unusual sensory experiences, such as burning or tingling, especially in the feet. Muscular weakness is the primary symptom that begins in the feet and later includes the legs and possibly even the hands. In all species examined, the hind limbs are always more severely affected than the forelimbs. The gait is high-stepping in bipedal species, with the feet slapping the ground. In quadrupeds, the hind limbs show waddling, lurching motions, accompanied by various degrees of ataxia. Actual clinical symptoms are not seen until 8 to 14 days after exposure to the toxic agent and then remain stationary for several weeks before beginning to improve. In mild cases, recovery may be almost or entirely complete, whereas more severe cases may lead to complete ataxia and death (Cavanagh, 1973; Geoffroy et al., 1960). The major group of organophosphates that can cause delayed neurotoxicity are the phenylphosphonothioates (Abou-Donia, 1979a, 1979b; Johnson, 1984). Examples are S,S,S-tributyl phosphorotrithiolate (DEF), a defoliant; o-ethyl o-[p-nitrophenyl]-phenylphosphonothiolate (EPN), an insecticide; and 2,4-dichlorophenyl methyl-Nisopropylphosphoramidothionate (DMPA), a herbicide. Single sublethal doses of 10–500 mg/kg or repeated doses of 0.1–50 mg/kg can elicit the response, characterized by ataxia and paralysis (Abou-Donia and Graham, 1979; ElSebae et al., 1980; Abou-Donia et al., 1986). The onset of irreversible nerve damage occurred at 10 or more days after initial exposure in most cases. Species differences are extreme among warm-blooded animals: humans, sheep, cats, dogs, pigs, water buffalo, ducks, pheasants, and chickens are sensitive; mice, rats, guinea pigs, rabbits, partridge, and quail are resistant to the effects (Francis et al., 1980; Hollingshaus et al., 1981; Abou-Donia et al., 1983). Leptophos, desbromoleptophos, cyanofenphos, EPN, and S-Seven (O-2,4-dichlorophenyl-O-ethyl phenylphosphorothioate [EPBP]) demonstrate potency in sensitive species. Impurities in many organophosphate pesticides may enhance the toxicity of the pesticide. Thus, a photolytic product of leptophos occurs as an impurity and is at least 4- to 10-fold more toxic than leptophos (Hollingshause et al., 1979; Sanborn et al., 1977). It is remarkable that this compound is a photodecomposition product and thus poses a more serious environmental hazard than the parent compound. Similarly, many organophosphate preparations (e.g., malathion) contain trialkylphosphate impurities. These compounds potentiate the neurotoxicity of the

parent compound by inhibition of carboxyl esterases, which detoxify the parent compound (Talcott et al., 1979a, 1979b). These impurities are inherently toxic themselves and produce acute, late acute, or delayed neurotoxic effects. The action of delayed neurotoxic organophosphates has been postulated to be the inhibition of a “neurotoxic esterase” (Johnson, 1975; Lotti et al., 1985). Effects on microtubule involvement in spindle formation have contributed to a hypothesis that phosphorylation of a protease inhibits or disrupts synthesis of microtubules (Seifert and Casida, 1982). But how such phosphorylation can result in demyelination of nerve tissue with consequent delayed onset, ataxia, or paralysis is unknown. Examination of potential effects on the nervous system is also an important aspect of the assessment of developmental toxicity (Somogyi and Appel, 1999). The stage of development of the nervous system at birth varies with the species involved. For example, the neonatal rat is at a stage of development most similar to that of humans at the beginning of the third trimester of pregnancy. Exposure of developing animals to a chemical may result in effects that differ quantitatively and qualitatively from those of exposure of adult animals. One reason is certainly the incomplete development of the enzymes catalyzing the detoxification of pesticides in young animals. Therefore, the applicability of the acceptable daily intake (ADI) values to infants and young children should be carefully evaluated. Because of current regulations regarding the tolerances of organophosphate and carbamate pesticides, very low levels are detected in foodstuffs. Therefore, it is rather unlikely that at these levels ataxia and other signs of delayed neurotoxicity may be induced in humans by frequent consumption of foods contaminated by these products. Nevertheless, knowledge of these effects is essential to proper assessment of the hazards posed by these compounds. In fact, it is precisely this knowledge that has led to current vigilance with respect to the toxic effects of these pesticides. Behavioral Effects It has been recognized for some time that the central nervous system is a primary site of pesticide toxicity and that the actions of pesticides on the central nervous system produce profound behavioral changes (Cannon et al., 1978; Clark, 1971; Larson et al., 1979; Levin and Rodnitzky, 1976); McMillan, 1982; Coats, 1987). Mammalian behavior is extremely sensitive to xenobiotic chemicals. Very low exposure levels can cause decreased learning and memory function, hyperactivity, altered aggressive and defensive behavior, or abnormal courtship, resulting indi-

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rectly in reduced reproduction. Pesticide effects on vertebrate behavior include changes in schedule-controlled responding induced by chlordimeform (Leander and MacPhail, 1980) and decamethrin (MacPhail et al., 1981), flavor aversions caused by chlordimeform (MacPhail and Leander, 1980), errors in discrimination and problem solving caused by toxaphene and endrin (Kreitzer, 1980), and impaired swimming in neonatal offspring of mothers dosed with dieldrin (Olson et al., 1980). Mirex and Kepone also have been shown to affect behavior of rats (Reiter et al., 1977; Dietz and McMillan, 1979), as have permethrin and deltamethrin (Bloom et al., 1983). Immunosuppression Numerous studies in animals suggest that exposure to certain pesticides or compounds associated with them may suppress the immunocompetence of the host, so that animals become more susceptible to infection. Some pesticides can cause either depression or stimulation of the immune response of vertebrates, depending on the type of chemical, duration of exposure, size of dose, and specific response monitored (Dandliker et al., 1980; McCorkle et al., 1980; Street, 1981; Allen et al., 1983). Dioxin, methyl parathion, carbofuran, hexachlorobenzene, trichlorfon, molinate, DDT, di-n-butyltin dichloride (DBT), and di-noctyltin dichloride (DOT) have all been shown to suppress the immunocompetence of the host, so that animals become more susceptible to infection (Concon, 1988). As in many biological phenomena, the interactions of various factors are exceedingly complex, and the effects are not easy to predict. Nevertheless, in this case, the hazards remain. Allergenicity Allergic reactions also can develop in individuals who become sensitized to specific pesticides or the adjuvants contained in the formulated products (Cushman and Street, 1982, 1983). Allergic symptoms are not necessarily always associated with dermatitis and other skin reactions, coryza, or rhinitis. Therefore, the relationships between pesticide ingestion in the diet and allergic symptoms may not always be recognized without thorough evaluation of specific cases. Pesticide Interactions Because pesticides are often used in combinations, agricultural workers and pest control specialists may encounter multiple exposures to combinations of two or more pesticides close in time. Although many types of pesticide interactions have been demonstrated, the number of potential interactions of pesticides with the tens of thousands

of other chemical and biochemical agents is astronomical. Premarket screening for all possible interactions is clearly impractical. Although many mechanisms of action for combination effects are poorly understood, those mechanisms may provide the only predictive capabilities for early warning against certain types of interactions. A true toxic interaction is the result of greater-than-additive toxicity of a combination that enhances the deleterious effect and is termed synergism or potentiation. An interaction that renders the substance(s) less toxic is termed antagonism. Quite often, potentiation can result from an inhibition of degradative enzymes by one or both chemicals; a larger-than-usual dose of intact toxicant circulates in the bloodstream as a result. Common enzyme systems affected include the MFOs and various esterases. Potentiation may be induced by chemicals within the same class, e.g., the cholinesterase inhibitors EPN and malathion (Murphy and Dubois, 1957; Frawley et al., 1957) or by unrelated pesticides such as thiuram and chlorfenvinphos (Wysocka-Paruszewska et al., 1980). Mirex and chlordecone have been demonstrated to enhance hepatotoxicity of chloroform (Hewitt et al., 1983) and carbon tetrachloride (Bell and Mehendale, 1985). Chemical reagents and by-products in technical or formulated preparations also can have biological consequences. Certain impurities in malathion can cause delayed toxic effects, whereas others in the mixture provide an antagonistic protective effect (Umetsu et al., 1981; Toia et al., 1980). Formulations of the herbicide 2,4,5-T can contain very small quantities of the very potent 2,3,7,8-tetrachlorodioxin, causing considerable controversy (Hanify et al., 1981; Chapman and Schiller, 1985). Estrogenicity Estrogens may be viewed toxicologically from two vantage points, i.e., as tumor initiators and as tumor promoters. The latter class may be more accurately considered as syncarcinogens (Concon, 1988). Thus, when considered in this context, the estrogenicity of certain pesticides adds another dimension to the potential health hazards of these substances. To establish whether a given compound is a typical estrogen, the activity of such a compound can be compared to that of estradiol with respect to a variety of parameters (Table 17.7), in both in vitro and in vivo studies. A popular in vivo test for estrogenic activity has been uterotropic activity, viz., the increase in uterine weight that follows single or multiple administration of the compound. This increase in uterine weight could be measured within 6 hr, primarily reflecting water imbibition, or after 24, 48, or

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Table 17.7 Characteristics Exhibited by Estrogenic Compounds In vivo • Increase in uterine weight (water imbibition, usually 6 hr; increase in DNA, RNA, and protein)a • Increase in uterine glycogen (18-hr test) • Increase in uterine enzymes (glycolytic and hexose monophosphate shunt, peroxidase, ornithine decarboxylases) • Increase in uterine progesterone receptor • Formation of uterine induced protein (IP) In vitro • Binding to uterine, liver, testes, or pituitary cytosolic estrogen receptor (direct assay) • Competitive inhibition of binding of 3H-estradiol to cytosolic estrogen receptor (indirect assay) • Translocation of uterine cytosolic receptor into nucleus • Induction of uterine progesterone receptor • Formation of uterine IP a

DNA, deoxyribonucleic acid; RNA, ribonucleic acid.

72 hr, reflecting true growth, i.e., increase in protein and deoxyribonucleic acid (DNA). Additional parameters that have been used to various extents to determine estrogen action in vivo include inhibition of the uptake of administered radiolabeled estradiol, elevation of uterine glycogen, increase in uterine ribonucleic acid (RNA) polymerase, elevation of uterine peroxidase, increase in carbohydrate metabolizing enzymes, increase in uterine ornithine decarboxylase activity, increase in progesterone receptor concentration, and elevation in the synthesis of the so-called induced protein (IP) (Kupfer and Bulger, 1982). The ability of the administered compound to diminish the levels of the cytosolic estrogen receptor and elevation of the levels of the nuclear receptor have also been used as indications of estrogenic action. Certain in vitro activities of compounds have been assumed characteristic of estrogens. These include competitive inhibition with the binding of radiolabeled estradiol to the uterine cytosolic estrogen receptor and binding of the compound to the cytosolic estrogen receptor and translocation of the receptor to the nucleus during the incubation with uteri (Jensen and DeSombre, 1973; Ruh et al., 1973). Estrogenic compounds have been shown in vitro to increase the levels of the progesterone receptor in an organ culture of uteri (Leavitt et al., 1977) and to increase the levels of the IP (Katzenellenbogen and Gorski, 1972). Examples of pesticides shown to be estrogenic are DDT (Bitman et al., 1978; Bulger and Muccitelli, 1978), methoxychlor (Bulger et al., 1978; Mitchell and West, 1978), and kepone (Eroschenko, 1979; Gellert, 1978).

The metabolites or analogs of o,p′-DDT with the ethane chain intact are estrogenic (Bitman et al., 1978). Hydroxylation and methoxylation of the benzene rings in the 3 or 4 position did not abolish estrogenicity. p,p′-DDE is not estrogenic (Bulger and Muccitelli, 1978); DDT has been found to be uterotropic in mice, rats, mink, and birds (Kupfer and Bulger, 1982). Like estradiol, it induces ornithin decarboxylase, decreases the uterine cytosol estrogen receptor, and elevates the nuclear receptor for this hormone as well as that of progesterone in the cytosol. Furthermore, Stancel and associates (1980) have found that like estradiol, DDT increases the synthesis of uterine-induced protein. The evidence also suggests that DDT itself and not as its metabolite is active. The weakly estrogenic activity of methoxychlor, in contrast, is attributed to one of its metabolites (Ousterhout et al., 1979). Only when incubated with rat liver microsomes does methoxychlor become highly estrogenically active (Nelson et al., 1978). Kapoor and colleagues (1970) have shown that methoxychlor is metabolized in rats and mice mainly to the mono- and bisphenol derivatives. In terms of their ability to inhibit estradiol binding to estrogen receptors, bisphenol and monophenol have about 1/30 and 1/100 the activity of diethylstilbestrol. The estrogenicity of kepone has been shown both in rats and Japanese quail (Gellert, 1978; Eroschenko, 1979). Uterine growth, persistent vaginal estrus, and structural abnormalities in the oviduct cells were some of the symptoms observed. All these three estrogenic pesticides are also carcinogenic in experimental animals. Thus, the evidence regarding the relationship between estrogenicity and carcinogenicity is further supported by these observations.

mans can be determined with the aid of common longterm carcinogenicity studies. In order to determine considerably lower risks, enormous numbers of animals would be necessary for a corresponding study (e.g., for an assumed risk of 1/106, more than 106 animals). Thus, in order to obtain statistically confirmed results, high doses of a carcinogen must be applied in animal experiments that commonly use relatively low numbers of animals (Somogyi and Appel, 1999). In these experiments, amounts up to a so-called maximal tolerable dose (MTD) are administered. Many of the pesticides, when tested at MTD in rodents in vivo, show a tumorigenic tendency in these species. Such observations, together with the epidemiologically proven finding of environmental influence on carcinogenicity, have created problems for scientists and regulatory agencies, for in practice it turns out to be very difficult to prove that any chemical present in the environment is in fact carcinogenic. Reasons for the difficulty in determining the safety with respect to carcinogenic potential include the following: 1.

2.

Carcinogenicity If a contaminant or a pesticide has been found to be an animal carcinogen, it is extremely difficult to show the dose safe for humans and to determine the risks at the doses to which humans are exposed. In addition, if an impurity present in a pesticide has been shown to be carcinogenic, it has to be removed or limited to such an extent that the exposure dose does not pose any intolerable risk to the consumer. The level of the impurity should be controlled by adequate analytical methods. The elucidation of the mechanism(s) of carcinogenicity is complex. In most cases, experimental studies with laboratory animals have dealt with the pathological evaluation of tumors. However, it is rather difficult to find a simple answer to the mechanism of carcinogenicity (Somogyi et al., 1993; Moolgavkar, 1990; Olin et al., 1995). Only very high risks that would not be tolerable for hu-

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3.

4.

The distinction between carcinogenesis and tumorigenesis is not always clear. It should be based on the potential to cause malignancy: i.e., uncontrolled somatic cell division including formation of malignant tumors is carcinogenesis and formation of benign tumors is tumorigenesis. For example, the term liver carcinoma is reserved for the former case, whereas hepatoma refers only to the latter. It is also actually impossible to distinguish between benign and malignant liver tumors in the mouse. The dose-effect relationships are not clearly established. Many types of dose-effect relationships that are mainly determined by the nature of the inducer and the tissue are also susceptible to other environmental and artificial factors. Moreover, it is not usually possible to establish a threshold value or minimal dose for induction of carcinogenesis. Species specificity makes it difficult to relate the effects or lack thereof of new chemicals on certain experimental animals to human safety. Species specificity involves differences in life span. Some stress the importance of nonhuman primate tests; others advocate the entire-life tests that are possible with mice and other animals that have shorter life spans. There is also a lack of adequate epidemiological data on human cases that could serve to establish species guidelines relative to other experimental animals. Although many chemicals are

epidemiologically implicated carcinogens, not all of them can cause carcinogenesis in animals under experimental conditions simulating the epidemiological ones (Matsumura, 1985). Despite these difficulties, the usefulness of animal experiments and epidemiological studies is apparent. In 1961, the National Cancer Institute (NCI) initiated a new program to test substances of environmental interest for possible carcinogenicity. In a study initiated in 1962, a few pesticides were among the compounds tested in Fischer rats (Weisburger, 1978). In 1964, tests on a large number of pesticides (fungicides, herbicides, and insecticides) began in two hybrid strains of mice (Innes et al., 1969). The results from that study, coupled with increased interest in pesticides due to the report of the Secretary’s Commission on Pesticides and Their Relationship to Environmental Health (1969), increased the emphasis on thorough studies of commonly used pesticides. Accordingly, a sizable number of such compounds have been tested. For the evaluation of the carcinogenic potential of a pesticide, the Environmental Protection Agency (EPA) in cooperation with the NCI has formulated the following guidelines. The EPA requires that the alleged carcinogen be tested in both sexes in at least two species of experimental animal. Guidelines for a testing protocol have been developed by the NCI. The test must be continued for duration close to the life span of the animal. The doses are chosen up to the MTD at which no overt toxic symptoms are observed. In reality, at the highest dose, moderate signs of toxicity such as body weight loss and changes in the liverbody weight ratio, are observed. Only positive results indicating a statistically significant increase in the cancer rate in relation to dose in both species are accepted as proof of carcinogenicity, because of the differences in cancer susceptibility among different animal species. In view of the scarcity of human cancer data, positive or suggestive data in human subjects are taken more seriously even if they are less complete than animal data. If there is clear-cut epidemiological evidence to show that a chemical is carcinogenic in humans, that evidence alone is enough to warrant its listing as a suspected human carcinogen. After a confirmatory test in experimental animals, the compound may be shifted to the category of definite human carcinogen if the results are positive. The EPA Cancer Assessment Group (EPA-CAG) also uses other types of circumstantial evidence in determining carcinogenicity potentials. Several kinds of in vitro tests are currently regarded as useful for this purpose. Two carcinogenicity tests used are the Ames test and the DNA repair assay, which show whether the chemical in question

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has the ability to affect DNA and its functions. Other methods include a promoter assay method designed to test the ability of compounds to promote the development of cancer and a transformation assay to assess the potential to transform cells. The transformed cells are recognized by their characteristic spindle shape as opposed to the generally round shape of normal cells, and by their tendency to pile up and form cell aggregates, as compared to normal cells in culture, which assume a uniform monolayer in the transformation assay. A positive result in any one of these tests does not immediately indicate the carcinogenic potential of a compound, but an accumulation of positive results may establish such potential. Because of the generally low incidence of human cancer, many efforts have been made to create a reasonable model to assess the probability of risk of carcinogenesis by exposure to low doses of carcinogens. The model originally offered by the EPA was a “one-hit” model, which was based on the assumption that even if only one molecule of DNA (or one cell) is altered by a chemical and starts the process of proliferation, this alteration is enough to initiate cancer in the animal. The model was criticized on several grounds, the most serious of which was that the assumption on which the theory was based was not sound. The model currently used by the EPA and NCI is the multistage model, which is based on the assumption that the basic behavior of a chemical interaction involving biological material is a function of dose and duration. In this model, the accuracy of the extrapolated data at low incidence levels is not a problem, since the basic theoretical background is different from that of the previous model, which relies on probability in a given population. In addition, in this model, many phenomena and interactions, including natural background incidence, can be taken into consideration as integral parts of the multistage reactions in the cumulative process of cancer development. In general, pesticides can be classified into two distinct groups, those that are carcinogenic after conversion to proximate carcinogenic metabolites and those that become carcinogenic by a nitrosation reaction or a chemical degradation to other forms. The majority of pesticides suspected to be carcinogenic belong to the chlorinated hydrocarbon group, although the question of their carcinogenicity is still not quite settled. Kemeny and Tarjan (1966) and Tarjan and Kemeny (1969) conducted a long-term multigeneration feeding study on the effects of DDT in mice maintained on a diet containing 2.8–3.0 ppm of p-p′-DDT, which corresponds to 0.4–0.7 mg/kg/day. They observed that the increased incidence of leukemia and tumors was statistically significant with respect to controls in the second and third

generations. By the fifth generation, the incidence of pulmonary carcinoma had increased 25-fold. However, no effects on reproduction were found. The meaning of such a multigeneration study is not certain; the fetus is subjected to both direct exposure to DDT and indirect stress through poisoning of the mother. Innes and coworkers (1969) screened a number of pesticides for one generation and listed chemicals that produced statistically significant increases in mouse hepatic tumor incidence. Although the dose employed for DDT was rather high (i.e., lower than the LD50 but much higher than the calculated daily intake in humans, which is approximately 0.5 µg/kg/day), this study at least established that DDT could cause hepatoma in the mouse. However, there is a large gap between such a finding and the establishment of carcinogenicity. Even though the carcinogenicity of DDT has not been satisfactorily proved, it is clear that tumorigenic— whether malignant or benign—effects intensify during continuous exposure of generations of mice to DDT. Tomatis (1970) found that tumors occurred in the second generation of mice (BALB/c strain) administered 2.8–3.0 ppm DDT. Tomatis and coworkers (1972) found that in a twogeneration experiment with the CF-1 minimum-inbred strain of mice the incidence of liver tumors, but not of lymphomas, osteomas, or lung tumors, increased at all levels (2, 10, 50, and 250 ppm in the diet) of DDT and also appeared at an earlier age than in controls. It would be unfair not to mention the studies indicating that DDT and its analogs are not carcinogenic and those showing that these compounds have antitumorigenic properties in certain cases. Ortega and associates (1956) and Ottoboni (1969) were not able to show that DDT has carcinogenic effects in the rat. Unfortunately, because these studies were conducted for a relatively short period, they do not resolve the extremely important question of whether this difference is species-specific. Although there is some evidence that DDT may increase the incidence of hepatic tumors in the rat (Fitzhugh and Nelson, 1947) at very high doses, the issue of the effect of DDT in the rat has not been settled. Laws (1971), in contrast, found that DDT had an antitumorigenic effect on the rate of success in transplanting an experimental ependymoma in the mouse. These animals were exposed to a 5.5-mg/kg/day dose of technical DDT (given in the diet at 33.3 mg/kg), and an ependymoma known to have a 100% take rate in mice was transplanted 1 week after the initial feeding of DDT. The incidence of subcutaneous tumors was 92.1% in the DDTtreated mice and 100% in controls. Of 89 animals receiving DDT and a transplant, in 7 no tumor developed throughout the experiments. Moreover, the DDT-fed mice lived longer. A few other studies also indicate that in DDT-

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treated animals cancer is less likely to develop in response to experimentally introduced chemical carcinogenic agents, probably because DDT has the ability to induce hepatic microsomal detoxification mechanisms (Okey, 1972). Studies on other chlorinated hydrocarbon pesticides are less extensive. BHC isomers have been examined by Japanese scientists. Nagasaki and colleagues (1971) fed mice 6, 66, and 660 ppm of technical BHC and found that hepatomas developed in 24 weeks in all 20 of those fed 660 ppm. Later, Nagasaki and coworkers (1972) compared the effects of four isomers of BHC and found that only α-BHC induced hepatoma in mice at 250- to 500-ppm levels in the diet after 24 weeks. Yellowish nodules up to 0.3–2.0 cm in diameter appeared at these doses of α-BHC; no carcinogenic effects were observed with the β-, γ- (Lindane), and δ-isomers. Independently, Goto and associates (1972) studied the effects of α-, β-, and γ-BHC and their possible metabolites, 1,2,4-trichlorobenzene, 2,3,5-trichlorophenol, and 2,4,5-trichlorophenol, on male mice. Their finding was essentially the same, that α-BHC is the most active analog in causing hepatoma in mice. They observed tumors of 0.5- to 1.5-cm diameter. Studies on the carcinogenicity of cyclodiene pesticides are also limited. According to Innes and associates (1969), mirex and Strobane (terpene polychlorinate) are definitely tumorigenic, whereas Telodrin (isobenzan) and Thiodan (endosulfan) are not at doses of 0.215 mg/kg (0.646 ppm) and 1.0 mg/kg (3 ppm), respectively, fed by stomach tube for up to 28 days or in the diet after 28 days. As for dieldrin and aldrin, Fitzhugh and colleagues (1964) studied their effects on rats for 2 years. They found 18 tumors (in 41 rats) at 0.5 ppm, 15 in 41 rats at 2 ppm, and 16 tumors in 40 rats at 10 ppm of aldrin or dieldrin in the diet. In contrast, Deichmann and coworkers (1967) and Walker and associates (1968) could not observe any increase in tumor incidence in rats fed aldrin (5 ppm) or dieldrin (0.1, 1.0, and 10 ppm) for 2 years. Aldrin and dieldrin are known to produce an increase of hepatic tumors in mice. There is also some evidence that aldrin, dieldrin, and endrin have mild antitumorigenic activities in the rat (Deichmann, 1970). Studies on the carcinogenicity of organophosphates and carbamates are less conclusive. Andrianova and Alekseev (1970) concluded that carbaryl is a carcinogenic agent in the rat. They either fed 30 mg/kg of carbaryl or administered 20 mg carbaryl via paraffin pellets implanted in the subcutaneous cellular tissue. Six tumors developed in 22 rats after 22 months as compared to only one tumor per 46 control rats. They state that the tumors observed had malignant characteristics. The lack of data on organophosphates is astonishing considering the importance of

this group of compounds as replacements for the more persistent chlorinated hydrocarbon pesticides. Epidemiological studies on the carcinogenic pesticides conducted thus far do not show a direct cause-effect relationship because of the multiplicity of possible causative agents. Furthermore, direct extrapolation of tissue susceptibility in animals to humans, as a basis of epidemiological studies, cannot be justified scientifically. Davies (1975) studied the adipose tissue concentration of DDT and its metabolites in relation to the incidence of different cancers. He found no significant differences in comparisons with matched controls based on race, sex, and socioeconomic status. In this case, blacks, older persons, and the less affluent had higher DDT levels. Again, such studies have inherent difficulties in obtaining reliable data for comparative purposes, even though it can be assumed that those with higher levels of DDT in the tissues had greater exposure to the pesticide. There have been no valid epidemiological studies regarding the possible carcinogenicity of chlorinated hydrocarbon pesticides in humans. Published studies of this sort (Hunter and Robinson, 1967; Jager, 1970; Laws et al., 1967; Ortelee, 1958; Versteeg and Jager, 1973) have involved an inadequate number of subjects and lacked sufficient latency periods or exposures. Citing the lack of epidemiological studies that can justify any conclusions, the International Agency for Research on Cancer (IARC, 1974) determined that this group of pesticides are noncarcinogenic in humans. Although the issue is still far from settled, the overall implications suggest possible relationships between the occurrence of cancer and exposure to certain pesticides. Because there are no valid scientific bases for establishing a safety threshold for carcinogens, the many carcinogenic pesticides that simultaneously contaminate our food and water collectively must be viewed with alarm. In this regard, the basic characteristics of the majority of pesticides can be summarized as follows: 1. 2.

3.

They cause an increased incidence of tumors in mice and rats in vivo. Tumor formation is most frequently observed in the liver and at very high pesticide doses at which animals show some sign of toxicity. Other target organs may include lungs, thyroid, duodenum, and stomach. With a few exceptions, such as toxaphene and technical chlordane, most of these tumor-causing pesticides do not increase frequencies of mutation as judged by microbial mutagenicity tests such as the Ames test.

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4.

They have been shown to be unreactive with DNA or possess physicochemical characteristics that indicate that they are not likely to react with DNA.

Mutagenicity Mutagenic effects represent the result of genetic impairments caused by introduced chemicals. Since actions that are mutagenic occur at the cellular level (particularly in chromosomal DNA and other cellular and nuclear constituents related to the function of cell division), they are also potentially carcinogenic with respect to somatic processes. However, the science of mutation or mutagenicity limits itself to detection of increases (or in limited instances, decreases) in the rate of genetic mutation above normal. Hence, mutagenicity is often studied independently of carcinogenicity. Numerous pesticides have shown potency as mutagenic agents in various screening techniques (Ames et al., 1975; Marshall et al., 1976; Shiau et al., 1981; Moriya et al., 1983). Mechanisms of changing genetic information include alkylation, substitution of base analogs, intercalations, as well as chromosome breakage and cross-linking. Some pesticides (e.g., toxaphene) are innately mutagenic (Hooper et al., 1979), whereas others require activation to a degradation product or a derivative, such as N-nitroso derivative of carbofuran (Nelson et al., 1981). Mutagenic potential of selected pesticidal chemicals is summarized in Table 17.8. An area of major concern involves the finding of nitrosamines as impurities in a variety of herbicides (Bontoyan et al., 1979; EPA, 1976; High Levels, 1976; Nitrosamines in pesticides, 1976; Nitrosamines in cutting oils, 1976; Oliver, 1979) and the postulation that various nitrogen-containing pesticides (including herbicides) and fertilizers may be nitrosamine precursors (Eisenbrand et al, 1975; Elespuru and Lijinsky, 1973; Elespuru et al., 1974; Fishbein, 1978, 1979; Oliver, 1979) via in situ and/or in vivo nitrosations. Many nitrosamines are among the most potent carcinogenic and mutagenic substances known (Montesano and Bartsch, 1976). In regard to the potential for nitrosamines in both pesticides and agricultural residues, two major, rather distinct problem areas can lead to human exposure and hence potential risk to consider. One area focuses on the concern that certain nitrogen-containing pesticides (e.g., carbamates, ureas, amides, and anilides) as residues in soil, water, and plants may be nitrosated by endogenous nitrate or by other nitrosating agents, e.g., NOx from automobile, tractor, or truck exhaust or other fuel consumption. The other area concerns the possibility that a wide variety of

Table 17.8 Mutagenicity of Selected Pesticidesa Pesticide

Assay method and effects

Carbaryl

Plant root tips Rat, three-generation teratogenic Mice sperm Plant root tips Rat sperm Marsupial somatic cell, chromosome aberration Rat, dominant lethal Drosophila spp. Salmonella and Serratia spp., dominant lethal Drosophila spp., recessive lethal Salmonella and Serratia spp., dominant lethal Drosophila spp. Salmonella and Serratia spp, (DDD) Onion root tips Bacterial species including Salmonella spp., Escherichia coli Sprouts, Crepis capillaries Albino rat, chromosomal changes Barley meiosis, no effect Chick embryo, teratogenic Salmonella and Serratia spp. Fungi, point mutation, reverse mutation Neurospora crassa Maize cells, chromosome break Mouse embryo, mild teratogenic Mice, dominant lethal Onion, root tips, chromosome break Allium cepa root tips Root tips of several plants Pisum sativum root cells Leghorn chick embryo, teratogenic Leghorn chick embryo, teratogenic Human hematopoietic cells, no effect on chromosomes Human hematopoietic cells and mice bone marrow cells, no effect on chromosomes Male mice, first 3 weeks mutagenic at low doses Allium cepa root tips, mitosis Barley, meiosis slight effect Mouse female, teratogenic Mouse embryo, mildly teratogenic Calf thymus DNA, reaction Male mice, first 3 weeks mutagenic at low doses Male mice, dominant lethal and translocation

DDT

DDA DDE, DDD, DDM Dichlorvos

Dieldrin Endrin DFP Ethylene dibromide Ethylene oxide

Fenthion Hempa Lindane

Malathion (technical 95%) Malathion Parathion, methyl Metepa Parathion Phosphamidon Sodium arsenate Systox (demeton) Trichlorfon Tepa a

Dose 0.5 and 0.25 saturation 100–500 mg/kg/day 105 mg/kg Saturated solution 50–70 mg/kg 10–50 ppm 80 mg/kg No effect below 0.14 mmol/L —b 0.14 mmol/L — No effect — — 3.2–6.5 mmol/L Vapona strip 10% Solution 0.25 mg/Testis 1000 ppm Soaked — — — 0.14 M 1 Part/20 parts air 40 and 80 mg/kg 25, 50, 125 mg/kg 4 times/week 12.5–50 ppm 0.00125%–2% Solid particles 250 ppm 3.99 mg/Egg, 6.42 mg/Egg 5.7 mg/Egg 23.50 µl/mL 5–100 mg/kg 0.782–100 mg/kg 0.01%, 0.005%, 0.0075% 1000 ppm Soaked, 500 ppm spray 45 mg/kg, Intraperitoneal injection 7 or 10 mg/kg 100 µg 0.156–20 mg/kg 2.5 mg/kg, Intraperitoneal injection

DDT, dichlorodiphenyltrichloroethane; DDA, dichlorodiphenylacetic acid; DDE, dichlorodiphenyldichloroethylene; DDD, dichlorodiphenyldichloroethane; DDM, p,p′-dichlorodiphenylmethane; DFP, diisopropyl fluorophosphate; DNA, deoxyribonucleic acid. b —, data not available. Source: Compiled from Matsumua (1985) and Fishbein (1978).

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pesticides that are applied to soil and plants may contain nitroso compounds as impurities. These may arise from three most probable routes of N-nitroso contamination: formation in the manufacturing process, formation during storage, and contamination of amines used in the manufacturing process (Oliver, 1979). The most important products thus far shown to contain nitrosamines are the dinitroaniline herbicides. The greatest focus has been on trifluralin, a preemergent soil-incorporated herbicide that is widely used on cotton and soybeans as well as on several other field crops, fruits, and vegetables to control broadleaf weeds and annual grasses. It should be noted that the current levels of di-n-propyl nitrosamine in trifluralin are at least an order of magnitude lower than the 150 ppm originally discovered, and it is anticipated that further decreases will result from further modifications of the production process. Pesticides N-nitrosated under in vitro and in vivo conditions in the laboratory have included the fungicide ziram (zinc dimethyldithiocarbamate), the insecticides carbaryl (1-naphthyl methylcarbamate) and propoxur (o-isopropoxyphenyl methylcarbamate), and the herbicides benzthiazuron [N-(2-benzothiazolyl)-N-methylurea], simazine [2chloro-4,6-bis(ethylamino)-s-triazine], and atrazine [2chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine]. Because of its possible effects on future generations, mutagenesis poses a far more serious threat to the future of the human race than carcinogenesis. Teratogenicity Effects on development of an embryo or fetus caused by maternal exposure have been demonstrated for a wide range of pesticidal chemicals (Longo, 1980). Types of effects include abnormal growth rates of offspring; incomplete development; skeletal, visceral, and biochemical anomalies; as well as abortions. Certain chlorinated hydrocarbons have demonstrated teratogenic activity, mirex (Khera et al., 1976), Kepone (Chernoff and Rogers, 1976), and DDT (Dean et al., 1980), among others. One of the most potent teratogens known is 2,3,7,8-tetrachlorodibenzodioxin (TCDD), an impurity in 2,4,5-T herbicide (Courtney and Moore, 1971). Although 2,4,5-T has been reported to be teratogenic at high doses (Hood et al., 1979), controversy persists primarily in regard to the low levels of the dioxin in the product (Hanify et al., 1981; Hay, 1982; Coats, 1987). Organophosphates—for example, dimethoate (Khera, 1979), monocrotophos (Jaffee and Mitrosky, 1979), methylparathion (Gupta et al., 1985)—have shown some effects but are implicated less commonly than the more persistent chlorinated hydrocarbons. The mercurial

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fungicides and ethylenethiourea can induce developmental abnormalities (Lewerenz and Bleyl, 1980). Mechanisms usually are considered identical or similar to those for mutagenic and carcinogenic effects. Detrimental influences on reproduction, aside from teratogenicity, can also result from exposure to some pesticides. The types of effects include decreased number, size, and growth rate of offspring; fetotoxicity; lowered sperm count; as well as behavioral abnormalities such as decreased libido. Chlorinated hydrocarbons have been shown to affect reproduction in many species. As described earlier, estrogenic effects are caused by o-p′-DDT (an impurity in DDT), Kepone, methoxychlor, and two of its phenolic degradation products, seemingly acting directly as an estrogen (Kupfer and Bulger, 1976; Bulger et al., 1985). Acephate decreases luteinizing hormone in mice (Rattner and Michael, 1985). Aldrin, dieldrin, hexachlorobenzene, and DDE (a degradation product of DDT) all induce some symptoms of reproductive dysfunction (Matsumura, 1985; McEwen and Stephenson, 1979). The fumigant dibromochloropropane (DBCP) has caused azoospermia and abnormal hormone levels in factory workers and experimental animals (Sandifer et al., 1979; Kluwe et al., 1983a; Rao et al., 1983). The mechanism of toxic action may act through alkylation of certain proteins by an oxidative metabolite of DBCP (Kato et al., 1980; Kluwe et al., 1983b). Carbaryl has been associated with abnormal sperm shapes in occupationally exposed men (Wyrobek et al., 1981). Benomyl fungicide has been shown to have deleterious effects on the male reproductive system in rats (Carter et al., 1984), as do triphenyltin compounds (Snow and Hays, 1983). It should be noted that no known human birth defect, has yet been unequivocally related to pesticide exposure under the conditions in which these chemicals are normally used. Epidemiological studies may provide some indications of a possible correlation. However, because of multiple exposures to various agents, a direct association is often difficult to establish. It is quite likely that pesticide combinations can enhance teratogenic potential. Even though no connection between human congenital birth defects and pesticide exposure has been established so far, the evidence that these chemicals are teratogens in animals should be sufficient grounds for considering these substances as teratogenic hazards for humans. 17.2.5

Residues in Food

Public concern over pesticide residues in food has been increasing since the 1980s. A national survey conducted by the Food Marketing Institute (1988) showed that approxi-

mately 75% of consumers are very concerned about pesticide residues in their food—a higher percentage than that of consumers worried about cholesterol, fat, salt, additives, or any other components. Contributing to such concerns have been the discovery of hazardous effects of certain pesticides, such as ethylene dibromide and chlordane, that were once deemed safe and publicized acute food poisonings caused by improperly used pesticides. Such was the case with aldicarb contamination of watermelon in the western United States and Canada in 1985 and the daminozide (Alar) controversy about apples and cherries in 1989 (OTA, 1988; Smith, 1990). The high level of uncertainty concerning the health effects of pesticide residues has further heightened consumer concern. To get information on residues in the food supply, several countries have established national monitoring programs for a wide range of pesticides (Hajslova, 1999). Information on food contamination can be obtained through two different but complementary approaches. The regulatory or commodity monitoring services commonly focus on raw agricultural commodities and measure levels of residues in individual lots of either domestic or imported foods to determine compliance with established tolerances or guidelines. In contrast, the total diet studies analyze food as consumed to determine the dietary intakes of pesticides. An indication of the effectiveness of measures introduced to reduce food residues is obtained in this way. The overall objectives of food contamination monitoring are to safeguard health, to improve the management of food and agricultural resources, and to prevent economic losses (van der Kolk, 1991; Galal-Gorchev, 1993; Hajslova, 1999). Monitoring data obtained within surveil-

lance programs (random sampling, no prior knowledge or evidence of illegal pesticide residues) are valuable for the identification of particular pesticide-commodity combinations that occur frequently. The pesticide residues in food in the United States found in one such survey are shown in Table 17.9. A summary of residue monitoring data sorted in order of incidence has been published by Hamilton and associates (1997); it was based on the data available from Australia, Brazil, Denmark, New Zealand, Sweden, and the United States. As an example, pesticides that were found in more than 10% of a particular commodity sampled are shown in Table 17.10. In the Hamilton and colleagues (1997) survey, grain protectants used on cereals show the highest incidence of residues. For fruits and vegetables, the only generalization that can be made is that those types with high surface area and “fuzzy and waxy” coatings and some root foods tend to have higher frequency of positive findings. Appraisal of the whole data set containing 208 items (combinations) shows that residues of acephate, benomyl/carbendazim, carbaryl, chlorothalonil, chlorpyrifos, cypermethrin, deltamethrin, dicofol, dimethoate, dithiocarbamates, endosulfan, fenitrothion, fenvalerate, malathion, parathion, permethrin, and vinclozolin were identified to account for most of the positive findings. This is not surprising since all of these pesticides are widely used the world over. Pesticide residues detected in major classes of foodstuffs are summarized in Table 17.11. Virtually every food group is contaminated with residues. Of all the major classes of compounds, the chlorinated hydrocarbons are the most commonly found contaminants, led by DDT and

Table 17.9 Pesticide Residues in Food in the United States in 1991 Samples with residues Food Grains and grain products Milk, dairy products, and eggs Milk and dairy products Fish, shellfish, and other meats Fish and shellfish Fruits Vegetables Other

Origin

Samples, no.

Below permissible level, %

Above permissible level, %

Domestic Import Domestic Import Domestic Import Domestic Import Domestic Import Domestic Import

495 396 809 216 536 611 2168 3481 3811 4311 462 918

40.8 25.5 12.5 10.2 41.6 23.2 50.9 34.1 30.6 28.3 19.5 17.9

0.8 2.3 0 0 0.2 0.2 0.5 1.3 1.3 3.3 0 3.5

From Food and Drug Administration Pesticide Program, Residue Monitoring 1991 (5th Annual Report).

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Table 17.10 Pesticide/Commodity Combinations Characterized by High Frequency of Incidence from the Combined Data of Several National Monitoring Programs Pesticide Carbaryl Fenitrothion Chlorothalonil Vinclozolin Vinclozolin Carbaryl Endosulfan Dithiocarbamates Chlorpyrifos Chlorpyrifos Endosulfan Endosulfan Chlorpyrifos Dithiocarbamates Endosulfan Permethrin Dithiocarbamates Carbaryl Endosulfan Permethrin Dicofol Permethrin Dithiocarbamates Endosulfan Dimethoate

Commodity

Samples tested, no.

Samples with residues, %a

Sorghum Cereal grains Celery Strawberry Kiwi fruit Oats Lettuce, leaf Tomato Tomato Peppers Melons Spinach Kiwi fruit Pome fruit Cucumber Lettuce, head Grapes Barley Lettuce, head Celery Citrus fruit Spinach Cucumber Common bean Peas

156 12,759 375 509 126 130 483 866 3,613 1,428 952 183 127 135 1,659 2,451 285 188 2,169 422 1,022 183 613 574 837

96.8 74.5 65.6 48.1 43.7 33.9 32.5 27.8 27.8 27.7 26.9 21.9 20.5 19.9 19.8 18.3 17.9 17.6 16.8 15.4 14.5 12.6 11.9 11.7 11.5

a Only examples with an incidence greater than 10% are listed. Source: Compiled from Hamilton et al. (1997) and Hajslova (1999).

its degradation products and metabolites, followed by dieldrin, hexachlorocyclohexane (BHC), and polychlorinated biphenyls (PCBs). DDT was previously found in almost all major classes of food; the ban of its use initiated in the United States (Pesticides, 1972) and earlier in other countries (Edwards, 1970) in part accounts for fewer reports of its detection in fruits and vegetables. However, the numerous reports of its presence in other foods, particularly meats and dairy products, reflect its persistence in the adipose tissues of animals as well as the abiotic environment. Products of animal origin as well as mother’s milk usually contain residues of chlorinated hydrocarbon pesticides. The residue content of mother’s milk is 10 to 30 times higher than that of cow’s milk. This difference appears primarily due to short lactation periods in humans in which to excrete many years of pesticide accumulation, whereas cows undergo intense lactation for a lifetime (Blanc, 1981). In recent years, the level of this pesticide and its derivatives in food has been declining.

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From May 1990 through July 1991, 806 milk samples from 63 metropolitan areas in the United States were collected and analyzed for pesticide residues by the FDA. In the samples from eight of the metropolitan areas, no residues could be detected. Pesticide residues appeared to contaminate 398 milk samples, however. The most frequently occurring residues were 4,4′-dichlorodiphenyltrichloroethane (p,p′-DDE) in 212 samples and dieldrin in 172 samples. The highest residue measured was 0.02 ppm p,p′-DDE (whole milk basis). These chlorinated pesticides have not been registered for agricultural use for about 20 years. Population exposures to pesticides in the diet can be assessed by a market basket total diet study conducted by the FDA at frequent intervals. The findings of the Total Diet Study 1991 on the occurrence of pesticides in food are summarized in Table 17.12. In general, residues present at or above 1 ppb could be measured. Malathion continues to be the residue most frequently found; it is used on a wide variety of crops, including in many post-

Table 17.11

Pesticide Residues Reported from Various Countries to Contaminate Major Classes of Foodstuffs

Food class

Pesticide residuesa

Total diet

Aldrin, arsenicals, BHC and isomers, DDT and metabolites,b dieldrin,b hexachlorobenzene, PCB,b other chlorinated hydrocarbons,b various organophosphatesb Aldrin, BHC and isomers, carbaryl, heptachlor, heptachlor epoxide, hexachlorobenzene,b lindane,b DDT and metabolites,b dieldrin,b endrin, PCB,b TDE, other chlorinated hydrocarbons, various organophosphates Aldrin, benomyl, BHC and isomers, biphenyls, captafol, captan, carbamates, carbaryl, carbendazim, carbon disulfide, chlorpyrifos, ethylene dibromide, ethylene thiourea, fenitrothion, formothion, lindane, DDT and metabolites, dicofol, dieldrin, dimethoate, dinocap, disulfoton, endosulfan, ethion, methyl bromide, parathion, o-phenylphenol, phosmet, trichlorfon, zineb, other chlorinated hydrocarbons, organophosphates, thiabendazole, imazalil, methamidophos, methidathion, vinclozoline, procymidon Acephate, aldicarb, aldrin, arsenicals, BHC and isomers, captafol, captan, carbamates, carbaryl, carbofuran, ethylene thiourea, funsulfothion, fenthion, folpet, hydroquinone, lindane, linuron, malathion, maneb, mercurials, dacthal, DCPA, DDT and metabolites, diazinon, dicamba, dieldrin, dimethoate, endrin, EPN, ethephon, ethion, ethoprop, methamidophos, methyl bromide, MSMA, parathion, phosalone, prometryne, pyrocatechol, TDE, terbutylazine, terbutryne, thiofanox, trichlormetaphos, zineb, ziram, other chlorinated hydrocarbons, organophosphates BPMC, chlordimeform, ethylene dibromide, fenitrothion, hexachlorobenzene, IBP, isoprocarb, malathion, MCPA, diazinon, MTMC, phthalide, piperazine, piperonjyl butoxide, PCB, propham, quintozene, triforine, other chlorinated hydrocarbons BHC and isomers, mercurials, DDT and metabolites, dieldrin BHC and isomers, chlordane, heptachlor, lindane, DDT and metabolites,b diazinon, dieldrin, dioxins, endrin, methoxychlor, 2,4,5-T, other chlorinated hydrocarbonsb BHC and isomers, hexachlorobenzene, other chlorinated hydrocarbons Parathion Various chlorinated hydrocarbons

Dairy products

Fruits

Vegetables

Cereals, grains, and grain products Fish Meat and meat products Poultry Wines Honey a

Not all the pesticide residues listed under each class of foodstuffs are present in each type of food. The list simply indicates the types of contaminant pesticides that have been found in that class of food in different countries. The large number of pesticide residues in fruits and vegegtables reflects the many different residues in these classes. BHC, benzene hexachloride; DDT, dichlorodiphenyltrichloroethane; PCB, polychlorinated biphenyl; TDE, tetrachlorodiphenylethane; DCPA, 2,2-dichloropropionic acid; EPN, o-ethyl-o-[p-nitrophenyl]-phenylphosphorothiolate; MSMA, monosodium methanearsonate; BPMC, 2-sec-butylphenyl methylcarbamate; IBP, Iprobenfos (s-benzyl-o,o-diisopropyl phosphorodithioate); MCPA, 2-methyl-4-chlorophenoxyacetic acid; MTMC, m-tolyl-N-methylcarbamate. b The most common pesticide residues found in that class. Source: Compiled from Concon (1988), Zabik (1987), and Hajslova (1999).

harvest uses on grains. From 1987 to 1991, the presence of malathion decreased from 23% to 18% and that of DDT from 22% to 10%. The actual analysis of the market basket surveys based on intakes of 19-year-old males, the theoretical maximal food consumers, showed that the fats and oils and the meat and poultry groups were most likely to contain pesticide residues. None of the intakes approached the acceptable daily intake (ADI); the highest measured intake was for dieldrin at 16% of the ADI (Jones, 1992). In summary, daily pesticide intakes averaged less than 1% of the ADI.

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In a similar survey of foods that an infant would conceivably ingest, no pesticide levels approached the ADI. Dieldrin was highest for this population group also: 48% of the ADI for infants and 36% for toddlers. For these young consumers, the foods most likely to contribute to residues would be fats, oils, and whole milk (Gartrell et al., 1985a). In infant and invalid diets monitored in a subsequent study (Georgii et al., 1989), average intakes were no more than 0.33% ADI and intakes of persistent pesticides continued to decrease. In addition, these data confirm that the levels of residues in food have not changed or have decreased since the previous data were gathered

Table 17.12 1991

Occurrence of Pesticides in Total Diet Study in

Pesticidea Malathion Chlorpyrifos-methyl DDT Dieldrin Endosulfan Methamidophos Chlorpyrifos Dicloran Acephate Diazinon Dimethoate Chlorpropham Heptachlor Lindane Omethoate Ethion Hexachlorobenzene Permethrin BHC, α-isomer Chlordane Parathion Quintozene Dicofol

Food items contaminated, no.

Occurrence, %b

167 97 93 73 67 58 51 44 42 42 34 28 24 22 22 21 20 16 13 12 12 12 10

18 10 10 8 7 6 5 5 4 4 4 3 3 2 2 2 2 2 1 1 1 1 1

a

Including parent compounds, isomers, metabolites, and related compounds. DDT, dichlorodiphenyltrichloroethane; BHC, benzene hexachloride. c On the basis of 936 items, a food item can contain several pesticides. Source: From Food and Drug Administration Pesticide Program, Residue Monitoring 1991 (5th Annual Report).

(Black, 1988; Gartrell et al., 1985a, 1985b; Georgii et al., 1989; McLoed et al., 1980). Similar data for private laboratories and from the Grocery Manufacturers of America show declines in pesticide levels (Brown, 1986). Many of the techniques presently used in food processing yield a considerable reduction of pesticide residue levels. Many types of residues are degraded to harmless products during processing as a result of heat, steam, light, and acid or alkaline conditions. In addition, major reductions of residue levels result from their physical removal by peeling, cleaning, or trimming of foods such as vegetables, fruits, meat, fish, and poultry. The reported values are, therefore, very likely higher than consumed values. The subject of commercial processing stages and their effects on pesticide residue levels in a variety of food groups has been comprehensively reviewed (Sissons and Telling, 1979; Zabik, 1987; Hajslova, 1999).

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The various studies described here and similar surveys globally indicate interesting trends with respect to the movement of pesticide chemicals through human populations. Following are some key points that have become apparent from the extensive surveys on pesticide residues in humans: 1.

2.

3. 4.

17.2.6

The chemicals found to accumulate in humans are mostly chlorinated hydrocarbons. These are stable and lipophilic and are detectable at very low concentrations. The levels of any given pesticide vary geographically and among various segments of the population. Changes in the levels of stable pesticides over time are rather slight. The major factor determining the distribution of pesticides in the body is fat content; however, there are a number of other factors influencing the final distribution of residues. Acceptable Daily Intakes

The World Health Organization/Food and Agricultural Organization (WHO/FAO) has recommended ADIs of pesticide residues, i.e., the maximal daily intake of a chemical which, during a lifetime, appears to be without appreciable risk. These values are updated at regular intervals. The judgment is based on toxicological, pharmacological, and biochemical data on experimental animals and, wherever available, on humans. The actual estimates of the ADI, if based on animal data alone, are usually 1% of the no-effect level of the most sensitive animal species tested. This 100-fold safety factor, although an approximation, results from the general approximate 10-fold difference in sensitivity between species and another 10-fold difference within species. The ADI is also applicable to the main pesticide metabolites if those detected in food are the same as those in experimental animals, and if the amounts present are of similar order of magnitude. If not, the ADI applies only to the parent compound, and a different value may be prescribed for the metabolite on the basis of a separate set of data. The ADIs of various pesticides according to the WHO/FAO Expert Committee on Pesticides are listed in Table 17.13. It should be noted that the ADI does not take into consideration multiple exposures to different pesticides or the influence of environmental factors. Thus, a low intake of pesticide may become toxicologically significant in the presence of similar toxicants, malnutrition, disease, and other factors.

Table 17.13 Food and Apriculture Organization/ World Health Organization Guidelines for Acceptable Daily Intake, Maximal Residue Limit, and Extraneous Residue Limit for Pesticide Residues in Human Foodsa ADI (mg/kg body weight)

Pesticide sec-Butylamine Captan Cartap Chlordimeform Chlorothalonil Coumaphos Cyanofenphos Cyhexatin DDT Dimethoate Diquat Ethiofencarb Fenamiphos Fenthiion Formothion Guazatine Lindane Omethoate Paraquat Parathion-methyl Phosmet Primicarb Propargite Tecnazene Thiophanate-methyl Trichlorfon Triforine

0.1 0.1 0.1 0.0001 0.03 0.0005 0.005 0.008 0.005 0.02 0.008 0.1 0.0006 0.0005 0.02 0.03 0.01 0.0005 0.002 0.001 0.005 0.01 0.08 0.01 0.08 0.01 0.02

MRL or ERL (mg/kg) 0.1 20 2 0.2 0.02 0.05–2 0.05–5 0.05–3 0.2–5 0.2 0.2 0.1–5 0.01–1 0.05–2 0.01–10 0.05–50 0.5–5 0.1–2 0.05–2 0.02–5

a

ADI, acceptable daily intake; MRL, maximal residue limit; ERL, extraneous residue limit. Source: From WHO (1979).

17.2.7

Residue Monitoring Program

The federal regulations governing pesticide use were first enacted as the Federal Insecticide Act (FIA) in 1910. The FIA was later replaced by the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) of 1947, which, as amended, remains the basis for regulating the use of pesticides today. Currently, federal jurisdiction over pesticide residues in foods is divided among four bodies: the EPA, the FDA of the U.S. Department of Health and Human Services, and the Food Safety and Inspection Service (FSIS) and Agricultural Marketing Service (AMS) of the U.S. Department of Agriculture (USDA). Their authority

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for this work arises primarily from five laws: FIFRA; Federal Food, Drug and Cosmetic Act (FFDCA); Federal Meat Inspection Act (FMIA); the Poultry Products Inspection Act (PPIA); and the Egg Products Inspection Act (EPIA). The EPA, under FIFRA, must register a pesticide before it can be distributed or sold in the United States. All pesticides that are legally sold and used on a domestically grown or imported food crop, therefore, must be registered with EPA for that crop prior to use. Compounds that may be deemed safe by the EPA but are not registered for a specific crop cannot be used legally on that crop, except under special state-approved circumstances. The most important health-related provision of the registration process is the requirement that pesticide manufacturers submit scientific data to the EPA demonstrating both effectiveness and lack of significant health risks for each crop use. The EPA also establishes tolerances for pesticide residues on raw commodities under Section 408 of the FFDCA. Enacted in 1954, this law stipulates that tolerances are to be set at levels deemed necessary to protect public health, while taking into account the need for “an adequate, wholesome, and economical food supply.” Section 408 thus explicitly recognizes that pesticides confer benefits and risks and that both should be taken into account in setting raw commodity tolerances. Pesticide residues that concentrate in processed foods above the levels authorized to be present in or on their parent raw commodities are governed by FFDCA Section 409, the law governing food additives. Under Section 409, such residues must be proved safe, i.e., show a “reasonable certainty” that “no harm” to consumers will result when the additive is put to its intended use. Consideration of benefits, however, is not authorized. Moreover, Section 409 contains the Delaney Clause, which prohibits the approval of a food additive that has been found to “induce cancer” (or, under the EPA’s interpretation, to induce either benign or malignant tumors, i.e., is oncogenic) in humans or animals. Thus, if any portion of a crop to which an oncogenic pesticide has been applied is processed in a way that will concentrate residues, the EPA’s policy is to deny not only a Section 409 tolerance for the processed food but also a Section 408 tolerance for residues of the pesticide in or on the raw commodity. Further, if the required Section 408 tolerances cannot be granted for a food-use pesticide, the EPA must also deny registration of the pesticide under the FIFRA. The aim of the Delaney Clause enacted in 1958 was to prevent cancer in humans. The decades since the 1970s witnessed a remarkable increase in the scientific knowledge about the causes of cancer and mechanisms of carcinogenesis as well as about analytical chemistry, per-

mitting accurate determination even of trace amounts of chemicals. Hence, in many quarters, it has been considered reasonable to abolish or modify the Delaney Clause against the background of current knowledge. The removal of the Delaney Clause has been effectively accomplished with the passage of the Food Quality Protection Act (FQPA), which became effective August 3, 1996. This act amends both the FFDCA and the FIFRA to provide a comprehensive regulatory scheme for pesticides. The new law (FQPA) establishes a health-based safety standard for pesticides in foods and uses “reasonable certainty of no harm” for cancer and noncancer health effects as the standard in processed foods and raw agricultural commodities. This is generally interpreted to mean that there is a “one-in-a-million” chance that the residue would cause cancer (a risk of less than one excess cancer per million persons exposed). The FQPA also establishes special protection for infants and children by mandating that the regulatory framework take into consideration the special susceptibility of children that is due to their eating habits and tighten the health standard (e.g., foods that children eat in large quantities need to be determined through a study of eating habits). One of the most important provisions of the FQPA requires the EPA to have positive evidence of safety for children before it can register or reregister a pesticide for use on food. The new law requires careful review of prenatal and postnatal risks as well as use of up to an additional 10-fold margin of safety for children when data are incomplete or cause concern. FQPA requires a number of other changes in pesticide regulations as well: new standards for pesticide tolerances, consideration of endocrine disruptors, consideration of a pesticide’s benefits, new reviews of minor use, and consideration of antimicrobial pesticides. Globally, since the inception of the World Health Organization (WHO) in 1949, food safety issues have represented one of the basic parts of its overall health mandate. Concern about the toxic hazards posed by pesticides, noted in documents published in 1953, initiated programs within WHO. The need to solve growing international problems resulting from pesticide application urged the Food and Agriculture Organization (FAO) to convene a Panel of Experts on the use of pesticides in agriculture in 1959. In 1961, a joint meeting of this FAO Panel and WHO Expert Committee on Pesticide Residues was held to implement these recommendations. The concept of permissible level calculated from ADI, the food factor, and the average weight of a consumer were accepted. Nevertheless, no conclusions were reached on a strategy to estimate internationally acceptable tolerances at that time.

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Significant differences in residue tolerances, therefore, existed even among countries of the same region, although it was accepted that the range of the residues actually remaining when the food is first offered for consumption, following “Good Agricultural Practice” (GAP), should be taken into account. The FAO Conference on Pesticides in Agriculture held in 1962 strongly stated the need to investigate the reasons for these differences and, if possible, to find a way to harmonize them. In response to this situation, the Codex Alimentarius Commission (CAC) was established in 1962 under the joint sponsorship of the FAO and the WHO to act as the body to protect human health from food-borne hazards. The first Joint Meeting of the FAO Committee on Pesticide Residues in Agriculture and the WHO Expert Committee on Pesticide Residues (referred to as JMPR) in 1963 studied the toxicological properties of a number of pesticides. In the same year, the FAO Working Party on Pesticide Residues identified the approaches essential for arriving at unified tolerances. Since 1966, JMPR has been establishing maximal residue limits (MRLs) for pesticides in food commodities. The forum for achieving agreement among member countries on international MRLs is provided by the Codex Committee on Pesticide Residues (CCPR), established in 1968. The process of international standards development in this field has been reviewed by Hajslova (1999). A large and complex system has thus been established for controlling the presence of pesticides as residues in foods. Nearly all elements of the agricultural system are actively involved in controlling the use of pesticides. Regulation thus takes the form of both premarket federal and state government controls as well as postmarket policing. The private sector also plays an effective role in regulation. Growers, food processors, and food manufacturers all have an economic interest in the safe use of pesticides. The use of pesticides in agriculture, however, is not without serious health and environmental risks or costs. Nevertheless, their residues in the human food chain are monitored by numerous agencies. The adequacy of testing of pesticides, however, remains questionable. Carcinogenic effects in humans may not appear until 20–30 years after exposure to the carcinogen. Similarly, mutagenic effects may surface generations after the initial exposure. Nor have the possible adverse effects of pesticide residues on behavior been thoroughly examined. Surprisingly, very few studies examine the possible adverse interactions among different pesticides and among pesticides and other components used during crop production. The cumulative effect of chronic low-dose exposure to a single pesticide and the additive effect of small quantities of many different pesticides have also not been thoroughly examined.

Multifaceted research in this field needs to continue to achieve the following objectives (Jones, 1992; Deshpande and Salunkhe, 1995): 1.

2.

3.

4. 5.

6.

7. 8.

Identify, isolate, and manufacture small selective pesticides that have only a single species as their target. Foster the use of biodegradable insecticides and growth regulators that have minimal adverse environmental impact. Determine the optimal time of pesticide application so that application coincides with times when the insect population is on the rise or when the pest is at its most vulnerable phase, such as during the reproductive cycle. Explore the use of natural predators, parasites, and pathogens. Isolate and identify naturally occurring pesticides in plants, such as pyrethroids, which are highly toxic to insects but seemingly have no effects on either rats or dogs. Currently, these naturally occurring and frequently target-specific products are very expensive. Utilize insect and pest traps that contain mating and feeding attractants (pheromones), oil, and other nonchemical means of pest control. Continue the search for genetically pest-resistant plants and genetically sterile insects. Emphasize control rather than kill of insect populations.

17.3 INDUSTRIAL CONTAMINANTS The majority of food contaminants of industrial origin are complex organic substances that are either products or byproducts of industrial chemical processes. In some instances, the contaminant of interest may be an impurity in the final product that arises during the manufacturing process. In other cases, these compounds are released to the environment as a result of human activity, eventually contaminating the food supply. One of the complexities involved with industrial contaminants is the multiplicity of substances involved. Over 43,000 chemical substances were listed by the EPA in its initial inventory of chemicals subject to the Toxic Substances Control Act. Under most conditions of use, these chemicals do not pose a threat to the safety of the food supply, but incidents such as those involving several polyhalogenated hydrocarbons (PHHs), such as polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs), indicate the potential hazard.

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Some of the key incidents that triggered the intense interest in the PHHs as occupational, environmental, and food contaminants and, more importantly, human health issues are listed in Table 17.14. Many of these incidents resulted from improper use or disposal with the result that animal feed or human food was contaminated. Alternatively, the material was released into the environment at relatively high concentrations. The early advantages of the PHHs in both agriculture and industry were quickly tempered by acute and chronic effects of these materials in the environment and in humans. During the late 1960s there was a growing awareness that the persistence, toxicity, and rapid redistribution of these chlorinated compounds at sites remote from any production or use meant that lowlevel, long-term contamination of all compartments of the environment and the food chain was inevitable. Their apolarity and lipophilicity also make PHHs bioaccumulate in fatty tissue through the food chain (Wells and de Boer, 1999). Initially, the food contamination studies included PCBs and PBBs in the United States and PCBs in Europe and Japan. By the mid-1980s, there was a growing realization, fueled by incidents like that at Seveso in 1973, that there was a secondary source of contamination from PHHs via the incineration of materials containing polyhalogenated waste. The polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and 2,3,7,8-tetrachlorodibenzo-p-dioxins/furans (TCDDs/ TCDFs), in particular, were added to the list of compounds for analysis in the market basket studies. Other PHHs such as chlorinated bornanes (CHBs, toxaphene) were included in the U.S. food survey programs and in German monitoring programs. However, current information on the polyhalogenated diphenyl ethers (PCDEs and PBDEs) and polychlorinated naphthalenes (PCNs) in food have only been generated by monitoring or research studies of individual research institutes. The chemical structures of important compounds of the PHH group are shown in Figure 17.3. In this section, the toxicological aspects of some of the major industrial contaminants that find their way into our food chain are briefly described. 17.3.1

Polychlorinated Biphenyls

First synthesized in 1881, PCBs are a complex mixture of different chlorobiphenyls and isomers, in which the isomers are two compounds with the same number of chlorine substituents on the biphenyl molecule but with the substitution occurring at different locations. Approximately 210 possible chemical isomers can be produced from the chlorination of biphenyls; these isomers range from the dichloro- to the nonachloro- derivatives (Kruell,

Table 17.14

Main Uses and Sources of Polyhalogenated Hydrocarbons Together with Examples of Incidents of Acute and Chronic Exposure to Humans

Polyhalogenated hydrocarbons (PHHs)

Abbreviation

Polychlorinated biphenyls

PCBs

Polychlorinated terphenyls

PCTs

Polychlorinated dibenzo-p-dioxins

PCDDs

Main uses/sources

Incidents

Dielectric in capacitors and transformers Hydraulic fluids, heat transfer fluids, additives in paints, carbonless copy paper

Yusho Rice Oil contamination, Japan, 1969 Yucheng, Taiwan 1979–81, Hudson river, General Electric PCB contaminated effluent Cow milk contamination from contaminated silage Seveso, Italy, 1976, trichlorophenol plant out of control

Impurities in pentachlorophenol (PCP), PCBs, Combustion product of PHHs

Polychlorinated dibenzofurans

PCDFs

Polychlorinated naphthalenes

PCNs

Polybrominated biphenyls

PBBs

Impurities in PCP, PCBs, combustion products of PHHs Impurities in PCBs, additives to rubber gas masks, insulation for detonators, insulation in cables Fire retardants

Polybrominated diphenyl ethers Polychlorinated diphenyl ethers Toxaphene (chlorobornanes)

PBDEs PCDEs CHBs

Fire retardants Impurities in chlorophenols Insecticide on cotton

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Vietnam 1962–71 impurity in agent orange defoliant, Missouri soil contamination, 1971 Yusho Rice Oil contamination, Japan, 1969, PCDFs as impurities Chloracne and acute yellow atrophy of liver Michigan cattle feed contamination with FireMaster BP6

Inhalation of “cotton dust” formulations

Reference Kuratsune (1980) Kashimoto and Miyata (1986), Horn et al. (1979) Fries and Marrow (1973) Wells and de Boer (1999)

Reggiani (1980)

Kuratsune (1980) Kimbrough (1980)

Landrigan (1980) WHO (1994) Becker et al. (1991) Deichmann (1973), Saleh (1991)

Figure 17.3 Chemical structures of some commonly occurring polyhalogenated hydrocarbons (PHHs).

1977). The commercial product was marketed as a mixture of several chloroderivatives as Arochlor (United States), Kanechlor (Japan), Phenochlor (France), and Colphen (Germany). The extreme stability and nonflammability made PCBs valuable in electrical transformers and capacitors and other electronic parts (JECFA, 1990). They were also used as vehicles for pesticide and paints, suspension agents for carbonless paper, and flame-retardants. About 9 million pounds of PCBs had been produced in the United States before their production was curtailed in 1974. The total volume produced globally for over 45 years was estimated at around 1.25 billion pounds, about 450 million pounds disseminated in the environment (Hecht, 1977). Much of this was discarded with no thought of environmental impact. In fact, the extreme chemical stability of these compounds caused many people to assume that they must be of low toxicity and of little consequence to the environment. This assumption was shown to be wrong by several incidents in the 1960s. In 1966, PCB contamination of wildlife was first discovered. Two years later, in Japan a poisoning outbreak affected 1000 persons and caused stillbirths and problems to infants whose mothers ate PCBcontaminated rice oil. The poisoning was given the name Yusho disease. During the search for a cause, it was learned that PCBs leaked out of the machinery during the

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heating of rice oil and contaminated it (Kuratsune, 1980). Since that incident, many investigators have found PCB residues in human populations, even those not occupationally exposed (Anderson, 1989; Jensen, 1989; Kimbrough and Grandjean, 1989). Several sources of PCB contamination of food have been located. One was recycled paper (which included PCB-containing carbonless paper) used to make cartons for food packaging. PCBs can migrate from packaging into food. Another source were PCB-containing coatings for silo interiors. Silage fed to cattle from these coated silos caused PCB contamination of the milk and meat. Both these sources of contamination have been virtually eliminated (Kreiss, 1985). Current human exposure to PCBs in food is primarily through fish and breast milk (Anderson, 1989; Belton et al., 1986; Jones 1992; de Boer et al., 1993; Leim and Theelen, 1997, Kannan, 1994). Bennet (1983) estimated that the mean daily intake of PCBs from food was between <0.1 and 1.9 µg/person/day. Between 1982 and 1984, the FDA estimated the average human PCB intake at 0.042 µg/day. For a 70-kg person the average intake was 0.6 ng/kg/day; for infants and toddlers, it was 1 µg/kg/day. Since the bans on manufacture and the restrictions on usage, there has been a decrease in the daily intake so that by 1987–89 the daily intake was 50 ng/day. The estimated daily intake of PCBs and dioxins in different countries is given in Table 17.15.

Table 17.15

Estimated Total Intake of Polychlorinated Biphenyls and Dioxins (Assuming 60 kg Person) in Different Countries

Country

Year of sampling

PCBs, µg/person/day

Year of sampling

Dioxin TEQs,b pg TEQ/person/day

Australia Canada

1990–92 1985

6.9 0.09

1989

Denmark Finland Germany

1988

33.6–126 92–140 290

1983 1987

14 4.1 93.5c

1989

93.5 23–96 44–67

India Italy Japan

1989

0.86

1977

3.3

260–480

Netherlands Norway New Zealand Sweden Spain Thailand United Kingdom United States

175 67 90 1982 1975 1990

54 7.5 6.8

1990

1.5 0.53

1980 1987

Vietnam

0.45 1.6 3.7

14–55 81–142

125 116 18–192

Reference Kannan (1994) Davies (1990), Gillman and Newhook (1991) Birmingham et al. (1989) Buchert (1988) WHO (1988) Georgii et al. (1989) Furst et al. (1990) Beck et al. (1989) Schery et al. (1995), Moser et al. (1996) Kannan et al. (1992) Di Domenico (1990) Matsumoto et al. (1987) Miyata (1991), Theelen (1991) Liem and Theelen (1997) Faeden (1991) Pickston et al. (1985) Vaz (1995) NEPB (1988) Jimenez et al. (1996) Kannan (1994) Duarte-Davidson and Jones (1994) MAFF (1992) Schaum et al. (1994) Schecter et al. (1994) Kannan et al. (1992)

a

PCB, polychlorinated biphenyl; TEQ, toxic equivalency; 2, 3, 7, 8-TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. TEQ, values expressed as toxic equivalency to 2,3,7,8-TCDD according to WHO guidelines. c Expressed as WHO-TEQ/day. b

17.3.2

Polybrominated Biphenyls

Unlike PCB contamination, PBB contamination of the food chain represented a localized problem. It is an excellent example of a serious hazard that arises from accidental contamination. In the early 1970s in Michigan, a flameretardant was inadvertently mixed with livestock feed. The PBBs responsible for this episode were a mixture of brominated biphenyls with average bromine content equivalent to about six bromine atoms per biphenyl molecule. Sold under the trade name FireMaster BP-6, they resulted in the contamination of meat, milk, and eggs sold in the Michigan area. The mistake was indeed costly, as 30,000 cattle, 6000 swine, 15,000 sheep, and 1.5 million chickens were destroyed, along with 5 million eggs (Biehl and Buck, 1987). Persons from quarantined farms and other groups with high exposures have been followed since the early 1970s. In all these studies, there were no findings that indicated that exposure to PBBs in any way affected the health

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of those exposed (Kimbrough, 1987). Although PBBs cause tumors in experimental animals, the levels used are over 10 times the highest measured human exposure. Thus, PBBs at the exposure levels encountered so far show no measurable chronic health effects. 17.3.3

Polychlorinated Dibenzo-p-Dioxins and Polychlorinated Dibenzofurans

PCDDs are present as impurities in pentachlorophenol (PCP) used as a wood preservative, and in herbicides such as agent orange, which is used as a defoliant. Both PCDDs and PCDFs are present as impurities in the high-temperature chlorination manufacture of other PHHs that have widely differing applications. These include chlorophenols, chlorobenzenes and associated derivatives, hexachlorophene, 2,4-D and 2,4,5-T, PVC, and epoxy resins (Jones and Alcock, 1996). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is the most toxic isomer of this group. Dioxins are also created by bleaching raw cellulose (paper

and cardboard) with chlorine and during the Kraft paper process (Fiedler et al., 1990). PCDDs and PCDFs are also formed in the incineration of material containing PCBs, PCNs, and PVC; the polybrominated dibenzodioxins (PBDDs) and polybrominated dibenzofurans (PBDFs) are produced from similar brominated analogs. These compounds are not manufactured and have no commercial value. PCDDs and PCDFs have been monitored intensely in foodstuffs in a number of different countries, particularly in Europe and the United States (Hallikainen and Vartiainen, 1997; Liem and Theelen, 1997). An overview of the mean values and/or the range of concentrations of PCDDs and PCDFs is given in Table 17.16. Many of the data for PCDDs and PCDFs in foodstuffs have been obtained from specific studies to investigate the transfer of these PHHs through particular routes of the food chain, such as the analysis of cow’s milk (Liem and Theelen, 1997; MAFF, 1997a, 1997b). These intense studies were undertaken because of the high concentration of these compounds in the milk fat, the rapid transfer from pasture to milk, and the wide cross section of the population, especially babies and infants, who have potentially high exposure to these compounds through the regular consumption of milk. The main route of dioxin exposure to food is via atmospheric deposition onto the plants in the proximity of incinerators that are consumed by the animals or into the water and taken up by benthic organisms and fish (Farland et al., 1994; Wells and de Boer, 1999). 17.3.4

Pentachlorophenol

PCP is an extremely effective pesticide and wood preservative. About 80% of the commercial production is used for wood preservation. Unlike many other contaminants, PCP does not resist environmental degradation, and thus residues in foods tend generally to be low and of little toxicological significance, except in cases of accidental misuse. Of major concern, however, is the presence of toxic isomers of the dioxin series in commercial PCP. Most PCP preparations are contaminated to some degree with dioxins, mainly the hexa-, hepta-, and octa- isomers. 17.3.5

Monocyclic and Polycyclic Aromatic Hydrocarbons

The diverse group of compounds commonly known as hydrocarbons range from the simple gaseous normal (n)-alkane, methane, to the poly-condensed aromatic ring systems, the polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene. They are ubiquitous in nature and are found in air, water, soil, and all living things and there-

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fore in food. These compounds exhibit a wide array of physical, chemical, biochemical, and carcinogenic properties and include volatile compounds; compounds with significant aqueous solubility (although all are lipophilic); compounds that are virtually insoluble in aqueous media, are relatively persistent in the environment, and can bioaccumulate in the food chain; compounds that may act as synergists or cocarcinogens or are well-established mutagens and carcinogens and those that are not mutagenic; and compounds that are relatively reactive chemically and biochemically, such as the PAHs (Moffat and Whittle, 1999). The PAHs have been described as the largest class of known environmental carcinogens (Grimmer, 1983) and are probably the most widespread environmental contaminants. The major origins of hydrocarbons in the environment can be summarized as biogenic, petrogenic, and pyrogenic. The biogenic hydrocarbons are those produced naturally by the biomass of plants, animals, and microorganisms on land and in the sea and as a result of degradative processes on the biomass. The petrogenic hydrocarbons include those naturally present in crude petroleum oils and gas, bituminous deposits, and deposits of oil shales and sands. The pyrogenic hydrocarbons are formed in natural combustion processes, mainly forest fires and combustion of oil and gas products and other fossil fuels. The groups of most interest from a food contamination viewpoint are the volatile monocyclic aromatic hydrocarbons (MAHs) and the PAHs. The structures of some of the important members of these two groups are shown in Figure 17.4. The probable human carcinogenic PAHs and their estimated relative potencies are listed in Table 17.17. PAHs that have been identified as the priority environmental pollutants by the EPA are listed in Table 17.18. Detailed descriptions of different aspects of hydrocarbons can be found in several excellent reviews (Whittle et al., 1977; Lee et al., 1981; Grimmer, 1983; Bjorseth and Ramdahl, 1985; Nevenzel, 1989; Seiber, 1990; Bartle, 1991; Gilbert, 1994; Tomaniova et al., 1997; Moffat and Whittle, 1999). Similarly to that of PHHs, the transfer of PAHs and other hydrocarbons to and through the food chain occurs via plants, aquatic animals, and beef and dairy cattle grazing on contaminated forage and formulated feedstuffs. Several aspects of the principles of concentration in the food chain that ultimately affect human exposure to PAHs as environmental contaminants have been reviewed (Hattemer-Frey and Travis, 1991; Seigneur et al., 1992; Butler et al., 1993; Giordano et al., 1994; EPA, 1995; Meador et al., 1995; Moffat and Whittle, 1999). The presence of PAHs in foods is well documented in several food basket studies (Dennis et al., 1983, 1991;

Table 17.16 Concentration of Polychlorinated Dibenzo-p-Dioxins and Polychlorinated Dibenzofurans in Foodstuffs from Different Countriesa

Country

Location

Austria

Urban

Belgium

Urban Rural

Canada Denmark Germany

Rural Rural Rural Urban

Japan Netherlands

Rural

Fish

Meat

8.5

0.19–0.56

29–34 8.1–39

1.2–7.2

31–43

0.9–2.6

0.87–0.33b 6.8–49

0.43–14

Dairy products

Milk

0.84 0.5–2.2 0.76–2.62

20.1–69.5 1–2.1 2.7–5.1 2.7–3.9 0.07 2.6 2 .32

1.4–1.8

a

0.8–3.2 1.9–6.5

0.015–0.23

0.7–2.5

0.13

0.7–2 1.2–13.5 0.18–0.22

New Zealand Norway

United States

0.05–0.07

0.61–1.75

Urban

Russia Sweden Switzerland United Kingdom

Fruits and vegetables

0.75 0.19–0.96b

0.97–1.7b 0.8–46c 2–10.4

0.33b 0.2–5 0.76–0.86

0.48 0.21 2.5–12.5

0.21 0.08 0.2–0.89

0.21 0.16 0.8–1.5

0.53–1.10

0.68–0.85

0.44–1.4

0.11b 0.28 –5 0.44–1.4 0.7–3.28 0.08 0.06

0.04

Cereals

0. 05

Reference Ris and Hagenmaier (1991) Ris (1993) Van Cleuvenbergen et al. (1993) Van Cleuvenbergen et al. (1993) Birmingham et al. (1989) Buchert (1988) Furst et al. (1990, 1992) Frommberger (1991) Furst and Wilmers (1995) Beck et al. (1989, 1990, 1994) Beck et al. (1990 Takayama et al. (1991) Liem et al. (1991), Liem and Theelen (1997) Traag et al. (1993) Liem et al. (1990, 1991) Buckland et al. (1990) Biseth et al. (1990) SNT (1995) Schecter et al. (1990, 1992) Hallikainen and Vartiainen (1997) Schmid and Schlatter (1992) MAFF (1992) MAFF (1995) Schecter et al. (1994a, 1994b; 1995) Cooper (1995)

Expressed as I-TEQ. Values are pg TEQ/g fat weight, except for vegetables (fresh weight) or as stated. I-TEQ, initial toxic equivalency values expressed as a summation of the total toxic equivalence of PCDDs/PCDFs; PCDDs, polychlorinated dibenzo-p-dioxins; PCDFs, polychlorinated dibenzofurans. b On fresh weight basis. c Freshwater fish.

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Figure 17.4 Chemical structures of monocyclic (benzene, toluene, and m-xylene) aromatic hydrocarbons (MAHs), and two-, three-, four-, five-, and six-ring polycyclic aromatic hydrocarbons (PAHs). Benz[a]anthracene, benzo[k]fluoranthene, benzo[b]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, and indeno[1,2,3-cd]pyrene are classified as possible human carcinogens by the International Agency for Research on Cancer. Fluoranthene, pyrene, chrysene, and benzo[g,h,i]perylene are suspected as acting as cocarcinogens.

Karl, 1997; de Vos et al., 1990; Lodovici et al., 1995). Data from one such survey are presented in Table 17.19. In this study, 33 items were chosen from among the most common components of the Italian diet. The estimated daily total PAH intake was about 3000 ng/person, and the carcinogenic PAHs contributed approximately half the daily intake (1400 ng/person). The highest total PAH concentrations were found in barbecued beef and pork and in pizza baked in a wood-burning oven. The next highest were found in leafy vegetable, chocolate, fruit, fried meat, cured meat, and cereal product items; the lowest were in potatoes, cooked fish, beverages, and eggs. However, cereal and milk products, meat, vegetables, and fruits contributed most to the dietary PAH intake because of the consumption pattern.

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For carcinogenic potency, the most significant factors from these dietary surveys appear to be the combined concentrations of benzo[a]pyrene and dibenz[a,h]anthracene. These surveys also indicate remarkably similar results in their overall estimates of exposure, ranging from more than a factor of about 5 and in the relative concentrations in the different types of foods, but differed according to the relative importance of the food items in the respective diets, e.g., consumption of oils and fats, coffee, cereals, sugar and sweets, and in the preparation and cooking of the products. Edible oils are clearly an important source of PAHs in the diet, either in food preparation or as food ingredients. There is very little information about the extent to which benzo[a]pyrene in food is available and absorbed from the gut. The availability is also strongly

Table 17.17 Probable Human Carcinogens and Estimated Relative Potencies

Polycyclic aromatic hydrocarbon Benzo[a]pyrene Benz[a]anthracene Benzo[b]fluoranthene Benzo[j]fluoranthene Benzo[k]fluoranthene Chrysene Cyclopenta[cd]pyrene Dibenz[a,h]anthracene Dibenzo[a,e]fluoranthene Dibenzo[a,e]pyrene Dibenzo[a,h]pyrene Dibenzo[a,i]pyrene Dibenzo[a,l]pyrene Indeno[1,2,3-cd]pyrene

Relative order-ofmagnitude potency 1.0 0.1 0.1 0.01 0.001 1.0

0.1

Source: Compiled from IRIS (1992) and EPA (1993).

influenced by dietary components (Stavric and Klassen, 1994). 17.3.6

Potential Human Health Hazards

The toxicological aspects of PCBs, PCDDs, and PCDFs have been extensively reviewed (Toxicological profile, 1989; Hong et al., 1993; Kimbrough, 1995; Liem and

Table 17.18 Priority Environmental Pollutants Identified in the Polycyclic Aromatic Hydrocarbon Family of Compounds Polycyclic aromatic hydrocarbon (PAH) Naphthalene Acenaphthalene Acenaphthalene Fluorene Anthracene Phenanthrene Fluoranthene Pyrene Benz[a]anthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Dibenz[a,h]anthracene Benzo[g,h,i]perylene Indeno[1,2,3-cd]pyrene

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Standard abbreviation NAP ANP ANY FLO ANT PHE FLU PYR BAA CRY BBF BKF BAP DBA BPE IDP

Theelen, 1997; Wells and de Boer, 1999). Among these classes of compounds, 2,3,7,8-TCDD is one of the most toxic compounds. Its potential to bioaccumulate in the food chain is a primary cause of concern. Furthermore, it is observed in many diverse segments of the environment. 2,3,7,8-TCDD has a very low oral LD50 value of 0.6 µg/kg body weight for the sensitive female guinea pig (Liem and Theelen, 1997). Similarly, 500 ng/kg in the diet was lethal to Rhesus monkeys within 2 months (Hong et al., 1989). Pohjanvirta (1991) observed large differences in sensitivity among test animals. The lethality of dioxin compared to that of other well-known poisons is presented in Table 17.20. The subchronic effects of these compounds in experimental animals include decreased body weights; increased liver weight; altered hematological and pathological changes in liver, thymus, and other lymphoid tissues; hair loss; and porphyria (Liem and Theelen, 1997; Kociba et al., 1976; Wells and de Boer, 1999). According to Holder and Menzel (1989), 2,3,7,8TCDD should be characterized as a multitarget carcinogen. It shows initiation by a hormonal mechanism and promotion of carcinogenic activities. Tumor promotion activity has been frequently found in the carcinogenicity studies of PCDDs and PCDFs (Flodstrom and Ahlbourg, 1989; Poland, 1991; Liem and Theelen, 1997). However, increase in the cancer mortality rate of humans was negligible in several of the well-documented cohorts made after the Seveso accident in which the local population were exposed to PCDDs and PCDFs after the accidental release at a chemical protection site (Bertazzi, 1991; Manz et al., 1991; Fingerhut et al., 1991). The induction of the hepatic microsomal mixedfunction oxidase system, cytochrome P450, on exposure to PHHs is the most commonly seen biological response. This type of enzyme induction is found in many organisms exposed to PCDDs, PCDFs, and PCBs and also in humans exposed to PCDFs through PCB-contaminated rice oil (Lucier, 1991; Liem and Theelen, 1997). Other responses include neurobehavioral changes, reproductive and immunotoxicological abnormalities, reduced hepatic vitamin A storage, vitamin K deficiency, and dermal effects. A number of other PHHs have toxicological effects very similar to those of PCBs and dioxins, although the degree of toxicity is normally lower than that of 2,3,7,8-TCDD. Among these, toxaphenes (polychlorinated camphenes) are known mutagens (Hooper et al., 1979). The toxicity of toxaphene is also being reviewed within the framework of the European Union (Wells and de Boer, 1999). One group that is particularly at risk with regard to toxaphene intake through food are the Inuit people in northern Canada (Quebec). Fish and marine mammals are an important part of the tra-

Table 17.19

Polycyclic Aromatic Hydrocarbons in Foodsa

Food product Vegetable Cauliflower Beet greens Squash Lettuce Tomatoes Potatoes Fruit Apples Peeled apples Citrus Cereal Bread Pasta Rice/corn Cooked fish Trout Cod Dried cod Dairy Milk and yogurt Cheese Meat Fried beef Fried pork Fried rabbit Chicken Beef liver Cured meats Eggs Barbecued Beef Pork Oils/fats Oliver oil Butter Chocolate Beverages Wine Beer Coffee Pizza

FLU

PYR

BAA*

CRY*

BBF*

BKF*

BAP*

DBA*

BPE

PAHb

CPAHc

2.45d 5.41 3.60 0.10 0.10 0.05

NDe 0.69 ND 0.43 0.40 0.42

0.07 0.25 2.39 0.96 0.01 0.03

0.24 3.66 1.65 0.84 0.13 ND

0.03 0.92 0.42 0.12 0.01 0.02

ND 0.14 0.10 0.08 ND 0.01

0.01 0.10 0.45 0.01 ND ND

ND ND 0.29 0.06 ND ND

ND 0.05 0.01 0.02 ND ND

2.79 11.21 8.91 2.62 0.63 0.52

0.34 5.06 5.30 2.06 0.14 0.05

2.36 0.26 0.05

3.46 0.45 ND

0.33 0.09 ND

ND ND 1.48

0.26 0.30 0.08

0.08 0.20 0.03

0.53 0.06 0.03

1.23 0.99 ND

0.02 ND ND

8.27 2.35 1.67

2.43 1.64 1.62

0.85 3.95 0.12

ND ND ND

0.31 0.03 0.06

1.88 1.88 0.52

0.04 0.04 0.03

0.02 0.02 0.03

0.02 0.02 0.02

ND ND 0.07

0.01 ND ND

3.12 5.93 0.86

2.26 1.98 0.73

0.43 0.02 0.38

ND ND ND

0.14 0.12 0.08

0.91 ND ND

0.13 0.26 0.03

0.01 0.03 0.02

0.03 0.01 0.03

0.11 0.15 ND

ND ND ND

1.75 0.58 0.53

1.32 0.57 0.15

0.47 0.46

ND ND

0.68 0.15

ND ND

0.15 0.09

0.02 0.02

0.34 0.01

ND 0.26

ND ND

1.65 0.99

1.18 0.53

1.00 0.55 2.06 0.17 0.29 2.68 0.10

0.01 5.38 ND ND ND ND ND

2.22 0.43 0.13 0.18 0.14 0.62 0.03

ND ND 1.18 ND ND ND ND

0.66 0.65 0.03 0.01 0.24 0.05 0.44

0.14 0.04 0.02 0.02 0.05 0.02 0.01

0.61 0.04 0.02 0.02 0.03 0.03 0.02

1.00 0.20 ND 0.20 0.16 ND ND

0.01 ND ND ND ND ND ND

5.66 7.27 3.43 0.60 0.90 3.41 0.59

4.64 1.35 1.37 0.43 0.61 0.72 0.49

10.81 1.85

1.27 5.29

0.53 0.50

24.66 5.36

1.20 0.40

0.61 0.08

1.45 0.12

1.53 ND

0.06 0.01

42.11 13.60

29.98 6.46

0.17 0.15 8.36

ND 1.77 ND

0.03 0.67 0.51

0.38 ND ND

0.26 0.02 0.48

0.06 0.03 0.11

0.10 0.02 0.33

ND 0.02 0.73

ND ND 0.01

1.01 2.67 10.53

0.84 0.75 2.15

0.09 0.05 1.18 0.18

ND ND ND 0.58

ND 0.11 0.10 9.11

ND ND ND 3.01

0.04 0.02 0.08 0.04

0.01 0.02 0.02 0.02

0.01 0.03 0.01 0.03

0.06 0.08 0.04 0.07

ND ND 0.01 0.06

0.20 0.31 1.45 13.10

0.12 0.26 0 .25 12.28

a

PAH, polycyclic aromatic hydrocarbon; FLU, fluoranthene; PYR, pyrene; BAA, benz[a]anthracene; CRY, chrysene; BBF, benzo[b]fluoranthene; BKF, benzo[k]fluoranthene; BAP, benzo[a]pyrene; DBA, dibenz[a,h]anthracene; BPE, benzo[g,h,i]perylene; CPAH, carcinogenic PAH. b Sum of the nine PAHs. c Sum of the six carcinogenic PAHs marked with asterisk. d Values expressed as ng/g product. e ND, not detected. f Pizza cooked in a wood-burning oven. Source: From Lodovici et al. (1995) and Moffat and Whittle (1999).

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Table 17.20 Dioxin’s Lethality Compared to Other Poisons in Decreasing Order of Toxicity

Substance Botulinum toxin A Tetanus toxin Diphtheria toxin Dioxin Bufotoxin Curare Strychnine Muscarin Diisopropylfluorophosphate Sodium cyanide

Animal

Minimum lethal dosea

Mouse Mouse Mouse Guinea pig Cat Mouse Mouse Cat Mouse Mouse

0.000000033 0.000001 0.0042 3.1 520 720 1,500 5,200 16,000 200,000

a Units: billionth of a mole per kilogram body weight. Source: From Rawls (1983).

ditional diet of the Inuit of northern Canada and Greenland. Breast milk of Inuit women contains three-fold higher toxaphene levels (0.3 mg/kg on a lipid weight basis) than the milk of Dutch and Swedish women (Vaz and Blomkvist, 1985; Stern et al., 1992; de Boer and Wester, 1993; Muir and de Boer, 1995). In contrast to PHHs, PAHs have very short biological half-lives in most species but may, nevertheless, have long-term effects. However, the impact of PAHs relative to that of all other xenobiotic chemicals is unclear. DNA adduct formation is a pertinent demonstration of exposure to PAHs through both diet and occupation (Rothman et al., 1993). Although there is also a direct relationship between the extent of cigarette smoking and the number of DNAbenzo[a]pyrene adducts, the relationship between DNAbenzo[a]pyrene adducts and the occurrence of lung cancer is less well defined (Walker et al., 1996). The more potent carcinogenic PAHs have, however, been shown to react more extensively with adenine residues in DNA (Dipple and Bigger, 1991). Sjogren and coworkers (1996) concluded that bacterial mutagenicity best reflects the cancer initiation potency of these chemicals, whereas the aromatic hydrocarbon receptor affinity reflects the promotive effect of some PAHs at the high doses applied in rodent carcinogenicity tests. They further concluded that initiation of carcinogenesis was provoked by reactive metabolites, whereas promotion was provoked by the parent PAHs. In summary, pesticides and chemical fertilizers are necessary to produce the quantities of food required to meet the needs of the world population. However, as noted earlier, their use should be integrated with other methods of pest management. Similarly, industrial chemicals have

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contributed greatly to current life-styles. Their risks versus benefits thus always should be assessed, and possible environmental impacts, especially as they affect the food chain, should be evaluated. Most studies have shown their residue levels to be low. However, continual surveillance is needed to ensure the safe use of these compounds. Cooking and/or processing can be an additional safety factor as many residues in food are reduced if the food is eaten after cooking and/or processing. Nevertheless, a reduction of human exposure to both pesticidal and industrial contaminants can only be beneficial. This can be achieved, in part, by reducing dietary exposure to these chemicals through changes in cooking practices and by reducing the overall concentration of these environmental contaminants in our food.

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Traag, W. A., Van Rhijn, J. A., van der Spreng, P. F., Roos, A. H., and Tuinstra, L.G.M. Th. 1993. Onderzoek naar polygechloreerde dibenzo-p-dioxinenb, dibenzofuranen en planaire gechloreende bifenylen in Nederlandse melk. Report No. 93.14, RIKILT-DLO. Umetsu, N., Mallipudi, N. M., Toia, R. F., March, R. B., and Fukuto, T. R. 1981. Toxicological properties of phosphorothioate and related esters present as impurities in technical organophosphorus insecticides. J. Toxicol. Environ. Health 7:481–497. Valtin, H. 1973. Renal Function Mechanisms Preserving Fluid and Solute Balance in Health. Little, Brown, Boston. Van Cleuvenbergen, R., Schoeters, J., Bormans, R., Wevers, M., De Fre, R., and Ryman, T. 1993. Isomer-specific determination of PCDDs and PCDFs in Flemish cows milk. Organohalogen Compounds 13:27–30. van der Kolk, J. 1991. The role of monitoring in decision making, In Pesticide Chemistry: Advances in International Research, Development and Legislation, ed. H. Frehse, pp. 485–491. VCH, Weinheim. Vaz, R. 1995. Average Swedish dietary intakes of organochlorine contaminants via foods of animal origin and their relation to levels in human milk. 1975–90. Food Addit. Contam. 12:543–558. Vaz, R. and Blomkvist, G. 1985. Traces of toxaphene components in Swedish breast milk analysed by capillary GC using ECD, electron impact and negative ion chemical ionization MS. Chemosphere 14:223–231. Versteeg, J.P.J. and Jager, K. W. 1973. Long-term occupational exposure to the insecticides aldrin, dieldrin, endrin and telorin. Br. J. Ind. Med. 30:201–202. Walker, A.I.T., Stevenson, D. E., Robinson, J., Ferrigan, L. W., and Roberts, M. 1968. Tunstall Laboratory Report TL/23/68, Shell Chemical Co., Sittingbourne, Kent, U.K. Walker, C. H., Hopkin, S. P., Sibly, R. M., and Peakall, D. B. 1996. Principles of Ecotoxicology. Taylor and Francis, London. Weisburger, E. K. 1978. Foreword. In A Rational Evaluation of Pesticidal vs. Mutagenic/Carcinogenic Action, eds. R. W. Hart, H. F. Kraybill, and F. J. de Serres. DHEW Publication No. (NIH) 78-1036, Bethesda, MD. Wells, D. E. and de Boer, J. 1999. Polychlorinated biphenyls, dioxins and other polyhalogenated hydrocarbons as environmental contaminants in food. In Environmental Contaminants in Food, ed. C. F. Moffat and K. J. Whittle, pp. 305–363. CRC Press, Boca Raton, FL. Whittle, K. J., Hardy, R., Holden, A. V., Johnston, R., and Pentreath, R. J. 1977. Occurrence and fate of organic and inorganic contaminants in marine animals. In Aquatic Pollutants and Biologic Effects with Emphasis on Neoplasia, ed. H. F. Kraybill, C. J. Dawe, J. C. Harshbarger, and R. G. Tardiff. Ann. N.Y. Acad. Sci. 298:47–79. WHO. 1979. Pesticide Residues in Food-1978. A report of the joint meeting of the FAO panel of experts on pesticide residues and environment and the WHO expert group on pesticide residues. World Health Organization, Geneva.

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in carbaryl-exposed employees. Environ. Health Perspect. 40:255–265. Wysocka-Paruszewska, B., Osicka, A., Brzezinski, J., and Gardowska, I. 1980. An evaluation of toxicity of thiuram in combination with other pesticides. Arch. Toxicol. 4 (suppl):449–451. Yarbrough, J. D., Chambers, J. E., and Robinson, K. M. 1982. Alterations in liver structure and function resulting from chronic insecticide exposure. In Effects of Chronic Exposures to Pesticides on Animal Systems, ed. J. E. Chambers and J. D. Yarbrough, pp. 25–59. Raven Press, New York. Zabik, M. E. 1987. Pesticides and other industrial chemicals. In Toxicological Aspects of Food, ed. K. Miller, pp. 73–102. Elsevier Applied Science, London.

18 Drug Residues

18.1 INTRODUCTION The use of drugs as additives in animal feeds has been approved since the early 1950s. Veterinary drugs are intended to maintain or improve the health of animals used for human food purposes. The food-producing animals in which veterinary drugs are approved for use comprise cattle, pigs, sheep, goats, poultry, and fish. The challenges of global population growth have led to increased efforts to find resources to enhance animal food production to its maximum. The developments in animal husbandry during the past 50 years have resulted in a proliferation of intensive production units, especially for pigs and poultry, where these animals are kept in limited space. Earlier such intensive units were prone to outbreaks of disease that could decimate them. It was soon found useful routinely to feed low levels of antibiotics prophylactically to animals to prevent the initial outbreak. The prophylactic treatment of animals with other drugs at low concentrations often improves the growth rates of these animals quite significantly, and hence, the practice grew, as did the use of hormones that are natural growth promoters. Currently, a variety of veterinary drugs and feed additives are routinely used for both therapeutic and prophylactic purposes in food-producing animals (Table 18.1). These range from antimicrobial agents to anthelmintics and ectoparasiticides. In addition, a number of drugs are available for zootechnical treatment, such as synchronization of estrus and anesthetics. Their use had a major impact in increasing the productivity and efficiency of livestock production for human food purposes. For example, the hormone diethylstilbestrol (DES) reduced the

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amount of feed required 10% to 20% by stimulating the growth of cattle, thereby saving approximately 8 billion pounds of cattle feed annually in the United States alone (ACS, 1980). Without its use, retail expenditures for beef would have increased by about $480 million per year. However, because residues that remain in edible tissues of animals may cause cancer in humans, the U.S. Food and Drug Administration (U.S. FDA) banned its use in livestock feed. The action was enlarged by the approval of other drugs that would replace DES for similar end uses. Additionally, antibiotics can be used effectively to retard spoilage and extend the storage stability of food products. It is estimated that more than 45% of the 2.1–2.5 million kg of antimicrobial drugs used in the United States annually is utilized for animal feed supplements (DuPont and Steele, 1987; Moorman and Koenig, 1992; Deshpande and Salunkhe, 1995). Over 100 million kg of these drugs is currently used worldwide annually for livestock production. Nearly 80% of poultry, 75% of swine, 60% of feedlot cattle, and 75% of dairy calves marketed or raised in the United States are estimated to have been fed an antimicrobial drug at some time during life. One survey estimated that the use of antibiotics in livestock production saved consumers more than $2 billion in 1973 (NAS, 1980). The widespread use and application of veterinary drugs as universal prophylactics, growth stimulants, and food and feed preservatives have thus provided significant economic benefits for both producer as well as the consumer. Intensive animal farming in developed countries thus has led to a substantial increase in the use of veterinary drugs and growth promoters since the 1980s. Concurrently, in recent years, public health concern over

Table 18.1 Selected List of Drugs Used in or Tested for in Animals Antimicrobials β-Lactams Benzylpenicillin, ampicillin, amoxicillin, cloxacillin, oxacillin, dicloxacillin, cephapirin, ceftiofur, other cephalosporins Tetracyclines Tetracycline, oxytetracycline, chlortetracycline, doxicycline Aminoglycosides Streptomycin, neomycin, gentamicin, lincomycin, bambermycin Macrolides Tylosin, erythromycin, spiramycin Sulfonamides Sulfamethazine, sulfadiazine, sulfamerazine, sulfadimethoxine, sulfamethiozole, sulfanilamide, sulfapyridine, sulfaquinoxaline Quinolones Oxolinic acid, nalidixic acid Fluoroquinolones Ciprofloxacin, enrofloxacin, danofloxacin, sarofloxacin Anthelmintics Mebendazole, benzimidazole, oxfendazole, thiabendazole Stilbenes Diethylstilbestrol (DES), hexoestrol, dienoestrol Resorcylic lactones Zeranol Thyrostats Methylthiouracil, phenylthiouracil, propylouracil, thiouracil, 1-methyl-2-mercaptimidazole Steroids Trenbolone, nortestosterone, methyltestosterone, estradiol, ethylestradiol, estrone Nonsteroidal antiinflammatory drugs (NSAIDs) Aspirin, paracetamol β-Agonists Clenbuterol, salbutamol, mabuterol, cimaterol, terbutaline Coccidiostats Nitrofurazone, ronidazol, nicarbazin, dimetridazol, furazolidon, dimetridazol Trace elements Arsenic, cadmium, lead Organochlorines/PCBs Dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE), dieldrin, polyhalogenated biphenyls (PCBs) Organophosphates Dichlorovos, malathion, parathion, coumaphos Carbamates/pyrethroids Cypermethrin, permethrin, deltamethrin Dyes Malachite green, leuco malachite green, gentian violet, methylene blue

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different aspects of drug use in veterinary medicine and the presence of drug residues in foods of animal origin has also grown rapidly. Residues are the amounts of the drug and/or its metabolites that are present in edible tissues of the treated animal at the time of slaughter or that have been passed into other edible products, such as milk and eggs. The safety of the consumer of food products originating from animals treated with veterinary drugs is of paramount importance. Whereas the medical use of drugs in humans involves voluntary treatment for short periods of time under controlled supervision, the exposure of consumers to traces of these drugs in the food supply is involuntary and uncontrolled. For example, antibiotic residues in dairy products have been shown to cause allergic reactions in certain individuals as well as technical problems due to starter culture inhibition in the dairy industry (Friend and Shahani, 1981; Deshpande and Salunkhe, 1995). Furthermore, the increased incidence of antibiotics in the food supply has been linked to the increased emergence of antibiotic-resistant microorganisms. In fact, concern about drug resistance persists despite the continuing efficacy of the oldest antibacterial drugs. A current example is Salmonella typhimurium DT 104, which is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines (Shearer, 1999). It is also suggested that evidence of further resistance of this strain to trimethoprim and fluoroquinolones is growing. In addition, there is concern about the emergence of methicillinresistant strains of staphylococci (MRSE), which is of particular concern when dealing with immunodepressed individuals. This organism is isolated from cattle, pigs, poultry, and a range of human foods. The number of isolates dramatically rose to more than 3000 by 1995 (Forsythe and Hayes, 1998). More strains of Salmonella hader have acquired resistance to ciprofloxacin (39.6%), a medically important antibiotic, than to any other Salmonella spp. serovar (1995). Ciprofloxacin resistance has also been reported in Campylobacter spp. in the Netherlands, Spain, and Austria (Jacobsreitsma et al., 1994; Feierl et al., 1994). It is also plausible that the veterinary use of enrofloxacin (another fluoroquinolone antibiotic) has resulted in the persistence and spread of ciprofloxacin-resistant Salmonella spp. in food animals. Initially, the use of enrofloxacin was limited to mainland Europe, but it was licensed for use in the United Kingdom in November 1993. The frequency of ciprofloxacin resistance in S. typhimurium DT 104 had risen from 0% to 14% by 1997 (Threlfall et al., 1997). These concerns have resulted in legislation and marketing authorization procedures for veterinary medicinal

products and feed additives at the national and international levels to safeguard animal and human health as well as the environment. In this chapter, public health concerns associated with veterinary drug residues in the human food chain are briefly addressed.

18.2 CLASSIFICATION OF VETERINARY DRUGS The term veterinary drug is defined as any substance applied or administered to any food-producing animal, such as meat- or milk-producing animals, poultry, fish, or bees, whether used for therapeutic, prophylactic, or diagnostic purposes or for modification of physiological functions, and for prevention and treatment of diseases in food-producing animals (FAO/WHO, 1984). The following are the major groups of veterinary drugs approved for use in foodproducing animals. 18.2.1

Antimicrobial Drugs

The antimicrobial drug group is certainly the most wellknown and extensive one and can be divided into antibiotics and antimicrobial drugs. Historically, antibiotics are defined as products from living organisms, which are not toxic to the producing organisms but are capable, at low concentrations, of inhibiting the growth of one or more microorganisms. Chemically synthesized compounds were classified as chemotherapeutic agents. Today that distinction is not valid since many antibiotics, such as chloramphenicol, are chemically synthesized, whereas some chemotherapeutic agents, notably quinine, are isolated from biological sources. There is also considerable literature related to the production of “natural” antibacterial components by lactic cultures used in the manufacture of fermented foods. A wide range of antibiotics, such as beta-lactams, tetracyclines, aminoglycosides, macrolides, and polymixins, are used for the treatment of infectious diseases in livestock production (Table 18.1) (Brander et al., 1982; Brander 1986; Deshpande and Salunkhe, 1995; van Leeuwen, 1999). Sulfonamides form a major class of antimicrobial drugs from which a range of compounds have been derived for fast-, medium-, and long-acting activity (The Merck Veterinary Manual, 1998). Sulfonamides are perhaps the most widely used group of drugs in animal husbandry, particularly in the swine industry to prevent respiratory disease.

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18.2.2

Antifungal Drugs

Several antifungal drugs are available, mostly for topical treatment. However, some, such as nystatin, are also used for treatment of gastrointestinal infections (Woodward, 1992). 18.2.3

Anthelmintics

A wide variety of drugs are used for the treatment of livestock animals against internal parasites. The presence of these parasites in animals might lead to distressing diseases and substantial economic loss (Bevill, 1988; van Leeuwen, 1999). Examples of this class of drugs include the benzimidazoles, such as thiabendazole, albendazole, febendazole, mebendazole, and oxfendazole (Table 18.1). These compounds are used for a wide range of parasitic infestations in cattle and sheep (Roberson, 1988). Levamisole and ivermectine are also widely used against gastrointestinal nematodes in, for instance, cattle, pigs, and sheep (Fink and Poras, 1989). 18.2.4

Ectoparasiticides

Organophosphates and synthetic pyrethroids are widely used against external parasites, such as fleas, lice, and ticks on cattle and sheep. Sheep “dips” containing these types of compounds are well known (van Leeuwen, 1999). 18.2.5

Steroid Hormones

Sex steroids like testosterone and estradiol are promoters of tissue growth and as such have been used in beef production as anabolic agents for several years. Synthetic steroid trenbolone and the nonsteroidal drug zeranol are also used for similar purposes. In Europe, the use of these substances for growth promotion was banned in 1988. However, their use for zootechnical purposes (e.g., synchronization of estrus, prevention of abortus) is still permitted (EEC, 1988). 18.2.6

Somatotropins

Somatotropins or growth hormones are naturally occurring peptide hormones found in all species. However, those used in dairy cattle to increase milk production are synthesized by using recombinant deoxyribonucleic acid (DNA) technology. There has been considerable controversy over the use of growth hormones, which is based on both economic considerations and animal and consumer safety issues (Juskevich and Guyer, 1990).

Administration of drugs to animals can be achieved by a variety of routes. They can be given orally via feed or water, injected subcutaneously or intramuscularly, or implanted. The route of choice is dictated by the desired effect of administration, the route of absorption of the drug, and the time required for the animal to dispose of the drug either as parent drug or as metabolites. This time is known as the residence time and the process of disposal of the drug as depuration.

been shown to affect water and nitrogen excretion (Steele and Beran, 1984). 18.3.3

Nutrient-Sparing Effect

Antimicrobial drugs have nutrient-sparing effects and act by depressing microorganisms in the intestinal flora that compete with the host animals for essential nutrients. This increases the nutrient availability through chelation and/or increasing their absorption from the gastrointestinal (GI) tract.

18.3 MODE OF ACTION 18.3.4 Antibiotics and other drugs are administered to livestock at therapeutic, prophylactic, or subtherapeutic concentrations. Therapeutic administration (200–1000 g drug/ton of feed, 220–1100 ppm) is used for disease treatment. Prophylactic doses (100–400 g/ton, 110–440 ppm) are used to prevent infectious diseases caused by bacteria or protozoa, whereas subtherapeutic administration is often used to increase feed efficiency and growth promotion. The FDA defines a subtherapeutic dose as ≥ 200 g (2.2–220 ppm) of the drug per ton of feed for 2 weeks or longer (NAS, 1980). Although the mechanisms of action by which veterinary drugs cause growth and feed enhancement are not well understood, it is likely that these drugs exert their effects on animals in at least four different ways (Franco et al., 1990; Moorman and Koenig, 1992; Deshpande and Salunkhe, 1995; Siddique, 1992). These are briefly described in the following. 18.3.1

Growth Promotion

Antibiotics, such as penicillins, inhibit the growth of many gram-positive organisms, a process that leads to development of increased numbers of Escherichia coli and other beneficial intestinal bacterial flora that play an important role in the synthesis of many essential vitamins and amino acids. Antimicrobial drugs when given in feeds on a continuous basis make the intestinal wall structure thinner and more adaptive. Therefore, the efficiency of absorption and utilization of nutrients is greatly enhanced. These types of drugs are primarily used to improve and contribute to the production of feedlot cattle, lamb, poultry, and swine (Steele and Beran, 1984; Deshpande and Salunkhe, 1995). 18.3.2

Metabolic Effects

Several reports suggest that veterinary drugs contribute to modification of metabolic reactions (Huber, 1971; Mandell and Sande, 1985). Tetracyclines, for example, have

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Disease Prevention

The effects of subtherapeutic feeding of antimicrobial drugs are quite pronounced. Response is greater in animals in contaminated and poor sanitation environments. The drugs suppress the microorganisms responsible for mild but unrecognized infections. Microbial production of growth-depressing toxins is also reduced. Antibiotics in feedlot cattle have been shown to reduce the incidence of liver abscesses from over 50% to about 18% with an increase in marketing weight (Siddique, 1992). Similar studies have indicated increase in weight gains and feed efficiency in other species of food animals.

18.4 SOURCES OF DRUG RESIDUES If drugs are properly used in compliance with requirements of the license authorized by the regulatory agencies, there should be no, or at the most, very low and acceptable, concentrations of residues. In several countries, the regulatory agencies have established tolerance limits for acceptable daily intake (ADI) and the maximal residue limits (MRLs) for the amount of the drug and/or its metabolites that might be present in animal products. Nonetheless, residues do occur and it is necessary to monitor their use in the human food chain. The concentration of residue that can be expected is a function of the degree of absorption of the drug from the GI tract, the dose given, and the withdrawal time. The most obvious reason for unacceptable residues is failure to observe the recommended withdrawal period for a given drug. This may be deliberate or accidental. In the former instance, there is a financial incentive for the farmer since disease in animals close to slaughter is undesirable. Since the sampling procedure for drug residue testing is often nationwide and quite random, the chances of catching such deliberate acts are slim. This circumstance often leads to a continuation of such practices by farmers.

Similarly, mistakes can be made with the feed close to slaughter, when animals should actually be on nonmedicated feed. Also, where single animals are being treated, poor treatment records and inadequate identification of the animal may result in such mistakes. Such accidents are more common in milk cows than in animals going to slaughter. Another source of drug residues can arise among swine if they are transferred to pens or are transported in vehicles in which medicated animals have been kept (Shearer, 1999). The new pigs can pick up residues from the leftover feed, layerage, or feces of the earlier pigs. A 1969 survey of animals slaughtered in Illinois, Indiana, Iowa, and Wisconsin indicated that 27% of swine slaughtered showed evidence of treatment with antimicrobials just prior to slaughter (Huber, 1971). Some 10% of the swine had residues, which were the result of feeding. It was assumed that these residues resulted from the lack of proper adherence to withdrawal periods or exceeding of the levels cleared for usage. In the beef animals sampled, 9% had positive findings for antimicrobials, 2% of which had penicillin residues; 17% of veal calves contained antibiotic residues, 7% attributed to penicillin; 21% of market

lambs had residue that tested positive, 4% with penicillin residues; and 26% of the chickens sampled contained residues, 6% of the birds with penicillin residues. Milk, traditionally a prime source of penicillin residues, had a relatively low incidence (0.5% of the samples tested) of antibiotic residues. This level was far below the levels found in a survey of market milk in the early 1950s. At that time 11% of the milk sampled contained antibiotic residues, primarily penicillin (Huber, 1971). A U.S. Department of Agriculture review of the incidence of residues occurring in carcasses indicated that 5.3% of 529 carcasses sampled had positive findings for antibiotic residues (Mussman, 1975). Chlortetracycline, oxytetracycline, tetracycline, streptomycin, neomycin, and erythromycin were the antibiotics included in the screening assays. Only 17 of 5301 samples (0.32%) yielded positive results for penicillin; 12 of 728 samples, or 1.6%, had positive findings for sulfonamides; nonspecific antimicrobial activity was found in 154 of the 5301 samples (2.9%). A similar sampling program for 1976–78 indicated that violative residues occurred in all animal species marketed (USDA, 1976–1978). The results of this survey are summarized in Table 18.2. The rate of violative antibiotic

Table 18.2 Percentage of Samples Exceeding Tolerances for Antibiotics and Sulfonamides Species 1976 Cattle Calves Sheep and goats Swine Chickens Turkeys Geese and ducks 1977 Cattle Calves Sheep and goats Swine Chickens Turkeys Geese and ducks 1978 Cattle Calves Sheep and goats Swine Chickens Turkeys Geese and ducks

Antibiotic samples

Violations, %

Sulfonamide samples

Violations, %

545 1378 70 247 155 258 160

1.3 8.6 7.1 1.6 0.6 0.0 0.6

476 327 100 1493 331 648 265

0.9 3.7 1.0 9.4 0.3 2.5 0.0

1739 1120 176 449 366 450 161

1.3 4.1 1.1 1.3 0.0 0.7 0.6

175 166 12 9461 1 445 206

2.3 3.0 0.0 13.1 0.0 0.9 0.5

1769 1409 210 1399 470 447 175

2.6 6.7 2.4 5.4 1.7 3.3 1.1

243 216 40 6687 119 443 148

0.8 2.8 15.0 9.7 0.8 4.4 0.0

Source: From USDA (1976–1978).

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residues was the lowest among poultry and cattle, whereas the highest rates occurred in swine and veal calves. The latter finding is not surprising since these animals are often raised on diets that are iron-deficient to give a light-colored product and supplemented with fairly high levels of antibiotics and sulfonamides (Katz, 1983). The high incidence of violative residues in swine is reflective of a recycling problem with sulfonamides. Swine are commonly fed a combination of antibiotics and sulfonamides. Although the medicated feeds are usually withdrawn 7–10 days before marketing, any residual medicated feed available to the animals causes a recycling of sulfonamides and the appearance of unwanted residues in the animal tissue. The first impact of the incidence of residues, whether violative or not, must be evaluated in the light of animals produced annually. Approximately 5 million broilers, 400 million laying hens, 175–200 million turkeys, 100 million swine, and 30 million steers are produced each year. Even small percentages of animals having violative residues indicate that large numbers of animals are marketed with drug residues (Feinman and Matheson, 1978; Katz, 1983; Deshpande and Salunkhe, 1995). Contamination of the feedstuff is yet another source. The contamination of laying poultry feeds with feeds medicated with the coccidiostats, nicarbazin, and lasalocid is not uncommon. In addition to deliberate or accidental acts, drug residues may still find their way into the human food chain in other ways. In this regard, the use of long-acting drugs is especially troublesome. Benzathine penicillin, for example, is injected as a salt that is relatively insoluble and releases penicillin slowly into the animal. A long withdrawal period for such drugs is recommended. However, it has been shown that this is inadequate around the injection site. If this area is sampled, violation of MRL is possible (Korsrud et al., 1994; van Leeuwen, 1999). Normally, the contribution of residues at the site of injection is not taken into consideration in assessing the toxicological risk for the consumer of residues in animal products and in setting MRLs. This is based on the condition that the MRLs set for residues of a veterinary drug in animal products reflect the risk of chronic exposure of the consumer. Furthermore, it is extremely unlikely that consumers are confronted regularly with meat that contains injection sites. However, this might still pose a potential problem for residue surveillance, since the meat inspectors collecting samples for chemical analyses cannot always differentiate between normal muscle tissue and the tissue that contains an injection site. Yet another source of drug residue contamination is the presence of “bound residues” (van Leeuwen, 1999).

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The drug residues or their metabolites may be bound to endogenous compounds such as amino acids, fatty acids, and nucleic acids. Reactive metabolites of certain drugs may even be bound covalently with cellular macromolecules. Such bound residues may not be easily extractable during normal chemical analyses and thus are not detected. The FDA currently permits the discount of bound residues as long as they are not bioavailable provided the drug does not show gastrointestinal carcinogenic properties (FDA, 1994; Brynes and Weber, 1996). Overdosing of animals in the belief that this may help produce a more rapid cure can also easily give rise to the need for extended withdrawal periods because the MRL would be exceeded at the normal period (Shearer, 1999). Van Dresser and Wilcke (1989) studied the problem of drug residues in food animals. Their analyses disclosed the following. 1.

2.

3.

4.

5.

6.

7.

Antibiotic residues were most often associated with streptomycin, penicillin, oxytetracycline, and neomycin. Sulfamethazine was, by far, the most frequently cited sulfonamide. Residues are being found predominantly in cows, veal calves, and market hogs (barrows and gilts). The cause of drug residue most frequently cited by the field investigators was failure to observe the withholding time for the drug. Almost half of these investigations revealed that the individual responsible for the sale of the animal did not know the proper withholding time for the drug. Failure to maintain adequate records was also a contributing factor. The producer was considered to be the responsible party in over 80% of the cases for which the responsibility was determined. Residues associated with injectable drugs were investigated most frequently. Long-acting and sustained-release products were most often associated with penicillin and oxytetracycline residues. The two most common sources of purchase for the drugs involved in the investigations were the feed/farm supply store and the veterinarian. Unapproved drug use was not a major cause of residues.

The possible routes by which drug residues contaminate various milk and meat products are listed in Table 18.3.

Table 18.3 Possible Sources and Routes of Drug Residues in Milk and Meat Products Extended usage or excessive dosage of approved drugs Poor records of treatment Increased frequency of intramammary antibiotic treatment on farms Failure to observe recommended label withdrawal time Prolonged drug clearance Treated animal identification problems Errors due to hired help Large herd size Increased frequency of use of medicated feed Contaminated milking equipment Multiple dosing Milker or producer mistakes: accidental transfer in bulk tank Products not used according to label directions Lack of advice on withdrawal period Withholding milk from treated quarters only Early calving or short dry periods Purchase of treated cows Use of dry cow therapy to lactating cows Source: Compiled from Booth and Harding (1986), Jones and Seymour (1988), McEwen et al. (1991), and Deshpande and Salunkhe (1995).

18.5 WITHDRAWAL TIME The withdrawal time is usually defined as the interval between cessation of administration of the drug and the moment that the residues have fallen below the MRL. A withdrawal time needs to be established for each formulation of the veterinary drug, for each target animal, and for each route of administration, using the highest recommended dose and the longest treatment period. The withdrawal time needs to be recorded after cessation of treatment of the animal before the animal can be slaughtered or before products like milk and eggs can be safely consumed (JECFA, 1991; van Leeuwen, 1999). Setting the withdrawal time is a crucial stage in the licensing procedure of veterinary drugs. The differences in formulation between similar drugs for the same therapeutic purpose might result in different withdrawal periods. Farmers and veterinarians then often choose, for obvious economic reasons, the product with the shorter withdrawal period so that an animal can be sent to slaughter earlier. Particular problems exist for withdrawal periods for products intended for use in dairy cattle and laying hens (van Leeuwen, 1999). Unlike residues in feed animals, which can be kept from slaughter to ensure that residues will deplete

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below the MRL, residues in milk and eggs do not deplete with time. Consequently, these products need to be discarded until the residue levels have fallen below the MRL, a procedure that can lead to considerable economic loss. Thus, in these cases, products with a short or even a zero withdrawal period offer considerable economic benefits. Determining the appropriate withdrawal times for fish produced at fish farms in open sea or in fresh water poses additional problems. Their metabolic rates, and thereby the depletion of residues, are partly dependent on their body temperature and thus on the temperature of the water in which they live. The warmer the water, the higher their metabolism and thus the depletion of residues; the cooler the water, the longer the time for residue depletion (Euroresidues, 1990). Therefore, residue depletion studies in fish are usually carried out at different temperatures in a range representative for the fish farming conditions. Because they are a function of time and temperature, withdrawal times in fish are expressed in degree days. Finally, where illegal drugs are used, there are no defined withdrawal periods. Therefore, it is very much a case of trial and error. An excessive withdrawal period may vitiate the benefits of the illegal use, so there may be a temptation to shorten this as much as possible. Hormones and β-agonists require protracted periods of treatment to exert their effect, and, equally, withdrawal times may be protracted. Hormones when permitted were supplied as implants to allow slow release. These implants used to be placed behind the ear, which was discarded. However, farmers may use implants in different points on the animal with the hope that they will not be looked for and detected.

18.6 ADVERSE EFFECTS OF DRUG RESIDUES Adverse effects of drug residues in the human food chain can be manifested in several different ways. Drug residues in human foods should be avoided for the following reasons: 1.

2. 3.

4.

Some residues can cause idiosyncratic reactions in ultrasensitive consumers, which can be extremely serious. Generally, drug residues above the prescribed tolerance limits are illegal. Some drug residues in fluid dairy products are capable of interfering with starter cultures used in processed milk products and cheese. Residues are generally indicative of the possibility that the food is from animals that had a serious infection.

5.

6.

Public awareness and concern regarding the natural wholesomeness of our food supply are increasing. Most importantly, drug residue contamination of human foods generally encourages the selection and spread of transferable multiple-resistance plasmids. This in turn leads to development of drug-resistant microorganisms that are pathogenic to humans.

Some of these adverse effects of drug residue contamination are described in the following sections. 18.6.1

Hypersensitivity and Allergic Reactions

One of the most important concerns related to residues is that of hypersensitivity and allergy in susceptible individuals. Although there are many drugs and antibiotics that can elicit a variety of allergic-type reactions, the majority of information is related to hypersensitivity and allergic reactions to antibiotics resulting from repeated treatment (Adams, 1975). Several commonly used antibiotics may also cause cardiovascular depression or respiratory difficulties or alter the metabolic breakdown of other drugs. Hypersensitive reactions are probably the most common cause of drug allergy. The symptoms may include urticaria, fever, bronchospasm, serum sickness, angioedema, and anaphylaxis, which can lead to death (Mandell and Sande, 1985; Deshpande and Salunkhe, 1995). There are many documented cases of humans’ having demonstrated immunopathological reactions as a result of exposure to antibiotic residues in foods. Penicillin has been on the top of the list of antibiotics in terms of its allergenic characteristics, in both veterinary and human clinical medicine. It is more or less accepted that up to 10% of the general population exhibits some form of adverse reactions to penicillin therapy (Adkinson, 1980; Katz, 1983; Deshpande and Salunkhe, 1995). Once an individual is sensitized to penicillin, for example, as little as 40 IU (0.024 mg) administered orally may elicit allergic reactions (WHO/FAO, 1969). Immediate hypersensitive reactions have been reported with consumption of foods of animal origin such as milk and meat containing antibiotic residues (Zimmerman, 1959; Nickas, 1975; Livingston, 1985; Okolo, 1986; van Dresser and Wilcke, 1989). In addition, second-generation penicillins, cephalosporins, have been found to cause diarrhea and to be nephrotoxic (Petz, 1978; Wade et al., 1978). Sensitivity reactions with sulfonamides may involve the skin and mucous membranes in the form of and including urticaria, petechial rashes, exfoliative dermatitis, and

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photosensitivity. In previously sensitized individuals, immediate reactions of the anaphylactoid type have been observed (Siddique, 1992). Serum sickness, which sometimes follows sulfonamide therapy, may be accompanied by fever, joint pain, conjunctivitis, bronchospasm, and urticarial eruptions (Weinstein et al., 1960; Mandell and Sande, 1985). The tetracyclines have been considered essentially free of allergic responses. Tetracycline allergy, skin rashes, and phototoxic dermatitis are the most common idiosyncratic reactions associated with these drugs. The most serious toxic effect of aminoglycosides (gentamicin, tobramycin, amikacin, netilmicin, kanamycin, streptomycin, and neomycin) is on the vestibular mechanisms in the inner ear responsible for maintaining balance. These antibiotics also exhibit nephrotoxicity and ototoxicity (Mattie et al., 1989). Like all toxicological responses, the allergic response of individuals to residues should be dose-related. The dose necessary for stimulating an allergic response depends always on the degree of sensitivity of the individual exposed. However, it should also be noted that, with the exception of the penicillins, such immunological reactions by humans to antimicrobial drugs used in animals are generally uncommon. In fact, such reactions from eating food contaminated with antibiotic residues have never been observed to cause anaphylactic reactions (Hewitt, 1975). The only adverse reaction of the hypersensitivity type has been associated with penicillin in milk, resulting in allergic reactions such as skin rashes, hives, asthma, and anaphylactic shock at concentrations as low as 0.003 IU penicillin/mL (Hewitt, 1975; Lindemayr et al., 1981). Allison (1985) has contradicted these arguments, suggesting that there is no evidence of an effect of oral administration (e.g., food consumption) on hypersensitivity. It was postulated that this was only a risk to extremely sensitive individuals and that hypersensitivity usually followed parenteral administration. In the United States, the FDA regulations prohibit measurable penicillin residues in milk offered for sale. There are at least three reasons behind the regulations: Milk containing measurable residues of penicillin has a strong potential of triggering allergic responses in highly sensitive individuals; it usually is from cows with an active infection of mastitis, and hence may contain high levels of pathogenic microorganisms; and residues also may interfere with the production of milk products by inhibiting the starter cultures. Antibiotic testing as well as other milk quality tests are performed routinely and frequently on all producers’ milk; financial penalties are assessed against those who default. Despite the huge numbers of producers, the milk is tested randomly at about weekly intervals.

In addition to withholding requirements, the U.S. FDA establishes tolerance limits for antibiotic residues in milk and meat tissues. The safe levels used by the FDA are only used as guides for prosecutorial purposes. They do not legalize residues found in milk that are below the safe level. For some drugs, no tolerance and/or safe level of the residue is allowed. The tolerance or safe levels for the current 23 FDA-approved drug residues in milk are summarized in Table 18.4. Residues persist in the milk supply, but the magnitude of the problem is not clear. Moorman and Koenig (1992) reviewed two studies, cosponsored by the Wall Street Journal and the Center for Science in the Public Interest (CSPI). They revealed the presence of antibiotics and sulfonamide residues in milk samples; of the samples collected in 10 major U.S. cities, 38% were contaminated with sulfamethazine (a suspected carcinogen), sulfa drugs, and other antibiotics. These findings were in contrast with results from an earlier FDA-sponsored survey, which

Table 18.4 Tolerances and/or Safe Levels of Animal Drug Residues in Milk Established by the Milk Safety Branch of the U.S. Food and Drug Administration Center for Food Safety and Applied Nutrition

Drug

Tolerance and/or safe levels, ppba

Ampicillin Amoxicillin Cloxacillin Cephapirin Neomycin Novobiocin Tylosin Erythromycin Gentamicin Dihydrostreptomycin Tetracycline Oxytetracycline Chlortetracycline Sulfachloropyridazine Sulfadimethoxine Sulfadiazine Sulfamerazine Sulfamethazine Sulfamethiozole Sulfanilamide Sulfapyridine Sulfaquinoxaline Sulfathiazole a

10 10 10 20 150 100 50 50 50 125 80 30 30 10 10 10 10 10 10 10 10 10 10

Parts per billion, µg antimicrobial/L fluid milk.

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found 1.5% sulfamethazine incidence level for samples collected from 23 cities. The issue of drug residues in foods has encouraged the development of numerous analytical test methods. The increasing use of drug-specific and rapid enzyme immunoassays by the livestock industry and stricter control by the regulatory agencies have resulted in a significant decrease in the amount of drug-contaminated food sold in the retail market. The data reported by the FDA Milk Safety Branch on drug residue test results from January to June 1992 confirm these findings (FDA, 1992). During this period, 1,828,020 farm trucks containing 59.5 billion pounds of milk from 38 states were screened by the industry for βlactam drug residues in accordance with the new requirements in Appendix N of the Pasteurized Milk Ordinance. Drug residues were found in 1505 farm trucks containing 45,610,408 pounds of milk (0.08%). In terms of the then average milk price ($11.64/cwt), the milk with drug residues was worth more than $5.2 million. Additional drug residue testing results reported by 50 of the 51 state regulatory agencies included the following: sulfonamides, 12 positive results of 26,818 samples from 20 states; tetracyclines, 7 positive results in 9196 samples from 10 states; gentamicin, 1 positive result of 1556 samples from 6 states; ivermectine, 2 positive results of 23 samples from 1 state. During the same period, state regulatory agencies reported that 4 of 52,618 (0.008%) grade A finished product samples had positive findings for drug residues. This compared to 0.02% (24 of 107,381 samples) that had positive findings in 1991 (FDA, 1992). Any residue found in milk, invariably, can be traced to the therapeutic use of treating mastitis. The feeding of medicated feeds to lactating animals is not the usual mode of contamination. Residues occur primarily from the lack of adherence to the disposal of milk for the prescribed time. In many instances, as soon as the classical clinical symptoms disappear, the less-than-scrupulous producer mixes the penicillin-containing milk together with large volumes of uncontaminated milk and ships the milk to the dairy. Dairies have instituted analytical screening systems to ensure that milk containing measurable residues is not accepted. 18.6.2

Development of Resistance by Microorganisms

The resistance of pathogenic microorganisms to antimicrobials and their transmission to humans are among the most important issues and concerns of contemporary drug therapy. The prevalence of drug residues in the human food chain thus merits our serious attention. As early as

1957, Smith and Crabb showed that the use of tetracycline in animal feeds increased the number of tetracycline-resistant E. coli in swine and poultry. A later study by Watanabe (1963) revealed that the resistance was transferable. Microbial resistance to antibiotics is not harmful per se but may create a public health hazard if the resistance interferes with the control of a given microorganism, especially a pathogen, in animals or humans. Since the two most widely used families of antibiotics in animal feeds, the tetracyclines and the penicillins (βlactam group), are also used for the treatment of microbial infections in humans, this became the central point of focus on feed antibiotics and human health risks. The two greatest concerns were (a) that the use of certain antibiotics in animal feeds could generate large numbers of resistance plasmids in the enteric flora of livestock and the genetic material might eventually encode antibiotic resistance in human pathogens, and (b) that the use of antibiotics in livestock feeds, particularly subtherapeutic or prophylactic, could ultimately induce a significant loss of antibiotic efficacy in human medicine. For this reason, the U.S. FDA required antibiotic sponsors to submit evidence that showed conclusively that the addition of antibiotics to animal feeds did not pose hazards to animal or human health (Gardner, 1973). The government guidelines for establishing a human health hazard were as follows: 1.

2.

3.

4.

If administration of antibiotics to animals significantly increased the animal reservoir of pathogenic gram-negative bacilli, which could be transferred to humans via the food chain, the antibiotics were hazardous to health. If antibiotic use significantly increased gramnegative bacilli in animals resistant to antibiotics used in human medicine, a potential hazard existed. If antibiotics enhanced the pathogenicity of gram-negative bacilli in animals by increasing the development and linkage of certain genetic elements with R factors (discussed later), and if these organisms could be transmitted to humans through the food chain, the antibiotic would not be permitted as a feed supplement. If ingestion of antibiotic residues in foods led to an increase of antibiotic-resistant pathogenic organisms in human flora, the antibiotics could not be used in animal feeds.

It is now a well-recognized fact that the human pathogenic bacteria are increasingly becoming resistant to antibiotics. Venkateswaran and associates (1988) isolated antibiotic-resistant salmonella organisms from raw meat

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and other food samples that had multiple drug resistance. In a similar study, the antibiotic-resistant Staphylococcus aureus was isolated from milk and meat of slaughtered animals and wounds of slaughterhouse workers (Vecht et al., 1989). It has been suggested that these highly resistant organisms can be transmitted to humans directly or through food prepared under unsanitary conditions. Experimental studies conducted by Corpet (1987) showed that continuous administration of small doses of ampicillin and chlortetracycline in gnotobiotic mice resulted in resistant E. coli. The evidence for these concerns is also derived from epidemiological studies of disease outbreaks. The Centers for Disease Control (CDC) estimated that 16% of the cases reported in the 1985 salmonellosis milk outbreak in Illinois were attributable to human consumption of antimicrobials (Ryan et al., 1987). The illness would not have occurred had the milk been contaminated with an antimicrobial-sensitive strain. The outbreak affected an estimated 200,000 people, making it the largest recorded outbreak of salmonellosis ever identified in the United States. Ryan and colleagues (1987) also found through patient surveys that ill persons who had been taking antimicrobials to which the Salmonella spp. was resistant consumed less milk than the other ill persons, suggesting the antimicrobials lowered the infectious dose of Salmonella spp. The causative organism was Salmonella typhimurium, which contained an antibiotic-resistant plasmid for ampicillin, tetracycline, carbenicillin, streptomycin, erythromycin, clindamycin, sulfisoxazole, sulfadiazine, triple sulfa (sulfabenzamide, sulfacetamide, and sulfathiazole), cefoperazone, mizlocillin, piperacillin, and kanamycin (Schuman et al., 1989). Similar outbreaks of salmonellosis in Great Britain in the mid-1960s were also traced to dairy calves that were exposed to low levels of antibiotics in feeds (Federal Register, 1977). The Swann Committee that was formed to study antibiotic resistance transfer and human health implications recommended that antibiotics used to treat human diseases should not be used as feed additives for growth promotion. The British law banning such drug uses was subsequently enacted in 1970. According to Moorman and Koenig (1992), if salmonellosis in livestock takes a septic form, several antibiotics that are currently available to the veterinarian may be rendered useless. Their conclusions were based on the practice of the nonjudicious exposure of livestock to subtherapeutic doses of antibiotics in feeds commonly used in the livestock industry in the United States. It may also further reduce the effectiveness of antibiotic treatment of infectious diseases in humans. Indeed, the CDC estimates

that 25% of the Salmonella spp. isolated from human infections are resistant to antibiotics and that antibiotic therapy of human salmonellosis is rarely the prescribed treatment (Linton, 1984; Holmberg et al., 1984; Deshpande and Salunkhe, 1995). The plasmid-mediated resistance was first recognized from studies on clinical bacterial isolates resistant to several gram-negative antimicrobial drugs that were able to transfer the genetic information encoding these resistances to other bacteria (Gustafson, 1991). This capability is mediated by means of extrachromosomal factors called R plasmids, or simply referred to as R factors or R+. These factors are small segments of circular DNA and have the capacity to reproduce themselves independently of the chromosome. They may determine resistance to more than one antibiotic. Plasmid-directed resistance to antibiotics involves the biosynthesis of specific enzymes that inactivate the antibiotic, such as penicillinase, which is active against both natural and semisynthetic penicillin varieties such as ampicillin. The plasmids may carry as many as nine drug-resistant genes as well as genes governing plasmid replication independent of the nuclear chromosomes. Genes controlling R+ transfers from one bacterium to another by conjugation are also contained in these plasmids. Transfer of R+ can occur in virtually all species of the Enterobacteriaceae as well as species of unrelated genera such as Pasteurella, Pseudomonas, and Vibrio, genera in which several species virulent to humans and animals belong. Thus, R+ transfer can occur not only with related strains or species, but also within unrelated species and, most importantly, between nonpathogens and pathogens. The seriousness of this phenomenon is reflected by the observations that already antibiotic resistance has emerged in, for example, Haemophilus influenzae, Salmonella typhi, Shigella dysenteriae, Neisseria gonorrhoeae, and Salmonella typhimurium (Concon, 1988; Deshpande and Salunkhe, 1995). These species are responsible for some of the most serious, epidemic-causing, and, in the case of first three, often fatal diseases before the advent of antibiotics. Certainly a direct consequence of the presence of drug residues in the human food chain is the proliferation of relatively high levels of resistant coliforms carried as part of the normal human intestinal microflora. Ferrando (1975) summarized the evidence related to the colonization of resistant E. coli from animals to humans. The pro evidence noted was as follows: 1.

Animals receiving antibiotics carried greater numbers of resistant E. coli than animals not fed antibiotics (the total number of the E. coli in the

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2.

3.

4.

animal intestine population is greater than that found in the humans). Salmonella spp. are transmitted regularly from animals to humans. Why shouldn’t E. coli be so transmitted? Experimentally, resistance determinants have been transmitted from E. coli of animal origin to E. coli of human origin and from E. coli to Salmonella spp. There are higher levels of resistant E. coli found in animal attendants and kitchen personnel than in the general population.

Evidence to the contrary can be summarized as follows: 1.

2. 3.

4.

5.

More antibiotics are used for humans than for animals, with the result that there is a high incidence of antibiotic-resistant E. coli in the human intestinal microflora. E. coli as well as Salmonella spp. levels are low in cooked meats. Colonization of the human intestinal tract requires large numbers of bacteria or optimal conditions for colonization. The transfer of resistance between strains and/or genera occurs at very low rates under normal intestinal conditions. There is a higher incidence of resistant E. coli in babies and in vegetarians than in adult meat eaters.

Anderson (1970) also suggested that the major cause of drug-resistant populations in humans primarily was the result of the overuse of antibiotics in human medicine rather than of agricultural usage. He did believe that there was a contribution from drug residues in the food chain and that there should be none. According to Silver and Mercer (1978), the increase in antibiotic resistance in Salmonella spp. will not lead to a widespread epidemic in the United States, since poor sanitation and lack of health care facilities contribute to the occurrence of epidemics. A greater human health hazard, in their opinion, is the large reservoir of R factors in the normal flora of animals, which can transfer resistance from nonpathogenic to pathogenic organisms. According to these researchers, the emergence of widespread resistance to penicillin in group A Streptococcus or Pnemococcus spp. would have far greater impact than resistance in Salmonella spp. or E. coli. Therefore, there can no longer be any doubt that the large-scale use of antibiotics at subtherapeutic doses in the feed of farm animals is partly responsible for the increasing presence of antibiotic-resistant strains of pathogenic and nonpathogenic bacteria. Even the existence of resistant

nonpathogens poses a serious health hazard because of their ability to transfer the R factors to pathogens. The factor can carry genes governing resistance to more than one antibiotic, and the greater the pool of R plasmids, the greater the probability of development of a wide variety of antibiotic-resistant pathogens. Humans and animal carriers alike from whom various diseases can be transmitted to the population at large may harbor such resistant pathogens. After reviewing data on drug-resistant plasmid transfer, the FDA published its intent to ban the subtherapeutic use of penicillin in 1977 (Federal Register, 1977). It also called for a restriction on the use of tetracycline and tetracycline-combination drugs. After much deliberation, the U.S. Congress requested the FDA to study further the relationship between antibiotic use and human health. As of July 1991, 23 antimicrobial agents were approved by the FDA for one or more uses as additives in poultry and livestock feeds (FDA revises, 1991). The regulations also call for specific withdrawal times after treatment of antibiotics for livestock prior to lactation or slaughter. For example, FDA regulations require the discarding of milk from treated cows for 96 hours after the last administration of the antibiotic. 18.6.3

It should also be noted that several lactic organisms also produce natural antibiotics in milk and milk products. For example, Streptococcus lactis produces nisin (Mattick and Hirsch, 1944); Lactobacillus bulgaricus produces bulgarican (Reddy and Shahani, 1971); L. brevis, lactobacillin (Kavasnikov and Sodenko, 1967); L. acidophilus, acidophilin (Shahani et al., 1976, 1977), acidolin (Hamdan and Makolajcik, 1974), lactobacillin (Wheater et al., 1951), and lactocidin (Vincent et al., 1959); and L. plantarum produces lactolin (Kodama, 1952). In many countries, nisin is permitted as a direct food additive. Nisin is inhibitory against several gram-positive streptococci, lactobacilli, clostridia, staphylococci, and bacilli (Hawley, 1957; Shahani, 1962), but has no effect on gram-negative organisms (Mattick and Hirsch, 1947). Its major use has been to prevent the growth and subsequent gas production by clostridia in cheese and processed cheese products. In France, for example, nisin-producing streptococci have been employed in the manufacture of processed cheese. Nisin is digested in the upper GI tract and has been shown to be relatively nontoxic and nonallergenic. Moreover, there appears to be no cross-resistance between nisin and other antibiotics (WHO/FAO, 1969).

Inhibition of Starter Cultures in the Dairy Industry 18.7 ANALYTICAL METHODS

As mentioned earlier, the primary cause of antibiotic residues in milk and milk products is the failure of producers to withhold milk from the market for a sufficient period after veterinary therapy for mastitis or other diseases in dairy cattle. Antibiotics are quite stable and remain in the milk even after manufacturing processes including pasteurization, drying, or freezing. The major problem associated with antibiotic residues in the fluid milk supply has been partial or complete inhibition of acid production by bacterial starter cultures used in the manufacture of dairy products such as cheese, buttermilk, sour cream, or yogurt. Shahani and Harper (1958) determined the minimal amount of penicillin and aureomycin needed to inhibit satisfactory growth of 19 stock cheese cultures. As little as 0.05–0.1 IU/mL penicillin and 1.0–3.0 µg/mL aureomycin completely inhibited the growth of most cultures studied. Whitehead and Lane (1956) also noted that during cheese manufacture, as little as 0.05 IU penicillin/mL milk considerably delayed acid production, and 0.5 IU/mL milk completely inhibited acid production. Low levels of antibiotics also affect the flavor and texture of the final product as well as increase the probability of growth of undesirable antibiotic-resistant coliforms (Mol, 1975; Friend and Shahani, 1981).

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The MRLs for almost all the veterinary drugs are generally in the microgram per kilogram (µg/kg [ppb]) range, with the highest values in the organ tissues, medium in muscle, and lowest in milk. Analysis of such very low concentrations of drugs in complex tissues poses very demanding problems for the analyst. Other major problems are the high numbers of samples and the fact that most are negative or drug-free. These factors have necessitated the demand for high-throughput screening methods that are sufficiently sensitive to monitor the MRL and produce a low incidence of false-positive results and zero false-negative result. Given the perishable nature of foods, such methods should also be rapid in providing the answer. When sample results are found to be positive by such screening methods, reanalysis using qualitative methods to determine which drug and how much of it is present is required. Finally, where MRLs are exceeded, confirmatory methods that unequivocally identify the analyte in question to allow legal proceedings to be taken are required. Antibiotics are readily detectable by microbial inhibition tests and drug-specific immunoassays (Deshpande, 1996). For complex samples, such as meats, appropriate sample extraction and cleanup methods are also required.

For confirmatory methods, mass spectrometry, either gas chromatography–mass spectometry (GC-MS) or liquid chromatography–mass spectrometry (LC-MS), is often employed. Rapid screening methods for testing MRLs in various foods are currently available commercially from a number of companies, both in the United States and in Europe.

18.8 LEGISLATION In the United States, the FDA has the mandate to determine whether a new veterinary drug is safe for the consumer under the conditions prescribed. It considers the cumulative effect of the veterinary drug on humans, the uncertainty factors deemed necessary for extrapolation from animal data to humans, and the probable consumption of residues of the drug. The current toxicological testing requirements for drug-related residues in animal products are well established and are laid down in a General Principles Document (U.S. FDA, 1994). In the European Union (EU), conventional veterinary drugs are dealt with under the same regulatory framework as human drugs, whereas medicated feeding stuffs fall under a separate regulation. The two major directives governing the veterinary drugs are Council Directives 81/851/EEC and 81/852/EEC (1981a, 1981b). These two directives ensure that the regulatory requirements for the health risk evaluation and the marketing authorization of veterinary medicinal products are the same throughout the European Union. In general, these principles are broadly similar to those applied by the U.S. FDA. In addition, the Codex Alimentarius Committee, a subsidiary body of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), proposes standards for food safety to national governments for acceptance into the prevailing national legislation. The primary aims of Codex are to protect the health of the consumer and to ensure fair practice in trade (van Leeuwen, 1997, 1999). In contrast to the U.S. FDA and the EU, the Codex has no legal authority. However, the proposed Codex standards are of great value in the worldwide harmonization of food standards, because many countries adopt these standards in their national legislation. Since 1989, the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) is responsible for the stepwise procedure leading to the acceptance of Codex standards for veterinary drugs. The Joint Expert Committee on Food Additives (JECFA) serves as a scientific advisory body to the CCRVDF.

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Thus the development and more widespread use of increasingly sensitive and reliable analytical test methods as well as increased surveillance and regulatory activity in the areas of antibiotic residue testing have resulted in significant lowering of the drug residue contamination of food in the retail market. Drugs certainly have an important role in safeguarding both animal and human health. The health consequence of chronic subtherapeutic use of broad-spectrum antibiotics for growth promotion in animals leading to the selection of multidrug-resistant pathogens, however, appears to be of far greater significance than the actual drug residues themselves in our food supply. Subtherapeutic uses of drugs, therefore, should be discontinued in light of these consequences and the availability of alternative measures. Thus it is toward a more defined therapeutic use of these agents that the present-day livestock industry should strive.

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