Reviews in Food and Nutrition Toxicity, Volume 4 (Reviews in Food and Nutrition)

REVIEWS IN FOOD AND NUTRITION TOXICITY Volume 4

REVIEWS IN FOOD AND NUTRITION TOXICITY Edited by Victor R. Preedy and Ronald R. Watson

Volume 1 Volume 2 Volume 3 Volume 4

REVIEWS IN FOOD AND NUTRITION TOXICITY Volume 4

Edited by Victor R. Preedy and Ronald R. Watson

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© 2005 by Taylor & Francis Group CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-3519-1 (Hardcover) International Standard Book Number-13: 978-0-8493-3519-8 (Hardcover) Library of Congress Card Number 2004047814 This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

Library of Congress Cataloging-in-Publication Data Reviews in food and nutrition toxicity / edited by Victor R. Preedy and Ronald Watson. p. cm. Includes bibliographical references and index. ISBN 0-8493-3519-1 (alk. paper) 1. Nutrition policy. I. Preedy, Victor R. II. Watson, Ronald R. (Ronald Ross). III. Title. TX359.A56 2004 363.8'561–dc22

2004047814

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Preface In this fourth volume of reviews, we present state-of-the-art chapters pertaining to the potential and actual harm that arises as a consequence of consuming food substances and other components in the diet. We emphasize the terms potential and actual, as very often there are threshold boundaries that need to be crossed before an innocuous substance becomes toxic and/or induces a cascade of cellular and pathological changes. However, as evidenced from the present chapters, there is some debate as to what these thresholds are, and this illustrates the fundamental need for constant scientific dialogue. The chapters in Reviews in Food and Nutrition Toxicity fulfill these scientific, academic, and intellectual needs. The first part of the present volume can be considered thematic in that five chapters cover chemical elements: heavy metals (mercury, lead, cadmium, and arsenic), selenium, arsenic, sulfur, and fluoride. The chapters on arsenic, sulfur, and fluoride are general and may be considered as overviews, whereas the two chapters on heavy metals and arsenic specifically pertain to their occurrence in breast milk and fish, respectively. There follows three chapters on bacterial and fungal components. These include contamination of ready-to-eat foods, T-2 mycotoxin, and aflatoxin B1. Finally, there are two very comprehensive reviews on cycad consumption and dietary lectins. As with previous volumes in Reviews in Food and Nutrition Toxicity, we believe that the present coverage will stimulate broad-based interests as well as specific applicability to other food or nutritional substances. It is difficult to highlight a single chapter for meritous mention, as they are all equally well focused and scientific stimulating. The present chapters are written by nationally and internationally recognized experts and essentially complement the previous three volumes to give wide coverage of food nutrition and toxicity in a holistic manner.

Editors Victor R. Preedy, Ph.D., D.Sc., F.R.C.Path., is a professor in the Department of Nutrition and Dietetics, King’s College, London. He directs studies regarding protein turnover, cardiology, nutrition, and, in particular, the biochemical aspects of alcoholism. Dr. Preedy graduated in 1974 from the University of Aston with a combined honors degree in biology and physiology with pharmacology. He received his Ph.D. in 1981 in the field of nutrition and metabolism, specializing in protein turnover. In 1992, he received membership in the Royal College of Pathologists based on his published works, and in 1993 a D.Sc. degree for his outstanding contribution to the study of protein metabolism. At the time, he was one of the university’s youngest recipients of this distinguished award. Dr. Preedy was elected a fellow of the Royal College of Pathologists in 2000. He has published more than 475 articles, which include more than 150 peer-reviewed manuscripts based on original research, and 70 reviews. His current major research interests include the role of alcohol in enteral nutrition and the molecular mechanisms responsible for alcoholic muscle damage. Ronald R. Watson, Ph.D., attended the University of Idaho but graduated from Brigham Young University in Provo, Utah, with a degree in chemistry in 1966. He earned his Ph.D. in biochemistry from Michigan State University in 1971. His postdoctoral schooling in nutrition and microbiology was completed at the Harvard School of Public Health, where he gained 2 years of postdoctoral research experience in immunology. From 1973 to 1974, Dr. Watson was assistant professor of immunology and performed research at the University of Mississippi Medical Center in Jackson. He was assistant professor of microbiology and immunology at the Indiana University Medical School from 1974 to 1978 and associate professor at Purdue University in the Department of Food and Nutrition from 1978 to 1982. In 1982, Dr. Watson joined the faculty at the University of Arizona Health Sciences Center in the Department of Family and Community Medicine of the School of Medicine. He is currently professor of health promotion sciences in the Mel and Enid Zuckerman Arizona College of Public Health. Dr. Watson is a member of several national and international nutrition, immunology, cancer, and alcoholism research societies. He is presently funded by the National Heart Blood and Lung Institute to study nutrition and heart disease in mice with AIDS. Dr. Watson has edited more than 35 books on nutrition and 53 scientific books and has contributed to more than 500 research and review articles.

Contributors Gabriella Augusti-Tocco Department of Cellular and Developmental Biology “La Sapienza” University Rome, Italy

Willy Baeyens Brussels Research Unit of Environmental, Geochemical and Life Sciences Department of Analytical and Environmental Chemistry Vrije Universiteit Brussel Brussels, Belgium

Tapan K. Basu Department of Agricultural, Food and Nutritional Science The University of Alberta Edmonton, Alberta, Canada

Marjan De Gieter Brussels Research Unit of Environmental, Geochemical and Life Sciences Department of Analytical and Environmental Chemistry Vrije Universiteit Brussel Brussels, Belgium Tony J. Fang Department of Food Science National Chung Hsing University Taiwan, Republic of China Hanne Frøkiær Biocentrum-DTU Biochemistry and Nutrition Technical University of Denmark Lyngby, Denmark Claudia Gundacker Center for Public Health Medical University of Vienna Vienna, Austria

Emanuele Cacci Department of Cellular and Developmental Biology “La Sapienza” University Rome, Italy

Erin L. Hawkes Graduate Program in Neuroscience University of British Columbia Vancouver, British Columbia, Canada

Thomas F.X. Collins Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration Laurel, Maryland

Ziad W. Jaradat Department of Biotechnology and Genetic Engineering Jordan University of Science and Technology Irbid, Jordan

Tanja Maria Rosenkilde Kjær Biocentrum-DTU Biochemistry and Nutrition Technical University of Denmark Lyngby, Denmark Lioudmila A. Komarnisky Department of Agricultural, Food and Nutritional Science The University of Alberta Edmonton, Alberta, Canada

Christopher A. Shaw Graduate Program in Neuroscience Departments of Ophthalmology, Physiology, and Experimental Medicine University of British Columbia Vancouver, British Columbia, Canada Robert L. Sprando Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration Laurel, Maryland

Ruggero Ricordy Institute of Molecular Biology and Pathology CNR Rome, Italy

Ujang Tinggi Centre for Public Health Sciences Queensland Health Scientific Services Brisbane, Australia

Jeff D. Schulz Graduate Program in Neuroscience University of British Columbia Vancouver, British Columbia, Canada

Bettina Zödl Center for Physiology and Pathophysiology Medical University of Vienna Vienna, Austria

Table of Contents Chapter 1 Heavy Metals in Breast Milk: Implications for Toxicity .........................................1 Claudia Gundacker and Bettina Zödl Chapter 2 Selenium Toxicity and Its Adverse Health Effects .................................................29 Ujang Tinggi Chapter 3 Arsenic in Fish: Implications for Human Toxicity.................................................57 M. De Gieter and W. Baeyens Chapter 4 Biological and Toxicological Considerations of Dietary Sulfur ............................85 Lioudmila A. Komarnisky and Tapan K. Basu Chapter 5 Fluoride – Toxic and Pathologic Aspects: Review of Current Literature on Some Aspects of Fluoride Toxicity..................................................................105 Thomas F.X. Collins and Robert L. Sprando Chapter 6 Bacterial Contamination of Ready-to-Eat Foods: Concern for Human Toxicity .....................................................................................................143 Tony J. Fang Chapter 7 T-2 Mycotoxin in the Diet and Its Effects on Tissues..........................................173 Ziad W. Jaradat Chapter 8 Aflatoxin B1 and Cell Cycle Perturbation.............................................................213 Ruggero Ricordy, Emanuele Cacci, and Gabriella Augusti-Tocco Chapter 9 Cycad Consumption and Neurological Disease....................................................233 Jeff D. Schulz, Erin L. Hawkes, and Christopher A. Shaw

Chapter 10 Dietary Lectins and the Immune Response ..........................................................271 Tanja Maria Rosenkilde Kjær and Hanne Frøkiær Index ......................................................................................................................297

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Heavy Metals in Breast Milk: Implications for Toxicity Claudia Gundacker and Bettina Zödl

CONTENTS Abstract ......................................................................................................................2 Abbreviations .............................................................................................................2 Introduction................................................................................................................2 Sources of Heavy Metals and Transfer in the Environment ........................3 Exposure Routes and Biological Half-Life...................................................4 Heavy Metals in Breast Milk ....................................................................................5 Exogenous and Endogenous Sources............................................................5 The Process of Milk Production ...................................................................7 Transfer of Heavy Metals into Milk .............................................................8 Mercury ................................................................................................9 Lead ......................................................................................................9 Cadmium ............................................................................................13 Arsenic ...............................................................................................14 Metal Concentrations in Breast Milk..........................................................14 Factors Influencing Milk Metal Contents ...................................................14 Mercury ..............................................................................................15 Lead ....................................................................................................15 Cadmium ............................................................................................15 Arsenic ...............................................................................................16 Toxicological Implications ..........................................................................16 Mercury ..............................................................................................17 Lead ....................................................................................................19 Cadmium ............................................................................................20 Arsenic ...............................................................................................20 Exposure Guidelines....................................................................................20 Conclusions..............................................................................................................21 References................................................................................................................22

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Abstract

Breast milk is unique as a matrix for biomonitoring, providing information about the metal body burden of women as well as the exposure of infants. The heavy metals mercury, lead, cadmium, and arsenic are widespread and persistent agents with significant dose-related toxicological implications at high exposure levels. However, the interrelationships under conditions of chronic exposure are not fully known. Metal in breast milk originates from exogenous sources, i.e., uptake via contaminated air, food, and drinking water, and endogenous release along with essential trace elements, which is characteristic for the reproductional period. Metal transfer into breast milk depends on the chemical form and the distribution of the metal in maternal blood fractions. Methylmercury is strongly bound to erythrocytes. A small quantity of methylmercury passes into breast milk and is easily absorbed by the suckling infant. Inorganic mercury is readily transferred into breast milk, but is not well absorbed by infants. Lead transfer is associated with casein. Human milk has a very low casein content; therefore, the excretion rate of lead is low. Because cadmium binds to metallothioneins, the mammary gland, like the placenta, is considered to serve as a barrier for cadmium and to protect the infant. Inorganic arsenic is not excreted in breast milk to any significant extent. The suckling infant may be exposed to toxic influences in a period of highest susceptibility. Metal toxicity is dependent on the chemical form involved, which determines the bioavailability, absorption rate, and retention time. The brain is regarded as the most important target organ of toxic impairment even at low doses. There is some epidemiological evidence that prenatal metal exposure (in particular, methylmercury exposure) correlates with neurodevelopmental deficits. Yet, it remains unclear whether and to what extent postnatal metal exposure through breastfeeding impairs the infant’s health. The toxicokinetics of arsenic among neonates and infants has been scarcely reported. As environmental and maternal conditions lead to significant differences in milk metal levels, all measures must be taken to avoid additional metal exposure of infants via breastfeeding.

Abbreviations

As: arsenic; Ca: calcium; Cd: cadmium; Hg: mercury; K: potassium; Mg: magnesium; Na: sodium; Pb: lead; Po4: phosphate; Zn: zinc

INTRODUCTION The American Academy of Pediatrics (AAP) firmly adheres to the position that breastfeeding ensures the best possible health as well as the best developmental and psychosocial outcomes for the infant. It is recommended that breastfeeding continue for at least 12 months, and thereafter for as long as mutually desired (AAP, 1997). There is no doubt that exclusive breastfeeding is ideal nutrition; yet it has to be considered that breast milk may contain pollutants, which implies the need to evaluate breast milk contents. Analyses of breast milk metal concentrations provide data about the metal burden in the woman’s body on the one hand, and metal exposure of neonates and infants via breastfeeding on the other. Therefore, breast milk is “unique as a matrix for biomonitoring, and analyses of breast milk for

Heavy Metals in Breast Milk: Implications for Toxicity

3

environmental chemicals as well as for nutrients are of wide scientific interest” (Needham and Wang, 2002). Among diverse environmental pollutants, heavy metals belong to the most harmful xenobiotics, as they are widespread and persistent agents with significant doserelated toxicological implications. The persistence of metals, i.e., that they are not degradable, is one of their most problematic features and a major factor in the ecotoxicological relevance of heavy metals. The toxicology of metals is related to approximately 80 elements, including those heavy metals that, per definition, exceed a density of 5 g/cm3. Heavy metals of relevance in this context are mercury, lead, and cadmium. Arsenic is usually regarded as a hazardous heavy metal although it is actually a semimetal. Humans are routinely exposed to environmental metal concentrations and accumulate metals accordingly, which results in a variety of health impacts. Heavy metals are known, or at least suspected, to possess an immunotoxic, mutagenic, carcinogenic, embryotoxic, and teratogenic potential. Their dose–effect relationships, however, are not fully known, especially under chronic exposure. Long-term, low-level metal exposure results in elevated metal burdens for the body. Such burdens are considered nontoxic as long as they are below health-based exposure guidelines; nonetheless they may impair human health. Women of reproductive age are subject to a process known as body clearance, which may be defined as the loss of essential and nonessential elements during pregnancy and lactation due to the high nutrient demand at this stage. Lactating women (and subsequently their offspring) are affected by heavy metal exposure not only via exogenous sources, i.e., environmental exposure, but also through endogenous metal release. Hence the infant may be exposed to toxic influences in a period of highest susceptibility due to rapid growth, immaturity of kidneys and liver, and the unique vulnerability of the myelinizing central nervous system (CNS) to neurotoxic exposure. Furthermore, in cases of maternal element deficiency, the risk of toxic effects for both the infant and mother may be higher (Vahter et al., 2002); yet very few data are available on the interrelationships of essential and nonessential trace elements in breast milk. Data on mercury, lead, cadmium, and arsenic transfer into and concentrations in breast milk are described in the following, as are the factors apparently responsible for increasing milk metal levels. Very few studies have been carried out on the distinct effects of metal exposure via breastfeeding, illustrating the difficulties in this concern: effects of postnatal exposure do not clearly separate from those of prenatal exposure.

SOURCES

OF

HEAVY METALS

AND

TRANSFER

IN THE

ENVIRONMENT

Heavy metals spread through natural and anthropogenic sources. Heavy metals are natural constituents of Earth’s crust, emitted by volcanic activity, forest fires, and rock weathering. Anthropogenic sources of heavy metals include various processing and manufacturing industries, mining, foundries and smelters, piping, waste disposal, and diffuse sources such as combustion of fossil fuels and by-products, constituents of products, and corrosion. Human activities throughout the last century have

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dramatically altered the biochemical and geochemical cycles of some heavy metals. Stumm and Keller (1984) presumed that, on a global scale, the anthropogenic emissions significantly exceed the natural emissions. Once released into the environment, metals move between the atmosphere, land, and water. Physical properties determine whether an element is predominantly transported by the atmosphere or the lithosphere, which is particle-bound aquatic transport for the latter. Relatively volatile heavy metals and those that become attached to airborne particles can be widely dispersed on a very large scale. The biosphere absorbs and accumulates various quantities of metals at certain trophic levels, depending on the environmental metal concentration, metal bioavailability, the feeding behavior, and the physiological state of organisms. In addition, some organic metal forms tend to accumulate along the food chain, e.g., methylated mercury. As a consequence, metallic elements are found in all living organisms and have potential toxicological implications for humans if the latter frequently consume species known to be accumulators of heavy metals, such as predatory fish, sea mammals, crustaceans, or shellfish.

EXPOSURE ROUTES

AND

BIOLOGICAL HALF-LIFE

The main exposure routes for humans are (1) inhalation of metal aerosols and metal vapor, (2) metal uptake through food and drinking water, (3) dermal metal absorption, and in case of the fetus, (4) uptake via the placenta. After a metal has been taken into the lung or into the gastrointestinal tract, it will be deposited on the walls of the airways or will be taken up in the mucosa of the gastrointestinal tract, and a certain fraction of the deposited amount will be transferred to the systemic circulation and distributed among tissue compartments throughout the body (Camner et al., 1986). Several chemical and physical characteristics of metals in exposure media, such as air, water, and food, are important for absorption, excretion, and retention of metals by humans, and determine the specific biological half-life of metals. Mercury is readily absorbed (especially methylmercury in the gastrointestinal tract) and distributed throughout the body. Biological half-life varies from a few days to months; the organs with the longest retention times are the brain and kidneys (Figure 1.1). Vahter et al. (2000) presumed that the half-life of methylmercury is longer in fetal blood than in maternal blood, about 2 months in the latter. About 10% of ingested lead is absorbed in the gastrointestinal tract. Infants and children may absorb as much as 50% of dietary lead. The main target organ of lead is the skeleton. The half-life varies among different tissue types. Lead retention in soft tissues is about 3 weeks, but in bone it may range from a few years to a few decades (Figure 1.2). Raghunath et al. (1997) reported retention times of 20 and 9 days for blood-lead and blood-cadmium in 6- to 10-year-old children, respectively. Gulson et al. (1999) described a longer lead half-life for infants than for mothers: 91 vs. 59 days. Cadmium predominantly accumulates in the kidney. On account of its low excretion rate, cadmium has a very long half-life of 10 to 30 years in the muscle, kidney, and liver (Figure 1.3). Organic and inorganic arsenic have been shown to be readily absorbed via the gastrointestinal tract, and also by inhalation (Figure 1.4).

Heavy Metals in Breast Milk: Implications for Toxicity

Invasion:

Hg:

Anorganic

•Anorganic •Organic

•Ingestion •Inhalation •Dermal

Organic

5

Absorption: Anorganic •1–7% •80–95%

Organic

•Ingestion •Inhalation •Dermal

Distribution via blood and lymph (mostly bound to plasma proteins)

Excretion: •Urine (60% of total elimination) •Feces (methyl-Hg) •Sweat •Saliva •Exhalation •Breast milk

Body Depots: •CNS (organic Hg) •Liver •Pancreas •Kidney

Target Organs: •Kidney •CNS (organic Hg-Minamata disease)

T1/2: •70–90 d •1–18 a in CNS (metallic Hg)

FIGURE 1.1 Mercury distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.) Note: a = annus; d = days.

Absorbed arsenic is widely distributed in the body; the highest levels are found in the hair, nails, and skin. The major part of arsenic in humans is eliminated within 10 days.

HEAVY METALS IN BREAST MILK EXOGENOUS

AND

ENDOGENOUS SOURCES

Metals circulating in the maternal bloodstream originate from endogenous (metals released from storage organs and tissues) as well as exogenous sources (metal uptake via inhaled air, food, and drinking water). It is assumed that the chemical similarity of nonessential and essential elements, for example, calcium and lead, leads to the incorporation of nonessential elements via the same routes into the same storage organs available for essential element uptake, e.g., the skeleton for calcium and lead. This is true also for the extraction process taking place in phases of high nutrient demand, which is characteristic of the reproduction period. During this time, metals are eliminated along with essential trace elements from the target organs. Mobilization of lead from bone is likely to occur during periods of altered mineral metabolism. Because calciotropic factors determine the uptake and storage of lead in this compartment, changes in calcium-related regulatory factors are likely to affect lead compartmentation (Silbergeld, 1991).

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Pb:

Invasion:

•Anorganic •Organic

Absorption

Anorganic

Anorganic •5–10% (children up to 50%) •50–80%

•Ingestion •Inhalation

Organic •Ingestion •Inhalation •Dermal

Organic •>90% •>90% •>90%

Distribution via blood (90% bound to erythrocytes)

Body Depots: •Bone [Pb3(PO4)2] (90–95% of body burden)

Target Organs:

Excretion: •Urine (75– 80% of absorbed Pb) •Feces (90% of oral uptake) •Breast milk •Sweat

•Kidney •CNS •Smooth muscle •Peripheral nervous system •Red bone mark

T1/2: •20–30 a in bone •20 d in soft tissues

FIGURE 1.2 Lead distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)

Cd:

Invasion:

Absorption:

•Ingestion •Inhalation

•1–7% •25–50%

Distribution via blood (95% bound to erythrocytes -most likely complexed to metallothioneins)

Excretion: •Urine •Feces •Breast milk •Placenta

Body Depots: •Kidney (50–75% of body burden) •Liver (metallothioneins) •Pancreas •Thyroid gland

Target Organs:

T1/2: >10 a

•Kidney •Lung (cancer) •Bone (Itai Itai disease) •Gonads

FIGURE 1.3 Cadmium distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)

Heavy Metals in Breast Milk: Implications for Toxicity

As:

Invasion:

Absorption:

•Ingestion •Inhalation •Dermal

•95% •30–60% •1–40%

Distribution via blood (95–99% in erythrocytes bound to globin)

7

Body Depots: •Hair, nails (keratin) •Erythrocytes •Thyroid gland •Liver, kidney •Epididymis

Excretion:

Target Organs:

•Urine •Feces •Sweat •Exhalation

•Skin (hyperpigmentary, cancer) •Lung (cancer) •Heart muscle •Liver •Kidney •

T1/2: 5d (except hair, nails, bone)

FIGURE 1.4 Arsenic distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)

Both endogenous and exogenous metal concentrations are responsible for the actual metal content in milk. Which of these sources is the more important, especially for lead, is under discussion (see, e.g., Gulson et al., 2003). Oskarsson et al. (1998) presume that the clearance of chemicals during lactation is the major factor. In fact, between 45 and 70% of blood-lead in adult females arises from long-term lead stores in the tissue (Gulson et al., 1995) and the mobilization of lead from the skeleton during the postnatal period is greater than that during pregnancy; this might be attributed to an inadequate calcium intake. Breast milk lead levels were related to a 5.6% bone loss and to significant bone turnover in a study conducted by Sowers et al. (2002).

THE PROCESS

OF

MILK PRODUCTION

Lactation, i.e., the production of milk by the mammary gland, is a highly complex procedure. It can occur only after a series of developmental processes have taken place in the breast of the pregnant woman. Interaction between the mother and the child is an essential aspect of the process. Unless the child starts suckling or the breast is emptied artificially, the secretion of milk will stop within a few days (Philip and WHO Working Group, 1988). The production of milk varies according to the infant’s demands, age, and ability to suckle. The normal output of mature milk ranges from 600 to 1000 ml per day, but may vary between 300 and 1200 ml per day. The process of milk production and ejection is triggered by the hormones progesterone, estrogen, prolactin, and oxytocin. Lactogenesis begins about 40 h after the birth. The foremilk colostrum is produced within 3 to 5 days after delivery; it is low in volume and fat content (2.9%). Over the next 2 to 6 weeks, the transitional milk matures and increases in fat content to about 4%; the major class of milk lipids

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are the triglycerides. Breast milk is composed of several other components including carbohydrates, proteins, and minerals, especially calcium (Needham and Wang, 2002). Milk is synthesized in the mammary alveolar gland, prior to which the components of milk and their precursors have to pass through a membrane that separates the blood flowing in capillaries from the alveolar epithelial cells of the breast. Alveolar secretory cells are involved in four processes that occur simultaneously: exocytosis; fat synthesis and excretion; secretion of ions, water, and proteins; and the transfer of milk components across the cell. Oskarsson et al. (1998) presumed that the exocrine pathway is quantitatively the most important. The major milk proteins casein and lactose, along with calcium and phosphate, form the so-called micelles, which are transported via Golgi vesicles to the apical membranes and then released into the milk alveoli. Small molecules such as sodium, potassium, chloride, and glucose can pass across the apical membrane.

TRANSFER

OF

HEAVY METALS

INTO

MILK

The transport of xenobiotics into milk is supposed to follow the same pathways as milk components and to proceed according to the principles of cellular metal uptake (Table 1.1). In general, there is a low transfer of toxic metals through milk when maternal exposure levels are low. However, knowledge concerning the lactational transport of metals and the potential effects of metals on milk secretion and composition is scarce (Oskarsson et al., 1998).

TABLE 1.1 Cellular Uptake of Heavy Metals Passive Transport

Active Transport

Diffusion Energy (ATP)-dependent via carrier-proteins Channel-mediated diffusion (e.g., Na+, K+, Ca2+) 2+ Carrier-mediated diffusion (e.g., Ca ) Simple diffusion Filtration Depends on: Similarity to essential metals (e.g., Ca-Pb, Zn-Cd) (chemical mimicry) Size and molecular weight Chemical bonding Lipophilic character Grade of ionization Transport through epithelial cells can take place: Transcellular (active or passive) Paracellular (passive through tight junctions of epithelial cells) Source: Data from Merian, 1984; Oehlmann and Markert, 1997.

Heavy Metals in Breast Milk: Implications for Toxicity

9

Experimental data have shown that each metal is distributed in a characteristic way between the milk fractions. Lead is almost exclusively found in the casein fraction, while cadmium and methylmercury are found in fat, and inorganic mercury is largely found in whey fractions (Oskarsson et al., 1998). In human milk, mercury is mainly bound to caseins (Mata et al., 1997). Thus, mercury possesses a greater ability to interact with milk proteins than with low-weight molecules. In contrast, it appears that cadmium and lead are equally distributed among milk components with high and low molecular weights (Coni et al., 2000). Mercury Findings of Sundberg et al. (1999b) showed serum albumin is a major mercurybinding protein in whey and plasma fractions of mice for both methylmercury and inorganic mercury. The authors suggested passive transfer from plasma into milk using albumin as a passive carrier. Following the administration of lead and mercury to lactating and nonlactating mice, metal elimination from plasma was significantly greater in lactating mice, while about 30, 8, and 4% of the administered dose of lead, inorganic mercury, and methylmercury, respectively, was excreted in milk (Oskarsson et al., 1998; Sundberg et al., 1998). The transfer of mercury from plasma to milk was found to be higher in dams exposed to inorganic mercury than to methylmercury. In contrast, the uptake of mercury from milk was higher in the sucklings of dams exposed to methylmercury than to inorganic mercury (Oskarsson et al., 1995). Almost all methylmercury delivered via milk was absorbed and the suckling pups had a very low elimination rate until lactational day 17. Sundberg et al. (1999a) concluded that, on account of differences in kinetics, lactational exposure to methylmercury is a greater hazard for the breastfed infant than is inorganic mercury. However, both inorganic and organic mercury can be excreted in breast milk and the demethylation that takes place in vivo is thought to play an important role in the lactational transfer (Abadin et al., 1997). In contrast to lipophilic chemicals such as the persistent organic pollutants, metals do not bind to fat and usually do not accumulate at higher concentrations in breast milk than in blood. Oskarsson et al. (1995) established that milk mercury levels are approximately 30% of the levels in blood. On account of the placental transfer of mercury, it may be concluded that prenatal mercury exposure is generally more important than lactational exposure. In contrast, Drexler and Schaller (1998) and Ramirez et al. (2000) found lower maternal blood-mercury levels compared to milk mercury concentrations (Table 1.2). Lead It has been suggested by Palminger Hallen et al. (1996) that lead is transported into milk via the same pathway as calcium because of its high affinity to casein. In lactating mice, lead was found to be associated with casein micelles inside the alveolar cells and the milk lumen, indicating that lead is excreted into milk, bound to casein, via the Golgi secretory system. Oskarsson et al. (1995) reported that tissue

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TABLE 1.2 Mercury, Lead, Cadmium, and Arsenic Concentrations in Breast Milk Country

μg/l Hg

SD/Range

Austria

7.70

11

Austria

0.85 <0.52 1.59

1.23 <0.52 1.21

Brazil*

5.7

0–24.8

Denmark

2.45

Germany

4.58 1.55 1.16 0.5 1.9 <0.2

Germany Germany

0.81

Germany

0.64

Mat B-Hg

Colostrum (1–3 days pp) Days 42–60 Days 97–293

Gundacker et al., 2002 Boischio and Henshel, 2000 Grandjean et al., 1995b Friedrich, 1986

Day 1 Day 3 Day 5 After day 15

0–20.3

Iraq**

0–11.7 <50–200

Iraq**

15–45

Italy Italy

<0.5 13.9

Japan**

63

Japan*

0.21

Philippines* Slovakia

36 1.6

18.2

Slovenia* Sweden*

11.8 0.6

1.2–37.4

No amalgam fillings 1–4 amalgam fillings 5–7 fillings >7 fillings 1 week pp 2 months pp Organic mercury Total mercury

Ref. Rossipal and Krachler, 1998

1.58

0.57 0.5 2.11 1.37

Notes

Klemann et al., 1990 Drasch et al., 1998

0.65

Drexler and Schaller, 1998

500–3250

Bakir et al., 1973

50–2390

Amin-Zaki et al., 1974 Clemente et al., 1982 Paccagnella and Riolfatti, 1989 Fujita and Takabatake, 1977 Sakamoto et al., 2002b Ramirez et al., 2000 Ursinyova et al., 1995 Kosta et al., 1983 Oskarsson et al., 1995

0–17.5 1.6–52.5

24

2.3

(continued)

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11

TABLE 1.2 (CONTINUED) Mercury, Lead, Cadmium, and Arsenic Concentrations in Breast Milk Country

μg/l Pb

SD/Range

Australia Austria

0.7 35.8

0.7 15

Austria Austria

3.4 2.30

0–20.4 2.9

Austria

2.40 0.90 1.6

3.3 1.7 1.7

China

4.7 52.7

Croatia Czechoslovakia

7.3 1.7

8.3 0–6.75

Egypt Germany

30.6 9.1

0–158 2.5

13.3 2.0 84.0 90.0 20.0

5.5 1.9–8.6 24 29 5

1.9 45.6 126.6 13.0 21.1 25.3 24.7 67.0

1.98 0–425 1–472 6

Great Britain Greece Greece India Italy Italy Malaysia Mexico Nigeria Saudi Arabia

7.7 5.2 13.4

Slovakia

4.2

Notes

Mat B-Pb <50 37

Colostrum (1–3 days pp) Days 42–60 Days 97–293

Nonexposed Occupational exposure

132

Kovar et al., 1984 Vavilis et al., 1997

149

Nashashibi et al., 1997 Tripathi et al., 1999 Guidi et al., 1992 Coni et al., 2000 Huat et al., 1983

Rural Urban 459

3.1–25

Gundacker et al., 2002 Li et al., 2000

101

Rural Urban

46–1300

Gulson et al., 1998 Plöckinger et al., 1993 Tiran et al., 1994 Rossipal and Krachler, 1998

Frkovic et al., 1997 Zahradnicek et al., 1989 Saleh et al., 1996 Sternowsky and Wessolowski, 1985

Rural Urban Urban Rural Urban

Ref.

Namihira et al., 1993 Vander Jagt et al., 2001 Younes et al., 1995

Women aged <36 Women aged >36 Ursinyova et al., 1995 (continued)

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TABLE 1.2 (CONTINUED) Mercury, Lead, Cadmium, and Arsenic Concentrations in Breast Milk Country

μg/l Pb

SD/Range

Slovenia Sweden

2.6 0.7

0.4

United States

6.1 5.6 5.9 4.3

Austria Austria

43 1.30

28–95 1.2

Austria

0.22 0.26 0.09

0.26 0.19 0.09

Croatia Czechoslovakia

2.54 0.31

2.06 0–1.08

Finland

1.7–3.1

Finland

2.00 1.50 1.60 0.10

Germany Germany

0.06 0.27 17.3

Slovakia

24.6 0.07 0.16 0.40 0.09 0.80 0.38 0.26 0.50

Slovenia* Slovenia

3 0.60

Great Britain India Italy Japan

32

1.5 months pp 3 months pp 6 months pp 12 months pp SD/Range

Germany

Mat B-Pb

Colostrum

μg/l Cd

Country

Notes

Notes

Mat B-Cd

Ref. Krachler et al., 1999 Palminger Hallen and Oskarsson, 1995 Sowers et al., 2002

Ref. Maruna et al., 1976 Rossipal and Krachler, 1998

Colostrum (1–3 days pp) Days 42–60 Days 97–293

Gundacker, unpublished data Frkovic et al., 1997 Zahradnicek et al., 1989 Vuori et al., 1983

1 month pp 3 months pp 6 months pp 1987

Kantol and Vartiainen, 2001

1993–1995 0–1.13 4.9 7.3

2.76 0.2 1.78 1.81

Müller, 1987 Sternowsky and Wessolowski, 1985

Rural Urban Nonsmokers Smokers Urban

Mat age >35 Mat age <35

1.0–5 Colostrum

0.5 1.5 0.7

Radisch et al., 1987 Kovar et al., 1984 Tripathi et al., 1999 Coni et al., 2000 Nishijo et al., 2002 Ursinyova et al., 1995 Kosta et al., 1983 Krachler et al., 1999 (continued)

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TABLE 1.2 (CONTINUED) Mercury, Lead, Cadmium, and Arsenic Concentrations in Breast Milk Country Sweden

μg/l Cd

SD/Range

0.06

0.04

Notes

Mat B-Cd 0.9

Nonsmokers Smokers μg/l As

SD/Range

Denmark*

3.0 2.8 3.4 1.6

2.3–4.8 1.9–5.5 2.3–5.5 0.1–4.4

Germany

<0.3

0–2.8

India*

0.7 0.5 1.3

Country Argentina*

Slovenia*

Palminger Hallen and Oskarsson, 1995

0.87 1.12

Notes 2.8 weeks pp 2.5 months pp 4.4 months pp Maximum of 8.7 μg/l

Ref.

Mat B-As 10

Tribal women Urban women 0.2–3.8

Ref. Concha et al., 1998

Grandjean et al., 1995b Sternowsky et al., 2002 Dang et al., 1985 Kosta et al., 1983

Abbreviations: SD = standard deviation; Mat B- = maternal blood contents are in μg/l; pp = postpartum; Mat age = maternal age. * Breast milk concentrations given as μg/kg fresh weight or ppb. ** Epidemic poisoning.

levels of lead in suckling pups were linearly correlated with lead milk concentrations. It was concluded that lead is transported into rat and mouse milk to a very high extent. Approximately 60 to 80% of lead is bound to casein in human breast milk; yet human milk has a very low casein content, and therefore the excretion rate of lead is low. Gulson et al. (1998) summarized ratios expressed as a percentage of lead concentrations in breast milk to whole blood. The ratios varied from 2.5 to 41.8%; however, values greater than 15% might indicate contamination during sampling or analysis. A linear relationship between breast milk and maternal whole blood has been reported; however, the relationship between plasma-lead and blood-lead is nonlinear, which might influence lead transfer into breast milk. Lead associated with erythrocytes is less toxicologically available (Schwartz, 1992). Cadmium Lactational transport of cadmium to suckling rat pups was investigated by Petersson Grawe and Oskarsson (2000). The authors established a high correlation between cadmium concentrations in pups’ kidneys and dams’ milk. The results indicated a low transfer of cadmium to the suckling pups, which might be due to the binding of cadmium to metallothionein in the mammary tissue. Cadmium was accumulated

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in the epithelium of the lactiferous ducts, and the uptake was low in the lumen, where milk is transported. Cadmium in the mammary cytosol was mainly present in the metallothionein fraction; metallothioneins have a detoxifying function known to be induced in cases of chronic cadmium exposure. The authors suggested that mammary metallothionein may either be synthesized in the mammary cells or originate from other tissues, e.g., the liver, and are transported by plasma to the mammary gland. In humans, milk cadmium concentrations were shown to be approximately 10% of corresponding blood levels of the substance (Radisch et al., 1987), which clearly reflects the burden of cadmium in the body (Honda et al., 2003). Cadmium is transferred in part to the next generation through breast milk (Nishijo et al., 2002). Arsenic Breast milk concentrations of arsenic have been scarcely reported. Because such exposure data for women are not frequently documented, the rate of arsenic excretion in breast milk cannot be estimated. Concha et al. (1998) revealed that inorganic arsenic is not excreted in breast milk to any significant extent.

METAL CONCENTRATIONS

IN

BREAST MILK

Comprehensive breast milk monitoring studies are available from several European countries, but not from the rest of the world. Obviously, most scientists have concentrated on lead, cadmium, and mercury, whereas very little research has been carried out on arsenic. Arsenic-contaminated groundwater is a serious health problem in many Asian and Latin American countries, but little information is available on arsenic exposure of nursing mothers and its health risks. Most likely on account of contaminated drinking water, arsenic levels in breast milk are much higher in Argentina than in European countries. There has been a remarkable decrease in breast milk metal concentrations over the last two decades in Austria, where lead contents have been steadily decreasing, most likely due to the mandatory shift from leaded to unleaded fuels in 1993. Similar reductions have been observed for lead in Italy and cadmium in Austria; methodological advances may have contributed to this phenomenon. Differences in metal content in milk throughout the world reflect varying environmental metal contamination as well as the numerous methods applied to collection, preparation, and measuring of breast milk.

FACTORS INFLUENCING MILK METAL CONTENTS In general, the time of breastfeeding, the mother’s age, and the local background concentrations determine metal concentrations of breast milk. Certain factors appear to specifically influence milk concentrations of mercury, lead, cadmium, and arsenic.

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15

Mercury Two factors are considered to be mainly responsible for increased mercury concentrations in breast milk: the uptake of organic mercury (methylmercury) primarily via fish consumption, and the uptake of inorganic mercury through dental amalgam (Oskarsson et al., 1996; Vahter et al., 2000). In the Faroe Islands, milk mercury concentrations were significantly related to the frequency of pilot whale dinners during pregnancy (Grandjean et al., 1995b). The toxicological implications of mercury exposure via dental amalgam have been widely discussed. Jones (1999) stressed that the amount of mercury released from dental amalgam is minimal; an individual would need 490 amalgam surfaces to meet the maximum exposure guidelines. Klemann et al. (1990) found no correlation between amalgam surfaces and mercury concentrations in breast milk. On the contrary, increased mercury excretion in breast milk correlated with the number of fillings (Vimy et al., 1997). Similar results were found by Oskarsson et al. (1996), Drasch et al. (1998), and Vahter et al. (2000). Drexler and Schaller (1998) concluded that the additional exposure to mercury from maternal amalgam fillings is of minor importance compared with maternal fish consumption. In Austria, factors significantly related to mercury levels in breast milk were the area of residence, prematurity, consumption of cereals, and vitamin supplementation; however, fish consumption appeared to have no influence on milk mercury levels (Gundacker et al., 2002). This might be confirmed by the findings of Oskarsson et al. (1999), where total mercury concentrations and fish consumption correlated in maternal blood but not in breast milk. Lead Environmental exposure, i.e., air pollution, contamination of the diet, and use of leaded paint, lead-glazed pottery, cosmetics, and the like, is the most important factor for increased body burden of lead. If the mother had long-term exposure in such environments, an additional source of lead would be from mobilization of skeletal stores during lactation (Gulson et al., 1998); hence the major sources of lead in breast milk are from the maternal bone and diet. Several authors have described higher metal contents in the breast milk of women living in urban vs. rural areas (Table 1.2), which is indicated as well by the Austrian data: the highest milk lead values were found in Linz, an industrial site, while lowest were found in the more rural area of Tulln (Gundacker et al., 2002). Cadmium Smoking is supposed to affect milk cadmium contents (Table 1.2), whereas dietary intake is the main source for nonsmokers. Honda et al. (2003) concluded that cadmium concentrations in breast milk are closely related to environmental exposure. Nishijo et al. (2002) found no correlation between cadmium in breast milk and maternal age, parity, and smoking.

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Arsenic Arsenic exposure of the general population occurs through food and drinking water. Although the high blood-arsenic levels in Andean mothers reflected their exposure through drinking water, their arsenic levels in breast milk were low, indicating that inorganic arsenic is not excreted in breast milk to any significant extent (Concha et al., 1998). Grandjean et al. (1995b) also reported very low breast milk levels of arsenic in the Faroe Islands. In Germany, arsenic soil and water contamination had no influence on breast milk levels (Sternowsky et al., 2002). Concentrations differed neither in samples obtained before and after nursing nor with respect to age of the infant.

TOXICOLOGICAL IMPLICATIONS Several metals are essential to life functioning, as they serve as enzyme activators or components of redox systems, and also as structural elements, stabilizers of biological structures, and components of control mechanisms (Friberg and Nordberg, 1986). Essential metals become toxic only when exposures to biota become excessive, i.e., when they exceed regulation capacities. Nonessential metals are usually toxic to organisms at much lower levels than are essential elements. The nature of biological responses to metal exposure is directly related to the quantity of exposure determined by dose–effect relationships. However, these relationships are not fully researched, especially under conditions of chronic low-dose exposure. Heavy metals are known, or at least suspected, to possess immunotoxic, mutagenic, carcinogenic, embryotoxic, and teratogenic potential. Toxicity of metals depends on the type of metal species involved, retention time, and excretion capacity. The principles of metal toxicity are described in Table 1.3. Interactions with other metals may increase toxicity via synergistic effects (e.g., between lead and cadmium) or via antagonistic relationships as is known for cadmium and zinc. Detoxifying agents such as metallothionein, which is a ubiquitous, cysteine-rich, metal-binding protein, may protect from adverse effects of metals. The synthesis of metallothionein is induced by various stimuli such as zinc, cadmium, and mercury. It has been assumed that metallothionein plays a role in the detoxification of heavy metals (Sato and Kondoh, 2002). Infants are highly susceptible on account of their rapid growth, immaturity of the kidney and the liver, and the vulnerability of the myelinizing CNS. Brain development begins early in fetal life and continues into adolescence. Interference with any stage of brain development may alter subsequent stages and result in permanent impairments (Stein et al., 2002). On account of the greater susceptibility of the fetus and the lower metal concentrations in breast milk compared to maternal blood, it has been suggested that prenatal metal exposure is more critical compared to postnatal exposure. However, Silbergeld (1991) stated that the effects of prenatal exposure cannot be clearly separated from those of postnatal exposure. In all, there is consensus that the additional uptake of xenobiotics via breastfeeding may increase the health risk for infants (Grandjean et al., 1994b).

Heavy Metals in Breast Milk: Implications for Toxicity

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TABLE 1.3 Some Mechanisms of Metal Toxicity Increased formation of reactive oxygen species results in oxidative stress, which causes damage to biomolecules such as proteins and nucleic acids; oxidative stress can also initiate lipid peroxidation, which can lead — after destruction of cell membrane — to cell death Displacement of essential metals like Ca2+, Mg2+, and Zn2+ (and therefore interference with essential metabolic compounds) Interactions with uptake and transport of essential metals (resulting in water electrolyte imbalances) Changes in molecular structures (cross-linking of DNA and RNA or binding to PO4 groups): mutations, cell death, carcinogenicity Disintegrating of hydrogen bonds: mutations, cell death, inactivation of enzymes, carcinogenicity Ability to form complexes with functional groups of enzymes (X), e.g., X-SH, (X-OH, X-NH2, X1NH-X2), and therefore inactivation of enzyme systems: • Inhibition of catalytic activity of enzymes • Interference with transport proteins • Failure of signaling pathways and signal cascading • Inhibition of DNA repair Source: Data from Merian, 1984; Oehlmann and Markert, 1997; Marquardt and Schäfer, 2003.

Mercury Mercury can cause neurologic and kidney impairment. The toxicity of mercury depends on the type of chemical forms involved, i.e., elemental mercury, inorganic mercury, and organic mercury compounds. Methylmercury is an especially potent neurotoxin. The hazards involved in the long-term intake of food containing methylmercury and in occupational exposure to methylmercury are due to the efficient absorption (90%) and the long retention time following accumulation of methylmercury in the brain (Berlin, 1986). Methylmercury adversely affects enzymes, cellular membrane function, and neurotransmitter levels (Schettler, 2001). The high susceptibility of the developing brain to methylmercury is well established; however, the lowest dose that impairs neurodevelopment is not known. Experimental results with guinea pigs showed that inorganic mercury and methylmercury were transferred to the offspring. The highest mercury contents were found in the kidneys but brain concentrations were significantly higher in the offspring of methylmercury-treated dams (Yoshida et al., 1994). Similar results were found by Sundberg et al. (1999a) for mice. Lactational exposure resulted in almost similar concentrations in the liver, kidney, and plasma, but higher concentrations in the brain (nearly 14-fold) and also a twofold higher mercury body burden in the methylmercury group. In suckling rats, the concentrations in the brain rapidly declined to one fifth of that at birth, suggesting that methylmercury transport by milk was limited while the brain and body volumes increased rapidly (Sakamoto et al., 2002a); reported abnormalities in the brain were attributed to highly accumulated methylmercury during gestation. Findings from Rossi et al. (1997) showed that during rat development a very low dosage of methylmercury exerted neurotoxic effects detectable in adulthood.

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Furthermore, it was noted that susceptibility is gender dependent, indicated by a significant decrease in spontaneous motility and rearing only in male rats. Several outbreaks of mercury poisoning have been described around the world: the largest occurred in Japan (Minamata disease) in 1953–1957 and in Iraq in the early 1970s, where extremely high breast milk levels up to 200 μg/l were measured (Bakir et al., 1973). The signs and symptoms of the mothers in Iraq were typical of methylmercury poisoning. The predominant exposure route for the children was through breast milk, in which approximately 60% of the total mercury was organic mercury. Pathological reflexes and delayed motor development in these infants were so high as to be considered significant even in the absence of a control group (AminZaki et al., 1981). Studies of prenatal exposure in Iraq suggested that maternal hair level above 10 ppm may be related to delayed developmental milestones and neurological abnormalities. As a result, long-term studies have been conducted to evaluate the neurodevelopmental effects of low-dose methylmercury exposure in early childhood. No adverse associations have been found in the Seychelles (maternal hair level 5.9 ppm), where exposure is mainly from fish consumption (Myers and Davidson, 1998). However, in the Faroe Islands (maternal hair level 4.3 ppm), where exposure is primarily from the consumption of whale meat and not fish, adverse associations have been reported (Grandjean et al., 1997). The main neuropsychological functions affected by prenatal methylmercury exposure were attention, language, and memory but deficits in visuospatial function were mainly related to postnatal exposures. These associations were stable after adjustment for confounders and exclusion of the children with the highest exposures. Despite highly significant effects on nervous system function, the deficits were subtle, and mercury exposure explained only a small part of the variation (Grandjean and White, 2001). Steuerwald et al. (2000) evaluated data from 200 term births in the Faroe Islands and found that prenatal exposure to methylmercury from contaminated seafood was associated with an increased risk of neurodevelopmental deficit. There is a noteworthy difference between the quantity of organic and inorganic mercury in milk and the toxicological implications. The share of inorganic mercury in total mercury can vary (Bakir et al., 1973; Skerfving 1988; Oskarsson et al., 1996). Several authors observed an efficient transfer of inorganic mercury from blood to milk (Oskarsson et al., 1996; Yang et al., 1997), which can be explained by the low erythrocyte/plasma ratio for inorganic mercury (about 1) compared to methylmercury (about 20), yet inorganic mercury is not well absorbed in the infant gastrointestinal system. In contrast, methylmercury does not enter breast milk at high rates because methylmercury is strongly attached to erythrocytes. The small amount that enters the breast milk is easily absorbed in the intestine of a nursing infant (Solomon and Weiss, 2002). Grandjean et al. (1994a) evaluated methylmercury transfer to infants via breastfeeding. Mercury concentrations in the infant’s hair increased with the length of the nursing period; the authors suggested a slow or absent methylmercury elimination during the first year of life. Surprisingly, infants who reached milestone criteria early

Heavy Metals in Breast Milk: Implications for Toxicity

19

had significantly higher mercury concentrations in the hair at 12 months of age, although early milestone development was clearly associated with breastfeeding, which was again related to increased hair mercury levels. Grandjean et al. (1995a) suggested that, if methylmercury exposure from human milk had any adverse effect on milestone development in these infants, the effect was compensated for or overruled by the benefits of nursing. Sakamoto et al. (2002) reported a declining risk of mercury exposure in infants during lactation, which might be due to low mercury transfer through breast milk and the rapid growth of infants after birth. They conclude that the offspring are at higher risk during the prenatal period. Lead Experimental data indicated a relationship between pre- and postnatal lead exposure and impairment of the offspring (Bogden et al., 1995): body weight and length of rat pups were reduced by lead exposure. Palminger Hallen and Oskarsson (1995) found that the lactational transfer after current or recent exposure to lead in dams is considerably higher than placental transfer, while the slight weight gain in the offspring was clearly attributable to lead exposure. Effects of lead on the growth and development of children indicated by stature, birth weight, gestational age, growth rates, and fetal death has been described by Schwartz (1992). Some authors evaluated the relationship between maternal metal loads (even at very low exposure levels) and the negative influence on gestational length, birth weight, and head circumference (e.g., Osman et al., 2000). Lead overloads create cerebral dysfunction and hematotoxic effects. While the weight of evidence supports the basic hypothesis that low-level exposure is associated with adverse neuropsychological function, remarkable little insight has been gained into fundamental aspects of these neurotoxic expressions, i.e., specific nature, severity, and persistence of the deficits and implications for adaptive capacities. More speculative yet are their neurochemical and neuropathological bases (Bellinger and Needleman, 1992). Lead is one of the first and perhaps best-understood examples of a common chemical that harms human brain development (Stein et al., 2002). Following improvements in study design, lead was found to influence IQ at progressively lower levels of exposure; thus, an increase in blood-lead from 10 to 20 μg/dl is associated with an average IQ loss of 2 to 3 points. It has been suggested that, in addition to declining IQ, low-level lead exposure is associated with impaired attention, hyperactivity, and antisocial and aggressive behavior. Some of these effects have been observed at very low levels of exposure and may occur in the absence of detectable IQ effects; hence, lead appears to have toxic effects at any level of exposure. In contrast, lead appeared to have no association with cognitive or psychomotor measures in Croatian school children (Prpic-Majic et al., 2000). Interaction between gender and prenatal lead exposure was observed. The impact of a given lead level was greater on boys at age 6 months as cited by Bellinger and Needleman (1992).

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Cadmium Continuous low-dose cadmium exposure via drinking water to lactating dams led to neurochemical disturbances of the serotonergic system in the offspring (Oskarsson et al., 1998). Cadmium treatment of lactating rats did not cause any detectable effects on milk synthesis, or on milk composition, and no histological lesions could be found (Petersson Grawe and Oskarsson, 2000). Thus the mammary gland, like the placenta, is presumed to act as a barrier that protects the infant. Hazardous cadmium levels lead to nephrotoxic effects, i.e., renal tubular dysfunction. Cadmium furthermore inhibits several SH-containing enzymes in the brain, while chronic cadmium exposure leads to reduced levels of norepinephrine, serotonin, and acetylcholine. Abadin et al. (1997) suggested that these changes may lead to adverse health effects, especially in the developing brain of infants. Although cadmium in breast milk is one of the major sources of cadmium exposure for infants, the effects on infant health have not been well described. No relation has been found between maternal exposure to cadmium and the concentration of cadmium in breast milk in previous European studies because of the low cadmium content of breast milk. Some authors reported decreased birth weight in humans exposed to cadmium (Nishijo et al., 2002), and also an inverse correlation between hair cadmium and verbal IQ and behavioral effects in children (Thatcher et al., 1982). Nevertheless, no study reports a link between breast milk cadmium levels and neurobehavioral effects in neonates. According to Abadin et al. (1997), this issue cannot be dismissed, because postnatal cadmium exposure through breast milk would unnecessarily increase the cadmium body burden of neonates, potentially contributing to the development of adverse health effects. Arsenic Skin lesions, skin cancer, and effects on the nervous system, as well as on the heart and circulatory system, are signs of impairment caused by arsenic exposure. It has been found that the methylated metabolites are less reactive, less toxic, and more rapidly excreted than inorganic arsenic as cited by Concha et al. (1998). Although the health effects of arsenic exposure among adults have been recognized, those among the younger generations including children, infants, neonates, and fetus have been scarcely reported. Although some data suggest potential effects on the skin and on the growth and development of children, the body of data as a whole is too premature to conclude whether or not arsenic poses a serious threat to younger generations (Watanabe et al., 2003). Although prenatal exposure to acute, very high doses of arsenic has resulted in miscarriage and early neonatal death (Concha et al., 1998), data concerning placental transfer of arsenic in cases of no apparent maternal toxicity are scarce.

EXPOSURE GUIDELINES Abadin et al. (1997) calculated screening levels for mercury, lead, and cadmium in breast milk, referring to an intake of 700 ml breast milk by an infant weighing 5

Heavy Metals in Breast Milk: Implications for Toxicity

21

TABLE 1.4 Background Levels and Minimal Risk Levels for Metals in Breast Milk

Background level Screening level

μg/l Hg

μg/l Pb

μg/l Cd

μg/l As

1.4–1.7a 3.5c

2.0–5.0a 20a

<1a 5a

0.2–2b

a

Abadin et al., 1997. Jensen, 1991. cAssuming that 20% of total mercury in breast milk is methylmercury. b

kg. Compared to cadmium levels, mercury and lead concentrations of breast milk more frequently exceeded these screening levels (Table 1.4). The screening level for total mercury is 3.5 μg/l, which corresponds to a daily intake of 0.5 μg/kg body weight. Via breastfeeding, the Austrian infant ingests an average of 0.3 μg/kg/day, the same intake as reported by Oskarsson et al. (1996) for Swedish infants. This exposure corresponds to approximately one half the tolerable intake for adults, the safety limit of 0.7 μg/kg/day recommended by the World Health Organization (WHO) (1972). Oskarsson et al. (1996) concluded that efforts should be made to decrease mercury burden in fertile women. In the study, 8% of Austrian breast milk samples (n = 165) exceeded the screening level of 3.5 μg/l; nevertheless, it should be noted that the minimum risk level (MRL), which was the basis for the risk assessment of Abadin and colleagues, and other guidelines refer to chronic exposure. It has to be amended that the assumption that 20% of total mercury in breast milk is in the form of methylmercury (Abadin et al., 1997) possibly underestimates the real situation. Oskarsson et al. (1996) found 49% of total mercury in breast milk was in the form of organic mercury, which was 60% in Iraq (Bakir et al., 1973) but only 20% in breast milk of women with a relatively high intake of fish (Skerfving, 1988). In fact, there is a narrow safety margin between no observed adverse effect level and lowest observed adverse effect level. Furthermore, the developing infant is more sensitive than the adult to adverse effects of metal exposure. Most recently, the WHO revised the provisional tolerable weekly intake for methylmercury, recommending that it be reduced to 1.6 μg/kg/day (former value 3.3 μg/kg/day) to protect the developing fetus sufficiently (WHO, 2003). Hence, further research on metal exposure via breastfeeding, in particular, methylmercury exposure, and the potential longterm effects will be necessary.

CONCLUSIONS Knowledge of heavy metal concentrations in breast milk is important to assess the health outcomes on infants. Such assessment is crucially important in pediatric practice, as well as in public health, women’s health, and environmental health

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research. However, data on metal toxicokinetics in breast milk and subsequent health effects are insufficiently available especially in the case of long-term, low-level exposure. The gaps in current knowledge concern the following aspects: •

• • • • • • •

Information on the nature and levels of heavy metals in breast milk, especially methylmercury concentrations, as well as arsenic and other insufficiently investigated elements Lactational transport of metals and essential elements Interrelationships of essential and nonessential trace elements and the potential effects on milk secretion and composition Precise uptake and excretion rates of suckling infants to develop appropriate pharmacokinetic models Toxicological implications of low-dose metal exposure of infants Gender-related vulnerability to (neuro)toxic impairment Significance of metal exposure via breastfeeding because the effects do not clearly separate from those of prenatal exposure Consistent methodological standards for collecting, preparing, and analyzing breast milk

Metal levels in breast milk appear to be decreasing, which is confirmed at least by the recent data available for lead and cadmium in some European countries. The levels and toxicokinetics of arsenic (particularly in neonates and infants) have been poorly researched. There is some evidence that offspring appear to be at higher risk during the prenatal period on account of (1) the greater susceptibility of the fetus and (2) the lower metal concentrations in breast milk compared to maternal blood (where mercury and lead are easily transferred via the placenta to the fetus). Moreover, the potential negative effects occurring in the lactational period might be compensated for by the benefits of nursing. Nevertheless, we cannot exclude adverse health effects caused by additional exposure through breast milk, especially for the most neurotoxic substances, i.e., methylmercury and lead. Metal exposure of the fetus and the suckling infant depends on the amount of metals accumulated by the woman over months (mercury) or even years (lead, cadmium). Therefore, all measures should be taken to avoid metal exposure in women of reproductive age.

REFERENCES Abadin, H.G., Hibbs, B.F., and Pohl, H.R. (1997) Breast-feeding exposure of infants to cadmium, lead, and mercury: a public health viewpoint, Toxicology and Industrial Health, 13: 495–517. American Academy of Pediatrics (1997) Breastfeeding and the use of human milk, Pediatrics, 100: 1035–1039. Amin-Zaki, L., Elhassani, S., Majeed, M.A. et al. (1974) Intra-uterine methylmercury poisoning in Iraq, Pediatrics, 54: 587–595.

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Selenium Toxicity and Its Adverse Health Effects Ujang Tinggi

CONTENTS Abstract ....................................................................................................................29 Abbreviations ...........................................................................................................30 Introduction..............................................................................................................30 Selenium Chemical and Physical Properties...........................................................31 Selenium in the Environment ..................................................................................32 Selenium Metabolism and Bioavailability ..............................................................33 Effects of Selenium Exposure .................................................................................35 Toxicity of Selenium Compounds...............................................................35 Selenium Toxicity in Animals .....................................................................36 Selenium Toxicity in Humans .....................................................................37 Respiratory Effects.............................................................................37 Neurological Effects...........................................................................37 Reproductive and Developmental Effects .........................................38 Immunological Effects .......................................................................38 Toxicity from High Dietary Selenium Intake....................................38 Death from Selenium Ingestion.........................................................39 Mechanisms of Selenium Toxicity ..........................................................................41 Assessment of Biomarkers for Selenium Status in Humans ..................................42 Conclusions..............................................................................................................46 Acknowledgments....................................................................................................47 References................................................................................................................47

Abstract

The major route of selenium exposure in humans is through diet. High dietary intakes of selenium have caused toxicity in humans after consuming selenium-rich food grown in seleniferous areas. Selenium status in a population varies widely and tends to reflect selenium levels in the soils and the produce of the region. Environmental contamination from the release of selenium from agriculture and industrial activities has caused diseases and death for wildlife and aquatic animals. Selenium toxicity in humans is rare. However, when it occurs, the clinical symptoms are similar to those of animals. Hair loss and nail deformity are common symptoms

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of chronic selenium exposure in humans. Selenium levels in hair and toenails are widely used to assess a long-term selenium status and exposure in humans, whereas levels in blood and urine are used for assessing a short-term selenium status. The enzyme activity of GSHPx, a selenoenzyme, is also widely used for assessing selenium status and exposure. However, GSHPx is sensitive only for detecting selenium deficiency and not overexposure. Other selenoproteins, such as selenoprotein P, may also be useful biomarkers for assessing selenium exposure in humans. In animals, selenium toxicity (selenosis) is well documented, in particular in the livestock grazing in seleniferous areas. The severity of this selenosis depends on the selenium compounds, route of administration, and the nutritional and health status of the animals. The symptoms of chronic selenosis in animals include rough coat, loss of fur, and lameness. In severe cases of acute selenosis, the symptoms are characterized by loss of appetite, hoof damage, abnormal movement, respiratory failure, and blindness; death may follow. Even though the effects of selenium toxicity are well documented, the mechanisms of action are not fully understood. There is a need for further research in this area, and in particular the elucidation of specific molecular and genetic biomarkers of selenoproteins for exposure and biological response. Research on the identification and characterization of selenium metabolites and pathways would also be useful in order to understand their toxicity and detoxification process.

Abbreviations

ALS: amyotrophic lateral sclerosis; GSH: glutathione; GSHPx: glutathione peroxidase; LOAEL: lowest observed effect level; NOAEL: no adverse effect level; RDI: recommended daily intake; RfD: reference dose; Se: selenium

INTRODUCTION Selenium has attracted tremendous interest because of the adverse health effects associated with the element in humans and animals. It is well documented that the element can cause both toxicity and deficiency diseases, and the gap between adequate and toxic levels is relatively narrow. When selenium was discovered in 1817 by the Swedish chemist Jöns Jakob Berzelius, it was appropriately named Selene, after the Greek goddess of the moon (Flohe et al., 1973). Like the two faces of the moon that symbolize both brightness and darkness, selenium can also be described as having two faces, reflecting its essentiality and toxicity (Tinggi, 2003). An early interest in selenium in the 1930s in animal health was particularly spurred by the report of its toxicity causing various disorders such as “alkali disease” and “blind staggers” to grazing animals in certain areas of the U.S. In the 1940s, selenium was recognized both as a toxic element and a carcinogenic agent; however, subsequent studies did not support its carcinogenicity (Franke, 1964; Nelson et al., 1943). IARC (1987) has also stated that there were insufficient data to suggest that selenium is carcinogenic to humans, and subsequently assigned selenium to Group 3, which is not classifiable as to its carcinogenicity to humans.

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The early description of selenium toxicity was probably first recorded by Marco Polo in his 13th-century travels through China, when he described “hoofs to drop off” in horses after grazing in areas of high-selenium-accumulating plants (Spallholz, 1994). Selenium toxicity to livestock in high-selenium areas has also been reported in other countries, including Colombia, Germany, Mexico, Canada, Ireland, and Australia (Reilly, 1996). In the 1950s, interest in selenium focused on its nutritional importance when it was discovered that selenium played a significant role in preventing liver necrosis in vitamin E–deficient rats and chicks (Patterson et al., 1957; Schwarz and Foltz, 1957). Later, in the 1970s, the nutritional importance of selenium in humans and animals was further established when it was found to be an essential component of GSHPx, an antioxidant selenoenzyme (selenium-containing enzyme) (Foster and Sumar, 1997; Rotruck et al., 1973). After the discovery of this GSHPx selenoenzyme, there have been several other selenoproteins (selenium-containing proteins) that have been identified and characterized, including the enzymatic roles of selenium in iodothyronine deiodinases, thioredoxin reductase, and selenoprotein synthetase (Behne and Kyriakopoulos, 2001; Kohrle, 2000). The specific functional role of these selenoproteins in human and animal health is the object of current research interest. This chapter reviews the effects of selenium exposure and its metabolism, bioavailability, and toxicity in humans and animals.

SELENIUM CHEMICAL AND PHYSICAL PROPERTIES Selenium belongs to the VIA group of the periodic table. It is classified as a nonmetallic element that lies between sulfur (S) and tellurium (Te). Some of its chemical and physical properties are summarized in Table 2.1.

TABLE 2.1 Some Atomic and Physical Properties of Selenium Properties

Values

Atomic number Atomic mass Stable isotopes Electronic structure Atomic radius Ionization energy Pauling electronegativity Melting point Boiling point Band energy gap

34 78.96 74Se, 76Se, 77Se, 78Se, 80Se, 82Se [Ar]3d104s24p4 0.14 nm 940.7 kJ/mol 2.4 217°C 685°C 178 kJ/mol

Source: Adapted from Greenwood and Earnshaw (1989).

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Selenium can exist in four oxidation states: 0, –2, +4, and +6. The oxidation state of –2 (selenide, Se–2) is the most common form that involves the structural inorganic complex and organic selenium compounds. In this –2 oxidation state, selenium can form both metallic and nonmetallic compounds such as FeSe, Al3Se2, NaSe, and H2Se. Most of these elements decompose in water or dilute acid to form hydrogen selenide (H2Se), a colorless and highly toxic gas. In many ways, the organic selenium compounds are analogues of organic sulfur compounds and can replace sulfur in cysteine and methionine to form selenocysteine and selenomethionine. However, there are major differences between these two elements; for example, selenols (R-SeH) (pKa = 5.2 for the selenohydryl group of selenocysteine) are stronger acids than thiols (R-SH) (pKa = 8.3 for the sulfhydryl group of cysteine). Furthermore, selenols are readily dissociated at physiological pH, which may be important for their catalytic role, whereas the corresponding thiols are mostly undissociated (Ursini and Bindoli, 1987). Some naturally occurring inorganic and organic selenium compounds are listed in Table 2.2.

SELENIUM IN THE ENVIRONMENT Selenium is naturally emitted into the atmosphere as volatile alkyl selenides (dimethyl selenide, dimethyl diselenide). These alkyl selenides are released from soils, sediments, and sewage sludge as a result of microbial and fungal activity. Plant species also contribute significantly to the release of volatile selenium compounds. Rice crops, for example, will release about 1500 μg Se/kg day on a dry weight basis (Terry and Zayed, 1994). Selenite and selenate are the predominant selenium species in soils. At higher pH, selenites are oxidized to more soluble selenates, which are readily taken up by plants to be metabolized into organic selenium compounds such as selenocysteine, selenocystathionine, and selenomethionine (Schrauzer, 2000). It is thought that selenomethionine in plant tissue is further methylated and catalyzed

TABLE 2.2 Naturally Occurring Inorganic and Organic Selenium Compounds Compound

Formula

Selenite Selenate Selenomethionine Selenocysteine Selenocystathionine Selenocystine Se-methylselenocysteine γ-Glutamyl-Se-methylselenocysteine Methylselenol Dimethylselenide Trimethylselenonium ion

SeO32– SeO42– CH3–Se–CH2CH2CH(NH2)COOH HSe–CH2CH(NH2)COOH HOOC(NH2)CHCH2–Se–CH2CH2CH(NH2)COOH HOOC(NH2)CHCH2–Se–Se–CH2CH(NH2)COOH CH3–Se–CH2CH(NH2)COOH CH3–Se–CH2(COOH)CH–NH–CO–CH2CH2CH(NH2)COOH CH3–SeH CH3–Se–CH3 (CH3)3–Se+

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into volatile dimethylselenium and released into the environment (Terry and Zayed, 1994). On a global scale, it estimated that the amount of released “natural” selenium is about 45,000 tonnes per year (Haygarth, 1994). The major anthropogenic sources of selenium in the environment include coalburning power plants, zinc-cadmium smelters, copper-refining plants, selenium rectifier plants, and agricultural runoffs. It is estimated that the amount of selenium released by anthropogenic means is in the range of 76,000 to 88,000 tonnes per year globally (Haygarth, 1994). The release of selenium in the environment has caused serious environmental problems to wildlife, particularly the pollution of waterways; the effect of this on aquatic environment is discussed later in the text.

SELENIUM METABOLISM AND BIOAVAILABILITY Even though there have been numerous studies on the metabolism of selenium in humans, its specific metabolic pathway in tissue is not well characterized. In animal studies, selenium is absorbed from the gastrointestinal tract and transported into the other tissues such as red blood cells and the liver, where it is metabolized to other forms of selenium compounds (Sunde, 1990). The absorption, transport, and distribution of selenium in the body is dependent on its chemical form, the amount ingested, and its interaction with other dietary components such as vitamins A, E, and C (Reilly, 1996). The predominant forms of selenium in the body are selenomethionine and selenocysteine residues. During protein synthesis, selenocysteine is incorporated into proteins following the action of specific codon for selenocysteine residue (Lee et al., 1996). Proteins that contain selenocysteine residues are referred to as selenoproteins, which can play an essential functional role in redox reactions as the active center of selenoenzymes (Suzuki and Ogra, 2002). In contrast to selenocysteine, selenomethionine is incorporated directly into proteins, not by its own specific codon but by the codon for methinyl residue, which does not discriminate between selenomethionine and methionine residues during protein synthesis (Lee et al., 1996; Sunde, 1990). The incorporation of selenomethionine into proteins this way is referred to as selenium-containing proteins. At present no specific biological function has been established for selenium-containing protein. However, it has been proposed that selenium-containing proteins may be used as a biological selenium pool for target organs when selenium availability is low. It has also been suggested that selenoprotein P, for example, which is rich in histidine, cysteine, and selenocysteine, and a predominant selenoprotein in plasma may play an important antioxidative role and assist in the detoxification of metals (Mostert, 2000; Patching and Gardiner, 1999). Because selenium can replace sulfur in protein synthesis from amino acids such as methionine and cysteine, it may resemble sulfur metabolism. Tracer studies in animals have shown that selenomethionine is metabolized, probably through the methionine metabolic pathways, with eventual formation of dimethylselenides, which are exhaled in breath, and trimethylselenonium ions, which are excreted in urine (Foster et al., 1986). It has recently been shown that selenium is also methylated to form selenosugar (Se-methyl-N-acetylselenohexosamine) in the low-toxic range

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Selenocysteine proteins

Selenomethionine proteins

Selenite Selenocysteine

Selenomethionine

GSSeSG

H2Se (Selenide)

CH3SeH

(CH3)2Se

+

(CH3)3Se

Selenate

Breath

Selenosugar*

GSSeH

Specific selenocysteine proteins

Urine

FIGURE 2.1 Pathways of selenium metabolism. *Selenosugar is a recently identified selenium compound in urine by Kobayashi et al. (2002).

and excreted in urine (Kobayashi et al., 2002). A proposed pathway of selenium metabolism in animal is shown in Figure 2.1. Various terms have been used to describe the bioavailability of elements (substances). The terms may depend on the ability of the elements to exert changes to physiological and homeostatic mechanisms that control their adsorption and that subsequently could result in either adverse health effects or improved growth and development (Stahl et al., 2002). In trace element nutritional studies, bioavailability is generally defined as a quantitative measure of the utilization of a nutrient under conditions to support the organism’s structure and physiological processes (Foster and Sumar, 1997). The rate of selenium absorption and its bioavailability depends on its chemical forms (Fairweather-Tait, 1997). The organic forms such as selenomethionine, which is a predominant form in foods, have a greater rate of absorption (95 to 97%) compared to selenite (44 to 70%) (Whanger, 1998). Even though selenomethionine is more bioavailable, it is not necessarily immediately available for functional selenoproteins. However, under optimal conditions, the absorption rate for selenomethionine is similar to selenate (95 to 98%) (Daneils, 1996). The bioavailability can also be affected by different forms of protein source, for example, the bioavailability of selenium from a fish diet is less than that from beef and wheat (Djujic et al., 2000; Meltzer et al., 1993; Yoshida et al., 2002). It is possible that the reduced bioavailability of selenium from fish could be the result of its interaction with other metals, such as cadmium and mercury. For example, mercury can form a mercury–selenide protein complex, which may play a significant role in detoxification process (Gailer et al., 2000; Sasakura and Suzuki, 1998). However, in recent studies, selenium from seafood has been shown to be as bioavailable as selenium from other high-protein sources (Bugel et al., 2001; Ornsrud and Lorentzen, 2002). There are wide variations in the reported values of selenium

Selenium Toxicity and Its Adverse Health Effects

35

bioavailability from different dietary sources. It has been suggested that treatment of a protein source during food processing may cause protein degradation and improve digestibility, and this may facilitate release of bound selenium with subsequent increase in bioavailability (Ornsrud and Lorentzen, 2002). One of the factors that may influence the final evaluation of bioavailability is the selection of appropriate parameters for the measurement. Windisch et al. (1998) have pointed out that the uncertainty of assessing selenium bioavailability and especially seleno amino acids is that the estimation is based on indirect measurements of parameters, such as GSHPx activities and selenium concentrations in blood and other tissue samples, and not on direct measurements in terms of quantitative bioavailability, which are not yet available.

EFFECTS OF SELENIUM EXPOSURE TOXICITY

OF

SELENIUM COMPOUNDS

In experimental animals, inorganic forms of selenium, such as selenites and selenates, are more toxic than the organic forms, such as selenomethionine, selenocysteine, and selenocystine (Hermann et al., 1991; McAdam and Levander, 1987). Other forms of organic selenium compounds such as dimethylselenide, trimethylselenonium ion, and selenobetaine are significantly less toxic in comparison with selenite salts (Spallholz, 1994). It has recently been shown that the toxicity of selenite is due to its ability to inhibit squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis (Gupta and Porter, 2002). Because of their lower solubility, elemental selenium and selenium sulfide (monosulfide and disulfide) are less toxic than selenites and selenates (Barceloux, 1999). Selenium in the form of hydrogen selenide is highly toxic, and it has been reported to cause respiratory distress to animals. An exposure of 0.02 mg/l of hydrogen selenide in air for 60 min has been reported to cause death to rats in 25 days. In humans, an exposure to hydrogen selenide is reported to cause irritation of the mucous membranes and may result in loss of the sense of smell for about a month (Wilber, 1980). In the last two decades, there has been increased interest in the development of new products for selenium compounds (organoselenides) because of their antioxidant activity and potential use in pharmacology and therapeutics (Chaudiere et al., 1992; Xie et al., 2001). However, few studies have investigated the toxic effects of some synthesized organoselenides. Nogueira et al. (2003) have investigated the toxic effect of diphenyl diselenide and found that the compound induced 100% seizures in mice and 50% death in 72 h after intraperitoneal administration at a dose of 65.5 mg/kg. However, when the compound was administered to rats, they did not show any seizure episodes in all doses tested. It appears that the toxicity of diphenyl diselenide depends on its route of administration and the susceptibility of animal species. When the compound was administered to both rats and mice by the subcutaneous route, neither animal showed signs of seizure, and the LD50 was higher than 156 mg/kg.

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SELENIUM TOXICITY

Reviews in Food and Nutrition Toxicity IN

ANIMALS

It is well documented that high-selenium areas (seleniferous) have caused severe selenium toxicity (selenosis) and death in animals. Chronic selenium toxicity, known as “alkali disease,” has occurred in livestock and domestic animals after they have consumed plants such as grasses and grains that contained 5 to 40 mg/kg selenium over an extended period of time (James et al., 1989). The disease is characterized by dystrophic changes in hooves, rough coat, loss of fur, and lameness. Another form of chronic selenium toxicity, “blind staggers,” is a result of animals consuming high quantities of selenium-accumulating plants where soil selenium levels are high (Raisbeck, 2000). These plant species (Astrgalus, Machaeranthera, Haplopappus, Stanleya), classified as primary indicator plants, can accumulate selenium to about 1000 mg/kg selenium. In severe cases of the disease, blindness can occur and death may follow as a result of starvation, respiratory distress, hoof damage, loss of appetite, and inability to move because of lameness and paralysis. Feeding pigs selenium-accumulating plant species of A. bisulcatus, at 25 μg/g selenium, has been shown to cause neurological dysfunction, paralysis, and death (Panter et al., 1996). In many parts of the world, selenium compounds are widely used in industry and agriculture, and the increased anthropogenic source of selenium is a cause for environmental concern. Elevated levels of selenium in the environment as a result of agricultural activities have caused deaths and deformities in water birds and fish species in the Kesterson National Wildlife Refuge in southern California (Ohlendorf, 2002). The deaths and deformities of embryos and chicks in the area were the result of selenium contamination in the waterways, which can be as high as 30 μg/l in the form of soluble selenates. Plant species readily take up soluble selenates, which are eventually metabolized into organic selenium compounds, including selenomethionine, and subsequently bioaccumulated in the wildlife’s food chain. Environmental selenium toxicity has also been reported in Belews Lake, North Carolina, which caused severe tissue pathology and reproductive impairment in fish species as a result of selenium contamination in wastewater released from a coal-fired electricitygenerating facility (Lemly, 2002). Acute toxicity of selenium in grazing animals is generally rare, because the animals are able to avoid feeds or plants that contain abnormally high levels of selenium. However, it has been observed that the clinical symptoms of acute poisoning include abnormal movement, severe diarrhea, distinct odor due to release of selenium selenide, and respiratory failure; death may follow, varying from a matter of hours to days (Raisbeck, 2000). Selenium toxicosis in pigs that caused paralysis and death after they fed on selenium-contaminated feed has also been reported (Davidson-York et al., 1999; Stowe et al., 1992). Consumption of contaminated feed containing 84 mg/kg selenium has caused acute poisoning in pigs, which inhibited flaccid paralysis, and death may follow within 1 to 2 h as a result of respiratory failure (Hill et al., 1985). Iron supplement contaminated with high concentration of selenium (19.8 mg/g) as a result of accidental mixing with sodium selenite has also been reported to cause acute poisoning and death in piglets (Sivertsen et al., 2003). It has also been reported that pigs given a diet containing selenium as selenite at 5 mg/kg or greater will endure weight loss, alopecia, and staggering gait (Kim and Mahan, 2001).

Selenium Toxicity and Its Adverse Health Effects

37

In experimental animals, selenium toxicity tends to affect species differently. In lambs, the reported LD50 for selenium, administered orally as sodium selenate, was 0.455 mg Se/kg body weight (Hopper et al., 1985). Selenium in the form of sodium selenite is also very toxic, and it has been reported to cause death to 12-week-old lambs after oral administration of 5 mg Se/kg (Smyth et al., 1990). The rate of selenium absorption from the gut of the lambs is different from other animal species and this may explain the greater susceptibility of lambs to selenium toxicity (Hopper et al., 1985). The reported LD50 values for other animals, orally administered with sodium selenite and expressed as mg Se/kg body weight, were 4.8 to 7.0 for rats, 1.0 for rabbits, 3.2 for mice, and 2.3 for guinea pigs (Cummins, 1971; Wilber, 1980).

SELENIUM TOXICITY

IN

HUMANS

Even though the effects of environmental selenium toxicity are well documented in animals, especially as a result of extensive agricultural activities, the incidents of similar toxic effects in humans are rare (Tinggi, 2003). However, the effects of selenium toxicity have been reported in workers who had hypochromic anemia and leukopenia and damaged nails while working for a long period of time in manufacturing of selenium rectifiers (Rosenfeld and Beath, 1964). Exposure to selenium compounds primarily through ingestion and inhalation has caused various health effects, as discussed in the following sections. Respiratory Effects The respiratory system is the most susceptible to injury when exposed to selenium and its compounds through inhalation. A number of reported cases of illness due to high selenium exposure have occurred in the workplace environment and, in particular, in industries that use selenium and its compounds in manufacturing. Chronic exposure of workers at a copper refinery has been reported to cause increased incidence of nose irritation and sputum when they were exposed to selenium in excess of 0.2 mg Se/m3 (Holness et al., 1989). However, a concurrent exposure to other elements, especially arsenic and tellurium, at this copper refinery could also be a contributing factor to the respiratory effects. It has also been reported that inhalation of large quantities of selenium dioxide can cause pulmonary edema, bronchitis, headache, and chill (Glover, 1970; Koppel et al., 1986; Wilson 1962). Neurological Effects There is limited information on the neurological effects caused by high selenium exposure in humans. It has been reported that there is a possible link between the occurrence of amyotrophic lateral sclerosis (ALS) and high selenium exposure (Kilness and Hochberg, 1977). It has also been reported that high exposure of selenium (selenate) in drinking water could enhance the risk of ALS, as shown in the cohort study of Italian women (Vinceti et al., 1996). An intake of high dietary selenium over a long period has also been observed to cause abnormalities of the nervous system in the Chinese population (Yang et al., 1983). In extreme cases, the

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clinical symptoms observed in this population included acroparesthesia, peripheral anesthesia, pain in the limbs, some paralysis, convulsions, and hemiplegia. Reproductive and Developmental Effects No conclusive evidence has been established regarding the adverse health effects of selenium compounds on reproduction and development in humans. A cohort study of Italian women exposed to selenium in drinking water did not show a significant difference in spontaneous abortions, in weight and length at birth, or in prevalence of congenital abnormalities (Vinceti et al., 2000). In 1999, the Health Council of the Netherlands committee for compounds toxic to reproduction produced a report on evaluation and classification of toxic effects of selenium and its compounds on human reproduction (HCH, 1999). The council concluded that the effects of selenium and its compounds on fertility could not be assessed due to the lack of appropriate data, and on the basis of data from animal studies, selenium could not be classified as a substance causing developmental toxicity in humans. However, regarding the effects during lactation, the council recommended that selenium and its compounds should be labeled with R64 (may cause harm to breastfed babies), which is based on Guideline 93/21 EEG of the European Community. Immunological Effects No evidence has been established in regard to adverse immunological effects as a result of high selenium exposure in humans. However, high selenium exposure, particularly from oral supplementation, may be important for immune response. In elderly subjects, oral supplementation of selenium-enriched yeast (100 μg Se/day) was reported to enhance lymphocyte response (Peretz et al., 1991). An oral selenium supplementation (400 μg/day) of sodium selenite in healthy subjects has also been reported to enhance the natural killer (NK) cell activity (Dimitrov et al., 1986; Kiremidjian-Schumacher et al., 1994). However, in another study, an increased selenium intake from a high-selenium diet in healthy subjects did not show any effects on the NK cell activity (Hawkes et al., 2001). Different forms of selenium may have different effects on immune functions, including NK cell activity, and this may explain the contradictory findings in these studies. Diet and selenium-enriched yeast, for example, contain various forms of organic selenium compounds (BNF, 2001; Ip et al., 2000). Toxicity from High Dietary Selenium Intake Selenium toxicity has also been reported in humans as a result of consuming foodstuffs from areas of naturally high selenium levels in soils. Endemic selenium toxicity in humans occurred in areas of high soil selenium in the People’s Republic of China, where the populations consumed foods with an average intake of 5000 μg Se/day over a long period (Yang et al., 1983). In most cases, the patients suffered from peripheral anesthesia and pain in the limbs, and in extreme cases the patients experienced convulsion, paresthesia, and hemiplegia.

Selenium Toxicity and Its Adverse Health Effects

39

Based on the study of Yang et al. (1989), supplementary intake of 600 μg Se/day in the form of selenite over an extended period could cause selenosis as characterized by brittleness of the nails. However, the symptom is reversible once the supplementation is discontinued. From the study of Yang et al. (1989), it has been calculated that the values for NOAEL and LOAEL are 15 and 23 μg/kg/day, respectively (Levander, 1994; Poirier, 1994). These values are calculated using the average body weight of 55 kg in this population and on the basis of a correlation between selenium blood levels and dietary intakes. The NOAEL value of 15 μg/kg/day is then used to calculate the RfD of 5 μg/kg/day (15/3) with an uncertainty factor of 3. The RfD is defined as an estimation of the daily exposure of the human population to a potential hazard that is likely to be without risk of deleterious effects during a lifetime (Levander, 1994). The Australian RDI for selenium in the adult male is 85 μg/day, which is equivalent to 1.2 μg/kg/day for an average adult male of 85 kg. This value of 1.2 μg/kg/day is about a 13-fold difference from the NOAEL value of 15 μg/kg/day. Other reported incidents of selenium toxicity in humans occurred in South America, as a result of consuming nuts from the tree species of the Lecythis ollaria (Coco de Mono’ or Monkey Coconut) (Reilly, 1996). The trees that bear these nuts are natural accumulators of selenium, and the nuts can contain significantly high levels of selenium (20 μg/g) in the form of selenocystathionine, which is highly toxic to humans (Reilly, 1999). The selenium poisoning is characterized by nausea and vomiting, diarrhea, immediate hair loss, nail deformity, and sometimes death. Death from Selenium Ingestion Fatality as a result of acute poisoning from selenium compounds in humans is very rare. However, accidental and intentional ingestion of selenium compounds has caused severe toxicity and death. Ingestion of selenic acid and sodium selenite has been reported to cause abdominal pains ranging from mild gastrointestinal disturbances to severe gastroenteritis, which is often accompanied by a garlicky odor of the breath (Gasmi et al., 1997). Acute selenium poisoning could develop into severe abdominal pains and death in a few hours as a result of cardiovascular failure. These symptoms were described in the death of the 22-year-old woman who took 20 ml of selenium solution in the form of sodium tetraoxoselenate (VI) (Lech, 2002). Reported cases of fatal incidents due to selenium intoxication in humans are shown in Table 2.3. Estimating the lethal dose of selenium for adult humans is difficult because the effect of selenium toxicity depends on its chemical form, route of administration, quantity consumed, nutritional status of individuals, and the interaction with other essential nutrients. There remains a concern that readily available forms of selenium supplements as health food products could pose a risk to the public from selenium toxicity if the products are not appropriately regulated (Tinggi, 2003). There has been a reported case of a 36-year-old man suffering from severe diarrhea, fatigue, and hair loss after taking selenium-containing vitamin tablets. These tablets were found to contain selenium between 500 and 1000 times (2.5 to 5 mg Se/tablet) more than the 5 μg Se/6 tablets as noted on the bottle label (Clark et al., 1996).

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TABLE 2.3 Reported Cases of Human Fatal Selenium Intoxication Age and Sex

Circumstances of Exposure

Clinical Symptoms and Cause of Death

Selenium Level

Ref.

22-year-old female

Ingestion of about 20 Developed severe diarrhea, Brain (2.29 μg/g), Lech, 2002 ml of sodium abdominal pain, and stomach (6.25 μg/g), tetraoxoselenate (VI) cardiovascular failure; intestine (4.37 μg/g), solution with suicidal postmortem findings: liver (4.28 μg/g), intent cerebral edema, kidney (3.48 μg/g), fibromatosis of the heart, lung (1.70 μg/g), hemorrhage of the lungs, blood (1.42 μg/g) necrosis of the gastric mucosa

22-monthold male

Developed ventricular Blood (12 μg/ml), Accidental ingestion fibrillation and pulmonary gastric content about 15 ml of gun edema; postmortem (270 μg/ml) bluing solution findings: hemorrhagic containing selenious acid (H2SeO3, 9.3%), gastritis, grayish nitric acid (NHO3, discoloration of 4.6%), and copper esophagus and gastric nitrate (Cu(NO3)2, mucosa 4.6%)

Quadrani et al., 2000

32-year-old male

Ingestion of unknown Coma, garlic odor of the Stomach content amounts of selenium breath, fascicular (520 μg/g), convulsions of muscles, liver (1.4 μg/g), peripheral cyanosis, lung (1.4 μg/g), pulmonary edema; blood (0.5 μg/g) postmortem findings: swelling of heart, kidney, and glottis, orange-brown discoloration of the gastric mucosa

Farago and Horvath 1987

17-year-old male

Ingestion of selenium dioxide (10 g) with suicidal intent

Koppel et al., 1986

Developed asystole and Blood (38 μg/ml) apnea, coma; postmortem findings: second-degree chemical burn of both esophagus and stomach, orange-brown discoloration, and garlic odor of the viscera

(continued)

Selenium Toxicity and Its Adverse Health Effects

41

TABLE 2.3 (CONTINUED) Reported Cases of Human Fatal Selenium Intoxication Age and Sex

Circumstances of Exposure

Clinical Symptoms and Cause of Death

44-year-old male

Accidental inhalation and ingestion during explosion while mixing selenic acid and caustic soda at workplace

Pulmonary edema, second- Brain (0.47 μg/g), degree burns, garlic odor stomach content of the breath, and (18.0 μg/g), circulatory failure; liver (5.4 μg/g), postmortem findings: kidney (1.53 μg/g), hemorrhagic alveolar lung (5.39 μg/g), edema of the lungs, plasma (18.4 μg/g), cyanosis of the brain urine (2.11 μg/g)

Schellman et al., 1986

40-year-old male

Ingestion (<90 ml) of gun bluing solution containing selenious acid (4%), copper sulfate (CuSO4, 2.5%), and hydrochloric acid (HCl, 1.7 M) with suicidal intent

Vomiting, diarrhea, cardiac Brain (1.2 μg/g), arrest, pulmonary edema; stomach content postmortem findings: (18.0 μg/g), necrosis of proximal renal liver (5.4 μg/g), tubules and dilation of kidney (14.4 μg/g), glomerulus in kidney, spleen (3.5 μg/g), hemorrhage of stomach lung (12.7 μg/g), mucosa blood (2.6 μg/g)

Matoba et al., 1986

52-year-old female

Ingestion of gun Vomiting, garlic odor of the Liver (0.79 μg/g), bluing solution breath, metabolic heart (3.36 μg/g), (30–60 ml) acidosis, respiratory muscle (0.14 μg/g), containing H2SeO3 distress, severe myopathy, serum (2.44 μg/g (2%), CuSO4 (2.5%), pulmonary edema; after 4 days), and methanol (10%) postmortem findings: urine (2435 μg/24 h with suicidal intent acute small bowel after 4 days) infarction, diffused lung, hemorrhage

Selenium Level

Ref.

Pentel et al., 1985

MECHANISMS OF SELENIUM TOXICITY There is much interest in the investigation of the mechanisms of selenium toxicity in both in vitro and in vivo model systems. Several studies have indicated that the effect of selenium toxicity is species dependent. High selenium levels in tissue have been shown to cause teratogenicity in aquatic birds and chicken, but this toxic effect has not been clearly demonstrated in mammals (Usami et al., 2002). The toxicity of selenium, particularly to aquatic birds that causes avian embryo malformations, could be the result of replacement of essential sulfur-containing enzymes and structural proteins with excess selenium analogues such as selenomethionine (Hoffman, 2002). Exposure to excess dietary selenium in mammals and birds has caused damage and deformity to fur, nails, hooves, and feathers, which all contain keratin (high in sulfur) (Spallholz and Hoffman, 2002). The selenite concentration could

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also change the structural chemistry of sulfur-containing proteins or enzymes as selenite interferes with the critical sulfhydryl groups by forming selenotrisulfides (Gupta and Porter, 2001; Park and Whanger, 1995). At present, the specific mechanisms of selenium toxicity have not been fully elucidated. However, it has been proposed that high levels of selenium in tissue may be due to its pro-oxidant activity in cellular systems. There is increasing evidence that the mechanism of selenium toxicity is the result of the formation of active oxygen species superoxide anion (O2–) after the reaction of selenium (selenite) with glutathione (GSH) (Spallholz, 1997). Much of this toxicity involves the generation of hydroxyl radical (OH•) after the reaction of O2– with peroxide (H2O2) (Halliwell, 1996). It is well established that excess H2O2 in mammalian cells causes DNA strand damage, which is the result of OH• attack. It has been shown that when GSH is added together with selenite to cell cultures, there is an immediate toxicity to the cells (Hu and Spallholz, 1982). Other selenium compounds that have been reported to generate O2– in vitro include selenocysteine, selenocystamine, diphenyl diselenide, and methylseleninic acid (Sphallolz 1997; Spallholz and Hoffman, 2002). It has been demonstrated that under aerobic conditions and in the presence of excess GSH, selenocystamine, for example, can continuously catalyze the production of superoxide, and this reaction has been described in detail by Chaudiere et al. (1992). Seko and Imura (1997) have also shown the reaction of selenite with GSH and then H2Se to produce superoxide. In the presence of peroxide and oxygen, this superoxide is further converted to a reactive hydroxyl radical through the process of Fenton chemistry. The increased production of superoxide from high selenium levels in tissue could exert its toxicity when the pro-oxidant activity exceeds its antioxidant defenses (Spallholz, 1997). In animal experimentation, subchronic selenium toxicity has been shown to cause oxidative stress in calves after daily oral administration with selenium selenite (0.25 mg/kg) as indicated by an increase in lipid peroxidation (Kaur et al., 2003). The pro-oxidant activity of selenium compounds generating active oxygen species may also be a mechanism of inducing apoptosis in cancer cells that could have potential in chemoprevention (Spallholz, 1994; Stewart et al., 1999).

ASSESSMENT OF BIOMARKERS FOR SELENIUM STATUS IN HUMANS Biomarkers are defined as indicators for signaling changes or events in biologic systems or samples, and these can be classified into markers of exposure, markers of effect, and markers of susceptibility (ICPS, 2001). The biomarkers that are widely used for the assessment of selenium exposure in human populations include levels in blood, nails, urine, and hair. The levels of selenium in these tissues are shown in Table 2.4. The levels of selenium in plasma, serum, and whole blood tend to indicate recent selenium exposure, whereas the levels of selenium in red blood cells are probably more sensitive for a long-term assessment of selenium status. It has been suggested that the reference values of selenium levels in the blood of healthy individuals are between 75 and 240 μg/kg for red blood cells, between 30 and 105

Selenium Toxicity and Its Adverse Health Effects

43

TABLE 2.4 Selenium Levels in Human Blood, Urine, Hair, and Nails from Several Countries Tissue Concentrationa

92 ± 15 (53–131) 118 ± 27 (64–173) 153 ± 21 41 ± 11 50–135 155 (81–225)

47–105 152 (115–278) 106 ± 4 92 ± 1 (60–130) 58.2 ± 18.3

131 ± 2 (60–210) 103 ± 30 61.8 ± 19.6 174 (110–280)

Country

Ref.

μg/l) Plasma (μ Australia Dhindsa et al., 1998 Italy Sesana et al., 1992 Japan Hojo, 1987 New Zealand Thomson and Robinson, 1980 Poland Wasowicz et al., 2003 United States Clark et al., 1984 μg/l) Whole Blood (μ Argentina Hevia et al., 2002 Australia Judson et al., 1978 China Xu et al., 1997 Germany Oster et al., 1988 Serbia Maksimovic and Djujic, 1997 Red Blood Cells Germany New Zealand Serbia United States

μg/l) (μ Oster et al., 1988 Thomson and Robinson, 1980 Maksimovic and Djujic, 1997 Meyer and Verrault, 1987

0.45 ± 0.06

μg/g) Nails (μ Finland Varo et al., 1994

0.54 ± 0.91

Greece

Bratakos et al., 1990

0.63 ± 0.12

Netherlands

van’t Veer et al., 1990

1.56 ± 0.58 (0.083–3.82)

United States

Longnecker et al., 1991

0.36 ± 0.17

μg/g) Hair (μ China Yang et al., 1983

0.42 ± 0.88

Greece

Bratakos et al., 1990

0.42 ± 0.10 (0.21–0.63)

Sweden

Muramatsu and Parr, 1988

0.64 ± 0.02

United States

Thimaya and Ganapathy, 1982

26 ± 12

μg/l) Urine (μ China Yang et al., 1983

24 ± 2

Greece

Bratakos et al., 1990

22 ± 2 (2–31)

Italy

Minoia et al., 1990

58 ± 26 (20–113)

Japan

Hojo, 1981

a

The concentration values are mean ± standard deviation and range.

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μg/l for serum, and between 80 and 140 μg/l for whole blood (Caroli et al., 1994). Foods are the major sources of selenium, and the levels in diets have a significant influence on the selenium status of individuals. Seafood and meat products contain relatively high levels of selenium, and relatively low levels are found in fruits and vegetables (Plessi, 2001; Tinggi, 1999; Tinggi et al., 1992). Selenium dietary intakes vary widely among countries and regions, which reflect the levels of selenium in soils and the produce of the regions (Combs, 2001). Table 2.5 and Table 2.6 show the levels of selenium intakes in selected countries and in selected foods, respectively. Blood platelet selenium levels have also been used to assess selenium status, and because they can contain higher levels of selenium than most other tissues and have shorter turnover, they are more sensitive in detecting changes to selenium status (Meltzer et al., 1993; Neve, 1991). However, the disadvantage of using blood platelets is that the cells are difficult to separate, which subsequently will affect the accuracy of the analysis. The other biomarker that has been widely used for assessing selenium status in humans is the measurement of GSHPx activity (Neve, 2000). GSHPx is a functional selenoenzyme that plays an important role in eliminating peroxides and thus protecting cells from oxidative damage. There are some limitations in using GSHPx as a biomarker. At low selenium status, there are significant correlations between GSHPx and selenium levels in plasma and whole blood. These correlations become less significant at higher selenium status, particularly when selenium levels in plasma or whole blood are equal to or greater than 100 μg/l. At these higher blood selenium levels, GSHPx activity reaches its saturation level or plateau, and this may suggest that selenium adequacy has reached its optimal level (Neve, 1991). Because GSHPx is more sensitive in detecting low selenium status, it is more suitable for assessing selenium deficiency in the population than for assessing selenium overexposure.

TABLE 2.5 Estimated Selenium Intakes in Several Countries μg/day) Selenium (μ

Country

Ref.

57–87 17–111 53–80 38–47 110 28–105 44 61–73 30–40 18–53 29–60 60–160

Australia Brazil China Germany Greece India Ireland Mexico Poland Turkey United Kingdom United States

Farady et al., 1989 Maihara et al., 2001 Zhang et al., 2001 Oster and Prellwitz, 1989 Bratakos et al., 1990 Dang et al., 2001 Murphy et al., 2002 Valentine et al., 1994 Wasowicz et al., 2003 Aras et al., 2001 BNF, 2001 Longnecker et al., 1991

10–300 7–220 37–1517 47–220 52–1132 ND–141 30–630 24–636 29–2000 110–265

Australia Egypt Finland France Greece Italy Mexico Slovak Republic United Kingdom United States

10–150 2–274 29–516 20–177 23–352 ND–1257 100–320 13–59 1–320 10–33

Cereals 120–500 223–397 156–1060 83–423 168–1450 747–1005 440–610 196–521 100–760 265–1090

Fruit 2.0–5 1–2 <1–8 <2–13 2–17 ND — 0.7–8 ND ND–10

Vegetables <1a–22 1–2 2–204 <2 6–44 ND —b 0.5–129 ND–20 19–165

Food Group Seafood

b

Low levels are expressed as less than the detection limit of the analytical method. Dash indicates no data were available for selenium content.

a

ND = Not detectable.

Meats

Country <1–110 6–11 22–278 6–19 11–129 ND 60–220 4–41 5–140 ND–43

Dairy Products

Ref. Tinggi et al., 1992; Tinggi, 1999 Hussein and Bruggeman, 1999 Eurola et al., 1991 Simonoff et al., 1988 Bratakos et al., 1987 Ciappellano et al., 1992 Wyatt et al., 1996 Kadrabova et al., 1997 Barclay et al., 1995; Thorn et al., 1978 Pennington et al., 1995; Zhang et al., 1993

TABLE 2.6 μg/kg, wet weight) in Several Countries Typical Contents of Selenium in Food Products (μ

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Moreover, the GSHPx method lacks standardization, the results produced can vary widely, and the method may not be a reliable index for a long-term selenium status (Diplock, 1993). The concentrations of other selenoproteins such as selenoprotein P, which is a predominant form of selenoprotein found in plasma, have recently been used to assess selenium status in humans, and may offer an alternative to GSHPx (PerssonMoschos et al., 1995). It has been observed that there is good correlation between selenium and selenoprotein P levels in blood plasma in humans after supplementation at 100 μg Se/day for 14 days (Hill et al., 1996). Greater increases in selenoprotein P than selenium and GSHPx in blood of New Zealand subjects have also been observed after supplementation with various levels of selenium compounds (Duffield et al., 1999). Hair, nail, and urine samples are widely used in epidemiological studies for human populations because they are less invasive and selenium levels in these samples tend to reflect long-term exposure (Longnecker et al., 1996; Vinceti et al., 2001). The selenium urine levels in humans are generally low (Table 2.3), except in selenium-rich areas where selenium levels can be as high as 330 μg/l without signs of toxicity (Yang et al., 1983). Renal clearance of selenium, predominantly in the form of trimethylselenonium ion from urine, a major route of excretion, appears to be metabolically regulated. Human subjects of low selenium status tend to excrete selenium at a much lower rate than subjects of high selenium status, which may suggest the presence of regulatory mechanisms for conserving selenium body levels (Neve, 1991). However, the excretion rates of 20 to 200 μg Se/day in humans are not associated with deficiency or toxicity problems (Alaejos and Romero, 1993). The levels of selenium in hair and nails are useful biomarkers for detecting the effect of excessive selenium exposure in humans, and any clinical signs of selenium toxicity will be indicated by loss of hair and nail deformity (Clark et al., 1996; Yang et al., 1983, 1989). Hair selenium levels can vary widely and may not be as reliable as the nail selenium levels, particularly the toenails, which have good correlation with the selenium dietary intakes (Longnecker et al., 1991; Yang et al., 1989). However, morphological changes in hair loss and fingernails may not provide specific effects of overexposure to selenium, and if these clinical signs are observed, other biomarkers for selenium status may be determined.

CONCLUSIONS As an essential trace element for animals and humans, selenium is ubiquitous in nature and its levels in the environment and biological tissues are generally low. At relatively higher levels, selenium can be toxic to animals, particularly in areas of naturally high selenium soils. Pollution of waterways as a result of selenium contamination from industrial and agricultural activities has caused death in wildlife and deformity in aquatic animals, particularly from teratogenicity, which tends to affect aquatic animal species more than mammals. Selenium is accumulative in tissues and subsequently will biomagnify in higher animals in the food chain. Selenium toxicity in humans is rare, and when cases of it occur, the symptoms are similar to those described in animals. The degree of selenium toxicity in experimental animals depends on its chemical

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forms, route of administration, and the health status of the individuals. The mechanistic mode of action of selenium toxicity has not been fully elucidated. However, there is increasing evidence that selenium toxicity could be the result of the pro-oxidative role of selenium in producing active oxygen species after reacting with GSH in cellular systems. This pro-oxidative role of selenium and its effect on DNA strand damage is an area of current research interest. Recently developed complementary DNA microarray technology may make it possible to further study the mechanisms of selenium toxicity at molecular levels by elucidating patterns of gene expression profiling for biological response and exposure to selenium toxic effects. These gene response patterns may provide new insight into the mechanisms of selenium-induced toxicity in human diseases and may be useful for the development of molecular biomarkers of exposure and for risk assessment in epidemiological studies. The mechanistic action of selenium toxicity could also depend on its molecular species, and this area of research has also attracted significant interest, in particular the determination, characterization, and identification of specific selenium metabolites as they undergo biotransformation in cellular systems. The identification of these metabolites could be an important step in understanding the mechanisms for toxicity and the detoxification process. The recent finding of selenosugar compound in urine and liver has indicated the ability of selenium to form various compounds in biological systems. An understanding of the effect of selenium toxicity at molecular levels may also help understanding of the important role of selenium in apoptosis and chemoprevention for cancer treatment.

ACKNOWLEDGMENTS The author is grateful to Professor M. Moore for his comments on the manuscript. Gratitude is also expressed to Emeritus Professor C. Reilly for providing references and suggestions on the manuscript. The support of the Department of Queensland Health is greatly appreciated. The views expressed in this manuscript are the author’s own and not the opinion of the Department of Queensland Health.

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Suzuki, K.T. and Ogra, Y. (2002) Metabolic pathway for selenium in the body: speciation by HPLC-ICP MS with enriched Se, Food Additives and Contaminants, 19: 974–983. Terry, N. and Zayed, A.M. (1994). Selenium volatilization by plants, in W.T.J. Frankenberger and S. Benson, Eds., Selenium in the Environment, New York: Marcel Dekker, 343–367. Thimaya, S. and Ganapathy, S.N. (1982) Selenium in human hair in relation to age, diet, pathological condition and serum levels, Science of the Total Environment, 23: 41–49. Thomson, C.D. and Robinson, M.F. (1980) Selenium in human health and disease with emphasis on those aspects peculiar to New Zealand, American Journal of Clinical Nutrition, 33: 303–323. Thorn, J., Robertson, J., and Buss, D.H. (1978) Trace nutrients. Selenium in British food, British Journal of Nutrition, 39: 391–396. Tinggi, U. (1999) Determination of selenium in meat products by hydride generation atomic absorption spectrophotometry, Journal of AOAC International, 82: 364–367. Tinggi, U. (2003) Essentiality and toxicity of selenium and its status in Australia: a review, Toxicology Letters, 137: 103–110. Tinggi, U., Reilly, C., and Patterson, C.M. (1992) Determination of selenium in foodstuffs using spectrofluorometry and hydride generation atomic absorption spectrometry, Journal of Food Composition and Analysis, 5: 269–280. Ursini, F. and Bindoli, A. (1987) The role of selenium peroxidases in the protection against oxidative damage of membranes, Chemistry and Physics of Lipids, 44: 255–276. Usami, M., Tabata, H., and Ohno, Y. (2002) Effects of methionine on selenium embryotoxicity in cultured rat embryos, Teratogenesis, Carcinogenesis, Mutagenesis, 22: 301–308. Valentine, J.L., Cebrian, M.E., Garcia-Vargas, G.G., Faraji, B., Kuo, J., Gibb, H.J., and Lachenbruch, P.A. (1994) Daily selenium intake estimates for residents of arsenicendemic areas, Environmental Research, 64: 1–9. van’t Veer, P., van der Wielen, R.P., Kok, F.J., Hermus, R.J.J., and Sturmans, F. (1990) Selenium in diet, blood, and toenails in relation to breast cancer: a case control study, American Journal of Epidemiology, 131: 987–994. Varo, P., Alfthan, G., Huttunen, J.K., and Aro, A. (1994) Nationwide selenium supplementation in Finland — effects on diet, blood and tissue levels, and health, in R.F. Burk, Ed., Selenium in Biology and Human Health, New York: Springer-Verlag, 199–218. Vinceti, M., Guidetti, D., Pinotti, M., Rovesti, S., Merlin, M., Vescovi, L., Bergomi, M., and Vivoli, G. (1996) Amyotrophic lateral sclerosis after long-term exposure to drinking water with high selenium content, Epidemiology, 7: 529–532. Vinceti, M., Cann, C.I., Calzolari, E., Vivoli, R., Garavelli, L., and Bergomi, M. (2000) Reproductive outcomes in a population exposed long-term to inorganic selenium via drinking water, Science of the Total Environment, 250: 1–7. Vinceti, M., Wei, E.T., Malagoli, C., Bergomi, M., and Vivoli, G. (2001) Adverse health effects of selenium in humans, Reviews on Environmental Health, 16: 233–251. Wasowicz, W., Gromadzinska, J., Rydzynski, K., and Tomczak, J. (2003) Selenium status of low-selenium area residents: Polish experience, Toxicology Letters, 137: 95–101. Whanger, P.D. (1998) Metabolism of selenium in humans, Journal of Trace Elements in Experimental Medicine, 11: 227–240. Wilber, C.G. (1980) Toxicology of selenium: a review, Clinical Toxicology, 17: 171–230. Wilson, H.M. (1962) Selenium oxide poisoning, North Carolina Medical Journal, 23: 73–75. Windisch, W., Gabler, S., and Kirchgessner, M. (1998) Effect of selenite, seleno cysteine and seleno methionine on the selenium metabolism of 75Se labeled rats, Journal of Animal Physiology and Animal Nutrition, 78: 67–74.

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Wyatt, C.J., Melendez, J.M., Acuna, N., and Rascon, A. (1996) Selenium (Se) in foods in northern Mexico, their contribution to the daily Se intake and the relationship of Se plasma levels and glutathionine peroxidase activity, Nutrition Research, 16: 949–960. Xie, Y., Short, M.D., Cassidy, P.B., and Roberts, J.C. (2001) Selenazolidines as novel organoselenium delivery agents, Bioorganic & Medicinal Chemistry Letters, 11: 2911–2915. Xu, G.L., Wang, S.H., Gu, B.Q., Yang, Y.Y., Song, H.B., Xue, W.L., Liang, W.S., and Zhang, P.Y. (1997) Further investigation on the role of selenium deficiency in the aetiology and pathogenesis of Keshan disease, Biomedical and Environmental Sciences, 10: 316–326. Yang, G., Wang, S., Zhou, R., and Sun, S. (1983) Endemic selenium intoxication of humans in China, American Journal of Clinical Nutrition, 37: 872–881. Yang, G., Yin, S., Zhou, R., Gu, L., Yan, B., Liu, Y., and Liu, Y. (1989) Studies of safe maximal daily dietary Se-intake in a seleniferous area in China. Part II: Relation between Seintake and the manifestation of clinical signs and certain biochemical alterations in blood and urine, Journal of Trace Elements and Electrolytes in Health and Disease, 3: 123–130. Yoshida, M., Abe, M., Fukunaga, K., and Kikuchi, K. (2002) Bioavailability of selenium in the defatted dark muscle of tuna, Food Additives and Contaminants, 19: 990–995. Zhang, X., Shi, B., and Spallholz, J.E. (1993) The selenium content of selected meats, seafoods, and vegetables from Lubbock, Texas, Biological Trace Element Research, 39: 161–168. Zhang, Z.W., Shimbo, S., Qu, J.B., Watanabe, T., Nakatsuka, H., Matsuda-Inoguchi, N., Higashikawa, K., and Ikeda, M. (2001) Dietary selenium intake of Chinese adult women in the 1990s, Biological Trace Element Research, 80: 125–138.

3

Arsenic in Fish: Implications for Human Toxicity Marjan De Gieter and Willy Baeyens

CONTENTS Abstract ....................................................................................................................57 Introduction..............................................................................................................58 Arsenic in Marine Organisms .................................................................................59 Origin of Arsenic in Fish and Shellfish ......................................................59 Chemical Forms of Arsenic in Fish and Shellfish ......................................60 Arsenic Concentrations in Fish and Shellfish.............................................65 Human Exposure to Fish Arsenic ...........................................................................70 Fate of Ingested Organic Arsenic................................................................71 Fate of Ingested Inorganic Arsenic .............................................................71 Toxicity to Humans .................................................................................................74 Concentrations vs. Legal Limits..................................................................74 Additional Cautions .....................................................................................75 Dose–Response Assessments ......................................................................76 Conclusions..............................................................................................................77 References................................................................................................................77

Abstract

Marine organisms can contain significant amounts of arsenic (As), which is an initially alarming observation with regard to human health. Extensive speciation studies have provided evidence for the presence of As in seafood in a variety of compounds. Although it is assumed that As is passed up the food chain via diet, and although the abundance of the several arsenicals in the marine ecosystem is well documented by now, the understanding of its exact origin and metabolical pathways remains hypothetical. Also, the origin of large differences in As concentration among fishes, both interspecies and intraspecies, remains to be elucidated. A reassuring finding is that the majority of the As in seafood is present in forms that are believed to be nontoxic. The fraction of toxic As generally remains reasonably low and, thus, is not believed to pose a direct threat to human health upon consumption of seafood. Nevertheless, formal norms for permissible levels of

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contamination are still ambiguous. Extensive research has also provided information about the fate of both the toxic and the nontoxic As compounds in the human body upon ingestion. Even though the majority of the ingested As is excreted in the urine, either unchanged or methylated, some cautions regarding possible health effects should be considered.

INTRODUCTION Toxic elements like arsenic (As) are generally regarded as accidental contaminants, although they are frequently found in plants and animals of different trophic levels. Certainly, As is widely distributed in the environment, both from natural sources and through anthropogenic applications. The As content of Earth’s crust averages 2.5 mg kg–1, ranking the element twentieth in abundance among the elements in Earth’s crust (National Academy of Science, 1997). The highest amounts of mineral As generally occur in ores. Arsenic can be associated with these ores as a minor compound, for example, in pyrite (FeS2) and sphalerite (ZnS) or as a major compound in arsenopyrite (FeAsS), orpiment (As2S3), and realgar (As4S4). Further, As is extensively used in industry, farming, and agriculture: small amounts are used in, among other industries, the glass and ceramics industry; lead arsenate, copper acetoarsenite, sodium arsenite, calcium arsenate, and organic compounds are used as pesticides; methylarsonic acid and dimethylarsinic acid are used as herbicides. Some phenylarsenic compounds such as arsanilic acid are used as a growth promoter for swine and as a food additive to combat the disease cecal coccodiosis in poultry. A relatively new but growing application is the use of chromated copper arsenate (CCA) as a preservative for wooden structures, such as pilings and docks, along the shoreline. They react with wood to create water-insoluble compounds, but studies have demonstrated leaching of As and copper into the marine environment (Brown and Eaton, 1999). In addition, uptake of As by algae growing on CCA-treated wood has been demonstrated (Weis and Lores, 1993). Pesticides and herbicides have a potential for contaminated runoff to rivers, estuaries, and the ocean (Mariner et al., 1996), but major pollution occurs only in unusual circumstances. Nevertheless, rivers are the main sources of As input into the marine environment. According to Chilvers and Peterson (1987), their estimated worldwide contribution to the ocean is 242,000 ton yr–1. Humans are exposed to many sources of As: via food, water, soil, and air, but exposure via diet is by far the most important. Smith et al. (1975) estimated the total daily intake of As from the Canadian diet at 0.025 to 0.035 mg day–1. Hamilton and Minski (1973) studied a diet based on fish and found an average intake of 0.1 mg day–1. This considerable difference likely results from variations in the amount of seafood in the investigated diets. Indeed, the Ministry of Agriculture, Fisheries and Food (1982) conducted a study of U.K. total diets and concluded that, from the nine categories of foodstuffs included in this study, fish is the most significant source of As (Table 3.1). The U.S. FDA (1993) indicated that, in most cases, fish and other seafood consumption accounts for 90% of the total human As exposure. Yet, in neither of the reports was a distinction drawn between exposures to inorganic and organic As. Nevertheless, this distinction is of major importance with respect to

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TABLE 3.1 Arsenic Content of Food Groups in U.K. Total Diets

Food Group

Estimated Weight Eaten (kg day–1)

Mean As Concentrations (mg kg–1)

Estimated Daily Intake μg) (μ

Cereals Meat Fish Fats Fruits/sugars Root vegetables Other vegetables Beverages Milk

0.23 0.15 0.02 0.08 0.17 0.18 0.11 0.12 0.40

<0.02 <0.03 2.71 <0.02 <0.02 <0.02 <0.02 <0.005 <0.01

<5 <5 54 <2 <3 <4 <2 <3 <4

Total

1.46

<81

Source: Ministry of Agriculture, Fisheries and Food, 1982.

metabolism and toxicity, because different arsenicals exhibit different toxicities and metabolisms. This chapter describes the results of several studies on origin, speciation, and concentration levels of As in fish and shellfish. In addition, the link between this knowledge of fish contamination with As and the response of the human body upon ingestion of As from fish is reviewed.

ARSENIC IN MARINE ORGANISMS Arsenic levels of marine organisms are generally much higher than those of terrestrial organisms. Further, among marine organisms, it is fish, crustaceans, and mollusks that contain the highest As concentrations. The amounts of As that fish accumulate directly from the water is nevertheless supposed to be very limited; instead, they are believed to accumulate As mainly via diet. Indeed, a number of other elements (e.g., mercury) are also translocated through the food chain, and thus an important question with regard to toxicity of As relates to the ability of organisms to concentrate As.

ORIGIN

OF

ARSENIC

IN

FISH

AND

SHELLFISH

Total As concentrations reported in phytoplankton and algae are markedly higher than those in water or sediments from the same study area. The brown macroalgae Fucus spiralis accumulates four times more arsenate (H3AsO4) than arsenite (H3AsO3) from equivalent concentrations in seawater (Klumpp, 1980). Indeed, the close relation between the chemical properties of As and phosphorus tends to cause dissolved arsenate to be taken up together with phosphate, leading to its accumulation

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via the cellular phosphate transport system. Microorganisms are thus able to accumulate substantial amounts of As, and this pathway is undoubtedly important for introduction of As in aquatic food webs. Nevertheless, microorganisms do not accumulate As to an unlimited extent, according to several studies that observed toxicity effects even at this level of the food chain. Sanders (1979) showed that a dose of As three times above that normally found in the ocean, combined with phosphorus concentrations below 0.3 nM, was enough to inhibit the growth of Skeletonema costatum. Sanders and Cibick (1985) observed a shift in dominant phytoplankton species, when As concentrations are one to ten times higher than normal. This demonstrates that increased As concentrations prevent the growth of certain phytoplankton species on the one hand and enhances the growth of other, more As tolerant species on the other hand, thus bringing about changes in the distribution of the species. In light of biomagnification, it would be logical that the accumulation of As vs. its level in water would be even more pronounced higher up the food chain. Certainly, for mollusks and crustaceans as well as for fish, diet is a far more important source of As than water (Fowler and Ünlü, 1978). However, in practice, biomagnification of As higher up the food chain is not perceived. Where molluscan species have been analyzed at the same locations as macroalgae, accumulated levels of As are broadly similar in the two groups. Nor is there a direct link between total As levels and dietary pattern. Shiomi et al. (1984) considered that carnivorous gastropods exhibit higher concentrations of As than herbivorous or planktivorous species, while Maher (1985) stated the opposite: that plankton-feeders accumulate greater amounts of As than either herbivores or carnivores. Also for fish, differences in As content could not be explained by looking only at their diet. De Gieter et al. (2002) concluded that fish species that feed primarily on larger fish contain less As than species that feed on benthic organisms and smaller fish. However, these authors also observed differences in As content among fish species that basically have the same feeding patterns; brill, for example, appeared to contain less As than megrim. The existence of a relationship between increased As concentrations and age or size of the fish is also not sufficiently established; Taylor and Bright (1973) could not find such correlation in groupers from the Bahamas, while Bohn (1975) did report this link in several species from the Arctic waters. Several food chain experiments, for example, autotrophic grazer–zooplanktonic grazer–guppy (Maeda et al., 1990), insect–crayfish–trout (Mason et al., 2000), or kelp–detrital feeding worms–whiting (Edmonds and Francesconi, 1981a), could not provide evidence for biomagnification of As throughout the food chain. These studies did, however, point out that instead of being biomagnified, As is metabolized by the organisms.

CHEMICAL FORMS

OF

ARSENIC

IN

FISH

AND

SHELLFISH

In reality, it is the metabolism specific to a certain species that influences the organism’s As content more than the trophic echelon to which it belongs. In the algae group, Hisikia fusiformis has seven times more arsenic than Undaria pinnitifida

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(Shinagawa et al., 1983). For polychaetes, Tharyx marioni has an As concentration 70 times greater than that of Perinereis cultrifera taken from the same site. As a result of the existence of this mechanism to metabolize As, the chemistry of the element within organisms is complex. While in the water column, As is mainly present as inorganic As, and macroalgae, mussels, shrimp, and fish, with a few exceptions, were found to exhibit consistently low concentrations of inorganic As. Sanders (1979) attributed this to possible detoxification mechanisms that regulate the concentrations of inorganic As. Indeed, studies with organisms at the bottom of the food chain indicated that, within organisms, inorganic As is transformed into a wide variety of derivatives (Andreae and Klumpp, 1979) (Figure 3.1). Lunde (1968) reported that oil from cod liver and herring contained primarily two arsenicals. Their structures appeared to be analogous to phospholipids. Aqueous extracts of herring, capelin, and mackerel, on the other hand, contained As in a water-soluble form (Lunde, 1969). Later, this same compound was also extracted from squid, shrimp, haddock, halibut, and mackerel (Lunde, 1975) and from mussels, lobster, and stingray (Edmonds and Francesconi, 1977). Edmonds and Francesconi (1981b) separated the compound from the tail muscle of the western rock lobster, Panulirus cygnus, and identified it as the zwitterion arsenobetaine (AB). Meanwhile, AB has been identified in a large range of marine animals: in fish, crustaceans, mollusks, echinoderms, and polychaetes. Moreover, most of the reports that describe the presence of AB in marine animals also note that this compound is present in proportions often larger than 80% of the total As. It became apparent that the only marine species that lacked AB were the algae. Lunde (1973) performed experiments on marine algae cultivated in seawater containing 74As, but could not find any AB. Instead, the 74As-labeled arsenate and arsenite were incorporated into various lipid- and water-soluble fractions. Irgolic et al. (1977) again pointed out that the lipid-soluble arsenicals, this time extracted from the macroalgae Tetraselmis chuii, appeared to be arsenolipids, As analogues of phospholipids. One of the water-soluble compounds was identified as dimethylarsinic acid (DMA). The prime detoxification in microorganisms is indeed believed to be based on the reduction and methylation of inorganic As (Cullen et al., 1989). Because these methylated derivatives are largely excreted by the organism, this offers a plausible explanation for the presence of the minor amounts of methylated As in the water column. From the brown algae Ecklonia radiata, two compounds were separated and characterized as containing the sugar moiety β-ribofuranoside and a specific side chain (Edmonds and Francesconi, 1981c). These two components together accounted for approximately 81% of the total As in the macroalgae. Shortly after, a second assessment of two arsenosugars was made, from the kidney of the giant clam, Tridacna maxima (Edmonds et al., 1982). Only one of them appeared to match the arsenosugars found in the brown algae. Since this discovery, the presence of arsenosugars has been confirmed in several algae and mollusks (Morita and Shibata, 1987; Shibata et al., 1987). Moreover, numerous types of arsenosugars have been identified (Figure 3.1). They are believed to be major As constituents of algae and minor constituents in mollusks. But, the arsenosugars in clams are postulated to be synthesized by symbiotic zooxanthellae in the mantle of the clam, rather than by the clam tissues themselves (Edmonds et al., 1982).

O OH

O R

R'

HO

CH3

As+ O

O

OH

OH

–SO3H –OH –OSO3H –OPO3HCH2CH(OH)CH2OH –SO3H

trimethylarsonioribosides

H3C

OSO3-

H3C

COO-

CH3

As+

CH3

CH3

As O

CH3

As+

-O

O P

O

O

OR

OH

OR

arsenocholine AC

H3C

CH3

dimethylarsinic acid DMA

HO

CH3

phosphatidylarsenocholine R= CO(CH2)nCH3

glycerylphosphorylarsenocholine R = H

arsenobetaine AB

CH3

FIGURE 3.1 Structure of As compounds present in the marine environment.

–OH R2 = –OH –OH –OH –NH2

dimethylarsinylribosides

HO

CH3

As

R1 =

O

CH3

tetramethylarsonium ion TeMA

trimethylarsine oxide TMAO

CH3

CH3

CH3

H 3C

As+

CH3

As+

As H3C

CH3

CH3

CH3 O

monomethylarsonic acid MMA

arsenite As(III)

arsenate As(V)

OH

O

OH

H3C

As

HO

As

O

As OH

H3C

HO

OH

OH

OH

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The discovery of AB as the major As form in marine animals led to the identification of several other As compounds, including arsenocholine (AC). But despite its wide distribution in gastropods, mollusks, and scallops, AC is present in much smaller quantities than AB (Maher et al., 1985). The compound soon attracted particular interest in that it appeared to be efficiently adsorbed from diet by yelloweyed mullet, transformed into AB, and accumulated in muscle tissue as AB (Francesconi et al., 1989). A strongly basic component, tetramethylarsonium ion (TeMA), was isolated from the clam Meretrix lusoria (Morita and Shibata, 1987). The clam appeared to contain this ion as well as significant amounts of AB, but while AB was dominant in almost all tissues of the clam, TeMA was dominant in the gill. Later, Cullen and Dodd (1989) suggested that TeMA is a common constituent of mollusks. Trimethylarsine oxide (TMAO) was reported to be present in small amounts in some fish (Norin et al., 1985). However, Edmonds and Francesconi (1987) found high concentrations of TMAO in estuary catfish. Indeed, the range of arsenicals encountered in the marine environment differs from one surrounding to another. Whereas in the water column, As is present as arsenite, arsenate, and in minor amounts as monomethylarsonic acid (MMA) and DMA, marine organisms contain a variety of arsenicals (Figure 3.2). Consequently, the cycling of As in organisms is far from straightforward. The characterization of the different arsenicals and their abundance are well documented by now, but the same cannot be said for the pathways of their synthesis. Francesconi and Edmonds (1983) hypothesized that there are in fact two As cycles in marine life, one for inorganic and one for organic As, and that the two cycles are linked at the level of sediment microbes and marine algae, where inorganic arsenic is converted into organic forms. Their proposed metabolical pathway is described in Figure 3.3. Seawater Major: As(V), As(III) Minor: MMA, DMA Notions of “hidden”As: AB, AC, TeMA, arsenosugars

Marine animals Marine algae Major: AB Major: dimethylated arsenosugars Minor: TeMA, arsenosugars, Minor: As(V), MMA, DMA,

Sediments Major: As(V), As(III)

TMAO, AC, As(V), As(III), trimethylated arsenosugars DMA

Minor: MMA, DMA Trace: TMAO

FIGURE 3.2 Abundance of the different arsenicals in the marine ecosystem.

As O

CO2-

S+

OH OH

N

CH3 O

N

arsenate

OH

reduction

N

O CH3

As

CH3 COOH

methylation H3C CH3

As

DMA

H3C

As

+

CH3 OH

methylation

arsenocholine Marine animals

oxidation

CH3

OH

arsenobetaine

COO-

CH3 As

HO

CH3

As

+

CH3

O

methylation

OH

dimethylarsinylethanol

O

reduction &

CH3

H3C

methylation

MMA

OH

As

OH

oxidation

dimethylarsinylacetic acid

N

NH2

OH

As

arsenite

HO

OH O

(C3–C4 cleavage)

anaerobic decomposition

(C3–C4 cleavage)

anaerobic decomposition

adenosylation

reduction &

FIGURE 3.3 Proposed pathway for the synthesis of different arsenicals from arsenate.

S-adenosylmethionine

H2N

HO

OH

CH3

As

OH

O

OH

N

N N

N

NH2

HO

CH3

As

CH3 O

OH

O R

R'

CH3

HO

CH3

+ As

O

OH

O OH

OSO3-

Marine algae

trimethylarsonioribosides

H3C

reduction & methylation

dimethylarsinylribosides

O

glycosidation

dimethylarsinyladenosine

H3C

O

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IN

FISH

AND

65

SHELLFISH

As mentioned earlier, the total As burden of fish and shellfish can mount to extreme levels. Table 3.2 summarizes some of these results. In fish, mollusks, and crustaceans from all over the world, large variations in As contamination, both interspecies and intraspecies, are observed. What exactly causes these differences is not well understood, but it can be hypothesized that they are related to (1) differences in diet and differences in the form of ingested arsenic, (2) seasonal changes, and (3) geographical area. Langston (1980) reported contamination of the mollusk Srobicularia plana along the southwestern coast of the U.K. and related the elevated As levels to contamination of the sediment. It seemed that seasonal variations, together with phytoplanktonic activity that changed the chemical form of dissolved As in the water column, greatly influenced the accumulation of As by the mollusks. An extensive study of fish inhabiting French coastal waters also indicated significant differences from one sampling area to another; for example, the As concentrations in samples of flounder ranged from 8.2 to 41.1 mg kg–1 dry weight (dw) and in samples of dogfish from 72 to 230 mg kg–1 dw (Michel, 1993). These variations appeared to be explainable only by local or seasonal variations in food sources. Another data set of this study showed a correlation between As level and the fish’s length, but this correlation did not appear to be very statistically significant. The observed increases in As concentration could be more easily explained by differences in diet between juvenile and adult fish. Nevertheless, certain comparabilities in As concentrations of species of the same scientific order do exist. Francesconi and Edmonds (1993) constructed a table in which they summarized As concentrations according to scientific order. The levels of As contamination of seafood, described in Table 3.2 and in Table 3.3, would at first sight pose a serious threat to human health upon consumption. However, this would be the case only if the As present were all in a toxic form. From Table 3.3, it is obvious that the major contribution to the total As content comes from AB; that this AB is believed to be nontoxic is thus a reassuring finding. Extensive toxicity study of the several arsenicals in the marine food chain show that the different forms exhibit different toxicities. Genotoxicity studies of AB produced consistently negative results and prove that the compound is quite stable (Jongen et al., 1985). AC and TMAO are equally of no or much less significance with regard to toxicity (Sakurai et al., 1996). Inorganic As, on the other hand, is highly toxic, as well as carcinogenic (Saha et al., 1999). DMA and MMA, despite that they are regarded as detoxification products of algae, are extensively used as herbicides, and thus have toxic potential of their own. A toxicity order — also evidenced from experimental LD50 values (Table 3.4) — can be set as decreasing with increasing degree of methylation. TeMA, the highest methylated arsenical, is an exception to this rule, as its toxicity to mice was observed to be higher than that of MMA and DMA (Shiomi, 1994). Few or no assays of toxicity have yet been made for arsenolipids, and with regard to arsenosugars this knowledge is also limited. Kaise et al. (1996) investigated the cytotoxicity of the arsenosugar 1-(2´,3´-dihydroxypropyl)-5-desoxyribosyldimethyl-arsine oxide on

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TABLE 3.2 Total Arsenic Concentrations in Fish, Mollusks, and Crustaceans Species Dogfish (Scyliorhinus canicula) Starspotted shark (Mustelus manazo) Thornback ray (Raja clavata) European conger (Conger conger) Atlantic cod (Gadus morhua)

Saithe (Pollachius virens) Pouting (Trisopterus luscus) Whiting (Merlangius merlagus) School whiting (Sillago bassensis) Ling (Molva molva) Angler (Lophius piscatorius) European seabass (Dicentrarchus labrax) Dab (Limanda limanda) European plaice (Pleuronectes platessa)

Lemon sole (Microstomus kitt)

Common sole (Solea solea)

Sand sole (Pegusa lascaris) Witch (Glyptocephalus cynoglossus) Megrim (Lepidorhombus whiffiagonis)

Location

Total As mg kg–1 ww

Ref.

Ostkante North Sea and Channel Japanese coast

5.62–10.78 21.3–64.0 33.6

Ballin et al., 1994 De Gieter et al., 2002 Hanaoka et al., 1999

Southern North Sea North Sea and Channel Northern North Sea North Sea and Channel Greenland Dogger bank Northern North Sea North Sea and Channel Northern North Sea North Sea and Channel North Sea and Channel North Sea and Channel North Sea and Channel Western Australia

31 6.2–35.9 12–30 2.4 1.97–4.33 3.65–7.08 15.3 3.1–7.0 1.4 1.8–5.7 2.5–5.4 1.7–2.1 4.0–6.5 3.2–14.5

Northern North Sea North Sea and Channel North Sea and Channel North Sea and Channel

6.0 2.1–8.5 4.1–13.7 1.1

Luten et al., 1982 De Gieter et al., 2002 Luten et al., 198 De Gieter et al., 2002 Ballin et al., 1994 Ballin et al., 1994 Luten et al., 1982 De Gieter et al., 2002 Luten et al., 1982 De Gieter et al., 2002 De Gieter et al., 2002 Luten et al., 1982 De Gieter et al., 2002 Edmonds and Francesconi, 1981a Luten et al., 1982 De Gieter et al., 2002 De Gieter et al., 2002 De Gieter et al., 2002

North Sea North Sea and Channel German Bight

2.4–12.5 6.5–21.4 7.39–12.72

Luten et al., 1983 De Gieter et al., 2002 Ballin et al., 1994

North Sea West coast Denmark North Sea and Channel Wadden Islands Dogger Bank North Sea and Channel North Sea West coast Denmark North Sea and Channel North Sea and Channel North Sea and Channel

3–166 17.5 7.7–26.0 25 46 14.9–76.1 6.2–10.2 10.4 4.1–48.8 4.1–34.8 9.4–49.3

Luten et al., 1982 Luten et al., 1982 De Gieter et al., 2002 Luten et al., 1982 Luten et al., 1982 De Gieter et al., 2002 Luten et al., 1983 Luten et al., 1982 De Gieter et al., 2002 De Gieter et al., 2002 De Gieter et al., 2002

North Sea and Channel

3.8–12.8

De Gieter et al., 2002 (continued)

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TABLE 3.2 (CONTINUED) Total Arsenic Concentrations in Fish, Mollusks, and Crustaceans Species Brill (Scophtalmus rhombus)

Turbot (Scophtalmus maximus)

Flounder (Platichthys flesus) Yellow-eyed mullet (Liza ramada)

Blue mussel (Mytilus edulis) Giant cupped oyster (Crassostrea angulata) Sea scallop (Placopecten magellanicus) Great scallop (Pecten maximus) Whelks (Buccinum undatum)

American lobster (Homarus americanus) Common shrimp (Crangon crangon)

Delta prawn (Palaemon longirostris) Edible crab (Cancer pagurus) Fiddler crab (Uca tangeri)

Location Southern North Sea West coast Denmark East coast England North Sea and Channel Southern North Sea West coast Denmark East coast England North Sea and Channel North Sea Guadalquivir Estuary (Spain)

Total As. mg kg–1 ww

Ref.

0.8 2.2 1.9 1.4–2.9 6.4 14.8 4.4 17.9 0.45–6.8 0.18–0.65

Luten et al., 1982 Luten et al., 1982 Luten et al., 1982 De Gieter et al., 2002 Luten et al., 1982 Luten et al., 1982 Luten et al., 1982 De Gieter et al., 2002 Luten et al., 1983 Suñer et al., 1999

1.6–5.3 1.67–2.44

Penrose et al., 1975 Suñer et al., 1999

2.1–9.4

Lai et al., 1999

North Sea and Channel North Sea

0.99–3.6 16.5–65.8

De Gieter et al., 2002 De Gieter et al., 2002

Crustaceans Northeast U.S. coast

6.5–26

Newfoundland Danish coast

3.8–7.4 9.2 ± 2.6

North Sea North Sea and Channel Guadalquivir Estuary (Spain) North Sea Guadalquivir Estuary (Spain)

2.0–6.8 5.2 0.80–1.33

Edmonds and Francesconi, 1981b Penrose et al., 1975 Francesconi et al., 1999 Vos et al., 1986 De Gieter et al., 2002 Suñer et al., 1999

36.8–40.6 0.76–1.76

De Gieter et al., 2002 Suñer et al., 1999

Mollusks Newfoundland Guadalquivir Estuary (Spain) Newfoundland

mammalian cell cultures. The compound was found to exhibit a toxicity 1/2800 that of sodium arsenite and 1/300 that of sodium arsenate. As a direct result, assessments of only the total As concentrations in fish cannot provide information about potential health impacts. For this purpose, knowledge of speciation is an absolute prerequisite. Unfortunately, until now, most studies have focused on the development of analytical techniques to accurately speciate As compounds in fish, instead of focusing on their effective assessment. Data about the abundance of toxic As substances in marine organisms are nevertheless listed in

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TABLE 3.3 Total Arsenic and Arsenobetaine Content of Marine Animals Animal

mg As kg–1 ww

% AB

Elasmobranchs Teleosts

Fish 3.1–44.3 0.1–166

94 – >95 48 – >95

Lobsters Prawns/shrimp Crabs

Crustaceans 4.7–26 5.5–20.8 3.5–8.6

77 – >95 55 – >95 79 – >95

Bivalves 1 Bivalves 2 Gastropods Cephalopods

Mollusks 0.7–2.8 1.0–2.3 3.1–116.5 49

Echinoderms Coelenterates Sponges

12.4 7.5 3.2–6.8

44–88 12–50 58 – >95 72 – >95 60 15 13–15

TABLE 3.4 Experimental LD50 Values of Arsenic Species Arsenic Species As(III) MMA DMA TeMA TMAO AC AB

LD50 (g/kg) 0.0345 1.8 1.2 0.89 10.6 >6.5 >10.0

Source: Shiomi, 1994. With permission.

Table 3.5. This table provides evidence for the relatively small amounts of toxic arsenicals in seafood. In fish, the concentrations of inorganic and other toxic As compounds generally stay below 5% of the total As content.

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TABLE 3.5 Toxic Arsenic in Fish, Mollusks, and Crustaceans Biota Species

Dogfish Thornback ray European conger Atlantic cod Saithe Pouting Whiting Ling Angler European seabass Dab European plaice Lemon sole Common sole Sand sole Witch sole Megrim Brill Turbot Herring Haddock Mullet Blue mussel Giant cupped oyster Great scallop Sea scallop Whelk

Common shrimp Delta prawn Fiddler crab Edible crab a

mg As kg–1 ww

As Species

Fish 0.046–0.60a 0.057–0.42a 0.028a 0.038–0.11a 0.039–0.057a 0.072–0.1a 0.051–0.094a 0.038–0.099a 0.036–0.20a 0.044a 0.034–0.46a 0.033–0.44a 0.02c 0.024–0.48a 0.49–0.57a 0.062–0.38a 0.13–0.40a 0.056–0.23a 0.031–0.087a 0.16a 0.03c 0.02c 0.01–0.03d

Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Inorganic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Inorganic Inorganic Inorganic

0.55 0.92 1.2 1.5 1.6 2.3 1.4 2.8 0.94 4.0 1.96 1.4 0.1 0.68 1.4 1.52 0.92 1.25 2.6 0.89 3.6 0.8 2.8

Mollusks 0.07b 2.01b 0.04–0.09d 0.036 –0.72a 0.6–0.7 dwe 0.065–0.43a 0.06–0.18c

DMA TMAO Inorganic Toxic Me4As+ Toxic Inorganic

1.6 47 2.4 9.1 7.5 0.42 1.3

Crustaceans 0.17a 0.03d 0.02–0.04d 0.04–0.22d 0.18–0.40a

Toxic Inorganic Inorganic Inorganic Toxic

3.3 0.74 2.2 8.0 0.77

% of Total As

Toxic As as the sum of As(V), As(III), MMA, and DMA (De Gieter et al., 2002). Kaise et al., 1988. cBrooke and Evans, 1981. dSuñer et al., 1999. eLai et al., 1999. b

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HUMAN EXPOSURE TO FISH ARSENIC FATE

OF INGESTED

ORGANIC ARSENIC

As early as 1919, Bang stated that the organic As contained in fish and other marine food was readily excreted by humans. It appeared to be excreted in the feces, but only to a very limited extent. Experiments showed that, in animals fed homogenized shrimp, fecal excretion was only 5 to 25% of the ingested As (Coulson et al., 1935). Excretion in human feces turned out to be even less pronounced (Charbonneau et al., 1980). One reason for this low fecel excretion can be proposed: rapid and almost complete absorption of As in the gastrointestinal tract. Subsequent to absorption in the gastrointestinal tract, As is transported by the blood to other tissue in the body. Siewicki (1981) conducted a study of rats fed witch flounder, with low (4.7 mg As kg–1), medium (15.8 mg As kg–1), or high (28.8 mg As kg–1) As concentrations. The rats fed medium and higher As diets exhibited higher retention of As in the liver and spleen than did the control group rats fed the low As diet. Retention of As in the erythrocytes was not observed in the rats fed fish; on the contrary, it was observed in rats fed 22.1 mg DMA kg–1. Vahter et al. (1983) administered 73As-labeled AB to mice, rats, and rabbits and demonstrated that this major arsenical in marine products is rapidly cleared from plasma and other tissue. Longest retention was observed in the cartilage, testes, and epididymis of the animals, and in rabbits, also in muscle. AB itself was the only arsenical that was detected in extracts of the tissue and 98% of the ingested AB dose was excreted unchanged in the urine. A parallel experiment with 73As-labeled AC concluded that the clearance of AC from plasma and tissue was somewhat slower than that of AB. Tissues with the longest retention were again the reproductive organs, prostate, epididymis, and testes, and the myocardium, liver, adrenal cortex, pancreas, dental pulp, and pituitary gland, but in contrast to AB, the major compound excreted in the urine after administration of AC was AB, indicating that AC was oxidized to AB (Marafante et al., 1984). In humans, the National Academy of Science (1977) mentions an estimated total human body content of As of between 3 and 4 mg. This As is widely distributed in the body, in liver, kidney, lung, spleen, skin, hair, and nails. The uterus, bone, muscle, and neural tissue have also been shown to accumulate As. Following the ingestion of seafood, however, there are no records of tissue distribution in humans. Despite this, it has been observed that the organoarsenicals in fish and shellfish are readily excreted in the urine in an unchanged form, thus indicating that the kidneys play a key role in the fate of As taken up from diet. Chapman (1926) ascertained that ingestion of 33 μg As by eating lobster led to the excretion of 74% of this dose in the urine in 48 h. From a similar experiment, Coulson et al. (1935) concluded that almost 100% of the ingested As is eliminated within 1 week. Freeman et al. (1979) observed urinary excretions by six volunteers fed flounder, varying from 64 to 90% of the ingested As after 9 days. The excreted As was moreover in the same chemical form as in the fish. A similar observation was made by Luten et al. (1982): after the consumption of plaice, 69 to 85% of AB was excreted in the urine. Consequently,

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consumption of seafood may result in substantial increases in As absorption by the human body, but also in increased elimination by the kidneys. The fate of the simpler organic As forms MMA and DMA seems to be similar to that of AB. Buchet et al. (1981) reported a study in which these arsenicals were ingested by volunteers; 75% of the ingested DMA was excreted unchanged in the urine within 4 days after exposure. From initial MMA, 78% of the dose was recovered in the same form, but 10% had been converted to DMA, suggesting some kind of methylation mechanism. Yamauchi and Yamamura (1984) observed an unchanged excretion of 90% of the trimethylarsinic acid from prawns in the urine within 60 h.

FATE

OF INGESTED INORGANIC

ARSENIC

Because the greater toxicological risks of As result from the presence of inorganic As in sources of exposure, the fate of ingested inorganic As has been studied more elaborately than that of organic As. The ingestion of water-soluble inorganic As was observed to proceed similarly to that of organoarsenicals; ingestion also results in high excretions in urine and only minor amounts in the feces. Arsenate and arsenite, as well, are rapidly absorbed by the gastrointestinal tract, and their presence in blood decreases shortly after exposure. The latter could not be confirmed in rats, as their erythrocytes seem to retain As, leading to much slower excretion in the urine (Vahter, 1994). In dogs, mice, rabbits, monkeys, and humans, however, as much as 90% of an administered dose is cleared from the blood with a half-time of 1 to 2 h. The half-times of the second and third phases have been estimated at 30 and 200 h, respectively (Vahter and Norin, 1980). Opposed to the pathway of organoarsenicals that causes AB, AC, DMA, and MMA to be largely excreted unchanged, inorganic As appears to be biotransformed and metabolized in vivo. Analyses of human urine from individuals exposed to inorganic As show the production of substantial amounts of DMA and MMA; 24 h after ingestion of 500 μg of inorganic As, 8, 5.3, and 9.3% of the dose is eliminated in the urine, respectively, as inorganic As, MMA, and DMA. Another 24 h later, an additional 2.3, 2.3, and 8.5% of the respective compounds is excreted (Buchet et al., 1980). Following exposure to inorganic As, elevated As concentrations in the liver, kidneys, lungs, and intestinal mucosa of rabbits and mice were noted. Vahter (1981) proposed that at low doses mammals are capable of methylating the inorganic As absorbed from the gastrointestinal tract. The mechanism for this methylation is believed to be the same mechanism as that suggested by Challenger (1945) for the methylation of As in microorganisms (Figure 3.4). It occurs via alternating reduction of pentavalent As to trivalent As and methylation. Subsequent to the reduction of As(V), As(III) is possibly bound to dithiol (Thompson, 1993); methylgroups are transferred from methyldonor S-adenosyl-methionine (SAM) to this As(III); and the resulting derivative is either transformed into MMA or used as a substrate for a second methylation to a dimethylated derivative, which later transforms into DMA. This reaction is believed to be mediated by the methyltransferases arsenite

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OH As

HO

O

HO

CH3

As

As

HO

CH3

OH

OH

OH

As(V)

As(III)

MMA(V)

CH3 As

H3 C

O

OH

red.

CH3 CH3

red.

H3C

As O

trimethylarsine

TMAO

red.

HO

CH3

As

CH3

OH

MMA(III)

CH3

CH3

CH3

As

CH3 CH3

red. O

OH

DMA(III)

As

CH3

OH

DMA(V)

FIGURE 3.4 Methylation pathway as suggested by Challenger (1945).

methyltransferase and MMA methyltransferase. Although they have not been fully characterized yet, they have been purified from liver of rabbits, hamsters, and rhesus monkeys (Zakharyan et al., 1995). The liver is thus believed to be the prime organ for As methylation. Arsenic methylating activity has also been detected in several other tissues of mice — in the testes, kidney, liver, and lung — but to a much lesser extent (Healy et al., 1998). Although microorganisms generally produce trimethylated derivatives, the end point of the methylation in mammals is DMA. It is this DMA that is readily excreted in the urine. Note that, since the liver is believed to be the main site of methylation, ingestion leads to a higher degree of methylation than does inhalation or parental administration (Vahter, 1981). Because inorganic As(III) is more toxic than As(V), the initial step in the reaction, the reduction of As(V) to As(III), might be interpreted as a kind of bioactivation of the toxicity of As. However, it has been shown that As(III) is taken up much more readily by the hepatocytes, where the methylation occurs, than is As(V) (Lerman et al., 1983). Additionally, both end products of the methylation, MMA and DMA, are less reactive with tissue constituents and are more easily excreted in the urine. Therefore, this methylation reaction is generally believed to be a detoxification mechanism for As. Some considerations do, however, oppose this assumed beneficial effect: •

The rate of methylation decreases with increasing dose concentration (Vahter, 1981). This can either be explained by an overload of the methylation capacity or by inhibition of the reaction at high concentrations of As(III). High As(III) concentrations were indeed observed to inhibit the second step of the methylation and, thus, the production of DMA. Increased inorganic As concentrations therefore allow and induce the buildup of significant amounts of inorganic As in the body tissue. Next to organs, high concentrations of As were measured in keratin-rich tissues such as skin, hair, and nails. These tissues contain several sulfhydryl groups and allow efficient binding of trivalent As. Sectional analysis of

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arsenatereductase As(V)

As(III)

2GSH

GS-SG

SAM arsenite-methyltransferase SAH

MMA(V)reductase MMA(III)

MMA(V)

SAM MMA-methyltransferase

SAM = S-adenosylmethionine SAH = S-adenosylhomocysteine GSH = reduced glutathione GS-SG = oxidized glutathione

SAH

DMA(V)

DMA(III)

FIGURE 3.5 In vivo methylation in mammals.

•

•

hair and nails indicated that the time of exposure to As can be demonstrated from peak concentrations, when the As entered the hair and nail roots via the bloodstream (Curry and Pounds, 1977; Pounds et al., 1979). The distribution of As in human organs following fatal acute intoxication by arsenic trioxide provided data on increased As levels in liver and kidney. These organs contained 7 to 350 times more As than blood (Benramdane et al., 1999). As mentioned above, the initial step in the biotransformation requires the reduction of As(V) to As(III) to allow uptake of As in the hepatocytes to occur more readily. Glutathione (GSH) and possibly other thiols serve as reducing agents. The first step in the methylation reaction is thus facilitated by the presence of reduced glutathione (Figure 3.5) and this step is at the same time believed to be the limiting step of the methylation. Significant differences in excretion pattern were observed in cases of liver insufficiencies. This difference is probably attributable to a reduced GSH content of the liver. Buchet and Lauwerys (1987) indeed reported reduced uptake of inorganic As by the hepatocytes of animals with low hepatic GSH. In addition to this observation, the authors also remarked that an increased amount of MMA was excreted. GSH is thus also believed to mediate the dimethylation step of the reaction. The existence of by-products from the reduction and methylation of inorganic As has recently been discovered. Suzuki et al. (2002) demonstrated the release of trivalent As containing MMA and DMA from the site of As methylation into body fluids. These intermediates, in particular

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MMA(III), are today known to be even more reactive and toxic to rat and human cells than both As(III) and As(V) (Styblo et al., 2000). Petrick et al. (2000) observed an MMA(III) toxicity to Chang human hepatocytes, 26 times greater than that of inorganic arsenite. Lin et al. (1999) estimated MMA(III) to be over 100 times more potent as an inhibitor of thioredoxin reductase than As(III). Also the potent cytotoxicity and genotoxicity of DMA(III) has been reported (Kenyon and Hughes, 2001). The formation of MMA(III) and DMA(III) can therefore be considered to represent toxification of inorganic As, rather than detoxification. Exactly to what extent this trivalent MMA and DMA contribute to the total toxicity following exposure to inorganic As remains to be elucidated, but their discovery leads to a changed understanding of the purpose of the methylation. Understanding the metabolism of As is thus a critical factor in the risk assessment of chronic As exposure.

TOXICITY TO HUMANS CONCENTRATIONS

VS.

LEGAL LIMITS

Some authors suggest that at low concentrations As might be an essential element for organisms (Uthus, 1994). These studies imply that As plays a physiological role in the methionine metabolism, which is contrary to the toxicity of As that has been sufficiently documented over the centuries. Arsenite’s toxicity is believed to arise from reaction with sulfhydryl groups, among others. The compound would thus inhibit sulfhydryl enzymes, necessary for cellular metabolism. Arsenate may replace phosphate in the ATP/ADP mechanism and thus inhibit oxidative phosphorylation. Arsenic compounds are also described as antagonistic to the essential trace elements iodine and selenium (Levander, 1977). Toxicity arising from ingestion of inorganic As is believed to manifest itself in systemic effects involving the skin, the cardiovascular system, and the neurological system. Additionally, the IARC (1980) concluded that there is enough evidence to associate the exposure to inorganic As with skin, lung, and bladder cancer and classified As as a so-called group 1 carcinogen to humans. DMA is equally shown to induce organ-specific lesions in the lungs of mice, rats, and humans (Kenyon and Hughes, 2001). The same authors also mention dose-dependent increases in urinary bladder tumors upon lifetime exposure to DMA from diet or drinking water. DMA is believed to act either as a cancer promoter or as a complete carcinogen in different animals. Despite this extensive knowledge of certain As compounds’ toxicities, it seems that little attention has been paid to the probability of exposure to the element through fish consumption. Since recent discoveries of major calamities involving As in drinking water in Bangladesh and West Bengal, several reports and committees have focused on setting or adjusting maximum permissible concentrations for drinking water, but studies on the bioavailability of inorganic As in various foodstuffs are not given high priority. This results in a set of ambiguous formal As norms for fish and shellfish; existing legal limits vary from 0.1 mg kg–1 in Venezuela to 10 mg kg–1 in Hong Kong. Also,

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in some countries, norms for As are related to total concentrations, while in other countries they express only the allowable inorganic As fraction. These discrepancies far from simplify judgments on the potential human risk related to seafood consumption and, thus, the meaning of these norms is not at all straightforward. The Joint FAO/WHO Expert Committee (1983) set a limit of 0.1 mg kg–1 wet weight (ww) for inorganic As in fish and seafood. In 1967, 50 μg kg–1 bodyweight (bw) was enforced as the tolerable daily intake (TDI) of As, but this norm dates from before epidemiological studies indicated that inorganic As might be carcinogenic to humans. Therefore, the committee arrived at an updated provisional TDI, specific for inorganic As, of 2 μg inorganic As kg–1 bw, thus a provisional tolerable weekly intake (PTWI) of 0.015 mg inorganic As kg–1 bw. Friberg (1988) stated that extensive consumption of seafood, in portions of 150 g day–1, might lead to ingested amounts of inorganic As that are high enough to affect the risk of cancer. Extrapolation of a 7-day-a-week consumption of 150 g day–1 of marine products, containing a realistic concentration of 10 mg kg–1 total As of which 5% inorganic, by a person of 60 kg, results in a weekly intake of 0.0087 mg kg–1 bw. When considering the FOA/WHO Expert Committee’s PTWI for inorganic As of 0.015 mg kg–1 bw, the toxic dose is thus not met. Conversely, this PTWI can produce either permitted amounts of seafood consumption or permitted levels of contamination. To arrive at the level of concern described as PTWI, the same person of 60 kg would daily have to ingest 128.6 μg inorganic As. Considering 150-g portions of fish, the inorganic As content of the fish would have to be 0.85 mg kg–1 ww. This concentration level is a lot higher than generally encountered in seafood.

ADDITIONAL CAUTIONS Nevertheless, PTWIs should be regarded with caution. Additional care should be taken when combining high levels of seafood consumption with other exposures to As. Several exposures to drinking water containing high levels of As have been recorded over the past decades in various countries. In Chile, Taiwan, West Bengal, India, Mexico, Argentina, China, and Bangladesh, thousands of people were exposed to high amounts of inorganic As of natural origin in drinking water (Mandal and Suzuki, 2002). This water often appeared to contain As in concentrations much higher than all existing drinking water limits. For example, in Bangladesh, 52 of 64 districts had well water As concentrations far above the Bangladesh drinking water standard of 50 μg l–1, and in 17 of the districts the maximum As level in groundwater exceeded 1 mg l–1. Another matter of concern is that there is inadequate information on the impact of storage, transport, and processing of seafood on As accumulation in the fish tissue. Until now, only limited consideration has been given to this issue: •

A comparative study of fresh and frozen fish pointed out that As concentrations in frozen species are higher than in fresh species (Juma et al., 2002).

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•

•

•

Vélez et al. (1995) showed that, converse to AB being the major arsenical in fresh seafood, this might not be the case in manufactured products. They found an AB content in frozen and canned products of 47 and 30% of the total As, respectively, vs. 80% in the fresh product. The DMA content was observed to be higher in preserved products than in fresh ones; 4% of the total As content in the processed vs. 1% in the fresh seafood was in the form of DMA. In a later study of canned seafood, the same authors demonstrated the possible transfer of AB and DMA from the tissue to the accompanying liquid (Vélez et al., 1997). These observations led them to believe that AB is likely to be solubilized in the intercellular liquids of the fish and released into the brine, and second, that AB might be degraded during processing. Mürer et al. (1992) showed that AB can be converted efficiently to inorganic As(V) upon ultraviolet radiation and speculated that this conversion might occur during storage or preparation of seafood. Reinke et al. (1975) discussed the rapid reduction of arsenate to arsenite after fish death, and also Hanaoka et al. (1993) observed the degradation of accumulated AB to inorganic As in shark’s muscle, after the death of the animal.

Thus, studies on concentrations, and especially speciation of As compounds, in processed seafood are absolutely necessary. The effects of transport, storage, and preparation on modifications in As speciation in seafood should be determined. This study may be used to recommend regulations for conservation and cooking in order to avoid or reduce the presence of toxic As species.

DOSE–RESPONSE ASSESSMENTS Doses responsible for effects after exposure to inorganic As have until now been deduced from incidents of mass poisoning. Fatal doses of ingested As(III) oxide have been reported in the range of 1 to 2.5 mg kg–1 bw. Additionally, it appears that ingestion of 3 mg As(V) daily over a period of few weeks may give rise to severe poisoning in infants and symptoms of toxicity in adults (WHO, 1981). A report of the NRC (1999) also cited noncancer effects from chronic ingestion of inorganic As, at doses of 10 μg kg–1 day–1 and higher. This report concludes that epidemiological studies are needed to characterize the dose–response relationship for Asassociated cancer and noncancer effects. Most laboratory animals, however, appear to be substantially less susceptible to As than humans. It has been reported that chronic oral exposure to 0.05 to 0.1 mg inorganic As kg–1 day–1 causes neurological and hematological toxicity in humans but not in monkeys, dogs, and rats (Byron et al., 1967). Also with regard to carcinogenicity, all models to test the effects of arsenicals in humans failed to mimic the actual human mechanism satisfactorily. Thus, quantitative dose-dependent data for animals should not be considered a reliable source for application to humans. Thus, one of the recommendations is to establish suitable animal models for As toxicity assessments.

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CONCLUSIONS Although extensive research has provided information either on the form of As in seafood or on its fate in the human body after consumption of this seafood, several questions that are of concern for the accurate assessment of potential dangers of exposure to As via the consumption of seafood remain: •

•

•

•

•

The exact origin and metabolical pathway of As in higher marine organisms remain hypothesized. Also the origin of several concentration differences, both interspecies and intraspecies, remains speculative to date. Further investigation of the type and concentration of As compounds in marine products are highly desirable. Several reports demonstrate the large differences in toxicity among the different arsenicals in marine animals. There is thus comprehensive knowledge that the majority of As in seafood is nontoxic. Nevertheless, many formal limits do not seem to take these notations into account and remain largely ambiguous. Formulations of maximum permissible concentrations for health regulations in seafood should recognize the chemical nature of As in seafood. Additionally, they should be lowered for populations exposed to other sources of As. The recent discovery of highly toxic trivalent by-products of the methylation reaction of inorganic As in mammals has changed the current assumptions of methylation as a detoxification mechanism for inorganic As. Understanding the metabolism of As in humans may thus be considered a critical factor in the assessment of the risk associated with chronic exposure to As. There is inadequate information on seafood processing effects on the concentrations and especially on the speciation of As in this seafood. The effects of transport, storage, and preparation on potential modifications in As content should be investigated. This study may be used to recommend regulations for conservation and cooking to avoid or reduce the presence of toxic As species. Reliable animal models and their transpositions to humans should be developed to assess the toxicity of As to humans.

REFERENCES Andreae, M.O. and Klumpp, D. (1979) Biosynthesis and release of organoarsenic compounds by marine algae, Environmental Science and Technology, 13: 738–741. Ballin, U., Kruse, R., and Rüssel, H.-A. (1994) Determination of total arsenic and speciation of arsenobetaine in marine fish by means of reaction — headspace gas chromatography utilizing flame-ionization detection and element specific spectrometric detection, Fresenius Journal of Analytical Chemistry, 350: 54–61. Bang, I. (1919) in H. Lund, Ed., Report by the Swedish Arsenic Commission, Vol. 11, Ohlssons Boktryckeri, 1–56.

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Benramdane, L., Accominotti, M., Fanton, L., Malicier, D., and Vallon, J.J. (1999) Arsenic speciation in human organs following fatal arsenic trioxide poisoning — a case report, Clinical Chemistry, 45: 301–306. Bohn, A. (1975) Arsenic in marine organisms from West Greenland, Marine Pollution Bulletin, 6: 87–89. Brooke, P.J. and Evans, W.H. (1981) Determination of total inorganic arsenic in fish, shellfish and fish products, Analyst, 106: 514–520. Brown, C.J. and Eaton, R.A. (1999) Leaching of copper-chrome-arsenic (CCA) wood preservative in sea water, Material und Organismen, 33: 213–233. Buchet, J.P. and Lauwerys, R. (1987) Study of factors influencing the in vivo methylation of inorganic arsenic in rats, Toxicology and Applied Pharmacology, 91: 65–74. Buchet, J.P., Lauwerys, R., and Roels, H. (1980) Comparison of several methods for the determination of arsenic compounds in water and in urine, International Archives of Occupational and Environmental Health, 46: 11–29. Buchet, J.P., Lauwerys, R., and Roels, H. (1981) Comparison of the urinary-excretion of arsenic metabolites after a single oral dose of sodium arsenite, monomethylarsonate, or dimethylarsinate in man, International Archives of Occupational and Environmental Health, 48: 71–79. Byron, W.R., Bierbower, G.W., Brouwer, J.B., and Hanse, W.H. (1967) Pathological changes in rats and dogs from two year feeding of sodium arsenate or sodium arsenate, Toxicology and Applied Pharmacology, 10: 132–147. Challenger, F. (1945) Biological methylation, Chemical Reviews, 36: 315–361. Chapman, A.C. (1926) On the presence of compounds of arsenic in marine crustaceans and shell fish, Analyst, 51: 548–563. Charbonneau, S.M., Tam, G.K.H., Bryce, F., and Sandi, E. (1980) in Proceedings of the 19th Annual Meeting of the Society of Toxicology, Washington, D.C., 9–13 March 1980, Abstr. 362, p. A121. Chilvers, D.C. and Peterson, P.J. (1987) Global cycling of arsenic, in T.C. Hutchinson and K.M. Meema, Eds., Lead, Mercury, Cadmium and Arsenic in the Environment, Scope 31, New York: John Wiley, 279–301. Coulson, E.J., Remington, R.E., and Lynch, K.M. (1935) Metabolism in the rat of the naturally occurring arsenic of shrimp as compared with arsenic trioxide, Journal of Nutrition, 10: 255–270. Cullen, W.R. and Dodd, M. (1989). Arsenic speciation in clams of British Columbia, Applied Organometallic Chemistry, 3: 79–88. Cullen, W.R., Hettipathirana, D., and Reglinski, J. (1989) The effect of arsenicals on cell suspension cultures of the Madagascar periwinkle (Catharanthus roseus), Applied Organometallic Chemistry, 3: 515–521. Curry, A.S. and Pounds, C.A. (1977) Arsenic in hair, Journal of the Forensic Science Society, 17: 37–44. De Gieter, M., Leermakers, M., Van Ryssen, R., Noyen, J., Goeyens, L., and Baeyens, W. (2002) Toxic and total arsenic levels in North Sea fish, Archives of Environmental Contamination and Toxicology, 43: 406–417. Edmonds, J.S. and Francesconi, K.A. (1977) Methylated arsenic from marine fauna, Nature, 265: 436. Edmonds, J.S. and Francesconi, K.A. (1981a) The origin and chemical form of arsenic in the school whiting, Marine Pollution Bulletin, 12: 92–96. Edmonds, J.S. and Francesconi, K.A. (1981b) Isolation and identification of arsenobetaine from the American lobster Homarus americanus, Chemosphere, 10: 1041–1044.

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Edmonds, J.S. and Francesconi, K.A. (1981c) Arseno-sugars from brown kelp (Ecklonia radiata) as intermediates in cycling of arsenic in a marine ecosystem, Nature, 289: 602–604. Edmonds, J.S. and Francesconi, K.A. (1983) Arsenic-containing ribofuranosides: isolation from brown kelp Ecklonia radiata and NMR spectra, Journal of the Chemical Society–Perkin Transactions I, 10: 2375–2382. Edmonds, J.S. and Francesconi, K.A. (1987) Trimethylarsine oxide in estuary catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) after oral administration of sodium arsenate; and as a natural component of estuary catfish, Science of the Total Environment, 64: 317–323. Edmonds, J.S., Francesconi, K.A., Healy, P.C., and White, A.H. (1982) Isolation and crystal structure of an arsenic-containing sugar sulphate from the kidney of the giant clam, Tridacna maxima, X-ray crystal structure of (2S)-3[5-deoxy-5 (dimethylarsinoyl)-βD-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulphate, Journal of the Chemical Society–Perkin Transactions I, 12: 2989–2993. Fowler, S.W. and Ünlü, M.Y. (1978) Factors affecting bioaccumulation and elimination of arsenic in the shrimp Lysmata seticaudata, Chemosphere, 7: 711–720. Francesconi, K.A., Edmonds, J.S., and Stick, R.V. (1989) Accumulation of arsenic in yelloweyed mullet (Aldrichetta-Forsteri) following oral-administration of organoarsenic compounds and arsenate, Science of the Total Environment, 79: 59–67. Francesconi, K.A. and Edmonds, J.S. (1993), in A.D. Ansell, R.N. Gibson, and M. Barnes, Eds., Oceanography and Marine Biology: An Annual Review, Vol. 31, UCL Press, London, 11. Francesconi, K.A., Hunter, D.A., Bachmann, B., Raber, G., and Goessler, W. (1999) Uptake and transformation of arsenosugars in the shrimp Crangon crangon, Applied Organometallic Chemistry, 13: 669–679. Freeman, H.C., Uhthe, J.F., Fleming, R.B., Oduse, P.H., Ackman, R.G., Landry, G., and Musial, C. (1979) Clearance of arsenic ingested by man from arsenic contaminated fish, Bulletin of Environmental Contamination and Toxicology, 22: 224–229. Friberg, L. (1988) The GESAMP evaluation of potentially harmful substances in fish and other seafood with special reference to carcinogenic substances, Aquatic Toxicology, 11: 379–393. Hamilton, E.I. and Minski, M.J. (1973) Abundance of the chemical elements in mans diet and possible relations with environmental factors, Science of the Total Environment, 1: 375–394. Hanaoka, K., Kogure, T., Miura, Y., Tagawa, S., and Kaise, T. (1993) Post-mortem formation of inorganic arsenic from arsenobetaine in a shark under natural conditions, Chemosphere, 27: 2163–2167. Hanaoka, K., Goessler, W., Yoshida, K., Fujitaka, Y., Kaise, T., and Irgolic, K.J. (1999) Arsenocholine- and dimethylated arsenic-containing lipids in starspotted shark Mustelus manazo, Applied Organometallic Chemistry, 13: 765–770. Healy, S.M., Casarez, E.A., Ayala-Fierro, F., and Aposhian, H.V. (1998) Enzymatic methylation of arsenic compounds V. Arsenite methyltransferase activity in tissues of mice, Toxicology and Applied Pharmacology, 148: 65–70. IARC (1980) IARC Monographs, Arsenic and Its Compounds, Vol. 23, Lyons: International Agency for Research on Cancer, 39–141. Irgolic, K.J., Woolson, E.A., Stockton, R.A., Newman, R.D., Bottino, N.R., Zingaro, R.A., Kearney, P.C., Pyles, R.A., Maeda, S., McShane, W.J., and Cox, E.R. (1977) Characterisation of arsenic compounds formed by Daphnia Magna and Tetraselmis chuii from inorganic arsenate, Environmental Health Perspectives, 19: 61–66.

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Biological and Toxicological Considerations of Dietary Sulfur Lioudmila A. Komarnisky and Tapan K. Basu

CONTENTS Abstract ....................................................................................................................85 Introduction..............................................................................................................86 Properties of Elemental Sulfur ................................................................................86 Occurrence of Sulfur in Nature...............................................................................88 Global Cycle ............................................................................................................89 Biological Role of Sulfur ........................................................................................91 Sulfur Metabolism ...................................................................................................92 Sulfur Deficiencies ..................................................................................................93 Sulfur Toxicity .........................................................................................................94 Toxicity Due to SO2 ....................................................................................94 Routes of SO2 Entry into Living Organisms.....................................95 Maximum and Threshold Limit Values of SO2 .................................95 SO2-Sensitive Subjects.................................................................................95 Diseases and Sulfur .....................................................................................96 Pathogenesis of SO2-Linked Toxicity .........................................................96 Clearance of SO2 from the Organism .........................................................98 Toxicity of Secondary Origin (Sulfur Metabolism)....................................98 Conclusion .............................................................................................................100 References..............................................................................................................100

Abstract

Essential amino acids, including methionine (a sulfur-containing amino acid), must be supplied through the diet, as humans and animals, except for ruminant animals, are unable to synthesize them. Sulfur is present in body tissues as part of the amino acids: methionine, cysteine, and taurine. The relative reductionoxidation state of the cell depends primarily on the precise balance between

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concentrations of reactive oxygen species (ROS) and the cysteine-dependent (thiol) antioxidant buffers, glutathione (GSH) and thioredoxin. These antioxidants have high affinity for ROS, thus protecting other intracellular molecules from oxidative damage. Dietary deficiency of sulfur is relatively uncommon, whereas its toxicity is increasingly recognized as a serious concern in our environment. Human activities, such as burning fossil fuels, refining petroleum, smelting sulfur compounds of metallic minerals into free metals, and other industrial processes, have a large impact on the atmospheric sulfur balance. The atmospheric sulfur is lost into water reservoirs, where some of the sulfur enters marine communities and soil as it moves through the food chain. The metabolic product, sulfur dioxide (SO2), is thus considered one of the major air pollutants worldwide. Sulfur toxicity is recognized to be a consequence of metabolic derangement of methionine. Its metabolic product, homocysteine, is thought to be an independent risk factor for arterial diseases.

INTRODUCTION Sulfur is a major inorganic element, essential to the entire biological kingdom because of its incorporation into amino acids, proteins, enzymes, vitamins, and other biomolecules. Unlike humans and monogastric animals, plants have the ability to synthesize sulfur-containing amino acids (e.g., methionine and cysteine) utilizing inorganic sulfur, and thereby become important sources for the element. The maintenance of sufficient levels of sulfur in the biosphere, however, involves the biogeochemical sulfur cycle (Figure 4.1). The primary components in this cycle are the lithosphere, the atmosphere, and the hydrosphere, which provide sulfur for the habitat of the biosphere. In living species, sulfur compounds undergo metabolic processes; sulfur metabolites are excreted to re-enter the biogeochemical cycle (Miller, 1998). Although sulfur is a dietary essential, its excess leading to toxicity is more of a concern than its dietary deficiency. Sulfur-containing food additives, for example, may increase indigenous sulfur in human and animal diets and may cause allergic reactions in sulfur-sensitive individuals. This chapter describes dietary sulfur as an important element to all living cells and delineates its potential toxicological characteristics.

PROPERTIES OF ELEMENTAL SULFUR Sulfur is uniquely classified within the group of macroelements, for it belongs to the group 6A of nonmetals in the periodic table of the chemical elements. Unlike other macrominerals, sulfur exists predominantly in organic molecules; there are only very small amounts of free metabolic sulfite (SO3=) and sulfate (SO4=). It is one of the most abundant elements in Earth’s crust and is cycled and metabolized through the lithosphere, hydrosphere, atmosphere, and biosphere in the biogeochemical cycle (Kellogg et al., 1972). Table 4.1 illustrates the major chemical and physical properties and oxidation states of sulfur. Sulfate (oxidation state +6) is the most stable and abundant form of sulfur available for use by living organisms in the

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ATMOSPHERE (hydrogen sulfide + oxidation = sulfur dioxide + hydration = sulfurous and sulfuric acids)

hydrogen sulfide

Precipitation (sulfurous and sulfuric acids)

dimethyl sulfide

LITHOSPHERE (sulfur compounds from volcanoes, mines, industries)

HYDROSPHERE (sulfur compounds from oceans, hot springs)

sulfates, sulfites

sulfates, sulfites decay, waste

decay, waste

BIOSPHERE Plants (reduced sulfur)

Animals

Humans

FIGURE 4.1 Simplified diagram of the global sulfur cycle. (Adapted from Komarnisky et al., 2003.)

biosphere. Table 4.2 lists the inorganic sulfur compounds available for biosynthesis of sulfur amino acids (SAAs) and other biological sulfur compounds. SAAs and other organic compounds found in living cells contain sulfur in the lowest oxidation state, –2 [–S–, –S–S–, or –SH (thiol) forms], found in sulfides. To be used for the synthesis of amino acids, sulfates or other sulfur compounds in the oxidation state greater than –2 must be reduced to sulfides. Essential amino acids, including methionine (a sulfur-containing amino acid), must be supplied through diet, as humans and animals other than ruminants are unable to synthesize them. Ruminants can obtain SAAs from the microbial protein synthesized in the rumen and have various populations of sulfur-reducing bacteria that can utilize plant amino acids and inorganic sulfur for the synthesis of their own proteins. Ruminants, therefore, can obtain proteins and amino acids from the microbial cells that have been subjected to the process of acid digestion and proteolytic hydrolysis in the postruminal part of the digestive tract (Komarnisky et al., 2003).

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TABLE 4.1 The Most Common Inorganic Sulfur Compounds Compound

Chemical Formula

Sulfur dioxide

SO2

Sulfur trioxide Sulfuric acid

Process Involved

Reaction State

End Product

Oxidation

Intestinal microbial fermentation

SO3 H2SO4

S + O2 or H2S + O2 SO2 + O2 SO3 + H2O

Oxidation Hydration

Sulfurous acid

H2SO3

SO2 + H2O

Hydration

Bisulfate ions

HSO4−

H2SO4 + H2O

Intestinal microbial fermentation Contact with moist mucous membranes, skin Contact with moist mucous membranes, skin Buffering system of body fluids

Sulfate ions

SO4=

Hydrogen sulfite ions Sulfite ions

HSO3−

Hydrogen sulfide

SO3= H2S

Ionization of sulfuric acid HSO4− + H2O Ionization of sulfuric acid H2SO3 + H2O Ionization of sulfurous acid HSO3− + H2O Ionization of sulfurous acid SO4= + 2e−, Reduction SO3= + 6e−

Body metabolic processes Buffering system of body fluids Body metabolic processes Intestinal microbial fermentation

Sources: Adapted from Komarnisky et al., 2003.

OCCURRENCE OF SULFUR IN NATURE In humans and animals, three forms of sulfur exist, predominantly as organic compounds. These include thiomethyl of methionine residues in protein, sulfhydryl disulfides of protein (cysteine–cystine residues), and compounds, such as ester- or amide-bound sulfates (e.g., glycosaminoglycansins, steroids, and xenobiotic metabolites). In its native state, elemental sulfur is present in metal ores or sulfide minerals. In fossil fuels, sulfur is found in a variety of complex organic compounds and as hydrogen sulfide (H2S). This sulfur is released into atmosphere as ubiquitous air pollutant sulfur dioxide (SO2). In plants, humans, and animals, sulfur occurs in various biological structures. This sulfur is released into environment as SO4=, SO3=, or H2S either in waste or after the decay of dead plants and animal and human carcasses. Table 4.3 summarizes the occurrence of sulfur in nature and outlines sources of dietary sulfur available for humans and animals. For example, in onions there are three main sulfoxides including methyl, propyl and 1-propyl, and 1propenyl, which give rise to the onion’s tear-producing effect. Onion flavor results from these organosulfur compounds arising from the enzymatic decomposition of the flavor precursors (Randle, 1997). Intact cells of the onion have no odor, but when cells are disrupted, the enzyme, allinase, hydrolyzes S-alk(en)yl sulfoxides to produce pyruvate, ammonia, and volatile sulfur compounds associated with flavor and odor.

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TABLE 4.2 Occurrence of Sulfur in Nature Sources

Volcanic deposits Subterranean deposits Hot springs, geysers Fossil fuels

Food Vitamins Amino acids Preservatives

Organic compounds Microorganisms

Forms Available Natural Gypsum, pumice stone Sulfur ore (So), metallic sulfides, nonmetallic sulfates Sulfurous water Coal, petroleum, natural gas Dietary Onion, cabbage, cauliflower, broccoli, oil of garlic, mustard, eggs Thiamin, biotin Methionine, keto-methionine, cysteine, cystine, homocysteine, cystathionine, taurine, cysteic acid Sulfites and sulfiting agents: sulfur dioxide, sodium, bisulfite, potassium bisulfite, sodium metabisulfite, potassium metabisulfite, sodium sulfite Biological Proteins, lipoic acid, coenzyme A, glutathione, chondroitin sulfate, heparin, fibrinogen, ergothionine, estrogens, ferredoxin Aerobic heterotrophic, Desulfovibrio, Desulfotomaculum, chemoautotrophic, photoautotrophic

Fertilizers

Industrial Phosphates and ammonium sulfate

Combustion of fossil fuels

SO2, H2S

Anthropogenic Sources: Adapted from Komarnisky et al., 2003.

GLOBAL CYCLE Large amounts of Earth’s sulfur are found as a part of metal ores and minerals. These forms of sulfur are available for plants and microorganisms to synthesize SAAs and other forms of sulfur compounds. Humans obtain SAAs by consuming plants or animal meats. In all living organisms, sulfur is found in organic (thiol) form as a constituent of some proteins, vitamins, and amino acids (Table 4.3). When organisms die and decompose, some of the sulfur is taken up by the microorganisms and some is released again into the soil as sulfate. Large volumes of sulfur gases (H2S and SO2) are emitted into the atmosphere via volcanic eruptions and the processing of fossil fuels (Hobbs et al., 1981). In addition, dimethyl sulfide (DMS), a metabolic waste product of marine phytoplankton (Hay and Kubanek, 2002), is released into the atmosphere from oceans. It has been estimated that the total natural flux of gaseous sulfur to the atmosphere is 65 to 125 Tg (1 Tg = 1012 g)

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TABLE 4.3 Chemical and Physical Properties of Sulfur Characteristics General

Appearance

Physical properties

Allotropic modifications

Structure of solid sulfur Chemical properties

Properties Symbol Atomic number Atomic weight Group Valence Color Smell Texture of solid sulfur Density at 20°C Boiling point Solubility: In water In carbon disulfide Flammability Rhombic Monoclinic Amorphous S8 rings Minimum oxidation number Maximum oxidation number Reactivity Oxidation state: +6 +5 +4 +4 +3 +2 0 –2

Description S 16 32.064 Nonmetallic oxygen element group 2, 4, 6 Light yellow Odorless, tasteless Brittle 2.06 g/cm3 444.6°C Insoluble Soluble Flammable Rhombic or octahedral crystals Needle-shaped crystals Noncrystalline (liquid) Ring molecules are composed of eight sulfur atoms –2 6 Highly reactive Sulfate (SO42–) Dithionate (S2O62–) Sulfite (SO32–) Disulfite (S2O52–) Dithionite (S2O42–) Thiosulfate (S2O32–) Elemental sulfur (So) Sulfide (S2–)

Source: Adapted from Komarnisky et al., 2003.

(Rodhe, 1989). Sulfur returns back to Earth as sulfates and sulfites as a result of encountering sulfur gases with humid atmosphere. Thus, sulfur circulates through the atmosphere, lithosphere, hydrosphere, and biosphere in a continuous global cycle. A simplified diagram of the sulfur cycle is presented in Figure 4.1. The activities of microorganisms are essential in the global cycling of sulfur. Sulfonates and sulfate esters are widespread in nature and make up over 95% of the sulfur content of most aerobic soils. Many microorganisms can use sulfonates and sulfate esters as a source of sulfur for growth, even when they are unable to metabolize the carbon skeleton of the compounds (Kertesz, 2000). From terrestrial ecosystems,

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sulfur is continually lost into the water reservoirs, where some of the sulfur cycles through marine communities as it moves through food chains.

BIOLOGICAL ROLE OF SULFUR Sulfur is present in body tissues as part of the amino acids methionine, cysteine, and taurine. The sulfur atoms in cysteine are responsible for the major covalent cross-links in protein structures. Disulfide bridges formed between two cysteine molecules are important in stabilizing protein conformation. Hair and fingernails have a high percentage of cysteine to facilitate strength and rigidity of shape. Sheep have wool of a lower quality when the nutritional level of cystine and high-sulfur protein contents is restricted (Campbell et al., 1975). Propionibacterium acne has a requirement not only for amino nitrogen but also for reduced sulfur that is satisfied by the constant availability of this substance in the form of sulfhydryl groups in the sebaceous follicle during keratinization. The relationship between P. acne and its nutritional substrate may give this organism a selective advantage in this ecological niche (Nielsen, 1983). Sulfur also has a structural function as a part of mucopolysaccharides and sulfolipids. It occurs in the iron–sulfur proteins of the coenzyme Q/cytochrome c reductase complex of the respiratory chain. Sulfur atoms are also important in ironcontaining flavoenzymes, such as succinate dehydrogenase and NADH dehydrogenase. Intracellular reduction-oxidation status is increasingly recognized as a primary regulator of cellular growth and development. The relative reduction-oxidation state of the cell depends primarily on the precise balance between concentrations of reactive oxygen species (ROS) and the cysteine-dependent (thiol) antioxidant buffers, glutathione (GSH) and thioredoxin. These antioxidants have high affinity for ROS, thus protecting other intracellular molecules from oxidative damage (Deplancke and Gaskins, 2002). Sulfur, as a part of the sulfhydryl groups, forms thioester linkages that are necessary for the activation of molecules such as acetate. Interconversions between disulfide (GSSG) and sulfhydryl groups in oxidationreduction reactions occur as a result of reduction and oxidation of the sulfurcontaining compound GSH: Glutathione reductase GSSG + NADPH + H+ → 2GSH + NADP+ Glutathione peroxidase 2GSH + H2O2 → GSSG + 2H2O Other forms of endogenous sulfur compounds such as aminoethylcysteine ketimine dimer (Pecci et al., 2000) and hypotaurine (a sulfinate found in various biological tissues) are also able to protect against the ROS-induced damage (Pecci et al., 1999).

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Lipid peroxidation is the primary mechanism by which food deteriorates during storage in the presence of oxygen. Free radicals including peroxil, alkoxyl, and hydroxyl have been implicated in the mechanism of lipid peroxidation. Natural sulfur-containing compounds such as cysteine, glutathione, and lipoic acid, as well as synthetic compounds including N-acetylcysteine and a-mercaptopropionylglycine, protect against oxidative stress in biological systems through the scavenging and reduction of various oxidants (Eiserich and Shibamoto, 1994). Cyanide (CN–), a potent respiratory poison, is released by the hydrolysis of cyanogenic glycosides. The metabolic process that detoxifies cyanide is catalyzed by the ubiquitous enzyme rhodanese. The latter transfers sulfur from various sulfur compounds, but mostly from thiosulfate (SSO3=), producing the nonpoisonous compound thiocyanate (SCN–), which is excreted from the organism (Josephy, 1997). CN– + SSO3= → SCN– + SO3= Some individuals have a diminished capacity to detoxify CN– to SCN– as a consequence of either a genetic predisposition, which occurs in patients with Leber’s optic atrophy, or a diet low in sulfur-containing amino acids (Calabrese, 1979). Taurine (2-aminoethanesulfonic acid), a sulfur-containing amino acid, is the most abundant intracellular amino acid in humans, and is implicated in numerous biological and physiological functions. In healthy individuals, the diet is the usual source of taurine, although in the presence of vitamin B6 it is also synthesized from methionine and cysteine. With the exception of cow’s milk, taurine is widely distributed in foods of animal origin but not plant sources (Kendler, 1989). Taurine has antioxidant and anti-inflammatory properties. It is involved in bile acid conjugation, cholestasis prevention, and antiarrhythmic, inotropic, and chronotropic effects, as well in modulation of calcium flux and neuronal excitability, osmoregulation, detoxification, and membrane stabilization (Lourenco and Camilo, 2002). Taurine is an essential amino acid for preterm neonates and is assured by breast milk. It is also suggested that patients requiring long-term parenteral nutrition, those with chronic hepatic, heart, or renal failure, including premature and newborn infants, are at risk for taurine deficiency and may benefit from supplementation (Lourenco and Camilo, 2002). Taurine may be also essential for patients in the postinjury state (Paauw and Davis, 1990).

SULFUR METABOLISM Methionine is the only dietary essential sulfur amino acid. From methionine are synthesized all other important sulfur compounds, including cysteine, cystine, glutathione, acetylcoenzyme A, thiamin, biotin, lipoic acid, and taurine. During sulfur metabolism, sulfur is oxidized from its organic form, sulfides (S=), to sulfites (SO3=) and sulfates (SO4=). Most of the ingested sulfur is excreted in the urine after oxidation to sulfate as free ion, SO4=. Production of sulfate proceeds through cysteine sulfonic acid to sulfinyl pyruvate, SO2, and finally SO4=. Sulfite oxidase (a molybdenumdependent enzyme) is the last step in this process. Inorganic sulfate is an end product

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93

ATP + SO 4= sulfurylase Adenosine-5´-phosphosulfate (APS) ATP-adenosine phosphosulfate kinase Adenosine-3´-phosphate-5´-phosphosulfate (PAPS) sulfotransferase

+R

R-SO3H (sulfate ester)

FIGURE 4.2 Role of SO4= in the biosynthesis of sulfate esters. R = phenols, steroids, indoles, hexosamine.

of sulfur amino acid metabolism, but it is also the cosubstrate for the biosynthesis of a wide array of complex sulfoesters (Figure 4.2).

SULFUR DEFICIENCIES Dietary sulfur deficiency is relatively rare, and hence there exists no recommended dietary intake for this element. Unlike in monogastric animals the rumen microbes in ruminants can use inorganic sulfur to synthesize sulfur-containing amino acids. Sulfur deficiency may therefore lead to methionine deficiency in polygastric animals. The sulfur requirements of these animals grazing on sorghum may thus be increased because of the need for sulfur in the detoxification of the cyanogenic glycosides found in most sorghum forages (Subcommittee on Beef Cattle Nutrition et al., 1996). It was also shown that calves fed the dietary urea-supplemented diets with added 0.15 or 0.30% of sulfur had higher weight gains and a trend toward improved efficiency compared with those with no added sulfur. Plasma total amino acid concentrations were also increased by the addition of sulfur to urea-supplemented diets compared to the basal urea diet (Hill et al., 1985). Although, in humans, primary deficiency of sulfur is relatively uncommon, there are some situations where a deficiency of secondary origin may exist. Newborn infants may thus be at risk of amino acid deficiency and toxicity, due to lack of small intestinal metabolism and metabolic immaturity. Impaired small intestinal metabolism (or lack of first-phase metabolism) alters the whole-body requirement for methionine, threonine, and arginine (Brunton et al., 2000). Considerable fractions of sulfhydryls in blood are present in erythrocytes (RBCs), which among others participate in intraorgan amino acid transport. The metabolism of SAAs and sulfhydryls is usually altered in patients with end-stage renal disease (ESRD). It was found that nutritional status influences plasma, but not RBC, concentrations of sulfhydryls in patients with ESRD. The study also shows that GSH concentrations in RBC and whole blood are related to hematocrit and not to nutritional parameters,

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indicating that anemia status rather than nutritional status determines RBC and whole-blood GSH levels in patients with ESRD (Suliman et al., 2002a). Dietary cyanide exposure from consumption of insufficiently processed bitter cassava roots may be a factor aggravating growth retardation (Banea-Mayambu et al., 2000) and paralytic disease konzo (Banea-Mayambu et al., 1997) in the Bandundu region, Democratic Republic of Congo (former Zaire). Cassava roots contain naturally occurring cyanogens that are associated with the seasonal outbreaks of these ailments. The signs of diseases mainly appeared in dry seasons when the diet lacked supplementary foods with sulfur-containing amino acids that promote cyanide detoxification (Tylleskar et al., 1991). Urinary linamarin, the cyanogenic glucoside and source of cyanide in cassava, was implicated in the development of konzo. This suggests that a specific neurotoxic effect of linamarin, rather than the associated general cyanide exposure resulting from glucoside breakdown in the gut, may be the cause of this disease (Banea-Mayambu et al., 1997).

SULFUR TOXICITY Awareness that SO2 is an environmental pollutant arose in the middle of 20th century (Amdur, 1974). The magnitude of SO2 toxicity may be even greater when present in combination with other air pollutants. Air pollution in the environment consists of a complex mixture of compounds, and various atmospheric conditions can alter the toxicity of air pollutants. In addition, the metabolism or detoxification of a single chemical in the body may be altered by the mixture of chemicals as well as by the pre-existing health state of the affected organism itself (Oehme et al., 1996). It is generally believed that the toxicity of SO2 in ambient air is significantly influenced by the coincident presence of particulates (Mehlman, 1983). For example, proteincontaining dust and SO2 may promote toxic allergic reactions (Sosedova and Benemanskii, 2000). It was shown that the surface of inorganic particles containing effluent gases, produced during combustion of fossil fuels, may interact with SO2 to form an irritant aerosol. The submicron fraction of this inorganic material may penetrate deep into the lung and cause serious health effects (Peoples et al., 1988). The studies on mice suggest that fine carbon particles can be an effective vector for the delivery of toxic amounts of SO4= to the periphery of the lung (Jakab et al., 1996). Sulfur toxicity may also be a consequence of deranged metabolism of sulfurcontaining amino acids, especially methionine.

TOXICITY DUE

TO

SO2

Sulfites and sulfating agents are sulfur-based preservatives that occur naturally or are used in the food-processing industry. They are used to prevent or reduce discoloration of light-colored fruits and vegetables, prevent black spots on shrimp and lobster, inhibit the growth of microorganisms in fermented foods such as wine, condition dough, and maintain the stability and potency of certain medications. Sulfites can also be used to bleach food starches, to prevent rust and scale in boiler water that is used to steam food, and even in the production of cellophane for food packaging. SO2, sodium bisulfite, potassium bisulfite, sodium metabisulfite,

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potassium metabisulfite, and sodium sulfite are the forms of these preservatives. Sulfites exert the antimicrobial properties through release of free SO2, which inhibits propagation of yeasts, molds, and bacteria by killing the microorganisms entirely or by blocking their ability to reproduce. Despite their broad use in the food industry, sulfites may produce deleterious health effects in sulfite-sensitive individuals, although clinical responses can vary. Sulfites can cause chest tightness, nausea, hives, or even anaphylactic shock. However, difficulty in breathing is the most common symptom reported by sulfite-sensitive people (Field et al., 1994). Sulfites, bisulfites, and metabisulfites are all dry chemical forms of the SO2 gas that may cause irritation in the lungs and a severe asthma attack for those who suffer from asthma. A person can develop sulfite sensitivity at any point in life; as yet, the mechanism for sulfur sensitivity is not fully understood. Routes of SO2 Entry into Living Organisms Mammals and birds are exposed to air pollutants by inhalation through the nose and mouth, as well via cutaneous or ocular routes (Oehme et al., 1996). People can be exposed to environmental pollutants through food (e.g., ingesting residues of pesticides used in agriculture), in the workplace (e.g., inhalation of chemicals used in industrial processes), and directly from the environment (e.g., breathing polluted air in cities). Maximum and Threshold Limit Values of SO2 In North America, the threshold limit values (TLV) of SO2 in atmospheric air have been established for humans: 2 ppm for a normal 8-h workday or 40-h workweek. It is assumed that workers may be repeatedly exposed to this amount of SO2 without adverse effects. The maximum concentration that should not be exceeded at any time during a 15-min exposure period is 5 ppm SO2 (Katzung, 1998). However, epidemiological studies have shown a direct relationship between moderate concentrations of air pollution and airway disease. Thus, during the summer of 1999 the respiratory symptoms of chronic cough and phlegm, wheeze, and shortness of breath observed in 3709 Chinese adults were associated with the median indoor concentrations of SO2 in Beijing: 14 μg/m3; Anqing City: 25 μg/m3; and rural Anqing: 20 μg/m3 (Venners et al., 2001).

SO2-SENSITIVE SUBJECTS Individuals with asthma are most sensitive to inhaled SO2, which is a common air pollutant found in the workplace (Riedel et al., 1992). Despite that there are considerable interindividual variations in response to SO2 in patients with asthma (Winterton et al., 2001), bronchoconstriction seems to be the most common sensitivity response (Lester, 1995). The bronchoconstrictive effect in asthmatic subjects may be produced by inhalation of air amended with 0.75 ppm or even lower concentrations of SO2 (Wiebicke et al., 1990). SO2-induced bronchoconstriction is mediated by parasympathetic pathways (Sheppard et al., 1980). The mechanism of this type of bronchoconstriction involves release of leukotrienes (Balmes et al., 1987; Gong, Jr.

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et al., 2001). Mast cells lying in the surface mucosa of the lung are thought to be less stable in asthmatic subjects and may also be implicated in the mechanism of response to SO2 (Rocchiccioli and Riley, 1989). It has been suggested that the genetic biomarker associated with the wild-type allele of the tumor necrosis factor-alpha (TNF-α) promoter polymorphism may be employed in identification of sensitivity to inhaled SO2 in subjects with asthma (Winterton et al., 2001). Human lungs develop throughout childhood until age 20. Children have higher metabolic rates than adults and therefore require more air to breathe to inhale more oxygen. Their lungs may thus be more affected by damage due to air pollution. Epidemiological studies have shown acute effects of ambient air pollution on the occurrence of respiratory symptoms in children having breathing disorders. In the Netherlands children susceptible to SO2 had bronchial hyperresponsiveness and relatively high serum concentrations of total IgE (Boezen et al., 1999).

DISEASES

AND

SULFUR

It was concluded from a variety of animal experiments that long-term exposure to SO2 alone did not cause cancer (Mehlman, 1983), and no clear evidence exists that SO2 or bisulfite causes mutagenicity in mammals (Pool-Zobel et al., 1990). However, it was found that workers exposed to SO2 at a sulfuric acid factory in Taiyuan City (northern China) had a higher number of lymphocytes with chromosomal aberrations compared to controls. Thus, these observations show that SO2 is a possible clastogenic and genotoxic agent (Meng and Zhang, 1990, 2002). Air pollution as a trigger for exacerbations of chronic obstructive pulmonary disease (COPD) has been recognized for more than 50 years, leading to the development of air quality standards in many countries, which have substantially decreased the levels of air pollutants derived from the burning of fossil fuels, such as black smoke and SO2 (MacNee and Donaldson, 2000). Subjects with diabetes mellitus are also at risk for SO2-induced toxicity. Exposure of chemically induced diabetic rats to 10 ppm of SO2 potentiated visual evoked potential (VEP) changes and lipid peroxidation caused by the increased release of free radicals (Agar et al., 2000). It has been suggested that the hypercalciuria induced by a high-meat diet is mainly caused by the high content of SAAs and may be reversed by the ingestion of potassium-rich foodstuffs (Kaneko et al., 1990).

PATHOGENESIS

OF

SO2-LINKED TOXICITY

SO2, a highly water-soluble gas, dissolves in water to form bisulfite (HSO3–), sulfite (SO3), and hydrogen (H+) ions. These reactions are described by the following equilibrium relationships: SO2 + H2O β ↔ HSO3– + H+ β ↔ SO3 = 2H+ The hydrolysis of SO2 occurs very rapidly in aqueous environments. As a consequence, within fluid-filled structures such as cells, tissues, and blood vessels, any

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effects of SO2 exposure must be due to the effects of bisulfite and/or sulfite anions. However, during inhalation of SO2 gas, SO2 itself will be present at the air–liquid interface, so the initial effects of SO2 in cells at the luminal surface of the airways could be due to the direct chemical effects of SO2 gas rather than to the effects of bisulfite or sulfite (Amdur, 1989). The latter two major hydrolysis products are present in roughly equal concentrations at physiologic pH as determined by the pKa of their equilibrium reaction (~7.2). However, at the pH level reported at the luminal surface of the airways (6.6), the ratio of bisulfite to sulfite is approximately 5:1. This may be unfortunate, because bisulfite is generally more chemically reactive than sulfite. Bisulfite is a nucleophile that reacts with many biomolecules through substitution at electrophilic sites (Neta and Huie, 1985). One of these reactions leads to the disruption of disulfide bonds and the production of thiosulfates (RSH) through the following reaction (Petering and Shih, 1975). R–S–S–R + HSO3– ↔ RSSO3– + RSH Because disulfide bonds are widely found in tissue proteins, it is possible that bisulfite formed at the airway surface during SO2 inhalation initiates bronchoconstriction by such an effect on surface proteins. In addition, sulfite ions (SO3=) in reaction with superoxides (O2•–) form very reactive bisulfite radicals (SO3•) and hydrogen peroxide (H2O2). SO3• participates in radical chain processes such as lipid peroxidation (Hippeli and Eltsner, 1995). The chemical relationship between SO2, SO3•, and SO3= has led to speculation that the bronchoconstriction that follows oral ingestion of sulfite-containing foods and beverages in some patients with asthma is mechanistically related to SO2-induced bronchoconstriction (Stevenson and Simon, 1984). The SO3• ion is converted to sulfate by sulfite oxidase, an enzyme found in the lung, the liver, and a variety of other tissues (Petering and Shih, 1975). Sulfite can also be converted to sulfate by nonenzymatic autooxidation, which occurs in the presence of oxygen. Although this reaction is usually slow, it can be catalyzed by trace metal ions. This reaction is also a source of free radicals that may contribute to the tissue toxicity of SO2 (Neta and Huie, 1985). The distribution, metabolism, and toxicity of sulfite in the respiratory tract and other tissues have been studied on sulfite oxidase-deficient rats. The animals were exposed to 10 and 30 ppm of SO2. The endogenously generated sulfite and Ssulfonate compounds (a class of SO2 metabolites) were accumulated in the respiratory tract tissues and in the plasma of these rats. In addition, their testes were severely atrophied and as a result were devoid of spermatogenic cells. In contrast, in normal, sulfite oxidase-competent rats exposed to the same concentrations of SO2, sulfite and S-sulfonate compounds were restricted to the airways (Gunnison et al., 1987). In addition, studies on five mongrel dogs exposed to 22 ppm and four dogs to 50 ppm of 35SO2 for 30 to 60 min suggested that the blood plasma contained more 35S, which was associated with α-globulin proteins (half of 35S), than erythrocytes, which contained intracellular sulfur. Most of the urinary radioactive sulfur was excreted in the form of inorganic sulfate (Yokoyama et al., 1971).

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CLEARANCE

OF

SO2

FROM THE

ORGANISM

Mucociliary clearance of the respiratory tract is an important defense mechanism against inhaled pathogens and depends on ciliary and mucous factors. Airway mucus, a complex airway secretion, functions as a renewable and transportable barrier against inhaled particulates and toxic agents. Inhalation of a number of air pollutants, including SO2 and cigarette smoke, may increase mucus secretion and alter mucus rheology (Samet and Cheng, 1994). Cilia, which line both the upper and lower airways, are covered by a thin layer of mucus. The rapid, coordinated beat of cilia propel particles trapped in the mucus layer to the pharynx. Although no evidence for direct nervous control of ciliary function has been demonstrated, it was suggested that adrenergic agonists might enhance it. Cilial defects may be either congenital (primary) or acquired (secondary) as a result of infection, toxins, or drugs (Verdugo et al., 1980). Degradation of the pulmonary surfactant dynamic interfacial properties due to inhalation of SO2-polluted air may result in a slowdown of the pulmonary clearance rate and an increase in the lung burden (Podgorski et al., 2001). For highly soluble gases such as SO2, the upper airways have been shown to be a very effective scrubber with much of the gas removed during a single pass. For example, it has been shown that, in nose-breathing rabbits inhaling 400 ppm SO2, less than 10 ppm reached the trachea. It was also noted that SO2 inhalation did not affect lung function in normal subjects, but induced bronchoconstriction in subjects with asthma. It was suggested that nasal breathing, which is often impaired in people with asthma, reduces the pulmonary effects of SO2 because this water-soluble gas is absorbed by the nasal mucosa (Peden, 1997). Sulfite oxidase, a mitochondrial molybdoenzyme in mammals, is essential for detoxication of the sulfite arising from metabolism of sulfur-containing amino acids, from ingestion of bisulfite preservatives, and from inhalation of SO2 (Coughlan, 1983). Endogenous sulfite is generated as a consequence of the body’s normal processing of sulfur-containing amino acids. Sulfites occur as a result of fermentation, and they also occur naturally in a number of foods and beverages. Despite that they have been safely used as food additives for a long time, sulfiting food preservatives have been found to be allergy-causative agents. Sulfite sensitivity occurs most often in adults with asthma and in preschool children (Lester, 1995).

TOXICITY

OF

SECONDARY ORIGIN (SULFUR METABOLISM)

Homocysteine (Hcy) is a sulfur-containing amino acid produced by the breakdown of methionine (Figure 4.3). Plasma Hcy levels can be elevated due to a variety of genetic and nutritional factors. Poor nutrition from diets low in folate and vitamins B12 and B6 can lead to hyperhomocysteinemia. Hyperhomocysteinemia is regarded as a public health problem of increasing importance likely to contribute to vascular disorders and premature mortality. Mildly elevated levels of Hcy have been implicated in a number of disease processes such as atherosclerotic vascular disease and adverse obstetrical outcomes. High levels of plasma Hcy are also associated with abnormal collagen cross-linking. Hyperhomocysteinemia in pregnancy is associated with preterm, premature rupture of membranes, an important public health concern,

Biological and Toxicological Considerations of Dietary Sulfur

Methionine CH2THF

Folate Cycle

SAM

DMG

THF

B12MS BHMT

S

Methionine Cycle

Homocysteine CBS

B6

DNA RNA Protein

Methyltransferase

Betaine

CH3THF

99

SCH3 SAH

Transsulfuration Pathway

Cystathionine B6

Cysteine

SO4=

FIGURE 4.3 Metabolic fate of homocysteine. THF: tetrahydrofolate; DMG: dimethylglycine; SAM: S-adenosylmethionine; CBS: cystathionine ß-synthase; BHMT: betaine homocysteine hydroxymethyltransferase. (Modified from Basu and Fisher, 2002.)

due to the effects of homocysteine on connective tissue integrity (Ferguson et al., 2001). Folate, cobalamin, pyridoxine, and riboflavin dietary deficiencies are currently regarded as causative factors of hyperhomocysteinemia, which may arise from the shrinking of endogenous nitrogen pools as a result of decreased protein intake or stress-induced increased losses. Raised total Hcy may result from the attempt of the malnourished or stressed body to preserve methionine homeostasis (Ingenbleek et al., 2002). Malnutrition, hypoalbuminemia, and diabetes mellitus in patients with chronic renal failure influence SAA levels, mainly plasma total Hcy, which should be considered when evaluating hyperhomocysteinemia as a cardiovascular risk factor (Suliman et al., 2002b). Homocystinuria, another inborn disease related to sulfur metabolism, is implicated as the result of enzyme cystathionine synthase deficiency (Poole et al., 1975). Intestinal gas is thought to be the cause of abdominal discomfort in infants. Gas release by infant feces is strongly influenced by an infant’s diet. It was found that the highly toxic sulfur gases hydrogen sulfide (H2S) and methanethiol (CH3SH) are high in soy-formula-fed infants and low in breastfed infants (Jiang et al., 2001). Mercaptides (sodium hydrogen sulfide and sodium methanethiol) and mercaptofatty acid (sodium mercaptoacetate) are reducing agents that help to maintain anaerobic conditions in the colonic lumen. Metabolic effects of sodium hydrogen sulfide on butyrate oxidation along the length of the colon closely resemble metabolic abnormalities observed in active ulcerative colitis. The increased production of sulfide in ulcerative colitis suggests that the action of mercaptides may be involved in the genesis of ulcerative colitis (Roediger et al., 1993). Problems associated with excess in dietary sulfur intake in ruminants are being increasingly recognized. Excessive levels of sulfur-containing compounds in domestic ruminant animals’ rations and clinical problems associated with low to moderate levels of exposure to dietary sulfur may be more common than previously thought

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(Olkowski, 1997). Subsequent excessive ruminal sulfide production is an important factor in the pathogenesis of polioencephalomalacia (PEM), without concurrent thiamine deficiency. Most cases of PEM were developed between 15 and 30 days after introduction to a high-sulfur diet. In cases where water is an important source of dietary sulfur, risk of PEM may increase during hot weather (McAllister et al., 1997).

CONCLUSION Sulfur is one of the most abundant chemical elements on Earth and is an important nutritive constituent in all biological systems. In nature, sulfur is commonly found in its most stable form as sulfate. The latter must be reduced to sulfite by microorganisms in order to be metabolized by animals and humans for the synthesis of SAA. As a constituent of certain amino acids, sulfur performs a number of functions in enzyme reactions and protein synthesis. Mammals acquire organic (thiol) forms of sulfur from their diets. Sulfur and sulfur compounds exhibit protective and antioxidative properties during a number of metabolic disorders in living cells. Anthropogenic emissions of sulfur have a large impact on the balance of this element in the environment, which in turn may influence health of plants, animals, and humans.

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Boezen, H.M., van der Zee, S.C., Postma, D.S., Vonk, J.M., Gerritsen, J., Hoek, G., Brunekreef, B., Rijcken, B., and Schouten, J.P. (1999) Effects of ambient air pollution on upper and lower respiratory symptoms and peak expiratory flow in children, Lancet, 353: 874–878. Brunton, J.A., Ball, R.O., and Pencharz, P.B. (2000) Current total parenteral nutrition solutions for the neonate are inadequate, Current Opinion in Clinical Nutrition and Metabolic Care, 3: 299–304. Calabrese, E.J. (1979) Possible adverse side effects from treatment with laetrile, Medical Hypotheses, 5: 1045–1049. Campbell, M.E., Whiteley, K.J., and Gillespie, J.M. (1975) Influence of nutrition on the crimping rate of wool and the type and proportion of constituent proteins, Australian Journal of Biological Sciences, 28: 389–397. Coughlan, M.P. (1983) The role of molybdenum in human biology, Journal of Inherited Metabolic Disease, Suppl. 1, 6: 70–77. Deplancke, B. and Gaskins, H.R. (2002) Redox control of the transsulfuration and glutathione biosynthesis pathways, Current Opinion in Clinical Nutrition and Metabolic Care, 5: 85–92. Eiserich, J.P. and Shibamoto, T. (1994) Sulfur-containing heterocyclic compounds with antioxidative activity formed in Maillard reaction model systems, in C.J. Mussinan and M.E. Keelan, Eds., Sulfur Compounds in Foods, Washington, D.C.: American Chemical Society, 247–251. Ferguson, S.E., Smith, G.N., and Walker, M.C. (2001) Maternal plasma homocysteine levels in women with preterm premature rupture of membranes, Medical Hypotheses, 56: 85–90. Field, P.I., McClean, M., Simmul, R., and Berend, N. (1994) Comparison of sulfur dioxide and metabisulfite airway reactivity in subjects with asthma, Thorax, 49: 250–256. Gong, H., Jr., Linn, W.S., Terrell, S.L., Anderson, K.R., and Clark, K.W. (2001) Antiinflammatory and lung function effects of montelukast in asthmatic volunteers exposed to sulfur dioxide, Chest, 119: 402–408. Gunnison, A.F., Sellakumar, A., Currie, D., and Snyder, E.A. (1987) Distribution, metabolism and toxicity of inhaled sulfur dioxide and endogenously generated sulfite in the respiratory tract of normal and sulfite oxidase-deficient rats, Journal of Toxicology and Environmental Health, 21: 141–162. Hay, M.E. and Kubanek, J. (2002) Community and ecosystem level consequences of chemical cues in the plankton, Journal of Chemical Ecology, 28: 2001–2016. Hill, G.M., Boling, J.A., and Bradley, N.W. (1985) Plasma amino acid profiles and growth of the bovine fed different sources of nitrogen and levels of sulfur, International Journal for Vitamin and Nutrition Research, 55: 439–446. Hippeli, S. and Eltsner, E.F. (1995) Biological and biochemical effects of air pollutants: synergistic effects of sulfite, Biochemical Society Symposia, 61: 153–159. Hobbs, P.V., Radke, L.F., Eltgroth, M.W., and Hegg, D.A. (1981) Airborne studies of the emissions from the volcanic eruptions of Mount St. Helens, Science, 211: 816–818. Ingenbleek, Y., Hardillier, E., and Jung, L. (2002) Subclinical protein malnutrition is a determinant of hyperhomocysteinemia, Nutrition, 18: 40–46. Jakab, G.J., Clarke, R.W., Hemenway, D.R., Longphre, M.V., Kleeberger, S.R., and Frank, R. (1996) Inhalation of acid coated carbon black particles impairs alveolar macrophage phagocytosis, Toxicology Letters, 88: 243–248. Jiang, T., Suarez, F.L., Levitt, M.D., Nelson, S.E., and Ziegler, E.E. (2001) Gas production by feces of infants, Journal of Pediatric Gastroenterology and Nutrition, 32: 534–541.

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Josephy, P.D. (1997) Sidebar: sulfate, sulfite, thiosulfate, and cyanide toxicity, in P.D. Josephy, Ed., Molecular Toxicology, Oxford: Oxford University Press, 130–131. Kaneko, K., Masaki, U., Aikyo, M., Yabuki, K., Haga, A., Matoba, C., Sasaki, H., and Koike, G. (1990) Urinary calcium and calcium balance in young women affected by high protein diet of soy protein isolate and adding sulfur-containing amino acids and/or potassium, Journal of Nutritional Science and Vitaminology (Tokyo), 36: 105–116. Katzung, B.G. (1998), Basic & Clinical Pharmacology, 7th ed., Stamford, CT: Appleton & Lange. Kellogg, W.W., Cadle, R.D., Allen, E.R., Lazrus, A.L., and Martell, E.A. (1972) The sulfur cycle, Science, 175: 587–596. Kendler, B.S. (1989) Taurine: an overview of its role in preventive medicine, Preventive Medicine, 18: 79–100. Kertesz, M.A. (2000) Riding the sulfur cycle — metabolism of sulfonates and sulfate esters in Gram-negative bacteria, FEMS Microbiology Reviews, 24: 135–175. Komarnisky, L.A., Christopherson, R.J., and Basu, T.K. (2003) Sulfur: its clinical and toxicologic aspects, Nutrition, 19: 54–61. Lester, M.R. (1995) Sulfite sensitivity: significance in human health, Journal of the American College of Nutrition, 14: 229–232. Lourenco, R. and Camilo, M.E. (2002) Taurine: a conditionally essential amino acid in humans? An overview in health and disease, Nutricion Hospitalaria, 17: 262–270. MacNee, W. and Donaldson, K. (2000) Exacerbations of COPD: environmental mechanisms, Chest, Suppl. 2, 117: 390S–397S. McAllister, M.M., Gould, D.H., Raisbeck, M.F., Cummings, B.A., and Loneragan, G.H. (1997) Evaluation of ruminal sulfide concentrations and seasonal outbreaks of polioencephalomalacia in beef cattle in a feedlot, Journal of the American Veterinary Medical Association, 211: 1275–1279. Mehlman, M.A. (1983) Current toxicological information as the basis for sulfur oxide standards, Environmental Health Perspectives, 52: 261–266. Meng, Z.Q. and Zhang, L.Z. (1990) Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of workers exposed to sulfur dioxide, Mutation Research, 241: 15–20. Meng, Z. and Zhang, B. (2002) Induction effects of sulfur dioxide inhalation on chromosomal aberrations in mouse bone marrow cells, Mutagenesis, 17: 215–217. Miller, G.T., Jr. (1998) Living in the Environment. Principles, Connections, and Solutions, 10th ed., Belmont, CA: Wadsworth. Neta, P. and Huie, R.E. (1985) Free-radical chemistry of sulfite, Environmental Health Perspectives, 64: 209–217. Nielsen, P.A. (1983) Role of reduced sulfur compounds in nutrition of Propionibacterium acnes, Journal of Clinical Microbiology, 17: 276–279. Oehme, F.W., Coppock, R.W., Mostrom, M.S., and Khan, A.A. (1996) A review of the toxicology of air pollutants: toxicology of chemical mixtures, Veterinary and Human Toxicology, 38: 371–377. Olkowski, A.A. (1997) Neurotoxicity and secondary metabolic problems associated with low to moderate levels of exposure to excess dietary sulfur in ruminants: a review, Veterinary and Human Toxicology, 39: 355–360. Paauw, J.D. and Davis, A.T. (1990) Taurine concentrations in serum of critically injured patients and age- and sex-matched healthy control subjects, American Journal of Clinical Nutrition, 52: 657–660. Pecci, L., Costa, M., Montefoschi, G., Antonucci, A., and Cavallini, D. (1999) Oxidation of hypotaurine to taurine with photochemically generated singlet oxygen: the effect of azide, Biochemical and Biophysical Research Communications, 254: 661–665.

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5

Fluoride — Toxic and Pathologic Aspects: Review of Current Literature on Some Aspects of Fluoride Toxicity Thomas F.X. Collins and Robert L. Sprando

CONTENTS Abstract ..................................................................................................................106 Abbreviations .........................................................................................................106 Introduction and Background ................................................................................106 Exposure ................................................................................................................107 Fluoride Compounds Used to Fluoridate Water ...................................................112 Acute Toxicity........................................................................................................114 Subchronic Toxicity...............................................................................................115 Dental Effects ............................................................................................115 Skeletal Effects ..........................................................................................117 Renal Effects..............................................................................................118 Pulmonary Effects .....................................................................................118 Hormonal Effects.......................................................................................118 Genetic Effects...........................................................................................119 Neural Effects ............................................................................................119 Reproductive Toxicity Aspects ..................................................................119 Correlation with Decreased Fertility in Humans ............................119 Correlation with Down’s Syndrome in Humans .............................120 Correlation with Male Reproduction and Male Offspring..............120 Correlation with Female Reproduction ...........................................129

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Developmental Toxicity Aspects ...............................................................132 Human Studies .................................................................................132 Animal Studies.................................................................................132 References..............................................................................................................134

Abstract

Humans are exposed to fluorides, which are ubiquitous compounds, primarily through water, food, dental products, and air. The use of fluoridated water in the preparation of foods and beverages at commercial establishments and at home, coupled with fluoride in dental products, has led to increased consumption of fluorides. Fluorides can produce wide-ranging effects on many tissues, organs, and systems in the body. Fluorides are toxic at high concentrations, but at low concentrations they are added to drinking water to prevent the formation of dental caries. In mammals, fluorides have a high affinity for teeth and bones. Consumption of excess fluoride during the time of tooth enamel formation in children can cause dental fluorosis, marked by discolored or “mottled” teeth. Skeletal fluorosis, marked by symptoms ranging from slight pain to crippling deformities, is an additive disease, for which daily consumption of high levels of fluoride for many years is usually required. In addition to teeth and bones, fluoride can also affect kidneys, lungs, and the nervous system, and it can disturb hormones and possibly change genetics. Treatment of male animals with high levels has indicated that fluoride can affect testicular production in mice and rabbits, but not always in rats. Treatment of female rats and rabbits with sodium fluoride during several generations failed to produce reproductive effects, but a high concentration of fluoride decreased bone ossification in rats.

Abbreviations

ATPase: adenosine triphosphatase; CDC: Centers for Disease Control; EPA: Environmental Protection Agency; F1 generation: first generation; FDA: Food and Drug Administration; FSH: follicular stimulating hormone; HSD: hydroxysteroid dehydrogenase; l: liter; LH: luteinizing hormone; mg/kg: milligrams per kilogram bodyweight; ml: milliliter; μg: microgram; NRC: National Research Council; NTP: National Toxicology Program; P generation: parental generation; PHS: Public Health Service; ppm: parts per million (= one milligram per liter); WHO: World Health Organization

INTRODUCTION AND BACKGROUND Much has been said and will continue to be said, and much ink has been used to praise or denounce fluoride. Proponents constantly laud its effectiveness in reducing dental caries, and opponents are equally steadfast in citing its toxic effects. For most trace elements in food or water such as chromium or zinc, the effect depends on the dose. Fluoride, however, is unusual among trace elements because the same range of exposure can produce beneficial or harmful effects, depending on the developmental stage of the individual and nutritional factors.

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Fluoride is the ionic form of the halogen fluorine, which is the most electronegative and reactive of all elements. Fluoride salts are readily formed. In nature, fluoride occurs chiefly in fluorspar (calcium fluoride) and cryolite (sodium aluminum fluoride), but it is widely distributed in other minerals. Volcanoes are a major source of hydrogen fluoride, a compound that dissolves readily in water to form hydrofluoric acid. Coal combustion, aluminum production plants, and phosphate fertilizer plants are the main anthropogenic sources of hydrogen fluoride (PHS, 2001). In general, exposure to hydrogen fluoride in the air is low, although persons living near industrial sources of the gas or workers in fluoride-processing industries may be exposed to higher levels of the gas in the air. Also, vegetables and fruits grown near these sources could contain fluoride from fluoride-containing dust settling on the plants (PHS, 2001). During ancient and medieval times, caries and periodontal disease occurred without the benefit of efficient treatments. With the ready availability of cheap sugar in Europe and North America, incidence of caries reached such high levels that a substantial number of young people lost their teeth because of them. In the early 1900s, particularly in the southwestern U.S., it was observed that people with mottled (i.e., discolored) teeth had fewer cavities than people without mottled teeth. Naturally occurring fluoride in the drinking water was identified as responsible for this hardening of tooth enamel. Prevention of caries on a public-health scale began in 1945 with the fluoridation of water in Grand Rapids, Michigan. Starting in the 1950s, fluoride was added to fluoride-deficient drinking water in many communities to bring the total level of fluoride to 1 mg/l (1 ppm). This level was considered the optimal level to reduce caries and to minimize the risk of dental fluorosis. In developed industrial countries where the incidence of caries has decreased substantially since then, emphasis in dentistry has shifted to cosmetic dentistry (Marthaler, 2002). Most recently, the variety and availability of teeth-whitening products appear to be expanding almost exponentially. The 1984 World Health Organization Guidelines (WHO, 1984) suggested that in areas with a warm climate, the optimal fluoride concentration in drinking water should remain below 1 ppm, and in cooler climates it could go as high as 1.2 ppm (Table 5.1). This difference is based on the fact that persons perspire more in hot weather and drink more water to compensate for the liquid lost. The upper limit of concentration was set at 1.5 ppm, a level considered a threshold between the benefit of resistance to tooth decay and the risk of dental fluorosis. The nutritional status also influences the rate at which fluoride is absorbed by the body. For example, calcium in the diet binds with fluoride and thus decreases the body’s retention of fluoride.

EXPOSURE For humans, the common sources of fluoride are water, food, dental products, and air. Fluoride concentrations and exposure vary among these sources. The fluoride concentration in fresh surface water is low, ranging from 0.01 to 0.03 mg/l (WHO, 1984). The fluoride concentration in groundwater fluctuates from less than 0.1 to more than 25 mg/l (WHO, 1984). Fluoride enters groundwater by natural processes,

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TABLE 5.1 Recommended Water Fluoride Concentrations Average Maximum Daily Air Temp.

Recommended Control Limits (ppm)

°F

°C

Lower-Upper

Optimum

50.0–53.7 53.8–58.3 58.4–63.8 63.9–70.6 70.7–79.2 79.3–90.5

10.0–12.0 12.1–14.6 14.7–17.7 17.8–21.4 21.5–26.2 26.3–32.5

0.9–1.7 0.8–1.5 0.8–1.3 0.7–1.2 0.7–1.0 0.6–0.8

1.2 1.1 1.0 0.9 0.8 0.7

Source: Data from NRC (1993).

and the concentration depends on the amount of leaching of rock. Waters from some areas of the Southwest have higher concentrations of fluoride than most areas in the East. On the basis of Environmental Protection Agency (EPA, 1985) estimates, more than 86% of people who are served by public water systems are exposed to fluoride levels of 1.0 mg/l or less. Approximately 0.4% are exposed to drinking water that is greater than 2.0 mg/l, particularly from groundwater sources. A person’s daily intake of fluoride from drinking water is a function of the person’s age, size, and the fluoride concentration of the water. In general, the fluoride intake from drinking water increases proportionally as the fluoride content of water increases. See Table 5.2 for adequate intake (AI) and upper-level (UL) intake. AI is based on fluoride

TABLE 5.2 Adequate Intake (AI) and Upper Limit (UL) of Fluoride Intake (mg/day) Age

AIa

ULb

Reference Weight kg (lb)

0–6 months 7–12 months 1–3 years 4–8 years 9–13 years, males 9–13 years, females 14–18 years, males 14–18 years, females 19 years and over, males 19 years and over, females

0.01 0.5 0.7 1.0 2.0 2.0 3.0 3.0 4.0 3.0

0.7 0.9 1.3 2.2 10.0 10.0 10.0 10.0 10.0 10.0

7 (16) 9 (20) 13 (29) 22 (48) 40 (88) 40 (88) 64 (142) 57 (125) 76 (166) 61 (133)

a

Fluoride intake of 0.05 mg/kg/day from all sources (for ages over 6 months). Fluoride intake of 0.10 mg/kg/day from all sources.

b

Source: Data from Food and Nutrition Board (1999).

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consumption of 0.05 mg/kg/day from all sources, and UL is based on fluoride consumption of 0.10 mg/kg/day from all sources (Food and Nutrition Board, 1999). The use of fluoridated water in the preparation of foods and beverages at commercial establishments or at home has led to increased consumption of fluoride. Increased fluoride content is seen in carbonated beverages prepared with fluoridated municipal water. The fluoride in foods and beverages contributes to the total fluoride daily intake. At the time the optimal level in drinking water was set, drinking water was the only source of fluoride. Now, the addition of fluoride in food during food preparation is causing the ingestion of fluoride in doses that could be detrimental. The concentration of fluoride available in foods tends to be below 0.05 mg/100 g (Taves, 1983), except in fluoridated drinking water, some beverages, and foods made with or cooked in fluoridated water (Food and Nutrition Board, 1999). When foods and beverages most commonly consumed by adolescents were analyzed for fluoride, no significant differences were seen between an optimally and a negligibly fluoridated community (Jackson et al., 2002). However, a significant difference was found between the two communities in the fluoride content of fountain beverages and in cooked or reconstituted foods prepared using local water. In some of the coal-burning areas of rural China, fluoride can be adsorbed by corn dried over unvented ovens burning high-fluorine coal (Zheng and Huang, 1989). Where fluoride in indoor air was adsorbed by food, the daily mean intake of fluoride from corn, wheat, chiles, potatoes, and vegetables was 37.6, 0.32, 5.77, 3.54, and 0.69 mg per person, respectively (Ando et al., 1998). In these areas of rural China, fluoride exposure was estimated to be 97% via food and 2% via inhalation (Ando et al., 1998). More than 10 million people in the Guizhou Province of China and surrounding areas suffer from dental and skeletal fluorosis (Ando et al., 1998; Finkelman et al., 1999). When 238 commercially available infant foods were examined (Heilman et al., 1997), fluoride concentrations ranged from 0.01 to 8.38 ppm, with the highest concentration found in infant foods containing chicken (Table 5.3). Foods made with mechanically deboned chicken may contribute significantly to total fluoride intake because the mechanical separation process removes attached meat from bone along

TABLE 5.3 Fluoride Concentrations in Infant Foods Type

N

Range (ppm)

Mean Concentration (ppm)

Fruits and desserts Vegetables Mixed foods Meats Chicken

88 48 42 19 6

0.01–0.49 0.01–0.42 0.01–0.63 0.01–8.38 1.05–8.38

0.10 0.12 0.21 1.46 4.49

Ref. Heilman Heilman Heilman Heilman Heilman

et et et et et

al., al., al., al., al.,

1997 1997 1997 1997 1997

Note: Ranges and mean concentrations of fluoride in infant foods, based on 203 samples.

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with a small fraction of finely powdered bone (Fein and Cerklewski, 2001). The bone fraction is likely to contain elements such as calcium and fluoride. Mechanically separated chicken, such as chicken sticks and luncheon meats that are likely to be consumed by young children, was tested for fluoride content, and elevated levels of fluoride were found, but the levels varied with the brands tested. Two brands of pureed chicken contained 2.82 and 5.58 μg fluoride/g, chicken sticks contained 3.61 μg fluoride/g, two brands of luncheon meat contained 1.60 and 2.35 μg fluoride/g, and two brands of Vienna sausage contained 1.45 and 2.18 μg fluoride/g. These levels are above the desirable intake levels for a child (Fein and Cerklewski, 2001). Foods made with mechanically separated turkey, however, contained much less fluoride than their chicken counterparts. It was proposed that turkey bones are more difficult to crush and powder during the mechanical separation process than are chicken bones (Grunder and MacNeil, 1973). Several studies have been done on the fluoride content of beverages such as juices, part-juice drinks, carbonated soft drinks, tea, and wine. When 43 fruit juices were examined, fluoride concentration was 0.15 to 6.80 ppm (Stannard et al., 1991). When 532 juices and juice drinks were analyzed for fluoride concentration, the concentration ranged from 0.02 to 2.80 ppm (Kiritsy et al., 1996). Fluoride levels in 332 soft drinks ranged from 0.02 to 1.28 ppm (Heilman et al., 1999). Some of the fluoride concentrations are summarized in Table 5.4. Within each type, the beverages are arranged in ascending order according to mean fluoride concentration. All the beverages had wide ranges of fluoride concentration, and in many cases the concentration was over 1 ppm. Studies by Kiritsy et al. (1996) and Heilman et al. (1999) were done as part of the Iowa Fluoride Study, and Stannard’s study (1991) was done to evaluate fluoride concentration in beverages obtained in the Boston area. When the same beverages were tested from different areas of the country, the concentrations were sometimes similar (e.g., apple juice) and other times quite varied (e.g., prune juice). This finding was not surprising because of the known production and distribution routes of juices and other beverages throughout the country. For some juices (e.g., apple juice), the fruit is grown and processed locally and shipped to locations throughout the country. For other juices, the juices or juice concentrates are purchased from other national and international locations, processed and/or reconstituted, and sent to locations throughout the country (Kiritsy et al., 1996). Any reconstitution alters the fluoride concentration based on the fluoride content of the water used for reconstitution. Individual companies also may have several sites of production. The fluoride content of the finished product then depends on the level of fluoride in the water. If the companies use water from several production sites with different levels of fluoride, the fluoride content may differ from one location to the next. The lowest fluoride values were found in juices that needed the least amount of water. Grape juice and other juices containing grape juice contained concentrations of fluoride that were higher than expected. By extracting the juice only from the insides of the grapes, a great reduction in the fluoride content occurred, indicating that the fluoride was on the skin (Stannard et al., 1991). By international agreement, the fluoride content of wine should not exceed 1 ppm (Burns and Gump, 1993). A study of California wines showed that the fluoride

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TABLE 5.4 Fluoride Concentrations in Beverages Type

N

Range (ppm)

Mean (ppm)

Ref.

Fruit nectar Pineapple Prune Orange Apple Apple Grapefruit Mixed fruit Red grape Pear Cranberry Mixed fruit Prune White grape Red grape White grape

7 12 2 88 8 83 34 119 37 8 45 11 10 10 5 3

Juices 0.05–0.27 0.15 0.03–0.88 0.16 0.18–0.26 0.22 0.02–1.85 0.37 0.16–1.30 0.47 0.03–2.64 0.54 0.05–1.30 0.55 0.03–2.02 0.57 0.05–2.45 0.74 0.16–1.64 0.75 0.04–1.73 0.81 0.22–2.60 0.96 0.05–2.13 1.18 0.15–2.80 1.45 1.25–2.60 1.81 1.95–6.80 3.92

Pepsi Cola Coca-Cola Royal Crown Cola Dr. Pepper/Seven-Up

104 79 2 47

Carbonated Soft Drinks 0.02–1.22 0.60 Heilman 0.02–1.10 0.70 Heilman 0.95–0.99 0.97 Heilman 0.70–1.28 1.02 Heilman

Rosé Red White

Lemonade Fruit drinks Tea

Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Stannard et al., 1991

et et et et

al., al., al., al.,

1999 1999 1999 1999

2 9 8

California Wines 0.90–1.28 0.96 0.23–2.80 1.05 0.41–1.50 1.09

Burgstahler and Robinson, 1997 Burgstahler and Robinson, 1997 Burgstahler and Robinson, 1997

17 8 5

Other Beverages 0.03–0.84 0.25 0.15–1.13 0.54 0.95–2.33 1.41

Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996

content of wines from five brands of California grapes ranged from 0.83 to 5.20 ppm (Table 5.4) (Burgstahler and Robinson, 1997). The elevated fluoride levels may be due to the use of cryolite as a pesticide in the vineyards. Where tea drinking is a daily occurrence, tea can contribute to the total fluoride intake. The tea tree can selectively absorb fluoride from soil and accumulate it in the leaves, and the concentration of fluoride is related to the age of the tree (Gupta, 1991; Xu, 1987). In Tibet and some parts of western China, brick tea is considered a necessity. The fluoride concentration of brick tea, which is made from old stems and leaves of the tea tree, is 200 to 300 times higher in fluoride than ordinary green

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or black tea, which is made from the tender leaves and buds (Cao et al., 1996). During the 1980s, brick tea–type fluorosis was found in groups of people living in remote west and north border districts of China (Cao et al., 2003). In Tibet and in the Sichuan Province of China, Tibetans with a long history of brick tea consumption were studied for total fluoride intake, dental fluorosis, and skeletal fluorosis (Cao et al., 1996). The fluoride intake of Tibetan children and adults was 5.49 and 10.43 mg/person/day, respectively. Of this intake, over 94% was from brick tea and zanba (roasted highland barley flour processed with brick tea water). Among the Tibetans older than 16 years of age, nearly one third had skeletal fluorosis. Among those older than 50 years of age, more than 50% had skeletal fluorosis. An epidemiological study in Tibet in 2001 showed that natural fluoride in water was very low, but foods processed with brick tea water (zanba and buttered tea) had fluoride contents of 4.52 and 3.21 mg/kg, respectively (Cao et al., 2003). The adult daily fluoride intake reached 12 mg, of which 99% originated from the brick tea–containing foods (Cao et al., 2003). Osteosclerosis-type skeletal fluorosis (overall increased bone matrix density) affected 74% of the persons studied, and ossification and tendon attachment calcification affected 63% (Cao et al., 2003). More than 90% of the toothpaste sold in the U.S. contains fluoride at concentrations of 1000 to 1500 ppm (Beltran and Szpunar, 1988; Whitford, 1987). Based on the amount of toothpaste used for each brushing, the number of brushings, and the amount swallowed, children can ingest more than 2 mg fluoride/day (Barnhart et al., 1974; Baxter, 1980; Brunn and Thylstrup, 1988; Dowell, 1981; Hargreaves et al., 1972). Fluoride in the air originates both from natural sources and from human activities (WHO, 1984). The natural sources of fluoride include dusts from soil and droplets of seawater dispersed by wind. The urban sources of fluoride in the air are generated by industries (Smith and Hodge, 1979). Intake of fluoride by air is considered negligible in the U.S. However, in some rural areas of China, the most important energy source is coal with high-fluoride content. Coal-burning stoves are used with or without chimneys for heating, cooking, and drying food for storage, and high concentrations of fluoride have been detected in indoor air (Ji, 1993). Fluoride in indoor air is directly inhaled by the residents, and it is easily absorbed in stored food. The combination of inhalation and ingestion can provide high fluoride intake. A correlation between high concentrations of airborne fluoride and a high prevalence of fluorosis has been observed in some rural areas of China (Ando et al., 1998).

FLUORIDE COMPOUNDS USED TO FLUORIDATE WATER In the United States, the regulation of fluoride in drinking water is the responsibility of the EPA. Under the Safe Drinking Water Act of 1974 (Public Law 93-523), the EPA sets primary and secondary maximum contaminant levels for natural levels of fluoride in drinking water. The primary maximum contaminant level of 4 mg/l is the level that drinking water is not allowed to exceed and the secondary maximum

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TABLE 5.5 Water Fluoridation Agents in the U.S. Agent

Chemical Formula

Community Size

Population Served (in millions)

No. of Utilities

Sodium fluoride Sodium fluorosilicate Fluorosilicic acid

NaF Na2SiF6 H2SiF6

1–10,000 10,000–25,000 25,000+

11.7 36.1 80.0

2491 1635 5876

Note: Of the 1992 population of 258.5 million, 144.2 million (55.8%) drank water treated with fluoridation agents. The figures in this table account for 88.6% of the population served by water fluoridation agents. The remaining 11.4% (16.5 million) are served by systems using unspecified chemical agents. Source: 1992 Census by the Centers for Disease Control (CDC, 1993).

contaminant level of 2 mg/l is the level that the EPA recommends that drinking water not exceed. At the time of the Fluoridation Census in1992 (CDC, 1993), three major fluoride chemicals were used for water fluoridation: sodium fluoride, sodium fluorosilicate (also known as sodium silicofluoride), and fluorosilicic acid (also known as hydrofluosilicic acid). Fluoridation agents are summarized in Table 5.5. Sodium fluoride is a by-product of the aluminum industry and the silicates are by-products of the phosphate fertilizer industry. Sodium fluoride, a white odorless salt with a solubility that remains constant at all water temperatures, is the additive of choice for small communities, i.e., with populations <10,000. Sodium fluorosilicate, a white odorless salt with a solubility that varies with the water temperature, is the additive used in mid-sized communities, i.e., with populations between 10,000 and 25,000. Fluorosilicic acid, a straw-colored, transparent, corrosive liquid used in concentrated aqueous solutions, is the additive used in larger community water systems, i.e., with populations >25,000. The methods and equipment used in fluoridation are described by Reeves (1996). At the time of the 1992 census, 62.1% of the U.S. population was drinking fluoridated water, and of this water 6.3% was naturally fluoridated (CDC, 1993). Thus, 55.8 million persons were drinking water treated with fluoridating agents. In the U.K., fluorosilicic acid and sodium fluorosilicate are the chemicals most commonly used. In Central and South America, sodium fluoride, sodium fluorosilicate, fluorosilicic acid, and calcium fluoride are used. Calcium fluoride can be used in tropical climates because the temperature of the drinking water is warm enough to dissolve the chemical. Fluoride is the active part of treated water. Sodium fluoride in water dissociates readily into free fluoride and sodium ions. It has been assumed that silicofluoride complexes in water would behave similarly to produce free fluoride ions and other fluoride compounds such as aluminum fluoride, and that the treated water, when consumed, would have no silicofluoride residues. There are, however, still unresolved

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problems concerning the fate of fluorosilicates added to drinking water (Urbansky, 2002). There are also possible problems in that the silicofluorides obtained from phosphate industry scrubbers could add a variety of impurities such as arsenic and lead, albeit at low levels.

ACUTE TOXICITY High concentrations of fluoride are toxic. Acute oral exposure to sodium fluoride can result in significant dysfunction, such as nausea, vomiting, abdominal pains, diarrhea, and death (Table 5.6). A fatal ingestion of sodium fluoride was reported as early as 1899 (Sharkey and Simpson, 1933). Hodge and Smith (1965) estimated the lethal dose for a 70-kg man was 5 to 10 g sodium fluoride, or 32 to 64 mg fluoride/kg bodyweight. One 3-year-old boy who swallowed 200 mg sodium fluoride for a dose of 16 mg/kg bodyweight died 7 hours after ingestion (Eichler et al., 1982). One 27-month-old child died 5 days after ingesting about 100 mg sodium fluoride (Whitford, 1990). When given orally, the lethal dose of fluoride in animals was 20 to 100 mg/kg bodyweight (Davis, 1961). The LD50 of sodium fluoride in Sprague-Dawley male rats was 101 mg/kg (Skare et al., 1986), and the LD50 values for female rats ranged from 52 to 31 mg/kg, depending on bodyweight (De Lopez et al., 1976). The LD50 value of sodium fluoride observed for mice was 44.3 mg/kg (Lim et al., 1978).

TABLE 5.6 Potential Adverse Effects of Excess Fluoride Organ or Organ System

Potential Adverse Effects Acute Dose

All systems

Teeth Bones Kidneys Lungs Hormones Neural system Male reproductive system Female reproductive system

Death Subacute Dose Dental fluorosis (mottled teeth) Skeletal fluorosis (joint pain, calcification of ligaments, osteoporosis, muscle wasting, neurological defects) Renal failure Congestion and histopathological changes Increased parathyroid hormone secretion, inhibition of thyroid hormones, decreased testosterone concentration Behavioral effects Testicular changes, decreased fertility Skeletal variations in offspring

Note: Potential adverse effects were described in one or more animal species.

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SUBCHRONIC TOXICITY Some of the potential adverse effects of subacute doses of fluoride are summarized in Table 5.6. Most fluoride is ingested orally, and 50 to 80% of the fluoride ingested may be absorbed from the gastrointestinal tract. The amount absorbed depends on the dietary concentrations of calcium and other substances with which fluoride may form insoluble and poorly absorbed compounds. The amount absorbed also depends on the individual features of absorption and excretion. For example, healthy, young, or middle-aged adults may retain approximately 50% of ingested fluoride and excrete the remaining quantity in the urine. Young children, however, may retain as much as 80% due to uptake by developing skeletons and teeth (Food and Nutrition Board, 1999). The uptake rate of fluoride is faster in the bones of children than in adults; hence fluoride is cleared faster from the bloodstream in children than in adults (PHS, 2001). Body fluid and tissue fluid concentrations are proportional to the long-term level of intake, and they are not homeostatically regulated (Food and Nutrition Board, 1999; Guy, 1979). Because the fluoride concentration is proportional to intake, it is the total amount of fluoride ingested on a daily basis, regardless of the source, that provides the difference between a beneficial, caries preventive effect and an undesirable dental, skeletal, or other effect. Once fluoride is absorbed into the body, it passes into the blood for distribution and partial excretion. In plasma, it may exist as a nonionic form or an ionic form (Guy, 1979). The ionic or free form is the most important form for toxicity. It has been shown to circulate unbound in plasma (Ekstrand et al., 1977; Taves, 1968), to complex with calcified tissues, to be distributed to the soft tissues, or to be excreted. Most of the ionic fluoride retained in the body enters the calcified tissues (bones and teeth), either by substitution for the hydroxyl or the bicarbonate ion in hydroxyapatite in bone or enamel to form fluoroapatite, or as an ionic exchange within the crystalline surface (McCann and Bullock, 1957). Approximately half of the fluoride absorbed each day is deposited in the calcified tissues, and the result is that more than 99% of the fluoride in the body is found in calcified tissue (Whitford,1983). Fluorosis refers to the toxic condition that results from exposure to excessive amounts of fluorine or fluorides. Because of the affinity of fluoride for calcified tissue, most of the toxic effects of fluoride are manifested primarily in teeth or bones, and the conditions are referred to as dental fluorosis or skeletal fluorosis. Dental fluorosis is usually considered an adverse cosmetic effect, and skeletal fluorosis is considered an adverse functional effect. Endemic or chronic skeletal fluorosis, seen in some parts of the world where high fluoride levels are found in drinking water, is characterized by bone, joint, and muscle pain, progressive ankylosis of various joints and crippling deformities (Gupta et al., 1993b). Such severe cases are rare in the U.S. (Bowen, 2002; NRC, 1993).

DENTAL EFFECTS Exposure to excessive levels of fluoride during the period of tooth development (birth to approximately 8 years of age) can lead to dental fluorosis. This condition is

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characterized by a mottling of tooth enamel that ranges from barely discernible flecks on enamel to obviously pitted and brown-stained enamel (NRC, 1993). The staining, characteristic of more severe forms of fluorosis, develops after tooth eruption, but is seen only when porous enamel has formed before eruption (Fejerskov et al., 1990). The tooth enamel maturation process consists of increased mineralization within the developing tooth and a concurrent loss of early-secreted matrix proteins. Excess fluoride available to the enamel during maturation disrupts mineralization and results in retention of excessive enamel proteins (NRC, 1993). Animal studies have indicated that the early-maturation stage is the period during which enamel is most sensitive to fluoride effects (Den Besten, 1986; Richards, 1990; Richards et al., 1986). In humans, severe fluorosis follows the breakdown of the enamel surface layers shortly after eruption and results in mineral uptake in the exposed hypomineralized subsurface lesions (Fejerskov et al., 1991; Thylstrup, 1983). Fluorosis may be severe in permanent teeth, but it is rarely reported in primary teeth except in areas of the world where high amounts of fluoride are ingested (Larsen et al., 1987; McInnes et al., 1982; Mann et al., 1990; Nair and Manji, 1982; Olsson, 1979; Thylstrup, 1978). The low degree of fluorosis in primary teeth was once believed to be due to the placental barrier preventing the passage of fluoride from maternal to fetal blood. However, additional evidence demonstrated that the placenta acts only as a partial barrier (Gedalia and Shapira, 1989). Fetal blood concentrations of fluoride are usually lower than maternal levels. Most fluoride in the outer enamel layer of teeth is deposited during the enamel maturation period before eruption. The maturation period lasts a short time in primary teeth (1 to 2 years), but permanent teeth take 4 to 5 years to mature. The combination of shorter maturation period for primary teeth and the lower blood fluoride concentrations during prenatal development probably is the reason for the low incidence of fluorosis in primary teeth (NRC, 1993). Fluoride intake by children aged 2 to 5 years is especially important because the front teeth are at the early-maturation stage, and during this period they are particularly susceptible to fluoride-induced changes. Dental fluorosis in permanent teeth appears to be a dose–response condition (Dean, 1942; Eklund et al., 1987; Fejerskov et al., 1990; Gedalia and Shapira, 1989; Larsen et al., 1987). Animal studies have shown that fluoride can disturb enamel maturation by several pathways, by spikes of fluoride caused by daily injections, by long-term administration of low doses of fluoride, or by a single high dose of fluoride. These studies have been summarized by the National Research Council (NRC, 1993). The role of fluoride as an anticaries agent was examined when an inverse relationship was noted in many areas of the country between the level of fluoride in the drinking water and the incidence of dental caries. A large amount of literature is available on this relationship (Dean, 1938; Newbrun, 1989; Whitford, 1983). Since the 1950s, the prevalence and severity of caries have been considerably reduced (NRC, 1993), and dentists can now concentrate some of their efforts on cosmetic dentistry such as teeth whitening procedures. Fluoride can act on dental enamel, dentin, and cementum while it is forming or after it erupts, through saliva or blood or by direct contact with fluoridated water or dental products. Fluoride replaces hydroxyl ions in the crystal lattice structure of enamel, forming fluoroapatite (Ten Cate, 1990; Whitford, 1983). This change in

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enamel is considered to enhance the resistance of the teeth to caries. The capacity of teeth to absorb fluoride into the enamel’s structure diminishes as the enamel matures (Weatherell et al., 1977). This quality makes it different from bone, in which there can be constant alteration. The concentration of fluoride in sound, mature tooth enamel averages 1700 ppm in people living in areas with drinking water concentrations of 0.1 ppm or less, and 2200 to 3200 ppm in areas with drinking water concentrations of approximately 1 ppm. At these fluoride levels, fluoride increases the enamel’s resistance to dissolution and decay (NRC, 1993). In areas with drinking water concentrations of 5 to 7 mg/l, enamel fluoride concentrations have been observed at 4800 ppm (Aasenden, 1974). People living in areas of high fluoride concentrations usually exhibit severe dental fluorosis, and their tooth enamel can become brittle enough to fracture. Teeth in this condition often require treatment to restore function (NRC, 1993).

SKELETAL EFFECTS Fluoride has a high affinity for bone, but the bond formed is a reversible one (Whitford, 1990). Fluoride in bone can be mobilized rapidly by interstitial ionic exchange or slowly as a result of the constant process of bone alteration. Bone alteration is more active in the young where bone is more hydrated and has a greater surface area than older bone. The greater surface area provides greater area for fluoride exchange. Fluoride deposition in bone has been found to be inversely proportional to age (Whitford, 1990). As fluoride deposition in bone decreases with age, fluoride levels increase in plasma (Parkins and Greenlimb, 1974). In bone, fluoride replaces the hydroxyl ion in hydroxyapatite and forms fluoroapatite, a compound with different physical and chemical properties. Continuous ingestion of high levels of fluoride can lead to skeletal fluorosis, a condition whose effects can range from increased bone density to crippling skeletal fluorosis, characterized by complete rigidity of the spine. Most cases have been reported from developing countries, particularly India, where drinking water sources in 15 states in India and 15 districts of Rajasthan contain over 1.5 ppm fluoride (Purohit et al., 1999). High levels of fluoride are necessary to cause fluorosis, but nutritional status and individual variation must also be considered contributors to this condition. Smith and Hodge (1979) described the preclinical and clinical stages of skeletal fluorosis. The preclinical stage, characterized by a slight increase in bone mass, is asymptomatic. Stage 1 of skeletal fluorosis is characterized by occasional stiffness or pain in the joints and some osteosclerosis of the pelvis and vertebral column. At Stage 2 and 3, the clinical signs are chronic joint pain, calcification of ligaments, osteosclerosis, possibly osteoporosis of long bones, muscle wasting, and neurological defects in severe cases. Crippling skeletal fluorosis might occur in persons who have ingested 10 to 20 mg fluoride/day for at least 10 years. From 1963 to 1993, only five cases of skeletal fluorosis were reported in the U.S. (NRC, 1993). As a compound with high affinity for bone, sodium fluoride has been investigated as a preventative or therapy for osteoporosis in postmenopausal women. The studies have been reviewed by NRC (1993) and the Food and Nutrition Board (1999), which found that fluoride therapy did not demonstrate a significant reduction in fractures.

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RENAL EFFECTS Most of the removal of fluoride that occurs from the body (approximately 50% of daily intake) is done by renal excretion (Whitford, 1990). The kidney cells are therefore a possible target of fluoride toxicity because they can be exposed to high concentrations of fluoride. Studies of renal effects have shown that structural renal changes occurred in rats exposed to fluoride and that there was a relationship between renal effects and pH of urine (Daston et al., 1985; Hodge and Smith, 1977; Greenberg, 1986; Manocha et al., 1975; Taylor et al., 1961; Whitford et al., 1976). In humans, the efficiency of the renal cells in removing fluoride from blood has been shown to be greater than for other halogens. Clearance values of 0.5 to 2.0 ml/min were observed for chloride and bromide (Whitford, 1990). Clearance values of 12.4 to 89.1 ml/min have been reported for fluoride (Jarnberg et al., 1983; Whitford, 1990). Two teen-age patients who suffered renal failure also suffered from fluorosis of teeth and bones (Juncos and Donadio, 1972). Renal failure and indications of chronic fluoride intoxication were reported in a person who ingested 2 to 4 l/day of mineral water containing 8.5 mg fluoride/l for 20 years (Lantz et al., 1987). Despite this observation, several epidemiological investigations have shown no human kidney disease from long-term exposure to fluoride at concentrations up to 8 mg/l (EPA, 1985). In another study of two communities, the renal status of persons who drank 8 mg/l and the status of persons who drank 0.4 mg/l was similar (Leone et al., 1954).

PULMONARY EFFECTS Excess fluoride in drinking water that causes dental mottling and skeletal fluorosis may also be responsible for lung damage. Rabbits fed 10 or 20 mg sodium fluoride/kg/day for 6 months showed gross lung changes (pale areas on the surface and dark brown congested areas in cross sections) and histopathological changes (alveolar hemorrhage, congestion, edema, etc.) (Purohit et al., 1999). The fluoride content of lung tissue homogenate was more than ten times greater in treated animals than in control animals, and the content was dose related. Lung damage was tested because patients of skeletal fluorosis in India may have been wrongly treated for tuberculosis.

HORMONAL EFFECTS In a study of the effect of high fluoride ingestion on serum parathyroid hormone, 200 children consumed water containing 2.4, 4.6, 5.6, or 13.5 mg fluoride/l (Gupta et al., 2001). High fluoride ingestion increased the level of parathyroid hormone secretion. The authors suggested that, due to the role of the parathyroid hormone in maintaining serum calcium levels, fluoride might play a role in toxic manifestations of fluorosis. Fluorides have also been implicated in the inhibition of thyroid hormones and in goiter formation (Jooste et al., 1999; Zhao et al., 1998). Decreased testosterone concentration was observed in patients with skeletal fluorosis, and in males without skeletal fluorosis who drank water with high fluoride concentration (Susheela and Jethanandani, 1996). The authors suggested that fluoride

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toxicity may cause adverse effects on the reproductive system of males living in endemic fluorosis areas. Testosterone levels in males in areas nonendemic for fluorosis were normal.

GENETIC EFFECTS The genetic toxicity of fluoride has been tested extensively in microbes, cultured mammalian cells, and animals. These test results have been reviewed by NRC (1993) and Zeiger et al. (1993), and they concluded that there is evidence that fluoride exposure can lead to chromosomal aberrations in in vitro test systems but that aberrations in in vivo systems are unresolved. The genetic toxicity of fluoride in human blood lymphocyte cells was determined after long-term exposure to various concentrations of fluoride in drinking water (Li et al., 1995). Subjects who ingested low-fluoride water had higher frequencies of sister chromatid exchanges than did subjects who ingested higher levels of fluoride. Skare et al. (1986) demonstrated that oral administration of up to 84 mg/kg of sodium fluoride to adult male rats did not induce DNA strand breaks in testicular cells when measured by alkaline elution. They also observed that although plasma fluoride levels were as high as 12 ppm, testicular fluoride levels were only 10 to 12% of the plasma levels and fluoride did not accumulate in the testis after five daily treatments. The authors concluded that sodium fluoride is unlikely to pose a hazard with respect to heritable genetic effects.

NEURAL EFFECTS When the effects of fluoride on the developing rat brain were tested, sex- and dosespecific behavioral changes were observed (Mullenix et al., 1995). Males were most sensitive to exposure at prenatal days 17 to 19, and females were most sensitive to exposure as weanlings and adults. The severity of the effect on behavior increased directly with plasma fluoride levels and fluoride concentrations in specific brain regions. The plasma fluoride levels were similar to those reported in humans exposed to high levels of fluoride. The molecular mechanism underlying brain dysfunction from chronic fluorosis was studied in rats that received either 30 or 100 ppm fluoride in their drinking water for 7 months (Long et al., 2002). At 100 ppm, but not at 30 ppm, the level of the nicotinic acetylcholine receptors (NAChR) α-4 subunit protein in the brains of the rats was significantly lowered. The expression of the α-7 subunit protein was significantly decreased by both 100 and 30 ppm. These decreases in the number of receptors may be an important factor in the mechanism of brain dysfunction in chronic fluorosis.

REPRODUCTIVE TOXICITY ASPECTS Correlation with Decreased Fertility in Humans Despite that fluoride has been added to municipal water supplies to diminish the occurrence of dental caries for more than half a century, there are limited data on

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the potential of fluoride to induce reproductive effects in humans and in animals. A statistically significant association was reported between decreasing total fertility and increasing fluoride levels in water (Freni, 1994). In the meta-analysis, birth data for more than 525,000 women (10 to 49 years old) living in areas with high fluoride drinking water levels (up to 3 ppm or higher) were compared with birth data for approximately 985,000 women living in adjacent areas with low fluoride drinking water levels. The results have not been duplicated by other investigators. Correlation with Down’s Syndrome in Humans The possible association between fluoride concentration in drinking water and the incidence of Down’s syndrome was proposed by Rapaport (1957, 1963). These studies have been used to support the claim that fluoridation of drinking water leads to increased incidence of Down’s syndrome. However, no support for this claim was found in other studies (Berry, 1958; Erickson, 1980; Erickson et al., 1976; Needleman et al., 1974). When the studies of correlation between fluoridation and Down’s syndrome were reviewed, Erickson (1980) found no correlation. A systematic review of the association of Down’s syndrome and water fluoride level was also done by Whiting et al. (2001). Based on a comprehensive literature search, they identified six studies worldwide for review. These were the same studies as the ones identified above. Of the six studies, four studies showed no association, and two were significantly associated. Based on their review, Whiting et al. (2001) concluded that the association between water fluoride level and Down’s syndrome was inconclusive. Correlation with Male Reproduction and Male Offspring Studies in Mice Table 5.7 provides a summary of some of the available studies in male mice. Kour and Singh (1980) exposed mice to 0, 10, 500, or 1000 ppm sodium fluoride in their drinking water for 30, 60, or 90 days. Microscopic effects were not observed in the testis of the animals receiving sodium fluoride at a concentration of 10 ppm, but necrotic seminiferous tubules were observed in animals from the 500 and 1000 ppm treatment groups. Chinoy and Sequeira (1989a) examined the effect of sodium fluoride exposure on the histology and histocytochemistry of the reproductive organs of male mice. Three groups received sodium fluoride, by gavage, at a dose of 10 mg/kg bodyweight/mouse/day and one group received sodium fluoride at a dose of 20 mg/kg/mouse for 30 days. The treatment of 10 mg/kg/mouse/day was withdrawn from two groups for 30 and 60 days, respectively, and the animals were used for recovery studies. Testicular effects characterized by a disorganization and denudation of cells of the germinal cells of the seminiferous epithelium were observed with some tubules showing a lack of sperm in the lumen in both treatment groups. Effects were also observed in the cauda epididymis, caput epididymis, and vas deferens of both treatment groups. The Leydig cells, the seminal vesicles, and the prostate were not affected by the treatment. Removal of treatment resulted in a complete recovery of these organs.

Kour and Singh

Chinoy and Sequeira

Chinoy and Sequeira

Shashi

1989a

1989b

1990

Authors

1980

Year

Duration

Subcutaneous injection

Feed

Gavage

0, 10, 500, 1000 ppm

Dose

100 days

5, 10, 20, 50 mg/kg/day

Two groups treated 30 days and 10, 20 euthanized; two groups treated 30 mg/kg/day days, then treatment withdrawn for 30 or 60 days

Two groups treated 30 days and 10, 20 euthanized; two groups treated 30 mg/kg/day days, then treatment withdrawn for 30 days

Drinking water 30, 60, 90 days

Route of Exposure

Rabbits

Albino mice

Swiss mice

Albino mice

Species

(continued)

Spermatogenic arrest and seminiferous necrosis; abnormal spermatocyte maturation and differentiation

Decreased: bodyweight, testicular succinic dehydrogenase, epididymides sialic acid, ATPase Increased: prostate and seminal vesicle weight, seminal vesicle fructose levels, prostate acid phosphatase, and total protein After treatment withdrawal, levels returned to normal

Testicular effects: denudation of germinal epithelium; Leydig cell, no effect Epididymal effects: caput epididymis — epithelial cell nuclear pyknosis and absence of luminal sperm Cauda epididymis: nuclear pyknosis, denudation of vas deferens, nuclear pyknosis, clumped stereocilia, and cellular debris, absence of sperm and increased lamina propria Treatment withdrawal: recovery of histoarchitecture

Lack of germ cell maturation and differentiation at 500 and 1000 ppm; necrotic seminiferous tubules after 90 days

Results

TABLE 5.7 Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

Fluoride — Toxic and Pathologic Aspects 121

Authors

Chinoy et al.

Chinoy et al.

Susheela and Kumar

Chinoy and Sequeira

Shashi and Kaur

Year

1991b

1991a

1991

1992

1992

Treated 30 days followed by withdrawal for 30 days; animals given ascorbic acid, calcium, or calcium and acorbic acid

Duration

Subcutaneous injection

Orally

Orally (?)

3.5 months

Two groups treated 30 days, then treatment withdrawn and 30–60 days; mating during withdrawal phase

18, 29 months

Direct injection Single injection in vas deferens

Feed

Route of Exposure

Swiss strain mice

Rabbits

Holtzman strain rats

Rabbits

Species

0, 5, 10, 20, 50 Rabbits mg/kg/day

10, 20 mg/kg/day

10 mg/kg/day

50 μg/50 μl

20, 40 mg/kg/day

Dose

Depletion of testicular structural, nuclear, and total proteins in all test groups; reduction of testicular DNA

Decreased sperm motility, count, and fertility Withdrawal of treatment: significant recovery in sperm count, motility, and fertility

29 months: seminiferous tubules: spermatogenic arrest, spermotagenic cells disrupted, tubules devoid of spermatozoa Epididymal effects at 18 and 29 months

Spermatogenic arrest, absence of spermatozoa in seminiferous tubule lumen, decreased cauda epididymal sperm count Cauda epididymis and vas deferens: sperm deflagellated or with tail abnormalities

Reduced fertility due to decreased sperm motility and sperm counts; abnormal sperm morphology; recovery pronounced in ascorbic acid treatment group; recovery most pronounced in animals supplemented with calcium and ascorbic acid

Results

TABLE 5.7 (CONTINUED) Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

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Kumar and Susheela

Kumar and Susheela

Sprando et al.

Sprando et al.

1994

1995

1996

1997, 1998

Single injection

20, 23 months

18 months

Drinking water Exposure: in utero through 14 weeks postlactation

Intratesticular injection

Orally

Orally

50 days treatment; 50 days treatment + withdrawal for 70 days; 50 days treatment + withdrawal for 70 days + supplements: ascorbic acid, calcium, or ascorbic acid + calcium

Narayana and Chinoy

1994

Gavage

Krasowska and Drinking water 6, 16 weeks Wlostowski

1992

0, 25, 100, 175, 150 ppm

0, 25. 100, 175, 250 ppm

10 mg/kg/day

10 mg/kg/day

10 mg/kg/day

SpragueDawley rats

SpragueDawley rats

Rabbits

Rabbits

Albino rats

100, 200 ppm Wistar rats

No spermatogenic or endocrine effects

No spermatogenic effects

(continued)

Loss of stereocilia, decreased height of pseudostratified columnar epithelium and increased diameter of caput and cauda epididymis Fragmented sperm in cauda epididymis

Acrosomal, nuclear, and flagellar abnormalities

Sperm acrosomal hyaluronidase and acrosin reduced, sperm acrosomal damage and deflagellation Cauda epididymal sperm count decreased, fluoride withdrawal, incomplete recovery Supplementation with ascorbic acid, calcium, or a combination of both: significant recovery from fluorideinduced effects

Testicular fluoride levels not increased, testicular zinc levels decreased, testicular effects resembled those of zinc deficiency

Fluoride — Toxic and Pathologic Aspects 123

29 days

NaF = sodium fluoride; HSD = hydroxysteroid dehydrogenase.

Gavage

Ghosh et al.

Duration

2002

Route of Exposure

Elbetieha et al. Drinking water 4, 10 weeks, then mated to untreated females

Authors

2000

Year

20 mg/kg/day

100, 200, 300 ppm

Dose

Wistar rats

Swiss mice

Species

Decreased testicular, prostate, and seminal vesicle weights with decreased 3- and 17β-HSD activities Decreased epididymal sperm count, dilated seminiferous tubules

Fertility affected by 10 weeks but not by 4 weeks of treatment Reduction in number of implantation sites and viable fetuses in females mated to NaF-treated males (200 ppm NaF) Increased seminal vesicle and preputial gland weights in mice exposed to 200 and 300 ppm NaF for 4 weeks but not 10 weeks

Results

TABLE 5.7 (CONTINUED) Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

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125

To determine if sodium fluoride induced biochemical changes in selected reproductive organs of albino male mice, Chinoy and Sequeira (1989b) fed sodium fluoride to groups of mice at doses of 10 or 20 mg/kg bodyweight/day for 30 days. Treatment was then withdrawn from selected groups for 1 and 2 months. At the end of the treatment period, testicular, epididymal (caput and cauda), vas deferens, seminal vesicle, and prostate weights were obtained. Significant changes in testicular cholesterol and serum testosterone concentrations were not observed. Testicular succinic dehydrogenase, epididymal sialic acid, and ATPase levels were decreased. Vas deferens glycogen, seminal vesicle fructose, prostate gland acid phosphatase, and total protein were increased. Chinoy and Sequeira (1992) fed male albino mice sodium fluoride at doses of 10 or 20 mg/kg bodyweight for 30 days. The treatment of 10 mg/kg/mouse/day was withdrawn from two groups for 1 and 2 months, and the animals were used for recovery studies. Normally cycling females were mated with treated males on the 31st day after treatment and, in the groups from which sodium fluoride was withdrawn, at the end of 1 and 2 months, respectively. Sperm motility and cauda epididymal sperm counts were decreased significantly in both treatment groups after 30 days of treatment and subsequently recovered after withdrawal from sodium fluoride treatment for 2 months. Additionally, fertility was almost absent in the treated animals but increased significantly after withdrawal of sodium fluoride treatment. Effects were observed on sperm head morphology, including effects on the acrosomal, post-acrosomal, and midpiece regions. Elbetieha et al. (2000) exposed 60-day-old male Swiss mice to sodium fluoride at concentrations of 100, 200, or 300 ppm in their drinking water for 4 or 10 weeks. Fertility was assessed by breeding the sodium fluoride–treated male mice to untreated females after the exposure period. Fertility was reduced in a dose-related manner in the 100, 200, and 300 ppm dose groups after 10 weeks of exposure but not after 4 weeks of exposure. The number of pregnancies resulting from mating treated males to nontreated females was 50, 45, and 36% for the 100, 200, and 300 ppm dose groups, respectively. Seminal vesicle and preputial weights were increased in mice exposed to 200 or 300 ppm sodium fluoride for 4 weeks, but not for 10 weeks. The authors concluded that long-term exposure to sodium fluoride adversely affected fertility in male mice. Studies in Rats Table 5.7 provides a summary of some of the available studies in male rats. Chinoy et al. (1991a) injected a single microdose (50 μg/50 μl) of sodium fluoride into the vasa differentia of the adult male albino rat resulting in an arrest of spermatogenesis and absence of spermatozoa in the lumina of the seminiferous tubules and a reduction in cauda epididymal sperm numbers, which consequently led to an impairment of fertility in experimental animals. Krasowska and Wlostowski (1992) exposed male Wistar rats to fluoride at concentrations of 100 or 200 ppm fluoride in their drinking water for 6 or 16 weeks. The results of this study indicated that although testicular fluoride levels increased, the increase was not associated with dose or time of exposure. The data also suggested that fluoride exposure to 100 or 200 ppm decreased significantly the

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concentrations of zinc in the testis, particularly in the 16-week treatment groups. Testicular iron and copper concentrations were not adversely affected by fluoride treatment. After 16 weeks of exposure, approximately 50% of the rats treated with 100 and 200 ppm of fluoride exhibited testicular histopathological effects in the germinal epithelium, reminiscent of zinc-deficient rats. The authors concluded the observed testicular effects were induced by a deprivation of testicular zinc resulting from high fluoride intake. Narayana and Chinoy (1994) examined the effects of sodium fluoride ingestion on sperm structure and metabolism. They also examined the effects of sodium fluoride withdrawal and the effects of administering fluoride with ascorbic acid or calcium alone or in combination on sperm structure and metabolism. The results of this study indicated that sperm hyaluronidase and acrosin were reduced after 50 days of treatment. Staining with alcoholic silver nitrate revealed acrosomal damage and deflagellation. These findings could account for the observed reduction in sperm motility. A reduction in cauda epididymal sperm count was attributed to an arrest of spermatogenesis. A recovery of the observed effects was not complete after withdrawal of sodium fluoride for 70 days. The administration of ascorbic acid and calcium alone or in combination appeared to reverse the fluoride-mediated effects. The authors suggested that the effect of sodium fluoride on sperm structure and metabolism in rats was reversible. Sprando et al. (1996) utilized intratesticular injections to characterize the effect of the short-term sodium fluoride exposure on spermatogenesis. One testis from each experimental animal was injected with sodium fluoride (50, 175, or 250 ppm) in vehicle (0.9% physiological saline). One testis from each control animal was injected with vehicle. Testicular effects were not observed at any of the doses utilized in this study and the authors concluded that spermatogenesis was not adversely affected by direct short-term exposure to sodium fluoride even at levels 200 times greater than those under normal conditions. Sprando et al. (1997) examined the potential of sodium fluoride to affect spermatogenesis and endocrine function in parental (P) and first (F1) generation male rats. In this study, male and female experimental rats received sodium fluoride in their drinking water at one of four concentrations (25, 100, 175, or 250 ppm). P generation male and female rats were exposed to sodium fluoride in their drinking water for 10 weeks, then mated within the same treatment groups. F1 generation male rats remained within the same treatment groups as their parents and were exposed to sodium fluoride in their drinking water for 14 weeks after weaning, at which time reproductive tissues were collected. Dose-related effects were not observed within the P and F1 treatment groups for testis weights, prostate/seminal vesicle weights, nonreproductive organ weight, testicular spermatid counts, testicular spermatid counts, luteinizing hormone (LH), follicular-stimulating hormone (FSH), or serum testosterone concentrations. Histological changes were not observed in testicular tissues from either the P or F1 generation males. The authors concluded that prolonged exposure to sodium fluoride in drinking water in the rat at the doses utilized in this study did not adversely affect spermatogenesis or endocrine function in the P and F1 generation male rats.

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In a further study of the rats treated throughout several generations (described above; Sprando et al., 1997), Sprando et al. (1998) obtained quantitative morphometric information on the testis of sodium fluoride–treated F1 generation male rats. No statistically significant changes were observed in the absolute volumes of the blood vessels, boundary layer, Leydig cells, lymphatic space, macrophages, tubular lumen, seminiferous epithelium, seminiferous tubules, interstitial space, and testicular capsule. These findings suggested that the volumetric composition of the various testicular components was not altered by exposure to sodium fluoride. When the number of Sertoli cell nucleoli was enumerated to detect tubular shrinkage resulting from germ cell loss or germ cell degeneration, the number of Sertoli cell nucleoli was not statistically different between control and treated rats. This result suggested that germ cell number in the seminiferous tubules was not significantly affected by sodium fluoride exposure. The mean diameters of the seminiferous tubules from the treatment groups were not significantly different from the control groups; this suggested that sodium fluoride treatment did not adversely affect spermatogenic activity in the sodium fluoride–treated animals. The length of the seminiferous tubule, seminiferous tubule length per unit volume, and the surface area of the seminiferous tubules from the treatment groups were not significantly different from the control groups, again suggesting that sodium fluoride exposure did not affect spermatogenesis in the treated animals. Ghosh et al. (2002) examined the effect of sodium fluoride on testicular steroidogenic and gametogenic activities in relation to testicular oxidative stress. Adult male albino Wistar rats were given sodium fluoride by oral gavage at a concentration of 20 mg/kg/day for 29 days. Decreases in testis, prostate, and seminal vesicle wet weight were observed without a concomitant decrease in bodyweight. Testicular Δ-5,3-β-hydroxysteroid dehydrogenase (HSD), 17-β-HSD, testosterone, and epididymal sperm counts were decreased in the fluoride treated group in comparison to the control. Dilated seminiferous tubules were also observed. Fluoride treatment was associated with oxidative stress as evidenced by an increase in conjugated dienes in the testis, epididymis, and cauda epididymal sperm. The authors concluded that fluoride, at doses encountered in contaminated areas, may exert toxic effects on the male reproductive system and these effects are associated with oxidative stress. Studies in Rabbits Table 5.7 provides a summary of some of the available studies in male rabbits. Shashi (1990) evaluated the relationship between infertility and the histological structure of the testes in albino rabbits following the subcutaneous administration of sodium fluoride at 5, 10, 20, or 50 mg/kg/day for 100 days. Effects were observed on spermatocyte maturation and differentiation in the experimental animals. Spermatogenesis ceased and necrotic seminiferous tubules were observed in the higher dosage groups. Chinoy et al. (1991b) assessed the effects of fluoride on metabolism and function of cauda epididymal sperm in rabbits. Male rabbits were fed either 20 or 40 mg/kg bodyweight sodium fluoride for 30 days, then cauda epididymal sperm was collected and assessed. Alteration in the specific activity of ATPase, acid phosphatase, succinate dehydrogenase, and protein was observed. Sodium and potassium levels were

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reduced. The reduced fertility observed in the treated animals was attributed to the reduction in sperm motility and counts and changes in morphology. After 30 days of treatment, selected groups of animals were withdrawn from fluoride treatment for 30 days. During the withdrawal phase, groups of animals were given ascorbic acid, calcium, or ascorbic acid and calcium. Recovery was more pronounced in the ascorbic acid–treated group than the calcium-treated group; however, if both ascorbic acid and calcium were administered together they interacted synergistically and recovery was most pronounced. Susheela and Kumar (1991) administered sodium fluoride orally at a concentration of 10 mg sodium fluoride/kg bodyweight to male rabbits for 18 or 29 months at which time the structure of the testis, epididymis, and vas deferens was studied using light and scanning electron microscopy. A disruption of spermatogenesis characterized by degenerating germ cells and seminiferous tubules devoid of germ cells and/or spermatozoa was observed in animals treated for 29 months. Histological effects were also observed in the ductuli efferentes and vas deferens in animals treated for 18 or 29 months. These effects were characterized by a loss of cilia on the epithelial cells lining the lumen of the ductuli efferentes and of stereocilia on the epithelial cells lining the lumen of the vas deferens. A spermatogenic arrest was observed only in animals treated for 29 months. Shashi and Kaur (1992) investigated the effect of fluoride toxicosis on testicular protein and DNA biosynthesis by exposing male albino rabbits to 0, 5, 10, 20, or 50 mg sodium fluoride via subcutaneous injections for 3.5 months in order to induce experimental fluorosis. The results of this study suggested that both testicular protein and DNA synthesis decreased as a result of fluorosis, further indicating that fluoride may interfere with RNA metabolism and consequently with the synthesis of specific testicular enzymes. Kumar and Susheela (1994) investigated the ability of sodium fluoride to disrupt spermiogenesis and induce defects in rabbit spermatids and epididymal spermatozoa. Male rabbits were treated with 10 mg sodium fluoride/kg bodyweight daily for 18 months. An ultrastructural examination of testicular spermatids and caput epididymal sperm revealed a wide variety of structural defects in the flagellum, the acrosome, and the nucleus of the spermatids and epididymal spermatozoa of fluoride-treated rabbits. These abnormalities included an absence of outer microtubules, complete absence of axonemes, structural and numeric aberrations of outer dense fibers, breakdown of the fibrous sheath, and structural defects in the mitochondria of the middle piece of the flagellum. Detachment and peeling of the acrosome from the flat surfaces of the nucleus were also observed. Kumar and Susheela (1995) utilized both light and scanning electron microscopy to observe the effect of chronic fluoride toxicity on the structure of the ductus epididymis, testis, and spermatozoa in rabbits. Rabbits were treated with 10 mg sodium fluoride/kg bodyweight/day for 20 or 23 months. Serum fluoride levels were significantly elevated in the sera of rabbits treated for both 20 and 23 months. Multiple effects were observed in the cauda and caput epididymis and testis in rabbits treated with sodium fluoride for 23 months. Epididymal effects included a loss of stereocilia, a significant decrease in the height of the pseudostratified columnar

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epithelium, and a significant increase in the diameter of both the caput and cauda ductus epididymis. Additionally, cauda and caput epididymal weights and the number of secretory granules were also reduced in comparison to the control animals. Testicular effects included decreased epithelial cell height and tubular diameter of the testis. The authors noted fragmentation of spermatozoa in the caput and cauda ductus epididymis of animals treated for 23 months but not in the testis and caput and cauda epididymis of the animals treated for 20 months. The authors concluded that sperm maturation might be adversely affected as a result of the structural changes observed in the caput and cauda ductus epididymis. Correlation with Female Reproduction Effect on Estrous Cycle Del Castillo (1928) reported suppression of the estrous cycle when 0.05 mg sodium fluoride per day was fed to two female rats. Phillips et al. (1933) reported that the estrous cycles of females treated with 430 ppm sodium fluoride in the diet were similar to those of the control animals (cycle of 4.5 to 6.5 days). In a second study in the same report, Phillips et al. (1933) reported that high levels of fluoride (greater than 25 mg/kg/day) suppressed estrus, but that the suppression was attributable to inanition, which occurred at high levels of fluoride. Maternal–Fetal Transfer of Fluoride Fluoride crosses the placenta and is found in fetal and placental tissue. Placental transfer has been documented in mice (Ericsson and Hammarstrom, 1964), rats (Theuer et al., 1971), rabbits (Nedeljkovic and Matovic, 1991), and in pregnant women (Caldera et al., 1988; Feltman and Kosel, 1955; Forestier et al., 1990; Gedalia et al., 1961; Malhotra et al., 1993; Shi and Zhang, 1995). In rabbits, fluoride content of bones and teeth of the newborn was significantly increased and dose dependent (Nedeljkovic and Matovic, 1991). When transplacental passage of fluoride was studied in 25 randomly selected neonates in India, fluoride concentration in cord blood was 60% of that in mother’s blood (Gupta et al., 1993a). Several analyses of human milk have shown that the daily fluoride intake of infants, from those drinking colostrum to 3-month-old infants drinking mature milk, ranged from 5 to 10 μg (Esala et al., 1982; Spak et al., 1983). This was the same regardless of the fluoride content of the water consumed. At 0.2 ppm fluoride, the daily fluoride intake was closer to 5 μg (Esala et al., 1982; Spak et al., 1983); at 1.0 or 1.2 ppm, the daily fluoride consumption was 7.3 to 8.5 μg (Esala et al., 1982). A survey of fluoride in human milk showed a strong correlation between fluoride level in milk and presence of fluoride in drinking water (Dabeka et al., 1986). In a study done in remote areas of Thailand, the fluoride content of human milk was 0.017 ppm, and there was no correlation between breast milk fluoride content and fluoride concentration in drinking water (Chuckpaiwong et al., 2000). Human breast milk tested from women living in Mangalore City, India, showed that a minimal amount of fluoride was in breast milk while infant formulae had higher fluoride levels (Rahul et al., 2003).

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Studies in Mice Table 5.8 provides a summary of some of the available studies in female rats and mice. Messer et al. (1973) gave female mice drinking water containing 0, 50, 100, or 200 ppm sodium fluoride and fed them a low-fluoride diet (0.1 to 0.3 ppm). After the first litter, the F1 animals were remated for up to four litters. At 50 ppm, the animals gained weight at the same rates in the F1 and F2 generations and bodyweight of pups was similar to controls. However, there was a progressive decrease in the number of litters produced by the control group and less than 50% of the animals in each generation produced four litters. At 100 ppm, the number of litters was reduced and six of nine litters were stillborn or eaten at birth. Third and fourth litter females were mated to produce a second generation. At 200 ppm fluoride, all had died by 20 weeks of age. Because the weight gain and health signs appeared normal in the control and 50 ppm groups, the investigators determined that the diet was nutritionally adequate except for fluoride, and thus impaired reproduction in the control group was due to fluoride deficiency. When the study by Messer et al. (1973) was repeated by Tao and Suttie (1976), they also noted impaired reproduction, but their results suggested that the diet was marginally deficient in iron and that fluoride improved iron utilization. Studies in Rats Schulz and Lamb (1925) fed high levels of sodium fluoride (equivalent to 500, 1000, 1500, or 2500 ppm) to rats for 9 months before mating. The two females given 500 ppm sodium fluoride each successfully reared a third generation of offspring. Four litters were reared (of six litters) from the animals given 1000 ppm, but the offspring grew at a slower rate than the control animals, and there were fewer third-generation offspring reared than in the control animals. The rats given 1500 ppm sodium fluoride produced four litters, of which only two litters lived, and the offspring grew slowly. In the group of seven rats given 2500 ppm, all died after 8 to 14 weeks. In the same report, Schulz and Lamb (1925) reported that they tested a second series of rats with 10 to 2500 ppm sodium fluoride, and that the animals exhibited no toxic effects until they were given at least 1000 ppm. They also reported that an unfavorable effect on reproduction began at 250 ppm. Unfortunately, the authors provided no information on the treatment or number of animals treated in the second series of studies. Lamb et al. (1933) and Phillips et al. (1933) fed 0 (basal diet) or 0.043% (430 ppm) sodium fluoride to rats, and the animals’ reproduction, growth, and estrous cycles were monitored for five generations. Based on lighter offspring in the treated animals at the time of weaning, Lamb et al. (1933) suggested that either the vigor of the young or the quality or quantity of the milk was affected by fluoride in the diet. Lamb et al. (1933) observed that in the third generation, the treated females produced only one litter each and then failed to produce subsequent litters; but they stated that they could not attribute this interruption to sodium fluoride, because it could have resulted from a chronic lung infection in these animals. Ream et al. (1983) tested the effects of fluoride on bone morphology. They dosed female rats with 0 (distilled water) or 150 ppm sodium fluoride in drinking water for 10 weeks prior to mating and during three successive pregnancy and lactation

Messer et al.

Tao and Suttie

Ream et al.

1973

1976

1983

NaF = sodium fluoride.

2001a Collins et al.

Lamb et al., Phillips et al.

1933

Route of Exposure

Drinking water

Throughout three generations

Three litters

Drinking water (with Same as Messer et al. addition of iron and copper to feed) Drinking water

0, 500, 1000, 1500, 2500 ppm NaF

Dose

Mice

Rats

Rats

Species

Rats

0, 25, 100, 175, Rats 250 ppm

150 ppm NaF

Same as Messer Mice et al.

0, 50, 100, 200 ppm NaF

Throughout five generations 0, 430 ppm

9 months prior to mating, and throughout the study

Duration

Drinking water (with Two generations with four low-fluoride diet) litters in first generation

Feed

Schulz and Lamb Feed

Authors

1925

Year

Results

25–250 ppm: no reproductive or developmental effects

No effects on reproduction Femurs from third litter pups were tested: no visible structural alterations

50–200 ppm: no effects

200 ppm: all dead by 20 weeks of age 100 ppm: growth retardation, reduced number of litters, 6/9 litters stillborn 50 ppm: no effect

Offspring lighter at the time of weaning

2500 ppm: fatal to 7 females after 8–14 weeks 1000 and 1500 ppm: females grew more slowly and produced smaller third-generation litters 500 ppm: no effects on reproduction

TABLE 5.8 Summary of Fluoride Effects on Female Reproductive Parameters in Rats and Mice

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periods. The investigators reported no effects of the compound on fertility or reproduction. The femurs of the 3-week-old third-pregnancy offspring were measured and no pathological changes in the femurs of the offspring were reported. In the early 1990s, the existing reproductive studies (many of which were summarized in the preceding text) were reviewed in several reports and were considered to be inadequate to determine potential reproductive or developmental hazards. These reviews included the report of the National Toxicology Program (NTP, 1990), the review of fluoride benefits and risks (PHS, 1990), and the report by the National Research Council (NRC, 1993). The inadequacies of the studies were attributed to insufficient or sometimes unknown numbers of animals per group and inadequate descriptions of the experimental procedures. None of the studies had been done according to currently accepted international guidelines. In response to the inadequacies demonstrated in the available studies, a multigeneration study was done at the U.S. Food and Drug Administration. Collins et al. (2001a) measured the effects of sodium fluoride ingestion at 0, 25, 100, 175, or 250 ppm in drinking water throughout three generations of rats. Low-fluoride diet was given to the treated animals throughout the study to minimize interference with the fluoride in water. Mating, fertility, survival, and offspring development were not affected.

DEVELOPMENTAL TOXICITY ASPECTS Human Studies Analysis of birth certificates for the period 1973–1975 showed that rates of congenital malformations (except Down’s syndrome) were similar in people ingesting fluoridated or nonfluoridated water (Erickson, 1980). An increased incidence of spina bifida was reported in sections of India where the fluoride content of drinking water is high (4.5 to 8.5 ppm) and this increase was associated with skeletal or dental fluorosis (Gupta et al., 1995). The investigators, however, did not examine the role of nutrients such as folic acid. Animal Studies Table 5.9 provides a summary of some of the available studies in female rats, mice, and rabbits. Pillai et al. (1989) gave 5.2 or 17.3 mg fluoride/kg bodyweight to mice daily on gestation days 6 to 15 (administered orally, as a single dose per day). At euthanasia on day 21, the treated mice showed no sign of pregnancy, and bodyweight and hemoglobin were decreased. The authors suggested that fluoride adversely affected implantation. Sprague-Dawley rats were given sodium fluoride in their drinking water at concentrations of 0, 50, 150, or 300 ppm on gestation days 6 to 15, and New Zealand rabbits were given 0, 100, 200, or 400 ppm sodium fluoride in the drinking water on gestation days 6 to 19 (Heindel et al., 1996). The number of live births, fetal bodyweight, sex ratio, and the incidence of external, visceral, or skeletal anomalies were similar in fluoride-treated and untreated rats and rabbits.

Pillai et al.

Collins et al.

Heindel et al.

Heindel et al.

Collins et al.

1989

1995

1996

1996

2001b

Drinking water

Drinking water

Drinking water

Drinking water

Gavage

Route of Exposure

Throughout 3 generations

Gestation days 6–19

Gestation days 6–15

Gestation days 0–20

Gestation days 6–15

Duration

0, 25, 100, 175, 250 ppm

0, 100, 200, 400 ppm

0, 50, 150, 300 ppm

0, 10, 25, 100, 175, 250 ppm

0, 5.2, 17.3 mg/kg/day

Dose

Rats

Rabbits

Rats

Rats

Mice

Species

250 ppm: decreased ossification of hyoid bone in F2 fetuses 25–175 ppm: no dose-related developmental effects

100–400 ppm: no reproductive or developmental effects

50–300 ppm: no reproductive effects

250 ppm: increased number of fetuses with three or more skeletal variations 10–175 ppm: no fetal effects

At gestation day 21, treated mice showed no sign of pregnancy, and bodyweight and hemoglobin decreased

Results

Note: Summary of studies of fluoride effects on reproductive parameters and on developing fetuses of mice, rats, and rabbits.

Authors

Year

TABLE 5.9 Summary of Developmental Toxicity Studies

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When pregnant rats were given fluoridated drinking water at 0, 10, 25, 100, 175, or 225 ppm throughout gestation, there was no effect on the development of specific bones, although there was an increase in the average number of fetuses per litter with three or more skeletal variations at 250 ppm (Collins et al., 1995). In a developmental toxicity study of pregnant rats that had been treated with sodium fluoride in utero and had continued to be given the same concentrations (0, 25, 100, 175, or 250 ppm) throughout gestation, decreased ossification of the hyoid bone was observed at 250 ppm (Collins et al., 2001b). The number of live births, fetal bodyweight, sex ratio, and the incidence of external and visceral anomalies were similar in fetuses of rats whose parents had also been exposed in utero and throughout their lifetime.

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Ekstrand, J., Alvan, G., Boreus, L.O., and Norlin, A. (1977) Pharmacokinetics of fluoride in man after single and multiple oral doses, European Journal of Clinical Pharmacology, 12: 311–317. Elbetieha, A., Darmani, H., and Al-Hiyasat, A.S. (2000) Fertility effects of sodium fluoride in male mice, Fluoride, 33: 128–134. EPA (Environmental Protection Agency) (1985) Drinking Water Criteria Document on Fluoride, Contract 68-03-3279, Cincinnati: Office of Drinking Water. Erickson, J.D. (1980) Down syndrome, water fluoridation, and maternal age, Teratology, 21: 177–180. Erickson, J.D., Oakley, G.P., Jr., Flynt, J.W., Jr., and Hay, S. (1976) Water fluoridation and congenital malformations: no association, Journal of the American Dental Association, 93: 981–984. Ericsson, Y. and Hammarstrom, L. (1964) Mouse placental transfer of F-18 in comparison with Ca-45, Acta Odontologica Scandinavica, 22: 523–538. Esala, S., Vuori, E., and Helle, A. (1982) Effect of maternal fluorine intake on breast milk fluorine control, British Journal of Nutrition, 48: 201–204. Fein, N.J. and Cerklewski, F.L. (2001) Fluoride content of foods made with mechanically separated chicken, Journal of Agricultural and Food Chemistry, 49: 4284–4286. Fejerskov, O., Manji, F., and Baelum, V. (1990) The nature and mechanisms of dental fluorosis in man, Journal of Dental Research, 69: 692–700. Fejerskov, O., Yanagisawa, T., Tohda, H., Larsen, M.J., Josephsen, K., and Mosha, H.J. (1991) Posteruptive changes in human dental fluorosis — a historical and ultrastructural study, Proceedings of the Finnish Dental Society, 87: 607–619. Feltman, R. and Kosel, G. (1955) Prenatal ingestion of fluorides and their transfer to the fetus, Science, 122: 560–561. Finkelman, R.B., Belkin, H.E., and Zheng, B. (1999) Health effects of domestic coal use in China, Proceedings of the National Academy of Sciences of the United States of America, 96: 3427–3431. Food and Nutrition Board (1999) Fluoride, in Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride, Washington, D.C.: National Academy of Science Press, 218–313. Forestier, F., Daffos, F., Said, R., Brunet, C.M., and Guillaume, P.N. (1990) Passage transplacentaire du fluor. Etude in utero, Journal de Gynecologie, Obstetrique et Biologie de la Reproduction, 19: 171–175. Freni, S.C. (1994) Exposure to high fluoride concentrations in drinking water is associated with decreased birth rates, Journal of Toxicology and Environmental Health, 42: 109–121. Gedalia, I. and Shapira, L. (1989) Effect of prenatal and postnatal fluoride on the human deciduous dentition. A literature review, Advances in Dental Research, 3: 168–176. Gedalia, I., Brzezinski, A., Bercovici, B., and Lazarov, F. (1961) Placental transfer of fluorine in the human fetus, Proceedings of the Society for Experimental Biology and Medicine, 106: 147–149. Ghosh, D., Das Sarkar, S., Maiti, R., Jana, D., and Das, U.B. (2002) Testicular toxicity in sodium fluoride treated rats: association with oxidative stress, Reproductive Toxicology, 16: 385–390. Greenberg, S.R. (1986) Response of the renal supporting tissues to chronic fluoride exposure as revealed by a special technique, Urologia Internationalis, 41: 91–94. Grunder, L.P. and MacNeil, J.H. (1973) Examination of bone content in mechanically deboned poultry meat by EDTA and atomic absorption spectrophotometric methods, Journal of Food Science, 38: 712–713.

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Bacterial Contamination of Ready-to-Eat Foods: Concern for Human Toxicity Tony J. Fang

CONTENTS Abstract ..................................................................................................................143 Abbreviations .........................................................................................................144 Food-Borne Disease Outbreaks and Ready-to-Eat Foods ....................................144 Microbiological Quality of Ready-to-Eat Food Products.....................................146 Bacillus cereus...........................................................................................147 Escherichia coli and Coliforms .................................................................151 Listeria monocytogenes .............................................................................154 Salmonella spp...........................................................................................155 Staphylococcus aureus...............................................................................158 Risk Assessment and Food-Borne Microorganisms .............................................159 Improvement of Microbiological Quality of RTE Foods through HACCP .........161 Conclusions............................................................................................................163 References..............................................................................................................164

Abstract

The increasing availability of ready-to-use (RTU) and ready-to-eat (RTE) foods reflects consumer demand for convenient foods. In addition to convenience, consumers are also looking for RTE foods that are fresh, healthy, safe, additive free, and nutritious. Microbiological data from food-borne disease outbreaks have indicated that microorganisms play a very important role of the incidence. In Taiwan, Republic of China, the most frequent causes were attributable to Vibrio parahaemolyticus, followed by Staphylococcus aureus and Bacillus cereus. Salmonella spp., Campylobacter, and Escherichia coli O157 were the causative agents for most food-borne illness in Scotland. In the years 1990 to 2000, the U.S. Centers for Disease Control and Prevention (CDC) reported a total of 8797 food-borne outbreaks in the U.S., with Salmonella responsible for 1138 outbreaks (13% of the total

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outbreaks). Detection of pathogens including B. cereus, E. coli, E. coli O157, Listeria monocytogenes, Salmonella spp., and S. aureus on various types of RTE foods, such as 18°C products, are discussed. Risk assessment is the estimation of severity and the likelihood of harm resulting from exposure to a hazard. Four steps, including hazard identification, hazard characterization, exposure characterization, and risk characterization are involved in the risk assessment process. Microbiological risk assessments of pathogens including L. monocytogenes, Salmonella spp., E. coli O157:H7, B. cereus, Staphylococcus aureus, Vibrio spp., Campylobacter, and Clostridium have been published. The relationship between the hazard analysis critical control point (HACCP) system and microbiological quality of RTE foods is discussed. Because microbiological quality can be improved by implementing HACCP, the incidence of food-borne disease could also be reduced by HACCP implementation.

Abbreviations

CCP: critical control point; CDC: U.S. Centers for Disease Control and Prevention; CFU: colony-forming unit; DOH: Department of Health (Taiwan, Republic of China); EAggEC: enteroaggregative Escherichia coli; EHEC: enterohemorrhagic Escherichia coli; EIEC: enteroinvasive Escherichia coli; EPEC: enteropathogenic Escherichia coli; ETEC: enterotoxigenic Escherichia coli; HACCP: hazard analysis and critical control point; GMP: good manufacturing practice; MAP: modified atmosphere packaging; MRA: microbiological risk assessment; MPV: minimally processed vegetables; RTE: ready-to-eat; RTU: ready-to-use SFP: staphylococcal food poisoning

FOOD-BORNE DISEASE OUTBREAKS AND READY-TO-EAT FOODS In the past, people have bought foodstuffs in grocery stores to prepare meals at home. However, a growing number of people are buying ready-to-use (RTU) or ready-to-eat (RTE) foods so that they do not have to spend time cooking. RTU vegetables are fresh-cut, packaged vegetables requiring minimal or no further processing prior to consumption; RTE food products are preprocessed or precooked foods that are ready to eat without further processing before consumption, although some of them are heated before eaten. As the demand for RTE foods increases, a greater variety of RTE foods are becoming available. Many street-vended foods, for example, are RTE foods prepared and sold by vendors on streets and in similar public places (Dawson and Canet, 1991; Ekanem, 1998). They provide a source of readily available, inexpensive, nutritional meals, while providing a source of income for the vendors (Bryan et al., 1992; Ekanem, 1998). Another example of RTE products is RTE vegetables. Many prepared RTE vegetables are packaged in bags, and there is an increasing market for this type of product. In the U.K., sales of salad vegetables between 1995 and 2000 increased from US $1.56 billion to $1.70 billion. Prepared salad sales also rose significantly during the same period, from $471 million to $592 million (Sagoo et al., 2003). Various fresh produce products, such as salad

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vegetables and minimally processed vegetables (MPV), which are fresh raw vegetables sold RTE, have now become available to consumers year-round in areas such as European Union and the U.S. (Aureli, 1991). In Taiwan, Republic of China, there has been a marked increase in the sales of 18°C RTE food products in recent years. The concept of 18°C RTE food products was originally developed in Japan and adopted by the food industry in Taiwan. In the production of such food products, controlling the processing conditions is emphasized. For example, in the case of 18°C box meals, the critical control point (CCP) includes controlling cooking time and temperature. In addition, after the box meals are packaged, they are vacuum-cooled to 18 ± 2°C within 5 min. The rapid cooling method retards the growth of contaminating microorganisms. In general, the shelf life of 18°C RTE food products is about 20 h when they were displayed in stores and kept at 18°C. Rice balls rolled in seaweed, sandwiches, sushi, box meals, and cold noodles are the most common 18°C RTE food products sold in convenience stores in Taiwan (Fang, 2000). Although 18°C RTE food products have become more popular in recent years, the microbial quality of these products needs to be considered because 18°C is a temperature at which most microorganisms grow well. RTE food products provide a source of readily available and nutritious meals for the consumers; however, questions have been raised about the safety and microbiological quality of these food products. Microbiological quality or data from foodborne disease outbreaks can provide us with valuable information. Many countries publish statistical data on food-borne illness annually. In Taiwan, the data have been available from the Department of Health (DOH) since 1981 (Department of Health, 2003). A total of 1873 outbreaks were cumulatively reported to DOH from 1991 through 2002 (Table 6.1). The most frequent causes were Vibrio parahaemolyticus (802 outbreaks), Staphylococcus aureus (169 outbreaks), and Bacillus cereus (126 outbreaks). Chang and Chen (2003) reported that from 1991 through 2000, 274 outbreaks of food-borne illness including 12,845 cases and three deaths were confirmed in central Taiwan. Of the 274 reported outbreaks, 171 (62%) were caused by bacterial pathogens. Bacillus cereus (41%, 113 of 274 outbreaks), S. aureus (18%, 49 of 274 outbreaks), and V. parahaemolyticus (16%, 43 of 274 outbreaks) were the main etiologic agents (Table 6.2). In Scotland, 8, 13, and 22 food-borne outbreaks were reported in the years 1996, 1997, and 1998, respectively (World Health Organization, 2000). Salmonella spp., Campylobacter, and Escherichia coli O157 were the causative agents for most of the food-borne illness in Scotland, accounting for 44, 23, and 19%, respectively (World Health Organization, 2000). Although the U.S. food supply is among the safest in the world, large numbers of food-borne illness outbreaks continue to occur. In 1990, the Centers for Disease Control and Prevention (CDC) documented 533 food-borne outbreaks; however, the number of outbreaks increased to 1417 in the year 2000 (Table 6.3). From 1990 to 2000, the CDC reported a total of 8797 food-borne outbreaks in the U.S., with Salmonella responsible for 1138 outbreaks (13% of the total outbreaks) (Table 6.3). Lindqvist et al. (2000) summarized the food-borne disease incidents in Sweden from 1992 to 1997. A total of 555 incidents, involving 11,076 ill people, were reported. Diseases are a persistent threat to public health worldwide. The three main causative agents involved have bacterial, chemical, or natural origins, with bacterial

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TABLE 6.1 Food-Borne Disease Outbreaks in Taiwan, 1991–2002 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaks

Vibrio parahaemolyticus

Staphylococcus aureus

Bacillus cereus

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

93 88 77 102 123 178 234 180 150 208 178 262

13 (12)a 23 (20) 33 (25) 34 (35) 37 (46) 59 (105) 68 (160) 57 (102) 50 (75) 40 (84) 29 (52) 33 (86)

25 (23)b 21 (18) 31 (24) 13 (13) 10 (12) 4 (7) 6 (14) 2 (3) 4 (6) 11 (22) 5 (9) 7 (18)

14 (13)c 18 (16) 16 (12) 12 (12) 9 (11) 4 (7) 6 (15) 7 (12) 8 (12) 2 (5) 5 (8) 2 (4)

Total

1873

43 (802)

9 (169)

7 (126)

a

Data in parentheses indicate the number of outbreaks caused by Vibrio parahaemolyticus. Data in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. cData in parentheses indicate the number of outbreaks caused by Bacillus cereus. b

Source: Department of Health (2003).

food-borne agents playing a leading role. Epidemiological data of the U.S. CDC between 1990 and 2000 reveal that bacterial pathogens accounted for 23% of total disease outbreaks (Table 6.4). In Taiwan, pathogens account for 58% of total incidents (Table 6.4). From 1981 to 1989, 622 outbreaks of food-borne illness were reported in Taiwan, and pathogenic microorganisms accounted for 80% of confirmed incidents (Chiou et al., 1991). Data collected from 1992 to 1997 in Sweden showed that, in 555 incidents, no disease agent was determined for 66% of the incidents. Bacterial agents were implicated in 25% and viruses in 8% of the incidents (Lindqvist et al., 2000).

MICROBIOLOGICAL QUALITY OF READY-TO-EAT FOOD PRODUCTS RTE foods, including 18°C products, provide a source of readily available and nutritious meals for consumers; however, assuring the safety and microbiological quality of these foods should become the first priority, especially because no heat treatment is given immediately before the foods are consumed. Investigations of the microbiological quality of various RTE or RTU food products, such as 18°C food products (cold noodles, box meals, rice balls rolled in seaweed, cone-shaped handrolled sushi, etc.) (Fang et al., 2002), vegetable salads (Albrecht et al., 1995;

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TABLE 6.2 Food-Borne Disease Outbreaks in Central Taiwan, 1991–2000 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaksa

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

16 20 20 18 24 36 46 39 25 30

Total

274

Vibrio parahaemolyticus 13 5 5 6 8 17 20 28 16 20

(2)b (1) (1) (1) (2) (6) (9) (11) (4) (6)

16 (43)

Staphylococcus aureus 31 40 40 22 13 6 11 3 24 23

(5)c (8) (8) (4) (3) (2) (5) (1) (6) (7)

18 (49)

Bacillus cereus 25 (4)d 35 (7) 40 (8) 28 (5) 58 (14) 44 (16) 57 (26) 54 (21) 28 (7) 17 (5) 41 (113)

a

In the total of 274 outbreaks, the number of outbreaks caused by bacterial agents were 171. bData in parentheses indicate the number of outbreaks caused by Vibrio parahaemolyticus. cData in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. dData in parentheses indicate the number of outbreaks caused by Bacillus cereus. Source: Chang, J.M. and Chen, T.H. (2003) Journal of Food and Drug Analysis, 11: 53–59. With permission.

Garcia-Gimeno et al., 1996; Kaneko et al., 1999; Odumeru et al., 1997; Sagoo et al., 2003), vacuum-packed vegetarian foods (Fang et al., 1999), bagged salad vegetables (Sagoo et al., 2003), cold and hot meals served by airlines (Hatakka, 1998a, b), cooked rice (Nichols et al., 1999), point-of-sale RTE rice (Nichols et al., 1999), street-vended foods (King et al., 2000; Kubheka et al., 2001; Mosupye and von Holy, 1999), RTE poultry stuffing from retail premises (Richardson and Stevens, 2003), hot-held foods (Chiou et al., 1996), catering dishes (Alberghini et al., 2000; Gillespie et al., 2000), sliced meat and meat products (Gillespie et al., 2000; Levine et al., 2001; Soriano et al., 2000; Tessi et al., 2002), and seafood (Hatha et al., 1998; Heinitz et al., 2000; Valdimarsson et al., 1998), have been reported. Some pathogens associated with RTE food products are discussed below.

BACILLUS

CEREUS

Bacillus cereus is an aerobic, spore-forming rod normally present in soil, dust, and water. It has been associated with food poisoning in Europe since at least 1906 (Jay, 2000a). This pathogen can be found in a number of food products, both fresh and processed. Bacillus cereus can give rise to two distinct forms of food-borne disease:

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TABLE 6.3 Food-Borne Disease Outbreaks in the U.S., 1990–2000 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaks

No. of Cases

Salmonella spp.

Staphylococcus aureus

Escherichia coli O157:H7

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

533 531 411 514 690 645 602 806 1,314 1,334 1,417

19,231 15,052 11,083 14,080 16,995 13,497 15,421 18,802 26,719 25,286 26,043

26 (138)a 23 (123) 20 (80) 19 (95) 14 (98) 15 (94) 13 (80) 11 (91) 9 (124) 9 (113) 8 (112)

2 (13)b 2 (9) 2 (7) 1 (7) 2 (13) 1 (6) 1 (8) 1 (10) 1 (15) 2 (18) 2 (22)

0.4 (2)c 1 (3) 1 (3) 3 (14) 4 (24) 4 (25) 2 (12) 1 (11) 2 (26) 2 (24) 2 (25)

Total

8,797

202,209

13 (1,148)

2 (128)

2 (169)

a

Data in parentheses indicate the number of outbreaks caused by Salmonella spp. Data in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. cData in parentheses indicate the number of outbreaks caused by Escherichia coli O157:H7. b

Source: Centers for Disease Control and Prevention (2003).

emetic and diarrheal syndromes. Cooked rice was first recognized as a cause of foodborne disease outbreak through contamination with B. cereus in 1971 (Mortimer and McCann, 1974). Since then, many outbreaks associated with this pathogen have been reported in Japan, Canada, Finland, the Netherlands, and the U.S. (Beckers, 1976; Khodr et al., 1994; Raevuori et al., 1976; Schmitt et al., 1976; Shinagawa et al., 1979; Terranove and Blake, 1978). Contamination may be introduced from boiled rice, mashed potatoes, and other cooked foods. Moreover, the incidence of B. cereus has been directly related to the temperature of storage and the length of time the food is kept before serving (Jaquette and Beuchat, 1998; Nichols et al., 1999). Bacillus cereus is emerging as an important food-poisoning organism because of its cosmopolitan distribution. A review by Granum and Lund (1997) indicated that B. cereus had become one of the more important causes of food poisoning in the industrialized world. This pathogen has usually been isolated from the samples of raw rice and thus can be considered part of its normal flora (Parry and Gilbert, 1980). The incidence of Bacillus in RTE foods varies widely, from 0 to 100% (Table 6.5). Mosupye and von Holy (1999) investigated the microbiological quality of RTE street-vended food products in Johannesburg, South Africa. In their study, 51 samples were taken for determination of the microbiological quality; B. cereus was detected in 22%. Kaneko et al. (1999) collected 196 samples from two food factories located in the suburbs of Tokyo to examine the bacterial contamination of RTE vegetables

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TABLE 6.4 Percentage of Total Food-Borne Disease Outbreaks Caused by Bacterial Pathogens Reported in the U.S. and Taiwan Percentage (%) of Total Food-Borne Outbreaks Caused by Bacteria Year

U.S.a

Taiwan, ROCb

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

37 33 29 31 25 25 23 19 20 17 16 NA NA

NAc 45 56 70 61 61 69 76 63 61 56 44 42

Average

23

58

a

The total number of food-borne illness outbreaks in the U.S. from 1990 to 2000 was 8797. bThe total number of food-borne illness outbreaks in Taiwan from 1991 to 2002 was 1873. cNA: Data not included. Source: Centers for Disease Control and Prevention (2003) and Department of Health (2003).

in the various processing steps including trimming, washing, slicing, soaking, dehydrating, blending, and packaging. High aerobic plate counts were found in most samples even after preparation. Bacillus cereus was detected at rates of 10 and 20% before and after preparation, respectively. Fang et al. (2002) investigated the microbiological quality of 18°C RTE foods. For 18°C RTE sushi, 18°C RTE cone-shaped hand-rolled sushi, 18°C RTE sandwiches, 18°C RTE rice balls rolled in seaweed, and 18°C RTE cold noodles, 18, 40, 54, 56, and 67% of samples were positive for B. cereus, respectively (Table 6.5). In one study, all mashed potatoes and skim milk tested were contaminated with B. cereus (Harmon and Kautter, 1991); similar results were found when cooked vegetables and salad were examined for incidence of B. cereus (Tessi et al., 2002). Bryan et al. (1992) isolated B. cereus from various potato items offered for sale by street vendors in Pakistan. They indicated that the source of contamination was thought to be human handling.

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TABLE 6.5 Bacillus cereus Contamination in Some RTE Products

Product

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–)

Ref.

4

0 (0)a

NAb

147

0 (0)

NA

12 4162 113

0 (0) 1 (29) 2 (2)

NA 2.0–6.0 <3.0

Harmon and Kautter, 1991 Richardson and Stevens, 2003 Tessi et al., 2002 Nichols et al., 1999 Fang et al., 1999

4162 99

3 (115) 3 (3)

2.0->7.0 <3.0

Nichols et al., 1999 Fang et al., 1999

180

4 (8)

<3.8

Hatakka, 1998a

83

5 (4)

<3.3

Hatakka, 1998a

240

5 (12)

<4.7

Hatakka, 1998a

38 108

5 (2) 6 (6)

2.0–4.0 <3.0

Mosso et al., 1989 Fang et al., 1999

86

11 (9)

2.0–3.0

Kaneko et al., 1999

22 27 85

18 (4) 19 (5) 20 (17)

2.3–3.5 2.0–4.0 2.0–3.0

Fang et al., 2002 Mosso et al., 1989 Kaneko et al., 1999

51

22 (11)

NA

Egg salad

4

25 (1)

NA

Beef gravy

4

25 (1)

NA

12 25

25 (3) 40 (10)

2.0–4.0 2.3–3.8

Mosupye and Von Holy, 1999 Harmon and Kautter, 1991 Harmon and Kautter, 1991 Mosso et al., 1989 Fang et al., 2002

34 12 50

44 (15) 50 (6) 54 (27)

NA NA 2.3–5.0

Tessi et al., 2002 Tessi et al., 2002 Fang et al., 2002

Chicken salad RTE stuffing from retail premises Cooked pastas Point-of-sale RTE rice Vacuum-packed vegetarian foods Precooked RTE rice Film-wrapped vegetarian foods Cold meals served on aircraft (dessert) Cold meals served on aircraft (salad) Cold meals served on aircraft (appetizer) Milk and milk products Unwrapped vegetarian foods RTE fresh vegetables (before operation) 18°C RTE foods (sushi) Sweet dessert RTE fresh vegetables (after operation) RTE street foods

Salad dressing 18°C RTE foods (coneshaped, hand-rolled sushi) Cooked meats Cooked cereals 18°C RTE foods (sandwiches)

(continued)

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TABLE 6.5 (CONTINUED) Bacillus cereus Contamination in Some RTE Products No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–)

18°C RTE foods (rice balls rolled in seaweed) 18°C RTE foods (cold noodles) Turkey gravy

52

56 (29)

2.3–5.5

Fang et al., 2002

15

67 (10)

2.3–4.3

Fang et al., 2002

4

75 (3)

NA

Noodles

8

88 (7)

NA

Cooked vegetables Mashed potatoes

8 10

100 (8) 100 (10)

NA NA

Skim milk

24

100 (24)

NA

Salads

35

100 (35)

NA

Harmon and Kautter, 1991 Harmon and Kautter, 1991 Tessi et al., 2002 Harmon and Kautter, 1991 Harmon and Kautter, 1991 Tessi et al., 2002

Product

Ref.

a

Data in parentheses indicate the number of positive samples contaminated by B. cereus. NA: Data not available.

b

ESCHERICHIA

COLI AND

COLIFORMS

Indicator organisms, such as E. coli and coliforms, have been used to determine an objectionable microbial condition of food, such as fecal contamination, the presence of potential pathogens or potential spoilage of foods, as well as the sanitary conditions of food processing, production, or storage (Gill and McGinnis, 2000; Rampersad et al., 1999; Russell, 2001). Coliforms include all aerobic and facultatively anaerobic Gram-negative nonspore-forming bacilli, which ferment lactose with gas formation within 48 h at 35°C (Banwart, 1989). The incidence of E. coli and coliforms in RTE foods, such as chocolate (wrapped or unwrapped), RTE cold dishes, cooked shrimp, point-of-sale RTE rice, Spanish potato omelet, RTE stuffing from retail premises, 18°C RTE foods (rice balls rolled in seaweed, sushi, cold noodles, sandwiches, cone-shaped hand-rolled sushi), RTE fresh vegetables, precooked RTE rice, RTE meats from catering premises, hotholding cooked foods, Spanish potato omelet (with meat), RTE foods and drinks, vacuum-packed vegetarian foods, RTE cold dishes, and film-wrapped vegetarian foods (Adesiyun, 1995; Chiou et al., 1996; Fang et al., 1999, 2002; Gillespie et al., 2000; Hatha et al., 1998; Kaneko et al., 1999; Lin et al., 1989; Nichols et al., 1999; Richardson and Stevens, 2003; Sagoo et al., 2003; Soriano et al., 2000; Torres-Vitela et al., 1995), have been investigated. Chiou et al. (1996) examined the microbial quality of 300 RTE food products, which were to be kept hot, sold in southern Taiwan. Their results indicated that the percentage of food products not meeting the microbiological standards accepted by Taiwan, Republic of China, regarding aerobic

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TABLE 6.6 Escherichia coli and/or Coliform Contamination in Some RTE Products

Product

No. of Samples

Percentage (%) of Positive Samples

Analyzed

E. coli

Coliforms

Range (E. coli/coliform) (log CFU g–)

Ref.

Wrapped chocolate

44

NAa

32 (14)b

1.0–3.0/NA

Unwrapped chocolate RTE cold dishes Cereal mixed products Instant cereal products Cooked shrimp Point-of-sale RTE rice Spanish potato omelet RTE stuffing from retail premises 18°C RTE foods (rice balls rolled in seaweed) RTE fresh vegetables 18°C RTE foods (sushi) Precooked RTE rice RTE meats from catering premises 18°C RTE foods (cold noodles) Hot-holding cooked foods Spanish potato omelet (with meat) 18°C RTE foods (sandwiches) RTE foods and drinks Vacuum-packed vegetarian foods RTE cold dishes

56

NA

29 (16)

NA/1.0–3.0

646 81

NA NA

78 (505) 7 (6)

NA NA

Torres-Vitela et al., 1995 Torres-Vitela et al., 1995 Lin et al., 1989 Fang et al., 1997

74

NA

3 (2)

NA

Fang et al., 1997

914 4162

0 (0)c 1 (38)

3 (28) NA

NA/1.0–2.0 <2.0–4.0/NA

Hatha et al., 1998 Nichols et al., 1999

114

2 (2)

NA

NA

Soriano et al., 2000

147

2 (3)

NA

2.0–5.0/NA

52

4 (2)

65 (34)

2.97–5.15/ 2.30–6.11

Richardson and Stevens, 2003 Fang et al., 2002

140

4 (6)

96 (134)

NA

Kaneko et al., 1999

22

5 (1)

68 (15)

Fang et al., 2002

6 (234) 6 (203)

NA NA

3.26–3.45/ 2.30–7.55 <2.0–7.0/NA <2.0–7.0/NA

15

7 (1)

60 (9)

0–2.3/2.3–5.45

Nichols et al., 1999 Gillespie et al., 2000 Fang et al., 2002

300

8 (24)

20 (61)

NA

Chiou et al., 1996

114

9 (10)

NA

NA

Soriano et al., 2000

50

10 (5)

88 (44)

2.3–4.0/2.9–9.6

Fang et al., 2002

293

10 (30)

NA

NA

Adesiyun, 1995

113

12 (14)

17 (19)

NA

Fang et al., 1999

496

15 (74)

NA

NA

Lin et al., 1989

4162 3494

(continued)

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TABLE 6.6 (CONTINUED) Escherichia coli and/or Coliform Contamination in Some RTE Products

Product 18°C RTE foods (cone-shaped, hand-rolled sushi) Film-wrapped vegetarian foods Unwrapped vegetarian foods RTE salad vegetable

No. of Samples

Percentage (%) of Positive Samples

Analyzed

E. coli

Coliforms

Range (E. coli/coliform) (log CFU g–)

Ref.

25

16 (4)

84 (21)

2.3–2.9/2.6–7.18

Fang et al., 2002

99

25 (25)

31 (31)

NA

Fang et al., 1999

108

47 (51)

50 (54)

NA

Fang et al., 1999

1.0–4.0/NA

Sagoo et al., 2003

3852

99.8 (3843) NA

a

NA: Data not available. Data in parentheses indicate the number of samples that were detected positive for coliforms. cData in parentheses indicate the number of positive samples contaminated by E. coli. b

plate count, coliforms, and E. coli were 18, 20, and 8%, respectively. Lin et al. (1989) indicated that the detection rate of E. coli and coliforms in RTE dishes was 15% (74 of 496 samples) and 78% (505 of 646 samples), respectively. Albrecht et al. (1995) evaluated the microbial contamination of vegetable ingredients (lettuce, tomatoes, broccoli, and cauliflower) in salad bars. They found that the total aerobic count and coliforms in these vegetables ranged from 5.51 to 6.63 log colony-forming units (CFU) g–1 and from 4.81 to 6.30 log CFUg–1, respectively. Fang et al. (1997) reported that in 155 instant cereal products retailed in Taiwan, coliforms were isolated from 6 of the 81 (7%) cereal mix products as well as from 2 of the 74 (3%) regular instant cereal products (Table 6.6). Fang et al. (2002) indicated that, in 164 samples of 18°C RTE food products that were purchased from convenience stores and supermarkets in central Taiwan, 8 and 75% incidences for E. coli and coliforms were detected, respectively. Among the five types (rice balls rolled in seaweed, sushi, cold noodles, sandwiches, cone-shaped hand-rolled sushi) of RTE food products tested, the highest incidence of E. coli (16%) and coliforms (88%) were found in hand-rolled sushi and sandwiches, respectively (Table 6.6). The occurrence of coliforms, especially such high values as >6 log CFU g–1 detected, indicates contamination and poor microbiological quality. The reason may be contaminated raw material, cross-contamination during preparation, or high storage temperature. Most 18°C RTE foods used in this investigation contain fresh vegetables; thus, the coliforms on the products reflect the initial microflora, which may contain Klebsiella and other genera commonly found, of the vegetables in the growing fields and recontamination during cutting and further processing. In addition to its role as an indicator, E. coli is also a food-borne pathogen, which was established in 1971 (Jay, 2000b). Five virulence groups of E. coli, including enteroaggregative (EAggEC), enterohemorrhagic (EHEC), enteroinvasive

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(EIEC), enteropathogenic (EPEC), and enterotoxigenic (ETEC), are recognized based on disease syndromes and characteristics, and also on their effect on certain cell cultures and serological groupings. Doyle and Schoeni (1987) reported the first published study on the prevalence of EHEC in meat. Cody et al. (1999) have reported on a food-borne outbreak caused by E. coli O157:H7 from unpasteurized commercial apple juice. In one study, the incidence of E. coli O157:H7 in beef, pork, poultry, and lamb samples were 3.7% (n = 164), 1.5% (n = 264), 1.5% (n = 263), and 2.0% (n = 205), respectively (Doyle and Schoeni, 1987). Tarr et al. (1999) examined 1400 samples of ground beef from retail stores in Seattle, Washington for E. coli O157:H7, and all of the samples did not contain this pathogen. Regarding RTE foods, no E. coli O157:H7 was detected in cooked meat patties (n = 452) (Levine et al., 2001), bagged salad vegetable (n = 3852) (Sagoo et al., 2003), and organic RTE (n = 55) (McMahon and Wilson, 2001). However, 1% of vegetable salad (n = 116) tested positive for E. coli O157:H7 (Huang et al., 1998) (Table 6.7).

LISTERIA

MONOCYTOGENES

Listeria monocytogenes is an established food-borne pathogen. Outbreaks of febrile noninvasive listeriosis have involved a number of food vehicles including chocolate milk (Dalton et al., 1997), cold-smoked rainbow trout (Miettinen et al., 1999), coleslaw (Schlech et al., 1983), corn and tuna salad (Aureli et al., 2000), dairy foods (Kvenberg 1988), imitation crab meat (Farber et al., 2000), smoked mussels (Misrachi et al., 1991), and vegetable products (Kvenberg, 1988). On the basis of data from the FoodNet active surveillance program, the CDC documented a listeriosis frequency of 3 cases per 1 million people for 2000 and 2001 (Centers for Disease Control and Prevention, 2001, 2002a, b). In Taiwan, few outbreaks of food-borne diseases caused by L. monocytogenes have been reported (Department of Health, 2003).

TABLE 6.7 Detection of E. coli O157 in Some RTE Products

Product Cooked meat patties RTE stuffing from retail premises Bagged salad vegetables Organic RTE vegetables Vegetable salad a

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–1)

452 3

0 (0)a 0 (0)

NAb NA

3852 55

0 (0) 0 (0)

NA NA

116

1 (1)

NA

Ref. Levine et al., 2001 Richardson and Stevens, 2003 Sagoo et al., 2003 McMahon and Wilson, 2001 Huang et al., 1998

Data in parentheses indicate the number of samples detected positive for E. coli O157. NA: Data not available.

b

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155

Kaneko et al. (1999) examined the bacterial contamination of 196 RTE vegetables from two food factories located in the suburbs of Tokyo. In their investigation, no L. monocytogenes was detected. Similar results were reported: no L. monocytogenes was found in Chinese pickle and fermented milk, coleslaw mix (4°C), green pepper (4°C), and RTE street foods (Table 6.8). For fresh soft cheese, RTE meats from catering premises, jerky, bagged salads, and luncheon meats, the positive samples were all less than 1% (Table 6.8). Incidences of this pathogen in RTE foods of between 1 and 10% were found in large cooked sausages; cooked poultry products; RTE salad vegetables; deli salads; salad/spreads/pâtés; cooked, roast, corned beef; fermented sausages; RTE deli products; small cooked sausages; smoked seafood; frozen cooked food; seafood salad; sliced ham and luncheon meats; coleslaw mix (10°C); green pepper (10°C); and chopped lettuce (4°C) (Table 6.8). Garcia-Gimeno et al. (1996) indicated that, of a total of 70 RTU-use mixed vegetable salad samples, 21 (30%) were observed to contain L. monocytogenes. In one study, a detection rate of 40% for L. monocytogenes in salad mix (10°C) was documented (Table 6.8).

SALMONELLA

SPP.

Among the Gram-negative rods that cause food-borne gastroenteritis, the most important are members of the genus Salmonella. They are widely distributed in nature, with humans and animals their primary reservoirs. Salmonella food poisoning results from the ingestion of foods containing appropriate strains of this genus in significant numbers. In the U.S., nontyphoidal Salmonella food-borne disease was estimated to account for a total of 1,341,873 cases, 15,608 hospitalizations, and 553 deaths annually (Mead et al., 1999). In England and Wales, 85 of 1518 (6%) foodborne general outbreaks of infectious intestinal disease were associated with the consumption of salad, fruit, and vegetables between 1992 and 2000 (O’Brien et al., 2000, 2001). The consumption of wholesale lettuce was linked to two notable outbreaks of Salmonella Typhimurium DT 104 infection and Salmonella Typhimurium DT 204b infection that affected 174 and 140 people, respectively, in England and Wales in 2000 (Public Health Laboratory Service, 2000). Levine et al. (2001) documented the Salmonella prevalence for RTE meat and poultry products from 1990 through 1999. They reported 0.1 to 1% of prevalence in these RTE products (Table 6.9), which indicated that the heat lethality processes used in manufacturing these products were generally effective in eliminating this pathogen and that postprocess contamination was not a significant problem. One study reported that the incidence of Salmonella in 22 of 566 raw shellfish examined, whereas only 1 of 774 samples of RTE seafood was positive for Salmonella (Table 6.9). Andrews et al. (1975) indicated that 60 of 539 oyster samples (Crassostrea virginica) tested positive for Salmonella; 85% of the positive samples contained the Salmonella serovars Derby, Infantis, or Newport. Because oysters are consumed raw, and are thus considered RTE food, it is obvious that Salmonella in oyster could contribute to Salmonella food-borne disease. For RTE cooked crab samples, 4 of 151 samples tested positive (3%) for Salmonella (Table 6.9). Serotypes isolated from these samples were Salmonella Enteriditis and Salmonella Paratyphi-B bioser java (Heinitz et al., 2000). For dried/salted fish and smoked fish, 25 of 778 (3%) and 10 of 255 (4%)

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TABLE 6.8 Listeria monocytogenes Contamination in Some RTE Products

Product Chinese pickle and fermented milk Coleslaw mix (4°C) Green pepper (4°C) RTE street foods Fresh soft cheeses RTE meats from catering premises Jerky Bagged salads Luncheon meats Large cooked sausages Cooked poultry products RTE salad vegetables Deli salads Salad/spreads/pâtés Cooked, roast, corned beef Fermented sausages RTE deli products Small cooked sausages Smoked seafood Frozen cooked food Seafood salad Sliced ham and luncheon meats Coleslaw mix (10°C) Green pepper (10°C) Chopped lettuce (4°C) Seafood RTE flesh foods Vegetables Salad mix (4°C) Chopped lettuce (10°C) RTU mixed vegetable salads Salad mix (10°C) a

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–1)

Ref.

12

0 (0)a

NAb

Wong et al., 1990

20 20 51

0 (0) 0 (0) 0 (0)

NA NA NA

2931 3494

0.2 (5) 0.4 (13)

0.04–2.0 <2.0–3.0

Odumeru et al., 1997 Odumeru et al., 1997 Mosupye and von Holy, 1999 Gombas et al., 2003 Gillespie et al., 2000

770 2966 9199 4262 6836 3852 8549 3932 5272 830 60

1 1 1 1 2 2 2 3 3 3 3

(4) (22) (82) (56) (145) (90) (202) (119) (163) (27) (2)

NA 0.04–3.0 0.04–4.0 NA NA 1.0–3.0 0.04–4.0 NA NA NA NA

6820 2644 45 2446 2287

4 4 4 5 5

(243) (114) (2) (115) (118)

NA 0.04–6.0 NA 0.04–3.0 NA

15 15 24 57 203 49 24 15 70

7 (1) 7 (1) 8 (2) 11 (6) 12 (24) 12 (6) 12 (3) 20 (3) 30 (21)

NA NA NA NA NA NA NA NA NA

15

40 (6)

NA

Levine et al., 2001 Gombas et al., 2003 Gombas et al., 2003 Levine et al., 2001 Levine et al., 2001 Sagoo et al., 2003 Gombas et al., 2003 Levine et al., 2001 Levine et al., 2001 Levine et al., 2001 Hudson and Mott, 1993 Levine et al., 2001 Gombas et al., 2003 Wong et al., 1990 Gombas et al., 2003 Levine et al., 2001 Odumeru et al., 1997 Odumeru et al., 1997 Odumeru et al., 1997 Wong et al., 1990 Hudson et al., 1992 Wong et al., 1990 Odumeru et al., 1997 Odumeru et al., 1997 Garcia-Gimeno et al., 1996 Odumeru et al., 1997

Data in parentheses indicate the number of samples detected positive for L. monocytogenes. NA: Data not available.

b

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TABLE 6.9 Salmonella sp. Contamination in Some RTE Products

Product Cold meals served on aircraft (appetizers) Cold meals served on aircraft (salads) RTE street foods RTE stuffing from retail premises RTE meats from catering premises Unwrapped chocolate Cooked meats, vegetables, cereals, and pastas Salads Salad/spreads/pâtés Large cooked sausages Cooked poultry products RTE salad vegetables Small cooked sausages Cooked, roast, corned beef Sliced ham and luncheon meats Jerky Fermented sausages Prepared seafood Cooked crab Dried/salted fish Smoked fish RTE foods and drinks Smoked fish Wrapped chocolate

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–1)

269

0 (0)a

NAb

Hatakka, 1998a

164

0 (0)

NA

Hatakka, 1998a

51

0 (0)

NA

147

0 (0)

NA

3494

0 (0)

NA

Mosupye and von Holy, 1999 Richardson and Stevens, 2003 Gillespie et al., 2000

56 66

0 (0) 0 (0)

NA NA

Torres-Vitela et al., 1995 Tessi et al., 2002

35 4202 4328 7020 3852 6996 5444 2293

0 (0) 0.1 (2) 0.1 (3) 0.1 (7) 0.1 (5) 0.2 (14) 0.2 (12) 0.2 (5)

NA NA NA NA <2.0 NA NA NA

Tessi et al., 2002 Levine et al., 2001 Levine et al., 2001 Levine et al., 2001 Sagoo et al., 2003 Levine et al., 2001 Levine et al., 2001 Levine et al., 2001

648 698 1550 151 778 156

0.3 (2) 1 (10) 2 (31) 3 (4) 3 (25) 3 (5)

NA NA NA NA NA NA

293 255 44

3 (10) 4 (10) 5 (2)

NA NA NA

Levine et al., 2001 Levine et al., 2001 Heinitz et al., 2000 Heinitz et al., 2000 Heinitz et al., 2000 Heinitz and Johnson, 1998 Adesiyun, 1995 Heinitz et al., 2000 Torres-Vitela et al., 1995

Ref.

a

Data in parentheses indicate the number of samples contaminated by Salmonella spp. NA: Data not available.

b

samples were positive for Salmonella, respectively (Table 6.9). Although Salmonella accounts for 31% of total food-borne deaths in the U.S. annually (Mead et al., 1999), the incidence of this pathogen in RTE foods was from 0 to 5% (Table 6.9).

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Many of the 20 or more species in the genus Staphylococcus cause disease in humans and animals, but S. aureus is the species involved in outbreaks of staphylococcal food-borne disease (Bergdoll, 1990). Staphylococcal food poisoning (SFP) occurs either as isolated cases or as outbreaks affecting large numbers of individuals. SFP results when food containing toxin produced by staphylococci is ingested. Large numbers of S. aureus must be present in food to produce enough enterotoxin to cause illness. The number of enterotoxigenic staphylococci frequently found in contaminated food is 1 × 106 CFU g–1 or more (Tranter, 1991). Humans are the most important reservoir of S. aureus; 40 to 50% of all healthy people carry this bacterium. Staphylococcus is most commonly found in the nose and throat; on hands and skin; and in infected cuts, abrasions, burns, boils, and pimples (Jay, 2000c). The presence of S. aureus in the food samples tested indicates improper handling and possible cross-contamination (Garcia et al., 1986; Snyder, 1998). This microorganism not only plays an important role in food-borne diseases in the U.S. (Bean and Griffin, 1990; Bean et al., 1990), but also in Taiwan, where S. aureus is the second most commonly found food-borne pathogen. The number of outbreaks in Taiwan due to this bacterium ranged from 11 to 24 annually, in the period 1986 to 1995 (Pan et al., 1997). The enterotoxin A-producing strains of S. aureus were the most frequently isolated strains during the outbreaks in Taiwan. Pan et al. (1997) have reported that 53% (8 of 15) of the S. aureus outbreaks in Taiwan in 1994 were associated with enterotoxin A. In general, staphylococci may be expected to exist, at least in low numbers, in any or all food products that are of animal origin or in those that are handled directly by humans. They have been found in a large number of commercial foods by many investigators. RTE street foods, RTE stuffing from retail premises, RTE meats from catering premises, cold desserts, appetizers, and salads served on aircraft, vacuumpacked vegetarian foods, 18°C RTE sandwiches, 18°C RTE cold noodles, 18°C RTE sushi, 18°C RTE cone-shaped hand-rolled sushi, RTE foods and drinks, filmwrapped vegetarian foods, 18°C RTE rice balls rolled in seaweed, unwrapped vegetarian foods, and RTE cold dishes are examples of RTE foods that have been examined for the incidence of S. aureus. In one study, 3494 RTE samples were tested for incidence of S. aureus; 111 (3%) samples tested positive (Table 6.10). A total of 164 18°C RTE food products, which were purchased from convenience stores and supermarkets in central Taiwan from 1999 to 2000, were examined by Fang et al. (2002) to determine the microbiological quality of these products. The 18°C RTE food products, manufactured by 16 factories, were divided into groups based on the type of food and their major ingredients. The positive samples for this pathogen in 18°C sandwiches, cold noodles, sushi, cone-shaped hand-rolled sushi, and rice balls rolled in seaweed were 12, 13, 14, 16, and 25%, respectively (Table 6.10). Lin et al. (1989) reported that, for 646 RTE cold dishes, S. aureus was detected in 345 samples (53%), which is the highest detection rate among the examples presented in Table 6.10.

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TABLE 6.10 Staphylococcus aureus Contamination in Some RTE Products

Product

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–1)

Ref.

51

0 (0)a

NAb

RTE stuffing from retail premises

147

1 (1)

2.0–3.0

RTE meats from catering premises

3494

3 (111)

Cold meals served on aircraft (desserts) Vacuum-packed vegetarian foods Cold meals served on aircraft (appetizers) Cold meals served on aircraft (salads) 18°C RTE foods (sandwiches) 18°C RTE foods (cold noodles) 18°C RTE foods (sushi) 18°C RTE foods (cone-shaped hand-rolled sushi) RTE foods and drinks Film-wrapped vegetarian foods 18°C RTE foods (rice balls rolled in seaweed) Unwrapped vegetarian foods RTE cold dishes

145

4 (6)

0–2.5

Mosupye and von Holy, 1999 Richardson and Stevens, 2003 Gillespie et al., 2000 Hatakka, 1998a

113 174

6 (7) 8 (14)

NA 0–3.5

Fang et al., 1999 Hatakka, 1998a

31

10 (3)

0–3.5

Hatakka, 1998a

50 15 22 25

12 13 14 16

(6) (2) (3) (4)

2.3–5.1 2.6–3.3 2.3–4.3 3.6–4.6

Fang Fang Fang Fang

293 99 52

20 (58) 20 (20) 25 (13)

NA NA 2.3–5.0

Adesiyun, 1995 Fang et al., 1999 Fang et al., 2002

108 646

29 (31) 53 (345)

NA NA

Fang et al., 1999 Lin et al., 1989

RTE street foods

<2.0–6.0

et et et et

al., al., al., al.,

2002 2002 2002 2002

a

Data in parentheses indicate the number of samples contaminated with S. aureus. NA: Data not available.

b

RISK ASSESSMENT AND FOOD-BORNE MICROORGANISMS Risk assessment is the estimation of severity and the likelihood of harm resulting from exposure to a hazard. The basic steps involved in conducting a risk assessment will be the same whether we are considering public health risk posed by chemicals or by microorganisms in food. The purpose of risk assessment is to derive a mathematical statement, based on the probability of certain events, of the chance of adverse health consequences from exposure to an agent capable of causing harm. The microbiological risk assessment (MRA), as described in an interim report (Advisory Committee on Dangerous Pathogens, 1996) or by the Codex scheme (Codex Alimentarius Commission, 1996), is depicted in Figure 6.1. The risk assessment process involves four steps (National Research Council, 1983):

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Statement of Purpose

RISK ASSESSMENT Hazard Identification

Exposure Assessment

Hazard Characterization

Risk Characterization

Risk Communication

FIGURE 6.1 The Codex Alimentarius risk assessment scheme.

1. Hazard identification: The qualitative indication that a substance may cause adverse health effects. 2. Hazard characterization (dose–response assessment): The evaluation of the adverse health effect qualitatively and quantitatively; to establish the relationship between the magnitude of the exposure and the probability of occurrence of an adverse health effect. 3. Exposure characterization: The qualitative and quantitative evaluation of the degree of exposure likely to occur. 4. Risk characterization: Integration of the above steps into an estimation of the adverse effects likely to occur in a population, to be used in decision making (risk management). Brown (2002) indicated that the aim of all MRA schemes is to reduce risk. This aim can be achieved by identifying realistic microbiological hazards and characterizing them according to the severity of their effects on consumers; by evaluating the impact of raw material contamination, processing, and use on the level of risk; and by communicating clearly and consistently, via the output of the study, the level of risk to the consumer. Although MRA is a new approach for food safety, articles regarding L. monocytogenes (Begot et al., 1997; Chen et al., 2003; Elliot and Kvenberg, 2000; Farber et al., 1996; Hitchins, 1996; Lindqvist and Westoo, 2000; Rocourt et al., 2003), Salmonella spp. (Berends et al., 1998; Brown, 2002; Whiting and Buchanan, 1997),

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E. coli O157:H7 (Cassin et al., 1998; Haas et al., 2000; Powell et al., 2001), B. cereus (Nauta et al., 2003; Notermans et al., 1997), Staphylococcus aureus (Lindqvist et al., 2002; Walls and Scott, 1997), Vibrio spp. (Rocourt, 1996; Rocourt et al., 2003), Campylobacter (Anderson et al., 2001; Duffy et al., 2001; Hartnett et al., 2002; Rosenquist et al., 2003; Teunis et al., 1997), and Clostridium (Barker et al., 2002; Fazil et al., 2002; Sumner and Ross, 2002) have been published. By performing risk assessment, mathematical models can generate a numerical estimate of risk, which may be used in decision making by comparisons with socially and politically accepted risk levels. The acceptable level or risk level should be based on what is technically feasible and economically achievable with current technology.

IMPROVEMENT OF MICROBIOLOGICAL QUALITY OF RTE FOODS THROUGH HACCP The causes of most food-borne illness are well understood. Most of these illnesses are preventable through proper food-handling techniques. Figure 6.2 shows the principal known factors contributing to food-borne illness in Washington State from 1990 through 1999 (Washington State Department of Health, 2003). Inadequate hand washing was the most common factor that caused the food-borne outbreaks to Washington, which accounted for 31% of the contributing factors. The total percentages of the factors add up to more than 100% because most outbreaks have more than one contributing factor. The principal known factors contributing to food-borne illness in Taiwan from 1996 to 2002 included cross-contamination between raw and cooked materials (34%), insufficient cooking treatment (27%), holding cooked foods too long at room temperature (12%), ill food workers (11%), and insufficient equipment cleaning (5%) (Figure 6.3) (Department of Health, 2003). Lindqvist et al. (2000) summarized food-borne disease incidents in Sweden from 1992 to 1997. In 330 (71%) of 464 outbreaks, no contributing factors were reported. For the identified factors, the major factors that contributed to the outbreaks were poor hygiene, knowledge, and handling (41 outbreaks); inadequate refrigeration (23 outbreaks); contamination by infected person (22 outbreaks); inadequate cooling (20 outbreaks); inadequate hot holding (9 outbreaks); and imported contaminated food (9 outbreaks) (Lindqvist et al., 2000). The strategy for prevention of food-borne illness caused by RTE cooked foods is based on (1) the initial microbial load (nature and quantity), (2) the severity of the heat treatment necessary to destroy pathogens and lower that microbial load, and (3) prevention of growth through temperature controls (Bryan et al., 1997; Buchanan, 2000; Langlois et al., 1997). HACCP, or the Hazard Analysis and Critical Control Point system, was developed originally as a microbiological safety system in the early days of the U.S. manned space program, as it was vital to ensure the safety of food for astronauts. At that time, most food safety and quality systems were based on end-product testing, but it was realized that this could fully assure safe products only by testing 100% of the product. Instead, it became clear that a preventative system was required that would provide a high level of food safety assurance, and the HACCP system was born. The HACCP concept has been used in the food industry for some time. Many

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Inadequate handwashing (31%) Not heating hot foods enough (24%) Inadequate refrigeration (20%) Slow cooling (20%) Cross-contamination (18%) Bare hand contact with food (13%) Ill food worker (13%)

13

18

13 31

20 20

24

FIGURE 6.2 The principal known factors contributing to food-borne illness in Washington State, 1990 to 1999. The total percentages of the factors sum to more than 100% because most outbreaks have more than one contributing factor. (From Washington State Department of Health, 2003.)

Cross-contamination between raw and cooked materials (34%) Insufficient cooking treatment (27%) Cooked foods are held too long in room temperature (12%) Ill food worker (11%) Insufficient equipment cleaning (5%)

12

27

11

5 34 FIGURE 6.3 The principal known factors contributing to food-borne illness in Taiwan (Republic of China), 1996 to 2002. (From Department of Health, 2003.)

research articles regarding this system can be found in various journals (Boccas et al., 2001; Bolton et al., 2001; Fang and Jeng, 2002; Gonzalez-Miret et al., 2001; Heggum, 2001; Hoornstra et al., 2001; Jeng and Fang, 2003; Mortimore, 2001; Panisello and Quantick, 2001; Soriano et al., 2002; Souness, 2000; Suwanrangsi, 2000; Taylor, 2001; Toh and Birchenough, 2000; Torres, 2000; Wallace and Williams, 2001). Soriano et al. (2001) investigated the microbial quality of Spanish potato

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omelet and pork loin before and after implementation of the HACCP system in university restaurants. The authors analyzed incidences of Clostridium perfringens, E. coli, E. coli O157:H7, L. monocytogenes, Salmonella spp., and Staphylococcus aureus. They reported that implementation of the HACCP system lowered the incidence of studied microorganisms. However, documented training in personal hygiene, good manufacturing practices (GMPs), cleaning and sanitation procedures, and personal safety in addition to rearrangement in the infrastructure of these establishments could improve yet more the microbial quality of the meals served. Ren et al. (1995) investigated the effect of implementation of HACCP system on the microbiological quality of 18°C box meals. After running the critical control point (CCP) decision tree of the HACCP system, five CCPs were chosen by the authors. They reported that at each CCP, corrective actions were immediately taken during 18°C box meal production. The bacteriological quality of the 18°C box meals was significantly increased (P < 0.05) by the corrective actions compared to that of traditionally manufactured box meals (Ren et al., 1997). By implementation of HACCP system, the shelf life of 18°C box meals was extended from 12 to 17 h, indicating the important role of a food safety control system in improving microbiological quality of foods.

CONCLUSIONS RTE foods provide a source of readily available and nutritious meals for the consumers. Tillotson (2002) claimed that half of U.S. food dollars go to ready-prepared RTE foods today; i.e., more people are eating out. To provide higher-microbiologicalquality RTE food products, it is important to collect and analyze all the information regarding food-borne outbreak surveys. As shown in this chapter, 23% (data collected from 1990 to 2000) and 58% (data collected from 1991 to 2002) of food-borne outbreaks were caused by bacterial agents in the U.S. and in Taiwan, respectively. In Washington State, the principal known factor that contribute to food-borne disease is inadequate hand washing (31%). However, cross-contamination between raw materials and cooked foods is the major factor, which accounts for 34%, contributing to food-borne outbreaks in Taiwan. For such uncooked RTE foods as salad vegetables, the prevention of contamination and bacterial growth lies in the application of good hygiene practice during growing and processing from farm to table, effective washing and decontamination, effective temperature control during storage and distribution, and the selection of appropriate packaging. Many RTE food products have been developed and the available combinations of technologies, such as modified atmosphere packaging (MAP), various cooking, processing, and packaging technologies, extend shelf life of these products. To provide good-microbial-quality RTE foods, more efforts from government and industry are needed. Although the government and industry have made tremendous progress in reducing pathogens in RET foods, there is obviously room for improvement. The mandatory implementation of HACCP systems in all RTE processing establishments should further improve the microbial quality and safety of all RTE products.

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CONTENTS Abstract ..................................................................................................................174 Abbreviations .........................................................................................................174 Introduction............................................................................................................175 Contemporary and Historical T-2 Toxin Outbreaks..............................................176 Structure of T-2 Toxin ...........................................................................................177 Modes of Action of T-2 Toxin...............................................................................178 Toxicity of T-2 Toxin.............................................................................................180 Metabolism and Elimination of T-2 Toxin in Animals.........................................181 Occurrence of Mycotoxins in Food and Feed ......................................................181 Effect of T-2 Toxin on Tissues ..............................................................................183 Effect on DNA and Chromosomal Abnormalities ....................................183 Effect of T-2 Toxin on the Circulatory System ........................................184 Effect of T-2 Toxin on the Cardiovascular System...................................188 Effect of T-2 Toxin on Skin ......................................................................188 Effect of T-2 Toxin on the Reproductive System .....................................189 Effect of T-2 Toxin on Liver and Spleen ..................................................189 Effect of T-2 Toxin on the Gastrointestinal Tract.....................................190 Effect of T-2 Toxin on the Brain and Neurotransmitters..........................191 Effect of T-2 Toxin on Lipid Peroxidation ...............................................193 Immunomodulation of T-2 Toxin ..............................................................195 Effect of T-2 Toxin on Humoral Immunity...............................................197 T-2 Toxic Effects on Cellular Immunity ...................................................198 Effect of T-2 Toxin on Host Resistance to Pathogens..............................199 Relation between T-2 Toxin and Apoptosis ..............................................201 Effect of T-2 Toxin on Cultured Cells (In Vitro) ......................................203 Is T-2 Toxin a Carcinogen? .......................................................................203 Conclusion .............................................................................................................204 Acknowledgments..................................................................................................204 References..............................................................................................................204

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Abstract

Trichothecene mycotoxins are a group of more than 300 toxins with only a few causing adverse effects in humans and animals. T-2 toxin, a trichothecene produced by Fusarium species, is prevalent worldwide in cereal crops, oil seeds, sugar beets, cereal-containing foods, and animal products such as eggs and milk. Similar to other trichothecenes, T-2 toxin is heat stable and cannot be destroyed by normal industrial processing. T-2 toxin is by far one of the most potent toxins among all known mycotoxins. The main toxic effect of T-2 toxin is the inhibition of protein synthesis, which arises from its direct binding to the 60S subunit of the 80S ribosome. This inhibition indirectly affects the synthesis of RNA and DNA. The effect of T-2 toxin in animals can be acute or chronic. Acute toxicity of T-2 toxin in animals results in weight loss, nausea, vomiting, abdominal pain and distention, diarrhea, bloody stools, dizziness, chills, inflammation, pharyngeal irritation, destruction of bone marrow, infertility, changes in brain neurochemistry, feed refusal, and lipid peroxidation. However, chronic T-2 toxicity can cause several adverse effects in multiple organs. These include hematological disorders such as neutropenia, thrombocytopenia, aplastic anemia, decreases in white blood cells count, and an increase in clotting time that might lead to coagulation problems. T-2 toxin ingestion for short periods of time causes edema and congestion in the gastrointestinal tract; however, feeding T-2 toxin for long periods cause intestinal necrosis to the epithelium and crypt cells of jejunum and ileum. T-2 toxin modulation of the immune system may be manifested by decreased activities of T and B lymphocytes, suppressed immunoglobulin production, and impaired macrophage activity. On the other hand, depending on the dose, route, and duration of administration, T-2 toxin can stimulate the immune system and superinduce the production of several cytokines. The inhibition of protein, RNA, and DNA synthesis is believed to be directly or indirectly responsible for immune suppression while the interference of the toxin with normal immune regulatory mechanisms might be responsible for immune stimulation. In vivo, T-2 toxin modulates the host resistance to certain bacteria. For example, it increases the host resistance to Listeria monocytogenes, while it decreases resistance to Salmonella typhimurium and has no apparent effect on host resistance to Mycobacterium bovis. The modulation of the immune system to several pathogens may predispose food animals to certain diseases that decrease productivity and might increase the period of shedding the microorganism. This might increase the susceptibility of animals and humans to the transmission of these pathogens. It is concluded that the health and welfare of the animals and to a lesser extent humans may be severely compromised by consumption of food and feeds contaminated with T-2 toxin. Therefore, it will be of great interest to genetically engineer crops resistant to Fusarium and to find methods to detoxify these toxins.

Abbreviations

3

H: titrated thymidine; ALAT: alanine aminotransferase; AMC: alveolar macrophages cells; AP: alkaline phosphatase; Apaf-1: apoptotic proteaseactivating factor-1; APTT: activated partial thromboplastin time; ASAT: aspartate aminotransferase; Aw: water activity; CHO: Chinese hamster ovary; DAS: diacetoxyscirpenol; DFF-40/ICAD: DNA fragmentation factor 45/inhibitor of caspase-activated-

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DNase; DNA, deoxyribonucleic acid; DON, deoxynivalenol; DOPAC: dihydroxyphenylacetic acid; EPR: electron paramagnetic resonance; FAO: Food and Agricultural Organization; GIT, gastrointestinal tract; GOT: glutamate oxaloacetate transaminase; GPT: glutamate pyrovate transaminase; GSH: glutathione; GST: glutathione-S-transferase; HB: hemoglobin; HIAA: hydroxyindoleacetic acid; HPA: hypothalamic-pituitary-adrenal; HPLC: high-performance liquid chromatography; HSV: herpes simplex virus; IC: inhibition concentration; IL: interleukin; IFN: interferon; IV: intravenous; JECFA: Joint Expert Committee on Food Additives; JNK: c-Jun-NH2–terminal kinase; KBD: Kashin-Beck disease; KLH: keyhole limpet hemocyanin; LD: lethal dose; LPS: lipopolysaccharide; MAPKs: mitogen-activated protein kinases; MCH: mean corpuscular hemoglobin; MCV: mean corpuscular volume; MDA: malondialdehyde; MHC: major histocompatibility; NIV: nivalenol; OTA: ochratoxin A; PARP: poly (ADPribose) polymerase; PGE2: prostaglanidin E2; PGF: prostaglandin F; PMTDI: provisional maximum tolerable daily intake; RBC: red blood cells; RNA: ribonucleic acid; SAPK: stress-activated protein kinase; SRBC: sheep red blood cells; TBARS: thiobarbituric acid reactive compounds; TXB2: thromboxane B2; WBC: white blood cells; WHO: World Health Organization; ZEN, zearalenone

INTRODUCTION Mycotoxins are low-molecular-weight secondary metabolites produced mainly by filamentous fungi, particularly molds (Pestka and Bondy, 1990; Corrier, 1991; Charoenpornsook et al., 1998; Galvano et al., 2001). These metabolites have no role in their growth and survival (D’Mello and MacDonald, 1997; Hussein and Brasel, 2001). Of more than 300 isolated mycotoxins, only a few of them including aflatoxins, ochratoxins, trichothecenes, tremorgenic toxins, and ergot alkaloids are considered of particular interest as they cause adverse effects in humans and animals. Ecological, environmental or storage factors are involved in the production of these toxins (Hussein and Brasel, 2001). Trichothecenes are a group of more than 180 alcohol or ester sesquiterpenoids produced by Fusarium, Stachybotrys, Myrothecium, Trichoderma, Trichothecium, and other fungal genera (Kimbrough and Weekley, 1994; Bondy and Pestka, 2000). In addition, they can be isolated from Brazilian shrubs Baccharis magapotomica and Baccharis cordifolia (Kimbough and Weekley, 1994). The trichothecenes produced by Fusarium, include; T-2 toxin, HT-2 toxin, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), fumonisins and more than 25 other trichothecenes (Rosenstein and Lafarge-Frayssinet, 1983). Fusarium fungi are saprophyte organisms that grow at temperatures between 2 and 35°C and ≥ 0.88 Aw are common to soil (D’Mello and MacDonald, 1997; Creppy, 2002) and are found in cereals grown in the temperate climate zones of Europe, North America, and Asia (Schoental, 1979; D’Mello and MacDonald, 1997; Thuvander et al., 1999; Creppy, 2002). All trichothecenes contain an olefinic bond at C9 and C10 and an epoxy ring at C12 and C13; therefore, they are characterized as 12,13-epoxytrichothecenes (Rosenstein and Lafarge-Frayssinet, 1983; Steyn, 1995). In addition, they are divided into four groups; A, B, C and D in which T-2 and HT-2 toxins belong to group A (WHO, 1990; Bondy and Pestka, 2000). T-2 toxin, is a heat stable trichothecene,

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and is considered to be one of the most potent toxic compounds produced by molds, particularly the Fusarium species (Hayes et al., 1980; Yarom, 1986; Schuster et al., 1987; D’Mello and MacDonald, 1997; Bilgrami et al., 1995; Creppy, 2002). A joint FAO/WHO expert committee on food additives (JECFA) has estimated the daily intake of T-2 toxin and HT-2 toxin to be 7.6 and 8.7 ng/kg body weight (Sudakin, 2003). However, the provisional maximum tolerable daily intake (PMTDI) was established by the JECFA to be 60 ng/kg body weight (Sudakin, 2003; Schollenberger et al., 2004). Intoxication of animals with T-2 toxin results in weight loss, nausea, vomiting, pharyngeal irritation, abdominal pain and distention, diarrhea, bloody stools, dizziness, chills, inflammation, destruction of bone marrow, infertility and lipid peroxidation (Tsuchida et al., 1984; Williams, 1989; Atroshi et al., 1997; Creppy, 2002). One of the major effects of T-2 toxin is to modulate the immune system of both humans and animals (Tai and Pestka, 1988b). An acute exposure to T-2 toxin results in severe damage to actively dividing cells in tissues such as bone marrow, lymph nodes, spleen, thymus and intestinal mucosa (Rosenstein and Lafarge-Frayssinet, 1983; Bondy and Pestka, 2000). Further, T-2 toxin has been reported to induce lymphatic necrosis or atrophy, and hematological symptoms such as anemia and leucopenia (Atroshi et al., 1997). Morphological and functional changes in membranes have also been observed in the heart (Yarom et al., 1983), red blood cells (Segal et al., 1983), and the liver (Tremel and Scinicz, 1984). In animals, high doses of T-2 toxin (0.5–15 mg/kg body weight) induced vomiting, gastroenteritis, pneumonia, dermatitis, and reduced feed intake (Muller et al., 1999). Human consumption of the trichothecene mycotoxins lead to several types of mycotoxicoses, including alimentary toxic aleukia, stachybotryotoxicosis, esophageal cancer, “akakabi-byo” (scabby grain intoxication or red mold disease) (Steyn, 1995; Rio et al., 1997; Thuvander et al., 1999; Froquet et al., 2001). Further, it is believed that T-2 toxin is a suspect cause of a bone disease called Kashin-Beck’s disease (KBD), in which T-2 toxin at high doses induces the production of interleukin-1-beta (IL-1) and IL-6, which are involved in degradation of the cartilaginous matrix (Tian Fu et al., 2001). It was widely believed that T-2 toxin was used in the yellow rain warfare agent exploited in Vietnam and Cambodia (Williams, 1989; Bilgrami et al., 1995). However, in a study conducted by Seeley and Nowicke (1985), they concluded that yellow rain was the feces of honeybees and not an agent of a chemical war or a T-2 toxin. The purpose of this chapter is to review the information to date regarding the occurrence of T-2 toxin in food and its effect on animals and humans.

CONTEMPORARY AND HISTORICAL T-2 TOXIN OUTBREAKS Although the first cases of mycotoxicoses were identified and reported in 1960, it appears that mycotoxins were responsible for several outbreaks of the disease throughout written history (Yiannikouris and Jouany, 2002). It is believed that T-2 toxin along with ZEN was responsible for the decline of the Etruscan civilization

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and for the Athenian crisis, which occurred in the fifth century B.C. (Yiannikouris and Jouany, 2002). In addition, it is believed that mycotoxins in the old Egyptian burials were responsible for the death of several archeologists in the last two centuries (Yiannikouris and Jouany, 2002). In recent history, T-2 toxin is believed to be responsible for several mycotoxicoses outbreaks worldwide. Although the toxic agent was never confirmed, it is believed that T-2 toxin was the agent responsible for the alimentary toxic aleukia, which reportedly caused the deaths of thousands of humans in the former Soviet Union between 1919 and 1947 (WHO, 1990; Steyn, 1995). The outbreak occurred in the Orenburg district near Siberia; 10% of the population in the district were fatally affected by consuming grains that were not harvested on time and were rained on for a long period of time, which allowed mold to grow and produce T-2 toxin and other trichothecenes (Steyn, 1995; Rio et al., 1997; Froquet et al., 2001). Major symptoms were severe leukopenia with depletion of immune cells, aplasia of bone marrow, and inflammatory lesions and hemorrhages of the digestive tract (Yang et al., 2000). Necrotic lesions of the oral cavity, esophagus, and the stomach were also reported. In Japan and Korea, in the period between 1946 and 1963, T-2 toxin among other mycotoxins caused a disease called scabby grain intoxication, which affected both humans and animals. However, no fatal cases were reported (WHO, 1990). A similar outbreak was reported in Kashmir in 1987 in which bread made from moldy flour was incriminated in the outbreak. In all, 97 people exhibited abdominal pain, throat irritation, diarrhea, bloody stools, or vomiting. T-2 toxin, among other mycotoxins detected in the flour, was found at concentrations of between 0.55 and 0.8 mg/kg (WHO, 1990). The most recent outbreak was reported by SPA-Associated Press on February 19, 1993. It reported that T-2 toxin-contaminated wheat was responsible for 24 deaths and ailments in several thousand people due to consumption of moldy wheat in southern Tajikistan (Steyn, 1995). T-2 toxin and other mycotoxins have also been implicated in several animal disease outbreaks throughout the world (Steyn, 1995).

STRUCTURE OF T-2 TOXIN T-2 toxin, [3α-hydroxy-4,15-diacetoxy-8α-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-ene] is a member of a family of trichothecenes, which includes, in addition to T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON, or vomitoxin), and NIV. These toxins are compounds containing sesquiterpene rings characterized by the presence of a double bond at the C-9 and C-10 and an epoxy ring at C-12 and C-13 (Figure 7.1) (Hussein and Brasel, 2001; Gutleb et al., 2002). However, these toxins have different constituents on carbon numbers 3, 4, 7, 8, and 15 (Hussein and Brasel, 2001). Among these toxins, T-2 toxin and DAS are the most potent toxins; both are soluble in polar solvents (Williams, 1989; Faifer et al., 1992; Atroshi et al., 1997; Hussein and Brasel, 2001). A comprehensive list of T-2 toxin characteristics is presented in Table 7.1.

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H

H3C

H

H

O OH

H

O H

O CH2 CH2 CH H3C

CH3

OH

CH CH2 3 O C CH3

O C CH3 O

O

FIGURE 7.1 Structure of T-2 toxin. (Adapted from Hussein and Brasel, 2001.)

MODES OF ACTION OF T-2 TOXIN T-2 cytotoxicity has been attributed directly or indirectly to its potent inhibition of protein, RNA, and DNA synthesis and to the breakage of cellular DNA resulting in radiomimetic-like lesions in lymphoid, hematopoietic tissues, and the gastrointestinal tract (GIT) (Ueno, 1977; Corrier and Ziprin, 1986a). This effect is likely to be linked to the 12- and 13-epoxytricothecene nucleus. In eukaryotic cells, the initiation, elongation, and termination of protein synthesis takes place on the ribosome and requires peptidyl transferase (Corrier, 1991). Furthermore, T-2 toxin binding could result in conformational changes in the ribosome itself, rendering it more susceptible to degradation (Rosenstein and Lafarge-Frayssinet, 1983) or it might result in the displacement of the ribosome binding molecule, thus inhibiting protein synthesis (Shifrin and Anderson, 1999). This finding was supported by the work of Witt (1988), who reported that the effect of T-2 toxin was inhibited by anisomycin, an antibiotic that binds to the ribosome suggesting that T-2 toxin binds to the 60S subunit of the 80S ribosome for its action in the inhibition of protein synthesis. T-2 toxin may also react with the thiol group of the peptidyl transferase enzymes required for protein synthesis rendering them ineffective (Karppanen et al., 1989; Thompson and Wannemacher, 1990; Bondy and Pestka, 1991; Corrier, 1991; Rizzo et al., 1994). Cundliffe et al. (1974) also showed that T-2 toxin caused disaggregation of the polyribosomes, which impaired the polypeptide chain initiation leading to inhibition of protein synthesis. Mitchison (1971) and later Thompson and Wannemacher (1990) proposed that inhibition of protein synthesis indirectly inhibits DNA synthesis, which is required to complete mitosis. This in turn results in an inhibition of cell division causing the cytotoxic radiomimetic-like effects of T-2 toxin. In addition to inhibiting the synthesis of cytoplasmic membranes, T-2 toxin at higher doses inhibits the synthesis of mitochondrial proteins by binding to the 70S ribosome (Pace et al., 1988). Altered intercellular communication and deregulation of calcium are also possible mechanisms for the action of T-2 toxin (Jone et al., 1987: Yoshino et al., 1996). T-2 toxin was found to interact with the protein or lipid components of the membrane; consequently, it altered membrane fluidity, which could have disrupted membrane

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TABLE 7.1 Summary of Information on T-2 Toxin Sourcea,b,c Molecular formulac Molecular weightc Melting pointc Occurrenced,e,f,g

Solubilityc Toxicityh,i,j,k,l,m LD50n Symptomso,p,q,r

Fusarium sporotrichioides, F. poae, F. equiseti, F. acuminatum, F. roseum, F. solani, F. tricinctum, and Trichoderma lignorum C24H34O9 466 151–152°C Corn, rice, sugar beets, wheat, rye, barley, oats, beans, soy beans, and other cereal-based products in addition to animal feed and animal products such as eggs and milk Soluble in polar solvents, such as chloroform, diethyl ether, ethyl acetate, and acetone Acute, chronic, cytotoxic, teratogenic, embryogenic, carcinogenic, and mutagenic Varies depending on route of administration and species; lowest was 0.7 mg/kg in rats administered IV and 7 mg/kg in rats administered intragastrically Causes weight loss, nausea, vomiting, pharyngeal irritation, abdominal pain and distention, diarrhea, bloody stools, dizziness, chills, inflammation, destruction of bone marrow, infertility, and lipid peroxidation

a

Hussein and Brasel, 2001. Creppy, 2002. c WHO, 1990. d Miller, 1994. e Mateo et al., 2002. f Bosch and Mirocha, 1992. g Galvano et al., 2001. h Norppa et al., 1980. i Lafarge-Frayssinet et al., 1983. j Schiefer et al., 1987. k Hood, 1986. l Rousseaux and Schiefer, 1987. m Pestka and Bondy, 1990. n See Table 7.2 for references. o Tsuchida et al., 1984. p Williams, 1989. q Atroshi et al., 1997. r Creppy, 2002. b

transport and function such as the transport of amino acids and nucleotides and the glucose activity of the Ca–K channel (Bunner and Morris, 1988). Mitochondrial electron transport is also inhibited by T-2 toxin by suppressing succinate dehydrogenase activity (Khachatourians, 1990). In rat liver, T-2 toxin exerts its action through the generation of free radicals, which cause lipid peroxidation (Sunja et al., 1984; Vila et al., 2002), or by inducing apoptosis in lymphoid organs and tissues.

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TOXICITY OF T-2 TOXIN The toxicity of T-2 toxin and its metabolites is measured by LD50, which is the amount of toxin needed to kill 50% of the laboratory animals receiving the toxin. The value of LD50 is different among animal species with the differences influenced by the route of administration and the susceptibility of the animal to the toxin. When T-2 toxin, for example, was given to mice by inhalation, the effect of the toxin was at least 10 times more than that for systemic administration and 20 times more toxic than topical administration (Sorenson, 1999). Table 7.2 shows the LD50 of T-2 toxin for several animal species. T-2 toxin is considered one of the most potent toxins produced by molds. For example, T-2 toxin is 421 times more toxic than deoxynivalenol (DON), a common trichothecene that has been extensively studied and widely distributed (Vila et al., 2002). Several toxic effects were linked to T-2 toxin; however, some of these effects were proved beyond doubt while others remain presumptive due to the lack of experimental evidence to prove the relation. Immunosuppression and cytotoxicity, as well as teratogenic and embryogeneic effects, have been linked positively to T-2 toxin. However, a clear association between T-2 toxin and mutagenic and carcinogenic effects awaits further investigation. T-2 toxin induced single-strand DNA breakage and minor chromosomal aberration in Chinese hamster ovary (CHO) cells (Norppa et al., 1980; Lafarge-Frayssinet et al., 1983), but did not induce any noticeable aberration in hamsters fed T-2 toxin for 6 weeks. Despite the observed chromosomal aberrations, T-2 toxin was not found mutagenic to Salmonella typhimurium, Saccharomyces cerevisiae, or Drosophila

TABLE 7.2 Lethal Dose Values of T-2 Toxin Administered to Laboratory Animals by Various Routes LD50 Values (mg/kg) Route

Rats

Oral Intramuscular Intraperitoneal Intravenous Subcutaneous Respiratory Intragastric

2.8–3.8a,b — 2.2b 0.7–0.9a 2.0c — 7.0c

a

Mice — — 5.0–5.2b,d — 6.4–8.0c — —

Hussein and Brasel, 2001. Middlesworth, 1986. c Williams, 1989. d Finsk and Fink-Gremmels, 1990. e Guerre et al., 2000. f Placinta et al., 1999. g WHO, 1990. b

Rabbits

Chicken

Guinea Pigs

— 1.1e — — — — —

3.6–5.25f — — — — — —

3.0–4.0c 1.0c — 1.0–2.0c 1.0–2.0c 3.3–4.3c 5.3c

Pigs 5.0c — — 1.21g–3.0c — — —

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(WHO, 1990). However, T-2 toxin was found to be both teratogenic and embryotoxic in mice, causing significant maternal mortality, fetal death, fetal bodyweight loss, and other malformations (Stanford et al., 1975; Hood, 1986; Rousseaux and Schiefer, 1987). In addition, T-2 toxin is a potent immunotoxin. The immunosuppression, cytotoxicity, and the question of whether it is a carcinogen are discussed later in the chapter.

METABOLISM AND ELIMINATION OF T-2 TOXIN IN ANIMALS T-2 toxin is hydroxylated via microsomal P-450 esterases in intestinal and hepatic tissues of rats, mice, pigs, cows, and chickens (Kobayashi et al., 1987). It is hydrolyzed to several products including HT-2, T-2 tetraol, and several other hydroxylated compounds such as 3´-OH HT-2 toxin (TC3) and 3´-OH T-2 toxin (TC-1), which are less toxic than the parent T-2 toxin (Bergmann et al., 1988). Initial hydrolysis of T-2 toxin and/or it hydroxylation to 3´-OH T-2 did not decrease its immunotoxicity while further hydrolysis to T-2 triol or to 3´-OH HT-2 toxin decreased its immunotoxicity significantly (Bondy and Pestka, 2000). Trichothecenes such as T-2, HT-2, T-2 triol, and some other metabolites are further detoxified by glucuronidation, which facilitates their excretion, and by reduction of the epoxy group responsible for their reactivity (Yiannikouris and Jouany, 2002). Also, certain lactic acid bacteria such as Propionibacteria and Bifidobacteria (used in yogurt production) have cell walls that bind mycotoxins and minimize their toxicity (Yiannikouris and Jouany, 2002). Therefore, consumption of yogurt particularly in areas containing foods contaminated with T-2 toxin and other mycotoxins could minimize their toxic effects.

OCCURRENCE OF MYCOTOXINS IN FOOD AND FEED Cereal grains and their products constitute a large proportion of human and animal food. When the temperature and humidity are appropriate for mold growth in these products or plants, a battery of mycotoxins could be produced. Fusarium species such as F. graminearum are widely distributed throughout the environment and are considered major contaminants of cereals as they are the main pathogens of cereal plants. Head blight wheat and barley that is caused by F. graminearum and ear rot in corn are known diseases caused by these pathogens (WHO, 1990; Placinta et al., 1999). Mycotoxins that affect humans and animals are found mainly in postharvest crops particularly cereal grains and forages. As they are lipophilic in nature, they tend to accumulate in the fat fraction of the plants (Hussein and Brasel, 2001). T-2 toxin has been reported in a wide range of plants and agricultural commodities. It has been found in wheat, corn, rice, barley, sorghum, oats, beans, soybeans, rye, peanuts, and safflower oil. Among these crops, wheat, corn, and barley are mainly affected as they constitute about two thirds of the world production of cereals (Miller, 1994; Mateo et al., 2002). In addition, T-2 toxin and its metabolite HT-2 toxin were

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found in processed cereal products from the above-mentioned cereals such as bakery products, particularly bread and crackers (Schollenberger et al., 2004). Interestingly, beets and beet fibers have also been found to contain T-2 toxin at concentrations ranging from 4 to 425 μg/g (Bosch and Mirocha, 1992). T-2 toxin can also enter the food chain through contaminated cereals and other foods despite the decrease in its concentration due to processing and fractionation of the cereal products (Schollenberger et al., 2004). Eggs and milk have been reported to be contaminated with mycotoxins as a result of feeding mycotoxin-contaminated feed to chickens and cows (Galvano et al., 2001). However, extraordinary high levels of T-2 toxin have been reported in India in two agricultural commodities: barley and peanuts containing 25 and 38.9 mg/kg of toxin, respectively (Table 7.3). Production of mycotoxins is strongly influenced by environmental conditions such as humidity, dryness, water activity (Aw), temperature, insect infestation, and microbial interaction (Mateo et al., 2002). It was found, for example, that T-2 toxin was produced in higher concentrations in corn compared to other crops at low temperatures and high Aw while T-2 toxin production in rice was favored by low temperatures (26°C) and low Aw (Mateo et al., 2002). On average, about 25% of the world’s food crops become contaminated with mycotoxins annually. However, this proportion is more severe in some parts of the world. In India, where temperature and humidity favors rapid mold growth, mycotoxins are present in high concentrations. For example, among 387 cereal samples

TABLE 7.3 Summary of Natural Occurrence of T-2 Toxin in Agricultural Commodities Worldwide Crop Wheat

Barley Corn

Grain

Oats Peanuts Sorghum Beets

Level (mg/kg)

Incidence

Country

Ref.

0.003–0.249 0.013–0.63 2.0–4.0 0.02–2.4 25.0 0.2–0.64 0.5–5.0 0.08–0.65 0.01–0.2 0.04–0.8 — 0.16–0.31 0.01–0.05 0.63–38.9 1.7–15 0.004–0.425

— — 3/12 12/24 — 5% 5/150 9/118 13/20 2/20 22% 5/55 — 6/87 4/84 —

Germany Romania India Poland — Canada Hungary Taiwan New Zealand Brazil Argentina Canada Finland India India U.S.

Curtui et al., 1998 Curtui et al., 1998 WHO, 1990 Placinta et al., 1999 WHO, 1990 Charmley et al., 1994 WHO, 1990 WHO, 1990 WHO, 1990 Placinta et al., 1999 Placinta et al., 1999 Stratton et al., 1993 WHO, 1990 WHO, 1990 WHO, 1990 Bosch and Mirocha, 1992

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tested for mycotoxins, about 51% were found to be contaminated with one or more mycotoxins (Fink-Gremmels, 1999). In Europe, about 20% of the crops grown for food or animal feed were reported to contain mycotoxins (Gutleb et al., 2002). However, T-2 toxin and its metabolite, HT-2, were detected in wheat at low levels, 0.003 to 0.25 and 0.003 to 0.02 mg/kg, respectively. DON, 3-ADON, and ZEN were also detected in combination with T2 toxin and its metabolites. In Germany, for example, T-2 toxin was found at levels of 3 to 249 μg/kg in wheat samples while in western Romania T-2 toxin was found at lower levels, 13 to 63 μg/kg (Curtui et al., 1998). In Poland, higher amounts of T-2 toxin were reported. Of 24 barley samples, 12 were found to contain high amounts of T-2 toxin or its metabolites, ranging from 0.02 to 2.4 mg/kg (Placinta et al., 1999). Interestingly, T-2 toxin and its metabolite HT-2 toxin were below detection limits in Asia, which could be due to the lack of accurate methods for its detection and quantification (Placinta et al., 1999). Surprisingly, in North America where most of the world’s grain is produced, grains are not heavily contaminated with T-2 toxin. For example, among 55 samples of grain tested in Canada, only 5 were contaminated with T-2 toxin at levels ranging from 0.16 to 0.31 mg/kg and two samples were contaminated with HT-2 toxin at levels from 0.12 to 0.44 mg/kg (Stratton et al., 1993). Further, in random samples taken from grains across Canada, only 4.9% of the tested corn samples were found contaminated with 0.2 to 0.64 mg/kg of T-2 toxin (Charmley et al., 1994). Similarly, the prevalence of T-2 toxin in South American grains is not high. In Brazil, among 20 grain samples tested there were only 2 samples contaminated with T-2 toxin at levels of 0.04 and 0.8 mg/kg. In Argentina, 20 to 22% of grain samples were found to contain either DAS or T-2 toxin. However, the lack of large volumes of information on the presence of T-2 toxin in food and feed could be attributed to the fact that regulatory issues pertaining to this toxin are not well defined, as is the case for the aflatoxins and DON (Placinta et al., 1999). Table 7.3 summarizes the information of the occurrence of T-2 toxin in different crops around the world.

EFFECT OF T-2 TOXIN ON TISSUES EFFECT

ON

DNA

AND

CHROMOSOMAL ABNORMALITIES

T-2 toxin inhibits protein synthesis, which indirectly affects RNA and DNA production and consequently causes abnormalities. When T-2 toxin was administered for 6 weeks to albino mice, it was reported to cause two types of chromosomal abnormalities: individual abnormalities, such as breakages, chromatid gaps, ring formation, and widespread fragmentation of chromosomes, and gross abnormalities. The gross abnormalities were the formation of polyploid and hypoploid, pulverization, stickiness, and clumping of the entire chromosomal material (Bilgrami et al., 1995). Rosenstein and Lafarge-Frayssinet (1983) reported that when T-2 toxin was administered to mice, DNA synthesis was clearly inhibited in the spleen, thymus, and bone marrow within a very short time. In addition, RNA and protein synthesis were also inhibited. This clearly indicates the susceptibility of lymphoid organs to T-2 toxin. However, this effect was reversed after clearance of the toxin from the

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body. These inhibitory effects have been attributed to the binding of T-2 toxin to subcellular components such as sulfhydryl groups and ribosomes (Atroshi et al., 1997) or to the suppression of peptidyl transferase activity, thus preventing chain initiation and elongation of proteins (Wang et al., 1998b). Moreover, DNA damage by T-2 toxin could be mediated by an increased level of intracellular calcium ion, which transduces the activation signals for endonuclease and proteases (Shokri et al., 2000). In addition to inhibition of DNA and protein synthesis, T-2 toxin introduced nicks in the DNA of lymphoid and hepatic cells, albeit to a lesser extent in the DNA of hepatic cells (Rosenstein and Lafarge-Frayssinet, 1983). Atroshi et al. (1997) reported that DNA-fragmentation was increased by 71% within 4 h of T-2 toxin administration in vitro and in vivo (into liver cells). It was also found that administering antioxidants prior to toxin administration prevented this fragmentation. Vitamin C was found to decrease the effect of T-2 toxin-induced damage. The protective mechanism of vitamin C is not understood. However, it might be due to its antioxidant activity, which decreases the formation of free radicals that are probably involved in chromosomal damage. Table 7.4 shows a summary of the major effects of T-2 toxin on tissues.

EFFECT

OF

T-2 TOXIN

ON THE

CIRCULATORY SYSTEM

Several hematological disorders have been reported in animals as a result of ingestion of T-2 toxin. Among these disorders are neutropenia, thrombocytopenia, aplastic anemia, and coagulation anomalies (Johnsen et al., 1988; Froquet et al., 2001). These disorders could have been caused by either direct destruction of circulating blood cells or altered hematopoiesis in the bone marrow at the level of proliferation or differentiation (Rio et al., 1997). Experiments performed on the effect of T-2 toxin on erythrocytes showed that T-2 toxin effect was dependent on the origin of the cells. For example, bovine erythrocytes are more resistant to T-2 toxicity than rat erythrocytes, which were severely damaged by the toxin. The difference in susceptibility was related to differences in the lipid components of the erythrocyte membranes particularly phosphatidylcholine and sphingomyelin (Holt et al., 1988; Khachatourians, 1990). A single dose of T-2 toxin given to rabbits caused a decrease in hematocrit, white blood cells (WBC) count, and serum alkaline phosphatase activity; prolongation of the activated partial thromboplastin time (APTT); and an increase in clotting time that might lead to hemorrhage (Gentry and Cooper, 1981). However, the hematocrit declined only for 2 days and went up to normal levels after day 4. Similarly, WBC counts as well as the APTT started to return to normal levels by the day 4 (Gentry and Cooper, 1981). The administration of T-2 toxin for several weeks appeared to cause a severe morbid effect on the circulatory system. Hayes et al. (1980) reported that when mice were fed T-2 toxin for up to 6 weeks they developed normochromic and normocytic anemia. The occurrence of these types of anemia could be due to the short life span of the mice red blood cells (RBC), which ranges from 65 to 20 days, as opposed to the human RBC life span of more than 100 days. However, the RBC counts were restored on withdrawal of the toxin from the diet, therefore, reversing the effect of the toxin. In addition to anemia, a decrease in the mean corpuscular volume (MCV)

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TABLE 7.4 Summary of the Major T-2 Toxin Effects on Tissues and Organs Tissue

Major Effect

Ref.

Chromosomes

Breakage, chromatid gaps, ring formation; fragmentation Rosenstein and Lafargeof polyploid and hypoploid, pulverization, stickiness, Frayssinet, 1983; and clumping of the entire chromosomal material Bilgrami et al., 1995

Circulatory system

Neutropenia; thrombocytopenia; aplastic anemia; coagulation anomalies; and hematocrit abnormalities

Froquet et al., 2001, Johnsen et al., 1988

Cardiovascular system

Cardiac insufficiency-induced systemic hypoxia; severe electrodynamic and ultrastructural changes; dilation and swelling of capillaries; hemorrhage; hypovolemic shock; hypotensive shocklike state

Yarom et al., 1986, 1987b; Borison et al., 1991

Skin

Dilated blood vessels; erythema and induration; ulceration and scab formation; severe cellular infiltration and vascular stagnation; increase in mast cell numbers; skin lesions; ischemic necrosis to epidermis

Yarom et al., 1987a; Cavan et al., 1988; Albarenque et al., 1999

Reproductive system

Decreased testosterone production, degeneration and necrosis of spermatogenetic cells, abortion

Fenske and FinkGremmels, 1990; Placinta et al., 1999

Lymphoid tissue

Decreased spleen weight; liver enlargement; thymus atrophy; liver necrosis; lipid peroxidation in liver

WHO, 1990; Velazco et al., 1996; Dugyala and Sharma, 1997; Vila et al., 2002

GIT

Increase in stomach size; increase in intestinal Hayes et al., 1980; propulsion; induction of pyknosis and karyorrhexis of Suneja et al., 1984; crypt epithelial cells; swelling of villi and necrotization; Taylor et al., 1989; ulceration of intestinal mucosa; hemorrhage; lesions in Williams, 1989 mucosa; hyperkeratosis and hyperplasia of squamous epithelium

Brain and neurotransmitters

Damage to the endothelial capillaries of blood vessels; Chi et al., 1981; Cavan altered entrance of tryptophan and tyrosine into brain; et al., 1988; disruption of monoamine metabolism by inhibiting MacDonald et al., monoamine oxidase; impaired wing positioning; 1988; Wang et al., hysteroid seizures and impaired righting reflex; 1998a, b increased dopamine; decreased norepinephrine; altered amino acid metabolism; hemorrhagic necrosis

Immune system

Severe damage to actively dividing cells in bone marrow, Rosenstein et al., 1979; Pestka and Bondy., lymph nodes, spleen, thymus, and Peyer’s patches; decreased B- and T-lymphocyte populations; prolonged 1990, 1994; Smith et al., 1994; Rafai et al., graft rejection; enhanced immune system; decreased 1995; Islam et al., IgM, IgA, and IgG production; superinduced cytokine 1998; Bondy and production; inhibition of mitogen-stimulated response Pestka, 2000 of both B and T lymphocytes; impaired phagocytic activity and proliferation; decreased CD3 and CD19

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during the period of toxin intake and a slight increase in both mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) have been reported by Hayes and coworkers (1980). Feeding pigs a diet containing 2 to 3 mg/kg T-2 toxin sharply reduced hematocrit and RBC counts. Further increases in the amount of administered toxin decreased leukocyte numbers and hemoglobin concentration (Rafai et al., 1995). However, prolonged administration of T-2 toxin abrogates its effect as indicated by the regeneration of hemopoietic tissue in which granulopoiesis and thrombopoiesis become active after 3 to 4 weeks, while lymphoid tissue stays depleted (Friend et al., 1983b). Acute T-2 toxicosis does not affect the RBC linage, but chronic exposure could lead to such an effect (Rio et al., 1997). Further, a single dose of T-2 toxin produces transient damage, while repeated doses can produce significant damage to the hematopoietic precursor cells (Faifer et al., 1992). At the organ level, the spleen size decreased at the beginning but increased again by the end of the 6-week period. Similarly liver size increased during the course of toxin administration while the thymus size decreased throughout the whole experiment (Friend et al., 1983b; Vila et al., 2002). The mechanism by which T-2 toxin caused this effect is not well understood. Nevertheless, it has been proposed that inhibition of either protein or DNA synthesis may have been caused by inhibition of the enzymes that are necessary for the synthesis of deoxyribonucleotides (Hayes et al., 1980). When T-2 toxin was administered to pigs by inhalation, it caused alteration to the cardiovascular system and tissue perfusion as indicated by the decreased body temperature (Rotter et al., 1994). Young et al. (1983) reported alterations in blood serum parameters following the exposure to mycotoxins. However, it was not clear if this effect was due to the toxin or merely due to altered feed intake. Also, Rafai and Tuboly (1982) reported a significant increase in the amounts of cortisol in the plasma of pigs fed 5 mg/kg of T-2 toxin. An increase in albumin levels and a decrease in the α-globulin fraction were also observed in pigs fed a diet contaminated with Fusarium (Rotter et al., 1994). When pigs were given a single dose of 0.6 to 4.8 mg/kg of T-2 toxin, early leukocytosis followed by a reduction in neutrophils, Tlymphocyte numbers, MCV, RBC count, and hemoglobin (HB) concentrations was reported (Rafai et al., 1995). These findings clearly suggest a disturbed hematopoiesis in these pigs. However, Friend et al. (1992), reported contradictory results to the above findings. They have reported that pigs fed up to 8 mg/kg of T-2 toxin showed no differences in leukocyte counts, morphology of lymphoid tissues, MCV, hematocrit, or HB concentrations. To measure the effect of T-2 toxin on the erythropoietic activity of the spleen and bone marrow, an iron-radioisotope was given to the animals after the administration of T-2 toxin and the fate of the isotope was followed. It was found that initially the uptake and incorporation of the radioisotope into the spleen, bone marrow cells, and erythrocytes was strongly inhibited by T-2 toxin in a dosedependent manner. This could have been caused by the sharp decrease in bone marrow cells (Faifer et al., 1992). The erythropoietic activity in spleen rapidly recovered while it stayed significantly reduced in the bone marrow (Velazco et al., 1996; Rio et al., 1997). This behavior was thought to be due to the migration of

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progenitors and stem cells from the bone marrow to spleen during this recovery phase (Velazco et al., 1996). Further, mice fed a diet containing 20 ppm of T-2 toxin for 6 weeks developed bone marrow hypoplasia (Hayes et al., 1980). Necrosis of bone marrow was also reported in guinea pigs after receiving a lethal dose of T-2 toxin. In a separate experiment, guinea pigs showed reduced systemic blood pressure and heart rate (Cavan et al., 1988). It is noteworthy here to emphasize that the bone marrow seems to be extremely sensitive to T-2 toxin. This might be due to the high fat content of the marrow and to the lipophilic nature of the toxin, which facilitates the uptake and concentration of the toxin (Froquet et al., 2001). In vitro, T-2 and HT-2 toxins were found to induce strong cytostaticity characterized by a decrease in the number of large blood cell progenitors (BFU-E) and an increase in small ones. In addition, T-2 toxin was cytotoxic for human megakaryocytes progenitors. However, total destruction of the cells was not observed and none of the trichothecene toxins induced any morphological changes in the cells (Faifer et al., 1992; Froquet et al., 2001). However, it is not ruled out completely that a chronic exposure to T-2 toxin cannot cause partial depletion of stem cells or hematopoietic precursor stem cells. Platelets and all other coagulation factors except fibrinogen were also affected by T-2 toxin administration to animals or animal cells (WHO, 1990). Studies have shown that T-2 toxin could impair platelet function both in vivo and in vitro. T-2 toxin especially appeared to inhibit platelet aggregation at concentrations between 5 and 500 μg/109 platelets in a dose-dependent manner even in the presence of activators (WHO, 1990). Nevertheless, the effect of the toxin on the platelet aggregation was simply reversed by eliminating the toxin (Mizutani et al., 1997). However, the actual mechanism of T-2 effect on platelet aggregation is not known. In contrast to the above studies, Gentry (1982) reported that circulating platelet numbers were not affected by T-2 toxin administration. Studies have shown that the administration of crude T-2 toxin to cows, rats, and calves caused an increase in the plasma prothrombin time, an increase in plasma clotting time, and produced hemorrhage in the intestine (Gentry, 1982; WHO, 1990). At the same time, the concentration of plasma fibrinogen increased within the first 24 h of T-2 toxin administration, which could reflect a response to the stress caused by the administration of the toxin (Gentry, 1982). Factors responsible for the increased incidence of hemorrhages may be suppressed prothrombin production, inhibition of platelet aggregation (Ueno, 1984), or direct physical damage of the blood vessels (Schoental et al., 1979). Another assumption is that T-2 toxin could inhibit the production of proteins including coagulation factors (Johnsen et al., 1988). In fact, Gentry (1982) reported that the activities of plasma factors VII, VIII, IX, X, and XI in rabbits were deceased by approximately 40% 6 hours after the toxin administration, which supports the above assumption. In addition, Gentry (1982) reported that a decrease in plasma factor XI leads to a prolonged APTT, which was reflected on the time of coagulation. When tested on human platelet progenitors (CFU-MK), T-2 toxin was found to be cytotoxic at a concentration of 10–7 mol (Froquet et al., 2001). In addition, it functions as an anticoagulant as it markedly reduced coagulation after 10 to 24 h of its administration. This effect was apparent from depletion of both prothrombin

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and its inhibitor, antithrombin III (Johnsen et al., 1988). Fibrinolysis was also affected by T-2 toxin as apparent from the consumption of antiplasmin (Johnsen et al., 1988).

EFFECT

OF

T-2 TOXIN

ON THE

CARDIOVASCULAR SYSTEM

As blood carrying T-2 toxin to all organs passes through the heart, it is inevitable that heart tissue will be affected by the toxin. Wang et al. (1998b) reported that T2 toxin administration to rats caused cardiac-insufficiency-induced systemic hypoxia. Ex vivo injections of T-2 toxin into isolated rat hearts induced severe electrodynamic and ultrastructural changes within 1 h of administration with the intramyocardial blood vessels being affected more than the myofibers (Yarom and Yagen, 1986). At 4 h after the toxin administration, the capillaries become widely dilated and the endothelium suffered extensive swelling. However, no hemorrhages occurred nor were the vessels torn. At 24 h later, more pathological changes were observed. The capillaries were widely dilated and their lumen contained red blood cells and some membrane debris. Further damage to the capillaries was observed as they were torn and their contents spilled out. Hemorrhages were also observed in the interstitial space around the small vessels (Yarom and Yagen, 1986). Borison and colleagues (1991) reported that cats died of hypovolemic shock when given T-2 toxin injection. The toxin caused internal hemorrhage as was apparent from bloody diarrhea. Further, they reported a 70% increase in hematocrit, which increased the viscosity of the blood and, therefore, decreased its rate of flow, thus predisposing the animal to hypotension and cardiac failure. Moreover, T-2 toxin affected the respiratory and blood pressure centers causing a hypotensive shocklike state. This condition could be due to the action of lysosomal enzymes secreted by the polymorphonuclear neutrophils damaged by the toxin (Yarom et al., 1987b). T-2 toxin also increased the concentrations of norepinephrine and epinephrine in heart, reaching a maximum value within 12 h of dosing (Yarom et al., 1987b; MacDonald et al., 1988). Certain morphological changes were observed in the aorta membranes of rats administered repeated doses of T-2 toxin (Yarom et al., 1983, 1987b; Schuster et al., 1987). This observation further supports the speculation that T-2 toxin could be considered as an environmental atherogenic agent (Yarom et al., 1987b).

EFFECT

OF

T-2 TOXIN

ON

SKIN

Skin is one of the most important sites for T-2 toxin to exert its deleterious effects. Studying T-2 toxicity through the skin is very important as farmers and grain workers could be exposed to this toxin via this route. The effect of T-2 toxin via skin starts minutes after the exposure with a threshold for its effect to be 0.5 mcg/cm2 (Sudakin, 2003). However, it takes 12 h for a dose-related inflammation characterized by increased amounts of erythrocytes in dilated vessels to appear (Yarom et al., 1987a; Cavan et al., 1988). Then, 24 h later, erythema and induration of the skin appears with an increased number of mononuclear cells at the site (Yarom et al., 1987a; Bhavanishankar et al., 1988). Skin shows ulceration and scab formation 2 to 3 days after administration of high doses (up 100 μg) of T-2 toxin. At this stage the

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subcutaneous layer shows severe cellular infiltration and vascular stagnation with an increase in the number of mast cells of which 40 to 70% appears to be degranulated. In addition, T-2 toxin was also found to induce skin lesions, which could be due to damage in the microvessels of dermis and subcutis. This damage interferes with the oxygen supply to tissues, causing an ischemic necrosis to the epidermis (Yarom et al., 1987a; Albarenque et al., 1999). At later stages, even the muscles beneath the skin become necrotic and infiltrated with polymorphonuclear granulocytes (Yarom et al., 1987a). Intracytoplasmic edema and apoptosis have been also reported (Albarenque et al., 1999). Interestingly, when the effects of other mycotoxins on the skin were compared, T-2 toxin was the most potent toxin followed by DAS, fusarenon D, and butenolide, which had the lowest potency (Bhavanishankar et al., 1988). On contact with skin, normal allergens cause wide opening of junctions between endothelial cells through the release of histamine and serotonin. However, on T-2 toxin exposure, the failure of these junctions to open suggests that T-2 toxin inhibits the release of both serotonin and histamine. This was also apparent from the failure of antihistamine drugs to prevent skin reaction (Yarom et al., 1987a). It appears that small blood vessels and mast cells are targets for the T-2 toxin; however, Albarenque and co-workers (1999) reported that no morphological changes were apparent in the mast cells. It has been suggested that T-2 toxin in general exerts its effect by damaging the actively dividing cells of skin via inhibition of protein synthesis (Johnsen et al., 1988).

EFFECT

OF

T-2 TOXIN

ON THE

REPRODUCTIVE SYSTEM

Actively dividing cells in testes and ovaries are very susceptible to damage induced by T-2 toxin (Chang and Mar, 1988). Further, T-2 toxin was found to decrease the production of testosterone from gerbil testicle cells in a dose-dependent manner. In addition, degeneration and necrosis of spermatogenetic cells have been reported in guinea pigs (Fenske and Fink-Gremmels, 1990). T-2 toxin has also been shown to cause infertility in pigs, and when administered parenterally during the last trimester of gestation, it caused abortion within 48 h (D’Mello et al., 1999).

EFFECT

OF

T-2 TOXIN

ON

LIVER

AND

SPLEEN

Liver, spleen, and thymus are considered vulnerable organs for T-2 toxin. Muscles and liver appear to retain higher amount of T-2 toxin for longer periods than other organs. When pigs were given between 0.4 and 0.1 mg of titrated thymidine (3H)toxin by gastric gavage, the retention of toxin by the body organs after 18 h varied; muscles retained 0.7%, liver retained 0.29 to 0.43%, while kidneys and bile retained 0.1% (WHO, 1990). Similarly, a high amount of T-2 toxin (1370 μg/kg) was recovered from the liver of chicken 18 h after an intraperitoneal injection of 3.5 mg/kg of the toxin (WHO, 1990). This trend was also seen in several other animals such as cows. The size of vital organs appeared also to be affected by the administration of T2 toxin. Vila et al. (2002) reported a decrease in the spleen weight after T-2 toxin

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administration, while liver weight increased. The increase in liver weight in rats could have been related to an increase in its hepatic lipid content after T-2 toxin administration, and it was likely caused by a reduced level of lipid metabolism due to inhibition of lipoprotein synthesis (Meloche and Smith, 1995). Earlier, Dugyala and Sharma (1997) reported an increase in the weight of spleen, especially after a high dose of T-2 toxin administration, and a decrease in thymus weight, which was related to cortical depletion of the thymocytes. Liver and intestine also developed necrosis after T-2 toxin administration. Hayes and co-workers (1980) reported that a single dose of T-2 toxin produced a striking reduction in spleen and thymus weight; however, continuous administration of the toxin markedly increased spleen weight. This appears to be due to an initial hypoplasia at the beginning, and a compensatory effect after the continuous exposure of the spleen to the toxin. Similarly, an initial hypoplasia was observed in the femoral bone marrow, but there was no compensatory effect after prolonged exposure. As a consequence, spleen erythropoietic activity was restored quickly while that of the bone marrow did not return to normal values until after a period of 3 weeks (Velazco et al., 1996). On administration of T-2 toxin, liver damage can be assessed by several biochemical changes that occur within a short time of the administration including a reduction in the drug-metabolizing capacity of the liver (Meloche and Smith, 1995). Furthermore, the increased production of alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), and alkaline phosphatase (AP) enzymes has long been used as a sensitive indicator of liver diseases, with AP as the most sensitive (Johnsen et al., 1988; Atroshi et al., 2000). In addition to the above enzymes, an increase in the concentration of glutamate oxaloacetate transaminase (GOT) and glutamate pyrovate transaminase (GPT) activities of the liver is a good indicator of liver damage. When, for example, T-2 toxin was administered to rats, GOT was increased by 90% 6 h after its administration while GPT was increased by 104% (Chang and Mar, 1988). In contrast, a 19% decrease in liver glutathione, another marker of liver damage, was observed after T-2 toxin treatment (Atroshi et al., 1997). Despite all the mentioned effects of T-2 toxin on liver, the liver is not considered a prime target for the toxin as it has high contents of glutathione-S-transferase (GST), which catalyze the reaction with glutathione thiol moiety in T-2 toxin, thus reducing its toxicity (Chang and Mar, 1988). T-2 toxin has been reported to induce secretion of high amounts of epinephrine in blood, causing a shock syndrome (MacDonald et al., 1988). Further, T-2 toxin treatment enhanced lipid peroxidation in liver as indicated by increased malondialdehyde (MDA) content in liver homogenates (Vila et al., 2002). Preadministration of antioxidants such as vitamin E and CoQ10 provided some protection against T-2 toxin and minimized liver damage (Atroshi et al., 1997; Vila et al., 2002).

EFFECT

OF

T-2 TOXIN

ON THE

GASTROINTESTINAL TRACT

T-2 toxin is a highly lipophilic substance that is readily absorbed from the gastrointestinal tract (GIT) (Cavan et al., 1988). As a protein inhibitor, T-2 toxin causes cytotoxic radiomimetic-like lesions in the rapidly dividing cells of the GIT (Corrier, 1991). The administration of T-2 toxin to mice caused an increase in the size of the

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stomach itself and an increase in intestinal propulsion that lasted for 4 days (Hayes et al., 1980). The effect on propulsion was hypothesized to be caused by either an increase in the production of prostaglandins and eicosanoids within the brain or simply by changes in mesenteric blood flow (Williams, 1989). In addition, T-2 toxin induced pyknosis and karyorrhexis of the crypt epithelial cells in mice small intestine along with a marked edema and swelling of the villi and necrotization and ulceration of the intestinal mucosa (Williams, 1989). Gastric mucosal hyperemia, hemorrhage, superficial mucosal necrosis, and subacute catarrhal gastritis were observed in rabbits orally given 0.5 mg T-2 toxin/kg/day. In addition, voluntary food intake was decreased by 60 to 70% even at lower T-2 toxin dose (Fekete et al., 1989). Feed refusal is one of the major syndromes of T-2 toxin administration of animals and birds. Ingestion of T-2 toxin by these animals or birds caused vomiting, which was dose dependent. The vomiting that accompanied the administration of T-2 toxin may be due to damage or irritation to the gastrointestinal mucosa. T-2 toxin ingestion for short periods of time in cattle and swine also caused edema and congestion in the GIT. However, feeding T-2 toxin for long periods caused intestinal necrosis, which was associated with an increase in lysosomal enzyme release (Suneja et al., 1984; Williams, 1989). T-2 toxin administration to swine also caused congestion and hemorrhage in the stomach and the intestine. In addition, necrosis to the epithelium and crypt cells of jejunum and ileum was observed (Williams, 1989). In turkey, lesions in the mouth were also reported after chronic exposure to T-2 toxin. In addition, Taylor et al. (1989) reported that the administration of T-2 toxin to rats caused various GIT lesions consisting of hyperkeratosis and hyperplasia of the squamous epithelium of the esophagus and stomach accompanied with ulceration and edema. The effect of T-2 toxin on the intestinal transport system, intestinal brush border enzymes, and lysosomal enzymes was studied in rats (Suneja et al., 1984). The uptake of glucose and tryptophan by everted intestinal segments decreased markedly after feeding rats diets containing T-2 toxin. Moreover, brush border sucrase, lactase, and (Na+-K+)-ATPase activities also decreased, which directly affected the uptake of sugar and amino acids, suggesting an interaction of the reactive site of T-2 toxin with the thiol group of these enzymes. However, there was no significant release of the lysosomal enzymes acid phosphatase and acid ribonuclease (Suneja et al., 1984). In addition, T-2 toxin administration induced some morphological changes in the intestine particularly at high concentrations. Transport of proteins was also inhibited due to either decreased protein synthesis or to the direct interaction with the epoxy group of the T-2 toxin (Suneja et al., 1984).

EFFECT

OF

T-2 TOXIN

ON THE

BRAIN

AND

NEUROTRANSMITTERS

T-2 toxin is known to be pathogenic to the circulatory system. It may cause dilation and swelling of the microvascular system, plasma membrane damage, and tearing of the blood vessel walls (Wang et al., 1998a). Its lipophilic nature might interfere with normal membrane functions including altering amino acid transport through the blood–brain barrier (Wang et al., 1998b). Similarly, the endothelial capillaries of the blood vessels in the brain could be damaged by T-2 toxin administration or

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it simply might disrupt the functional integrity of this barrier by altering the entrance of tryptophan and tyrosine into the brain (Wang et al., 1998a, b). In addition, it may disrupt monoamine metabolism in the brain by inhibiting synthesis of monoamine oxidase, an enzyme that breaks down neurotransmitters (Wang et al., 1998b). When T-2 toxin was administered to chickens, it impaired their wing positioning, caused hysteroid seizures, and impaired righting reflex (Cavan et al., 1988; Wang et al., 1998a). Cavan et al. (1988) also demonstrated that the brain neurotransmitter dopamine was increased while neurotransmitter norepinephrine was decreased following a T-2 toxin dose in chickens. However, MacDonald et al. (1988) reported that after an acute dose of T-2 toxin, norepinephrine was not affected while dopamine was increased. In another study, they reported that pigs given T-2 toxin by the intravascular route showed a dramatic increase in both norepinephrine and epinephrine concentrations. Chi et al. (1981) also reported an increase in norepinephrine in chicken brains 24 to 48 h after an acute dose of T-2 toxin. They related this to the inhibition of dopamine — hydroxylase after the administration of T-2 toxin. In addition, an acute ingestion of T-2 toxin produced an increase in the tryptophan and serotonin and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), which was followed by a gradual increase in dopamine levels while its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) decreased (MacDonald et al., 1988). It appears that the altered metabolism of amino acids might account for the observed perturbations in brain neurochemistry. The other possible reason for the change in neurotransmitter concentrations could be due to the inhibition of the synthesis of monamine oxidase, which oxidizes serotonin, therefore, elevating its concentration. The high concentration of serotonin and tryptophan is believed to be responsible for the loss of appetite and, thus, food refusal in animals exposed to T-2 toxin (Cavan et al., 1988; Meloche and Smith, 1995). Manipulation of the precursor amino acids in the diet may, therefore, provide a basis for overcoming feed refusal in animals. For example, introducing diets that elevate amino acids (e.g., leucine) that compete with the transport of tryptophan through the brain–blood barrier would reduce tryptophan transport and/or might reduce the ratio of tryptophan to the large neutral amino acids in the brain. This, in turn, reduces the production of serotonin (5hydroxytryptamine, 5-HT) and its metabolite 5-HIAA (Cavan et al., 1988; Meloche and Smith, 1995). T-2 toxin, in addition to affecting the neurotransmitters in the brain, affects the levels of plasma eicosanoids as it induces a transient increase in the release of 6keto-prostaglandin F (PGF) and thromboxane B2 (TXB2) from the brain cortex (Shohami and Feuerstein, 1985). In addition, prostaglandin E2 (PGE2) release was elevated in the cortex and hypothalamus in a dose-dependent manner. This probably reflects an increase in arachidonate turnover concomitant with an increase in the activity of PGE2. On the other hand, prostacyclin and thromboxane A2 did not seem to be affected after a short-term application of T-2 toxin (Shohami and Feuerstein, 1985). Further, an acute injection of solid T-2 toxin directly to the brains of rats caused hemorrhagic necrosis in the brain tissue by forming intrinsic prostaglandins that might be responsible for the damage (Bergmann et al., 1988).

T-2 Mycotoxin in the Diet and Its Effects on Tissues

EFFECT

OF

T-2 TOXIN

ON

193

LIPID PEROXIDATION

Lipid peroxidation is defined as the attack of the unsaturated bonds of membrane phospholipids by highly reactive polyunsaturated fatty acid hydroxyl free radicals (Hoehler et al., 1998; Atroshi et al., 2000). It was hypothesized that T-2 toxin is taken up by the membrane bilayer as an analogue to cholesterol as both possess a similar charge shift electrophoresis pattern (Khachatourians, 1993). Once taken up by the bilayer, T-2 toxin and its metabolites induce lipid peroxidation by generating free radicals that cause damage to cell membranes (Leal et al., 1999). Much of the free radicals are scavenged by glutathione, a cellular antioxidant; however, it is believed that T-2 toxin binds to the SH-group of this antioxidant, therefore, diminishing its capacity to minimize cellular damage exerted by these free radicals (Ueno and Matsumoto, 1975). The liver as a detoxification apparatus in the body (Schuster et al., 1987) has an elaborate antioxidant defense system that can metabolize regular doses of toxins. However, at a high dose of T-2 toxin, liver necrosis and damage to other tissues such as spleen, bone marrow, thymus, and kidney is inevitable (Shokri et al., 2000). T-2 toxin administration leads to a pronounced increase in thiobarbituric acid reactive compounds (TBARS) in liver homogenates of T-2 toxin-treated rats (Tsuchida et al., 1984). Activities of some other hepatic enzymes such as catalase, superoxide dismutase, cytochrome P-450 and GST have been shown to be markedly decreased as a result of the lipid peroxidation induced by the administration of T-2 toxin (Rizzo et al., 1994). In addition, Rizzo et al. (1994) reported an increase of 79% in the concentration of TBARS in rats treated with T-2 toxin and given antioxidant supplement of selenium and vitamin E. However, when rats were given the same amount of T-2 toxin but no antioxidants, TBARS concentrations were increased to 268%. This was interpreted as an indicator of the presence of lipid peroxidation products such as 2alkenals, 4-hydroxy-alkenals, and malondialdehyde (MDA), which have been reported to be present in the liver after acute exposure to T-2 toxin (Karppanen, 1989; Rizzo et al., 1994; Leal et al., 1999; Guerre et al., 2000). Concomitant with the TBARS increase, there was a decrease in hepatic glutathione (GSH) (Leal et al., 1999). Segal et al. (1983) also reported that T-2 toxin stimulated lipid peroxidation in rat livers. However, Schuster and co-workers (1987) reported contradictory results, and they concluded that thiobarbituric acid amounts were not increased after treating their rats with T-2 toxin, therefore, casting doubts about the role of T-2 toxin in lipid peroxidation. The above authors, however, used subjective methods that measured the evolved hydrocarbon gases or TBARS. Looking for another marker that might give more reliable data about the role of T-2 toxin in lipid peroxidation, Vila et al. (2002) studied the presence of MDA in mice livers using an accurate high-performance liquid chromatography (HPLC) method and reported that MDA levels were always higher for toxin-treated mice than the control (Figure 7.2). Moreover, the MDA levels were inversely related to the amount of vitamin E in the diet (Figure 7.3). When studying the effect of T-2 toxin on yeast cells, Hoehler et al. (1998) reported a 51% decrease in protein synthesis concomitant with a 100% increase in the amount of MDA produced by the decomposition of lipid peroxides. In addition,

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nmol MDA/g of liver

4

3

2

1

0 0

10

20

30

40

50

Time (hours) after toxin challenge FIGURE 7.2 MDA content of liver from mice challenged orally with 6.25 mg/kg bodyweight () compared with MDA content of liver from control mice (♦). (From Vila, B. et al., 2002, Journal of Food and Chemical Toxicology, 40: 479–486. With permission.)

1 0.9 0.8

ng of MDA

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 60

500

Vitamin E IU 0 mg toxin

1 mg toxin

2 mg toxin

3 mg toxin

4 mg toxin

FIGURE 7.3 Effect of vitamin E supplementation of diet (60 or 500 IU/kg diet) and dose of T-2 toxin administration to mice (0, 1, 2, 3, or 4 mg/kg bodyweight for each group) on liver MDA determined 48 h after the toxin challenge (bars denote standard deviation 4 mice/treated). (From Vila, B. et al., 2002, Journal of Food and Chemical Toxicology, 40: 479–486. With permission.)

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electron paramagnetic resonance (EPR) spin trapping studies showed that free radical production was promoted by toxin and that vitamin E effectively quenched the EPR signal of the spin adducts. These studies further suggest that T-2 toxin stimulates lipid perodixation by promoting free radical production (Hoehler et al., 1998). However, lipid peroxidation might not be the only mechanism for T-2 toxin-induced damage. In some cases the production of free radicals may be secondary to the initial toxic mechanism of T-2 toxin. Antioxidant treatment can significantly reduce T-2 toxin-induced lipid peroxidation. Lycopene, a red carotenoid pigment (antioxidant) found abundantly in tomato (up to 50 mg/kg) that exhibits the highest singlet oxygen quenching activity, and leutin, another highly reactive antioxidant, were found to reduce the cytotoxic effects of T-2 toxin in experimental animals (Leal et al., 1999; Atroshi et al., 2002). This was apparent from the higher levels of GSH enzyme detected when T-2 toxin was administered in conjunction with the lycopene than with T-2 administered alone (Leal et al., 1999). Selenium and vitamins E and C are also antioxidants that minimize or even quench the effect of T-2 toxin against membrane damage by acting as free radical scavengers (Atroshi et al., 1995; Hoehler et al., 1998; Vila et al., 2002). Some other mycotoxins might work in a synergetic manner with T-2 toxin in inducing lipid peroxidation. Lipid peroxidation, for example, was enhanced when T-2 toxin was administered with ochratoxin A (OTA), while DON and OTA when administered together inhibited lipid peroxidation (Muller et al., 1999). These data collectively indicate the role of T-2 toxin in the formation of free radicals. Despite the reports on the induction of lipid peroxidation by T-2 toxin, others (Schuster et al., 1987) reported no effect of trichothecenes on lipid peroxidation.

IMMUNOMODULATION

OF

T-2 TOXIN

Ingestion of T-2 toxin can cause clinical syndromes that range from fatal acute toxicity to such non-life-threatening syndromes as slow growth rates or merely redness of the skin upon exposure. Immunosuppression is one of the syndromes that occur after ingestion of lower levels of T-2 toxin, which greatly affects the wellbeing of humans and animals and alters its resistance to pathogens. Mycotoxininduced immunosuppression could affect either the humoral or the cellular components of the immune system. T-2 toxin has been known to cause severe damage to actively dividing cells in bone marrow, lymph nodes, spleen, thymus, and Peyer’s patches (Pestka and Bondy, 1990, 1994; Bondy and Pestka, 2000; Rafai et al., 1995). This extensive damage is believed to be due to early suppression of protein synthesis as inferred from the lack of [14C] leucine incorporation into circulating proteins in serum of toxin-treated rats (Thompson and Wannemacher, 1990). In addition, T-2 toxin was found to cause reduction in circulating numbers of B and T lymphocytes as well as circulating immunoglobuline (IgG) and immunoglobuline M (IgM) antibody levels (Islam et al., 1998). The extreme sensitivity of these lymphoid organs could be attributed to the presence of only minimal amounts of detoxification enzymes such as glutathione transferases or due to the presence of receptors for this toxin on the surface of the cells in these organs (Rosenstein and Lafarge-Frayssinet, 1983).

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Rafai et al. (1995) reported that a single intravenous (IV) injection of 1.2 to 4.8 mg/kg of T-2 toxin caused massive necrosis in all lymphoid tissues in pigs. Even a single dose of 0.6 mg/kg of T-2 toxin was enough to cause necrosis in some cells of spleen, tonsils, and lymph nodes. This damage caused atrophy of these lymphoid tissues especially thymus tissues in several laboratory animals, cattle, and sheep (Hayes et al., 1980; Tomar et al., 1988; Corrier, 1991). Smith et al. (1994) reported that low levels of T-2 toxin decreased thymic cells with a delay in thymic differentiation and maturation that was dose dependent, while high toxin doses resulted in severe thymic depletion. They also reported that fetal liver hematopoietic cells appeared to be a target for T-2 toxin, which contributed to thymic atrophy; however, at the adult level, the toxin especially targeted the B-cell progenitors in the bone marrow. This was apparent from the profound decrease in bone marrow cells (70%) upon exposure to T-2 toxin (Smith et al., 1994). Spleen cells were also severely affected by T-2 toxin administration with the decrease in B cells in 74% of the control animals. Similarly, Holladay et al. (1993) documented that T-2 toxin passed through the placenta to the fetus and caused fetal thymic atrophy and immunosuppression, which was believed to have occurred at the progenitor level. T-2 toxin also exerts similar effect on the bursa of fabricus, spleen, and thymus tissues in chickens and turkeys. When T-2 toxin was administered to pigs, the size of lobules and the width of the lobular cortex of the thymus were found to decrease in a dose-dependent manner (Rafai et al., 1995). In addition, a decrease in the size of the T- and B-dependent zones of the spleen white pulp was apparent as the size of the spleen decreased significantly. These results appeared to be in accordance with those previously reported by Smith et al. (1994). It has been hypothesized that T-2 toxin-induced injury to the immune system is caused by apoptosis, which causes a marked decrease in both B- and T-lymphocyte populations, and to the atrophy that occurs in lymphatic tissues (Nagata et al., 2001). Prolonged graft rejection was also observed in mice following a T-2 toxin treatment (Rosenstein et al., 1979). In addition, Pestka and Bondy (1994) and Islam et al. (1998) reported that chronic exposure to T-2 toxin suppresses the immune system; therefore, impairing the tumor defense mechanisms, leading to the growth of certain cancers that otherwise would not grow. The immunosuppressive effects of T-2 toxin are believed to be caused by an inhibition of protein synthesis and consequently RNA and DNA that is widely reflected on the atrophy of the lymphoid organs (Thuvander et al., 1999). In an in vitro experiment, Mekhancha-Dahel et al. (1990) reported that T-2 toxin inhibited DNA synthesis in peripheral blood lymphocytes. Another possible mechanism is that T-2 toxin-associated immunomodulation may occur through the hypothalamicpituitary-adrenal (HPA) axis (Taylor et al., 1989). In this mechanism, it was proposed that the ingestion of T-2 toxin at 2.5 mg/kg causes ulceration and disruption of the nonglandular gastric mucosa resulting in lymphocytic infiltration, epithelial proliferation, and hyperkeratinization. This in turn might lead to absorption of endotoxin, which triggers the stimulation of the HPA axis. These changes increase blood levels of circulating epinephrine, norepinephrine, and corticosteroids (stress-related hormones), which are known to cause thymic involution. This leads to adrenocortical

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hyperfunction and a decrease in T-dependent antibody response, thus modulating the immune response (Taylor et al., 1989). In contrast to T-2 toxin-induced immunosuppression, low doses of T-2 toxin can enhance the immune system. Otokawa et al. (1979) demonstrated that it enhanced the delayed-type hypersensitivity to sheep red blood cells (SRBC) in mice, which could be due to a T-2 toxin effect on suppressor cells that regulate cell-mediated responses. It also enhanced the blastogenic responses of T and B lymphocytes and the production of IL-1 and IL-2. In addition, in certain instances, T-2 toxin enhanced the immune system causing hypersensitivity and autoimmune-like disorders (Pestka and Bondy, 1994). The effect of T-2 toxin on both humoral and cellular immune system is discussed thoroughly in the following sections.

EFFECT

OF

T-2 TOXIN

ON

HUMORAL IMMUNITY

T-2 toxin is believed to induce apoptosis in antibody-producing organs, which ultimately leads to their atrophy. This atrophy affects the humoral immune response causing a decrease in IgM, IgA, and IgG production, which, in turn, impairs the immune system of mammals. However, depending on the dose and the route of injection, T-2 toxin was found to either stimulate or suppress humoral immunity. Humoral immune response of pigs fed T-2 toxin was significantly lower than that of control pigs (Rafai et al., 1995). Repeated intraperitoneal (IP) doses of T-2 toxin to mice decreased T-lymphocyte-dependent antibody response to SRBC, horse serum, or Escherichia coli while the T-lymphocyte-independent antibody response to E. coli antigens was not altered (Tomar et al., 1988; Pestka and Bondy, 1990; Rafai et al., 1995). Rosenstein and his group (1981) also reported a decrease in Tlymphocyte-dependent response due to thymus atrophy in mice exposed to T-2 toxin but, unlike others (Tomar et al., 1988; Pestka and Bondy, 1990; Rafai et al., 1995), they observed an increase in the T-independent response to an antigen. These observations demonstrate that T-2 toxin does not affect the B cells directly; rather, it exerts its suppression on T-helper cells. Similarly, macrophages also are not a target for T2 toxin (Dugyala and Sharma, 1997). T-2 toxin also affects the spleen and the number of antibody-producing cells. Tomar et al. (1988) showed that when mice were fed 15 ppm T-2 toxin they had a reduction in weight gain and in total number of cells per spleen. Consequently, the antibody response was decreased compared to the control. In contrast, Cooray and Lindahl-Kiessling (1987) and later Bondy and Pestka (2000) reported that mice given T-2 toxin by oral gavage at 0.75 mg/kg/day for 21 days showed an increase in antibody-producing cells in the spleen, which was thought to be mainly of the isotype IgA producing cells. This could be due to super induction of T-helper-2 cytokines (Pestka and Bondy, 1994). This increase in IgA production is considered significant as up to 40% of people having glomerulonephritis and approximately 10% of those require kidney dialysis have been reported to have elevated titers of IgA (Pestka and Bondy, 1990). This observation might explain a high incidence of kidney failure in some parts of the world; however, a detailed investigation is warranted.

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Interestingly, when T-2 toxin was coadministered with OTA, the humoral response of the animals to keyhole limpet hemocyanin (KLH) antigen was not influenced by this combination (Muller et al., 1999) indicating that OTA and T-2 toxin have antagonistic effects. Although a combination of T-2 toxin with OTA and DON increased phagocytosis and apoptosis, the increase did not exceed the effect for OTA alone, indicating a separate mechanism for each (Muller et al., 1999).

T-2 TOXIC EFFECTS

ON

CELLULAR IMMUNITY

Similar to the effect of T-2 toxin on humoral immune response, exposure to T-2 toxin either enhances or suppresses B- and T-lymphocyte mitogen proliferation in a dose-dependent manner (Berek et al., 2001). T-2 toxin appears to affect mostly Tcell-mediated functions and delayed-type hypersensitivity as apparent from the reduced major histocompatibility (MHC) class II antigen presentation (Shokri et al., 2000). In addition, the lymphoid organs appeared to be the target for T-2 toxin by themselves. For example, thymus size was decreased by 48% in animals treated with T-2 toxin. A marked depletion of lymphocytes in thymus and spleen was also observed (Corrier and Ziprin, 1986a). It has also been shown that T-2 toxin inhibits the mitogen-stimulated response of both B and T lymphocytes at an inhibition concentration (IC50) of 1.5 ng/ml of human lymphocytes. Of other trichothecenes, DAS has an IC50 of 70 ng/ml and DON has an IC50 of 140 ng/ml. These values indicate that T-2 toxin is at least 50 times more toxic than DAS and 100 times more toxic than DON. T-2 toxin impairs phagocytic activities in several different small and large animals. This was demonstrated in in vitro experiments by the impaired migrationchemotaxis test and/or phagocytosis of macrophages and other polymorphonuclear cells of several mammals including humans (Dugyala and Sharma, 1997). In addition, high doses of T-2 toxin (0.5 to 15 mg/kg) caused suppression of phagocytosis and lymphocyte proliferation (Muller et al., 1999). Plastogenic response of leukocytes to stimulation by homologous antigens phytohemagglutinin and concanavalin-A decreased significantly in pigs fed T-2 toxin for at least 7 days (Rafai et al., 1995). The amount of T-2 toxin required to inhibit lymphocyte blastogenesis (proliferation) was found to be at least ten times less than the amount needed for inhibition of protein synthesis (Pestka and Bondy, 1990, 1994). In addition, T-2 toxin inhibited proliferation of mitogen-stimulated bovine peripheral blood mononuclear cells at a very low T-2 toxin concentration in a dosedependent manner (Tomar et al., 1988; Charoenpornsook et al., 1998; Berek et al., 2001). This inhibition could be explained by the binding of T-2 toxin to the 60S subunit of the 80S ribosome, which disrupts the protein and DNA synthesis. Nevertheless, the decrease in T-2 toxicity appears to be dependent on the nature of substitution of acyl groups at C3, C4, C8, and C15. Therefore, the substitution of the acyl groups by keto or hydroxyl groups minimizes T-2 toxicity (Pestka and Bondy, 1994). In addition, hydrolysis of the acetyl groups of T-2 toxin at the C4 position to HT-2 decreases its lymphotoxicity by 36-fold. Therefore, it appears that T-2 toxicity is dependent on the nature of modification on these carbons (Pestka and Bondy, 1990; Madhyastha et al., 1994).

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Contrary to the suppression effect, T-2 toxin was found to stimulate the production of IL-1 by macrophages and IL-2 by murine rat spleenocytes or human tonsil lymphocytes (Pestka and Bondy, 1990; Ouyang et al., 1995). Moreover, Dugyala and Sharma (1997) reported that T-2 toxin induced the production of IL-2, IL-3, and interferon-gamma (IFN) in addition to an increase in cell-mediated immunity. Further, Ouyang et al. (1995) reported that trichothecenes in general superinduce IL-2, IL-4, and IL-5 production by 2- to 15-fold in a dose- and time-dependent manner. A possible explanation for the enhancement of IL production could be that T-2 toxin suppresses a control protein that is involved in the degradation of these interleukins (Pestka and Bondy, 1990). Another explanation is that, concomitant with the enhancement of ILs, there was a general decrease in the proliferation of cells, which might have resulted in a decrease in IL receptors and, therefore, decreased IL consumption (Ouyang et al., 1995). Or simply, T-2 toxin administration induces stress to the macrophages and lymphocytes, which provokes them to produce high amounts of these cytokines to counteract this stress, as in the case of viral infections or tissue damage. T-2 toxin at a concentration >8 ng/ml superinduced chondrocytes to produce IL1 and IL-6. These two cytokines were found to cause degeneration of cartilaginous matrix causing Kashin-Beck disease (Tian Fu et al., 2001). However, at lower concentration (≤4 ng/ml), T-2 toxin inhibited the production of these two cytokines (Tian Fu et al., 2001). These findings were supported by results from a previous in vitro study by Wright et al. (1987) who reported that T-2 toxin is highly toxic to human chondrocytes causing more than a 50% reduction in DNA per flask. Therefore, it is clear that the enhancement or suppression of T-2 activity depends on the dose and the time of administration (Forsell and Pestka, 1985; Pestka and Bondy, 1990). T-2 toxin also affects several lymphocyte subsets with CD4+ and CD8+ T cells the most sensitive, particularly in the mesenteric lymph nodes. CD3 and CD19 populations decreased 24 h after the toxin administration with CD3 cells most affected. However, in Peyer’s patches the toxin effect was equal on both CD3 and CD19 (Nagata et al., 2001). As for the numbers of B cells in the Peyer’s patches, IgA+ B cells were most affected followed by IgM+ and IgG+ producing cells (Nagata et al., 2001).

EFFECT

OF

T-2 TOXIN

ON

HOST RESISTANCE

TO

PATHOGENS

The ability of an animal to counteract an infection depends mainly on the presence of mononuclear-phagocytic cells called macrophages, which are regarded as the first line of defense of the immune system (Kidd et al., 1995). Macrophages scavenge microorganisms and present them to T and/or B lymphocytes to induce either the humoral or the cellular immune response. Depending on route of administration and the dose of T-2 toxin, it might either act as an immunosuppressant or immunostimulant to macrophage activity (Corrier, 1991; Bondy and Pestka, 2000). Treatment of mice with a single dose of T-2 toxin 7 days before a challenge of Listeria monocytogenes increased their resistance and therefore their survival. The increase in mice survival was found to be T-2 toxin dose-dependent with best results at ≥2 mg/kg of T-2 toxin (Corrier and Ziprin, 1986a;

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Ziprin and McMurray, 1988). The treatment of mice with T-2 toxin may have stimulated the lymphocytes to produce macrophage-activating lymphokines that stimulated macrophages, which efficiently cleared bacteria (Corrier and Ziprin, 1986a). In addition, the extensive depletion of lymphocytes in thymus and spleen may have decreased the regulatory T-cell population thus increasing macrophage activity (Corrier and Ziprin, 1986a). However, at lower doses of T-2 toxin, immunosuppression occurred, which could be due to depletion of T-lymphocytes and the failure of sensitized T-cells and macrophages to clear the host bacteria (Corrier and Ziprin, 1986b). Contrary to the effect of T-2 toxin on the resistance of mice to Listeria infection, it markedly increased the susceptibility of mice to Salmonella typhimurium infection over a wide range of doses. In this experiment the LD50 of S. typhimurium was decreased fivefold (Tai and Pestka, 1988a, b). In addition, the number of the organisms recovered from the spleen and liver increased over time and reached a plateau at day 9, which might be the time required for the cellular immune system to develop and combat the infection (Tai and Pestka, 1988a). The effect of T-2 toxin on host resistance was best illustrated by Tai and Pestka (1988b) who reported a decrease in the LD50 of S. typhimurium from 5 ∞ 106 to only 5 cells per mouse. In another experiment, Tai and Pestka (1990) reported severe immune suppression to S. typhimurium when mice were given a single T-2 toxin dose preinoculation and several doses postinoculation of the pathogen. In addition, mice treated with T-2 toxin in this way exhibited pathological lesions in liver, spleen, and kidney (Tai and Pestka, 1990). In contrast, Ziprin and McMurray (1988) reported no significant effect of pretreatment of mice with a single dose of T-2 toxin on the resistance to S. typhimurium. When lipopolysaccharide (LPS), a surface component of Gram-negative bacteria, was coadministered with T-2 toxin, the susceptibility of mice to the bacteria was enhanced (LD50 was decreased by 14-fold), suggesting that the impaired resistance to these pathogens might be directed toward LPS (Tai and Pestka, 1988b; Islam et al., 2002). Another possible mechanism is that T-2 toxin inhibits protein synthesis and thus decreases the production of the proteins necessary for T-2 toxin detoxification (Tai and Pestka, 1988b). In addition, inhibition of protein synthesis might impair the ability of mice to repair the damage exerted by LPS, which is an endotoxin, thus increasing lethality (Tai and Pestka, 1988b). Taylor et al. (1991) and Islam et al. (2002) also suggested that T-2 toxin treatment enhances LPS absorption, which stimulates host cells to produce a battery of mediators including the proinflammatory cytokines (tumor necrosis factor-alpha, or TNF-α, IL-6, and IL-1), reactive oxygen species, and prostaglandins, which in itself produces the pathologic effect. When T-2 toxin was administered 7 days before a challenge with Mycobacterium bovis, there was a higher proliferation of bacteria in the spleen of subject mice as opposed to the control mice, which had minimal proliferation. However, there was no apparent effect of T-2 toxin administration on the proliferation of bacteria in lungs (Ziprin and McMurray, 1988). Obviously, damage to the lymphocyte components of the immune system could be the reason behind the decreased resistance of mice to M. bovis. Contrary to chronic administration, it was demonstrated that the suppression effect on Listeria and mastitis pathogens occurs if T-2 toxin was administered after

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the pathogen challenge, whereas if it was administered shortly prior to the challenge it enhanced the immune system. This was explained by the increased migration of macrophages and an enhanced phagocytic activity (Corrier et al., 1987, 1991; Cooray and Jonsson, 1990), or by the depletion or impaired function of the T-suppressor cell population. Further, this enhancement might also be due to the ability of T-2 toxin to induce IL-2, IFN, and IL-3 and to an increase in the cell mediated immunity (Dugyala and Sharma,, 1997). However, this enhancement was followed by leukocytopenia, lymphopenia, necrosis, and depletion of lymphocytes in the thymus and spleen. The effect of T-2 toxin on the resistance to a given bacterial infection, therefore, depends on the nature of the host and pathogen, the toxin dose, and the route of toxin administration (Ziprin and McMurray, 1988). The resistance of the host to viruses is also affected by T-2 toxin. Friend et al. (1983a) reported that mice were highly susceptible to herpes simplex virus-1 (HSV1) virus following the administration of 20 ppm T-2 toxin for 2 to 3 weeks. The immunosuppression was severe as 70 to 100% of treated mice developed brain and liver lesions with about 61% mortality whereas the effect in nontreated mice was much less dramatic. However, the T-2 toxin-induced immunosuppression in mice was not sufficient to induce reactivation of latent HSV-1 infection (Friend et al., 1983b). It is noteworthy that the immunosuppression induced by high doses of T-2 toxin appeared to protect mice against the overreaction of the immune system that occurs as a result of the HSV-1 virus. This was clear from the absence of the necrotizing meningoencephalomyelitis that results from HSV-induced inflammation to the spinal cord and the brain (Friend et al., 1983a). The modulation of the immune system to several pathogens as caused by T-2 toxin may predispose food animals to certain diseases that decrease productivity and might increase the period of shedding the microorganism, which might increase animal-to-human transmission of these pathogens.

RELATION

BETWEEN

T-2 TOXIN

AND

APOPTOSIS

Apoptosis is a Greek term meaning the “falling off” leaves from a tree. Physiologically, it is defined as programmed cell death or cell suicide, which is a normal process that happens during embryonic development as well as in maintaining tissue homeostasis (Kerr et al., 1972). Contrary to cell necrosis, apoptosis involves cellular self-destruction of the genomic DNA into 200 base pair (bp) pieces by the action of endonucleases (Islam et al., 1998). Apoptosis starts with changes in plasma membranes, chromatin condensation, nuclear and DNA fragmentation, and loss of mitochondrial inner membrane potential, which predispose to blebbing of the plasma membrane, and finally the apoptotic cell becomes fragmented into apoptotic bodies that are cleared by the macrophages (Islam et al., 1998; Carrasco et al., 2003). The morphological changes are believed to be caused by a family of cysteine-containing proteases called caspases (Miller and Marx, 1998; Carrasco et al., 2003). Administration of a single dose of T-2 toxin to rodents induces apoptosis in the thymus, bone marrow, liver, and spleen (Ihara et al., 1997; Islam et al., 1998). Neither endogenous glucocorticoid nor TNF-α appeared to play any role in T-2 toxin-induced thymocyte apoptosis (Islam et al., 1998). Instead, T-2 toxin-derived metabolites play a critical

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role in inducing thymic apoptosis. However, among all the metabolites only 3´OHT-2, which is a hydroxylation product of T-2, was equal in toxicity to that of T-2 toxin. There are different theories explaining the apoptosis induced by trichothecenes. One of the possible mechanisms of apoptosis is that T-2 toxin causes DNA damage as a secondary effect of the inhibition of protein synthesis. This DNA injury will either transcriptionally or posttranscriptionally activate a nuclear phosphoprotein, p53. This protein functions as a mentor of the cell, directing it whether it should go toward repair or apoptosis based on the extent of the damage (Wyllie, 1997). Nagase et al. (2001) reported that T-2 toxin-induced apoptosis involves activation of enzymes called caspases through the release of cytochrome c from the mitochondria. Cytochrome c binds to the apoptotic protease activating factor-1 (Apaf-1), which activates procaspase-9, which in turn activates caspase-9 that activates caspase-3, a key effecter in the apoptosis pathway (Carrasco et al., 2003). The activation of caspase3 results in proteolytic degradation of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation factor 45/inhibitor of caspase-activated-DNase (DFF-40/ICAD) leading to apoptosis (Nagase et al., 2001; Carrasco et al., 2003). Shifrin and Anderson (1999), however, suggested that T-2 toxin, which strongly inhibits protein synthesis but does not activate the c-Jun-NH2–terminal kinase (JNK)/p38 kinases, induces a three- to fourfold increase in caspase activity. A higher level of caspase activation requires both an inhibition of protein synthesis and the activation of JNK/p38 kinases. Therefore, the authors suggested that T-2 toxin does not induce apoptosis via this pathway since it is a strong protein inhibitor yet it does not cause activation of JNK/p38 kinases, while the less toxic trichothecenes (T-2 tetraol and DON) did activate the JNK/p38 kinases and cause apoptosis. These results were contradictory to results obtained by Yang et al. (2000) who reported a close correlation among protein synthesis inhibition, activation of stress-activated protein kinase (SPAK)/JNK, and p38 mitogen-activated protein kinases (MAPKs) and induction of apoptosis in macrophages. The reason for these contradictory results could be due to the concentrations used by each group. Shifrin and Anderson (1999) used 3.8 μM of T-2 toxin, which was 400 times more than the amount used in the study conducted by Yang et al. (2000). This high dose of T-2 toxin must have induced a swift MAPK activation and apoptosis that damaged all the cells and, by the time readings were taken (3 h later), there was neither DNA laddering nor any MAPK signal. Furthermore, contrary to results reported by Shifrin and Anderson (1999), Islam et al. (1998) showed that exposure to T-2 toxin caused severe thymic atrophy in mice through apoptotic cell death. These results were inferred from the amount of DNA cleaved into a ladder in response to the administration of three trichothecenes (DAS, NIV, and T-2 toxin) in which T-2 toxin appeared to be the most potent in inducing apoptosis. In another possible mechanism of apoptosis, it was found that T-2 toxin induces lipid peroxidation, which generates H2O2 that activates p38 MAPK and SPAK/JNKs, which leads to apoptosis (Yang et al., 2000). From the above results, it appears that the role of T-2 toxin in the induction of apoptosis and the relation between the magnitude of apoptosis and the potency of protein inhibition are controversial. This controversy appears to stem from different experimental systems and the quantities of T-2 toxin used in these experiments.

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Nevertheless, T-2 toxin plays a role in apoptosis as inferred from the atrophy of the lymphoid organs upon exposure to this toxin.

EFFECT

OF

T-2 TOXIN

ON

CULTURED CELLS (IN VITRO)

Cultured human or animal cells provide an indispensable tool for several types of studies including but not limited to pathogenicity, toxicity, teratogenicity, and carcinogenicity. The toxicity of T-2 toxin can be evaluated using an appropriate cell line. For example, alveolar macrophages cells (AMC), one type of cells obtained from rat lungs, have been used to study respiratory disorders in grain workers who might be exposed to mycotoxins including T-2 toxins. When the AMC were exposed to as little as 0.1 μM of T-2 toxin, an increase in chromium leakage was observed. This leakage was found to be proportional to the number of dead cells in the culture and occurred in a dose-dependent fashion (Gerberick and Sorenson, 1983). The toxicity of T-2 toxin was compared to that of three other trichothecenes (DAS, NIV, and DON). When tested on human lymphocyte cells in vitro, T-2 toxin was the strongest inhibitor of mitogen-stimulated lymphocyte proliferation. In addition, it inhibited stimulated lymphocyte from producing IgA, IgG, and IgM (Thuvander et al., 1999).

IS T-2 TOXIN

A

CARCINOGEN?

The carcinogenicity of T-2 toxin is not as well established as it is for aflatoxins. However, there are some reports linking T-2 toxin to the appearance of tumors in some animals. Schoental (1979) reported the appearance of tumors in several rat organs after the administration of 5 to 8 doses of T-2 toxin. In addition, chronic administration of T-2 toxin to rats induced leukemias, which could be due to damage of single-stranded DNA (Rosenstein and Lafarge-Frayssinet, 1983). In a study conducted by Schiefer and co-workers (1987) statistically significant differences were found in the incidence of pulmonary adenomas and hepatic adenomas in males given 3 mg/kg and in controls while there was no difference in the incidence among females. The results were not conclusive as controls also developed adenomas. It appears that male mice might have developed the adenoma due to other factors and the low T-2 toxin level might have enhanced their immune system to prevent it, while those with high doses or left without treatment had weaker immune systems and, therefore, developed the adenomas. A good relationship exists between the carcinogenicity of certain chemicals and the degree of DNA damage that it caused (Atroshi et al., 1997). In addition, failure to activate apoptosis after DNA damage might lead to the development of cancer (Wyllie, 1997). Thus, the appearance of leukemia in rats after prolonged exposure to T-2 toxin could be due to the DNA damage exerted by the toxin. However, other studies (Ueno et al., 1992) reported that in vivo carcinogenicity tests were negative as they have tested for the presence of hepatocarcinogenicity in rats and found it clear of GST-positive liver cell foci. The authors proposed that T-2 toxin might act as a carcinogen indirectly by suppressing the immune system, allowing the tumor cells to escape destruction by the immune system and eventually developing into a tumor (Lafarge-Frayssinet et al., 1990).

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The effects of T-2 toxin were reported to resemble those of bracken toxins, methylnitrosourethane and methylnitrosourea. These toxins induce tumors in the digestive tract, pancreas, and brain and cause and a variety of lesions in rats. In addition they all cause depigmentation when applied directly on hair (Schoental, 1979). Although a single dose may not be enough to induce tumors of any type, several doses may induce them. Furthermore, Schoental (1979) reported that the genetic predisposition or some viruses may be a factor in causing tumors. However, certain mycotoxins appear to determine when and whether a dormant condition becomes an actual tumor. Based on the current literature available, it seems that the role of T-2 toxin as a carcinogen is controversial and further studies are needed.

CONCLUSION This review has provided an update of information on T-2 toxin and its effect on humans and animal health. T-2 toxin is a potent toxin that has been implicated in human and animal toxicosis throughout history. Cereal crops, oil seeds, sugar beets, cereal-containing foods, and animal products such as eggs and milk are the main products that could be contaminated with T-2 toxin. At the cellular level, the ability of T-2 toxin to inhibit protein synthesis predisposes it to several pathological manifestations such as inhibition of DNA and RNA synthesis, inhibition of proliferation of certain immune cells, and inhibition or induction of apoptosis. However, at the level of the whole animal, T-2 toxin causes several pathological effects, including effects on the nervous system, GIT, skin, circulatory system, and especially on the immune system. The relation of T-2 toxin to these pathological effects is well documented. Nevertheless, its role as a carcinogen is still controversial. It has been speculated that genetic predisposition or some viruses may be a factor in causing tumors. However, mycotoxins appear to determine when and whether a dormant condition becomes cancerous.

ACKNOWLEDGMENTS Professor Emeritus R.R. Marquardt, Dr. Israr Khan, and Dr. Tahhan Jaradat are thanked for their valuable comments on the manuscript. The author also would like to thank the editors, Dr. Victor R. Preedy and Professor Ronald R. Watson, for the invitation to submit this chapter.

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Aflatoxin B1 and Cell Cycle Perturbation Ruggero Ricordy, Emanuele Cacci, and Gabriella Augusti-Tocco

CONTENTS Abstract ..................................................................................................................213 Abbreviations .........................................................................................................214 Introduction............................................................................................................214 AFB1 Cytotoxicity on Human Cell Lines.............................................................216 AFB1 and Cell Cycle Progression: Inhibitory Proteins p53, p21, and p27 .........222 Inactivation of INK4/ARF Locus and AFB1 Carcinogenetic Action ...................225 Epigenetic Events in AFB1 Carcinogenesis: Hypermethylation of Specific Genes .......................................................................................................226 Conclusions............................................................................................................228 References..............................................................................................................228

Abstract

Aflatoxins are a group of highly toxic metabolites, studied primarily because of their negative effects on human health. They represent food contaminants, produced by strains of fungi, such as Aspergillus flavus and A. parasiticus. Among them aflatoxin B1 (AFB1) is the most frequent food contaminant and the major target of AFB1 toxicity is the liver. A large number of studies on aflatoxins have been undertaken with the aim of identifying the mechanism of their toxicity and eventually designing appropriate protocols for the removal of the toxic derivatives from contaminated food. AFB1 exposure causes alteration of several specific cellular activities; among these, impairment of the cell cycle progression mechanism appears particularly relevant, considering the carcinogenetic action of the toxin. Direct analysis of AFB1 effects on cell cycle progression has been performed on cell lines; the results of this study point to a role for p53 in the response to AFB1. Considerable attention has been dedicated to investigation of the interference of AFB1 with molecular components of cell cycle checkpoints. These studies are reviewed; they strongly suggest that p53, p27, p16, and p19 functions are impaired by AFB1. DNA methylation appears to be a major mechanism for cell cycle inhibitory control inactivation.

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Abbreviations AFB1: aflatoxin B1; BrdUrd: bromodeoxyuridine; CDKs: cyclin-dependent kinases; cip/kip: CDK inhibitory protein/kinase inhibitory protein; CpG: cytosine-guanine dinucleotide; dTR: thymidine; Gadd45: growth arrest and DNA damage inducible; HCC: hepatocarcinoma; HepG2: human hepatoma cell line; INK4: inhibitor of cyclin-dependent kinase 4; INK4/ARF: inhibitor of CDK4/ADP ribosylation factor; LOH: loss of allele heterozygosis; Mdm2: mitochondrial distribution and morphology; MGMT: O6 methylguanine methyltransferase; pRB: retinoblastoma protein; ROS: reactive oxygen species; SK-N-MC: human neuroblastoma cell line; SK-N-SH: human neuroblastoma cell line; Sp1: regulatory protein interacting with specific DNA sequences INTRODUCTION Aflatoxins are a group of highly toxic metabolites that have been widely studied, mainly because of their negative effects on human health. They are produced by strains of fungi, such as Aspergillus flavus and A. parasiticus, which represent possible sources of food contamination, mainly as a result of improper storage of cereals. Among them, aflatoxin B1 (AFB1) is the most frequent food contaminant (Table 8.1); it is known to exert extremely toxic effects on animals and humans when contaminated food is ingested (Table 8.2 and Table 8.3). Because of the alimentary intake of the toxin and its presence in the digestive tract, the major target of AFB1 toxicity is the liver. In fact, acute aflatoxicosis causes liver dysfunction and gastrointestinal bleeding, and AFB1 is also known for its potent hepatocarcinogenic action (Massey et al., 1995). The presence of the toxin has been reported in various human tissues and organs (Chao et al., 1991), and AFB1 exposure is also associated TABLE 8.1 Aflatoxin Presence in Food Aflatoxin B1 B 1, B 2, G 1, G 2

M1 M2 All listed aflatoxins

Food Eggs Liver Cocoa beans Coconut, copra, and copra meal Green coffee Pistachio nuts Soybeans Dairy products Liver Milk Corn, cottonseed, and groundnuts

Source: Adapted from Heathcote and Hibbert (1978).

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TABLE 8.2 AFB1 Toxicity in Different Animal Species Animal Species

LD50 (mg/kg bodyweight)

Mouse Rat Guinea pig Pig Trout Sheep Chicken

9.0 5.5–17.9 1.4–2.0 0.6 0.8 2.0 6.3

Note: The LD50 for the listed animal species was evaluated after a single oral dose administration of the toxin. Source: Adapted from Heathcote and Hibbert (1978).

TABLE 8.3 Effects of Aflatoxins on Mammalian Tissuesa Liver is the main target of aflatoxin toxicity: Hepatocarcinoma Bile duct hyperplasia Damage of liver parenchyma Parenchymal necrosis in the periportal region AFB1 impairs synthesis of fibrinogen and causes gastrointestinal hemorrhage. Aflatoxins diminish resistance to infection by interfering with the immune system, although the precise mechanisms are poorly understood. Aflatoxins induce necrosis of kidney tubules; the kidney damage is a transient effect and tissue regeneration takes place in a few days. Hemorrhagic lesions have been found in lungs and adrenal glands. Skin lesions are induced by an intraepidermal vesiculation and consequent epidermal necrosis. Aflatoxin poisoning can be responsible for some cases of human encephalopathy, possibly caused by metabolic consequences of hepatic damage. Aflatoxins have been found to induce teratogenic effects; although not many reports on this are available, the toxic effect on the fetus may be secondary to maternal liver damage. Transplacental carcinogenesis caused by aflatoxins has also been reported. a

For a review, see Heathcote and Hibbert (1978).

with an acute syndrome characterized by encephalopathy and extensive necrosis of the brain (Reye et al., 1963). This finding has prompted a study to examine whether

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neurons can be a primary target for AFB1 toxicity (Bonsi et al., 1996), although the mechanism allowing the toxin to cross the blood–brain barrier is not known. A large number of studies on aflatoxins have been undertaken with the aim of identifying the mechanism of their toxicity and eventually designing appropriate protocols to remove the toxic derivatives from contaminated food. AFB1 can readily cross the plasma membrane and interact with many proteins responsible for specific cell functions. Alterations of specific cellular activities have been described, such as changes in protein phosphorylation level (Viviers and Schabort, 1985), protein kinase C (Mistry et al., 1995), Ca2+ ATPase (Adebayo et al., 1995), and cyclic nucleotide phosphodiesterase activities (Bonsi et al., 1999). The role of the impairment of these cell activities in AFB1 toxicity has not been established, as they have been studied in different experimental systems and a general picture of their involvement in AFB1 toxicity is lacking. It is pertinent to mention that the mentioned proteins are mainly involved in signal transduction pathways, which are largely interconnected; thus malfunction of one of the proteins can cause significant alterations of cell physiology and possibly survival. On the other hand, the ability of the toxin to form covalent adducts with DNA (Figure 8.1) and thus impair DNA, RNA, and ultimately protein synthesis has been well established (Meneghini and Schumacher, 1977; Yu, 1981; Jackson and Groopman, 1999). Both in experimental animals and in humans the formation of guanine adducts has been observed following exposure to AFB1 (Wogan, 1992) and the ability of the toxin to directly induce DNA damage has been related to its well-known carcinogenetic action (Foster et al., 1983; Bailey et al., 1996).

AFB1 CYTOTOXICITY ON HUMAN CELL LINES The report that in acute aflatoxicosis brain function can be impaired is of interest, because in the adult nervous system neurons are in fact postmitotic; thus a toxin acting mainly via DNA damage should not be expected to cause severe functional alterations in this system. On the other hand, AFB1 has been reported to cause acute but reversible functional alterations in isolated organs (Luzi et al., 2002), which supports the existence of multiple cellular targets for AFB1 toxic action. These considerations prompted evaluation of AFB1 neurotoxicity on cultured cells. Cytotoxicity assays were performed on human neuroblastoma cell lines and on chick postmitotic spinal cord motor neurons (Bonsi et al., 1996). The cytotoxic response of these cells was compared to HepG2, a cell line isolated from a human hepatocarcinoma used as a control, because liver is the main target of AFB1. Data obtained from this experimental system (Table 8.4) clearly demonstrated that AFB1 is capable of a direct interaction with neuronal cells and causes a dose- and timedependent decrease in cell survival. Comparing the sensitivity of the different neuronal cells examined to AFB1, it became evident that SK-N-MC cells were more susceptible to AFB1 treatment than SK-N-SH or spinal cord neurons. SK-N-SH survival was also evaluated under serum starvation, a culture condition that transiently retards cell cycle progression. Under these conditions AFB1 cytotoxicity was considerably lower with respect to cultures where cell proliferation was allowed by the presence of serum; in addition, the survival rate of cell cycle arrested cells is

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O

O

O 9 8

O

OCH3 O Aflatoxin B1 Cytochrome P450 O O O

O OCH3 O O Aflatoxin B1-8,9-epoxide

Nucleic acid adducts O

O

O HO O N+ O

HN H2N

N

O

OCH3

NDNA

8,9-Dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1

FIGURE 8.1 Metabolic activation of AFB1 and binding to DNA of the epoxide derivative. AFB1 is converted by cytochrome P-450 to its 8,9-epoxide derivative, which primarily reacts with guanine giving origin to AFB1-N7-guanine DNA adducts. This DNA adduct formation causes the C:G to A:T transversion, a genetic change that is in turn responsible for the mutagenic action of the toxin.

very similar to that observed for spinal cord postmitotic neurons. These data together suggest that the cellular response to AFB1 could be modulated by the state of cell cycle progression at the time the cells come in contact with the toxin. The wellknown capacity of AFB1 to form stable DNA adducts, which can be repaired by specific cellular mechanisms (Hoeijmakers, 2001), but which are also a possible source of mutations, poses the question of whether AFB1-dependent cell cycle impairment in neuronal cells could be a primary mechanism of AFB1 neurotoxicity. A comparative analysis of cell cycle perturbation in the presence of AFB1 on the three different cell lines thus appeared to be of interest. A study based on cytofluorimeter analysis (BrdUrd/DNA content) of the cell lines used in the previous study was performed to establish if AFB1 could alter cell cycle distribution of treated cells; the results of this study are summarized below. At the end of a 24-h treatment with increasing concentrations of AFB1, SK-NMC showed an accumulation of cells in S phase; cell distribution tended to return to control values after 48-h recovery in the absence of AFB1 (Figure 8.2). In contrast

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TABLE 8.4 Correlation between Cytotoxicity and Proliferative State Cell Type SK-N-MC SK-N-SH Spinal cord motoneurons

Cell Cycle State

Survival (%)

Proliferating Proliferating Resting Postmitotic

14 58 72 76

Note: Human neuroblastoma cell lines SK-N-MC and SK-N-SH maintained in cultures in the presence of 10% serum were exposed to 10 mg/ml AFB1 for 48 h. In SKN-SH cultures serum was omitted in a parallel set of dishes to stop proliferation (resting). Motoneurons were obtained by dissociation of E8 chick embryo spinal cord and maintained in cultures in serum-free medium. They represent a postmitotic neuron population. Viable cells were determined as described by Mossman (1993) and cell survival expressed as percentage of AFB1 treated vs. untreated cultures.

to the behavior of SK-N-MC, both SK-N-SH and HepG2 cell lines showed a tendency to reduced cell numbers in S phase and the altered distribution persisted when toxin was removed. The different behaviors of the cell lines (SK-N-MC vs. SK-N-SH and HepG2) was further demonstrated by biparametric analysis, after DNA synthesizing cells had been labeled with BrdUrd (bromodeoxyuridine) at the end of toxin treatment (Figure 8.3). In contrast to the behavior of SK-N-MC, both SK-N-SK and HepG2 cell lines showed a different modification of S phase traverse after 24 h of treatment with AFB1. In fact, AFB1 caused a dramatic decrease in the cell numbers labeled by BrdUrd and distributed in the S phase region. The difficulty of traversing S phase was not reversed after 48-h recovery in absence of AFB1; while in the case of SK-N-MC the increase in BrdUrd-positive cells in S phase was restored to control levels after 48 h of recovery in the absence of AFB1. Experimental observations based on the [3H]-thymidine incorporation assay (Figure 8.4) on the three cell lines under similar experimental conditions confirmed the data derived from cytofluorimetric analysis. AFB1 brings about a persistent arrest of DNA synthesis in SK-N-SH and HepG2 cell lines, whereas SK-N-MC cells appear capable of resuming DNA synthesis and thus reestablishing normal cell distribution in the cycle phases after the toxin has been removed.

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80 60 40

SK-N-MC

20 0

9.6 μM

control

19.2 μM

32 μM

80 70 60 50 40

SK-N-SH

30 20 10 0

19.2 μM

control

80 μM

160 μM

80 70 60 50 40

HepG2

30 20 10 0

9.6 μM

control

G1

19.2 μM

S

32 μM

G2

FIGURE 8.2 AFB1 effects on cell cycle distribution. Distribution in the cell cycle phases of control cultures and cultures treated with AFB1 was evaluated by cytofluorimeter analysis, based on DNA content/cell. Histograms showing percent of SK-N-MC, SK-N-SH, and HepG2 cells in G1, S, and G2 phases after 24-h treatment with various doses of AFB1 and 48-h recovery time. The results are the mean of three different experiments. Bars indicate the standard deviation. The reported data show that AFB1 causes SK-N-MC cells to accumulate in S phase, whereas the S phase in the other two cell lines appears to be nearly empty.

FIGURE 8.3 AFB1 and S phase progression. The ability of toxin-treated cells to resume DNA synthesis, after toxin had been removed was evaluated by a bivariate analysis of BrdUrd incorporation (ordinate) and DNA content (abscissa) in SK-N-MC, SK-N-SH, and HepG2 cells. Cultures were treated for 24 h with increasing toxin concentrations (as indicated in Figure 8.2); after a 48-h recovery time cells were collected for cytofluorimeter analysis. This analysis shows a significant labeling of S phase region for SK-N-MC cell, indicating their ability to recover from the toxin action and traverse the S phase; for the other two cell lines the S phase region remains poorly (SK-N-SH) or not (HepG2) labeled, indicating their inability to overcome the DNA synthesis block caused by the toxin.

HepG2

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SK–N–MC

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48 rec.

time (hours)

HEPG2

DPM / well

20000

10000

DMSO AFB

1

0 0 rec.

24 rec.

48 rec.

time (hours)

FIGURE 8.4 [3H] Thymidine (dTR) incorporation. The amount of [3H] dTR incorporated by SK-N-MC, SK-N-SH, and HepG2 was determined (intermediate concentrations reported in Figure 8.2), in order to evaluate the level of DNA synthesis. The cultures treated with or without AFB1 for 24 h were incubated with 1 μC/ml [3H] dTR for 2 h, after 0, 24, 48 h from the end of treatment. The data shown are the mean values + SEM of three independent experiments. The reported data demonstrate that [3H] dTR incorporation is inhibited by AFB1. A partial recovery of DNA synthesis at 48 h can be observed in SK-N-MC cells only.

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AFB1 AND CELL CYCLE PROGRESSION: INHIBITORY PROTEINS P53, P21, AND P27 The current model of cell cycle control maintains that the transition between different cell cycle stages is regulated at checkpoints (for recent reviews, see Vermeulen et al., 2003; Galderisi et al., 2003). Different checkpoints are in turn regulated by a family of protein kinases, namely the cyclin-dependent kinases (CDKs), and their obligate activating partners, the cyclins. Other proteins involved in mammalian checkpoint regulation have been discovered to work as tumor suppressors. The major proteins involved in cell cycle progression and their functions are listed in Table 8.5. One of these inhibitors is p21, a protein that binds to and inhibits a wide variety of cyclin–CDK complexes (Hunter and Pines, 1994). p21 transcription is in turn activated by p53 (Hunter and Pines, 1994). Increased levels of p53 in response to DNA damage appear to be an essential step to enforce the p21-mediated arrest of the cell cycle in G1 or S phase (van Gijssel et al., 1997) and for the induction of other gene expression, such as Gadd45, which in turn may activate DNA repair mechanisms (van Gijssel et al., 1997).

TABLE 8.5 List of the Major Proteins That Take Part in the Control of Cell Cycle Progression and Their Main Function in the Process Cyclins

pRB

E2F p53

p27

INK4a/ARF

p16 p19 Mdm2

Proteins that associate with their respective CDKs and are key components of cell cycle progression machinery. The complexes cyclin–CDKs are holoenzymes that phosphorylate various proteins in successive steps of the cell cycle, driving the cell through the whole cycle. Retinoblastoma protein. The ipophosphorylated form of the protein binds to the transcription factor E2F, inhibiting transcription of a number of genes required for the transition of the cell from G1 to S phase. Transcription factor promoting expression of genes that encode proteins required for DNA synthesis. Oncosuppressor protein that exerts a negative control on cell cycle progression. This protein is a transcription factor that is capable of blocking the cell in G1 phase, promoting transcription of p21 protein, a CDKs inhibitor. A member of cip/kip protein family that acts either positively or negatively on cell cycle progression, respectively, through stabilization of cyclin D/CDK4 complex and inhibition of cyclin E/CDK2 complex. Locus encoding two different proteins involved in cell cycle control. Through an alternative splicing, the locus encodes an inhibitor of CDK4 (p16 protein) or an inhibitor of Mdm2 (p19), a protein modulating p53 action. Protein that specifically inhibits CDK4, the counterpart of cyclin D; it prevents pRb phosphorylation and cell cycle progression. Protein involved in the control of p53 level, forming binary or ternary complexes together with p53 and/or Mdm2 proteins. Protein involved in the control of p53 level causing p53 delocalization, ubiquitinazation, and degradation.

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Although direct data on cell cycle perturbation by AFB1 are scarce, considerable attention has been paid to the interference of the toxin with molecular components of cell cycle checkpoints. In the experimental system used in the experiments reviewed above, Western blot analysis has shown increased levels of p53 and p21 expression in SK-N-SH cells but not in SH-N-MC following AFB1 exposure (Figure 8.6). This finding has suggested that the lack of expression of proteins involved in the control of cell cycle and specifically in the control of DNA integrity, such as p53 and p21, may be responsible for the higher ability of SK-N-MC to resume progression through the cell cycle when the toxin is removed, regardless of possible DNA damage caused by the toxin (Ricordy et al., 2002). The role of p53 mutations in carcinogenesis is well known (Hsu et al., 1991), and they are likely to play an important role also in AFB1-induced carcinogenesis. As a matter of fact, a spectrum of p53 mutations in liver cancer cells related to AFB1 exposure has been described. Aguilar et al. (1993) reported that in geographical areas with a high degree of AFB1 contamination about 50% of hepatocarcinoma (HCC) mutations at p53 locus could be detected, while the percentage of p53 mutations decreases to 20% in areas with a low toxin contamination. Similar results have been also reported by Lunn et al. (1997). The most frequent p53 mutation in liver cancer cells related to AFB1 exposure is a G:C to T:A transversion at codon 249 (Essigman et al., 1983; Aguilar et al.,1994). The mechanism for codon 249 selective preference by AFB1-induced mutations is not known; Chan et al. (2003) have related the high frequency of mutations to the presence of cytosine-guanine dinucleotide (CpG) methylated sites. Cytosine methylation has been proposed to increase the reactivity of guanine residues to AFB1, so that the toxin treatment results in an increase of AFB1-induced p53 mutations at specific CpG sites after CpG methylation. The high frequency of p53 mutations related to AFB1 exposure supports the hypothesis that this toxin exerts its carcinogenic effects through interference with regulatory mechanisms of cell cycle progression and possibly also interferes also with apoptosis induction. Further support comes from a recent paper dealing with the relationship between AFB1-related HCC and the expression of some proteins that play a pivotal role in cell cycle regulation (Yang et al., 2003). Despite previous results that strictly correlate p53 mutations and HCC (Aguilar et al., 1993), mice deficient in p53 do not show an increase of HCC incidence; however, loss of one allele of p53 makes mice very responsive to AFB1 exposure in terms of HCC development (Ghebranious and Sell, 1998). In addition, p53-deficient mice display increased hepatocyte proliferation, as shown by H3 thymidine uptake and immunological staining for proliferating cell nuclear antigen (Dumble et al., 2001). Yang et al. (2003) reported that the increased hepatocyte proliferation in p53-deficient mice is dependent on lower levels of p27kip1, one of the inhibitors of CDK; they also suggested that the higher susceptibility to AFB1 hepatocarcinogenesis of p53-deficient mice is dependent on the higher proliferation ability of hepatocytes (Yang et al., 2003). The lower level of p27 in livers of p53deficient mice is an interesting observation in the light of the current model of cell cycle progression through the G1 phase and subsequent initiation of DNA synthesis, which are cooperatively coordinated by a number of CDKs whose activity is in turn regulated by CDK activators and CDK inhibitors.

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pRB

Cell division

E2F p Cyclin-D

pRB

Spindle Cell cycle genes

CDK4 E2F Cyclin-E

G1 M

CDK2 Other cell cycle genes Cyclin-A G2

Cyclin-B

CDK2 S

CDK1

cdc25

FIGURE 8.5 Cell cycle molecular network. The figure outlines the major regulatory systems controlling cell cycle progression so far identified and the timing for their action. To enter mitosis cells must orderly traverse G1, S, and G2 phase. The progression from one phase to the next is dependent on a set of protein kinases (CDKs) activated by their interaction with cyclins, as indicated in the scheme. Other genes playing an important role for cell cycle progression are cdc25 and pRB; the former is a phosphatase, which selectively dephosphorylates specific tyrosine residues of CDK1, and the latter is a protein that interacts with transcription factors (E2F), regulating transcription of genes needed for DNA synthesis (e.g., DNA polymerase). RB/E2F interaction occurs when RB is dephosphorylated and blocks E2F interaction with its target genes. RB is phosphorylated by CDK4/cyclin D complex, which is active toward the end of G1, and by CDK2/cyclin E complex.

The specific molecular determinant for cell entry in S phase is phosphorylation of pRb by CDK4 and CDK2; phosphorylated pRb leads to the activation of the transcription factor E2F, which ultimately promotes transcription of a number of proteins required for S phase entry and progression (Figure 8.5). The CDK inhibitor p27 is part of the cip/kip (CDK inhibitory protein/kinase inhibitory protein) family, which inhibits cyclin E/CDK2 and cyclin A/CDK2, and forms a complex with cyclin D/CDK4. The association between cyclin D/CDK4 and p27 results in a sequestration of the inhibitor away from cyclin E/CDK2, allowing phosphorylation of pRb by CDK2 to proceed (Figure 8.6) (Sherr and Roberts, 1999; Albrecht et al., 1999). Although the relationship between p53 expression and p27 levels in p53-deficient mice is still unclear, in the light of the present knowledge of cell cycle regulation it seems conceivable that AFB1 damage leading to cancer in hepatocytes is, at least in part, dependent on cell cycle distribution at the time of exposure to the toxin.

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p16 Ink4a

mitogens

Cyclin-D

pRb

E2F

p19Arf

Mdm2

p53

CDK4 S phase firing p27 Cyclin-E

p21Cip1

CDK2 Cell cycle arrest

FIGURE 8.6 CDK-inhibitor network. The figure describes a second group of gene products (CKIs), which counteract mitogen activation of CDK4/cyclin D and other CDK/cyclin complex activity. These proteins act as inhibitors of CDKs, thus blocking cell cycle progression and causing cell cycle exit and maintenance of a nonproliferative state. They belong to two families: one (INK4) specifically inhibits CDK4 (as p16 and p19); the second (CIP/KIP) shows a large spectrum of CDK inhibition (as p21 and p27). The transcription factor E2F is able to activate transcription of p16 and p19 and p21 transcription is activated by p53, a known tumor suppressor gene. This regulation of CKI transcription represents a feedback system that cooperates with the positive regulators described in Figure 8.5, to ensure a multiple control system for cell division.

INACTIVATION OF INK4/ARF LOCUS AND AFB1 CARCINOGENETIC ACTION The possibility that AFB1 exerts its carcinogenic effects because of its interference with regulatory mechanisms of cell cycle progression is further supported by recent experiments on the relationship between AFB1 exposure and inactivation of the INK4a/ARF locus (Tam et al., 2003). In human cells this locus encodes two proteins, p16Ink4a and p14Arf, homologous to mouse p16Ink4a and p19Arf, respectively. The presence in the locus of two separate promoter regions leads to two different transcripts; they are distinguished by an alternative exon (1α and 1β, respectively) and share two common exons (2 and 3), which, however, are read in an alternative frame (Quelle et al., 1995; Sharpless and DePinho, 1999). The p16 protein is a member of the CDK INK inhibitory family and specifically inhibits the kinase activity of CDK4, preventing its association to cyclin D; this blocks pRB phosphorylation and the subsequent transition of the cells from G1 to S (Sherr, 2001). On the other hand, induction of expression of p19Arf induces cell cycle arrest in the G1 and G2 phase, likely acting on p53 level through the p53 destabilizing factor Mdm2 and allowing

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accumulation of the p53 tumor suppressor protein (Sherr, 1998; Sherr and Roberts, 1999). p16 and p19 are considered oncosuppressor proteins because both contribute, via different mechanisms, to inhibition of cell proliferation in response to oncogenic stimuli (as discussed by Tam et al., 2003; Lin et al.,1998; Serrano et al.,1995); their inactivation leads to a loss of cell cycle control and to abnormal cell growth. Inactivation of the INK4a/ARF locus has been reported as a frequent molecular alteration in human cancers, including HCC (Sharpless and DePinho, 1999). Three different, but not alternative pathways leading to inactivation of the locus have been identified. The first mechanism is the loss of allele heterozygosis (LOH), which is frequently found in human and experimental animal tumors (Wiseman et al., 1994; Herzog et al., 1996; Wong et al., 1997). A second mechanism is mutation in exons 1α, 1β, and 2, which, however, is a rare event and occurs mainly in melanoma (Walker and Hayward, 2002). A third mechanism is promoter silencing by hypermethylation of CpG sites in p16Ink4a and p19Arf, which has been reported in human tumors; hypermethylation of p16Ink4a promoter often occurs in experimental animals (Swafford et al., 1997; Patel et al., 2000); p19Arf hypermethylation has also been reported in murine lymphoma (Melendez et al., 2000). The relevance of the INK4a/ARF locus perturbation for AFB1 carcinogenesis has been reported by Tam et al. (2003) in lung tumors induced in mice by aflatoxin treatment.

EPIGENETIC EVENTS IN AFB1 CARCINOGENESIS: HYPERMETHYLATION OF SPECIFIC GENES Patel et al. (2000) investigated the exon 1α region of p16Ink4a promoter, which contains a CpG island frequently methylated in human and rodent tumors. They focused attention on CpG sites –1 to –7 relative to the translation start codon in exon 1α of the mouse p16Ink4a gene, which appears as the most frequently methylated region of the p16Ink4a promoter and is responsible for altered transcription (Patel et al., 2000). This study revealed at least partial methylation of the p16Ink4a promoter exon 1α in 84% of the AFB1-induced lung tumors. A decreased expression of p16Ink4a correlates well with the percentage of methylated CpG sites (Gonzalgo et al., 1998) and the percent methylation at individual CpG sites (i.e., ≥50%) (Patel et al., 2000). In the mentioned study a markedly lower (>50%) methylation at 25% or more of the CpG sites was detected in 10 of 61 (16%) AFB1-induced tumors, and the frequency of methylated CpG sites was shown to increase with tumor progression in malignancy (from adenoma to adenocarcinoma) (Tam et al., 2003). These data indicate that inactivation of p16Ink4a by hypermethylation plays a role in AFB1-induced tumors. The observation of an increasing frequency of CpG site methylation in tumors progressing from adenoma to adenocarcinoma suggests that the methylation level for gene repression, and consequent loss of protein expression, may reach the required threshold very early in the tumor progression process. The results showing a hypermethylated status of the p16Ink4a promoter, not only in mouse lung adenocarcinomas but also in adenomas, are consistent with results from other species and tumor types, indicating that p16Ink4a methylation is an early event in tumorigenesis (Belinsky et al., 1998).

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In addition, Zhang et al. (2002) reported a close relationship between p16Ink4a hypermethylation and HCC. They show a significant association between methylation status and AFB1–DNA adducts, suggesting that exposure to the toxin may lead to modifications in methylation patterns. This conclusion finds further support in the results of epidemiological studies on human HCC in different geographic regions: p16 aberrant methylation was observed with higher frequency when tumors in China and Egypt populations were compared to tumors in the U.S. or Europe. p16 aberrant methylation was also observed in cases with concurrent hepatitis or cirrhosis compared to tumors not related to toxin exposure and altered liver function (Shen et al., 2002). These data together point to environmental factors as possible agents of altered methylation patterns of cell cycle regulatory genes in HCC. Although the mechanism is not known, inflammatory processes, associated with hepatitis and cirrhosis, as well as oxidative stress following the generation of DNA adducts due to environmental agents as AFB1, may play a role and influence DNA methylation (Shen et al., 1995). The methylation status of the p19Arf promoter, the other product of the INK4a/ARF locus has also been studied by Tam et al. (2003). They observed hypermethylation in 88% of AFB1-induced lung tumors, as opposed to a lack of methylation in normal lung cells, and to a low frequency of methylation reported in γ-radiation-induced mouse primary lymphomas (Melendez et al., 2000). Unlike p16, concordance between protein expression and hypermethylation of CpG sites on p19Arf promoter is low; however, it becomes prominent (86%) when hypermethylation of transcription factor binding sites is considered. Point mutations in the promoter region of p19Arf also contribute to the altered protein expression, as shown by 2 mouse lung tumors of 43 examined carrying a G>T or C>A transversion in positions within an Sp1 transcription factor consensus sequence. These mutations correlate with decreased protein expression since the altered chromatin conformation prevents RNA transcription. The relevance of epigenetic mechanisms, as DNA methylation, in AFB1 carcinogenicity is further stressed by recent data on the O6-methylguanine-methyltransferase (MGMT) gene (Zhang et al., 2003), showing a significant correlation between AFB1–DNA adducts and hypermethylation of MGMT. This enzyme plays a crucial role in DNA repair processes, specifically removing alkyl groups from the O6 position of guanine. Alkylation in this position can lead to a conversion of a guanine–cytosine to an adenine–thymine base pair, since the O6-methylguanine is able to pair with thymine during DNA synthesis. Hypermethylation of the MGMT gene is associated with a loss of MGMT protein causing maintenance of alkyl groups on DNA. In addition, a significant correlation between the methylation status of MGMT gene and p53 mutations in HCC is also observed. These results suggest that epigenetic inactivation of MGMT plays an important role in the development of HCC and, more generally, that exposure to environmental carcinogens may cause altered methylation of genes involved in cancer development. Finally, it must be mentioned that in addition to direct DNA damage, due to guanine adduct formation, the ability of the toxin to induce oxidative stress has also been considered. Evidence for aberrant methylation of the p16Ink4a gene has been reported to be related to oxidative stress induced by exposure to carcinogens that

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elicit chronic inflammatory processes (Belinsky et al., 2002). Furthermore, these data suggest that activation of inflammatory mediators due to environmental factors may contribute to cancer initiation through oxidative stress and the formation of 8hydroxydeoxyguanosine-DNA adducts. In rats exposed to AFB1 Shen et al (1995) show a dose-dependent increase of 8-hydroxydeoxyguanosine, which is one of the most representative forms of oxidative DNA damage induced by reactive oxygen species (ROS) (Kasai et al., 1987; Floyd et al., 1988).

CONCLUSIONS Increasing knowledge of the multiple components participating in the regulation of cell cycle progression has provided the basis for investigating the mechanism of AFB1 cell cycle perturbation and carcinogenetic action. The recent data reviewed above allow the following conclusions to be drawn: 1. Silencing of the inhibitory system components following AFB1 exposure appears to be the major mechanism of cell cycle progression impairment; p53 and CKI as p21, p27, p16, and p19 are the main gene products whose function is altered by AFB1 exposure. 2. The molecular mechanism of inactivation of the inhibitory system appears to be gene methylation; this has been shown for specific genes such as p16Ink4 and, possibly more interestingly, for the enzyme MGMT, which is involved in DNA repair of guanine adducts. 3. Finally, recent findings on the possible role of oxidative stress induced by AFB1 exposure as a cofactor of aberrant methylation of gene as p16Ink4 allow new questions on the interaction of AFB1 and other environmental factors in additional mechanisms of cell cycle perturbation to be posed.

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Lin, A.W., Barradas, M., Stone, J.C., van Aelst, L., Serrano, M., and Lowe, S.W. (1998) Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling. Genes Dev., 2: 3008–3019. Lunn, R.M., Zhang, Y.J., Wang, L.Y. et al. (1997) p53 mutations, chronic hepatitis B virus infection, and aflatoxin exposure in hepatocellular carcinoma in Taiwan. Cancer Res., 57: 3471–3477. Luzi, A., Cometa, M.F., and Palmery, M. (2002) Acute effects of aflatoxins on guinea pig isolated ileum. Toxicol. Vitro, 16: 525–529. Massey, T.E., Stewart, R.K., Daniels, J.M., and Liu, L. (1995) Biochemical and molecular aspects of mammalian susceptibility to aflatoxin B1 carcinogenicity. Proc. Soc. Exp. Biol. Med., 208: 213–227. Melendez, B., Malumbres, M., de Castro, I.P., Santos, J., Pellicer, A., and Fernandez-Piqueras, J. (2000) Characterization of the murine p19(ARF) promoter CpG island and its methylation pattern in primary lymphomas. Carcinogenesis, 21: 817–821. Meneghini, R. and Schumacher, R.I. (1977) Aflatoxin B1, a selective inhibitor of DNA synthesis in mammalian cells. Chem. Biol. Interactions, 18: 267–276. Mistry, K.J., Krishna, M., and Bhattacharya, R.K. (1995) Signal transduction mechanism in response to aflatoxin B1 exposure: phosphatidylinositol metabolism. Chem. Biol. Interactions, 98: 145–152. Mossman, T. (1993) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. J. Immunol. Methods, 65: 55–63. Patel, A.C., Anna, C.H., Foley, J.F., Stockton, P.S., Tyson, F.L., Barrett, J.C., and Devereux, T.R. (2000) Hypermethylation of the p16 (Ink4a) promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines. Carcinogenesis, 21: 1691–1700. Quelle, D.E., Zindy, F., Ashmun, R.A., and Sherr, C.J. (1995) Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 83: 993–1000. Reye, R.D.K., Morgan, G., and Baral, J. (1963) Encephalopathy and fatty degeneration of the viscera. A disease entity in childhood. Lancet, 91: 749–752. Ricordy, R., Gensabella, G., Cacci, E., and Augusti-Tocco, G. (2002) Impairment of cell cycle progression by aflatoxin B1 in human cell lines. Mutagenesis, 17: 241–249. Serrano, M., Gomez-Lahoz, E., DePinho, R.A., Beach, D., and Bar-Sagi, D. (1995) Inhibition of ras-induced proliferation and cellular transformation by p16INK4. Science, 267: 249–252. Sharpless, N.E. and DePinho, R.A. (1999) The INK4A/ARF locus and its two gene products. Curr. Opin. Genet. Dev., 9: 22–30. Shen, H.M., Ong, C.N., Lee, B.L., and Shi, C.Y. (1995) Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis, 16: 419–422. Shen, L., Ahuja, N., Shen, Y., Habib, N.A., Toyota, M., Rashid, A., and Issa, J.P. (2002) DNA methylation and environmental exposures in human hepatocellular carcinoma. J. Natl. Cancer Inst., 94: 755–761. Sherr, C.J. (1998) Tumor surveillance via the ARF-p53 pathway. Genes Dev., 12: 2984–2991. Sherr, C.J. (2001) The INK4a/ARF network in tumour suppression. Natl. Rev. Mol. Cell Biol., 2: 731–737. Sherr, C.J. and Roberts, J.M. (1999) CDK inhibitors: positive and negative regulators of G1phase progression. Genes Dev., 13: 1501–1512. Swafford, D.S., Middleton, S.K., Palmisano, W.A., Nikula, K.J., Tesfaigzi, J., Baylin, S.B., Herman, J.G., and Belinsky, S.A. (1997) Frequent aberrant methylation of p16INK4a in primary rat lung tumors. Mol. Cell. Biol., 17: 1366–1374.

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Tam, A.S., Devereux, T.R., Patel, A.C., Foley, J.F., Maronpot, R.R., and Massey, T.E. (2003) Perturbations of the Ink4a/Arf gene locus in aflatoxin B1-induced mouse lung tumors. Carcinogenesis, 24: 121–132. van Gijssel, H.E., Maassen, C.B., Mulder, G.J., and Meerman, J.H. (1997) p53 protein expression by hepatocarcinogens in the rat liver and its potential role in mitoinhibition of normal hepatocytes as a mechanism of hepatic tumour promotion. Carcinogenesis, 18: 1027–1033. Vermeulen, K., Van Bockstaele, D.R., and Berneman, Z.N. (2003) Cell cycle and apoptosis. Cell Prolif., 36: 131–149. Viviers, J. and Schabort, J.C. (1985) Aflatoxin B1 alters protein phosphorylation in rat livers. Biochem. Biophys. Res. Commun., 129: 342–349. Walker, G.J. and Hayward, N.K. (2002) p16INK4A and p14ARF tumour suppressors in melanoma: lessons from the mouse. Lancet, 359: 7–8. Wiseman, R.W., Cochran, C., Dietrich, W., Lander, E.S., and Soderkvist, P. (1994) Allelotyping of butadiene-induced lung and mammary adenocarcinomas of B6C3F1 mice: frequent losses of heterozygosity in regions homologous to human tumor-suppressor genes. Proc. Natl. Acad. Sci. U.S.A., 91: 3759–3763. Wogan, G.N. (1992) Aflatoxins as risk factors for hepatocellular carcinoma in humans. Cancer Res., 52: 2114–2118. Wong, D.J., Barrett, M.T., Stoger, R., Emond, M.J., and Reid, B.J. (1997) p16INK4a promoter is hypermethylated at a high frequency in esophageal adenocarcinomas. Cancer Res., 57: 2619–2622. Yang, C., Sun, M., Ilic, Z., Friedrich, T.D., and Sell, S. (2003) Reduced expression of p27kip1 and increased hepatocyte proliferation in p53-deficient mice. Mol. Carcinogenesis, 36: 15–22. Yu, F.L. (1981) Studies on the mechanism of aflatoxin B1 inhibition of rat liver nucleolar RNA synthesis. J. Biol. Chem., 256: 3292–3297. Zhang, Y.J., Ahsan, H., Chen, Y., Lunn, R.M., Wang, L.Y., Chen, S.Y., Lee, P.H., Chen, C.J., and Santella, R.M. (2002). High frequency of promoter hypermethylation of RASSF1A and p16 and its relationship to aflatoxin B1-DNA adduct levels in human hepatocellular carcinoma. Mol. Carcinogenesis, 35: 85–92. Zhang, Y.J., Chen, Y., Ahsan, H., Lunn, R.M., Lee, P.H., Chen, C.J., and Santella, R.M. (2003) Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation and its relationship to aflatoxin B1-DNA adducts and p53 mutation in hepatocellular carcinoma. Int. J. Cancer, 103: 440–444.

9

Cycad Consumption and Neurological Disease Jeff D. Schulz, Erin L. Hawkes, and Christopher A. Shaw*

CONTENTS Abstract ..................................................................................................................234 Abbreviations .........................................................................................................234 Introduction............................................................................................................235 Cycad Ethnobotany................................................................................................236 Introduction................................................................................................236 Cycad Phylogeny and Taxonomy..............................................................236 Cycad Products as a Food Source.............................................................238 Medicinal Uses of Cycad ..........................................................................239 Processing Cycad Seeds ............................................................................239 ALS-PDC: History, Symptomatology, Neuropathology, and Etiology ................240 History of ALS-PDC .................................................................................240 Clinical Features of ALS-PDC..................................................................240 Neuropathology of ALS-PDC ...................................................................242 Etiology of ALS-PDC ...............................................................................245 Cycad Toxicity.......................................................................................................246 Searching for Cycad’s Toxins ...................................................................246 Cycad Consumption and Acute Toxicity ..................................................247 “Slow Neurotoxin” Theory........................................................................247 Sterol Glucosides: Causal Agents in the Development of ALS-PDC? ....249 Consumption of Cycad by Other Species (Natural and Experimental) ...252 A Mouse Model of ALS-PDC...............................................................................254 Behavioral and Neuropathological Features of ALS-PDC in Mice .........254 Validity and the “Four Dimensions” of Our Model .................................259 The “Time Course” Project .......................................................................260 Sex, Age, and Genetics: Important Variables............................................261 Limitations of the Current Model .............................................................263 Conclusions............................................................................................................263 Acknowledgments..................................................................................................263 References..............................................................................................................264 * Jeff D. Schulz and Erin L. Hawkes are equal coauthors.

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Abstract

Amyotrophic lateral sclerosis–parkinsonism dementia complex (ALS-PDC) has been termed the “Rosetta Stone” of neurological disease, due to its component amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease symptomatology and neuropathology. The largest and most-studied geographic focus of ALS-PDC is that of the island of Guam in the South Pacific. The consumption of the seeds of a local, indigenous species of cycad, Cycas micronesica, is thought to play a causal role in the development of ALS-PDC. Cycad contains many toxins, and traditional processing methods have therefore been developed with the aim of removing them. Water-soluble, cycad-specific toxins such as cycasin, macrozamins, and β-methylamino-L-alanine are removed by washing the cycad. This refutes early cycad theories of ALS-PDC that these toxins are causal in the development of ALS-PDC. Since cycad consumption still appeared to be linked to ALSPDC, our laboratory has reexamined the cycad hypothesis. By feeding mice processed cycad flour, we have created a valid mouse model of ALS-PDC. This model allows us to analyze ALS-PDC in “four dimensions”: its behavioral deficits, biochemical changes, and morphological or pathological outcomes through time. Our “time course” project is an attempt to delineate the rate, type, and extent of disease progression as the subjects move from normal central nervous system state, through preclinical neurological damage, to clinical diagnosis, and, ultimately, arrive at the end state. This information, in conjunction with data on the effects of various genetic conditions, sex, and age, will allow us to template-match our findings against the human experience of ALS-PDC. This will allow us to distinguish between the disease’s causal, coincidental, and compensatory (successful or failed) components, and thus provide us with targeted therapeutics with which we can move from palliative to preventative care for patients suffering from ALS-PDC as well as those diagnosed with amyotrophic lateral sclerosis, Parkinson’s disease, or Alzheimer’s disease.

Abbreviations

6-OHDA: 6-hydroxy-dopamine; AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; ALS-PDC: amyotrophic lateral sclerosis–parkinsonism dementia complex; AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ApoE: apolipoprotein E; BMAA: β-methylamino-L-alanine; BOAA: β-N-oxalylamino-L-alanine; BSSG: β-sitosterol–D-glucoside; CDK-2: cell division protein kinase 2; CNS: central nervous system; Erk-1: extracellular signalregulated kinase 1; LDH: lactate dehydrogenase; MAM: methylazoxymethanol; MAMAL: methylazoxymethanol aldehyde; MND: motor neuron disease; MPTP: 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MRI: magnetic resonance imaging; mya: million years ago; NFT: neurofibrillary tangle; NMDA: N-methyl-D-aspartate; PD: Parkinson’s disease; PDC: parkinsonism dementia complex; PET: positron emission tomography; PKC: protein kinase C; PSP: progressive supranuclear palsy; Rsk-1: ribosomal S6 kinase 1; SG: sterol glucoside; SNpc: substantia nigra pars compacta; SOD-1: superoxide dismutase 1; TH: tyrosine hydroxylase; TUNEL: transferase-mediated dUTP nick end labeling

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INTRODUCTION Neurodegenerative disease begs the classic question of what roles the environment and genetics have to play in the development, course, and end state of such diseases as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). Each of these disorders has been linked to both environmental toxicity and genetic susceptibility. Furthermore, these three neurodegenerative diseases generally have onset in the latter years of life, suggesting that age, too, plays a key role. Picture three interlocked circles, then: a Venn diagram of genetics, environment, and age (Figure 9.1). If sufficiently severe, any one of these may become an important variable in the development of neurodegenerative disease. However, the more common scenario is one where two, or even all three, of these factors interact (shaded areas). This interaction can compound susceptibility. For example, a genetic vulnerability increases with age, when neurons are less able to resist or repair damage dictated by genetics. This allows the development of disease where each factor alone cannot evoke a disease state but, when occurring in concert, they are able to provoke dysfunction. Moreover, this representation of neurodegenerative disease allows for the effects of both time-restricted additive and long-term cumulative factors.

Environment Low

Toxicity

Genetics

High

High

Low Susceptibility

Old

Age Young

FIGURE 9.1 Venn diagram representing three key factors influencing the development of neurodegenerative disease. The probability of developing neurological disorder is least in unshaded areas, increased in gray areas, and most likely in the central blackened area. This corresponds to having one, two, or all three factors, respectively, interacting to provoke disease. High environmental toxicity, e.g., high intake of a food that contains (neuro)toxins, and high genetic susceptibility, e.g., genetic mutation such as the SOD-1 mutation that causes familial ALS, both increase the probability of neurodegenerative disease. Age is likewise an important variable in the development of neurological disorder, with older subjects more vulnerable than younger ones.

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While not denying the role of genetics and age, our focus in this chapter is on environmental insult, and the neurodegenerative disease of interest is ALS-PDC (amyotrophic lateral sclerosis–parkinsonism dementia complex). ALS-PDC is a rare disorder virtually endemic to the island of Guam. Nevertheless, it has been termed the “Rosetta Stone” of neurological disease (L.T. Kurland, personal communication); in light of the fact that this disease has clinical and neuropathological similarities to the classic forms of ALS, PD, and AD, research on ALS-PDC should give insight to the disease processes of its component features (ALS, PD, and AD). Genes “for” the classic forms of each of these neurodegenerative disorders have been mapped, and age is a vulnerabilizing factor obvious to even the general public. Our interests and hypotheses, however, concern the role of a specific environmental factor, namely, the consumption of cycad seeds and their (neuro)toxins, and its significance in the development of Guamian ALS-PDC. Cycads are palm-like gymnosperm plants whose seeds, which contain toxic compounds, are traditionally eaten in Guam. Specific preparation techniques remove some of these toxins, but residual (neuro)toxins are thought to play a causal role in the development of ALS-PDC. Feeding processed cycad to mice creates a mammalian model of ALS-PDC. We can therefore examine the role of this environmental toxin alone (i.e., in wild-type mice), as represented by the unshaded part of environment’s circle (Figure 9.1). Furthermore, we can consider issues corresponding to the overlap of the other factors’ circles with that of the environment; that is, genetically vulnerable mice (e.g., transgenic SOD-1 mice that develop ALS) or older mice are predictably more susceptible to developing ALS-PDC. In sum, we are interested in demonstrating the role of a specific environmental insult — a nutritional toxicity — in the development of a neurodegenerative disorder, and determining how this factor works alone as well as in concert with genetic susceptibility and/or age.

CYCAD ETHNOBOTANY INTRODUCTION Many societies have discovered that the seeds, stem, roots, and leaves of the palmlike cycad tree can be used as a source of dietary starch. Because cycad contains virulent toxins, traditional processing techniques have been developed to remove these substances. Nevertheless, cycad consumption has been implicated as a causative agent of the development of a rare neurodegenerative disorder, ALS-PDC, on the island of Guam.

CYCAD PHYLOGENY

AND

TAXONOMY

At times referred to as the “living fossils” of the plant world, cycads have changed little over the millions of years of their existence (Audhali and Stevenson, 2003). Cycad fossils dating as far back as the Paleozoic era (270–280 mya) have been found, but it is the Jurassic period (210–145 mya) that has been heralded as the “Age of the Cycads.” In fact, the fossil record shows that cycads have been present

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FIGURE 9.2 Map of the western Pacific showing the three foci of ALS-PDC. The species of cycad that is indigenous to each area is also noted. (Adapted from Garruto et al., 1985; Jones, 2002.)

on every continent and at every latitude. There are three extant families (all of which arose more recently, during the Tertiary period, 50–60 mya), and 19 extinct genera. The 102 described species of the genus Cycas are distributed across parts of Australia, Asia, and Africa, with individual species often restricted to specific geographical areas (Figure 9.2). Jones (2002) describes Cycas seeds as distinctly ovoid, oblate, ellipsoid, or rounded, the outer sarcotesta (integument) green, yellow, brown, orange, or red, smooth, and may or may not have a spongy flotation or fibrous layer. Of interest here is the fact that all cycads contain virulent toxins, such as the azoxyglycosides cycasin and macrozamins, and the nonprotein amino acid, β-methylamino-L-alanine (BMAA). Endemic to Micronesia, the Marianas Group, and the Western Caroline Islands, including the island of Guam, C. micronesica is the species of cycad that has been exploited by the indigenous Chamorro population of Guam as a food source (Audhali and Stevenson, 2003). Cycas micronesica belongs to the Rumphiae subsection of the genus Cycas, and is therefore characterized by the presence of a spongy flotation layer between the sarcotesta and the seed kernel (Jones, 2002). In the past,

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C. micronesica has been incorrectly identified as C. circinalis (Audhali and Stevenson, 2003), a species that lacks a spongy flotation layer and is not found in the Western Pacific but in India and Burma. Review of the literature is therefore at times difficult, but the fact that C. micronesica is confined to a relatively small geographic area — and in which C. circinalis is not present (Figure 9.2) — can allow the assumption that any reference to cycads in Micronesia, the Marianas Group, and the Western Caroline Islands (including the island of Guam) will be referring to C. micronesica. In this chapter, references to C. circinalis that are more likely referring to C. micronesica will be referred to as C. micronesica.

CYCAD PRODUCTS

AS A

FOOD SOURCE

For as many as several thousand years, cycad seeds, stem, roots, and leaves have been exploited as a traditional food source by various indigenous populations around the world (Jones, 2002). Since seeds are a more renewable source than stems, seeds are the most commonly ingested part. These edible cycad products are rich in starch, but also contain high amounts of toxic bioactive compounds that must be removed before ingestion. Cycad products are eaten in Australia and surrounding islands (including Guam), Asia (Malaysia, the Philippines, Assam, Indonesia, Sri Lanka, Japan, Andaman, Aru, Kei Islands, Fiji, Senegal, Nicobar Island, Moluccas, India, Ryukyus [Okinawa]), the Americas (Florida, Mexico, Central and South America), and Africa (Audhali and Stevenson, 2003). However, they are more often resorted to as a “famine food” than a diet staple (Beardsley, 1964) due to their toxicity and the extensive processing required to remove these toxins. They are particularly used as a food source following a fire as cycads are more likely to survive fire than other plants. Only a subset of cycad species are traditionally consumed by human populations (Jones, 2002), perhaps because different cycad species have differing concentrations of the typical cycad toxins. This subset includes at least four Cycas species (media, micronesica, revoluta, and rumphii), two Dioon (edule and spinalosa), and one of each of Encephalartos (caffer) and Zamia (integrifolia). Interestingly, it is only the consumption of C. micronesica that has been repeatedly shown to be linked to the development of human neurological disorder. It is therefore not surprising that, while C. micronesica is indigenous to the Marianas Group of islands in Micronesia (Jones, 2002), the main geographic focus of the neurodegenerative disorder ALS-PDC that is thought to be caused by the ingestion of these cycad seeds is similarly focused within this area (specifically, the island of Guam). The consumption of cycad as a food source among indigenous populations has decreased substantially with the adoption of a more Westernized diet, a decrease that has been mirrored by the decrease in cases of ALS-PDC (Zhang et al., 1990). Interestingly, the occupation of Guam by the Japanese during World War II forced the people of Guam to rely on cycad due to food shortages, and this change in diet was later found to correspond to a transient increase in the number of cases of ALS-PDC (reviewed by Plato et al., 2003). Today, cycad is eaten only by certain indigenous populations, such as the Bantu peoples of South Africa, and the consumption of these species of cycad has not been found to be related to ALS-PDC (Norstog and Nicholls, 1997).

Cycad Consumption and Neurological Disease

MEDICINAL USES

OF

239

CYCAD

A variety of medicinal uses of cycad seeds, stems, and leaves have been documented in various parts of the world (Audhali and Stevenson, 2003). In both China and New Guinea, a pulp or paste made from raw, unwashed seeds is used for skin complaints, such as cuts and ulcerous wounds. Cycads are also thought to “increase strength” (Kii Peninsula of Japan) and “regulate energy flow” (China), and in both these regions it is used as a natural remedy for stomach troubles. Various other medicinal uses of cycad products have been noted in China, including pain relief, the treatment of hypertension, and as a cure for liver cancer, rheumatic colds, and kidney problems. Although cycad may in fact ameliorate some ailments, it cannot be ignored that these practices also expose the patient to cycad’s toxic compounds. This may explain the focus of motor neuron disease on the Kii Peninsula of Japan, where cycad is not eaten but is used medicinally to a much greater degree than in other areas of Japan (Iwami et al., 1993).

PROCESSING CYCAD SEEDS The use of cycad seeds as a food source has required the development of processing traditions to render the naturally toxic cycad safe for human ingestion. In fact, it has been suggested that this “taming” of cycad was the first example of humans developing specific and effective techniques that render a highly toxic product safe for human consumption (Jones, 2002). These processing methods effectively remove, neutralize, or destroy toxic compounds that can otherwise cause acute or protracted illness, or even death, in humans. Although different regions use varying processing methods, they typically involve one or more of the following after removing the endosperm from the sarcotesta: washing, fermentation, cooking, and aging (Audhali and Stevenson, 2003). In Australia, seeds are cooked and then leached in running water overnight, or are dried and then leached in running water for 3 to 5 days. Similar cooking (Andaman, Aru, Fiji, and Kei Islands), drying (India), and leaching (Moluccas) procedures are used in parts of Asia. Alternatively, the seeds can be aged, as is sometimes done in Australia. Skilled harvesters of cycad seeds can reportedly selectively pick only those seeds that have low toxin levels. On the island of Guam, the traditional processing of C. micronesica seeds can take up to a month (Lister and Hill, 2003). The seeds are soaked for 7 to 9 days, cut into smaller pieces, and soaked for another week. After another slicing and weeklong wash, the seeds are cut into even smaller pieces and rinsed with water until the water residue is no longer milky. Finally, the cycad chips are dried and ground into a fine flour. The flour is then made into such products as “fading/frederico” tortillas, or is used to thicken soup. However, these traditional processing techniques are less than ideal. While they remove toxins that are water soluble and/or altered by heat, water-insoluble thermotolerant toxins remain. Because the former are associated with more acute illness, it was likely assumed that, because washing and cooking cycad prevents such acute toxicity, it rendered cycad safe for ingestion. Consequently, water-insoluble

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and heat-resistant toxins whose clinical effects are not seen until many years, or even decades, after exposure would not have been easily linked to cycad consumption. It is therefore not surprising that although traditionally prepared cycad has had many of its toxins removed or destroyed, the ingestion of processed cycad products is still associated with the development of ALS-PDC (Kurland, 1988).

ALS-PDC: HISTORY, SYMPTOMATOLOGY, NEUROPATHOLOGY, AND ETIOLOGY HISTORY

OF

ALS-PDC

Guam was first recognized as having a remarkable concentration of neurodegenerative disorders in the early 1950s. Shortly after World War II, a U.S. Navy pathologist serving on Guam presented the first formal report of a high incidence of ALS among the indigenous Chamorro population (Zimmerman, 1945). Initially, only ALS was recognized to be highly prevalent on the island, but early surveys of the native population soon revealed a large population of cases with predominantly parkinsonian symptoms and dementia (Hirano et al., 1961a). Mulder, Kurland, Hirano, and colleagues referred to the collection of these symptoms as ALS-PDC (Kurland et al., 1954; Hirano et al., 1961a, b). In 1954, Kurland estimated that the prevalence of ALS was between 50 and 100 times that in the continental U.S. Later reports estimated that nearly 25% of adult deaths among the Chamorros, between the early 1950s and 1980, were due to ALS and PDC (Kurland, 1994). Following the identification of this unique cluster of neurodegenerative disease, teams of neurologists, pathologists, and epidemiologists became interested in understanding Guamian neurodegeneration in the hopes that it would provide insight into understanding neurological disease worldwide.

CLINICAL FEATURES

OF

ALS-PDC

ALS-PDC garnered considerable attention because its component features so closely resembled the primary neurodegenerative disorders occurring throughout the world: ALS, parkinsonism similar to PD, and dementia reminiscent of AD. The similarities were immediately apparent upon examination of patients, and later postmortem studies revealed similar neuropathological changes underlying the observed clinical symptoms. The following sections summarize the clinical and neuropathological manifestations of ALS-PDC; comparing them to features of ALS, PD, and AD. ALS is a fatal paralytic disease characterized by loss of spinal cord motor neurons resulting in progressive muscle weakness and atrophy. Hyperreflexia, fasiculations, and spasticity are also typically present. Disease onset is insidious; an increasing paralysis leads to death on average 3 years after diagnosis. Guamian ALS is similar to classic ALS in its form and presentation (Kurland and Mulder, 1954; Rodgers-Johnson, 1986). As in classic ALS, the most common features at diagnosis are atrophy and muscle weakness. The only significant differences are slight increases in the average age of onset and disease duration (Elizan et al., 1966). Additionally, a review of clinical trends from 1950 through 1979 found an increase

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in the age at onset for ALS as well as a shorter duration of illness (Rodgers-Johnson et al., 1986). Nevertheless, from a clinical perspective Guam ALS and classic ALS are nearly identical disorders (Elizan et al., 1966). As the name implies, PDC can manifest with parkinsonian features and cognitive decline reminiscent of Alzheimer’s dementia. The parkinsonism aspect of the disease includes the classic features of tremor, rigidity, and bradykinesia (slow movement) (Hirano, 1961a). PDC is often associated with a disturbance of speech and gait apraxia (impairment of the ability to execute complex coordinated movements), however, the classic festinating (forceful) gait associated with PD is not observed (Lilienfeld et al., 1994). The motor dysfunction also includes markedly impaired fine motor movements as well as facial masking with “reptilian” stare and infrequent blinking. At death, 85% of patients have developed bradykinesia, 75% rigidity, and 65% tremor (Elizan et al., 1966; Rodgers-Johnson et al., 1986). While these proportions of symptoms are somewhat different than classical PD, in which tremor is a hallmark feature, the classic parkinsonism features are present. The dominant cognitive feature of PDC is a progressive mental deterioration not unlike that of AD. Hirano and colleagues (1961a) note that in many PDC patients an “organic mental syndrome” is the dominant clinical feature, while the parkinsonian syndrome is less conspicuous. In a group of 72 patients with PDC, 29% initially presented with dementia and only later did some develop features of parkinsonism (Elizan et al., 1966). In other cases, parkinsonian symptoms occurred initially, and only later did dementia appear (Elizan et al., 1966). Dementia is therefore considered to be a ubiquitous feature of PDC, eventually appearing in all patients who initially present with parkinsonism features. As described by Elizan et al. (1966), the most frequent signs of dementia at initial presentation are memory deficits and disorientation with regard to time, place, and person. Difficulty with simple calculations and reasoning become increasingly severe with disease progression. In addition, personality changes, including apathy, irritability, and aggression, have been noted in one third of patients (Elizan et al., 1966). Olfactory deficits similar to those observed in AD and PD are present in virtually all patients with PDC (Doty et al., 1991). The clinical distinctions between ALS and PDC become less clear given that patients often express features of both ALS and PDC, in varying combinations and severities. In a study of 104 cases of ALS, Elizan and associates (1966) observed that 5 patients subsequently developed the total clinical picture of PDC, whereas 27 of the 72 previously mentioned PDC cases eventually developed ALS. Taken together, this strongly suggests that ALS and PDC on Guam are not distinct disease entities, and instead points toward a common pathology and etiology. Conventionally, neurodegenerative diseases such as ALS, PD, and AD have been thought of as distinct disorders, arising from different etiologies and expressing with unique behavioral and neuropathological indicators (Table 9.1). The apparent overlap of symptoms found in ALS-PDC is in keeping with recent research describing significant commonalities across these disorders. For example, patients with AD may show tremor, a hallmark of PD (Yokoyama et al., 2002). Similarly, patients with PD and ALS may show losses of cognitive function, similar to that in AD (Vaphiades et al., 2002; Aarsland et al., 2003). Gait abnormalities, typical of PD, in

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TABLE 9.1 Comparison of Behavioral Changes Occurring in the Major Neurodegenerative Diseases and ALS-PDC ALS-PDC

ALS

PD

AD

Memory deficits Disorientation with regard to time, place, and person Personality changes Olfactory deficits

   

*

* * * 

   

Motor Muscle weakness, atrophy Tremor Bradykinesia Postural instability Gait disturbance

    

Cognitive

*  

    

*Feature is not present in all cases.

elderly persons have been shown to be a significant predictor of the risk of developing dementia (Verghese, 2002). Finally, recent clinical observations have defined a new disorder referred to as ALS-plus, which combines symptoms of ALS with dementia and/or parkinsonism (Zoccolella et al., 2002).

NEUROPATHOLOGY

OF

ALS-PDC

Just as Guamian ALS is similar to classic ALS from a clinical perspective, the spinal cord pathology of the Guamian variant of ALS is also comparable to the more classic ALS found throughout the world (Hirano et al., 1967). The typical neuropathological features of ALS are loss of spinal and cortical motor neurons that innervate skeletal muscle resulting in muscle weakness and atrophy (Figure 9.3A). The notable differences in Guamian ALS include an abundance of neurofibrillary tangles (NFTs) composed of the microtubule-associated protein Tau throughout the central nervous system (CNS) (Rodgers-Johnson et al., 1986). Tau regulates the assembly and stability of microtubules in a manner dependent on its level of phosphorylation (Brich et al., 2003). Interestingly, abnormally high levels of hyperphosphorylated Tau are associated with various neurological pathologies including AD (Lee et al., 2001) and to a lesser degree PD (Ishizawa et al., 2003). Further differences are present in other CNS regions of Guamian patients with ALS. In a study of ALS patients lacking symptoms of PDC, there was significant neuronal loss and presence of NFTs in the hippocampus — a pathology normally associated with AD and dementia — in 46% of the cases (Rodgers-Johnson et al., 1986). Additionally, a more recent study found a reduced uptake of 6-fluorodopa in the striatum of Guamian patients with ALS, a sign of dopaminergic cell loss that is the hallmark pathology of PD (Snow et al., 1990). While these changes are not

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FIGURE 9.3 The basic neuroanatomy of the major neurodegenerative diseases. (A) Cross section of the spinal cord showing the region of motor neuron cell loss in the ventral horn that occurs in ALS. (B) In Parkinson’s disease neuron loss occurs in the substantia nigra pars compacta. These cells send projections to the basal ganglia, the loss of which results in motor complications. (C) In Alzheimer’s disease there is cell loss throughout cortical regions resulting in decreased brain volume.

features of classical ALS, their presence could indicate preclinical, concomitant PDC that has not progressed to a clinically detectable point (Snow et al., 1990). This is in keeping with numerous studies of neurodegenerative disease demonstrating that significant neuron loss occurs before clinical symptoms appear. For example, the symptoms of PD only become apparent when more than 50% of nigral dopamine neurons are lost (McGeer et al., 1988), and for ALS, some estimates of spinal alpha motor neurons loss at diagnosis approach 70% (Arasaki and Tamaki, 1998). The neuropathological changes in PDC are also similar to those observed in PD and AD, with a few exceptions. PD is characterized by a loss of dopamine-containing neurons in the substantia nigra pars compacta (SNpc) and their terminals, which project to the striatum (Figure 9.3B). Similar changes have been documented in the nigral-striatal system in PDC. Not only is there a dramatic loss of dopaminergic cells in the SNpc (Hirano et al., 1961b), but Snow and colleagues (1990) have shown there is a dramatically reduced uptake of 6-fluorodopa in the striatum of patients with PDC: further evidence of a PD-like lesion. However, one of the hallmark pathologies of PD (albeit, a criticized one: Calne and Eisen, 1989), Lewy bodies (intracellular inclusions of the aggregated protein α-synuclein), is not ubiquitous in PDC. In contrast to PD, in which virtually all patients display such α-synuclein pathology (Calne and Eisen, 1989), only 37% of patients with PDC show such

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pathology (Forman et al., 2002). (It is due to this distinct lack of Lewy bodies, as well as differences in clinical manifestation, that the parkinsonian aspect of PDC is not considered to be true PD but rather a form of parkinsonism.) With regard to neuropathological features of the dementia component of PDC, there is marked cortical atrophy, and NFTs are present in numerous brain regions including the hippocampus, entorhinal cortex, basal forebrain, and the neocortex (Hirano et al., 1961b; Kurland, 1994), all of which is similar to the neuropathological changes observed in classical AD (Hyman et al., 1984) (Figure 9.3C). It is interesting to note that the ultrastructure and immunohistochemical profile of the NFTs appears to be identical to that in AD (Kurland, 1994). However, there are differences in the pattern and distribution of NFTs. In PDC, there is a strong predilection for tangles in cortical layer 3, whereas in AD they are mainly found in layer 5 (Hof et al., 1994). Additionally, NFTs are present to a greater degree subcortically in PDC than in AD, where they primarily occur in cortical regions (Hirano et al., 1961b). Our understanding of the role of NFTs in dementia and neurodegeneration is furthered by a study of the occurrence of NFTs in Chamorros who died without any clinically apparent neurological disease. Chen and associates (1981) found that 57% of Chamorros between the ages of 40 and 59 years, and 95% of Chamorros older than 60 had extensive NFTs. These results could either mean that these people died before the symptoms of PDC were expressed at clinically detectable levels, or that NFTs are merely a background feature unrelated to neurodegenerative disease in the Chamorro population. We believe that the former view warrants further consideration given that the extent of NFT formation in neurologically intact Chamorros was often equal to what is seen in end-stage AD (Perl et al., 2003). Similar to the overlap of clinical symptomatology in classic forms of neurodegenerative disorders previously described, postmortem analysis has demonstrated that some of the hallmark features of these diseases may also cross conventional boundaries. As an example, NFTs, characteristic of AD, have been identified in some cases of PD and ALS (Arima et al., 1999; Kokubo et al., 2000). Similarly, αsynuclein, the major component of Lewy bodies in PD, was originally isolated from patients with AD (Lucking and Brice, 2000). Some (Calne and Eisen, 1989) have gone so far as to suggest that since AD, PD, and ALS are all characterized by the accumulation of cytoskeletal debris (NFTs, Lewy bodies, etc.) these neurodegenerative disorders may be best described as “cytoskeletal disorders.” A common denominator at the level of neuropathology may thus link these three diseases (Table 9.2). The coexistence of features of Guamian ALS and PDC within individual patients points to a common underlying etiology and pathogenesis. Furthermore, the clinical and pathological similarities between Guamian ALS-PDC and the classical forms of ALS, PD, and AD suggest that an understanding of the etiology and progression of Guamian neurodegeneration might shed light on neurodegenerative disease throughout the world. As symptomatic and neuropathological overlap is uncovered, it becomes more reasonable to assume that shared features of ALS, PD, and AD point to a shared etiology and/or pathology. In this view, ALS-PDC may well serve as a neurological Rosetta Stone, the decipherment of which would unlock clues to neurological disease worldwide.

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TABLE 9.2 Comparison of Neuropathological Changes Occurring in the Major Neurodegenerative Disorders and ALS-PDC Neuropathology

ALS-PDC

ALS

Motor neuron loss Dopamine deficiency Cell loss in the SNpc Lewy bodies Neurofibrillary tangles Cortical atrophy β-Amyloid plaques

   *   *

 * *  *

PD

   * *

AD

*   

*Feature is not present in all cases.

ETIOLOGY

OF

ALS-PDC

During the initial investigations into the etiology of ALS-PDC, investigators had hope that straightforward casual factors would be readily unearthed. For example, the Chamorro population was relatively homogeneous in genetic background, facilitating genetic analysis and identification of genes implicated in the disorder (Plato et al., 2003). In spite of this, detailed genetic surveys and analysis failed to identify a genetic basis for the disease (Reed et al., 1975; Lilienfeld et al., 1994). Further evidence against a genetic etiology came from studies of a Chamorro population living on the nearby island of Saipan (80 miles north of Guam). They had virtually the same genetic background as Chamorros on Guam, yet there was no evidence of an increased incidence of neurodegenerative disease, a finding that is strongly indicative of an environmental agent playing an etiological role (Yanagihara et al., 1984; Lilienfeld et al., 1994). The apparent lack of a genetic basis of the disorder is in keeping with observations regarding the classic forms of ALS, PD, and AD in which the majority of cases do not show a familial pattern of inheritance. For example, in a study of identical twins, Tanner and associates (1999) show that genetic factors do not play a major role in causing idiopathic PD. Similarly, the majority of ALS cases (approximately 95%) are sporadic in nature, with no obvious genetic basis or familial pattern of inheritance (Mitchell, 2000). Finally, three genes have been identified as responsible for the rare early-onset form of AD; however, this accounts for only 5% of all cases (Rocchi et al., 2003). Investigators therefore rapidly focused on potential environmental toxins, screening hundreds of potential factors, including the ionic (mineral and heavy metal) composition of soil and groundwater, native food products, and industrial materials associated with military activity. Most of these environmentally based hypotheses were discredited, although a few gained some support through further investigations. One hypothesis that has garnered support for some time is that ALS-PDC is triggered by nutritional deficiencies of calcium and magnesium, which leads to

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secondary hyperparathyroidism that then facilitates the entry of calcium and toxic heavy metals, such as aluminum, into the brain (Yanagihara et al., 1984). However, this was later refuted by a study that showed that patients with ALS-PDC have no indications of abnormalities in calcium metabolism, have normal parathyroid hormone levels, and have levels of heavy metals in blood and urine samples that are statistically similar compared to that of controls (Ahlskog et al., 1995). Further work demonstrated that monkeys fed a low-calcium, high-aluminum diet develop neurodegenerative changes comparable to those of early ALS and PD in the spinal cord, brainstem, substantia nigra and cerebrum (Garruto et al., 1989). Some of the neurons in these affected areas accumulate aluminum at concentrations 200 times that of unaffected cells (Perl et al., 1982). However, an examination of a large number of NFT-bearing and non-NFT-bearing neurons from deceased patients with AD revealed no significant difference in aluminum content (Markesbery et al., 1990). Furthermore, despite early studies suggesting low environmental levels of calcium and high levels of aluminum (Garruto et al., 1980), Zolan and Ellis-Neill (1986) report adequate calcium and magnesium content of water and food grown in soil near areas of the highest prevalence of ALS-PDC. In the search for a different environmental etiology, Kurland (1988) was led to investigate the consumption of a local food product: cycad. Cycad consumption parallels the occurrence of ALS-PDC in several respects. First, cycad has been consumed by the Chamorros as a dietary staple and occasional famine food, but the level of consumption rose to far greater than normal levels during World War II and the harsh conditions during the Japanese occupation of the island in the 1950s. In support of the cycad hypothesis, the incidence of ALS-PDC peaked within several years of the war and declined dramatically as cycad consumption lessened during the post-war years, during which Guam was largely “Westernized” and cycad became a less significant part of the Chamorro diet (Kurland, 1988). Second, as previously noted, there is a distinct absence of ALS-PDC on the nearby island of Saipan, where cycads had been removed from the island and the Chamorros of Saipan only very rarely consumed cycad products (L.T. Kurland, personal communication). Kurland (1994) therefore reasoned that the striking differences in the incidence of ALS-PDC on these two islands could be a result of cycad consumption. Following these observations, cycad was considered by many to be the key etiological factor in ALSPDC, sparking a flurry of investigations into the biology of cycads and the identification of cycad toxins.

CYCAD TOXICITY SEARCHING

FOR

CYCAD’S TOXINS

Once cycad had been identified as the most probable candidate in the search for an environmental cause of ALS-PDC, many researchers focused on the isolation and identification of cycad’s toxins. The sixth international cycad conference in 1972, for example, included almost a dozen presentations on the topic of the neurotoxicity of cycad derivatives and related compounds. Most of these (e.g., Jones et al., 1972; Sanger et al., 1972) focused on cycasin, an acutely toxic compound present in all

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species of cycad. By the mid-1980s, another, slower-acting toxin, BMAA, had been identified and experimentally linked to neurological disease resembling that found on Guam (Spencer et al., 1987). As the toxins removed by traditional processing and those surviving such preparation are more precisely identified, the precise etiological portrait of ALS-PDC will become clearer.

CYCAD CONSUMPTION

AND

ACUTE TOXICITY

Unprocessed cycad contains appreciable amounts of acutely toxic compounds. Although indigenous populations, such as the Chamorros of Guam, had “tamed” cycad’s toxicity by adopting extensive processing methods, early European travelers had no such understanding. It follows, then, that when Europeans began exploration of areas to which cycads were native they naively consumed unprocessed cycad, with deleterious consequences. Norstog and Nicholls (1997) describe how, for example, members of Captain James Cook’s crew ate cycad seeds and became violently ill with vomiting and vertigo. During periods of wartime scarcity (e.g., the Boer War, American Civil War, and World War II) soldiers ate cycad seeds with similar results. Such acute cycad toxicity is caused by azoxyglycosides present in the seed, leaf, and stem of cycad plants (Norstog and Nicholls, 1997). These bioactive compounds can account for as much as 5% of the plant’s dry weight (DeLuca et al., 1980), and are composed of a sugar moiety (cycasin: glucose; macrozamins: glucose and xylose) that is joined via a β-glucoside linkage to methylaoxymethanol (MAM). This aliphatic azoxy side chain is highly toxic, but the sugar–MAM complex is itself innocuous. This allows for the safe storage of large amounts of azoxyglycosides in cycad tissues. Toxicity arises when these azoxyglycosides are cleaved by the relatively common enzyme, β-glucosidase. The freed MAM is itself toxic, but can also be converted to an aldehyde form (MAMAL) by the enzyme alcohol dehydrogenase (an enzyme found in the liver); MAMAL then decomposes, yielding destructive methylcarbonium ions. These azoxyglycosides are specific to, and ubiquitous in, this plant order (DeLuca et al., 1980). The cycad plants themselves, then, must have evolved some way of preventing the toxic cleavage of their azoxyglycosides, which would otherwise endanger their own survival. The answer to this problem is twofold (Norstog and Nicholls, 1997). One, there is evidence that cycads sequester these toxins in specialized storage cells, thus physically separating them from β-glucosidase. Second, they may not in fact have β-glucosidase enzymes. Since humans are not similarly protected from azoxyglycoside toxicity by virtue of their physiology, cycad processing that removes virtually all traces of these water-soluble toxins has been adopted.

“SLOW NEUROTOXIN” THEORY In addition to the presence of acutely toxic compounds, cycad also contains toxins that are instead associated with the development of more chronic, long-term illness. BMAA is one such slow-acting cycad toxin. A nonprotein but analogous amino acid,

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it is found in all cycad genera (highest concentrations are in the genus Cycas; Audhali and Stevenson 2003). When the analogous amino acids are incorporated during protein synthesis, protein function may be severely disturbed. Furthermore, BMAA is structurally very similar to glutamate (Audhali and Stevenson, 2003). Agonism at glutamatergic N-methyl-D-aspartate (NMDA) receptors may therefore cause extreme ionic flux, including calcium ions, across the cell membrane, resulting in cell death by excitotoxicity. This profile of BMAA, coupled with negative findings regarding genetic, viral, and environmental (mineral deficiencies, cycad’s azoxyglycosides, etc.) causes of ALS-PDC, initially led some researchers to propose that BMAA was the major causative agent in the development of ALS-PDC. Early work by Spencer and associates (1987) included exposing macaques to synthetic BMAA by gavage. Over time, these primates developed an ALS-like motor neuron disease with parkinsonism symptoms, and it appeared that the elusive toxin had been found. Traditional washing of cycad before consumption, however, removes, on average, 87% of the total BMAA content (Duncan et al., 1990). In fact, 50% of samples tested by Duncan and colleagues had virtually all — greater than 99% — of traces of BMAA removed, and others have reported BMAA levels in processed cycad flour to be in the range of 0.00 to 18.39 μg/g of flour (Kisby et al., 1992). Spencer and colleagues’ (1987) experiments were therefore not comparable to the human situation. Furthermore, their use of much higher doses of BMAA than any human would ever encounter led not to the chronic formation of NFTs (as is found in human ALSPDC cases) but instead to subacute chemical encephalitis (Spencer et al., 1987). Since then, the “BMAA theory” has been superseded by other etiological explanations of ALS-PDC. Nevertheless, a similar link between another nonprotein amino acid and the development of a long-term neurological disease has been found in India, and this has made the BMAA theory of cycad toxicity appear, at least superficially, more credible than it in fact may be. BOAA (β-N-oxalylamino-L-alanine), a neurotoxin structurally and functionally very similar to BMAA — i.e., a glutamate agonist; the main difference between BMAA and BOAA is that the former is an agonist at NMDA glutamate receptors, while the latter is an AMPA (α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor) glutamate receptor agonist — is present in grass-peas (Lathyrus sativus, L. cicera, and L. clymenum), and the consumption of grass-pea has been conclusively linked to the development of an ALS-like fatal motor neuron disorder, lathyrism (Spencer, 1995). Grass-pea must constitute a sizable proportion of the diet in order to initiate the development of lathyrism (Kessler, 1947). BOAA is also present in cycad, but at such low concentrations that it is unlikely to contribute to the development of ALS-PDC (Ross and Spencer ,1987). Konzo, another progressive motor neuron disorder, occurring mostly in Africa, is symptomatologically and neuropathologically similar to lathyrism. Again, consumption of a specific food (in this case, cassava, also known as yucca or manioc) has been found to be causal in the development of a specific neurological disease (Tylleskär et al., 1994). While cassava is eaten by many societies around the world, konzo is restricted to central and eastern Africa. Upon closer examination, it becomes obvious that konzo only develops when heavy or even exclusive cassava use occurs

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on a background of low protein intake (Selmar, 1994). Cassava’s disease-provoking neurotoxins, however, are cyanogenic glucosides, not BMAA or BOAA-like toxins. On the other hand, it is interesting to note that cyanide gas may be given off during the first few days of soaking cycad seed chips (Lister and Hill, 2003). Along with lathyrism, konzo nevertheless represents another geographically limited focus (unusually high prevalence) of motor neuron disease with nutritional toxicity as its supposed primary cause. This reminds us that nutritional toxicity may require other predisposing variables, such as lack of other diet alternatives, in order for an observable disease state to be provoked. Other foci of neurological disease similar to Guamian ALS-PDC also exist, such as Guadeloupean parkinsonism in the French West Indies (Caparros-Lefebre et al., 2002), MND on the Kii peninsula of Japan (Iwami et al., 1993), and a form of ALS-PDC among the Auyu and Jakai people of West New Guinea (Gajdusek and Salazar, 1982). These disorders may or may not be linked to cycad consumption and/or medicinal use, or to other similar environmental toxicities (Spencer et al., 1991). This information is summarized in Table 9.3.

STEROL GLUCOSIDES: CAUSAL AGENTS

IN THE

DEVELOPMENT

OF

ALS-PDC?

Although cycasin, macrozamins, and BMAA have all been shown to be toxic, and despite the fact that early research pointed to these compounds as causal agents in the development of Guamian ALS-PDC, it is unlikely that these are in fact the neurotoxins causally responsible for this disease. The major evidence against this link is that traditional washing of cycad effectively removes virtually all traces of these water-soluble compounds. Thus, if we are to explain the apparent link between cycad consumption and the development of ALS-PDC, we must reexamine the socalled “cycad hypothesis” and look for (neuro)toxins that remain unchanged by the traditional preparation. That is, the toxin or toxins must be insoluble in water, able to survive normal cooking temperatures, and sufficiently lipophilic to be able to cross the blood–brain barrier (Table 9.4). Recent work in our laboratory has shown that several sterol glucosides, present in processed cycad seeds, may be at least partly responsible for the neuronal damage underlying the symptomatology and neuropathology of ALS-PDC. Experiments using cortical wedge preparation and assays for lactate dehydrogenase (LDH) activity in cortical slices revealed biological activity and cell death, respectively. When the most active fractions were analyzed, it was found that they contained significant amounts of three sterol glucosides (SGs): β-sitosterol-β-D-glucoside, campestral or dihydrobrassicasterol β-D-glucoside, and stigmasterol β-D-glucoside (Khabazian et al., 2002). Further supporting our hypothesis that these SGs are the neurotoxic agents in washed cycad, both isolated and synthesized SGs provoke similar cellular reactions, as shown by Khabazian and colleagues (2002). In cortical wedge preparations they both give depolarizing responses that can be selectively blocked by the NMDA receptor antagonist D-AP5 and the noncompetitive antagonist MK-801. Rapid cell death is evidenced by LDH release. Although SG exposure leads to a significant release of glutamate, it does not compete with either glutamate or NMDA in

Upper motor neuron degeneration causing ALS-like symptoms Upper motor neuron degeneration causing ALS-like symptoms Progressive supranuclear palsy (PSP) and atypical PD

Lathyrism

Konzo (cassavism, mantakassa)

Guadeloupean parkinsonism

French West Indies

Central and Eastern Africa

India, Bangladesh, Ethiopia, and Nepal

Guam, West New Guinea, Kii Peninsula of Japan

Geographic Focus

Tropical plants such as Annonaceae

Cassava (yucca/ manioc; Maniot esculenta)

Grass-pea (Lathyrus sativus, L. cicera, and L. clymenum)

Cycad (Cycas micronesica).

Causal Nutritionally Toxic Plant

Quinolines, acetogenins, and rotenoids (mitochondrial complex I inhibitors)

Cyanogenic glucosides

BOAA (-N-oxalylamino-L-alanine) (AMPA glutamate receptor agonist)

Sterol glucosides (-sitosterol-Dglucoside, dihydrobrassicasterolD-glucoside, and stigmasterol-Dglucoside)

Proposed Causal Toxin(s) Ref.

Caparros-Lefebre et al. (2002)

Selmar (1994); Tylleskär et al. (1994)

Kessler (1947); Spencer (1995)

Spencer et al. (1991); Iwami et al. (1993); Caparros-Lefebre et al. (2002); Khabazian et al. (2002)

Note: Each disorder has a distinct geographic focus. The proposed causal toxins and the plants from which they are derived are also noted.

Combines features of classical ALS, PD, and AD

Description

ALS-PDC

Disorder

TABLE 9.3 Summary of Several Neurodegenerative Diseases Thought to Be Caused by Nutritional Toxicity

250 Reviews in Food and Nutrition Toxicity

Aglycone sterols innocuous Degeneration of specific CNS neuronal Proposed toxicity: disrupts glutamate populations leading to the development transporter function of ALS-PDC In vitro: neurotoxic glutamate release results in neuronal death

Undetermined

β-Sitosterol-β-D-glucoside (BSSG), dihydrobrassicasterol, β-D-glucoside, and stigmasterol β-D-glucoside

Note: See text for references.

Sterol glucosides

Degeneration of anterior horn neurons of the lumbar spinal cord and upper motor neurons causing an ALS-like motor neuron disease (“lathyrism”)

Agonism at AMPA glutamatergic receptors; also inhibits mitochondrial complex I in motor cortex and lumbar spinal cord

Very low amounts

Nonprotein amino acid β-Noxalylamino-L-alanine

BOAA

Iceburg lettuce, cocoa butter, rice bran, etc.

Chickling pea (Lathyrus sativus, L. cicera, and L. clymenum)

None known

Acute: hepatotoxicity; gastrointestinal Specific and problems ubiquitous to Protracted: carcinogenic, mutagenic, species of cycad teratogenic, may lead to the development of neurological disease ALS-like motor neuron disease with parkinsonism symptoms

Nonprotein amino acid β-methylamino-L-alanine

BMAA

Occurrence in Other Food Sources

Acute: hepatotoxicity; gastrointestinal Specific and problems ubiquitous to Protracted: carcinogenic, mutagenic, species of cycad teratogenic, may lead to the development of neurological disease

Physiological Effects

Raw: ~0.05% of dry weight Agonism at NMDA glutamatergic Processed: virtually receptors; also chelates of divalent eliminated metal ions (e.g., zinc)

Azoxyglucoside: disaccharide Raw: with cycasin, up to 5% Sugar–MAM complex innocuous; (glucose and xylose) joined via of dry weight cleaved by -glucosidase -glucoside linkage to MAM Processed: virtually Free MAM toxic; free MAM also eliminated decomposes into other toxic compounds

Macrozamin

Sugar–MAM complex innocuous; Cleaved by -glucosidase Free MAM toxic; free MAM also decomposes into other toxic compounds

Toxicity

Azoxyglucoside: glucose joined Raw: with macrozamin, up via -glucoside linkage to MAM to 5% of dry weight Processed: virtually eliminated

Composition

Amount Present in Raw and Processed Cycad

Cycasin

Toxin

TABLE 9.4 Cycad Toxins

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competition binding assays. Exposure to NMDA or SG fractions changes the expression of some (CDK-2, PKC-β) but not all (Erk-1, Rsk-1, Cot, PKB-2) protein kinases. Taken together, these results suggest that cycad’s SG content is sufficiently neurotoxic to be considered a potential causal agent in the development of ALS-PDC. It is only in their glucosidated forms that these plant sterols are toxic (Khabazian et al., 2002). Aglycone sterols, such as β-sitosterol, may in fact have (neuro)protective effects (Bi et al., 2000). It is therefore not surprising that the unbound forms of these compounds are more abundant in nature than are the glucosidated forms. Nevertheless, SGs are present in a number of plants besides cycad, some of which are also exploited nutritionally, medicinally, or recreationally. Rice bran, for example, contains the same three SGs as cycad (Fujino and Ohnishi, 1979), as also does cocoa butter (cocoa beans Lome Tongo; Staphylakis and Gegiou 1985) and iceburg lettuce (Lactuca sativa L.; Knapp et al., 1968). Used in traditional herbal medicine, juniper (Juniperus macropoda) and Chinese boxthron (Lycium chinense) are among the plants that contain the highest amounts of BSSG (Duke, 2003). Tobacco leaf and smoke likewise contains these compounds (Kosak and Swinehart, 1960; Wright et al., 1962; Kallianos et al., 1963).

CONSUMPTION EXPERIMENTAL)

OF

CYCAD

BY

OTHER SPECIES (NATURAL

AND

There have been many accounts of cycad being eaten by other species in the wild. Because these animals do not have the benefit of socially transmitted processing methods, they are therefore exposed to both the toxins that are removed by human processing (e.g., azoxyglycosides, BMAA) and those that survive processing (e.g., sterol glucosides). Despite this exposure, not all species show signs of illness. While some species, such as sheep and cattle, experience the gastrointestinal problems and liver damage caused by cycad’s azoxyglycosides, others species, such as kangaroos, consume cycad without acute illness (Norstog and Nicholls, 1997). Norstog and Nicholls go on to suggest that a deciding factor for whether an animal will show signs of acute toxicity appears to be whether the species is native to environments in which cycad is available (as is the case for kangaroos in Australia), or was instead introduced to the area by humans (as were sheep and cattle in Australia). Some animals, such as cattle, may show little or no signs of acute distress upon the ingestion of raw cycad, but will instead experience a hind-limb paralysis. This “zamia stagger” syndrome occurs in response to the appreciable amounts of BMAA that are present in unwashed cycad (Hall, 1954; Hall and McGavin, 1968). The subclinical effects of this “slow-acting neurotoxin” (Spencer et al., 1987) builds over time until it is manifested as neurological disease. Many experiments have been conducted over the decades in which the effects of being exposed to cycad or any of its toxic derivatives have been explored in laboratory. Rats, for example, develop microencephaly when exposed to MAM prenatally (Rabe and Haddad, 1972) or experience hormonal changes when injected with MAM later in life (Malevski et al., 1972). Subcutaneous injection of cycasin

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into newborn mice induces lesions of the CNS (Sanger et al., 1972). While Campbell and colleagues (1966) failed to find behavioral deficits or changes in neuronal morphology in adult rats fed MAM/cycasin from raw cycad seeds, others (Albretsen, 1998) found that dogs fed raw cycad seeds develop liver and gastrointestinal pathologies as well as neurological disorders including ataxia, coma, and seizure. Also, as mentioned above, primates fed sublethal but significant doses of BMAA will, over time, develop an ALS-like syndrome (Spencer et al., 1987). There are thus several examples of both natural and experimental exposure to cycad and its toxins, with exposure and subsequent ingestion resulting in the development of either acute gastrointestinal illness or protracted neurological illness. However, not all species react negatively to the ingestion of cycad; in fact, some species actually sequester the toxins and use them for their own protection (Jones, 2002). Zamia butterflies, for example, consume cycad as caterpillars and then store cycad toxins such as cycasin and macrozamin, which imparts to them a chemical protection at later developmental stages. The larval forms of other insects — moths, beetles, flies, weevils — also feed on cycad and, like Zamia butterflies, sequester cycad toxins as a protective measure. Assumedly, these species prevent the cleavage of azoxyglycosides and thus prevent MAM-releasing decomposition. Although it is thought that cycad consumption is invariably poisonous to vertebrates, several vertebrate species do feed on cycad seeds (Jones, 2002). In Australia, for example, the native brown rat Rattus fuscipes ingests the kernels of Macrozamia, and in Mexico the same is done by rodents that eat the kernels of Dioon species. “Flying fox” bats native to Guam regularly ingest the juice squeezed from the sarcotesta of cycad seeds, apparently avoiding toxic consequences by sequestering the toxins (Cox and Sacks, 2002). (Human consumption of flying foxes in Guam may therefore result in ingestion of biomagnified amounts of cycad toxins, with deleterious effects.) Other mammalian cycad-eating species include other rodents (rats, mice, squirrels), bears, peccaries, baboons, monkeys, hyrax, possums, kangaroos, and wallabies. Parrots, cockatoos, mockingbirds, oilbirds, hornbills, cotingas, crows, cassowaries, and emus also consume cycad. Again, a critical factor in cycad tolerance is nativity to the region in which the cycads grow (Norstog and Nicholls, 1997). Any species that can consume cycad without deleterious effect must have a resistance to its toxins. Humans have, over the centuries, developed behavioral strategies in the form of traditional processing practices to protect them from cycad’s toxins. Likewise, some species avoid cycad toxicity behaviorally (Jones, 2002); many animals will carry the seeds away before eating the sarcotesta. Other animals swallow the seeds whole, only to void the kernel (minus the ingested sarcotesta) soon afterward (as is done by crows, cassowaries, emus) or to defecate it whole a day or so later (African elephants). Alternatively, they might only ingest the less toxic sarcotesta, with the seed kernels discarded. These strategies ensure that the more toxic kernel is not ingested. Other species have, over much longer time periods, evolved physiological protection such as compartmentalization or detoxification (Jones, 2002). This physiology has yet to be precisely defined.

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A MOUSE MODEL OF ALS-PDC Given the work from our laboratory showing the neurotoxicity of processed cycad seeds, along with the correlation between cycad consumption and ALS-PDC, we performed the critical in vivo experiments that would definitively link cycad consumption to ALS-PDC: feeding mice washed cycad. Such experiments provide us with an animal model of the human situation, in which cycad is ingested only after extensive processing, and would establish cycad as having a causal role in ALSPDC. Because processing removes virtually all of the azoxyglycosides and BMAA (Duncan et al., 1990) this model allows us to determine the effect of cycad’s other toxins, such as its sterol glucosides. It has been shown repeatedly that animals, such as laboratory rodents and primates, are susceptible to cycad’s water-soluble toxins (azoxyglycosides: Sanger et al., 1972; BMAA: Spencer et al., 1987), but the crucial in vivo experiments involving the ingestion of processed cycad have been neglected. For this reason, we have undertaken these in vivo studies to give evidence that cycad plays a causal role in ALS-PDC. The cycad used to induce ALS-PDC in mice (CD-1 males) was prepared in the same way as done by the Chamorros on Guam (i.e., washed, dried, ground into flour, and mixed with water to form a dough). Cycad constituted approximately one fourth of their daily intake by weight. Control mice were fed pellets identical in weight and similar in nutritional content made of commercial-grade processed white flour. To assess neurological effects of washed cycad consumption, we employed a battery of behavioral assays to monitor changes in motor, cognitive, and olfactory function. After animal sacrifice, the neuropathological consequences of cycad consumption were investigated using a variety of histological indices of CNS neurodegeneration.

BEHAVIORAL

AND

NEUROPATHOLOGICAL FEATURES

OF

ALS-PDC

IN

MICE

We found that consumption of washed cycad led to both behavioral and neuropathological outcomes that in many respects mirror features of ALS-PDC. Behavioral analysis revealed a marked, progressive decline in both motor and cognitive function. For example, performance of the leg extension reflex, a marker of motor neuron (dys)function (Barneoud and Curet, 1999), became progressively worse during the feeding protocol, reaching significance from controls 14 days following the initiation of cycad consumption (Figure 9.4A). These deficits continued to increase following the cessation of cycad feeding, suggesting that either initial cycad exposure unleashes secondary biochemical cascades that lead to neurodegeneration, or that the putative toxin in cycad remains in the CNS for prolonged periods after initial exposure. This is in keeping with studies reviewed by Kurland (1988) that suggests a minimum incubation period of 10 years following toxin exposure is required before symptoms develop. Deficits in gait and locomotion were investigated by measuring the average distance between ipsilateral rear paw prints (de Medinaceli et al., 1982). We found that cycad-fed animals perform poorly when compared to control animals (Figure 9.4B). With regard to cognitive function, cycad-fed animals perform worse on the Morris water maze, a test of spatial memory (Morris, 1984) and the radial arm maze, a test of working and reference memory (Figure 9.4C and D). These results suggest

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FIGURE 9.4 Motor and cognitive effects of cycad feeding. (A) Leg extension deficits in cycad-fed vs. control mice as a function of time (days). (B) Gait length in the same animals as a function of time (days). Circles indicate right stride length, triangles indicate left stride length. (C) Morris water maze performance following relocating the hidden platform to a new position. (D) Radial arm data showed significant learning and memory deficits in cycadfed mice on reference memory tasks. Significance: *P < 0.05, **P < 0.001, #P < 0.0001, ANOVA.

that our model indeed includes hallmark features of all three neurological components of ALS-PDC: motor deficits, gait disturbances, and decline in cognitive function (symptoms of ALS, PD, and AD, respectively) (Table 9.5). Histological analysis revealed regions of neurodegeneration consistent with the observed behavioral deficits, as well as with regions of neurodegeneration found in ALS-PDC, AD, PD, and ALS (Wilson et al., 2002). With regard to cognitive function, apoptotic cells, as revealed by caspase-3 and TUNEL immunohistochemistry, are observed in various cortical and hippocampal regions. Additionally, there are significant decreases in cortical and hippocampal volume as measured in sections through relevant brain areas, results indicative of cell loss and atrophy. These results have been duplicated using magnetic resonance imaging (MRI) technology enabling a 40× greater resolution as compared to standard MRI images (Wilson et al., 2004). While this work has been done on postmortem tissue, future experiments using living animals will facilitate the tracking of volume changes during the feeding protocol and the induction of neurodegenerative disease, allowing us to correlate changes in specific regions with motor and cognitive deficits. Cycad-fed mice also exhibit neuropathology related to the observed motor impairments and similar to that of ALS-PDC. There is 30% loss of motor neurons in the ventral horn of cervical spinal cord regions (Wilson et al., 2002). MRI analysis

Mouse walks length of tunnel with painted hind feet Scoring: distance between consecutive ipsilateral paw prints measured Mouse held upside down by tail will reflexively extend hind limbs Scoring: retraction of limb(s) indicates motor neuron dysfunction Mouse grasps wire with front paws and hangs, or balances on top of the wire; three repeats Scoring: latency to fall Mouse grasps grid and hangs upside down by all four limbs; three repeats Scoring: latency to fall Mouse tries to stay on top of rotating rod Scoring: latency to fall

Leg extension

Wire hang

Grid hang

Rotarod

Description

Gait length

Test

Motor neuron function: motor coordination, balance, motor learning, sensory function

Motor neuron function: neuromuscular strength in limbs; grasping reflex

Motor neuron function: neuromuscular strength and balance; grasping reflex

Hind limb motor neuron function

Gait length: indicative of basal ganglia integrity

Function Evaluated

ALS: motor neuron dysfunction as evidenced by impaired motor coordination

ALS: progressive weakness in all four limbs

ALS: progressive weakness in upper limbs

ALS: progressive motor neuron dysfunction leading to impaired lower limb function

PD: gait length is reduced AD: gait abnormalities predict dementia

Analogy to Human Symptomatologya

Crawley (1999)

Adapted from Takeda et al. (2003)

Dean et al. (1981)

Barneoud and Curet (1999)

de Medinaceli et al. (1982)

Ref.

TABLE 9.5 Summary of Seven Behavioral Tests Used in Assessing Various Functions in Mice That Are Analogous to Human Symptomatology of ALS, PD, and AD

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Mouse is habituated to a scent, then exposed to a novel scent Scoring: number of sniffs of novel vs. familiar scent; three repeats

Olfactory Olfaction: scent detection and discrimination between novel and familiar scents

Cognitive function: spatial, reference, and working memory

PD: deficits in olfaction AD: deficits in olfaction

AD: cognitive deficits such as memory loss ALS: similar to AD PD: similar to AD Tillerman, J. (personal communication)

Adapted from Morris (1984) and Olton and Samuelson (1976)

As seen in the classic forms of ALS, PD, and AD. Because ALS-PDC has symptomatological similarities to these diseases, the information presented here is also applicable to ALS-PDC.

a

Note: References indicate researcher(s) responsible for developing the test. See text for references regarding human symptomatology.

Mouse swims in water to enter maze arms, where one of the four arms has reward platform; three repeats Scoring: number of correct arm entries

Water maze

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FIGURE 9.5 Effect of cycad feeding on tyrosine hydroxylase immunoreactivity. (A) Quantification of striatal TH staining shows a decrease of 30% in cycad-fed mice (N = 4 mice/group, p < 0.05, two-tailed t-test). Sections of the striatum showed decreased expression of TH in cycad-fed mice (B and C).

has also demonstrated a significant decrease in ventral horn volume (Wilson et al., 2004). Furthermore, some motor neurons exhibit a highly altered morphology suggestive of a dysfunctional state. Similar changes are seen in ALS-PDC and classic ALS (Shaw et al., 2002). With regard to neuropathological features of parkinsonism, cycad-fed mice show a substantial dopaminergic deficit, along with cell death in the substantia nigra. Tyrosine hydroxylase (TH), the enzyme responsible for converting L-dopa to dopamine and therefore a specific marker for dopaminergic neurons, was significantly reduced in the striatum of cycad-fed mice (Schulz et al., 2003) (Figure 9.5). The striatum receives dopaminergic innervation from the SNpc, the primary region of cell loss in PD. While we have yet to see significant cell loss in the SNpc, the loss of dopaminergic projections, combined with the parkinsonism-like behavioral deficits, suggests we are seeing the early stages of disease, which given time and continued toxin exposure will develop into more overt end-stage behavioral and neuropathological outcomes reminiscent of the classic neurodegenerative disorders ALS, PD, and AD. Analysis of additional biomarkers related to neurodegeneration reveals further evidence that our animal model mimics key aspects of human neurodegenerative disease. One of the earliest changes observed in cycad-fed animals is a downregulation of glutamate transporter in various regions of the CNS (Wilson et al., 2003). This appears as spotty “patches” devoid of the transporter. Interestingly, these patches are usually centered on capillaries, the likely site of ingress of the cycad toxin into the CNS (Khabazian et al., 2002). If toxins enter the CNS via the blood supply, they would affect cells surrounding the blood vessels before altering adjacent regions. In accordance with this notion, the small patches centered on capillaries may reflect relatively early stages of toxin action, and with continued cycad exposure there may be an increase in patch size, ultimately culminating in an overall loss of

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glutamate transporter as observed in end-state human neurological disease (Wilson et al., 2003). With regard to neuronal cell death, a decrease in glutamate transporter is highly significant, as glutamate transporters remove glutamate from the synapse, thus preventing overstimulation and subsequent excitotoxic cell death of the postsynaptic neuron (Trotti et al., 2001; refer to Wilson et al., 2003). There is mounting evidence that excitotoxic cell death has a role in human neurodegenerative disorders, such as ALS (Rothstein et al., 1995) and AD (Masliah et al., 1996) further supporting the notion that the murine model of ALS-PDC closely resembles early stages of human disease. A proposed timeline of events leading to cell death for ALS-PDC in mice has been described in Shaw and colleagues (2002). Based on our work to date, we speculate that the initial event in cycad-induced neurodegeneration is the ingress of cycad toxins into the CNS through the vascular system. By still unknown means, but likely involving abnormally increased protein kinase C (PKC) phosphorylation (Khabazian et al., 2002), glutamate transporter levels decrease, leading to decreased uptake of glutamate and a concomitant increase of glutamate in the synaptic space. The increase in extra-synaptic glutamate causes overactivation and subsequent death of adjacent neurons. In this view, downregulation of glutamate transporters is an early event in neurodegenerative cascades, suggesting that other pathologies observed in human postmortem, disease end-state tissue such as NFTs and inflammation are “downstream” events that occur later in the disease process. This view is supported by recent work with a mouse model of ischemia in which a decrease in glutamate transporters precedes neuronal cell death in the hippocampus (Raghavendra Rao et al., 2000). Given that our mouse model of ALS-PDC mimics many key aspects of human neurodegenerative disorders we propose that it is a valid and valuable tool for further investigations into the nature of neurodegenerative disease. While the cognitive and motor deficits, along with the neuropathological changes induced by cycad consumption, demonstrate that an ingested toxin can be responsible for such disorders, current work is aimed at understanding the interaction of other factors related to neurodegeneration such as age and genetic susceptibility.

VALIDITY

AND THE

“FOUR DIMENSIONS”

OF

OUR MODEL

A model is defined as any experimental preparation developed for the purpose of studying a condition in the same or different species (Geyer and Markou, 1995). Standard criteria that any model must meet are etiological, construct, and predictive validity. We believe that the etiological validity of our model supersedes that of several other current models of neurodegenerative disease. Many animal models of neurological disease, such as the α-synuclein overexpression model of PD (Kirik et al., 2002), rely on disease induction that is relatively sudden or severe. In contrast, our model mimics the human situation quite closely: Daily consumption of processed cycad constituting a small but significant proportion of total dietary intake provokes the gradual emergence of ALS-PDC. That our model links specific behavioral deficits with appropriate underlying neuronal dysfunction and/or death indicates that construct validity — the accuracy with which a test measures what it is intended to

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measure — is also satisfied. With regard to predictive validity (the ability of a test to predict a criterion that is of interest to the investigator), we hope to be able to use the time course data to accurately predict future stages of the disease by looking at early markers, as well as to use behavioral testing to predict biochemical neuropathology, and vice versa. Ideally, we seek to analyze ALS-PDC in “four dimensions” (Shaw and Wilson, 2003). The behavioral deficits, biochemical changes, and morphological or pathological outcomes we see in our animals occur within a fourth dimension of time, and these four dimensions together allow us to more fully understand neurodegenerative disease. It is this fourth dimension of time that confers added etiological validity to our model. Many animal models of neurodegenerative disease, such as the 6-hydroxy-dopamine (6-OHDA) lesioning of the substantia nigra model of PD (Oiwa et al., 2003), only concern states that correspond to late or even end-state phases of the disease. In contrast, we seek to include the rate, type, and extent of disease progression as the subjects move from normal CNS state, through preclinical neurological damage, to clinical diagnosis, and, ultimately, arrive at the end state. Arrival at this end point must be slow enough to allow us to catch as many of the events from insult to end state as possible. Moreover, a very rapid induction of ALSPDC (e.g., compare MPTP-induced PD; Renkawek, 1986) would be a less etiologically valid model of human neurodegenerative disease, as it would be less likely to accurately reflect the actual series of events that occur in human patients.

THE “TIME COURSE” PROJECT Our realistically slow, cycad-based model has therefore allowed us to begin piecing together the disease progression of ALS-PDC in mice. By sacrificing animals at a number of time points over the entire course of the disease, our “time course” project will provide us with windows of what biochemistry and neuronal changes underlie behavioral correlates over time. As mentioned, the downregulation of glutamate transporters, for example, is a change that occurs very early on in the disease progression (Wilson et al., 2003), while neuronal apoptosis, as measured by TUNEL, appears later on (Wilson et al., 2002). Over the course of neurodegenerative disease, phases of protective measures (e.g., the heat shock response), immunological response involving microglial activation, as well as phases of neurodegeneration will all appear at different time points in the variously affected areas of the CNS. As we uncover this temporal sequence, we will become more able to distinguish among the disease’s causal, coincidental, and compensatory (successful or failed) components (Shaw et al., 2002). Current limitations include the inability to detect disease clinically until as many as two thirds of the neuronal population in question have degenerated and the reliance on postmortem tissue for insight into neurodegenerative disease. As such, most models fail to identify ways in which intervention could halt, or even prevent, neurological damage; instead, intervention has been largely palliative in nature for these patients. A model such as ours that includes the progressive nature of neurodegenerative disease will allow us to go beyond this merely palliative “treatment” and develop targeted therapeutics that have the potential to actually halt disease progression. As depicted schematically in Figure 9.6, it

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FIGURE 9.6 Schematic timeline of putative stages in sporadic forms of neurological disease. The schematic represents an idealized timeline starting from a condition of an intact (“predisease”) nervous system. Clinical diagnosis (“disease onset”) occurs in most cases once behavioral symptoms have become overt, which generally occurs when approximately 50 to 70% of neurons have died. This threshold is represented by the horizontal line.

is critical that we begin understanding the earliest pathological changes of neurological disease in order to provide optimal diagnosis and intervention. To move from mouse model to human experience of neurodegenerative disease we must transpose our data (Shaw et al., 2002; Shaw and Wilson, 2003). This process of template matching, dependent on the predictive validity of the model, involves specific knowledge of the disease in all of its four dimensions, particularly its progressive nature. As we expand our knowledge of the behavioral deficits, biochemical changes, and morphological or pathological outcomes of our cycad-fed mice, we will be increasingly able to move from mouse to human. Scans (e.g., MRI: Wilson et al., 2004; PET: Snow et al., 1990) and biomarker detection processes based on, and employed in conjunction with, our time course model, may in the future be used to identify early stages of neurodegenerative disease in human patients.

SEX, AGE,

AND

GENETICS: IMPORTANT VARIABLES

As we seek to apply information gleaned from our model to human populations, we must address three variables of significant effect: sex, age, and genetics. Although the fundamental disease processes will be similar for these different populations, there are also significant differences, known and unknown, between them. The sex ratio of Guamian ALS and PDC was skewed toward males in the 1950s and 1960s, but had changed to comparable rates in males and females by 1980 (Plato et al., 2003). Furthermore, neither mean age at onset nor duration of illness is the same for males and females suffering from ALS or PDC (Rodgers-Johnson et al., 1986). While we as yet know little about sex differences in ALS-PDC, current work in our laboratory addresses this issue. Age, too, is a variable we must be careful to include in our model-based analysis of ALS-PDC. It is, along with genetics and environment, one of the three main factors responsible for the development of neurodegenerative disease. As shown in Figure 9.1, increasing age is associated with increasing risk of disease. Because we do not as yet understand the “four dimensions” of this factor — how it affects the

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biochemical, physiological, and behavioral manifestations of ALS-PDC over time — our laboratory has undertaken crucial “age-dependent” studies. This work extends our model of ALS-PDC, and furthers its etiological and predictive validity. We predict that younger animals will have better resistance, stronger compensatory mechanisms, and more efficient repair processes compared to older animals and will consequently be less susceptible to developing ALS-PDC. However, the relationship between age and susceptibility may not be linear. For example, both very young and very old animals are more prone than middle-aged mice to develop the disease. In addition to this increased susceptibility, we expect that more susceptible animals will also develop more severe and/or accelerated neurodegeneration. Although a paucity of clear familial patterns of inheritance suggests that a single genetic defect is not the basis of the majority of these cases of neurological disease, to disregard the role of genetics would be a gross oversimplification. In fact, genetic susceptibility is, along with age, a factor that at times plays a significant etiological role in disease development and progression. This genetic susceptibility, however, ranges from decreasing the risk of developing neurological disease to minor changes in an organism’s ability to respond to the toxic insult (e.g., an attenuated heat shock response that subsequently leads to an accumulation of tangled, “unfolded” proteins) to very specific mutations that are sufficiently disruptive and therefore directly cause neurological disease (e.g., ALS caused by an SOD-1 mutation). We have begun to examine gene–environment interactions using the cycadinduced model of ALS-PDC with apolipoprotein E (ApoE) knockout mice. ApoE is the major lipoprotein of the CNS and is involved in the transport of plasma lipids and in the redistribution of lipids among cells (Mahley, 1988). Over the past decade, numerous studies have linked various ApoE isoforms to both protection from and susceptibility to neurodegenerative disease. For example, ApoE4 is associated with an increased risk of AD and poor outcome following acute head injury or stroke (Slooter et al., 1997) whereas ApoE2 decreases the risk of developing certain neurodegenerative disorders (Buee et al., 1996). We found that cycad-fed mice with a complete knockout of ApoE alleles develop no significant motor deficits compared to cycad-fed wild-type mice that display deficits (as previously described). These results lend support to the notion that the cycad toxins are sterol glucosides. A failure to transport these toxins to the brain, potentially induced by an ApoE knockout, could explain the absence of normally observed neurological outcomes of cycad consumption. Currently, the effects of specific ApoE allele additions or deletions on the outcome of cycad consumption are being investigated. We are thus currently expanding our murine model of ALS-PDC to include the variables of genetic vulnerability, sex, and age. Moreover, since sex and age effects together predict that different groups will have different outcomes, in that younger age is associated with better prognosis (Mukai et al., 1982), and sex is related to age at onset (Reed et al., 1975), we also seek to combine these variables. Together with our considerations of genetically based neurological disease (e.g., SOD-1, which causes ALS, ApoE) and the fact that genetic susceptibility may either increase or decrease the risk of developing disease (Figure 9.1), this creates a much richer model of ALS-PDC.

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LIMITATIONS

OF THE

263

CURRENT MODEL

As is inevitably the case when using an animal model to study human disease, our current murine model of ALS-PDC has its limitations. Some of these can be readily addressed by manipulating a few key variables (e.g., age), and current work in our laboratory is beginning to address these issues. We believe that our model is valid and will continue to provide us with significant insight into the “Rosetta Stone” of neurological disease, ALS-PDC. One issue our current model does not address is the fact that there are some case studies of individuals who, as children, suffered an acute and severe reaction to cycad consumption, never ate it again, and yet developed full-blown ALS-PDC decades later (Calne and Eisen, 1989). Our age-dependent studies do acknowledge this interaction of age and toxin exposure, but we have yet to examine the interaction between the degree of initial insult and incubation time. As yet, we cannot disentangle the effects of initial vs. protracted exposure to cycad. Another drawback to our current model is our dependence on the use of cycad, and not the pure toxin(s). We do know that while processed cycad is relatively free of azoxyglycosides and BMAA, and that sterol glucosides remain, it nonetheless still contains as yet unidentified compounds, any of which may prove to be causal — either alone, or in concert with other toxins — to the development of ALS-PDC. A cleaner animal model of cycad-induced ALS-PDC would therefore involve the exposure of the animals to single specific toxins.

CONCLUSIONS This chapter summarizes the evidence linking cycad consumption to the development of a unique neurological disorder. This example of nutritional toxicity may well provide insight into related neurodegenerative disorders, leading to the conception of ALS-PDC as a neurological Rosetta Stone in relation to classical forms of ALS, AD, and PD. Specifically, our model of ALS-PDC furthers our understanding of the behavioral and neuropathological components of the disease progression in mice, and we believe that this information can be transposed to neurological disease in humans. As earlier phases of the disease are linked to later clinical manifestations, and as initiating events are likewise linked to these early phases, we are able to “reverse-engineer” neurological disease and suggest interventions that will prevent, halt, or reverse the disease progression. Moreover, insight into the mechanisms of cycad (neuro)toxicity may shed light on similar nutritional toxicities around the world.

ACKNOWLEDGMENTS This work was supported by grants from the ALS Association, Parkinson’s Disease Foundation, Scottish Rite Charitable Foundation of Canada, Natural Science and Engineering Research Council of Canada, and the U.S. Army Medical Research and

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Material Command (DAMD17-02-1-0678) (to C.A.S.). The authors thank J. Wilson, M. Wong, J. Avent, D. Pow, and the late L.T. Kurland for technical assistance, discussions, and the use of primary data. Thanks to C. Melder for comments on the manuscript. Cycad seeds were provided by our colleagues on Guam, Drs. U. Craig and T. Marler to whom we are very grateful.

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Dietary Lectins and the Immune Response Tanja Maria Rosenkilde Kjær and Hanne Frøkiær

CONTENTS Abstract ..................................................................................................................271 Introduction............................................................................................................272 Plant Lectins ..........................................................................................................273 Lectins in the Human Diet ....................................................................................273 Toxicity and Biological Effects of Lectins in Foods ............................................274 Mitogenicity of Plant Lectins................................................................................276 Immunomodulatory Effects of Plant Lectins ........................................................277 Gut-Associated Lymphoid Tissue and Oral Tolerance .............................278 Interaction of Dietary Lectins with Immune Function .............................280 Generation of Specific Antilectin Immune Responses....................280 General Immunomodulatory Effects................................................283 Modulation of Immune Function by Dietary Lectins in Disease.........................285 Involvement in the Initiation of IgE-Mediated Allergy ............................285 Involvement in Other Immunologic Diseases...........................................287 Conclusion .............................................................................................................288 Acknowledgments..................................................................................................289 References..............................................................................................................289

Abstract

Human food contains lectins, and lectins are therefore consumed in their native form when foods are eaten raw or when foods containing lectins that are heat stable are eaten. Because lectins are resistant to digestion, they reach the small intestine in an active form. Some lectins are toxic, giving rise to diarrhea, and have on a few occasions been found to cause death. Lectins have many biological activities as they bind to carbohydrate on various cells. Many receptors are membrane-integrated glycoproteins and function as receptors for hormones and cytokines or are involved in cell–cell recognition. By binding to these receptors, lectins may mimic a natural ligand of a receptor or inhibit binding of a natural ligand and thereby evoke a variety of systemic and local effects, such as cell division and growth, cell maturation, and cell death. The use of lectins in immunology as polyclonal activators has long been recognized as their binding to receptors on immune cells induces

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mitogenesis. Dietary lectins may be immunomodulatory and affect both the innate and the adaptive immune response. Some lectins are able to induce a lectin-specific immune response, while others function as adjuvants, giving rise to an immune response against coadministered proteins. Inflammatory cytokine production, oral tolerance, production of antibodies, and apoptosis are some of the immune functions influenced by dietary lectins. Dietary lectins might be involved in induction of diseases with an immunological background, such as allergy and autoimmune diseases. To exploit fully the effect of lectins on the immune function, further studies are required, especially in relation to diseases potentially related to the ingestion of lectins. Moreover, the potential use of lectins in cancer therapy and in drug and vaccine delivery systems clearly emphasize that further studies on the influence of lectins on the immune system are required.

INTRODUCTION Several dietary components are known to affect various functions of the immune system and to interfere with immune regulatory circuits. The immunobiological activity of carbohydrate-binding proteins of vegetable origin, the plant lectins, has long been recognized. Some plant lectins have been shown to be able to modulate important immune mechanisms, such as inflammatory reactions and effector functions. Lectins are proteins or glycoproteins of nonimmune origin, which bind specifically to the glycan part of glycoconjugates (e.g., glycoproteins, glycolipids, oligosaccharides, and polysaccharides) in a sugar-specific manner. They often have two or more binding sites per molecule and tend to agglutinate cells to which they bind. They are abundant in living matter, whether of plant or animal origin. Legume lectins are probably the most famous lectin family. Lectins have been defined by their ability to bind specifically to carbohydrate and by their cell-agglutinating properties (Goldstein et al., 1980). An updated definition has lately been proposed; plant lectins have been redefined as “plant proteins possessing at least one noncatalytic domain, which binds reversibly to a specific mono- or oligosaccharide” (Peumans and Van Damme, 1995, 1998). A redefinition was necessary because monovalent lectins have been identified that cannot precipitate glycoconjugates or agglutinate cells (Peumans and Van Damme, 1998). By binding to specific carbohydrates on cell surfaces, lectins can elicit multiple changes in cell and body metabolism. Lectins bind specifically to different terminal carbohydrates and thus elicit various effects on the cell. The immune system serves to protect the host from external dangers; yet, inappropriate responses of this system can lead to disease. Common among these dysfunctions of the immune system are allergies and autoimmune diseases. It has often been suggested that dietary lectins might be involved in initiating these inappropriate immune responses. Lectins may modulate the immune response in different ways, for example, by being an antigen by themselves or by functioning as an adjuvant, leading to an immune response against coadministered proteins. The purpose of this chapter is to look at the immunotoxicological aspects of plant lectins upon ingestion; the use of lectins in targeted delivery of drugs and in

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cancer therapy is not discussed in this chapter unless relevant to orally ingested lectins and the immune response.

PLANT LECTINS Because the definition of lectins includes a wide range of proteins, plant lectins are divided into four major families of structurally or functionally related proteins: legume lectins, monocot mannose-binding lectins, chitin-binding lectins, and type2 ribosome-inactivating proteins. In addition to these four large families, three other families are now also recognized (Van Damme et al., 1998). Another way of classifying lectins is based on their structure, and they fall into three classes: merolectins, hololectins, and chimerolectins. Merolectins consist of only one carbohydrate-binding domain, and they are hence incapable of agglutinating cells. Hololectins, the class in which the majority of plant lectins belong, have at least two domains, but they are composed only of carbohydrate-binding domains, as opposed to chimerolectins. The latter consist of one or more carbohydrate-binding domains and, in addition, an unrelated domain with a well-defined biological activity such as enzymatic activity. Several hundred plant lectins have been isolated; some selected plant lectins with their botanical and common names of the plant source and their carbohydrate specificity are listed in Table 10.1. The listed lectins are selected based on their relevance in connection to commonly consumed foodstuff; some are, however, selected due to their relevance in connection to immunological research. Some lectins recognize simple sugars like glucose, mannose, galactose, N-acetylgalactosamine, N-acetylglucosamine, or fucose, whereas other lectins have much higher affinity for oligosaccharides. Lectin from red kidney bean (Phaseolus vulgaris agglutinin, PHA) is probably the most commonly investigated lectin with respect to biological effects and the most widely used lectin in immunological research. PHA is a tetrameric glycoprotein composed of two different subunits designated L for lymphocyte reactive and E for erythrocyte reactive. Five different isolectins originate from all possible tetrameric combinations of the two subunits (Felsted et al., 1977). PHA binds to terminal galactose terminally positioned on complex carbohydrates (Green and Baenziger, 1987). With regard to dietary lectins, two other important lectins are soybean agglutinin (SBA) and wheat germ agglutinin (WGA). SBA is a tetrameric protein that recognizes N-acetylgalactosamine, whereas WGA recognizes N-acetylglucosamine and sialic acid and is composed of two subunits (Van Damme et al., 1998).

LECTINS IN THE HUMAN DIET Plant and animal material used as foodstuffs contains lectins, some of which are denaturated by cooking. There are, however, still active lectins in the diet as when foodstuffs are eaten raw (uncooked) and some lectins are still active after cooking or processing (Peumans and Van Damme, 1998). In general, most lectins are inactivated by heat treatment such as that involved in commrcial processing or household

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TABLE 10.1 Selected Plant Lectins, Their Plant Source with Botanical and Common Names and Their Carbohydrate Specificitya Lectin (Abbreviation) Concanavalin A (ConA) Jacalin (JAC) Lens culinaris agglutinin (LCA) Lima bean lectin (LBL) Lycopersicon esculentum (LEA) Peanut agglutinin (PNA) Phaseolus vulgaris erythroagglutinin (PHA-E) Phaseolus vulgaris leukoagglutinin (PHA-L) Pisum sativum agglutinin (PSA) Ricinus communis agglutinin I (RCA-I) Soybean agglutinin (SBA) Vicia faba agglutinin (VFA) Viscum album agglutinin (VAA-I) or mistletoe lectin (ML-I) Wheat germ agglutinin (WGA)

Source Botanical Name

Common Name

Sugar Specificityb

Canavalia ensiformis Artocarpus integrifolia Lens culinaris Phaseolus lunatus Lycopersicon esculentum Arachis hypogeae Phaseolus vulgaris

Jack bean Jack fruit Lentil Lima bean Tomato Peanut Kidney bean

Man/Glc Gal/GalNac Man/Glc GalNac Chitobiose Gal Complex

Phaseolus vulgaris

Kidney bean

Complex

Pisum sativum Ricinus communis I Glycine max Vicia faba Viscum album

Pea Castor bean Soybean Fava bean Mistletoe

Man/Glc Gal GalNac/Gal Man/Glc Gal

Tritium aestivum

Wheat germ

GlcNac, sialic acid

a

Based on information taken from Rüdiger and Gabius, 2001; Van Damme et al., 1998; Wu et al., 1988; Liener, 1997. b Gal: galactose, GalNac: N-acetylgalactosamine, Glc: glucose, GlcNac: N-acetylglucosamin, Man: mannose.

cooking. This, however, still leaves foodstuffs like fruit juices, tomatoes, raspberries, garden peas, salad ingredients, spices, dry cereals, and roasted nuts in which lectins are consumed in an agglutinative-active form. Nachbar and Oppenheim (1980) found, by a survey of the literature in combination with their own work, that 82 different edible plants contained agglutinative-active lectin.

TOXICITY AND BIOLOGICAL EFFECTS OF LECTINS IN FOODS Lectins bind with high affinity to oligosaccharides, which are absent in plants, but are abundant in bacteria as polysaccharides of bacterial cell walls and in animals as constituents of glycoproteins in cell membranes (Peumans and Van Damme, 1996; Van Damme et al., 1998). Many of the membrane-integrated glycoproteins function as receptors for hormones and cytokines or are involved in cell–cell recognition. Lectins may therefore mimic a natural ligand of a receptor or inhibit binding of a

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natural ligand and thereby evoke a variety of systemic and local effects, such as cell division and growth, cell maturation, and cell death. Binding of lectins to different cells may cause cell division and growth characterized by hyperplasia (an abnormal increase in cell division) or hypertrophy (excessive growth due to increase in the size of the cell). Lectins are often detected by their ability to agglutinate red blood cells; this biological activity is, however, not central with respect to most lectins found in human food and animal feed. On the other hand, a very important in vivo biological activity is the striking biological activities that some dietary lectins have on gut function. Such lectins react with the surface epithelium of the digestive tract, and are in some cases mitogenic for enterocytes (Banwell et al., 1993; Otte et al., 2001). PHA is a powerful growth factor for the gut and by interacting with the brush border epithelial receptors induces extensive proliferation of epithelial cells. It has been shown that PHA reversibly induces hyperplastic and hypertrophic growth of the small bowel (Bardocz, 1996). In addition to its role as a growth factor for the gut, PHA also induces enlargement of the pancreas (Pusztai et al., 1995). Many lectins are very potent exogenous growth signals; some can even mimic the action of major metabolic hormones and growth factors. The effects of some lectins on the gut and on other parts of the body are especially important because lectins account for a relatively large fraction of plant protein. Further, because legume lectins are highly resistant to proteolytic digestion and bind to carbohydrate structures on enterocytes, a larger proportion of lectins is absorbed intact compared to other proteins (Jørgensen et al., 1998; Pusztai et al., 1989). Peanut lectin (Arachis hypogeae agglutinin, PNA) can be identified in peripheral venous blood of humans after ingestion of peanuts (Wang et al., 1998); intact lectin is thus absorbed and lectins may therefore display systemic effects. Another consequence of binding of dietary lectins to epithelial cells is increased endocytosis and shortening of the microvilli (Pusztai, 1993). An increase in gut permeability allows increased passage of both dietary and bacterial antigens to the periphery (Greer and Pusztai, 1985; Liener, 1997). Raw or improperly cooked red kidney beans can cause intoxication in humans (Rodhouse et al., 1990) and diarrhea associated with weight loss in animals (Banwell et al., 1984). The toxicity of red kidney beans is mainly due to PHA (Carvalho and Sgarbieri, 1998), which as described above affects the integrity of the intestinal wall leading to changes in intestinal permeability and intestinal cell hypertrophy. Other lectins have also been shown to be toxic: ingestion of lectin from Jack bean known as Concanavalin A (ConA, Canavalia ensiformis agglutinin) has caused enteric signs and symptoms (Freed and Buckley, 1978), and relatively high concentrations of WGA are antinutritive in rats (Pusztai et al., 1993). WGA was shown to retard growth of rats and was a potent growth factor for the gut. Moreover, WGA caused pancreas enlargement and thymus atrophy. Also SBA, which is known to bind strongly to human (Koninkx et al., 1992) and rat (Pusztai et al., 1990) enterocytes and hereby change the permeability of the intestinal wall, has antinutritive effects that are similar to WGA (Pusztai et al., 1997). The immunomodulatory effects of lectins are discussed in a later section.

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MITOGENICITY OF PLANT LECTINS Mitogenic agents are capable of inducing mitosis and cell division. Lectins are able to induce cell division in different kinds of cells and as mentioned some plant lectins are mitogenic toward enterocytes. The in vitro mitogenicity of lectins is typically measured as their ability to induce proliferation of lymphocytes from lymph organs or blood. Lectins are used to induce proliferation in experimental immunology; the best described and most used are PHA and ConA. They are used as polyclonal activators irrespectively of antigenic specificity, and the frequency of responding cells is very high. After prolonged contact with lectins, lymphocytes proliferate and become mature effector cells that secrete cytokines and may exert effector functions such as cellular cytotoxicity and antibody production (Kilpatrick, 1999). However, not all plant lectins are mitogenic; they can be grouped as mitogenic, nonmitogenic or antimitogenic. Actually, WGA has been found to be nonmitogenic (Muraille et al., 1999), antimitogenic (Barrett et al., 1983), and mitogenic for either T cells or B cells (Kilpatrick, 1995), probably dependent on the concentration of the lectin or the purity of the examined cells. Antimitogenic lectins inhibit the performance of mitogens in co-culture experiments. Whether a lectin is mitogenic or antimitogenic might also depend on the position of the sugar moiety to which the lectin binds. If the carbohydrate to which the lectin binds is located in close proximity to the binding site of the receptor, binding of the lectin does not lead to activation, but the lectin may act as an antagonist due to sterical hindrance of ligand binding. Some of the so-called antimitogenic lectins might fall into this category. Examples of lectins that have been found to be antimitogenic are potato and tomato lectins (LEA) (McCurrach and Kilpatrick, 1988). Lectins shown to be nonmitogenic may also influence cells of the immune system. For example, WGA has been found to be nonmitogenic, but at the same time the lectin was able to induce secretion of interleukin-12 (IL-12) and interferon-gamma (IFN-γ) (Muraille et al., 1999). Some lectins need the presence of antigen-presenting cells (APCs) to exert their mitogenicity toward lymphocytes. The presence of APC is controversial, but probably crucial. T cells recognize foreign epitopes in the context of histocompatibility antigens on APCs. More precisely, foreign proteins are taken up by APC, which processes the protein. The processed antigen is presented to the T cell on the surface of the APC bound to major histocompatibility complex (MHC). Cell types that express MHC antigens, and therefore may act as APCs, include dendritic cells (DCs), macrophages, and B cells. DCs are regarded as professional APCs, capable of activating naïve T cells. Most work on the mitogenicity of lectins was done decades ago, when the involvement of DCs in the initiation of an immune response was not recognized. It was believed that lectins preferentially activate T cells to mitogenesis. Now that it is recognized that T cells can be divided into different subsets (Th1, Th2, and Tr cells) and that DCs are important in initiating immune responses, many of the experiments done decades ago with various lectins should be reconsidered or redone with purified T-cell subsets in combination with different APCs. Antimitogenesis of lectins could, for example, be a consequence of activation of regulatory T cells, Tr cells, secreting suppressive cytokines.

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The mitogenicity of a lectin can differ depending on the animal from which the cells originate, as well as the immune compartment. We found that PNA is mitogenic toward murine cells from Peyer’s patch (PP), but not toward cells from lymph nodes, mesenteric lymph nodes, and spleen, whereas SBA is only slightly mitogenic toward cells from the spleen and PSA is mitogenic toward cells from all tested organs (Frøkiær et al., 1997). The variation in cell responsiveness toward different lectins is not surprising, as cell surface saccharides change during development and differentiation and cells from different immune compartments are at different developmental stages. For the same reason lectins have been used to separate and identify lymphoid cells. As an example, PNA is known to bind to immature B cells in germinal centers (Rose et al., 1980) and has been used to separate mature and immature thymocytes (Holladay et al., 1993). Reviews on the mitogenic action of lectins have been published (Heegaard and Müller, 1988, Kilpatrick, 1999), However, knowledge of the mechanisms by which lectins induce mitogenesis remains in its infancy. It has been shown that binding of some lectins to the T-cell receptor induces mitogenesis (Chilson and Kelly-Chilson, 1989). Likewise it is possible to stimulate T cells to proliferation by monoclonal antibodies against the CD3 part of the T-cell receptor. However, now that it is recognized that complete T-cell activation requires two signaling events, the involvement of APC in lectin-mediated mitogenesis should not be neglected. Both recognition of peptide/MHC complexes by antigen-specific T-cell receptors and the signal delivered by co-stimulatory molecules on APCs are normally required to induce Tcell activation and proliferation (Oosterwegel et al., 1999). The required co-stimulation can be provided through ligation of B7.1 or B7.2 (CD80 or CD86) with their counterreceptors CD28 or cytotoxic T-lymphocyte-associated protein-4 (CTLA-4). The ligation leads to activation or inactivation. Interaction of B7 with CD28 is a positive signal for T cell proliferation, whereas ligation of B7 with CTLA-4 is an inhibitory signal leading to anergy (Wells et al., 2001). CD28 on T cells is the primary co-stimulatory receptor for activation of naïve T cells. No other cell surface receptor can fully replace CD28, but other co-stimulatory pathways might have effects at different stages of T-cell activation, on different subsets of T cells or contribute to the development of different effector functions (Watts and DeBenedette, 1999). Moreover, as many mitogenic lectins — ConA, lentil agglutnin (LCA), PHAE, PHA-L and pea agglutinin (PSA) — bind to APC populations and of these PHAE and PHA-L, even showed a preferential binding to APC compared to T cells (Muraille et al., 1999), APCs may play a predominant role in induction of mitogenesis by lectins.

IMMUNOMODULATORY EFFECTS OF PLANT LECTINS To be able to discuss in detail the influence of dietary lectins on the immune response a brief introduction to mucosal immunology is presented.

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GUT-ASSOCIATED LYMPHOID TISSUE

AND

ORAL TOLERANCE

The gut-associated lymphoid tissue (GALT) is a major component of the mucosal associated lymphoid tissue (MALT). In MALT there are more antibody-producing plasma cells than in all other lymphoid tissues and organs together (Brandtzaeg et al., 1998). MALT has the ability to both mount an immune response and to induce specific hyporesponsiveness, also termed mucosal tolerance. Pathogens and other potential harmful substances are eliminated by an active immune response in the mucosal immune system, whereas harmless food antigens usually lead to immunological hyporesponsiveness (Strobel and Mowat, 1998). GALT consists of three components: the PP, the immune cells in lamina propria (LP), and the intraepithelial lymphocytes (IEL) (Figure 10.1). PP are predominately found in the small intestine and are lymphoid follicles composed of a specialized follicle-associated epithelium, a subepithelial dome area overlying B-cell follicles that contain germinal centers (Figure 10.1). Ingested antigens and microorganisms are transported from the gut lumen via specialized M cells present in the follicleassociated epithelium. M cells have a pronounced capacity to transport a wide variety of substances to the subepithelial dome region. M cells have a pocket area on the basolateral side of the epithelium, in which different immune cells are present. DCs Dendritic cell T cell B cell Enterocyte Plasma cell Macrophage

Bacteria food protein M cell

FAE

Dome area Germinal center IEL

Follicle

Gut lumen

IEL Interfollicular region

Peyer’s patch

Lamina propria

FIGURE 10.1 Schematic representation of the general structure and major cell populations of the GALT, which is composed of PP, intraepithelial cells (IEL), and cells in LP. The intestinal epithelium, composed of a single layer of enterocytes, lines the intestinal tract and is the surface between the inside and the outside of the body. M cells are present in the follicle-associated epithelium (FAE) overlying organized lymphoid follicles. Antigens are absorbed from the gut lumen through M cells in FAE or through enterocytes to LP. After uptake by the M cell, antigen may be presented by DCs to the T cells in the dome area or DCs take up antigen and migrate to interfollicular regions or into the B cell follicle. Some dietary lectins are able to bind to glycan on the luminale side of enterocytes or M cells; binding destroys the epithelium and results in enhanced uptake of lectins and other luminal content, e.g., other food proteins or bacteria.

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are the main APC here and they bind bacterial products by their toll-like receptors, which are a part of the innate immune defense. The effector sites of the mucosal immune system are the LP and the IEL (Figure 10.1): The main effector site is the LP, whereto mature T and B cells migrate following induction in PP. IgA secretion is one of the primary defenses against pathogens at mucosal surfaces (Brandtzaeg et al., 1998). Orally ingested soluble proteins are usually tolerated by the mucosal immune system. In general, soluble antigens are less potent mucosal antigens than particulate antigens (Strobel and Mowat, 1998). This may result from different routes of entry and the cell types involved in antigen presentation and processing. Particulates are probably absorbed by M cells overlying PP and after presentation by DC induce active immunity. In comparison, uptake of soluble antigens across the villi may be followed by a suppressive response after presentation by DCs in LP. This is, however, still highly uncertain as DC might work in a tolerogenic or immunogenic way (Finkelman et al., 1996; Hackstein et al., 2001; Lutz and Schuler, 2002). Moreover, not all soluble antigens are poor mucosal antigens. For example, cholera toxin (Holmgren et al., 1993) and different lectins (Kjær and Frøkiær, 2002; Lavelle et al., 2000) are able to induce an active immune response when delivered by the oral route. The mechanisms of oral tolerance are far from resolved, but it may be helpful to look closely at the antigen from the point of absorption to induction of oral tolerance or immunity. Antigen may be absorbed either through enterocytes or through M cells, but may also be taken up by DCs directly from the gut lumen by their dendrites (Kaiserlian and Etchart, 1999). After uptake by the M cells, antigen may be presented by DCs to T cells in the pocket area of the M cell in the subepithelial dome of PP, or immature DCs may take up antigen and migrate to interfollicular regions or into the B cell follicles to initiate immune responses or unresponsiveness at these sites (Figure 10.1). Antigen might instead be absorbed via enterocytes presented to T cells by DCs in LP of an intestinal villus, or immature DCs may transport antigen from LP via blood vessels or lymphatics to mesenteric lymph nodes, where presentation to T cells and activation and differentiation of T cells are completed (Garside and Mowat, 2001). As the DC differentiates, acquired antigen is processed and peptides are expressed in association with MHC class II and adhesion molecules such as ICAM-1 and co-stimulatory molecules such as B71 (CD80) and B7-2 (CD86) and CD40 are upregulated. Both B7-1 and B7-2 bind to the ligand CD28 and CTLA-4 on T cells. Presentation of antigen to the T cell can lead to activation of T cells that produce transforming growth factor-beta (TGFβ) and/or IL-10; these T cells are the regulatory cells, Tr1 or Th3 cells (Figure 10.2). Activation of Tr1 or Th3 results in specific active suppression of antigen-specific immune responses known as oral tolerance. Alternatively, oral tolerance is induced by development of anergy of specific T cells or by deletion of specific T cells. The mechanisms may be different depending on the administered dose, but there may also be overlapping of systemic and local immune responses due to absorption of antigen to the lymph or blood, giving rise to both local and systemic responses. DCs, T cells, and B cells are known to migrate out of PP and home to the LP of distant mucosal tissues. Accordingly, an immune response activated in PP leads to

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IL-12

Th1

IL-4 Th2

IL-4 IL-5 IL-9 IL-13

Naive T cell

Dendritic cell

IFN-γ IL-2

IL-10

Tr

TGF-β IL-10

Cell-mediated immunity Intracellular pathogens

Immunopathology: Autoimmunity

Humoral immunity Parasitic infections

Immunopathology: Atopy/ allergy

Tolerance Tolerance to harmless antigens

FIGURE 10.2 DCs control the differentiation of an immune response. Th cells can differentiate into at least three different kinds of Th cells; Th1, Th2, Tr, dependent on the initial stimuli. Tr cells include Th cells called Tr1 and Th3. IL-4 is dominant in directing the development of Th2 responses, whereas IL-12 produced by APCs is an important factor in driving Th1 development. Th1 cells, which produce IL-2 and IFN-γ, are associated with cellmediated immune responses, whereas Th2 cells, which secrete IL-4, IL-5, IL-9, and IL-13, are responsible for humoral immunity. Resistance to intracellular pathogens is linked to the induction of Th1 responses, whereas extracellular pathogens and particularly parasitic infections typically trigger Th2-dominated responses. Autoimmune diseases are often related to Th1 responses against self-components and allergic reactions involving IgE and mast cells are due to the development and activation of allergen-specific Th2 cells. The regulatory T cells are immune suppressive and secrete IL-10 and TGF-β and are involved in induction of tolerance against harmless antigens, like food proteins and autoantigens.

effector functions in LP of the gut, but also in other mucosal sites such as the respiratory and urogenital systems (McGhee et al., 1999).

INTERACTION

OF

DIETARY LECTINS

WITH IMMUNE

FUNCTION

Lectins may interfere with the immune system in various ways. Dietary lectins may lead to generation of a specific antilectin response, but they may also modulate immune responses against coadministrated proteins, the so-called adjuvant effect. Moreover, lectins may also be able to polarize the immune response toward certain effector functions. In addition, dietary lectins may give rise to a mucosal (local) or a systemic response, Table 10.2 and Table 10.3 summarize the influence of dietary lectins on immune function. Another way of categorizing the immunomodulatory effects of lectins is by differentiating between effects on the innate or the adaptive immune response, although it is sometimes difficult to distinguish between these two arms of the immune system. Innate immunity provides a first line of defense against microorganisms and is essential for the control of common bacterial infections. Adaptive immunity has evolved to specifically recognize and provide enhanced protection upon reinfection, called immunological memory. Generation of Specific Antilectin Immune Responses Lectins are highly immunogenic and capable of inducing a specific immune response after oral administration, in contrast to other dietary proteins, which usually give

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TABLE 10.2 Influence of Dietary Lectins on Immune Function Divided into Putative Systemic and Mucosal (local) Immune Responses and Further Divided into Lectin-Specific Responses and General Immunomodulating Effects Generation of LectinSpecific Response

Immunomodulating Effects

Systemic response

Specific IgG, IgM, and IgE antibodies

Adjuvant activity (immune response as opposed to tolerance) Polarizing effect (Th1 or Th2 polarization) Release of histamine and other bioactive mediators (allergy symptoms) Increase in NK cell activity

Mucosal response

Specific IgA antibodies

Adjuvant activity (immune response as opposed to tolerance) Release of histamine and other bioactive mediators (allergy symptoms)

NK: natural killer.

TABLE 10.3 Influence of Dietary Lectins on Immune Function Divided into Specific and General Immunomodulation and Further Divided into Effects Directly on Immune Cells (direct effects) and Effects Mediated through Interaction of Lectin with Non-Immune Cells (indirect effects)

Generation of lectinspecific immune response

Immunomodulation

Direct Effects

Indirect Effects

Cross-binding of lectins to B-cells (Tcell-independent antibody response, mitogenesis)

Binding of lectin to red blood cells (particulate antigen) Binding of lectin to enterocytes (increased absorption)

Cross-binding of Fc receptors on mast cells (histamine release) Binding to APCs or T cells (mitogenesis, polarization, and adjuvant effects) Increase in NK cell activity (increased cytotoxicity) NK: natural killer; APCs: antigen-presenting cells.

Intestinal degeneration (increased and/or altered absorption)

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rise to a specific downregulation of the immune response, due to induction of oral tolerance. Orally ingested lectins may after absorption affect the immune system systemically; however, it is also important to consider the local mucosally induced immune response, as the mucosa is where orally ingested lectins initially encounter the immune system. Natural plasma antibodies against dietary lectins can be detected in humans (Tchernychev and Wilchek, 1996) and various plant lectins are capable of inducing a specific IgG response in mice (de Aizpurua and Russell-Jones, 1988, Kjær and Frøkiær, 2002), and in some cases also a lectin-specific IgA response (de Aizpurua and Russell-Jones, 1988). LEA and PHA showed high immunogenicity after nasal administration, and LEA also gave rise to a specific mucosal IgA response, whereas nasally administrated PHA only demonstrated an IgA response systemically and not mucosally (Lavelle et al., 2001). The immune response against dietary lectins thus seems to be a natural consequence of lectin ingestion, but whether this in any way has an adverse influence on the immune system is not fully elucidated. Also, in light of the intensive research in use of lectins as drug and vaccine delivery systems and as potential antileukemia agents (Gabor and Wirth, 2003; Lavelle, 2001), the immune response to orally ingested lectins needs further research. Relatively few molecules have been identified that are able to induce a strong immune response when delivered by the oral or other mucosal route. This is, as discussed above, attributed to induction of oral tolerance against mucosally administrated soluble antigens. Lectins are some of the few proteins that when given by the mucosal route induce an antibody response. Although still sparsely documented, the type of immune response (local vs. systemic, tolerance vs. immunity) may be strongly dependent on the site of absorption in the gut. Uptake across PP might induce immunity, whereas absorption through enterocytes might induce tolerance. Apart from indications that the binding activity of lectins can confer immunogenicity, there are very few investigations on what determines mucosal immunogenicity of lectins. Proteins with a strong tendency to bind to epithelial cells, such as Escherichia coli pilus antigen and certain other bacterial lectins, do not induce oral tolerance (de Aizpurua and Russell-Jones, 1988). Likewise, we have shown that it is not possible to induce oral tolerance against lectin from red kidney beans (PHA), which also bind to epithelial cells (Kjær and Frøkiær, unpublished observations). Many different plant lectins have been shown to be mucosal immunogens in rodents (de Aizpurua and Russell-Jones, 1988; Lavelle et al., 2000). In humans, banana lectin was found to induce a strong specific antibody response, especially of the IgG4 isotype (Koshte et al., 1992). PHA is highly orally immunogenic; in fact, orally applied PHA mounted an antibody response at the same level as if given parenterally (de Aizpurua and Russell-Jones, 1998; Kjær and Frøkiær, 2002). An increase of intraepithelial lymphocytes and jejunal lamina propria cells was observed in response to orally administrated PHA (Banwell et al., 1993), suggesting that absorption of PHA leads to T-cell proliferation in GALT. Lavelle et al. (2000) have investigated the mucosal and systemic immune response to a number of mucosally administrated plant lectins. VAA-I showed the highest mucosal specific immune response. None of the used lectins — PHA, WGA, Ulex europaues agglutinin I (UEA-I), LEA and VAA-I — gave rise to a strong

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mucosal response in the absence of a systemic response (Lavelle et al., 2000). WGA and UEA-I induced a stronger response when administrated by the oral route as compared to intranasal administration (Lavelle et al., 2000). General Immunomodulatory Effects Innate immunity provides a rapid host antimicrobial defense in which neutrophils, DCs, and macrophages are involved. The innate immune system recognizes pathogens by means of certain conserved structural features of the microbe; these molecular patterns are called pathogen-associated molecular patterns (PAMPs) (Medzhitov and Janeway, 1997). Among the receptors recognizing pathogens are the Toll-like receptors that play an essential role in innate host defense (Takeda et al., 2003). Very few studies have focused on the involvement of dietary lectins in innate immunity; however, as many receptors on mammalian cell surfaces are glycoproteins, some lectins may well be able to bind to PAMPs, and thereby either initiate an inappropriate immune response or block receptors like antimitogenic lectins block the function of the receptor and thereby prevent an immune response. As some lectins are able to induce secretion of proinflammatory cytokines (Hajto et al., 1990; Muraille et al., 1999) and as innate immunity also plays an important role in determining how the adaptive clonal immune response reacts to pathogen-derived antigens (Manickasingham et al., 2003), lectins would be expected to have an effect on the innate immunity. Of interest, the galactoside-specific mistletoe lectin (Viscum album agglutinin, VAA-I, also abbreviated ML-I) has been shown to stimulate natural killer cell number and activity in vivo and in vitro (Hajto et al., 1989, 1998). VAA-I has also been shown to enhance the secretion of proinflammatory cytokines in cultures of human peripheral blood mononuclear cells in vitro (Braun et al., 2003; Hajto et al., 1990) and to induce elevated activation markers on the surface of monocytes and macrophages (Hajto et al., 1998), which indicates activation of the innate immune system. Adjuvants work by activating cells of the innate immune system (e.g., macrophages, natural killer cells, and DCs) to become more stimulatory and to produce cytokines and inflammatory mediators, which in turn upregulate many of the components needed to stimulate lymphocyte responses. As mentioned, lectins display adjuvant activity; co-administration of lectins with other proteins leads, in many cases, to an immune response against the co-administered protein. As an example, Korean mistletoe (V. album coloratum) is a potent adjuvant when co-immunized subcutaneously with keyhole limpet hemocyanine (KLH) (Yoon et al., 2001), and ConA complexed to ovalbumin (OVA) has been shown to enhance the formation of anti-OVA IgE in mice upon immunization (Gollapudi and Kind, 1975). Nasal and subcutaneous administration of ConA has been found to induce IgE antibodies against ConA and against a hapten conjugated to ConA (Mitchell and Clarke, 1979). PHA has been reported to affect the OVA-specific IgE response in mice injected with OVA and PHA (Astorquiza and Sayago, 1984). Oral ingestion of jackfruit lectin (jacalin, JAC) provoked an enhanced IgE response toward both OVA and the lectin after parenteral challenge with both proteins

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(Restum-Miguel and Prouvost-Danon, 1985). More recent studies have also demonstrated that lectins affect the immune response against mucosally administered OVA (Lavelle et al., 2001; Watzl et al., 2001). Plant lectins are thus not only mucosally immunogenic but also have mucosal adjuvant activity, as tested by coadministration of OVA and different lectins (Lavelle et al., 2001). In the study by Lavelle et al. (2001) no connection between immunogenicity and adjuvanticity of the investigated lectins was found. These data suggest that lectins may influence mucosal immunity against other co-administrated proteins, and might hence influence the onset of allergy. In fact, PHA can, if administered together with ovomucoid (OM), abrogate the induction of oral tolerance against OM (Kjær and Frøkiær, 2002), which might be the first step in initiation of an adverse immune response. Activation of the immune system leads to different effector functions. The adaptive immune response is divided in two: the humoral and the cell-mediated immune response. Humoral immunity is the antibody-mediated response and cellmediated immunity is handled by cytotoxic T cells. Cytotoxic T cells are involved in destruction of foreign cells such as cancer cells and virus-infected cells. PHA, ConA, pokeweed mitogen (PWM), and WGA have been shown to inhibit the cytotoxic activity of human thymocytes and peripheral lymphocytes against target cells (Toribio et al., 1985). The action of lectins in connection to cytotoxicity may be dual: First, lectins may activate T cells to become killer cells. Second, lectins may mediate contact between the killer cell and the target cell (Heegaard and Müller, 1988). Some plant lectins are cytotoxic and capable of inducing apoptosis in immune cells. VAA-I is cytotoxic for human lymphocytes, monocytes, and murine thymocytes, and cell death is induced by apoptosis in all cell types (Hostanska et al., 1997). WGA was found to be cytotoxic against normal lymphocytes in vitro, but the cytotoxicity was much higher against cultured leukemic cell lines (Ohba et al., 2003). Such cytotoxicity might thus indirectly influence the immune response as a loss of immune cells could influence the delicate balance between cells in the immune system. Lectins may also affect the immune response through indirect effects on nonimmune cells. Interaction of some lectins with carbohydrate on the intestinal wall leads to lesions, severe disruption, and abnormal development of the microvilli (Banwell et al., 1993; Koninkx et al., 1992; Pusztai et al., 1990). Lectins may therefore either impair the capacity of the epithelial cells to absorb nutrients from the gut or increase the permeability of the mucosal barrier to macromolecules. This change in absorption may influence oral tolerance as induction of oral tolerance depends on the absorption route, the absorbed amount, co-absorbed components, or danger-signals due to the absorption of components that are usually not absorbed, e.g., bacteria. Translocation of viable bacteria has been shown to occur under three circumstances: disruption of the ecological equilibrium allowing intestinal bacterial overgrowth, deficiencies in the host immune system, and increased permeability of the intestinal barrier (Berg, 1999). As increased intestinal permeability has been observed after lectin feeding (Greer and Pusztai, 1985) and as lectins are able to induce bacterial overgrowth in lectin-fed animals (Banwell et al., 1983), lectins

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might be able to induce translocation of intestinal bacteria from the gut lumen to the periphery. The polarization or differentiation of an immune response is controlled by many factors (Bajenoff et al., 2002; Bellinghausen et al., 1999; Moser, 2001), and the effector functions and immunopathological effects are quite diverse for the various responses (Figure 10.2). It is well accepted that both human and rodent helper T cells can be divided into at least three subsets based on their cytokine and effector profile (Figure 10.2). Th1 cells secrete IL-2, IFN-γ, and tumor necrosis factor (TNFβ), while Th2 cells secrete IL-4, IL-5, IL-9, and IL-13 (Abbas et al., 1996; Coffman et al., 1993). Regulatory T cells (Tr1) secrete IL-10 and TGF-β (Groux et al., 1997; Tsuji et al., 2003). The differentiation of T helper cells toward a Th1-like phenotype is promoted by IL-12 secreted by DC in particular (Moser and Murphy, 2000), while the differentiation toward Th2 cells is promoted by IL-4 (Abbas et al., 1996). Lavelle et al. have found that mucosal administration of PHA, WGA, and UEA-I gave rise to IgG1 antibodies indicative of a Th2 response (Finkelman et al., 1990), whereas PHA gave raise to an IgG2a response indicative of a Th1 response. However, the IgG2a response was very weak and only one mouse responded (Lavelle et al., 2000), and the results were not reproducible in another study by the same group (Lavelle et al., 2001). They found that intranasal immunization with LEA and VAA-I induced lectin-specific antibodies of the IgG1, IgG2a, IgG2b, and IgA isotype, whereas PHA induced only IgG1 and IgA in serum (Lavelle et al., 2000, 2001). An important outcome of their study was, when using isotype antibodies as markers of Th1/Th2 immunity, that lectins are more Th2 skewing toward a Th2 response than is cholera toxin (Lavelle et al., 2000), which is known to be a potent Th2 skewing immunogen (Xu-Amano et al., 1993). The ability of lectins to promote polarization is relevant in connection to certain diseases, such as allergy, which is a Th2-driven disease and certain autoimmune disease that are Th1 driven (Figure 10.2). Lectins might be able to polarize the immune response against co-administered proteins either directly or by allowing microorganisms that might polarize the immune response to be absorbed.

MODULATION OF IMMUNE FUNCTION BY DIETARY LECTINS IN DISEASE INVOLVEMENT

IN THE INITIATION OF IGE-MEDIATED

ALLERGY

The immune system serves to protect the host from infections and cancer; yet, inappropriate responses of this system can lead to disease. Common among these dysfunctions of the immune system are allergies and asthma. Simply said, allergic reactions are inappropriate responses to usually harmless antigens that result in an immune response mainly of the IgE class of antibodies. In relation to nutrition, inappropriate immune responses to antigens present in the gut lumen may explain the pathogenesis of gastrointestinal diseases such as gluten-sensitive enteropathy, inflammatory bowel disease, and food allergy. The immunological response generated in such conditions seems to be secondary to a loss of oral tolerance to a particular food protein or other luminal antigens (Strober and Fuss, 1999). Food allergies are a group of disorders characterized by abnormal

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or exaggerated immunological response to a specific food protein. Food allergy differs from food intolerance, as food intolerance does not have an immunological background (Schreiber and Walker, 1989). Food allergies may be divided into two clinical distinct types (Hill and Hosking, 1995; Sampson, 1997). The first type of food hypersensitivity is the classic type I, IgE-mediated immediate hypersensitivity, which can involve various organs and can provoke systemic symptoms such as systemic anaphylaxis, or more localized anaphylaxis such as hay fever, asthma, food allergies, and eczema, as well as gastrointestinal symptoms such as vomiting and diarrhea. The second type of food hypersensitivity is a type IV, delayed-type cellmediated allergic reaction (non-IgE-mediated hypersensitivity), in which intestinal symptoms occur after a few days of exposure to the allergen. The mechanism underlying IgE-mediated hypersensitivity is initiated by an event leading to activation of allergen-specific B cells that differentiate to IgE-secreting plasma cells. The allergen-specific IgE bind to Fc receptors on mast cells or basophils. Upon second exposure to the allergen, cross-linking of the bound IgE triggers mast cell or basophil degranulation and release of histamine and other pharmacologically active mediators (Figure 10.3). The abrogation of oral tolerance against a co-administered protein, when feeding PHA (Kjær and Frøkiær, 2002), and the interconnection between allergy and loss of oral tolerance indicate that ingestion of lectins may influence allergy induction against another co-ingested protein. However, there have been only a few investigations on the influence of lectins on allergy induction and no general conclusion can be drawn from these investigations, due to the use of different animal models and different lectins. In one of these studies it was found that PHA does not induce IgE production in Sprague-Dawley rats (Haas et al., 2001). In another study using Brown Norway rats, which are high IgE responders, it was found that WGA suppressed IgE production, but at the same time stimulated mast cell mediator release (Watzl et al., 2001). In a study using mice, orally ingested jacalin increased levels of IgE toward OVA (Restum-Miguel and Prouvost-Danon, 1985). The early production of IL-4 and IL-12 are key elements controlling differentiation of Th cell and skewing the immune response toward a Th1 or Th2 response (Abbas et al., 1996; Figure 10.2). Th cell differentiation is controlled by exogenous and endogenous factors of which the antigen, the kind of APC involved, and the genetic background are believed to be the most important factors. It has been hypothesized that lectins, due to their ability to induce basophils and mast cells to release histamine (Bach and Brashler, 1975; Busse et al., 1986; Hook et al., 1974; Siraganian and Siragania, 1975) and produce IL-4 and IL-13 (Haas et al., 1999b), may contribute to early IL-4 production and hence might influence allergy induction (Haas et al., 1999a). ConA, LCA, PSA, castor bean agglutinin (RCA-I), and PHA-E have been shown to be able to bind with high affinity to IgE from allergic patients. The same five lectins induced release of histamine when leukocytes from allergic patients were stimulated. WGA bound to IgE with low affinity, but still induced release of histamine (Shibasaki et al., 1992). As dietary lectins are able to bind to human IgE, they might induce symptoms like those of allergy by cross-linking IgE molecules on mast cells (Figure 10.3).

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A. Allergen cross-linkage of cell-bound IgE

Allergen IgE Fc receptor specific for IgE

Mast cell

B. Antibody cross-linkage of cell-bound IgE

IgE

Mast cell

C. Lectin cross-linkage of cell-bound IgE Lectin

IgE

Mast cell

FIGURE 10.3 Schematic diagrams of mechanisms that can trigger degranulation of mast cells. Cross-linkage of cell-bound IgE induces degranulation of mast cells and the release of histamine and other pharmacologically active agents that mediate allergic manifestations. Cross-linkage can be mediated by the allergen that initiated the IgE response (A), by an antibody that recognizes the IgE antibodies (B), or by a lectin that recognizes carbohydrates on the Fc part of the IgE molecule (C).

INVOLVEMENT

IN

OTHER IMMUNOLOGIC DISEASES

Allergies and asthma are not the only dysfunctions of the immune system. Another severe dysfunction is autoimmunity. In autoimmunity the immune system malfunctions by losing track of self and nonself, which permits an immune attack on the host. Autoimmunity is often associated with Th1-driven responses (Figure 10.2). Adjuvanticity of lectins is poorly investigated in connection to Th cell polarization and this is an area that needs further investigation.

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That immune status is affected by dietary lectins is clearly demonstrated by the findings that thymus and spleen are significantly reduced in size in rats fed high amounts of lectin (Banwell et al., 1993; Greer et al., 1985; Huisman et al., 1990; Pusztai et al., 1993). As discussed by Cordain et al. (2000) and Freed (1999), lectins in the diet may be related to certain autoimmune diseases. This could be accomplished by dietary lectin interaction with enterocytes and lymphocytes, which in turn facilitates the translocation of both dietary and gut-derived bacterial antigen, leaving a constant antigenic load in GALT. Freed (1999) hypothesizes that lectins may be involved in diseases such as insulin-dependent diabetes, rheumatoid arthritis, and IgA nephropathy. Another disease arising from immune dysfunction is celiac disease. Celiac disease, also called gluten-induced enteropathy, is caused by an immunologic reaction to the gluten fraction of wheat, manifested as degeneration of the intestinal epithelium causing malabsorption. The underlying mechanisms, however, are not fully established. Also in celiac disease it has been hypothesized that WGA or other carbohydrate-binding peptides are involved in the development of the disease (Auricchio et al., 1990; Weiser and Douglas, 1976), but the hypothesis has yet to be proved. Nevertheless, a higher level of wheat lectin–specific antibodies has been detected in blood from individuals suffering from celiac disease compared to healthy individuals, indicating at least a secondary involvement of the lectins in the disease (Fälth-Magnusson and Magnusson, 1995; Sollid et al., 1986).

CONCLUSION As a consequence of their recognition of carbohydrate on the surface of mammalian cells, lectins have many biological activities. Although some lectins have been recognized as toxic, giving rise to diarrhea and on a few causing death, the number of toxic plant lectins is relatively low and, due to their generally low heat stability, acute toxic reactions toward plant lectins are not common. A less-recognized biological effect of lectins is their potential immunomodulatory influence. Lectins may directly or indirectly affect the immune response. By binding to surface glycans on gut epithelial cells, lectins may cause increased endocytosis and in some cases shortening of the microvilli and increased gut permeability. These effects may result in increased absorption of either the lectin molecules specifically or dietary antigens in general, as well as in an altered route of absorption, both effects that may alter the immune recognition and response to dietary proteins. By binding directly to carbohydrate on the surface of immune cells, dietary lectins can influence the immune system systemically as well as locally at the gastrointestinal mucosa. They may act as antigens or function as adjuvants. If a lectin has the potential to function as an adjuvant, ingestion may lead to an active immune response against co-ingested harmless dietary proteins, which usually give rise to a specific downregulated immune response. Lectins may also modulate the immune system by acting on the polarization of the immune response, resulting in adverse reactions such as loss of oral tolerance against otherwise harmless dietary antigens, which may eventually lead to allergic

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or inflammatory reactions. The literature on immunomodulatory effects of dietary lectins is limited; the important long-term effects of dietary lectins on the immune functions are especially sparsely elucidated. Therefore, the effects of dietary lectins on oral tolerance induction or abrogation, allergies and autoimmune diseases, and infections are in need of further investigation.

ACKNOWLEDGMENTS The authors thank Trine L. Mikkelsen for competently polishing the text and critical reading of the manuscript. Financial support from the Center for Advanced Food Studies is also gratefully acknowledged.

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Index A Abortion, see Fetal toxicity Acute toxicity, see also specific toxins cycads, 247 selenium, 36 Additives, sulfur-containing, 86 Adrenal cortex, arsenic compounds in, 70 Aflatoxin B1, 213–228 carcinogenesis, 225–228 epigenetic events, hypermethylation of specific genes, 226–228 inactivation of INK4/ARF locus and, 225–226 cell cycle progression, inhibitory proteins p53, p21, and p27, 222–224, 225, 226 cell line cytotoxicity, 216–221 effects on tissue, 215 foods found in, 214 metabolic activation, 214, 216, 217 toxicity in different species, 215 Age, and neurogenerative diseases, 235, 261–263 Allergy and hypersensitivity lectins and, 285–286, 287 sulfite, 95 sulfur dioxide and, 95–96 ALS-PDC (amyotrophic lateral sclerosisparkinsonism-dementia complex), see Cycads Amino acids beta-methylamino-L-alanine (BMAA), 237, 251, 252, 253 sulfur-containing, 91, 93 Amyotrophic lateral sclerosis (ALS), 37–38 Amyotrophic lateral sclerosis-parkinsonismdementia complex (ALS-PDC), see Cycads Animal toxicity, see also specific toxins selenium, species differences in, 30, 36, 37 sulfur-containing amino acids in ruminants, 93 Antagonisms, heavy metal, 16 Antioxidants, and tricothecene, 193, 194, 195 Apoptosis, tricothecene and, 179, 185, 189, 201–203 Arsenic in breast milk

concentrations in breast milk, 14 exposure guidelines, 20–21 exposure routes, distribution routes and half-life, 4–5, 7 factors influencing levels in milk, 16 toxicological implications, 20 transfer of metals to milk, 10–13, 14 in fish, 57–77 human exposure, 70–74 origins, forms, and levels in marine organisms, 59–69 toxicity in humans, 74–77 organic compounds, structure of, 62 Aspergillus species, aflatoxins, see Aflatoxin B1

B Bacillus cereus, 147–151 Bacteria, tricothecene-binding, 181 Bacterial contamination of ready-to-eat foods, 143–163 disease outbreaks, 144–146, 147, 148, 149 improvement of microbiological quality of products, 161–163 microbiological quality of products, 146–151 Bacillus cereus, 147–151 Escherichia coli and coliforms, 151–154 Listeria monocytogenes, 154–155, 156 Salmonella spp., 155, 157 Staphylococcus aureus, 158, 159 risk assessment and food-borne microorganisms, 159–161 Bacterial infection, tricothecene effects in host resistance to pathogens, 199–201 Behavioral and neuropathological features, ALS-PDC, 254–259 Beta-methylamino-L-alanine (BMAA), 237, 251, 252, 253 Biomagnification, arsenic, 60 Bisulfite, 96–97, 98 Bladder tumors, arsenic and, 74 Blood-brain barrier, aflatoxin B1 and, 216 Blood clotting aflatoxin B1 and, 215 tricothecene and, 184, 187, 188 Blood/plasma/serum levels

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selenium, 44, 43 sulfite-oxidase-deficient rats, 97 Bone, see Skeletal system Breast milk fluoride in, 129 heavy metals in, see Heavy metals in breast milk

Concanavalin A, see Lectins Corticosteroids, 196 Cultured cells aflatoxin B1 toxicity, 215, 216–221 selenium effects, 42 tricothecene toxicity, 203 Cyanogenic glycosides, 93, 94 Cycads, 234–264 ALS-PDC, 240–246 clinical features/symptomatology, 240–242 etiology, 245–246 history, 240 neuropathology, 242–245 ethnobotany, 236–240 medicinal uses, 239 phylogeny and taxonomy, 236–238 products as food source, 238–239 seed processing, 239–240 mouse model of ALS-PDC, 254–263 behavioral and neuropathological features, 254–259 limitations of, 263 sex, age, and genetics, 261–263 time course project, 260–261 validity of, 259–260 toxicity, 246–254 acute toxicity, 247 consumption by non-human species, 252–254 discovery of toxins, 246–247 slow neurotoxin theory, 247–249, 250 sterol glucosides, 249, 251–252 Cycasin, 237 Cytotoxicity, aflatoxin B1, 216–217

C Cadmium in breast milk concentrations in breast milk, 14 exposure guidelines, 20–21 exposure routes, distribution routes and half-life, 4, 6 factors influencing levels in milk, 15 toxicological implications, 20 transfer of metals to milk, 10–14 selenium and, 34 Calcium heavy metals and, 17 and lead distribution, 5, 7 tricothecene and, 178–179 Carcinogenetic potential aflatoxin B1, 225–228 epigenetic events, hypermethylation of specific genes, 226–228 inactivation of INK4/ARF locus and, 225–226 arsenic, 74 tricothecene, 203–204 Cardiovascular system, tricothecene and, 184, 185, 186–188 Cassavism, 250 Cell culture, see Cultured cells Cell cycle aflatoxin B1 and, see Aflatoxin B1 tricothecene and, 178 Cellular immunity, see Immune response Central nervous system ALS-PDC, see Cycads heavy metals and cadmium, 20 lead, 6, 19 mercury, 5, 17–18 susceptibility in infants, 16 mycotoxins and aflatoxin B1, 215 tricothecene, 185, 191–192 Coagulation aflatoxin B1 and, 215 tricothecene and, 184, 187, 188 Coco de Mono’, 39

D Dementia, ALS-PDC, see Cycads Detoxification, heavy metals, 16 Developmental stage, fluoride effects, 106 Developmental toxicity fluoride in animals, 132–134 in humans, 132 heavy metals cadmium, 20 lead, 19 mercury, 17–19 selenium, 38 Diet, see also Nutritional status; specific toxins lectins in, 273–275 selenium in, 34–35, 38–39, 44, 45 sulfur in

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299

deficiencies, 86 excess, 99–100 sulfur-related diseases, 96 Disulfide bonds, 97 DNA and chromosomal abnormalities aflatoxin B1 and, 216, 217, 218 hypermethylation of genes, 226–228 teratogenicity, 215 heavy metals and, 17 tricothecene and, 180, 183, 184, 185 carcinogenic potential, 203–204 teratogenicity, 189 Dosage, exposure and intake levels arsenic, 74–77 selenium, 35, 37, 39 sulfur dioxide, 95 Down’s syndrome, fluoride and, 120 Drinking water arsenic in, 14, 75 fluoride in, 107, 108–109, 112–114

Eicosanoids, tricothecene and, 192 Embryotoxicity, see Fetal toxicity Encephalopathy, aflatoxin B1 toxicity, 215 Endocrine system, see also Reproductive system/reproductive toxicity fluoride and, 114, 118, 129 testosterone, see Testosterone tricothecene and, 196 Environmental factors, neurodegenerative disease development, 235 Enzymes fluoride effects, 127, 128 heavy metals and, 17 selenium-containing, 31 Epigenetic events, hypermethylation of specific genes, 226–228 Epinephrine, 190 Escherichia coli and coliforms, microbiological quality of food products, 151–154 Estrous cycle, fluoride and, 129 Ethnobotany, cycad, 236–240 medicinal uses, 239 phylogeny and taxonomy, 236–238 products as food source, 238–239 seed processing, 239–240

Fetal toxicity aflatoxin B1, 215 arsenic, 20 tricothecene, 180, 181, 189 Fibrinogen, aflatoxin B1 and, 214 Fish and shellfish, arsenic in, 57–77 human exposure, 70–74 inorganic, fate of, 71–74 organic, fate of, 71 human toxicity, 74–77 additional cautions, 75–76 concentrations versus legal limits, 74–75 dose-response assessments, 76–77 marine organisms, 59–69 chemical forms, 60–64 concentrations, 64–69 origins/sources, 59–60 sources of, 58–59 Fish and shellfish, selenium in, 214 Fluoride toxicity, 106–134 acute toxicity, 114 compounds used to fluoridate water, 112–114 exposure, 107–112 sources of fluoride in environment, 107 subchronic toxicity, 115–134 dental effects, 115–117 developmental toxicity in animals, 132–134 developmental toxicity in humans, 132 genetic effects, 118–119 hormonal effects, 118 neural effects, 119 pulmonary effects, 118 renal effects, 118 skeletal effects, 117 subchronic toxicity, reproductive system effects, 119–132 decreased fertility in humans, 119–120 Down's syndrome in humans, 120 female reproduction, 129–132 male reproduction and male offspring, 120–129 Food additives, sulfur-containing, 86 Food chain arsenic in, 59–61 selenium in, 36 Food preservatives, sulfur-containing, 94–95 Fungal toxins, see Aflatoxin B1; Tricothecene mycotoxin

F

G

E

Fertility, see Reproductive system/reproductive toxicity

GALT (gut-associated lymphoid tissue), lectins and, 278–280

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Gastrointestinal system aflatoxin B1 toxicity, 214 arsenic compounds in intestinal mucosa, 71 GALT, lectins and, 278–280 sulfur dioxide routes of entry, 95 tricothecene and, 185, 190–191 Gender differences ALS-PDC, 261–263 lead effects, 19 Genetics ALS-PDC, 261–263 fluoride effects, 118–119 neurodegenerative disease development, 235 Glutathione, 42, 86, 91, 93–94 Glutathione peroxidase, 44, 46, 91 Glutathione-S-transferase, 190 Gonads, cadmium distribution and half-life, 6 Guadeloupean parkinsonism, 250 Gut-associated lymphoid tissue (GALT), lectins and, 278–280

Hematological toxicity arsenic, 76 tricothecenes, 184, 185, 186–188 Homocysteine, 98–100 Homocystinuria, 99 Hormones, see Endocrine system; Reproductive system/reproductive toxicity; Testosterone Host resistance to pathogens, tricothecene and, 185, 199–201 Humoral immunity, tricothecene and, 185, 197–198 Hypermethylation of genes, aflatoxin B1, 226–228 Hypothalamic-pituitary-adrenal axis, tricothecene and, 196

H Hair, see Skin, hair, and nails Hazard Analysis and Critical Control Point systems, 161–163 Heart, arsenic compounds in, 7, 70, 74 Heavy metals, selenium and, 34 Heavy metals in breast milk, 2–22 concentrations, 14 exposure guidelines, 20–21 exposure routes and biological half-life, 4–5, 6, 7 factors influencing milk metal contents, 14–16 arsenic, 16 cadmium, 15 lead, 15 mercury, 15 milk production process, 7–8 sources, exogenous and endogenous, 5, 7 sources and transfer in environment, 3–4 toxicological implications, 17–20 arsenic, 20 cadmium, 20 lead, 20 mercury, 19 transfer of metals to milk, 8–14 arsenic, 10–13, 14 cadmium, 10–14 lead, 9, 10–13 mercury, 9, 10–13

I Immune response aflatoxin B1 and, 215 lectin immunomodulation, 277–285 in disease, 285–288 general effects, 283–285 gut-associated lymphoid tissue and oral tolerance, 278–280 IgE-mediated allergy, 285–286, 287 interactions with immune function, 280–285 miscellaneous, 287–288 specific antilectin immune responses, 280, 282–283 selenium and, 38 sulfur dioxide hypersensitivity, 95–96 tricothecene and, 185, 195–203 apoptosis, 185, 201–203 cellular immunity, 185, 198–199 host resistance to pathogens, 185, 199–201 humoral immunity, 185, 197–198 immunomodulation of toxin, 185, 195–197 Infants fluoride in diet, 109, 129 heavy metals in breast milk, see Heavy metals in breast milk heavy metal susceptibility, 16 sulfur clearance, 98, 99 Infertility, tricothecene and, 189 Inhibitory proteins p53, p21, and p27, aflatoxin B1 and, 222–224, 225, 226 INK4/ARF locus inactivation, aflatoxin B1, 225–226 Intestinal mucosa, see Gastrointestinal system

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K Kashin-Beck disease, 199 Keratin, selenium and, 41–42 Kidney disease sulfur-containing amino acids in, 93–94 sulfur metabolism in, 99 Kidneys aflatoxin B1 toxicity, 215 fluoride effects, 114, 118 heavy metals and arsenic, 7, 70, 71 cadmium, 6, 20 lead, 6, 20 mercury, 5, 17 susceptibility in infants, 16 Konzo, 250

L Lactic acid bacteria, tricothecene-binding, 181 Lathyrism, 250 Lead breast milk concentrations in breast milk, 14 exposure guidelines, 20–21 exposure routes, distribution routes and half-life, 4, 6 factors influencing levels in milk, 15 toxicological implications, 20 transfer of metals to milk, 9, 10–13 Lectins, 271–289 in human diet, 273–275 immunomodulatory effects, 277–285 general effects, 283–285 gut-associated lymphoid tissue and oral tolerance, 278–280 interactions with immune function, 280–285 specific antilectin immune responses, 280, 282–283 immunomodulatory effects in disease, 285–288 IgE-mediated allergy, 285–286, 287 miscellaneous, 287–288 mitogenicity, 276–277 plant, 273, 274 toxicity and biological effects, 274–275 Lecythis ollaria, 39 Lipid peroxidation, see Oxygen radicals/oxidative stress Listeria monocytogenes microbiological quality of food products, 154–155, 156

tricothecene effects in host resistance to pathogens, 199–201 Liver aflatoxin B1 toxicity, 214, 215 arsenic compounds in, 70, 71 heavy metals and arsenic distribution and half-life, 7 mercury distribution and half-life, 5 susceptibility in infants, 16 tricothecene and, 185, 189–190, 194, 201 apoptosis, 201 Lungs/respiratory system fluoride and, 114, 118 heavy metals and arsenic, 7, 70, 71 cadmium, 6 selenium and, 37 sulfur dioxide and clearance from organism, 98 routes of entry, 95–96 sulfur-related diseases, 96

M Macrozamins, 237 Magnesium, heavy metals and, 17 Mantakassa, 250 Medical conditions, sulfur metabolism in, 93–94, 99 Medicinal uses, cycads, 239 Mercaptides, 99 Mercury breast milk concentrations, 14 exposure guidelines, 20–21 exposure routes, distribution routes and half-life, 4, 5 factors influencing levels in milk, 15 toxicological implications, 19 transfer of metals to milk, 9, 10–13 selenium and, 34 Metabolism aflatoxin B1 and, 214, 216, 217 arsenic and, 74 heavy metals and, 17 selenium-containing enzymes, 31 Metal-binding proteins/metallothioneins, 16 Metals, see Arsenic; Heavy metals in breast milk; Lead Methionine deficiency, 93 Methylation of genes, aflatoxin B1, 226–228 Microbiological quality of food products, 146–151 Bacillus cereus, 147–151

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Escherichia coli and coliforms, 151–154 Listeria moncytogenes, 154–155, 156 mycotoxins, see Aflatoxin B1; Tricothecene mycotoxin Salmonella spp., 155, 157 Staphylococcus aureus, 158, 159 Mitogenicity, lectins, 276–277 Monkey coconut, 39 Mycotoxins, see Aflatoxin B1; Tricothecene mycotoxin

testicular, fluoride and, 127 tricothecene and, 185, 190, 193–195, 202 p53, p21, and p27, aflatoxin B1 and, 222–224, 225, 226

N Nails, see Skin, hair, and nails Neonatal death, arsenic and, 20 Neonates, amino acid deficiencies, 93 Nervous system cycad consumption and, see Cycads fluoride and, 114, 119 heavy metals and arsenic and, 74, 76 cadmium, 20 lead, 6, 19 mercury, 5, 17–18 susceptibility in infants, 16 selenium toxicity, 37–38 tricothecene and, 185, 191–192, 196 Neurobehavioral abnormalities ALS-PDC, 254–259 cadmium and, 20 Neurotoxins, see also Central nervous system; Nervous system cycads, see Cycads neurodegenerative disease development, 235 Nutritional status and fluoride effects, 106 heavy metals and, 17 selenium in, 31 sulfur deficiencies, 86 excess, 99–100 homocysteine levels, 98

O Oxygen radicals/oxidative stress heavy metals and, 17 selenium and, 42 sulfur and food lipid peroxidation, 92 role of, 86 sulfur-containing antioxidants, 91 sulfur dioxide and, 96

P Pancreas, 5, 70 Parkinsonism, ALS-PDC, see Cycads Pathogens, tricothecene and host resistance, 185, 199–201 PHA, see Lectins Phosphorus, arsenic and, 59–60 Phylogeny, cycads, 236–238 Phytohemagglutinin, see Lectins Pituitary gland, arsenic compounds in, 70 Placental transfer, fluoride, 129 Plants cycads, see Cycads lectins, see Lectins selenium in food chain, 36 Polioencephalomalacia, 100 Prenatal exposure, heavy metals, 16, 18 Processing/processed foods arsenic levels, 74–75 bacteria in, see Bacterial contamination of ready-to-eat foods cycads, 239–240 lectins in, 273–274 Prostaglandins, tricothecene and, 192 Protein synthesis, aflatoxin B1 and, 216

R Red blood cells arsenic distribution and half-life, 7 selenium levels, 42, 43, 44 sulfhydryl groups, 93 tricothecene and, 184, 185, 186, 190 Renal disease sulfur-containing amino acids in, 93–94 sulfur metabolism in, 99 Reproductive hormones fluoride effects, 118–119 testosterone, see Testosterone Reproductive system/reproductive toxicity arsenic, 7, 70 cadmium, 6 fluoride, 118–132 acute toxicity, 114 decreased fertility in humans, 119–120 Down's syndrome in humans, 120 female reproduction, 129–132

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male reproduction and male offspring, 120–129 selenium, 38 tricothecene, 185, 189 Respiratory system, see Lungs/respiratory system

S Salmonella spp. microbiological quality of food products, 155, 157 Seed processing, cycads, 239–240 Selenium, 29–47 biomarkers for, 42–46 chemical and physical properties, 31–32 environment, 32–33 metabolism and bioavailability, 33–35 toxicity in animals, 36–37 mechanisms of, 41–42 of specific compounds, 35–36 toxicity in humans, 37–41 dietary intake, high levels, 38–39 fatalities from ingestion of, 39–41 immunological effects, 38 neurological effects, 37–38 reproductive and developmental effects, 38 respiratory effects, 37 and tricothecene, 195 Selenoprotein P, 46 Skeletal system cadmium distribution and half-life, 6 fluoride, and, 114, 117 lead distribution and half life, 6 Skin, hair, and nails aflatoxin B1 toxicity, 215 arsenic in, 7, 20, 70, 72–73, 74 selenium and, 36, 39, 43, 46 tricothecene and, 185, 188–189 Slow neurotoxin theory, cycad toxicity, 247–249, 250 Spleen arsenic compounds in, 70 tricothecene and, 185, 186, 189–190, 186, 201 Staphylococcus aureus, microbiological quality of food products, 158, 159, 158, 159 Sterol glucosides, cycads, 249, 251–252 Sulfite, 96–97 Sulfite oxidase, 92–93, 97, 98 Sulfite-oxidase-deficient rats, 97 Sulfur, 85–100 biological role, 91–92

chemical and physical properties, 90 deficiencies, 93–94 elemental sulfur properties, 86–88 global cycle, 87, 89–91 metabolism, 92–93 natural occurrence, 88–89 of secondary origin, toxicity of, 98–100 selenium and, 33, 41–42 sulfur dioxide toxicity, 94–100 clearance from organism, 98 diseases and, 96 maximum and threshold limit values, 95 pathogenesis, 96–98 routes of entry, 95 sulfur dioxide-sensitive subjects, 95–96 Synergisms, heavy metal, 16

T Taxonomy, cycads, 236–238 Teeth arsenic compounds in, 70 fluoride effects, 114, 115–117 Teratogenicity aflatoxin B1 toxicity, 215 tricothecene, 180, 181 Testosterone fluoride effects, 118–119, 125 tricothecene and, 189 Thioredoxin, 86 Thymus, tricothecene effects on apoptosis, 201, 202 Thyroid, arsenic distribution and half-life, 7 Tricothecene (T-2) mycotoxin, 174–204 effects in specific tissues, 183–204 apoptosis, 185, 201–203 brain and neurotransmitters, 185, 191–192 carcinogenic potential, 203–204 cardiovascular system, 185, 188 cellular immunity, 185, 198–199 circulatory system, 184, 185, 186–188 cultured cells, 203 DNA and chromosomal abnormalities, 183, 184, 185 gastrointestinal tract, 185, 190–191 host resistance to pathogens, 185, 199–201 humoral immunity, 185, 197–198 immunomodulation of toxin, 185, 195–197 lipid peroxidation, 185, 193–195 liver and spleen, 185, 189–190 reproductive system, 185, 189

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skin, 185, 188–189 fungal sources, 175 metabolism and elimination of, 181 modes of action of, 178–180 occurrence of, 181–183 outbreaks, contemporary and historical, 176–177 structure of, 177–178 toxic effects, 176, 180–181

W Wheat germ agglutinin, 276 White blood cells lectins and, see Lectins tricothecene and, 184, 185, 186 Wildlife, selenium in food chain, 36

Z V Vitamin E, 193, 194, 195

Zinc, heavy metals and, 17