Advances in Environmental Control Technology: Health and Toxicology (Advances in Environmental Control Technology)

HEALTH and TOXICOLOGY

m/«A^

Advances in Environmental Control Technology Series

ENCYCLOPEDIA Ol^ ENVIRONMENTAL CONTROL TECHNOLOGY Volume 1: Thermal Treatment of Hazardous Wastes Volume 2: Air Pollution Control Volume 3: Wastewater Treatment Technology Volume 4: Hazardous Waste Containment and Treatment Volume 5: Waste Minimization and Recycling Volume 6: Pollution Reduction and Contaminant Control Volume 7: High-Hazard Pollutants Volume 8: Work Area Hazards Volume 9: Geotechnical Applications • Leak Detection • Treatment Options

ADVANCES IN ENVIRONMENTAL CONTROL TECHNOLOGY Storage Tanks Ecological Issues and Environmental Impact Assessment Health and Toxicology

Gulf Publishing Company Houston, Texas

HEALTH and TOXICOLOGY

Advances in Environmental Control Technology Series

Paul N. Cheremisinoff, Editor Nicholas P. Cheremisinoff, Associate Editor in collaboration M. Bacis P. Beggs F. Y. Bois M. Charretton P. Chiang B. Cole P. Curson P. Dingle J. W. Dobrowolski J. L. Domingo T. Driscoll

I. Ebihara L. Gerhardsson L. Giusti M. Hirata N. Hisanaga P. Hrelia J. Indulski Y. Ishikawa A. Karjalainen A. Leclerc P. Leghissa

with—

J. Leigh K. P. Li D. Luce W. Lutz M. A. McGeehin G. Mosconi A. Oskarsson J. S. Reif K. Sakai C. Sala E. Shibata

D.T.H.M. Sijm A. Sisko S. Skerfving R. Song M. A. Subramanian J. Tolls A. Trevisan G. Varadaraj R. Vincent S. C. Yang S. P. Yang

HEALTH and TOXICOLOGY

Advances in Environmental Control Technology Series

Copyright © 1997 by Gulf Publishing Company, Houston, Texas. All rights reserved. Printed in the United States of America. This book, or parts thereof, may not be reproduced in any form without permission of the publisher. Gulf Publishing Company Book Division P.O. Box 2608 D Houston, Texas 77252-2608 10

9 8 7 6 5 4 3 2 1 Library of Congress Cataloging-in-Publication Data

Health and toxicology / Paul N. Cheremisinoff, editor ; in collaboration with M. Bacis . . . [et al.]. V. < > ; p. cm. — (Advances in environmental control technology) Includes bibliographical references and index. ISBN 0-88415-386-X (alk. paper) 1. Environmental toxicology. I. Cheremisinoff, Paul N. II. Bacis, M. III. Series. RA1226.H43 1997 615.9'02—dc21 96-40506 CIP ISBN 0-87201-238-7 (Series) Printed on Acid-Free Paper (oo) IV

CONTENTS CONTRIBUTORS TO THIS VOLUME

vii

(For a note about the editor, please see page x) PREFACE 1. Environmental Risk Factors on Cancer and Their Primary Prevention I W. Dobrowolski 2. Respiratory Function Changes from Inhalation of Polluted Air Shieh-Ching Yang and Sze-Piao Yang

xi 1 47

3. Biological Markers of Early Health Effects in the Assessment of the Risk of Cancer in People Exposed to Environmental Carcinogens .. 53 JanuszA, Indulski and Waldemar Lutz 4. Sick Building Syndrome Peter Dingle

67

5. Respiratory Findings of Construction Workers Exposed to Asbestos Dust 93 Isamu Ebihara, Mamoru Hirata, Naomi Hisanaga, Eiji Shibata and Kiyoshi Sakai 6. Asbestos Exposure and the Risk of Lung Cancer in Urban Populations Anti Karjalainen andAnttila Sisko

127

7. Emphysema and Lung Mineral Content in Coalworkers J. Leigh, T. Driscoll, and B. Cole

137

8. Sinonasal Cancer and Wood Dust Exposure A. Leclerc and D. Luce

171

9. Chromium Accumulation of Chromate Workers' Bronchi Yuichi Ishikawa and Eiju Tsuchiya

191

10. Benzene Toxicokinetics in Humans Frederic Yves Bois 11. Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons in Biological Systems Kuang-Pang Li, Ping Chiang, and Raxia Song

207

219

12. Occupational Exposure to Organic Solvents During Paint Stripping and Painting Marc Charretton and Raymond Vincent

251

13. Occupational Exposure to Metallic Cobalt G. Mosconi, M. Bacis, P. Leghissa, and C. Sala

307

14. Climate and Chronic Respiratory Disease Paul Beggs and Peter Curson

329

15. Effects of Acid Precipitation on the Environment and on Human Health Lars Gerhardsson, Staff an Skerfving andAgneta Oskarsson

355

16. Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms G. Varadaraj and M. A. Subramanian

365

17. Bladder Cancer and Water Disinfection Methods Michael A. McGeehin, John S. Reif

377

18. Metal-induced Development Toxicity in Mammals Jose L. Domingo

395

19. Genetic Evaluation of Pesticides in Different Short-Term Tests Patrizia Hrelia

415

20. Pesticide Residues in Food Peter Dingle

433

21. Biological Monitoring by Means of Urinary Samples and Problems Concerning Concentration-Dilution of Spot Urine 453 Andrea Trevisan 22. Causes of Artifacts in Sorption Studies with Trace Elements Lorenzino Giusti

461

23. Bioaccumulation of Surfactants J Tolls and D. T H. M. Sijm

493

INDEX

501

VI

CONTRIBUTORS TO THIS VOLUME M. BACIS, Department of Occupational Medicine, Ospedali Riuniti Bergamo, Bergamo, Italy PAUL BEGGS, Climate Impacts Centre, School of Earth Sciences, Macquarie University, New South Wales 2109, AustraUa FREDERIC YVES BOIS, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 MARC CHARRETTON, Caisse Regionale d'Assurance Maladie Rhone-Alpes, Laboratoire de Chimie du Val Rosay, F-69370 St Didier au Mont d'Or, France PING CHIANG, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854 B. COLE, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, Australia PETER CURSON, Climate Impacts Centre, School of Earth Sciences, Macquarie University, New South Wales 2109, AustraUa PETER DINGLE, Murdoch University, Murdoch, Western Australia 6150 J. W. DOBROWOLSKI, Committee for Prot. Public Health, Inst. Manage, and Prot. Environ., PoHsh Acad. Sci., Mickiewicza 30, Paw, C 4, Pok, 117 30 059 Krakow, Poland JOSE L. DOMINGO, School of Medicine, Rovira i VirgiU University, 43201 Reus, Spain T. DRISCOLL, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, AustraUa IS AMU EBIHARA, The Institute of Science of Labour, Kawasaki, Japan LARS GERHARDSSON, Department of Environmental and Occupational Medicine, Lund University Hospital, S-221 85 Lund, Sweden LORENZINO GIUSTI, School of the Environment, Sunderland University, Sunderland, SR2 7BW, UK MAMORU HIRATA, Osaka Prefectural Institute of Public Health, Osaka, Japan NAOMI HISANAGA, National Institute of Industrial Health, Kawasaki, Japan PATRIZIA HRELIA, Department of Pharmacology, University of Bologna, Bologna, Italy

YUICHI ISHIKAWA, The Cancer Institute, Department of Pathology, Toshimaku, Tokyo, Japan JANUSZ A. INDULSKI, The Nofer Institute of Occupational Medicine, Lodz, Poland ANTI KARJALAINEN, Finnish Institute of Occupational Health, Topeliuksenkatu 41 aA, Helsinki, Finland A. LECLERC, Unite 88 INSERM-HNSM, 14 rue du Val d'Osne, 94410 SaintMaurice, France P. LEGHISSA, Department of Occupational Medicine, Ospedali Riuniti Bergamo, Bergamo, Italy J. LEIGH, Worksafe Australia, National Occupational Health and Safety Commission, GPO Box 58, Sydney, New South Wales 2001, Austraha WALDEMAR LUTZ, The Nofer Institute of Occupational Medicine, Lodz, Poland KUANG-PANG LI, Department of Chemistry, University of Massachusetts Lowell, Lowell, MA 01854 D. LUCE, Unite 88 INSERM-HNSM, 14 rue du Val d'Osne, 94410 Saint-Maurice, France MICHAEL A. MCGEEHIN, Centers for Disease Control, Atianta, GA 30341-3724 G. MOSCONI, Department of Occupational Medicine, OspedaU Riuniti Bergamo, Bergamo, Italy AGNETA OSKARSSON, Department of Food Hygiene, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden JOHN S. REIF, Colorado State University, Boulder, CO KIYOSHI SAKAI, Nagoya City Public Health Research Institute, Nagoya, Japan C. SALA, Department of Safety and Hygiene, Chemical Division USL 16, Lecco, Italy FIJI SHIBATA, Nagoya University School of Medicine, Nagoya, Japan D.T.H.M. SUM, Environmental Chemistry Group, Research Institute of Toxicology, NL-3508 TB Utrecht, The Netherlands ANTTILA SISKO, Finnish Institute of Occupational Health, TopeHuksenkatu 41 aA, Helsinki, Finland STAFFAN SKERFVING, Department of Environmental and Occupational Medicine, Lund University Hospital, S-221 85, Lund, Sweden

RAXIA SONG, Department of Physics, Beijing University, Beijing, People's Republic of China M. A. SUBRAMANIAN, P.G. & Research, Department of Zoology, Chikkaiah Naicker College, Tamil Nadu, India J. TOLLS, Environmental Chemistry Group, Research Institute of Toxicology, NL3508 TB Utrecht, The Netherlands ANDREA TREVISAN, Istituto di Medicina del Lavoro, Universita di Padova, Padova, Italy G. VARADARAJ, P.G. & Research, Department of Zoology, Chikkaiah Naicker College, Tamil Nadu, India RAYMOND VINCENT, INRS, Institut National de Recherche et de Securite, Service Evaluation et Prevention du Risque Chimique, BP 27, F-54501 Vanoeuvre Cedex, France SHIEH-CHING YANG, National Taiwan University Hospital, No. 7, Chung-Shan S. Rd., Taipei, Taiwan, Republic of China SZE-PIAO YANG, National Taiwan University Hospital, No. 7, Chung-Shan S. Rd., Taipei, Taiwan, Republic of China

IX

ABOUT THE EDITOR The late Paul N. Cheremisinoff, P.E., was a professor of civil and environmental engineering at the New Jersey Institute of Technology. Professor Cheremisinoff had more than 40 years of experience in research, design, and consulting for a wide range of government and industrial organizations. He was author and co-author of numerous papers and books on energy, resources, and the environment, and was a licensed professional engineer. He was a member of Sigma Xi and Tau Beta Pi, and a Diplomate of the American Academy of Environmental Engineers.

PREFACE This volume, entitled Health and Toxicology, contains twenty-three chapters prepared by an international group of scientists and engineers. The volume is designed to provide an extensive overview and reference on human physiological responses to various forms of pollution. Extensive discussions can be found on asbestos, carcinogenic and mutagenic poisons, poison inhalation hazards, and a wide range of industrial poisons. The volume is not organized as a collection of clinical data and information, but rather as a general working reference for engineers, scientists, industrial hygienists, and toxicologists, as well as environmental health and safety managers. With the advent of ISO 14000 and EMAS standards, the industrial community is taking on a more aggressive role in dealing with environmental and health and safety issues, fueled by the concept of Responsible Care. Industry managers and technologists must be technically versed in the health-care issues associated with industry pollution and human exposure. The information presented in this volume has been compiled and presented by many experts. These individuals should be congratulated for their efforts. A heartfelt thanks is also extended to Gulf Publishing Company for their fine production of this series. During the course of the production of the volume Ecological Planning Issues and Health and Toxicology, the editor, Paul N. Cheremisinoff, P.E., passed away. It was his vision and diligence that made this important series a reality. This volume represents in many ways an epitaph to a respected scientist and engineer who devoted his career to environmental problems. Nicholas P. Cheremisinoff, Ph.D. Associate Editor

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CHAPTER 1 ENVIRONMENTAL RISK FACTORS ON CANCER AND THEIR PRIMARY PREVENTION J. W. Dobrowolski Committee for Prof. Public Health Inst. Manage, and Prof. Environ. Polish Acad. Sci. Mickiewicza 30, Paw C 4, Pok, 117 30 059 Krakow, Poland

CONTENTS LASER MICROANALYSIS AS A NEW TOOL FOR PRIMARY PREVENTION, 11 ELECTROCHEMICAL POTENTIAL AND BIOLUMINESCENCE FOR DIAGNOSIS, 12 CHEMILUMINESCENCE AND PHOTON EMISSION AS METHODS FOR EARLY DETECTION OF CANCER RISK, 15 PHAGOCYTIC ACTIVITY, 16 SMOKING, 17 AIR POLLUTION AND OTHER RISK FACTORS, 17 SEAFOOD, 18 MERCURY, 18 SALTS AND MINERALS, 22 CONCLUSIONS, 23 Short Review of the Environmental Risk Factors of Cancer Incidence, 24 Emotional Stress as a Risk Factor, 29 STAGES OF CARCINOGENSIS, 36 APPENDIX 1: CANCER RISK FACTORS AND PREVENTION, 40 APPENDIX 2: FIGURES, 42 REFERENCES, 44 To evaluate the influence of the quality of human environment on public health (including cancer incidence), it is imperative to perform interdisciplinary case studies involving epidemiological surveys supported by the application of new, highly sensitive methods of multi-factorial analyses of the natural environment, including the food chain. Both field and experimental studies are required for better understanding of the long-term effects of low concentrations of various pollutants, including their synergistic effects. Ecotoxicology and medical elementology (knowledge about the role of elements in health and diseases) are new and very promising sciences for the environmentally oriented prevention of diseases. With this background, special attention should be paid to the complex studies, carried out

2

Health and Toxicology

in recent years, on environmental deterioration and risk factors for the incidence of cancer, congenital malformations, and so-called "diseases of civilization," which are incurable at present. According to experts of the WHO/IARCAVEEC, over 80% of proliferative diseases are caused by environmental factors. There are two basic fields of study. The first one deals with the assessment of health hazards of long-term employment under the influence of pathogenic factors. One of the case studies in this area is team research carried out by Samal et al., who evolved laboratory tests for determining the relation between the time spent while working under hazardous conditions (with a minimal radiation load) and an increase in the number of thrombocytes in the whole blood accompanied with an increase in the activity of serum lactate dehydrogenase. The second field of study is connected with the relationship between the geographic pattern of the incidence of some selected diseases—e.g., cancer and the quality of the local living environment. It is necessary to take into consideration both geochemical and other natural human-made predisposing factors. Geochemical Environment and Disease Patterns is useful for the interpretation of spatial differentiation of the rate of cancer incidence and for nutritional prevention of neoplastic diseases. Epidemiological and ecological investigations are related to the areas under comparison, justifying their description in quantitative terms. Jansson applied the Monte Carlo technique for verification of geographical clusters characterized by increased mortality rates in cases of all selected malignant neoplasms. He defined "cluster" as an area consisting of one or more subareas, in which it is possible to move from every point to any other point without leaving the area. Jansson considered this method to be effective for the evaluation of spatial clustering of various types of cancer. Besides, it is an objective way to confirm or reject subjective assumptions of the significance of higher incidence of these diseases in a particular area. Following the principles of ecotoxicology, model studies were conducted with particular attention to the analysis of elements in a trophic chain (soil, water, plants, animal feed, cattle blood, cow milk, and human blood) in various regions of Poland, taking into consideration control areas comparable with "clusters of neoplasmatic diseases" with respect to similar climatic and other natural conditions [3, 6, 5]. This can be illustrated with results of an interdisciplinary pilot project from a control village-R and a neighboring administrative unit in rural region village-P as a "cluster" with high incidence of various types of human neoplasms and lymphatic leukemia in cattle. Different analytical methods (e.g., AAS, NA, PIXE) were used in this study. By application of the scanning electron microscope and electron microbeam, the quantity of oligo and trace elements was measured in various fractions of human and animal blood cells. Significant differences were observed in the levels of some elements. The model study showed the influence of an excess or a deficiency of these elements in soil, water, cultivated plants, animals, and human food on the elements' concentration in the human body. Some of the results are shown in Table 1.

Environmental Risk Factors on Cancer and Their Primary Prevention

3

It will be seen that: (a) Greatest differences were found in relation to the levels of Mg in drinking water and Co in cow milk; the values were significantly lower in cluster areas. (b) Elevated amounts of Zn, Pb, Cu, Mn, Ni, Hg, Br, and Rb were detected in the natural environment and local food in cluster areas. The most distinct differences were noted in the concentration of elements in bran. The latter can, therefore, be recommended as a good indicator of disturbances in elemental levels of the trophic chain. (c) Mean amounts of Mg were found to be lower and that of Zn higher in both cattle and human cells (lymphocytes and erythrocytes) in the cluster areas. According to some studies about regional differences in biogeochemical properties and patterns of distribution of neoplastic and cardio-vascular diseases related to some elements, the amount of water soluble oligo and trace elements is of special importance. Increasing numbers of scientific reports are now supporting previous concepts about the health implications of environmental geochemistry [6]. Haviland, as early as 1869, reported a higher incidence of tumors in magnesium-deficient regions of France [1]. Many studies conducted in various parts of the world indicate a correlation between geochemical environment and the frequency of neoplasms, sclerosis, myocardial infarction, hypertension, apoplexy, urolithiasis, and some other diseases. From a practical point of view and for prophylactic purposes, studies about the concentration of elements in drinking water in relation to the health status, seem to be particularly interesting. Progress of knowledge about the biological function of trace and ultra trace elements is followed by research developing studies on nutritional prevention of some kinds of carcinoma. The role of selenium in primary prevention of cancer and leukemias is of special importance in this respect [38]. A reverse correlation between selenium intake and the incidence of cancer and leukemias is well documented [1, 6, 38]. Against this background, it is interesting to note that in "clusters" of human cancers and leukemia enzootica, the levels of selenium in drinking water were found to be below 0.000 ppm. A protective effect of sub-toxic doses of selenium administered in drinking water has been reported [6]. This author did not find any increase in the levels of selenium in lymphocytes (deficient in this element) of leukemic patients even after administration of 300g of sodium selenate daily for 10 days [6]. It is well known that selenium has preventive properties against neoplasms in early stages of carcinogenesis. The element acts not only as an anti-oxidant and a free radical scavenger, but also as an immunostimulative factor. This is indicated by increased titer values of antibodies against some bacteria and toxicogenic mold antigens in response to supplementation of experimental diet with sodium selenate [1]. Radio-protective properties of selenium were discovered by Badilo and co-workers in organic selenium compounds, and by Aleksandrowicz and CzyzewskaWazewska in inorganic selenium compounds. These facts may be of special interest

Health and Toxicology Table 1 Comparative epidemiological and elementological studies in control and cluster areas [4]

Study Number 1 1. 2.

3.

4.

5.

Parameters of study II Mean index for the incidence of lymphatic leukemia in cattle (per 100,000 animals) Drinking water: 2.1. Magnesium (ppm) 2.2. Manganese (ppm) 2.3. Iron (ppm) 2.4. Copper (ppm) 2.5. Nickel (ppm) 2.6. Lead (ppm) 2.7. Zinc (ppm) 2.8. Rubidium (ppm) 2.9. Strontium (ppm) 2.10. Nitrite (mgN/I) 2.11. Nitrate (mgN/I) Soil: 3.1. Manganese (ppm) 3.2. Iron (ppm) 3.3. Copper (ppm) 3.4. Nickel (ppm) 3.5. Lead (ppm) 3.6. Zinc (ppm) 3.7. Rubidium (ppm) 3.8. Zirconium (ppm) 3.9. Ytterium (ppm) 2.10. Nitrite (mgN/I) Cattle feed: 4.1. Manganese (ppm) 4.2. Iron (ppm) 4.3. Copper (ppm) 4.4. Nickel (ppm) 4.5. Lead (ppm) 4.6. Zinc (ppm) Cow milk: 5.1. Manganese (ppm) 5.2. Iron (ppm) 5.3. Copper (ppm) 5.4. Nickel (ppm) 5.5. Lead (ppm)

Control area village-R III

43.5 46.50 0.06 20.28 0.26 0.02 0.03 0.71 0.02 0.47 N.D. 0.04 204.34 193,019.21 11.76 3.16 23.90 29.57 94.91 N.D. 161.60 2,326.47 2.46 0.37 0.50 132.43 0.157 2.522 0.552 0.045 0.112

Cluster area village-R IV

781.3 6.90 0.08 0.17 0.32 0.12 0.21 0.90 0.03 0.51 0.0005 0.76 502.96 14,310.04 16.55 15.27 43.22 42.89 67.92 198.29 16.93 760.57 103.53 12.89 5.96 8.58 ' 202.97 0.065 1.882 1.893 0.205 0.762

Environmental Risk Factors on Cancer and Their Primary Prevention

6.

7.

8.

9.

10.

5.6. Zinc (ppm) 5.7. Selenium (ppm) 5.8. Rubidium (ppm) 5.9. Strontium (ppm) Cattle blood: 6.1. Manganese (ppm) 6.2. Iron (ppm) 6.3. Copper (ppm) 6.4. Nickel (ppm) 6.5. Lead (ppm) 6.6. Zinc (ppm) 6.7. Rubidium (ppm) 6.8. Selenium (ppm) 6.9. Strontium (ppm) Catde lymphocytes: 7.1. Magnesium (wt%) 7.2. Calcium (wt%) 7.3. Zinc (wt%) Human blood: 8.1. Manganese (ppm) 8.2. Iron (ppm) 8.3. Cobalt (ppm) 8.4. Copper (ppm) 8.5. Nickel (ppm) 8.6. Zinc (ppm) 8.7. Selenium (ppm) 8.8. Rubidium (ppm) 8.9. Strontium (ppm) Human lymphocytes: 9.1. Magnesium (wt%) 9.2. Calcium (wt%) 9.3. Zinc (wt%) Human erythrocytes: 10.1. Magnesium (wt%) 10.2. Calcium (wt%) 10.3. Zinc (wt%)

5.655 0.020 1.013 1.673

8.055 N.D. 1.680 1.003

1.150 709.000 1.500 0.103 0.318 4.600 0.600 0.107 0.310

2.983 606.667 3.483 0.653 2.383 14.817 0.800 N.D. 0.803

0.295 0.860 0.197

01168 0.445 0.265

0.790 319.700 0.063 1.113 0.060 2.860 0.147 0.857 0.360

1.000 210.233 N.D. 2.317 0.083 3.393 N.D. 0.230 0.430

0.270 0.827 0.110

0.220 0.393 0.180

0.280 0.843 0.137

0.207 0.627 0.203

Figures represent mean values for the elements estimated by various methods of analyses (including X-ray microanalyses in dry blood cells). Note different ratios of antagonistic elements. N.D. = non-detectable.

because ionizing radiation is recognized as a well-known leukosogen and carcinogen. Selenium is a non-specific protective agent involving inhibitory effects against di-methyl-benzene-anthracene (DMBA)-induced mammary tumorigenesis, as well as growth of transplanted leukemic cells. A negative correlation has been observed between the level of selenium in human blood and cancer mortality in various populations. This kind of reverse cor-

6

Health and Toxicology

relation is particularly seen in epidemiological data about leukemia, cancers of breast, anus, kidneys, prostate gland, pancreas, and lungs [38]. It is significant to note that in the USA and New Zealand, the mean values for human blood Se levels ranged between 0.08 to 0.2 ppm, and at the same time a high incidence of leukemia (7.0 and 6.6 per 100,000) with a very high mortality from breast cancer (23.3 and 16.9 per 100,000) was reported among the inhabitants of these countries. In countries having soil rich in selenium (e.g., Venezuela), elevated levels of this element were reported in human blood (0.81 ppm) with a low incidence of leukemia (2.8 per 100,000) and low mortality rate from breast cancers (2.6 per 100,000) [38]. Excellent reviews about links between selenium and cancer, and about the relation of this element to general health are available. According to these reports, decreased concentrations of selenium were found in the blood of almost all investigated cancer patients. Further selenopurines were shown to be highly effective for treating lymphomas. Shamburger discovered that anti-oxidants (e.g., sodium selenite, vitamin E, and vitamin C) inhibit DMBA-phorbital and DMBA-croton oilinduced papillomas in mice. Protective effects of selenium in relation to butter-yellow carcinogens (liver cancer caused by N9-methyl-p-dimethyl-aminobenzene) were also found. Similar results were obtained with respect to the relation between nutritional intake of selenium and prevention of liver cancer. Some mycotoxins (e.g., aflatoxins) are well-known hepatocarcinogens. In many regions of the world characterized by seasonal humidity, toxicogenic molds and intoxication of food with mycotoxins are very common. Therefore, it may be interesting to note that statistically significant differences were observed in the degree of contamination with toxicogenic molds (e.g., Aspergillus flavus and Penicillum meleagrinum) in the living rooms of people suffering from leukemias and those of other patients. These workers had much higher titer of antibodies against A. flavus antigen in peripheral blood of leukemic patients (suffering from chronic lymphatic leukemia, granulocytic leukemia, and acute leukemia) as compared to the values among their healthy relations. In some clusters of human cancer and bovine leukemia, a higher occurrence of the above-mentioned molds was reported in houses where leukemic patients lived and in "leukemic" breeding centers of cattle, compared with the houses of a control group. A similar correlation was later reported in a "leukemic cluster" of children in the USA. Some recent studies may be helpful in decreasing the risk of cancer due to indoor pollution with the toxicogenic molds and other pathogenic microorganisms. Good mycostatic and bacteriostatic effects were observed by fortification of paint with 150^/g sodium selenate or even 15mg/g sodium selenite. Such additives of the emulsion paint, covering rearing boxes, did not elicit any symptoms of chronic toxicity in mice, rats, or hamsters. Protective effects of selenium were also detected against the effects of in vitro cultures of human lymphocytes contaminated with mycotoxins. The aflatoxin Bl (0.5g/ml culture medium) decreased transformation of phytohaemoaglutine (PHA)-treated lymphocytes to 71.4% [6].

Environmental Risk Factors on Cancer and Their Primary Prevention

7

Selenium-caused neutralization of the inhibitory effect of this cancerogenic mycotoxin on blastic transformation is one more proof of the protective effects of Se on the immunological system. Our studies reveal neutralization of aflatoxin B1 (5g/ml) by sodium selenate (the same concentration) in respect to its embryotoxic and teratogenic effects. The element protects lysosomal enzymes of granulocytes against benzeneinduced inhibition of their activity [1]. Supplementation of diet with 0.2, 0.5, 1.0, and 2.0 ppm of Se proportionally reduced the incidence of DMBA-induced breast cancer by 44, 70, 76, and 85%, respectively. The protective effect was observed only during the period of initiation of mammary tumorigenesis by DMBA [38, 39]. Basic mechanisms involved in protective effects of selenium against chemical carcinogens seem to be: (a) competition with these carcinogens for protein binding sites, (b) enhancement of the immune response, and (c) normalization of DNA activity in neoplastic cells. Experimental data thus support a working hypothesis about an inverse relationship between the geographic pattern of neoplasms and regional deficiencies of selenium in soil, water, and food. Antagonistic elements give a real chance for the nutritional prevention of cancer. In a study about the ecotoxicology of cancer/leukemic clusters, pollution of the trophic chain with mercury was detected [6]. The protective effect of selenium against methyl mercury toxicity is well documented [38]. Coincidence of pollution of the natural and living environment with toxicogenic molds and mercury in feed, combined with the deficiency of selenium in human and animal food, may markedly increase the risk factor connected with these pathogens. Proper proportion between antagonistic elements is also very important [6]. In areas described as neoplastic clusters, this proportion was quite different from that in control regions (e.g., a deficiency of selenium and iron and the excess of zinc). Neutron activation analysis indicated a decreasing concentration of rubidium with an increased concentration of selenium in experimental mice, especially under acute intoxication with selenium. Many studies indicate a strong chemical competition between selenium and zinc [38]. Disturbance of the optimal ratio of antagonistic elements in the diet can aggravate the biological effects of a deficiency of essential trace elements. This aspect should be taken into consideration, but usually it is overlooked. Some reports give estimates of daily selenium intakes by US adults to be 60-160g. These data are not accompanied by information about average intakes of antagonistic elements (e.g., Zn, As). A similar situation exists in relation to the recommended daily intake of selenium in different age groups (Table 2). Ratios between selenium and other elements are not included in the assessment of human exposure to inhalatory and ingestion pathways of environmental seleni-

8

Health and Toxicology Table 2 Recommended daily intake of selenium after Schrauzer, 1978 [6, 38] Age (years)

RDA of Se (g)

0.0-0.5 0.5-1.0 1.0-3.0 4.0-6.0 7.0-adult

10-40 20-60 20-80 30-120 50-200

.

um [1, 6, 38]. State-of-the-art studies on ecological prophylaxis recommend increased intake of dietary selenium in many regions of the world. Nutritional risk factors involved in this respect are connected not only with the natural geochemical, climatic, and other conditions, but also with contemporary agriculture, food industry, and some new trends in nutrition and food habits (e.g., higher consumption of fat, meat, and purified sugar, and lower consumption of seafood, fiber cereals, and other traditional food in modem industrialized societies). Computerized Pattern Recognition is a new tool in cancer study and nutritional prevention in relation to geochemistry and food. Because of the multifactorial etiology of many diseases, including cancer, application of computerized pattern recognition seems to be very promising for the future [6]. This new method was used for studying changes in elements, antagonistic to selenium, for the detection of lung cancer. Based on the results of the analysis of the hair samples, it has been concluded that there were three distinguishable areas corresponding to elemental patterns characteristic for healthy people and groups with early lung cancer. The rate of accuracy of prediction of the early lung cancer was estimated as 86%. Such studies are of special importance in working environments with high health hazards. There are new perspectives of the personal approach to prevention based on elemental Analysis in Normal and Pathological Cells by the application of X-Ray microanalysis under SEM. Some other methods may also be useful for early recognition of risk factors. Observation of human blood cells under scanning microscopy connected with an X-ray microanalyzer made possible comparative studies on the quantity of elements present within the normal and pathological cells. This new technique was developed and patented by the Polish Academy of Science under Polish patent number 199102 [6]. A modification of the method relating to the preparation of blood cells for measurement of oligo and trace elements was recently introduced. The method of X-ray microanalysis—for elements from atomic number 4 (beryllium) to atomic number 92 (uranium)—has relative detectability of elements to be as high as 10-14 to 10-15 (higher detectability is for heavier elements). Application of this method of cell monitoring revealed significant changes in the amount of some elements in blood cells of leukemic patients (Table 3).

Environmental Risk Factors on Cancer and Their Primary Prevention

9

Table 3 Elemental changes in lymphocytes of leukemic patients after Dobrowolski et al. 1989,1990 [6] Elemental Content (wt %) Group

Mg

Zn

Ca

Healthy subjects Patients with chronic lymphatic leukemia Patients with acute lymphatic leukemia

0.23 0.94 0.20

0.13 0.02 0.10

0.91 0.30 1.55

Table 3 shows that the level of magnesium is increased in human blood cells in patients suffering from chronic lymphatic leukemia (CLL) with concurrent decrease in levels of zinc and calcium in these cells. Not only was the absolute amount of these elements drastically changed, but also the ratio between calcium and magnesium was recognized as very symptomatic for neoplastic diseases. This was confirmed by our investigations. Application of scanning microscopy coupled with energy dispersive X-ray microanalysis (SEMQ) has permitted detection of alterations in Mg/Ca ratios in groups exhibiting increased incidences of sudden coronary death syndrome, cardiac arrhythmias, and other chronic diseases. Under SEMQ, it became possible to discover crystal-like structures (corresponding to the diameter of red cells) observed only in peripheral blood of leukemic patients, especially those suffering from acute leukemia [6]. They are characterized by a high amount of sulphur and a very high concentration of calcium (more than 20%). Coincidence was observed between the degree of pathological changes in erythrocytes and an increasing level of calcium. Hypercalcaemia is well known in cancer and leukemic patients as evidenced from the findings of conventional analyses of blood samples. This study provided a proof of correlation between morphological and chemical alterations. A significantly elevated content of iron was detected by the same method in lympho-reticular cells of one patient with sideroblastic anemia. It was retrospectively analyzed as a preleukemic case [6]. Correlation between some morphological differences of B cells, T cells, and intermediate forms in peripheral blood of newborn children and phosphate marker of T cells was reported. Age-dependent changes in the concentration of nine elements, particularly in blood cells of premature and mature newborn children, were also reported. Therefore, it is absolutely essential that the groups of healthy subjects and patients under comparison are of similar age. Quantitative investigations (using standard cytochemical techniques including radionucleides in autoradiography) for some essential elements in blood cells (e.g., lymphocytes) and activity of selected enzymes, which are activated by these elements, may be very promising in the future. Another interesting use of the method of cell monitoring of these elements is that it gives an idea about the biogeochemical and ecotoxicological back-

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Health and Toxicology

ground. A correlation between the concentration of selected elements in the natural environment (and the trophic chain in particular) on their content in the blood cells of healthy persons and cattle was described. Such correlation was not found in neoplastic (e.g., leukemic) cattle. Differences between the amounts of some selected elements in the blood cells of healthy and leukemic cattle living in the same living center were found to be higher than those between normal animals from different regions. The average content of magnesium in cattle lymphocytes was 0.29 wt % in a magnesium-rich area. The values in a low magnesium area were 0.16 wt % in healthy cattle and 0.37 wt % in leukemic stock [6]. Similar changes were found in relation to calcium, viz: in the region having Ca-rich soil, mean Ca content in cattle lymphocytes was 0.86 wt %. In low Ca areas, the corresponding mean values were found to be 0.45 wt % in healthy catde and 0.66 wt % in leukemic animals. Significantly increased amounts of magnesium in lymphatic cells of both humans and livestock suffering from chronic lymphatic leukemia seems to be rather surprising because epidemiological data indicate a tendency for a higher incidence of leukemia in regions having lower concentrations of magnesium in soil. Such correlation is particularly evident in cases of lymphatic leukemia in cattle as indicated by Karaczkiewicz's study in Poland. Deficiency of magnesium in animal feed leads to hypomagnesemia and increased morbidity because of concurrence of this disease in livestock. This trend may be interpreted against the background of studies pertaining to the influence of a magnesium-deficient diet on lymphatic glands (particularly the thymus gland) of experimental animals [1, 3]. A tendency toward accumulation of magnesium and calcium in non-physiological amounts, in the lymphatic cells of human subjects and cattle with chronic lymphatic leukemia, might be related to pathological adaptation of neoplastic cells in endoecological and ecotoxicological circumstances. It might be interpreted in light of multifactorial etiology of leukemia and biocybemetic models [5]. It is worthwhile to mention that in samples of the whole blood, analyzed by the PIXE method, deficiency of selenium and excess of antagonistic trace element rubidium was detected in lymphatic cells of cattie suffering from leukemia enzootica bovina (e.g., level of Se in healthy cattle was 0.11 ppm while it was below 0.00 ppm in leukemic animals; levels of Rb in normal and leukemic groups were 0.60 ppm and 0.81 ppm, respectively). An even wider range of mean concentrations was observed for strontium in analogous cells (healthy animals: 0.31 ppm Sr; leukemic cattle: 0.80 ppm Sr). In addition to the deficiency of selenium in blood cells of leukemic subjects, its assimilability is also very poor. Administration of optional physiological doses of selenium was not found to increase the amount of this element in peripheral cells [6]. Deficiency of zinc was also detected in some neoplastic cells, but application of Zn may stimulate the growth of cancer. Magnesium compounds may, however, be used for supplementation of Mg pool in deficiency states. It is effective for treating even advanced forms of some tumors in animals but in cases of leukemia, Mg supplementation is effective only to induce remission at the very early phase of the

Environmental Risk Factors on Cancer and Their Primary Prevention

11

disease [IJ.This is another reason which emphasizes the importance of early detection of risk factors of neoplasms and ecologically oriented preventive activity, with particular reference to the amount of essential elements in the diet.

LASER MICROANALYSIS AS A NEW TOOL FOR PRIMARY PREVENTION One of the methods useful for quantitative analysis of essential elements (e.g, Ca and Mg) in small samples of biological material, as well as for the estimation of enzymes by these elements, is laser microanalysis introduced by R. Kozik. This method was also applied for early detection of risk related to low levels of some chemical pollutants in water [3, 6]. The method allowed simultaneous analysis of selected elements along with the enzymes activated by them. Large-scale screening studies may be necessary because a reverse relationship was observed between the level of Se in selected populations and the frequency of incidence of some neoplasms. Lower mean levels for this element were reported in whole blood of patients suffering from early stages of fibroblastic diseases and breast cancer as compared to those in healthy women both in Japan and the USA [1, 37, 38]. Decreased concentrations of Se and Zn along with increased levels of Mn and Cu were observed in blood of patients having stomach cancer [6, 37]. Similar assessment, like the one described for Se, may be recommended for ecological prevention of elemental disturbances due to human exposure by ingestion and inhalatory pathways. Evaluation for such exposures was carried out for representative values of environmental Se in air, soil, diet, and human organism. For estimation of environmental risk factors (due to excess or deficiency of trace elements), computer simulation of concentrations in the natural environment may be useful. Inhibitory effects of selenium compounds were found against many mutagens (including MNH) and chromosomal aberrations. Therefore, deficiency of Se in the areas of cancer clusters, accompanied by the excess of Hg in the trophic chain, seems to be important as it might increase the risk factor of chromosomal breakage by this heavy metal. A new cytochemical technique for the study of oxidative enzymes has recently been introduced as a highly sensitive indicator of intoxication with Hg and other harmful elements. Using this technique, Niwelinski and coworkers (unpublished work) found a correlation between the amount of total Hg in placenta and decrease of activity of these enzymes, as well as frequency of congenital malformations in some regions of South Poland [6]. A positive correlation (r = 0.637) was found between the incidence of human leukemias in rural regions and the intensity of utilization of seed dressings containing mercury. Mean concentration of this element was significantly higher in the hair samples of patients suffering from acute leukemia than in their healthy relations Hving in the same houses [6]. A positive, but weaker correlation (r = 0.345) was observed between the incidence of lymphatic leukemia in cattle and the amount of similar fungicides used in various rural areas in Cracow region.

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Health and Toxicology

ELECTROCHEMICAL POTENTIAL AND BIOLUMINESCENCE FOR DIAGNOSIS Application of different methods of high sensitivity could increase the possibility of early detection of cancer and other diseases. One of the newest methods introduced by Tomassi and improved by Janczarski uses powder electrodes for the measurement of electrochemical potential in blood samples [6]. The mean ratios of potential observed between blood plasma and blood cells were as follows: healthy subjects: 0.83; chronic lymphatic leukemia: 1.01; acute lymphatic leukemia: 1.06; and hemolytic anemia: 1.10. Changes in electrophoretic properties of lymphoblastic cells of patients with chronic lymphoblastic leukemia were reported. Differences were also observed in Verdet's coefficient (reflecting the magnetic rotation of polarized light by serum globulins), as well as in fluorescence, Raman dispersion, and optical activity in the infrared light. In the case of Zn deficiency, characteristic for lymphatic cells in chronic lymphatic leukemia patients, irradiation with lowintensity He-Ne laser light enhanced the intensity of bioluminescence much more than that seen in lymphocytes containing a normal amount of Zn and other essential elements. The results are presented in Table 4. Laser biostimulation could be used for nutritional prevention by stimulation of accumulation of some essential trace elements—e.g., selenium, iron, zinc [6] and Dobrowolski et al., in press). The pathological cells are more sensitive to red light. Irradiation has been suggested of both trace element-deficient solutions and the blood cells flowing out of the organism, in separate apparatus using laser light corresponding to the wavelength of

Table 4 Effect of trace element content of saline and laser stimulation on bioluminescence of lymphocyte suspensions after Dobrowolski et al. 1989,1990 [6]

Groups and sub-groups

Bioluminescence (mean number of impulses in 40 seconds)

1. Normal lymphocytes 1.1 Before irradiation 1.2 After irradiation (a) Cell suspension in trace element-deficient saline (b) Cell suspension in trace element-rich saHne 2. Lymphocytes from patients of bone aplasia 2.1 Before irradiation (a) Cell suspension in trace element-deficient saline (b) Cell suspension in trace element-rich saline 2.2 After irradiation

96,000 189,000 109,000 35,000 38,000 94,000

Environmental Risk Factors on Cancer and Their Primary Prevention

13

the spectrum of elements for possible increase in the activity of element-dependent enzymes [6]. Short exposure to He-Ne laser or 25 mW intensity was found to be specially effective for increasing the number of erythrocytes and hemoglobin level in rats suffering from Sarcoma-45 and Roux Sarcoma. Optimal activity of blood serum of these animals after laser irradiation became similar to that of normal serum under infrared light. It was accompanied by higher levels of albumin and globulins. Irradiation of pig ovary in vitro, with red light of 2.8 mW laser for 60 seconds, was found to significantly increase the activity of delta-5, 3-beta-hydroxy-steroid-dehydrogenase (HSD) as well as estrogen and progesterone levels. In control cultures of granulosa cells from porcine ovary, estrogen and progesterone concentrations were highest on the first day. In laser-stimulated cultures, peak progesterone levels were observed during 2-3 days when the estrogen levels dropped. In non-irradiated cultures, activity of delta 5, beta 3 HSD was gradually reduced, while in laser beamirradiated cultures, it rose in a manner similar to the progesterone levels. Hyperplasia and epithelization of granulosa cells was detected only in the experimental cultures. Stimulation of ovarian function, induced by 25 mW laser irradiation, was also put to gynecological application. There are new perspectives of application of laser light biostimulation for normalization of female endocrinological activity and elimination of some of risk factors of common neoplasms. Adequate use of low intensity laser beams could inhibit the growth rate of some species of toxicogenic molds, producers of well-known carcinogens. In relation to the nutritional aspects of medical elementology, it is interesting to note that treatment of seeds in two varieties of tomato increased bio-accumulation of selenium in the fruits. The results, which were particularly significant after supplementation of the standard soil with non-toxic doses of sodium selenate, are presented in Table 5. An increase in Se levels was accompanied with the decrease in Rb and Zn concentrations owing to an antagonistic relation between Se, Rb, and Zn. Laser photostimulation could be also a new method for higher accumulation of iron in the potato bulbs (Dobrowolski et al., unpublished report, 1995).

Table 5 Effect of soil treatment on selenium content of tomato fruits after Dobrowolski et al., 1989,1990 [6] Selenium Content (Mean values mg/kg dry weight) Groups

Untreated soil Soil treated with sodium selenate

Subjects: Venture tomato

Open-air tomato

0.0 2.6

0.1 2.3

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Health and Toxicology

The results of comparative studies of normal and cancerous (leukemic) cells showed changes in bioluminescent, electrochemical, and metabolic properties. On this background, the concept of oncogenesis created by Klimek is a new trial of interpretation [32, 33]. Taking into consideration thermodynamic aspects of tumor origin on the presumption of the appearance of dissipative structures in biological systems, one could have a quite new starting point of neoplastic transformation of cells. Amaya (in press report) further developed the Polish concept of multifactorial, environmental carcinogenesis and thermodynamics of the irreversible processes. Amaya's hypothesis is based on decreasing dormant gene information during the transformation of a normal gene to a malignant one. The cell is recognized as a very complicated feedback system of genotype enzymes and their products. Mutated genes control synthesis of new isoenzymes having properties that differ from the original (e.g., in relation to optimal conditions for maximum activity, sensitivity to environmental factors as also to controlling effectors, etc.). The molecular anvil model of enzymes was recommended by Amaya for explaining changes in chemical composition of cytoplasm in transformed cells. Changes in response of cells to environmental factors can cause marginal oscillations of the material system, including genes and final products resulting from enzymatic reactions. Changes in cell metabolism involve heating effects necessitating high-sensitivity equipment for quick detection of neoplasmatic transformation of some cells in early stages. Recently, some new methods were recommended for early diagnosis of common diseases, including cancer. Good results were reported by the application of a method based on Vincent's research. A computer program was developed for data pertaining to examination and interpretation of pH, redox, and conductivity characteristics of saliva, urine, and blood. The importance of immunological examination (using complex parameters) was suggested for the detection of risk factors of neoplasms. Immunoelectrophoresis and scanning electron-microscopy, as well as cytochemical studies of immunologically active blood cells, especially subfractions of B- and T-lymphocytes, became useful. Blood sedimentation velocities of some elements (e.g., Se, Zn, Fe, and Cu) were recognized as markers of cancer activity. A simple technique of holistic blood diagnosis was reported by Aurus-Blank and Blank. According to the authors, the application of the test enabled the detection of cancer, its stage of development, metabolic disturbances, disorders of various organs, and stress conditions of the body, as well as measurement of the control brought about by the therapeutic measures. Auroscopy was recognized by experts as more convenient than other conventional diagnostic methods for better detection of early stages of cancer. Free radicals are unstable and their recombination is associated with chemiluminescence. Intensity of this process is proportional to the concentration of superoxides in the examined biological material. Ottolengi described the catalytic function of Fe2+ in the origin of superoxides in biological membranes. Oxidation of bivalent iron is stimulating these processes. A feedback relation exists between the consid-

Environmental Risk Factors on Cancer and Their Primary Prevention

15

eration of SH-groups, Fe^"^, Fe^"^ and lipid superoxides in living cells and the enzymes involved in regulation of ROOH-levels [6]. CHEMILUMINESCENCE AND PHOTON EMISSION AS METHODS FOR EARLY DETECTION OF CANCER RISK (ASSOCIATED EXCESS OF FREE RADICALS) Recombination of free radicals in the presence of O2 and Fe^"^ may be associated with photon emission. Bivalent iron has a key role in three stages of lipid oxidation: namely, in the initiation of the process, its multiplication, and its inhibition. The last one is connected with reactions between iron and slow radicals. Excess of iron was detected in pre-leukemic state. The similarity between hemoglobin (containing iron) and chlorophyll (containing magnesium) was described by Polish scientists Marchlewski and Nenchi. Experimental studies revealed that when the temperature of chloroplasts was increased, bivalent cations of magnesium were released. This process is accompanied by photon emission. Ultraweak luminescence has been detected in all investigated higher plants and animals. Photon emission is more intense in higher plants and animals. Photon emission is more intense in higher organisms than in simpler ones. Living cells exhibit marked increases in photon emission before mitosis and amplify a secondary radiation. There are great differences between living cells in spectral range of photon emission, as well as in the intensity of this ultraweak radiation. A new concept of biologically useful information is based on photon storage in DNA and a control system of photon emission of a different wavelength than the control cells [6]. It is interesting to note that a decrease in free radical scavengers—e.g., selenium—in the human body is associated with an increased risk of incidence of breast cancer. Deficiency of some elements may be compensated by pathological mechanisms leading to an excess of these elements in particular cells, e.g., during cancer development. A higher level of anti-oxidants in neoplastic cells seems to be related to a higher intensity of chemiluminescence according to the following equation: ImH -H RO2

k2

In + ROOH

where ImH = anti-oxidant In = mild inhibitor of free radicals. Concentration of anti-oxidants is especially high during early stages of cancerogenesis. Therefore, the measurement of photon emission is seen mainly in the lower part of the spectrum (blue and green light). The chemiluminescence method also allows indirect estimation of excited molecules by a single oxygen measurement of longer red light emission. Exposure of experimental animals to leucosogens results in a significant increase in the amount of free radicals in the spleen and liver. First

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Health and Toxicology

pathological changes in the body, like increases in weight of the spleen and number of leucocytes in peripheral blood, are observed only when the highest concentration of free radicals is reached. By application of the single photon-counting technique, a much higher photon emission was reported in the leucocytes of leukemic patients than in ones among healthy subjects. The highest intensity of chemiluminescence is characteristic of people with acute leukemia. In a logarithmic scale, opposite trends were found referring to changes of chemiluminescence with time, in relation to lymphocytes and blood serum of patients with lymphatic leukemia and control subjects [6]. Leukemic and cancer clusters exhibit deficiency of cobalt in the trophic chain [6]. After supplementation of suspension of lymphocytes and the blood serum with trace amounts of cobalt chloride, the above-mentioned differences in chemiluminescence rapidly decreased. A quite opposite situation exists with respect to the application of divalent cations. The differences between photon-emission of the tested material collected from healthy and leukemic persons are substantially increased under the influence of bivalent iron, H2O2 and ultraviolet (UV) light. These factors, UV light in particular, multiplied the chemiluminescence of control blood serum and lymphocytes to a much higher degree than the comparable leukemic materials. As a result of this stimulation, the intensity of photon emission from control blood serum became much lower than that from serum of leukemic patients. The same stimulant factors slightly changed the lower intensity of photon emission from healthy lymphocytes into the much higher intensity of photon emission in leukemic lymphocytes. Simulated chemiluminescence, therefore, appears to be a very useful screening test for early detection of the incidence of leukemia or cancer. Chemiluminescence may also be helpful in estimating risk factors, e.g., sensitivity to ionizing radiation. Concentration of singlet molecular oxygen is related to the oxygen-dependent radical damage to cells. Low-level chemiluminescence emitted from rat hepatocytes (wavelength beyond 600 nm), may serve as a monitoring tool for continuous measurement of simulative effects. PHAGOCYTIC ACTIVITY Another important parameter of homeostatic ability is phagocytic activity. In the presence of anti-Escherichia coli antibodies, guinea pig alveolar macrophages released more H2O2 than in their absence. The polymorphnuclear leucocytes have a high activity of myeloperoxidase, opposite to the alveolar macrophages. As it is known that the efficiency of delay chemiluminescence from polymorphnuclear granulocytes reflects their bactericidal activity, this method was applied for comparative study of blood cells from healthy persons and some patients. A significantly higher mean value of chemiluminescence was observed in the control group compared to patients suffering from acute lymphoblastic leukemia [6]. It was observed after 45 minutes of stimulation with latex particles. Phagocytic ability of neurophils may be

Environmental Risk Factors on Cancer and Their Primary Prevention

17

increased by optimization of concentration of some bivalent ions, e.g., Ca^"^ and Mg^"^ at 10"^m were stimulatory while Ba^"^ exhibited inhibitory effects at all concentrations. Levels higher than 10"^m inhibited phagocytic activity. SMOKING One of the well-documented risk factors of respiratory tract cancers is smoking. A positive correlation was found between the number of cigarettes smoked and mutagenicity of Salmonella typhimurium in urine. Even passive smoking (equivalent to two cigarettes) caused an increase in urinary excretion of mutagens in Wistar rats within 24 hours of exposure. These results were obtained by mammaUan microsome tests. In supplementation to the application of measurements of changes in chemiluminescence of the expired air smokers, a highly sensitive single-photon counter was introduced for estimation of ultraweak light emission in blood samples of smokers and non-smokers. Significantly higher photon emission in the blood plasma of smokers was reported. Heavy smokers showed increased levels of some harmful elements (e.g., Cd and Pb), especially if the tobacco plant was cultivated in polluted areas [6]. AIR POLLUTION AND OTHER RISK FACTORS A new chemiluminescence method is also very useful for indirect estimation of other risk factors to public health. The excited molecules causing the luminescence are associated with soot particles emanating from the exhaust of a combustion engine-driven power generator. The long lifetime of these particles and their ability to transfer energy seems to increase the activity of potentially carcinogenic compounds. Enzymatic processes in the human body could increase cancer risk by hydroxylation of weak carcinogens and mutagens, mainly in respect of higher concentration of K-region epoxides. Benzene extracts of these soot particles could produce chemiluminescence and fluorescence studies. This fraction contains epoxides of poly cyclic hydrocarbons produced during the combustion process. The mutation rates in these extracts were three times greater than expected by considering only the concentration of mutagenic poly cyclic hydrocarbons. The highest dose-dependent increase in mutagenicity by the Ames system was described for the 4-7 ring polycyclic hydrocarbons among all fractions of the petrol engine exhaust condensate of a passenger car. Traffic input on the air pollution is related also to increased concentration of N-oxides. A simple colorimetric method developed by Amaya for analysis of personal exposure to NO2 is recommended for field study about the time and spatial differentiation of this risk factor. More sensitive methods—e.g., laser and chemiluminescence studies—may be applied for measurement of N-oxides and Pb to study their impact on blood levels and human health. Interdisciplinary studies about the levels of Pb in air, soil, water, the food chain, and human blood samples from inhabitants of different regions were carried

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Health and Toxicology

out by the Monitoring and Assessment Research Center, London. An excellent review relating to the impact of air lead and other pathogenic pollutants on blood lead in man and a methodological report about quality control in the analysis of Pb, Cd, and other ecotoxins in human blood are periodically released by this center. For ecologically oriented prevention of diseases, multifactorial analysis about the interactions between metals and carcinogens seems to be of special importance. Metal oxide particles were shown to modify carcinogenic effects of benzo(a)-pyrene and nitrosamines. Enhancement of nickel and asbestos carcinogens by polycyclic hydrocarbons was reported. Opposite/inhibitory effects were described for zinc and cadmium, or nickel and manganese, or selenium and zinc, as well as arsenic. SEAFOOD The application of natural food supplements' reach in deficient elements is widely recommended for nutritional prevention of health hazards. Seafood is a common source of necessary elements. The anti-carcinogenic effects of an edible seaweed, Laminaria angustata, which is popular in Japan, may be related to its elemental content. Antimutagenic effect of these extracts was observed against a breast carcinogen on Ames/mammalian microsome system. Anti-tumor activity of various seaweed extracts tested on experimental animals was recendy reviewed. A correlation between anti-tumor effects of various fractions of Semicarpus sp. and concentration of some trace elements was reported. Application of selenium-rich sea animal food exhibited better protective effects against acute and sub-acute methyl mercury poisoning in rodents than observed after the use of similar amounts of total selenium administered as sodium selenite. Sea bass—Lateolabrax japonicum—a common fish found in the seas surrounding Japan, has 2.5 times more seleniumconcentrating ability than mercury, especially in the liver and muscles. Thus, this species may be recommended for lowering the retention rate of mercury in the human body [6]. Some other products are also good for this purpose, e.g., crustaceans, muscle meat, some varieties of corn-like yellow IV for direct use by people, and halophytes as an animal feed in some regions. Some mutants of yeast concentrate high levels of selenium, and these are widely used in the form of pills in the USA [38]. A good natural source of selenium is pollen from plants growing in areas rich in this element [6]. MERCURY Mercury contamination of the natural environment is a high health hazard. Different kinds of food additives for supplementation of diet with selenium can increase human resistance against environmental pathogens, including some carcinogens and teratogens like mercury. Intoxication with mercury is a risk factor for higher incidence of leukemia and Minamata disease. The first recognized case of

Environmental Risk Factors on Cancer and Their Primary Prevention

19

this civilization disease was reported in Minamata (Japan) by Dr. Hosokawa in 1953 [6]. Nine years later, similar disease was reported from Nigata, and then reports from countries such as Iraq and Canada followed. Nutritional contamination with organic mercury is highly neurotoxic, especially for young children and prenatal development. The conditions were described in detail by Harada. During early stages of poisoning with organic mercury, the highest concentration of this metal was found in the kidney, and at later stages in the liver and brain. Four to six weeks after the affliction, mercury content decreased significantly in the kidney and liver, although it still remained high in the brain. Introduced by Harada, neurological tests are, therefore, useful for detection of early symptoms of Minamata disease, associated also with increased risk of neoplastic changes of some blood cells. According to Janicki, contamination of rural environments with pesticides containing mercury is associated with elevated frequency of adult leukemia [6]. Patients suffering with acute leukemia have a higher mean concentration of mercury in the body (as it was found by Dobrowolski, Janicki, Krasnicki) than their healthy relatives living in the same places. Mercury accumulation for prolonged periods is pathogenic for phagocytes, microghal cells, neurons, and epithelial cells of Henle's loop. Monitoring of Hg and Se in these cells (micro- and macro-phagocytes in particular) appears to be promising for the prevention of acute intoxication. Epithelial cell linings from fetal lung tissue of golden hamsters and also from human trachea and bronchi are recommended for testing chemical toxicity. This material is useful for establishing more accurate correlations between in vitro and in vivo data. Present in the cell membranes, sulphydryl groups have high affinity for mercury and its compounds. Cell membranes, therefore, are the first site of damage by Hg and other heavy metals. Exposure to Hg compounds may raise the concentration of metalothioneine-like protein in kidneys. Referring to the cases of coincidence of environmental contamination with mercury and cadmium, it should be borne in mind during interpretation that protein binding is enhanced by the presence of cadmium in these organs. Despite the stated lack of critical enzymes for mercury intoxication, decreased histochemical activity of some enzymes (e.g., NADPH-dehydrogenase and cytochrome oxidase) was observed with increased placental levels of Hg. Experimental poisoning of pregnant rats with methyl mercury chloride induced hyperplasia of labyrinth in placenta and reduced activity of some lysosomal enzymes. This intoxication was, however, associated with an increased number of arthritic follicles in the ovaries and subsequently increased activity of some lyposomal enzymes. This indicates that histochemical reactions are organ-dependent. According to an epidemiological study, the highest frequency of dysarthria in the Cracow region was observed in the westem part (1.38%), which corresponds to the highest mean amount of Hg in the placenta (8 ppm). The lowest incidence of such congenital malformations was detected in the northern part of the regions (0.61%,) corresponding with lowest level of Hg in the placenta (4 ppm). The highest incidence of all kinds of congenital malformations

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Health and Toxicology

(5.33%) was reported in the town of Oswiecim, which has a large chemical plant using mercury. The corresponding value for Cracow City was 1.04%, and for the non-polluted town of Myslenice 0.84%. Epidemiological differences are associated with differences in mean levels of mercury in the human placenta. In view of the protective role of selenium against mercury intoxication, adequate control of concentration of both these trace elements is desirable. Umbilical cord Se levels among the inhabitants of the Minamata region were almost constant for 39 years despite tremendous increase in Hg concentration [6]. The amount of these and other trace elements varies considerably in human food including cow and human milk from various regions. Experimental studies using radionuclides indicate that heavy metals can penetrate the placenta. As a result of bio-accumulation, higher Hg, Cd, and Fe concentrations were observed in the cord blood of newborn children as compared to the blood levels in their mothers. This increases the risk, particularly because of the higher sensitivity of fetuses and infants compared to the adults. In the region of the metallurgical center at Miasteczko Slaskie, increased levels of Pb and Cd (more than 30 g/dl) were reported in 15% of mothers and 24% of newborn children, in contrast to only 1.2% in control rural areas. Increased concentration of these metals in the placenta is accompanied by the tendency for higher incidence of gestoses and premature births as well as higher mortality of the newborn. Newborn children contained more Hg in blood than their mothers. It is surprising to note that some of such children from Greenland had more than 170 g/1 Hg, which is equivalent to the blood levels of victims with congenital Minamata disease. The relationship between total Hg in the child and the mother is approximately linear. This can be expressed by the equation: Hg in child (g/1) = 6.35+1.46 Hg in mother (g/1). Blood Se relationship between the child and mother is, however, non-linear. Mean level of the Se exceeded that of Hg on a molecular basis [6]. This seems to be important for preventing Hg intoxication. The main source of Se is marine food, which is subject to seasonal variation in chemical composition. Average amount of Se in cord blood was 100 g/1 in summer and 300 g/1 in winter. Differences in the concentration of Se and Hg depended on factors like dietary intake and age. Both blood and hair samples are good indicators of Hg level in the body. Seasonal variation in placental Hg content has also been reported by Niwelinski et al. from some rural areas in Poland [6]. Methyl-mercury aggravates toxicity of some pesticides. Nutritional intoxication with organic mercury followed by exposure to phosphamides increases chances of teratogenic effects in experimental rodents. It is, therefore, necessary to take into consideration the interaction of many pollutants and other factors to plan measures for preventive action. Some environmental pollutants are risk factors of both higher rates of cancer and congenital malformations incidence. One example of this coincidence is contamination of the human food chain with mercury [6]. Therefore, primary prevention has to be related to both aspects of the health hazard [3]. Exposure to lead is indirectly involved in higher cancer incidence. Nowadays, no region in the world is free from health hazards [6]. Lead levels in blood samples

Environmental Risk Factors on Cancer and Their Primary Prevention

21

from inhabitants of Greenland were found to be similar to those of people from industrial regions. Experimental studies in rodents have shown that radioactive lead crosses the placental barrier in significant amounts even if blood Pb level in the pregnant female is low. Calcium cations lower the concentration of Pb in erythrocytes followed a linear-dose response pattern. These cations also reduce the rate of Pb transport between blood plasma and bones. Calcium may usefully be applied for dietary supplementation in children from similar uptake kinetics in adults. One of the important aspects of a global UNEPAVHO project on assessment of exposure to Pb and Cd through biological monitoring is quality control in the analysis of these trace elements in blood. A significant correlation exists between the increase in amount of lead in erythrocytes (above 40g/dl) and decrease in activity of Na/K ATP-ase in cell membranes. Elevated Pb levels in these human cells is recognized as symptomatic for anemia as a result of Pb hemotoxicity. Activity of erythrocyte delta-aminolevulic and dehydrase and its change by heat treatment was suggested as an index of Pb exposure. Field studies conducted in the regions of big metallurgical centers revealed coincidence of high Pb and Cd levels in blood among the inhabitants of some areas. The influence of pollution of the living areas was found to be more important than that of the working places. The data for whole blood correlated with those for single lymphocytes. Increased amounts of Pb and other heavy metals in these blood cells is associated with immunosuppresive effects as risk factors of manifestation clinical symptoms of cancer. Bioindicators are necessary for early detection of risk factors. By the application of X-ray microanalysis, a correlation was detected between Se and Pb in the air, and Se or Pb levels in blood cells—e.g., red cells investigated under scanning electron microscope from blood samples collected from humans of low exposition and from industrial workers. Such investigation appears to be particularly useful while selecting proper blood donors under elevated risk of environmentally born contamination—e.g., by heavy metals. Another highly sensitive bioindicator is human placenta. Wide variations in concentration of some trace elements were observed even in a protective zone of one metallurgical center in Poland viz., 0.01-0.38ppm Pb, 0.015-0.055ppm Cd, 5.1-11.7 ppm Zn. For the prevention of nutritional intoxication (e.g., with heavy metals), analytical studies about phyto- and zooindicators are also recommended. Dog hair analyzed for Pb, Cd, and Zn was found a useful zooindicator for determining spatial differences in mean amount of trace elements. In samples collected 2 km from a mining and metallurgy center, the average amount in air samples was 13-15 ppm Pb and 1.47 ppm Cd. In the next zone 8 km away, the mean levels were 7.32 ppm Pb and 0.67 ppm Cd. Zinc levels in these samples did not show significant differences. Phyto- and zooindicators seem to be very useful in estimating the degree of environmental pollution in the area and the production of uncontaminated food. In the most polluted area, the frequency of cattle intoxication was 13 times higher than that in the control area. This was associated

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with a higher incidence of metabolic diseases (3.5-fold) and reproductive disorders (5.9-fold). Some precautions are, therefore, necessary for breeding of animals in highly polluted regions. Strict control over the quality of food is absolutely essential for nutritional prevention of diseases. In any case animal breeding in such areas is not economical. An eminent expert in ecotoxicology—Rene Truhaut from the French Academy of Sciences and Descartes University in Paris—recommends analysis of essential oligo and trace elements as complex in the human trophic chain. These studies should be supported by field and laboratory experiments. Application of human and other mammalian tissue cultures seems to be of special importance for evaluating biological effects of environmental pollutants, including their carcinogenic, cytotoxic, and immunosuppressive effects in vitro (on animals) and in vivo, as well. In this context, valuable experimental data is available about the influence of elements at cellular level, including chemiluminescence studies in human blood cells after treatment with industrial dusts [9]. SALTS AND MINERALS Mineral waters and salts are traditional and important food additives. Proper amounts of essential oligo and trace elements in the human diet is important not only for physiological functions of the body, but also for reducing bioaccumulation of toxic elements. Soft water may increase health hazards due to pollution of drinking water with heavy metals. Deficiency of essential minerals in drinking water is associated with higher risk of heart attack and other cardiovascular disorders. Therefore, supplementation studies were carried out, using water rich in Mg and other essential elements, in a group of people with a tendency toward deficiency of these elements in their bodies. After long-term application of Mg-rich mineral water instead of tap water, a statistically significant increase was observed in mean levels of Mg in the whole blood and single blood cells of steel workers [6]. This mineral water was also used for production of bread and special cakes for persons working at high temperatures, with good results. Referring to observations of Hawiland and others, optimalization of magnesium amounts in the human body could also be useful for nutritional prevention of neoplasms. Some animal experiments and later clinical studies were undertaken to assess effects of application of rock salt rich in essential elements, instead of commonly used purified table salt containing NaCl only. After using this natural salt from a Wieliczka mine (in a normal dose of 2.5 g per day), a significant increase in serum immunoglobin level (from 113 to 240.4 mg%) was observed after two weeks of application of the same salt. X-ray microanalysis revealed increased concentrations of oligo and trace elements in single blood cells of the patients under this experiment. The amount of the following elements increased in human lymphocytes: Fe from 0.15 to 0.26%, Zn from 0.17 to 0.25%, and Mg from 0.18 to 0.27%. A similar

Environmental Risk Factors on Cancer and Their Primary Prevention

23

tendency was detected in cattle blood cells under this experiment. The amount of the following elements increased in cattle lymphocytes: Fe from 0.15 to 0.26%, and Zn from 0.18 to 0.27%. The rock salt added to animal feed caused retarded frequency of spontaneous leukemic symptoms in AKR mice and decreased aflatoxin B-1, reducing mortality of these mice by 35%. This beneficial Wieliczka salt is rich in Fe and contains elements such as Mn, Se, Co, Cr, and Li. Deficiency of Fe, Se, and Co observed in the food chain of "cancer cluster" could be a starting point for searching about natural food additives as sources of these elements. Another Indian way is widescale fortification of table salt with iron compounds and vitamin C for better availability of Fe. This is recommended to prevent Fe-deficiency anemia. Supplementation of the human diet with high amounts of vitamin C was recommended by American Nobel Prize winner Louis Pauling. CONCLUSIONS 1. A systems approach to the relation between the quality of the whole human environment and pattern of incidence of selected diseases seems to be necessary for assessing the influence of complex external factors (with special reference to the excess or deficiency of particular elements in different regions) on the public health. 2. Interdisciplinary field studies should be supplemented with experimental research on various species of mammals. These should include investigations on synergistic or antagonistic relations of elements. 3. In "clusters" of neoplastic diseases, higher levels of Ni, Pb, Hg, Mn, Cu, Zn, and Rb, and lower levels of Mg, Fe, Se, and Co were observed in the food chain. Ecotoxicological studies were followed by the measurement of the quantity of these elements in biological samples (including a cellular level). 4. Risk of health hazards related to the deterioration of the environment is increased by the deficiency of essential elements. Higher incidence of cancer was associated with concurrent pollution of residences with toxicogenic molds and selenium deficiency in food. 5. Studies on the interdependence of different elements (e.g.. As, Cd, Hg, Se, Zn, Mg, Ca, and Pb) and other factors affecting pathogenesis of common diseases have received considerable attention in recent years. The deficiency of selenium in the body is associated not only with a higher risk of cancer, but also of myocardial infarction and Keshan cardiomyopathy, as well as with higher sensitivity to mycotoxins, benzenes, ionizing radiation, and some heavy metals. 6. The application of newer methods of analysis enables the measurement of trace amounts of both necessary and harmful elements in health and disease (even in single cells by use of X-ray microanalysis or PIXE analysis together with SEM observations of these cells) and also early detection of health hazards due to the deterioration of the human environment.

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7. Nutritional prevention of diseases (including diseases of civilization) require constant control of elemental composition of trophic chain and modification of the human diet with widescale application of natural food additives (e.g., essential element-rich rock salts, plants, bee products, pollen, etc.). 8. Area characteristics, by higher incidence of human cancer and cattle leukemia, have elevated levels of different carcinogens, including precursors of nitrosoamines in drinking water. Therefore, there is recommended feedback between permanent control of quality of water and food (as a part of environmental and ecotoxicological monitoring) and development of technical activity focused on primary prevention of cancer risk factors (e.g., adequate use of efficient wastewater treatment, improvement of sanitary conditions, etc.), as well as education toward environmental health (nutritional prevention in particular). Short Review of the Environmental Risk Factors of Cancer Incidence The general recommendation for primary prevention is a reduction of the rate of emission of the carcinogens at source, minimizing exposure as well as biological effects of the risk factors Risk factor

I. Ionizing radiation [1].

II. Non-ionizing radiation [1].

Ultraviolet radiation [1] from sunlight as well as for therapeutic uses sometimes concomitant with medicine, e.g., furocoumarins [16]. UV A, B, C [7, 16, 28].

III. Radon-222 and manmade fibers (glasswool, glass filaments, rockwool, ceramic fibers) as indoor pollutants [19].

Preventive measures

Fortification of food with radioprotectors, free-radical scavengers, etc. Supplementation of diet with additives stimulating activity of immunological system, antioxidants, etc. Use of protective glasses, and creams. Irradiation of the skin with the photoreactivating light [2].

Prevention of ozone layer depletion by air protection (including cutting down traffic output of NOx, etc.). Effective system of ventilation, selection of proper construction materials [35].

Environmental Risk Factors on Cancer and Their Primary Prevention

IV. Silica, wollastonite, attapulgite, sepiolite, talc, erionite [18]. V. Asbestos fibers [9]. VI. Beryllium and beryllium compounds as well as cadmium and Cd compounds are carcinogenic to humans; methyl mercury is possibly carcinogenic [5, 30]. VII. Carbon black particles and tar-pitch aerosol [10]. VIII. Chromium (VI) is carcinogenic, nickel [24] and lead [6] are possibly carcinogenic. IX. Occupational exposure in petroleum refining is probably carcinogenic; gasoline and residual fuel oils are possibly carcinogenic [21]. X. Exposure to gasoline engine exhaust and to diesel engine exhaust is probably carcinogenic; respirable particles are risk factors of lung or bladder tumors and also childhood cancers [24]. XI. Exposure to polynuclear aromatic compounds in coal gasification, coke production, steel founding, and aluminum production is associated with elevated risk of cancer [13]. XII. Occupational exposure to some organic solvents and pigments is possibly carcinogenic to adults, and parental exposure increases risk of leukemia and for child cancer [23]. Tobacco smoking, including passive XIII. exposure, is a major cause of cancer of respiratory system [15]. XIV. Chewing of betel and tobacco leaf, and areca-nut is possibly carcinogenic, depending on concentration of N-nitroso compounds [14]. XV. Chlorophenoxy herbicides and chlorophenols pesticide are possibly

as above. as above. Hermetization of production, substitution of materials, etc.

as above.

as above.

25

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XVI.

XVII.

XVIII.

XIX.

XX.

XXI.

cancerogenic [17]; a fungicide captafol is probably carcinogenic; some insecticides (e.g., DDT, atrazine, dichlorvos, heptachlor) are possibly carcinogenic, and workers exposed to them are under higher risk of lung cancer, multiple myeloma, and tumors of B-cell origin [27]. Exposure to N-nitroso compounds, and aromatic amines in the chemical and rubber industries are risk factors of bladder cancer [11]. Mycotoxins (e.g., aflatoxins, ochratoxin A) are carcinogenic [4, 29]. Chinese-style salted fish [29] and pickled vegetable concentrations after fermentation elevate risk for esophageal and gastric cancer [ibid]. Halogenated by-products of chlorination of drinking water are potentially carcinogenic [26]. Coffee is possibly carcinogenic to the urinary bladder [25] and coincidence of occupational exposure to Nnitro compounds could increase chance of incidence of this cancer. Drinking hot beverages is probably associated with higher incidence of esophageal and oral tumors [2]. Alcohol overconsumption is a risk factor of cancers of the oral cavity, pharynx, larynx, esophagus, and liver [20]. Application of estrogens to adult women increases rate of endometrial cancer [12].

Coincidence of some environmental risk factors (e.g., mycotoxins and nitrosoamines, or polycyclic aromatic hydrocarbons and aromatic amines, as well as some metals) could increase risk factors of incidence of some tumors. Another point for common action for primary prevention is elimination of exposure to carcinogenic factors both in working places and residences as well as in the outdoor

Environmental Risk Factors on Cancer and Their Primary Prevention

27

environment and in food (e.g., contact with mycotoxins is possible in buildings contaminated with toxicogenic molds as well as by food imported from countries of high temperature and humidity, and contact with aromatic amines could be in working places and through food additives). Some behavioral patterns may also contribute to higher incidence of particular types of cancer. Synergistic effects of tobacco smoking and occupational exposure is of special importance in this respect. Knowledge about related risk factors seems to be important for engineers, teachers, and decision makers. There are some new data about environmental etiology of some neoplastic diseases. Correlations between rate of incidence of the following carcinoma and factors have been established: • leukemia and ionizing radiation • skin cancer and exposure to UV (from sunlight) • lung, pleura cancer and asbestos • urinary bladder cancer and aromatic amines • liver cancer and aflatoxins • esophagus, pharynx, oral cavity cancer and alcohol • lung, larynx cancer and cigarettes (After Kacki E., Stemczynska J. [31]) The following information is a starting point for a data bank about the system approach to environmentally oriented primary prevention of neoplastic diseases based on risk factors of human leukemia (after Aleksandrowicz et al. 1982 [1]). A. Risk factors and markers of genetic-racial-family and ecological origin: 1. Enhanced development of leukemia in offspring of older mothers. 2. Chromosomal aberrations in somatic cells in Down's syndrome and Fanconi's anemia chromosomal abnormalities. 3. Sex chromosomes in Klinefelter, Turner, Louis-Barr, Kostmann's, and Bruton syndromes. B. Risk factors and markers connected with viruses: 1. Latent infection with oncorna virus. 2. Oncorna virus reservoirs. 3. Consumption of food infected with oncorna virus (milk and meat of leukemic cattle and poultry products). 4. Vaccine made from infected animals. C. Risk factors and markers of immunological origin: 1. The early deaths of siblings among children suffering from frequent upperrespiratory tract infection. 2. Impairment of delayed hypersensitivity reaction to common antigens. 3. The positive hypersensitivity reaction to application of human leukemia antigen. 4. Occurrence of specific antibodies to human leukemia antigen.

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5. Elastic transformation of lymphocytes in vitro upon exposure to human leukemia antigen. 6. Increased level oi Aspergillus flavus and Penicillum meleagrinum antibodies. 7. Alpha-feto-protein. 8. CEA—carcino-embryonic antigen. 9. Low level of E-rosette forming cells. 10. Impairment of lymphocyte response to PHA and Con A. 11. Risk factors and markers of enzymatic origin: a. decreased activity of alkaline phosphatase in granulocytes of chronic granulocytes leukemia. b. increased activity of muramidase—in chronic granulocytes leukemia. c. decreased activity of muramidase—in acute myelogenous and chronic lymphatic leukemia. d. elevation of IgM levels in polyclonal and monoclonal gammopathies. e. decreased activity of terminal deoxynucleoitidyl transferase in acute myelogenous and chronic granulocytic leukemias and high TdT values in most cases of acute lymphoblastic leukemia. f. decreased activity of serum RNA-se. g. decreased activity of antineoplastons. h. decreased 5' nucleotidase activity in chronic lymphocytic leukemia lymphocytes, i. reverse transcriptase level as risk factor. D. Risk factors and markers connected with the level of essential elements in blood and tissues: 1. Copper level increased in blood cells and serum in acute myelogenous leukemia. 2. Calcium level increased in blood cells and serum in acute myelogenous leukemia. 3. Zinc level subnormal in serum of leukemia and lymphoma patients and markedly decreased in peripheral leukocytes of chronic granulocytic leukemia and other neoplastic diseases. 4. Magnesium level decreased in serum and increased in leukocytes in leukemia selenium levels decreased in serum. E. Risk factors and markers connected with the level of vitamins in blood and tissues: 1. Vitamin C level decreased in blood cells. 2. Vitamin E level decreased. 3. Vitamin B-12 level increased. F. Environmental risk factors. Physical, chemical and biological immunosuppressors as leukemia risk factors: 1. Ionizing and non-ionizing radiation: a) Ionizing radiation—there are significant differences between wooden buildings and buildings constructed from prefabricates (reinforced concrete) with regard to the extent of emitted radiation.

Environmental Risk Factors on Cancer and Their Primary Prevention

29

b) Non-ionizing radiation—non-ionizing radiation in the range of 10-100 W/m^ may induce a significant hazard with respect to leukemic morbidity. c) Electromagnetic field and microwaves exceeding values of 1.0 mW/cm^. 2. Chemical leukemogens. 3. Biological leukemogens. G. Electronic risk markers: 1. The lowering of intracellular lymphocyte potential. H. Clinical risk factors: 1. Fewer unknown causative agents. 2. Therapy-resistant anemia and granulocytopenia. 3. Primary polycythemia. 4. Hemochromatosis. 5. Herpes zoster. 6. Urticaria. 7. Meningitis. 8. Decreased viability of granulocytes. 9. Decrease of phagocytic activity of granulocytes. 10. Decrease migrational capacity of granulocytes. Emotional Stress as a Risk Factor [1] Nowadays it is believed that the vast majority of neoplasms is caused by environmental risk factors. At the same time, an increase of cancer incidence has been observed, especially in heavily contaminated areas. Circumstances mentioned above cause common interest in the prospects of more efficient primary prevention of neoplasmatic diseases [1, 3, 5]. Etiology of neoplasms is multifactorial. Thus, to learn about environmental risk factors of cancer, it is not enough to investigate single factors and their biological effects. There is a need to create data banks of different scientific disciplines that can be fundamental to permanent updating of our knowledge. This synthesis makes a foundation for the development of related interdisciplinary case studies [4, 6]. They should provide not only deeper knowledge on actual risk of neoplasms for the population and help to predict future trends, but first of all define conditions necessary for the efficient, environmentally oriented prevention of cancer. This disease makes a potential threat for all. This implies the need for active involvement of everybody in the common action. That demands the popularization of the knowledge, both of environmental risk factors as well as various practical opportunities of permanent preventive action toward individuals as well as institutions, including the local, national, and international levels. The knowledge and skills necessary for this action can be used better if regarded as a necessary element for all the programs of formal and informal education. For moral reasons, the orientation for environmental risk factors of cancer and their primary prevention is a necessary part of training of decision makers (includ-

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Health and Toxicology

ing post-graduate courses). Different forms of open universities and distance education (TV satellite broadcasts) can make partnership between politicians and local communities easier. This partnership is to be focused on cancer prevention. This particularly refers to the consolidation and integration of local environmentally oriented efforts with solving global problems indirectly connected with environmental risk factors or cancer (e.g., prevention of ozone layer depletion or ecotoxicological causes of food contamination, including transboundary pollution). Updating and promotion of the knowledge provides motivation for common action. To make it effective, it seems necessary to create an efficient system of permanent exchange of scientific and practical information (international system of email and satellite communication), considering also the experience connected with socio-technical methods such as legal regulations, economic instruments, and cooperation between decision makers and the public on a regional and international scale. This refers to the links between primary prevention of mutations and cancer incidence with the sustainable development related not only to the promotion of environmentally sound technologies and system of management, but also the prevention of the introduction into less-developed countries technologies and equipment that are outdated and harmful to the environment or exporting wastes to these countries. Such wastes can be a source of contamination of the natural environment and the human food chain with carcinogens or co-carcinogens. That refers to, e.g., the export of outdated cars to less developed countries, where they are widely used and not only cause enormous waste of petrol resources, but also contaminated environment with strongly carcinogenic poly-aromatic carbohydrates. An essential element of primary prevention of cancer is proenvironmental education of the society by encyclopedias, textbooks, and mass media, particularly in countries of lower levels of environmental and hygienic awareness. Nowadays the use of effective means of visual propaganda for the increase of cigarette consumption, which is high anyway, becomes a bigger and bigger problem in Central and East European countries. The societies of rich countries, because of the increase of knowledge on the high risk of neoplasms in the respiratory system, caused by smoking, reduced cigarette consumption radically. Thus, tobacco companies intensify their activities in poorer countries. Their land is usually more contaminated by traditional high energy-consuming technologies (with carcinogens as well). Association of pollution from industry and transport with mass addition to tobacco gives a synergistic effect, i.e., increases the incidence of deaths caused by lung cancer and similar neoplastic diseases. International preventive actions such as "Europe against cancer" are developed mainly in rich countries. In less-developed countries, the lack of information on actual environmental risk factors is widespread. This refers also to persons with elevated risk levels such as radiologists and nuclear physicists. Usually the estimation of risk factors is limited to the measurements of exposure on ionizing radiation only. But according to the founder of ecotoxicology—Rene Truhaut, expert of WHO, FAO, EC, etc.—the ability of keeping homeostasis in an organism, including the risk of cancer incidence, is influenced by a synergistic

Environmental Risk Factors on Cancer and Their Primary Prevention

31

effect of physical, chemical, biological, and other factors that directly or indirectly cause neoplastic diseases. Thus, at the same working exposure the risk can be different depending on total exposure to all carcinogens, co-carcinogens, immunosuppressors, and promoters of carcinogenesis out of work. That refers not only to the content of harmful ingredients in food (potentially pathogenic), but also the content of protective ingredients in the diet of a particular person as well as individual biological susceptibility, which is defined genetically. These rules imply both the necessity of the development of interdisciplinary studies combined with the development of multifactorial risk analyses based on estimation of personal exposure. There are differences both in total exposure to complex environmental carcinogens as well as individual biological susceptibility. Thus, there is a trend to combine the assessment of the exposure on external factors with the biological monitoring of many different physiological, biochemical, biophysical, and other parameters characterized by an individual reaction. To minimize the risk of the occurrence of irreversible morphophysiological changes, it is necessary to develop interdisciplinary studies in larger and larger teams and combine them with the promotion of the methods of early detection of risk factors of cancer incidence. Unfortunately, present scientific policy usually prefers financing fragmentary research that does not provide adequate premises to estimate the risk. This has a negative influence on the effectiveness of the primary prevention of cancer. The future trend of cooperation in this field seems to be also the integration of basic and applied studies, and extensive scientific cooperation of the scientists and the whole society. The present state of knowledge resulting from the synthesis of epidemiological data referring to spatial differentiation of cancer incidence and the results of environmental monitoring (including multifactorial emission measurements) and ecotoxicological control of food contamination (together with experimental data) already provides some scientific premise for the active participation of everybody in the primary prevention of tumors. The basic trend in contemporary environmental management and reduction of related health hazards is the elimination of pollution at the source. Thus under democratic conditions, the knowledge of the society on environmental risk factors of cancer can generate adequate decisions in policy referring to better environment and life quality. That refers to the preferences in the modernization of technologies or the construction of vehicles or other equipment so that the emission of pollution induces changes in DNA and stimulates the increase of cancer incidence. Because the majority of environmental mutagens also have carcinogenic properties, such action is to protect the health of both present and future generations. The necessary condition of the effective primary prevention of environmentally born health hazards is a system approach to the elimination of all risk factors related to environmental deterioration. That demands the integration of present activities on the field of legal regulations, administrative management, education of the whole society, and the propagation of model-comprehensive solutions. For example, the achievements of American scientists are very valuable. They dis-

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Health and Toxicology

covered that increased incidence of some neoplasms could be linked with the deficiency of selenium in some populations and they suggested supplementing diet with selenium by using selenium-rich natural products like mutants of yeast, which were introduced by G. N. Schrauzer from California University [38]. There is the need for the development of more complex methods of primary prevention of cancer and their promotion on the international scale. Interdisciplinary research developing studies in this field were initiated in Poland by Julian Aleksandrowicz from the Copernicus University of Medicine in Cracow. Related case studies were done in small areas of very high incidence of leukemia and other neoplastic diseases called "cancer houses" and "cancer clusters" in villages. Statistically significant higher incidence of some species of toxicogenic molds in indoor environments of cancer patients was discovered. At the same time, the deficiency of a protective factor (i.e., selenium) in the natural environment and food chain in cancer clusters was reported. During the experimental studies, our team found preventive effects of selenium in relation to human lymphocytes of in vitro cultures under immunosuppressive effects of one mycotoxin—aflatoxin Bl produced by the above-mentioned mold was found. Complementary results of this research indicate the cause-result relation and imply practical recommendation for preventive measures. Thus, we also conducted a team study with Bolestaw Smyk from the University of Agriculture in Cracow and his team. The study referred to the opportunities to prevent contamination of indoor environments by adding nontoxic mycostatic agents to the paints. My team initiated research on the influence of laser light stimulation of yeast and cultivated plants for increasing accumulation of selenium and some of their essential elements and vitamins, which are deficient in the food chain when there is higher incidence of cancer [4, 5]. Our interdisciplinary research can find a feedback system between the application of new research techniques for early detection of environmental risk factors on basic studies, including synergistic effects of different pathogens, and research studies for a system approach to primary prevention of environmental risk factors of cancer (leukemia). The common base (submolecular level) for many environmental carcinogens is the elevation of the concentration of free radicals. Thus, we investigated the shape of the curve and intensity of photon emission from neoplastic cells and related normal cells of in vitro culture, finding some differences. There is a growing recognition of the role of free radicals in pathology. Nutritional prevention is based on the supplementation of the human diet with rich "free radicals scavengers"—natural additives (including some selenium compounds and vitamins). Premature delivery of otherwise normal neonates is a problem in both developed and developing countries. What is more, there are common thermodynamic roots of cervical cancer and premature delivery. The results of treatment of cervical carcinoma from 1950 to 1986, obtained from all over the world, show no practical improvement in the 5-year survival rate for stages I (75%-79%), II (53%-58%), and III (28%-33%). Still, the survival rates depend on the stage of cancer at the

Environmental Risk Factors on Cancer and Their Primary Prevention

33

moment of its detection, not on the method of treatment. It means that, therefore, therapeutic methods have reached their apogee and indicate that medicine has exhausted its possibilities. Medicine, as no other field of human activity, requires from gynecologists the specific responsibility for conscientious constant acquisition of the general knowledge, which always has determined man's approach to cancer. The decisive improvements on it can be achieved only by simultaneous medical and psychological cooperation throughout childhood and youth, since adult cervical cancer is called the "cancer of premature sexual activity." That is why researchers should play a more important part in conquering "cancer of mothers" and preventing "cancer of early sexual life." Cancer can be promoted by many non-specific factors, including such gynecological ones as: 1. Faulty prediction and determination of birth date; 2. Instrumental instead of possible natural labor; 3. Reduction rather than prolongation of lactation; 4. Acceptance of early sexual life; 5. Wrong hormonal therapy; 6. Long-lasting prescription of pills, especially the same ones; 7. Infrequent diagnosis and incorrect therapy of hypothalmic-conditioned abortions and premature deliveries; 8. Clinical, coloposcopic, and cytologic false negative diagnosis of cervical cancer; 9. Eradication of neoplastic lesion without normalization of its near-environment; and 10. Too radical and aggressive treatment of precancerous states. For example, the unexpected diagnosis of cervical intraepithelial neoplasia (CIN) in women seeking professional help due exclusively to infertility is a major emotional trauma, additionally intensified by direct medical manipulation of the uterine cervix. Eradication of cervical pathological changes, particularly cervical conization or amputation, additionally decreases fertility. In order to minimize this jeopardy, it is possible to introduce medical treatment by immunotherapy in such cases. Cryosurgery, CO-laser therapy, and radical electrocautery are all effective in eradicating CIN, but not the cancerogenic. This local ablative therapy must be followed by medical restoration of the body's defense mechanism to prevent the recurrence of the disease. Only immunotherapy as a means of treating the whole body can alone prevent and cure the dissipathogenic states, as a final cause of cancerogenesis of any part of the body, not only the uterine cervix. Neoplasms are biological dissipative structures, and their self-organization in the body is an alternative to the death of its subcellular structures, cells, and tissues whose equilibrium state has been disturbed for too long. This concept not only explains the previous difficulties in the elucidation of both the nature of oncogene-

34

Health and Toxicology

sis and the course of neoplastic diseases but also unifies in thermodynamic terms all known theories of neoplasia. It demonstrates their compatibility with our present knowledge of neogenesis as well as with the description of prerequisites for carcinogenesis put forward by sciences such as physics, chemistry, biology, medicine, sociology, and ecology. For many scientists, such terms as "cancer," "neoplasm," and "tumor" seem interchangeable and carry the same meaning as for the general public. Similarly, for many years physicists were not able to determine unmistakably states far-fromequilibrium as proved by the history of final development of the second principle of thermodynamics by Prigogine. The principle has allowed us to comprehend the origin of so-called dissipative structures as self-organizing formations in states farfrom-equilibrium. The equilibrium state among all systems ensures proper functioning of the body as a unit. This is known to biologists as physiological equilibrium. One sees readily how difficult it is not only to delineate borders between these structures within the organism, but even to imagine them since collectively they constitute one entity, being a higher biological dynamic-spatial organization. Similarly, any of these structures consists of basic microsystems, which are subcellular formations. In turn, each fundamental microsystem is made up of atoms and molecules which together demonstrate biological properties and, like any system, it has characteristic internal features and proficiency of external work. As existence itself, the body's growth and development depends on the equilibrium between particular systems. Therefore, each system, including individual cells, is affected by the balance between its component microsystems. From a thermodynamic point of view, all systems ranging from a fundamental microsystem to a social community—i.e., a system consisting of human beings— are subject to the principles of thermodynamics. Their interrelationships may attain the following three states: equilibrium, near-equilibrium, or far-from-equilibrium. The concept of equilibrium is a state of dynamic balance in which two or more opposing processes simultaneously cancel each other's effects. This state is also known as physiological equilibrium, dynamic equilibrium or simply, steady state. The claim, whereby equilibrium structures (systems) remain stable and resistant to minor disturbances, is fundamental for our line of reasoning. Also, near-equilibrium systems spontaneously tend to attain equilibrium, while the formation of a new structure is not possible. In the two cases, both the entire organism and its elementary microsystems are characterized by the same feature, i.e., they must exist themselves and be capable of performing work directed outside for the benefit of their environment. In equilibrium (health) or near-equilibrium (any disease except cancer), the system remains capable to perform outside work and to respond to the state and requirements of its milieu. If the system attains a state far from equilibrium with the environment, then its capability of external work becomes, for various reasons, limited or virtually nonexistent. The very survival of the system and the preservation of its internal fea-

Environmental Risk Factors on Cancer and Their Primary Prevention

35

tures are of greater importance. In persistent far-from-equilibrium states, the system is either destroyed or it may evolve into another formation. This is known as a dissipative structure. To exist, it takes from the environment excessive amounts of energy and matter. Cancer constitutes such a biological dissipative structure. Any disease, to a greater or lesser extent, brings the organism to the bifurcation point at the thermodynamic branch of life, which is individually determined for each man. That branch is the finite set of states in which a given human lives and functions. Beyond that point is death, or a violent attainment of thermodynamic balance with the environment, i.e., achieving the same temperature, humidity, or partial pressure of mass as the world. Apart from death, far-from-equilibrium states may lead to a spontaneous organization of dissipative structures. Man as an entity may in this manner change only his personality if the state concerns exclusively psychic and/or emotional processes. A body part which is far from equilibrium in a dissipathogenic state (the bifucation point at the thermodynamic branch) may survive by means of either returning to near-equilibrium or equilibrium, or self-organization into a neoplasm (cancer). Otherwise it must perish (death). Cancer is firstly a local process and it may be restricted or destroyed by a still-efficient regulatory and defense mechanism of the body. In other cases it becomes a generalized disease, and only this stage of its development is commonly known, affecting our idea of neoplasms. Starting at dissipative subcellular structures, the organism is able to suppress any threat to its internal equilibrium. This happens continuously in our body and is thus one of the significant inherent features of life. The precancerous states evolve into cancer more frequently than unaltered tissue. One cannot claim, however, that the currently observed precancerous lesion will in fact develop into cancer. Currently such conditions may be observed in situ, without their removal from the body employing magnetic resonance of atomic nuclei. The conditions are known as dissipathogenic changes. If the organism as a unit does not efficaciously respond to the most primary, though universal signal, manifested as a locally increased requirement for dissipation of matter and energy, the tumor will continue to develop. Its development may still be arrested. Not infrequently, cancer may remain at this stage of development for several years without giviiig rise to any symptoms and complaints. As it disturbs the physiological equilibrium within the body, it is not able to suppress the cancer single-handedly. Medical intervention is required and the cancer's efficacy is nearly complete at this stage. The latency period in the formation of neoplasms is well known. The concept is founded essentially upon the idea that a carcinogen need not act throughout the entire period of carcinogenesis. What we encounter here is a disorder in body homeostasis to which particular cells respond with their whole range of potential states for survival (cellular thermodynamic branch) including a neoplasm. Exposure to radiation may produce mutations in the cellular genome. In case of persistent cellular mutation, the process is irreversible.

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Health and Toxicology

On its own, however, it does not produce or incite cancer until the cell attains the dissipathogenic state, far-from-equilibrium, as the ultimate cause of carcinogenesis. The course of neoplastic disease depends on the reaction of the organism to the formation and development of the neoplasm as an individual biological system capable of existence, and further growth only if it remains in a state far from the internal equilibrium of the body. Accordingly, the suppression of such a state alone is sufficient to achieve a cure. The sooner the removal from the body of the neoplasm as the source supporting the state far from the equilibrium of its environment by means of taking up and dissipating matter and energy, the faster and easier the cure. The phenomena of molecular fluctuation play a decisive role in nature, which is ruled by the second principle of thermodynamics augmented by these nonlinear events. Far from equilibrium, a system assumes a statistical character, being dependent on the probability of occurrence and the extent of fluctuation. Unlike stable states, i.e., the ones that return to normal after sUght perturbations, fluctuations are not suppressed in unstable states or processes, and so they may considerably increase in an area far from equilibrium. The system may then experience various considerable changes associated with the fluctuation, which has further unsettled the unstable state of the system. The system may also attain a stable stationary state of different dynamic-spatial structure. In the latter case, dissipative structures with a higher degree of spatial and/or temporal organization are formed. They become stable stationary states of the systems whose entropy decreases while the very existence ensures irreversible excessive dissipation of matter and energy. They are part of a complex comprising an open system and its environment, together determined by the second principle of thermodynamics. STAGES OF CARCINOGENESIS Stage 1-a: Intracellular Dissipative Structures When subcellular dissipative structures evolve within a cell, there is an internal cause of neoplastic transformation. Survival may give rise to a tumor as a clone of a single cell. In unicellular organisms, such alterations are known as mutations. Cancer is not observed in these organisms as it requires a multicellular system to thrive. Tissues require cell transformation to increase their chances of surviving those cells capable of self-organization or immortal cells. Stage 2-b: Dysplasia Differentiation of the daughter cells may be incomplete, attaining merely the G state. Accumulation of such cells in the tissue produces a disturbance, mostly within the tissue structure manifested as a varying degree of dysplasia. Simultaneous neoplastic transformation of distant cells occurs when dissipathogenic conditions coexist due to cellular transformation by external forces. The ulti-

Environmental Risk Factors on Cancer and Their Primary Prevention

37

mate picture in Stage 2 is similar, corresponding to the concept of tissue dysplasia in contrast to dysplastic changes affecting a single cell. Stage 3-c: Neoplastic Tissue (Tumor) In this stage, the tissue (tumor) is made of transformed cells separated from the rest of the body by systemic barriers. The process involves the formation of extracellular substances as well as new vascular and neural networks. Unlike changes in the first two stages, at this level of organization cancer persists in 80 to 90% of developed dissipative structures. Stage 3-d: The Stochastic Phenotype of a Cancer Cell A cell, being an open system, comprises a number of states that make up its thermodynamic branch ranging from thermodynamic equilibrium with the body environment to a far-from-equilibrium state. Beyond the bifurcation point, it may exist only in its new cancerous form. The most likely state in given conditions starts a new thermodynamic branch of a newly formed cell. This is the essential difference between a neoplastic cell and surrounding cells in its environment, although in its structure the former may resemble the latter to a varying degree. A cell's dissipathogenic condition does not constitute a neoplasm but corresponds to the bifurcation point at its thermodynamic branch. A cell within this thermodynamic branch but in a case of dissipathogenic state may return to equilibrium, perish (death), or change into a potential dissipative structure, thus starting an entirely new thermodynamic branch. The last possibility demonstrates that a neoplasm (cancer) is directly in line with a biological system finding itself in a dissipathogenic condition. When the boundary of stability found at a certain critical distance from the state of equilibrium is crossed, any open system may, and under certain circumstances must, become a new dissipative system. In its earliest stage, cancer represents an additional and easily received universal signal to the remaining organism by scattering matter and energy. More specialized means of contact, including via highly differentiated hormones, is possible though less commonly and efficiendy decoded. It involves mobilizing the entire organism to suppress dissipathogenic conditions when cancer formation preserves a state far from the thermodynamic equilibrium in a tissue or organ. A system whose birth, growth, or development has been affected by unfavorable disorders is infrequently capable of removal, suppression, or repair by means of natural homeostatic mechanisms. Such disorders either facilitate or actually lead to deviations from a state of physiological equilibrium and thus promulgate initiation of dissipathogenic conditions. The evolving neoplasm further accelerates unfavorable body entropy and precipitates death of the entire organism, acting therefore, according to the principle of suppression of evolutionarily adverse intrasystemic changes, by means of destroying the organism.

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Initiation is the self-organization of biological subcellular structures, cells, and tissues into a new dissipative structure. As shown above, this is a long-term process. The onset of initiation facilitates processes of reversal or suppression. The final effect depends on the preservation of the dissipathogenic condition that induces carcinogenesis. Induction means above all the ability to alter the state of a system by changing the fields in which the system is found. The dissipathogenic condition is produced and determined by many factors. Further removal from the initial state of equilibrium may only have two alternative outcomes, (1) destruction, or death, and (2) self-organization, or cancer. Initiation of the latter process is effected through induction at the end of the present thermodynamic branch of the system. Promotion means aiding cancer to preserve in its environment the conditions of the far-from-equilibrium state at the start of its own new thermodynamic branch. Even natural hormones, which support tissue growth, or hyperplasia, may act as promoters. Certain tumors grow as long as there is such hormonal stimulation. Others disappear and are known as hormonally conditioned, but throughout a certain point growth ceases to be affected by hormones without perceptible cellular changes. Due to a different history, i.e., different origin and course of progression, any neoplasm is a separate biological entity unlike other neoplasms. In thermodynamic terms, progression refers to that stage in neoplasm development when acting as an independent biological system it may, unaided, remain in a state far from the dynamic equilibrium of its biological environment, such as whole or part of the organism. The rate at which its cells grow and differentiate is affected by environment and the cellular division cycle at the site where changes develop. There are two extreme situations in the cell cycle. A rapid growth of undifferentiated cells, and slow growth of well-differentiated cells. The points of growth arrested by promotion factors remain a challenge. In the latter instance, however, initiating damage merely affects the capability of terminal differentiation, while promotion begins to operate at the point of growth and differentiation integration, thus covering all points of cell self-regulation. Cancer seems to be a regulator of the lifespan of individual organisms. It is a mechanism involved in species survival, which includes the elimination of organisms poorly adjusted to prevailing environmental conditions. That final phase of its action is seen as neoplastic disease with a full range of consequences. A neoplasm as such is the first important signal informing the body of the necessity of self-defense at a purely biological level, versus so-called "apoptosis" (programmed death of body). Powerful protective and repair mechanisms are active in the organism at the subcellular level, cells, tissues, organs, and systems (immune, nervous, endocrine and humoral). Such structures, by means of additional dissipation of matter and energy, automatically become a warning signal launching distant mechanisms of defense and

Environmental Risk Factors on Cancer and Their Primary Prevention

39

repair. In a multicellular organism, structures are continually killed, or destroyed and subsequently removed at subcellular and cellular levels, precisely to maintain the life of tissues and hence the whole body. Thousands of cells perish every day in our body, including newly developed neoplastic cells. Additionally, the very formation of dissipative structures in cell organelles leads to their destruction together with the cells in which they are contained, so that the latter do not manage to evolve into overtly cancerous cells. Thus, through neoplasia nature hastens the death of our abnormal cells, allowing us to live longer. We can benefit from this generous stage in the biology of neoplasia, mainly, by avoiding that which weakens the body's defense. The cigarette smoker, alcohol or drug abuser, the unhygienic, etc., will respond poorly to these first neoplastic warning signals. Between the ages of 20 and 65, the incidence of cancer rises a hundred-fold, but the same increase in human mortality is observed a decade earlier, meaning that with age the body's powers of self-defense decrease. Intentional psychic attitude is another major form of self-defense against neoplasms. Knowledge of the cancer's essence frees one from the fear of its mysterious character, incurability, and possibilities of contagiousness and inheritance, offering a unique chance of purely human self-help when the neoplasm has already developed. Before a neoplasm defeats the body's defensive mechanisms, it has been restricted to an area long enough to be diagnosed and removed, although it might be subjectively asymptomatic. This is the task for health education, properly trained medical and nursing personnel, well-equipped health facilities, and rehabilitation centers. Social community self-defense is of utmost importance for controlling cancer. Improvement and widespread introduction of appropriate diagnostic methods of early stages in biological development of neoplasms should be instituted. The most important thing is to eradicate the disease prior to the beginning of pathological symptoms and signs. It is necessary to continue research into the body's natural defense mechanism and to pursue more recent modalities of therapy, such as: interferon, monoclonal antibodies, immunochemotherapy, and computerized techniques of hormone therapy. The community must protect the natural and indoor environment, limiting the conditions that favor the formation and development of social evils such as dependence on alcohol and drugs, tobacco smoking, industrial pollution, undernutrition, etc. In addition, it is necessary to set up community facilities to provide adequate care to people suffering from neoplastic diseases, with stress on emotional, as well as physical support. Review of environmental risk factors of neoplasms could be a starting point for primary prevention focused on elimination of related health hazards at the sources, as well as on fortification of diet with protective additives, etc.

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APPENDIX 1: CANCER RISK FACTORS AND PREVENTION Causes and Secondary Factors

Time Scale

Genetic predisposition or susceptibility Exposure to mutagens; malnutrition

Years before birth Conception and pregnancy

Prevention

Genetic counseling; Diet adequate in free radical scavengers Perinatal examination; minimalization of exposure; Supplementation of nutrition with protective factors—e.g., antioxidants, radioprotectors, free-radical scavengers, etc.

Childhood UV-light; Carcinogens; Virus infection Lack of hygiene; Smoking, drugs. Alcoholism

Prenatal supervision; Education Adolescence Adulthood Old age

Improvement of living standard; Regular self-inspection; Early treatment; No smoking; Proper diet; Regular medical examinations (including NMR use) Avoidance of fats, sugar, excessive meat, alcohol, etc.

After G. N. Schrauzer, [38] modified

Active prevention involves the cooperation of scientists with persons dealing with education, agriculture, farming, the food industry, and various areas of technology (including civil engineering technology to protect the indoor environment). This refers to the elimination of potential risk factors from the whole human environment and the optimization of the composition of drinking water and food. In these activities everyone can participate as a consumer. The particular role, however, belongs to engineers, farmers, and decision makers. These professional groups have the strongest influence on the natural environment. Thus, as far as the elimination of the sources of contamination with carcinogens is concerned, their significance can be greater than the one of physicians or biologists, although biomedical studies are basic, both in relation to motivation as well as verification of effectiveness of common action for the elimination of environmental cancer risk factors. These activities are the main reasons for the management of environ-

Environmental Risk Factors on Cancer and Their Primary Prevention

41

ments suitable for healthy work, living conditions and rest, the promotion of environmentally friendly technologies of production, transport, and consumption models (including diet). Knowing synergism of many environmental risk factors of cancer, one must conclude the need of integrated action for the improvement of the whole human environment. This refers both to individual decisions (modifying habits, e.g., giving up smoking, modifying diet regarding increase of the consumption of pollutant-free fruit and vegetables rich in antioxidants, fiber, whole complex of essential elements, vitamins, etc.) as well as the cooperation of institutions on local and global scale. Apart from this action, there is prevention related to environmental carcinogenesis including chemical, radioactive, biological, and other contamination. Both primary and secondary sources of this contamination should be considered in land reclamation, proper management of heavily contaminated areas, and the monitoring of food on a large scale. Another direction is the fortification of soils and diet with protective substances that diminish the input (permeability) of carcinogens into the food chain and human organisms. One more area of primary prevention is creating conditions for proper interpersonal relationships, because psychological factors play an important role in the distortions of neurohormonal function and lowering the effectiveness of some protective mechanisms against carcinogenic process [9]. The interference of physical carcinogenic factors with the state of immunosuppression and the distortion of homeostasis in the organism can be regarded as frequent circumstances increasing the risk of the appearance of cancer symptoms. This can be confirmed in the contemporary interpretation of a carcinogenic process as a whole-organism phenomenon seen from the point of view of information theory [6] and thermodynamics [31, 32]. Cell-type specific gene mutation could be induced by some pollutants, e.g., 7,12-dimethylbenz (a) anthracene (DMBA). The type of tumors/transformed cells depends on the mode of administration of carcinogens in experimental mice, including transplacental exposure [36]. There are also some data about the interaction of genotoxic and non-genotoxic mechanisms in multistage carcinogenesis [39]. In addition to the accumulation of genetic changes during this process, some non-genotoxic effects could indirectly generate genetic changes, e.g., by active oxygen species, cytosine methylation, and stimulation of mitosis [39]. Discovery of the inhibition of gap junctional intercellular communication by different tumor-promoting agents is a new field of study about the contribution of non-genetic changes in tumor progression [34]. Disruption of feedback system and homeostatic properties seems to be related to disturbances in the exchange of information between some elements involved in spatial-temporal order within integrated biological systems from cellular to organismal level of organization [5]. Carcinogenesis can be interpreted as the result of so-called "environmental noises" that can induce as very important mutations in genetic code, but also disturb the transfer of information necessary for the synthesis of both structural

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elements and enzymes, neurotransmitters, and hormones [8, 32, 33]. Changes appearing in single cells can be interpreted as the appearance of biological dissipative structures. However, when their range transgresses certain threshold value, irreversible pathological changes including possible death can take place. This happens with the transgression of a certain value of the distortion of information transfer and disturbance of bioenergetic processes, which causes the increase of entropy in the system [32, 33]. Closer discussion of both theoretical premises for this interpretation and some examples of interdisciplinary case studies could be achieved with more active participation of experts from different disciplines focused on primary prevention of cancer. There is a certain analogy from the biocybernetic point of view, between the integration of different levels of structure and function of human organism starting from an individual cell [6], primary prevention of cancer starting from the level of ecosystem, and ending on the level of biosphere [4]. For the effective prophylaxis it seems to be necessary to integrate individual and common efforts including both sphere of social and technical activity for an environmentally friendly model of civilization. APPENDIX 2 PAPER BY DOBROWOLSKI AND KLIMEK ANNEX

Model of the influence of external and internal risk factors for leukemia development Output (leukemic effect)

Input (risk factors)

innmunological j reactivity

Xl

U2

genetic factors

X2

U3

oncorna vinjses

X3

-•y

Basic data for mathematical approach to environmental leukemogenesis. (After Kwiatkowski/from Aleksandrowicz et al.)

Environmental Risk Factors on Cancer and Their Primary Prevention

43

In Vitro Assay Template Correct Substrates: dATP + [a-32p] dTTP Incorrect Substrate:[3H] dGTP DNA Polymerase Mg2+ - Exogenous Agents (Metal Cations)

Known Carcinogens and/or Mutagens Ag Be Cd Co Cr Cu Mn Ni Pb As* Se* *Not Tested

Increased Misincorporation

No Change in Fidelity

Ag Be Cd Co Cr Cu Mn Ni Pb

Al Ba Ca Fe K Rb Mg Na Sr Zn

Fidelity assay screen for mutagens and/or carcinogens. (After Loeb et al., from Schrauzer, 1978 [37])

(After M. and R. Klimek gestational calculator, from Klimek, 1992 [33])

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REFERENCES 1. Aleksandrowicz, J. and Czyzewska-Wazewska, M., "Selenium as radioprotective substance," Haematologica, 17: 61-62, 1976. 2. Aleksandrowicz J., Skotnicki, A. B. et al.. Leukemia ecology, Natl. Library Med., Natl. Sci. Foundation, Washington, D.C., NCSTEI, Warsaw 1982. 3. Amaya, K., "Report on cancer origin," International Conference on Primary Prevention, Krakow, 1995. 4. Badiello, R., Trenta, A., Matti, M., Marettini, M., "Azione radioprotective dei seleno-derivati. Affesto della selenourea in v/vo," Med. Nucleare Radiobiol Latina, 10: 1-12, 1967. 5. Dobrowolski, J., "Studies on photoreactivation," Acta Biol Cracoviensa, ser. zool. 18:211-220, 1975. 6. Dobrowolski J. W., "New aspects of environmental protection against developmental malformations and the cancer incidents," Scientists for better environment, pp. 517-527, Fukushima, Y. et al., eds., Sci. Council of Japan, HESQ Asahi Press, Tokyo, 1976. 7. Dobrowolski, J. W., Smyk, B., "Environmental risk factors of cancer and their primary prevention,"/. Env. Pathol. Toxicol. Oncol, 12(1): 55-57, 1993. 8. Dobrowolski, J. W., Tadeusiewicz, R., "Interpretation of cancerogenesis from the point of view of information theory," (in Polish), Gin. Pol, 57(12): 111-111, 1986. 9. Dobrowolski, J. W., Vohora, S. B., eds., "Ecologism in health protection," (in Polish), Ossolineum, Wroceaw, Krakow, 1989. 10. Dobrowolski-unpublished 11. Ferenczi, L. Z., Hill, G. B., Scrimger, J. W., "Ultraviolet radiation and the incidence of cancer of the skin in Alberta," Medecine/Biologie/Environment, September-December, pp. 48-53, 1982. 12. Gupta, D., WoUmann, H. A., Fedor-Freybergh, P. G., eds., Pathofisiology ofimmune-neuroendocrine communication circuit.. Mattes Verlag, Heidelberg, 1994. 13. Harada, M., "Minamata disease," Report, Kumamoto University, 1976. 14. Heinrich, U., "Carcinogenic effects of solid particles," Toxic and carcinogenic effects of solid particles in the respiratory tract, Mohr U. et al., eds., pp. 69-73 ILSI Press, Washington, D.C., 1994. 15. Heinrich, U. et al., "The carcinogenic effects of carbon black particles and tar-pitch concentration aerosol after inhalation exposure of rats." Ann. occup. Hig. (38) suppl. 1: 351-356, 1994. 16.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 4, 1974. 17. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 21, 1979. 18. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 34, 1984. 19. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 37, 1985.

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20. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 38, 1986. ll.IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 40, 1986. 22. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 41, 1986. 23.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 42, 1987. 24. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 43, 1988. 25. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 44, 1988. 26. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 45, 1989. 27. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 46, 1989. 28. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 47, 1989. 29. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 49, 1990. 30. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 51, 1991. 31. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 52, 1991. 32. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 53, 1991. 33.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 55, 1992. 34. lARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 56, 1993. 35.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans, WHO International Agency for Research on Cancer, vol. 58, 1993. 36. Janczarski, I. Personal communication, 1980. 37. Janicki, K., Dobrowolski, J. W., Krashicki, K., "Correlation between contamination of the rural environment with mercury and occurrence of leukemia in men and cattle," Chemosphere 16: 253-257, 1987. 38. Jansson, N. K., "Topographical study in cancer in Denmark," Ninth International Cancer Congress Proceedings, Abstract 1017, Tokyo, 1966. 39. Kacki, E., Stempczynska, J., "Correlational research into the environmental etiology of cancer." pp. 607-611, European Centre for Pollution Research, London, 1993. 40. Karaczkiewicz, M., "Remarks concerning the geographical distribution of bovine leukemia in Poland," Przegl Let, 11: 391-394, 1970. 42. Klimek, R., "Cause, Predisposing Factors and Host-defense," (in Polish, English summary), PWN, Warszawa, 1985.

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43. Klimek, R., Peri-natal Psycho-Medicine, DReAM, Cracow 1992. 44. Kozik, R., Personal communication, 1975. 45. Krasnicki, K., Personal communication, 1986. 46. Krutovskikh, V. A. et al., "Inhibition of rat liver gap junction intercellular communication by tumor-promoting agents in vivo. Association with Aberrant Localization of Connexin Proteins," Laboratory Investigation, 72 (5), 571-577, 1995. 47. Marchlewski, Necki. 48. Niwelinski, J., Zamorska, L. "Report on contamination of human placenta and cytochemical changes of some enzymes activity," International Conference on Elements in Health and Disease, Adana, 1990. 49. NNAS, RNAE, CIOM, Indoor pollutants, National Academy Press, Washington, D.C., 1981. 50. Otto, H., "Vergleich der Krankheltsblider der menschlichen und tierischen leukosen, identische und differente symptoma," Z Arztl. Fortbil, 57: 461, 1963. 51. Samal, J. et al.. Report on environmental risk factors of cancer at International Conference on Ecology and Cancer, Brussels, 1982. 52. Sasaki, K. et al., "Cell-type-specific rat mutations but no microsatellite instability in chemically induced mouse skin tumors and transformed 3T3 cells," Cancer Res. 55, 3,513-3,516, 1995. 53. Schrauzer, G. N., ed., "Inorganic and nutritional aspects oicdLWcox'' Advances in experimental medicine and biology. Plenum Press, New York, London, 1978. 54. Schrauzer, G. N., "Trace elements in cancer diagnosis and therapy. A review," Trace Element-Analytical Chemistry in Medicine and Biology, Vol. 4, P. Bratter, P. Schramel, eds., pp. 403^17, Walter de Gruyter Co., Berlin, New York, 1987. 55. Shamberger, R. J., "Relationship of selenium to cancer. Inhibitory effects of selenium on cancerogenesis," Journal of National Cancer Institution., 44: 931-936, 1970. 56. Smyk, B., Aleksandrowicz, J., "The association of neoplastic diseases and mycotoxins in the environment," Tx. Rep. Biol. Med., 31: 715-726, 1973. 57. Tomassi, J., Personal communication, 1977. 58. Truhaut, R., Dobrowolski, J. W., "Ecotoxicology and primary prevention," Introduction to the Human Environment, Team textbook, J. W. Dobrowolski, B. Bhatia, D. Banerjee, New Delhi Press. 59. Vincente, de R., Personal communication, 1986. 60. Vohora, S. B., Dobrowolski, J. W., eds.. New horizons of health aspects of elements, Hamdard University, New Delhi, 1990. 61. Yamasaki, H., "Non-genotoxic mechanisms of carcinogenesis: studies of cell transformation and gap junctional intercellular communication," Toxicology Letters 77:55-61, 1995.

CHAPTER 2 RESPIRATORY FUNCTION CHANGES FROM INHALATION OF POLLUTED AIR Shieh-Ching Yang and Sze-Piao Yang Pulmonary Function Laboratory National Taiwan University Hospital No. 7, Chung-Shan S. Rd. Taipei, Taiwan, Repubhc of China

CONTENTS FORMS OF AIR POLLUTANTS, 47 Effects of SO2 and NO2 on Lung Function, 48 URBAN CONCENTRATIONS OF AIR POLLUTANTS, 48 PULMONARY FUNCTION PARAMETERS, 49 BRONCHIAL PROVOCATION TEST, 50 PULMONARY EFFECTS OF AIR POLLUTANTS IN ASTHMATICS, 51 CONCLUSION, 51 REFERENCES, 51 Because of civilization and increased industrial activities, the effects of air pollution on health and diseases have attracted much attention in recent years. Air pollution means a state resulting from manmade wastes produced so rapidly that they accumulate in concentrations that cannot be dispersed by the normal selfcleansing propensities of the atmosphere. Air pollution is thought to be one of the most important risk factors for respiratory diseases, particularly for bronchial asthma and chronic obstructive pulmonary disease (COPD). However, a direct causal relationship is not easy to prove because air pollutants do not occur as individual entities but in combination. In addition, the concentration and duration of exposure to air pollutants required for inducing an adverse pulmonary effect have not yet been determined. FORMS OF AIR POLLUTANTS Components of air pollution are usually inhomogeneous and may include suspended particulates, metallic contaminants (lead), chemicals, and irritating or toxic gases such as sulfur dioxide (SO2), nitrogen dioxide (NO2), ozone and carbon monoxide (CO). Particulate pollutants may appear in the forms of fog, smoke, or dust, according to their size and physical properties. Small-sized particles (less than 1 ^m) have catalytic effects on other air pollutants, particularly the oxidation of SO2, so as to produce irritation to the lower respiratory tract. Dust, like carbon or silica, are large particles that can produce pneumoconiosis if heavily exposed. 47

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Polycyclic aromatic hydrocarbons (PAHs) are chemicals generated mainly by automobile fuel combustion, and they are well-known carcinogens. Effects of SO2 and NO2 on Lung Function The adverse effects of SO2 and NO2 on respiratory function have been investigated. Inhalation of high concentrations of SO2 (4-6 ppm) induced bronchoconstriction and caused an acute reduction in maximal expiratory flow (MEF) in normal subjects. This response can be blocked by treatment with atropine, suggesting that it is mediated by parasympathetic reflex pathways. Sulfur dioxide itself appears to cause only mildly irritating effects. The oxidation of SO2 leads to a much more prominent injurious effect in the small airways. At higher concentrations (i.e., 10-15 ppm), SO2 can inhibit mucociliary clearance of the respiratory tract. Such a concentration, however, is 300-fold higher than those observed in an urban polluted atmosphere. In a clinical setting, it was reported that there was a threefold increase in acute asthmatic episodes of adults on days characterized by high, compared to low, sulfate content in the residential environment. A significant change in pulmonary function of COPD patients after exposure for 2 hr to 0.8 ppm or less of SO2 was also found. Studies showed that asthmatic patients were sensitive to SO2 at concentrations as low as 0.5-1 ppm. Therefore, persons with bronchial hyper-reactivity are more likely to develop bronchoconstriction as a result of inhaling concentrations of SO2 that are well tolerated by normal subjects. Nitrogen oxides are generated mainly by power plants and automobile fuel combustion. The effects of NO2 on pulmonary function is incompletely understood. Nitrogen dioxide in high doses (40 ppm) is a prominent airway irritant. Results of studies on the effects of exposure to low levels ( 0 . 1 ^ ppm) of NO2, however, have been controversial. It was found that exposure to 4 ppm NO2 for 75 min., accompanied by intermittent exercise, had no effect on pulmonary function. On the contrary, exposure of asthmatics to only 0.1 ppm NO2 for 1 hr resulted in increased airway reactivity. Increased airway reactivity to carbachol challenge in normal humans was also observed after exposure to 1.5 ppm NO2 for 3 hrs. Pulmonary epithelial injury associated with mast cell influx and/or mediator release were probably the underlying mechanisms. Moreover, increased capillary permeability might occur, which leads to focal pulmonary edema. URBAN CONCENTRATIONS OF AIR POLLUTANTS The polluted air is collected for a specific site within the city and compressed into 40-L sample bags. The concentrations of air pollutants in the sample are determined by various methods. For example, total suspended particulates (TSP) and size-fractionated particulate samples are collected by using high-volume samplers equipped with acid-washed quartz-fiber filters. Particle-size can be viewed directly

Respiratory Function Changes from Inhalation of Polluted Air

49

with a scanning electron microscope. Analysis of atmospheric SO2 is performed with the colorimetric method. The NO2 concentration can be measured with a spectrophotometer and a midget-fritted bubbler used with the absorbing reagent. The reagent is a mixture of sulfanilic acid, N-(l-naphthyl)-ethylenediamine dihydrochloride, and acetic acid. The carbon monoxide (CO) concentration is usually determined with an infrared CO analyzer or a gas chromatograph. Urban concentrations of SO2 and NO2 are usually in the range of 0-1,000 ppb and 100-1,000 ppb, respectively. The concentrations of air pollutants may be influenced by time and site of sample collection, and by meteorologic factors such as humidity, wind velocity, temperature, and barometric pressure. Tunnel air collected during traffic jams in rush hours has much higher levels of pollutants than the average urban air. Typical air pollutant concentrations of a sample collected from a large city are shown in Table 1. Table 1 Components of air pollution and their concentrations* Pollutant

Concentration

S02,ppb 124.0 NO2, ppb 502.0 CO, ppm 3.6 TSP, [ig/M^ 227.0 ^Sample collected from Lin-Shan South Road Tunnel of Taipei, Taiwan, in August, 1993. PULMONARY FUNCTION PARAMETERS More than fifty parameters are concerned in a complete pulmonary function testing. For epidemiologic studies and for determining the adverse effects of air pollutants on respiratory function, however, it is possible to employ less than 10 parameters. Of these, some are volume parameters, e.g., forced vital capacity (FVC) and functional residual capacity (FRC); some are flow parameters, e.g., forced expiratory volume in one second (FEVl) and peak expiratory flow rate (PEFR); the remaining may be a resistance parameter (airway resistance. Raw) or a diffusion parameter (diffusing capacity of the lung for carbon monoxide, DLCO). Most flow parameters and some of the volume parameters, such as FVC, can be simply determined with a maximal expiratory flow volume maneuver. An automated spirometer is often used for this purpose. The instrument requires periodical calibrations, and its accuracy for volume and flow measurements should be within ±3-5%, or less than 150 ml. Determination of FRC and Raw requires an integrated multifunctional lung function analyzer or a body-plethysmograph. Helium, nitrogen, and carbon monoxide are used as the reference or test gases.

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The standard procedures for a flow-volume determination are described briefly as follows: Subjects are seated and wear nose-clips. Once adjusted to breathing through the mouthpiece, the subject exhales to residual volume (RV) and then inspires to total lung capacity (TLC), and then rapidly expires to RV again with maximal effort. Time-volume and flow-volume curves are recorded simultaneously. Up to three trials are performed, and the average of two technically acceptable tests is reported. The tests have to agree within 5% of each other to be considered acceptable. BRONCHIAL PROVOCATION TEST The assessment of airway hyper-reactivity is an increasingly popular but difficult task for a pulmonary function laboratory. The test is often called a "challenge" and uses several kinds of stimulants such as methacholine, histamine, carbachol, cold exposure, and specific antigens like pollens and distilled water, to provoke airway changes. A number of techniques exist for using each stimulant, but no agreement has been reached on which stimulant or technique best evaluates bronchial hyperreactivity. The major indications for a bronchial challenge are to diagnose asthma and document the severity of bronchial hyper-reactivity. Methacholine and histamine are the two most commonly used stimulants for a bronchial provocation test. The results of challenges with methacholine and histamine have good correlation and reproducibility. It is generally agreed that equal concentrations of methacholine and histamine produce similar results in asthmatics. However, histamine is associated with more side effects, e.g., flushing, throat irritation, and headache, especially at higher doses. Consequently, methacholine has become the agent of choice in clinical practice. Response to bronchial provocation tests is measured as change or lack of change in pulmonary function. Simple spirometry produces FEVl; the body plethysmograph produces Raw and specific conductance by panting or quiet breathing. Although it is recommended that FEVl be included in all bronchial challenge tests, FEVl may not be the most sensitive measurement. Furthermore, repeated expiratory efforts during simple spirometry can cause bronchoconstriction. Apparently, the sensitivities among various lung function parameters to detect a change after bronchial challenge are unequal. Airway resistance and specific conductance are more sensitive parameters than FEVl. This is why, in some cases, Raw and specific conductance are measured with a body box or newer devices. The "Astograph" is a new tidal breathing method for the bronchial provocation test. Bronchial hyper-reactivity is examined by directly writing the dose-response curve of respiratory resistance (Rrs) during the continuous inhalation of methacholine in stepwise incremental concentrations. Respiratory resistance is measured by the forced oscillation method. The concentrations of methacholine tested are from 0.01 to 25 mg/ml. The instrumentation also consists of 12 nebulizers capable of generating aerosols with a particle size of less than 5 |Lim. Aerosols are delivered

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from each nebulizer for 1 min in sequence and inhaled by the subject until Rrs reach twice the baseline values. In that case, challenge is terminated and a bronchodilator is administered immediately. The test results are thereby considered positive. The cumulative dose of methacholine at the point where Rrs starts to increase prominently is calculated and expressed as methacholine units, i.e., 1 unit equals 1 min inhalation of 1 mg/ml methacholine. This oscillation technique is characterized by its advantage to proceed the challenge during quiet tidal breathing and to complete the test during a short period of time. The cumulative dose of methacholine instead of PD20FEV1 (the provocative dose that causes a 20% fall in FEVl) is calculated. PULMONARY EFFECTS OF AIR POLLUTANTS IN ASTHMATICS Many pertinent studies concerning the pulmonary effects in asthmatic subjects after acute inhalation of low concentrations of air pollutants had been done in the 1980-1990s. It is difficult to make direct comparisons among these studies because of differences in clinical characteristics of the subjects, type and concentrations of pollutants, exposure time and conditions, and lung function measured. Polluted tunnel air is inhomogeneous and has higher component concentrations than that of ambient air. In addition, subjects with a hypersensitive airway may exhibit an exaggerated response to short-term exposure of such low levels of air pollutants. Moreover, bronchial challenge aggravates the pulmonary functional response to air pollutants. It is not a surprise, therefore, that by using polluted tunnel air and bronchial provocation with methacholine, it is possible to demonstrate directly the adverse effects of polluted urban air to subjects with airway hypersensitivity. CONCLUSION Although a cross-sectional study using short-term exposures to low levels of air pollutants may be insufficient to cause a fall in lung function of the general population, exposure to polluted tunnel air may trigger respiratory symptoms in certain subjects such as those who have underlying bronchial hyper-reactivity while riding motorcycles. Careful selection of subjects and lung function parameters are essential to a successful demonstration of the unfavorable effects of polluted air. REFERENCES Brunekreef, B., Kinney, P. L., Ware, J. H., et al. "Sensitive subgroups and normal variation in pulmonary function." Health Perspect., 1991; 90:189-93. Chai, H., Farr, R. S., Froehlich, L. A., et al. "Standardization of bronchial inhalation procedures." /. Allergy Clin. Immunol^ 1975; 56:323-27.

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Frampton, M. W., Morrow, P. E., Cox, C , Gibb, F. R., Speers, D. M., Utell, M. J. "Effects of nitrogen dioxide exposure on pulmonary function and airway reactivity in normal humans." Am. Rev. Respir. Dis., 1991; 143:522-27. Koenig, J. Q., Pierson, W. E., Horike, M. "The effects of inhaled sulfuric acid on pulmonary function in adolescent asthmatics." Am. Rev. Respir. Dis., 1983; 128:221-25. Koenig, J. Q., Covert, D. S., Marshall, S. G., Belle, G. V., Pierson, W. E. "The effects of ozone and nitrogen dioxide on pulmonary function in healthy and asthmatic adolescents." Am. Rev. Respir. Dis., 1987; 136:1,152-1,157. Linn, W. S., Solomon, L. C , Trim, S. C , et al. "Effects of exposure to 4 ppm nitrogen dioxide in healthy and asthmatic volunteers." Arch. Environ. Health, 1991; 46:296-99. Orehek, J., Massari, J. P., Gayrard, P., Grimaud, C , Charpin, J. "Effect of shortterm, low-level nitrogen dioxide exposure on bronchial sensitivity of asthmatic patients." J. Clin. Invest., 1976; 57:301-07. Takishima, T., Hida, W., Sasaki, H., Suzuki, S., Sasaki, T. "Direct-writing recorder of the dose-response curves of the airway to methacholine: clinical application." Chest, 1981;80:600-06. Xu, X. P., Dockery, D. W., Wang, L. H. "Effects of air pollution on adult pulmonary function." Arc/i. Environ. Health, 1991; 46:198-206. Yamaguchi, S., Kano, K., Shimojo, N., et al. "Risk factors in chronic obstructive pulmonary malfunction and 'chronic bronchitis' symptoms in Beijing district: a joint study between Japan and China." J. Epidemiol. Comm. Health, 1988; 43:1-6. Yang, S. C , Yang, S. P. "Respiratory function changes from inhalation of polluted dk:' Arch. Environ. Health, 1994; 49: 182-87. Zeidberg, L. D., Prindle, R. A., Landau, E. "The Nashville air pollution study: L Sulfur dioxide and bronchial asthma. A preliminary report." Am. Rev. Respir. Dis., 1961;84:489-503.

CHAPTER 3 BIOLOGICAL MARKERS OF EARLY HEALTH EFFECTS IN THE ASSESSMENT OF THE RISK OF CANCER IN PEOPLE EXPOSED TO ENVIRONMENTAL CARCINOGENS Janusz A. Indulski and Waldemar Lutz The Nofer Institute of Occupational Medicine Lodz, Poland The continuing chemicalization of human life associated with the development of civilization is responsible for a steady increase in health risk due to chemical pollution of the environment. Large quantities of industrial and household chemical wastes are deposited (often in an uncontrolled manner) in the vicinity of human habitation places. Toxic chemicals permeating the soil, water, or air pose a real threat to the people living in the polluted areas. In the majority of cases, the effects of exposure to environmental pollutants take the form of prolonged exposure to such concentrations of the pollutants that do not result in acute forms of diseases. The majority of these effects, however, lead to some biological changes in the organisms of the exposed people and often contribute to increased incidence of chronic disease. As no overt clinical manifestations are evident, the only way to detect minute pathological changes resulting from prolonged exposure to environmental chemicals is by detecting them on the cellular level, or in the cellular metabolism products present in the biological fluids available for testing. The idea of biomarkers [1,2,3,4,5] has become very useful in detecting the adverse effects of environmental pollution on the human organism and taking suitable preventive measures. The concept of a biomarker is associated with laboratory testing of physiological fluids or cells from a person exposed to toxic chemicals; the result of the test, expressed in the biochemical or cellular terms, points either to the presence or content of the toxic chemical agent, or to adverse health effects caused by the chemical agent in the human organism. The concept of the biomarkers also covers an estimate of the individual susceptibility to harmful agents [3]. By integrating the concepts of exposure, health effect, and individual susceptibility, enhanced analysis of the complex problem of health risk in a person exposed to environmental toxic factors has become possible. The measurements performed directly on the material taken from exposed people makes it possible to eliminate speculation from the estimations of the associated health risks. This has been made possible because the effects of toxic chemicals are not related to their concentrations in the polluted environment but to the quantities of the chemicals that have permeated the relevant tissues and organs, causing specified toxic effects. The concept of biomarkers also made it possible to eliminate errors resulting from separately assessing the effects of occupational and communal environment.

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as well as to the lifestyle factors, such as tobacco smoking, alcohol drinking, and dietetic habits. The concept of biomarkers tends to highlight the assessment of health condition, or possible risks to health condition, rather than the assessment of the conditions of external chemical pollution [1]. The biomarkers measure individual hazard from exposure to environmental toxicants. They are indices of abnormalities within different biological systems (at the level of the whole body or of particular organs, tissues, and cells) that may be induced by hazardous agents of various chemical or biological natures. The biomarkers make it possible to monitor the processes within the organism from the moment of toxic agent penetration to the development of clinical symptoms. Biomarkers can be compared to windows which allow us to look into a "black box" and track the fate of the toxic agent and the effects it produces in the exposed organism. It is worth pointing out, however, that the very notion of biomarker refers not only to the data on the particular laboratory test results—e.g., toxic agent concentration or enzymatic activity—but also to the fact that it may provide tentative information about the possible concentration of the agent or the enzymatic activity within those tissues which are inaccessible for testing. Biomarkers can supply measurable data on the rate of absorption of the environmental contaminant and answer whether the critical dose leading to eventual molecular, subcellular, and cellular changes has reached the critical organ. The nature of these abnormalities is determined both by the type of the toxic agent and the quantity that penetrates the target organ as well as the actual duration of exposure [6]. At present it seems that the assessment of chemical exposure of the individual people living in the specified environment is more essential than the assessment of the chemical contamination present in that environment. The assessment can be performed either by estimating the quantity of the absorbed toxic agent with the aid of exposure biomarkers, or by assessing biochemical changes occurring in the cells of the exposed organism with the aid of health effect biomarkers. In view of the fact that we cannot carry out a complete analysis of environmental chemicals entering the system or determine their toxic potential, it seems reasonable to undertake measurement of the resulting health effects in exposed individuals along with routine evaluation of exposure based on measuring the toxic agent concentration. Complicated and costly as it is, the performance of health effect assessment seems highly justified from the point of view of social and economic needs. In many a case, the excessive expenditure connected with the relocation of inhabitants of the hazardous territories can thus be avoided. This may also help to prevent panic among the population of contaminated areas. Moreover, the health effects may form the grounds for undertaking relevant preventive measures. The latter, in turn, increases the probability that the health effects of exposure, when detected early, will still be reversible and the discontinuation of exposure and the appropriate treatment will inhibit their transformation into permanent clinical effects [7]. Health effect biomarkers are indicators that provide information on the changes in the organism which took place as a result of exposure to an environmental toxic

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agent. This biomarker group includes, in a measurable way, these phenomena which constitute either the individual stages of the pathogenic process or the manifestations of the disease itself. The biological response of the organism to environmental toxic agents includes a relatively wide range of changes, from temporary and reversible (which do not produce any noticeable health effects) to permanent changes associated with increased disease incidence and mortality. This wide spectrum of changes may be arbitrarily divided into several categories: a) Metabolic disturbances which remain within the physiological limits; b) Physiological dysfunctions not accompanied by any detectable morphological changes in tissues; c) Pathological changes in tissues, but not accompanied by overt manifestations of disease; d) Clinically overt pathology. Environmental medicine and environmental toxicology are interested primarily in these biomarkers that enable the detection of changes occurring in the organism which are classified to the first three categories. These biomarkers are referred to as pre-clinical or early health effect biomarkers [3]. In evaluating the adverse effects of a chemically polluted environment on the human organism, most research concentrates on finding biomarkers of the carcinogenic process. The research refers both to the search for the exposure and the health effect biomarkers. In the latter case, particular attention is paid to the biomarkers of early stages of carcinogenesis. It is intended to find such biomarkers that would make it possible to detect those stages of carcinogenesis which occur many years in advance of clinically overt cancer forms [8]. The principal stages of the carcinogenic process have already been studied in some detail, and it is known that the process involves many stages, including both cellular genetic as well as epigenetic changes [9]. The following stages can be distinguished in the carcinogenic process: initiation, promotion, conversion, and progression. During the first (initiation) stage, DNA is damaged as a result of exposing a cell to carcinogenic agents. This change is hereditary and it is transferred to descendant cells. In the next (promotion) stage, numerous agents are involved, which do not necessarily lead to further direct changes in the genetic material of the cell, but by acting rather as gene activators and changing the phenotype of the initiated cells, cause selective and clonal cell expansion. The initiation process itself seldom leads to the formation of cancer cells when not followed by the promotion stage. On the other hand, in order for the promotion stage to occur in the process of carcinogenesis, it must be preceded by the initiation process. The promoting agents can be described as those that display no, or very little, carcinogenic activity when they occur alone. When they are, however, combined with agents causing genetic changes in the cells (initiating agents), they remarkably enhance their mutagenic properties. During the stage of the carcinogenesis conversion process, the benign forms of cancer are transformed into malignant ones. Now it seems that this stage

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requires some phenomena to occur which cause changes in DNA. Thus, both the initiation and the third stage of the carcinogenesis process require participation of chemical (or physical) agents that produce changes in DNA structure. Progression, the final stage of the carcinogenesis process, leads to the development of the clinically detectable form of cancer. Each of the above stages of the carcinogenesis process can be monitored with the aid of suitable biomarkers (Figure 1). The initiation stage, although short-lasting, can be relatively easily monitored by employing currendy available biochemical (e.g., carcinogen adducts with DNA or proteins) or cytogenetic (chromosome aberrations, sister chromatid exchange, presence of micronuclei) techniques. For example, the formation of the carcinogen/DNA adducts is detectable with the sensitivity of 1 adduct per lO'^ to 10^^ DNA nucleotides [10]. The new possibilities in detecting DNA structure changes attributable to the carcinogenesis initiation process have been opened up by the PCR (Polymerase Chain Reaction) techniques. The information supplied by the biomarkers in evaluating the process of initiation, unfortunately, plays a limited role in cancer prevention. It demonstrates that carcinogenes genotoxically affect the cells of the exposed people in the chemically polluted environment, but because the initiation process is very short-lasting and practically irreversible, detection of the initiation process does not offer any opportunity for effective intervention [11]. The biomarkers of early health effects are essential in the prevention of the neoplastic disease induced by environmental carcinogenic agents. They provide information supplementary to that obtained by using the exposure biomarkers. From the data obtained by assessment of these biomarkers, it can be concluded whether the exposure to environmental carcinogenic factors has triggered cellular mechanisms capable of causing neoplastic transformation. The biomarkers of early health effects represent the intermediate stages of the carcinogenesis process, between the initiation and the conversion, and the clinically overt stages. Thus, the cellular process capable of being indicated by the biomarkers of early health effects corresponds to the promotion stage. Detecting those processes is extremely important, as the promotion process lasts many years and seems to be reversible. Therefore, some preventive action can be taken to stop development of cancer [12]. The biomarkers of early health effects, from this point on referred to as promotion biomarkers, can be used not only to detect the early stages of the carcinogenesis process, but also to determine the efficiency of preventive steps taken [12]. The cellular changes, which involve activation of protooncogenes and their transformation into oncogenes, as well as deactivation of the suppressor genes, seem to play a dominant role in the promotion process. The knowledge of the influence of environmental carcinogenes on the cellular genome is, in fact, limited only to the process of protooncogene activation, and litde is known about their influence on the deactivation of suppressor genes. Nevertheless, the data collected thus far help us understand the mechanism that may lead to the neoplastic transformation of cells initiated by environmental carcinogenes. It seems likely that activation of

METABOLISM

ENVIRONMENTAL GENOTOMC CHEMICAL

m cn. 0 c

MALIGNANT TUMOR

2 0

(CLINICAL DISEASE)

FURTHER GENETIC CHANGES

Metabolder m body *id and excreia (INTERNAL DOSQ

-

MUTATIONS, MUTATIONS, Mtpkficalion, Gene Mtpkficalion, tramlocatwn, &tic &hc loss, deletion, CEA, TPA deletion, (EARLY BIOLOGICAL EFFECT ON STRUCTURE AND FUNCTION LEVEL)

I EXPOSURE BlOMARKERS

DNA andprotein adduce TWOLOGICALLY EFFECTIVE DOSG A

MU TATIONS Glycophorm, r(ls HPRT, HLA, Glycophorin, oncogenes,p 53 gene, onkoprotcm onkoprotcins SCE's, micronuclei mrcronuclez (EARLY BIOLOGICAL EFFECT ONMOLECUUR LEVEL)

f

I

-.3

CELL PROLIFER4 T I 0 CLONAL EXPANSION

EARLY HEALTH EFFECT BIOMARKERS

Figure 1. A simplified diagram of the cascade of events from exposure to environmental gentoxic chemicals to malignant tumor (clinical disease), showing the relationship among exposure markers and early health effect biomarkers.

8

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some protooncogenes and related qualitative and quantitative changes in oncoproteins, involved in the control of growth and division of cells, is a basic mechanism used by environmental carcinogenes to evoke this transformation process [1,12,13]. The possibility of observing the trend of those changes through assessment of protein products of the oncogenes and suppressor genes in the easily obtainable biological material (blood, serum, urine) has proved to be extremely useful from the point of view of occupational medicine and environmental health. It provides new diagnostic methods in the assessment of carcinogenic effects of the occupational environment and human habitation places. Knowledge of the fact that oncoproteins can appear in biological fluids long before (many months or even years) clinical symptoms of cancer occur provides new opportunities for taking more effective preventive measures [14,15,16]. The measurements of oncoproteins in plasma or urine in order to assess the risk of cancer under conditions of occupational or environmental exposure to chemical carcinogenes have some limitation associated with the high cost of one measurement that requires highly specific monoclonal antibodies. The measurement of oncoproteins in very easily available saliva-material has been carried out in persons occupationally exposed to asbestos and in smokers, in other words, in groups with a high risk of lung cancer. The level of ras oncoprotein p21 was measured in saliva cells. In some persons the p21 test was positive; however, they did not show any clinical symptoms of the cancer [14]. Very interesting studies on the measurement of oncoproteins in blood serum as a biomarker of early cancer changes were carried out by Brandt-Rauf et al. [17]. The authors examined 18 workers employed in a foundry and exposed to polycyclic aromatic hydrocarbons (PAHs)—well-known occupational and environmental carcinogenes, in view of the presence of oncoproteins in blood serum. A high level of PAH adducts from DNA had been earlier found in the peripheral blood lymphocytes of those persons. This confirmed that those workers belonged to the group of a particularly high risk of malignant disease. The studies showed, among others, the presence of fes oncoproteins in the blood serum of a worker employed in the system of an eight-hour shift under condition of exposure to benzo(a)pyrene in concentration in ambient air exceeding 0.2 |ig/m^. In the serum of another worker also employed in a foundry and exposed to slightly higher concentrations of benzo(a)pyrene (0.05-0.2 iig/m^), fes and ras oncoproteins were detected. The level of adducts in lymphocytes of these two workers was doubled in comparison with workers whose blood serum was free from oncoproteins. It should be stressed that according to data presented by the authors, none of those workers showed clinical symptoms of neoplasm of the respiratory pathways or other organs. Another study was performed in persons working in fire brigades and involved in anti-fire campaigns in chemical plants. Out of 33 persons covered by the study, oncoproteins (corresponding in their properties with B-transforming growth factor) were found in the blood serum of 14 workers. It is worth mentioning that in none of

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the controls (non-occupationally exposed to chemical carcinogenes) was the presence of these oncoproteins detected [14]. A similarly high percent of persons with oncoproteins present in their blood serum was found in those exposed to polychlorinated biphenyles (PCBs) [18]. This group was consisted of 16 workers involved in purification of transformer oil. The presence of fes oncoproteins was detected in six persons. It should be emphasized that all of them were smokers. Ras oncoproteins and sis oncoproteins were found separately in two workers. Moreover, in two workers free from fes oncoproteins, the presence of ras oncoproteins was detected. The highest blood level of PCBs was found in the worker with fes and ras oncoproteins detected in his blood serum. Data presented by Brandt-Rauf [14] on the significance of oncoprotein expression in the prognosis of malignant disease proved to be interesting. Studies were performed on samples of blood serum collected from 46 workers exposed to asbestos and silica dusts. The samples were kept in a blood bank. Fourteen workers developed cancer from the time of blood collection (including nine cases of the respiratory neoplasm). Usually, there was a period of 14 months between the blood collection and the development of cancer. In the group of nine persons with lung cancer, in as many as seven the presence of at least one oncoprotein was detected in blood serum collected earlier. One can draw a conclusion that the detection of increased expression of oncogene or its mutation through the measurement of oncoproteins in blood serum can be a useful biomarker in assessing the risk of cancer in populations exposed to occupational and environmental carcinogenes. The changes that lead to protooncogene activation and suppressor gene deactivation are not the only changes that lead to the neoplastic transformation. It seems, however, that the former correspond to the earliest stage of the promotion [19, 20]. Inhibition of intracellular communication by inhibiting specific intercellular junctions seems to be extremely important in the complex mechanism of the transformation of a normal cell into a neoplastic one [21]. Studies have indicated that the ability of forbol esters to promote carcinogenesis (cell canceration) is revealed by interaction with proteins responsible for the formation of intercellular junctions and cell adhesion [22]. It seems interesting to note here that the carcinoembryonic antigen (CEA), a neoplastic marker known in oncology for many years, acts a protein responsible for intercellular adhesion [23]. CEA belongs to the immunoglobulin super-family that includes also the adhesive protein of nerve cells. It is thought that the changes in the number of the sialic acid included in the saccharic component of the glycoproteins (to which CEA and neuronal adhesive protein belong) are correlated with the loss of the adhesive properties of those proteins [24]. When evaluating the role of CEA as a biomarker of the promotion stage within the neoplastic process, attention should be paid to the fact that the genes for coding that protein are located in chromosome 19, in the vicinity of the genes that code the betagrowth transforming agent, or the protein kinase [25]. The possible relationship between elevated CEA concentration in the serum and the carcinogenic process is supported not only by observations in the oncology clinics, but also by the data

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from studies on populations that do not display clinically overt signs of cancer process. In the latter case, elevated CEA concentrations in blood serum are found in people characterized by the occurrence of the agents which are known to increase the risk of neoplastic disease, such as age, tobacco smoking, alcohol drinking, and occupational exposure to pollutants [26]. Studies published in 1986 by Pluygers et al. [27], which involved a large population of 2,000 people said to be clinically healthy, have supported those suggestions. The authors have demonstrated that during a 5-year observation, the incidence of cancer in the people in whom elevated values of CEA concentration in blood serum were found during the screening tests was ten times higher than among the people with normal CEA values. A number of studies have confirmed that staying in an environment containing toxic chemical substances (not necessarily considered to be carcinogenic) contributes to increasing the frequency of occurrence of elevated CEA concentrations in the blood serum of the exposed people. This refers both to chemical pollutants present in the communal and in the occupational environments. Thus, Schilpkorter et al. [28] observed higher values of CEA concentrations in non-smoking males residing close to industrial plants as compared with those non-smoking males who resided some distance from the industrial region. Pluygers et al. [29] presented data on the behavior of CEA concentrations (simultaneously, TPA concentrations were also determined) in people residing in close vicinity to refuse dumps. According to the authors, 10.3% of the tested population had elevated values of CEA concentration. Page et al. [cit. from 30] were some of the first authors who attempted using the assessments of CEA in blood serum to assess the risk of occupational cancer. They reported that in as much as 48% of the studied population working under conditions of occupational exposure to vinyl chloride, increased values of CEA concentration had been detected. During the study, overt clinical symptoms of neoplastic disease were not detected in any of the studied people. Those observations were then confirmed by Anderson et al. [31], who performed a similar study on a group of 1,115 factory workers exposed to vinyl chloride in a vinyl chloride polymerization plant. Elevated values of CEA concentration were also found to occur in people occupationally exposed to asbestos dust [32]. During the XXII International Congress on Occupational Medicine in the Chemical Industry held in 1987 in Sydney, Australia, Pluygers et al. [33] presented interesting reports on the application of tumor marker assessments, including CEA, to determine the risk of neoplastic disease in employees (subdivided into non-smokers and smokers) of three chemical plants (the type of the chemical exposure was not specified). Evidently the highest percentage of people with CEA concentration above the cutoff (53.3%) was detected in the group of exposed smokers. Therefore, it can be concluded that tobacco smoking under conditions of occupational exposure to potentially carcinogenic chemicals increases the risk of incidence of neoplastic disease by many times. In 1991, Plugers et al. [12] presented data suggesting that the assessment of CEA concentration in blood serum, in combination with several other tumor markers

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(TPA, ferritin, hyalouronic acid), may be used for evaluating the risk of neoplastic disease in people occupationally exposed to asbestos dusts. The authors report that assessment of CEA concentration (and of other markers quoted above) can be helpful in evaluating the effectiveness of preventive procedures aimed at reducing the risk of incidence of asbestos-induced cancer. The observations made heretofore indicate, however, that the use of assessment of CEA concentration in blood serum as an indicator of the risk of neoplastic disease cannot serve as a universal test for various exposures to chemical carcinogenes and for various cancer types. It is thought that the assessment of CEA concentration should be accompanied by the assessment of another tumor marker, tissue polypeptide antigen (TPA), known for some years already in clinical oncology [34]. TPA, a protein of the cellular cytoskeleton, is recognized by antibodies to cytokeratins and is a constituent of protein intercellular junctions. Functionally, TPA is related to the family of the adhesive proteins known as cadherins, participating in cell-cell intercellular interactions. According to Pluygers et al. [12], assessment of blood serum TPA concentration as a marker of neoplastic disease risk is particularly useful under the conditions of exposure to these carcinogenic chemicals that display their genotoxic and mutagenic properties by forming highly active free radicals. Studies by Lutz and Krajewska [35] and Lutz et al. [36] have revealed that under conditions of exposure to carcinogenic aromatic amines, assessments of TPA concentration in blood serum performed to evaluate the risk of bladder cancer are more useful than that of CEA. Somewhat earlier, Kumar et al. [37] demonstrated that TPA can be used as a biomarker of early bladder epithelium cell changes, which can but do not necessarily lead to neoplastic transformation. The two discussed tumor markers, CEA and TPA, are not the only markers used for early detection of neoplastic disease risk. Nevertheless, at the current state of our knowledge on tumor markers, it can be said that CEA and TPA are universal biomarkers that complement each other and should be assessed simultaneously. Assessment of TPA and CEA concentration in blood serum can be supplemented by assessment of those tumor markers specific to a given exposure to chemical carcinogenes or to the type of neoplastic disease they can induce. For example, under conditions of exposure to asbestos dusts, assessment of ferritin hyaluoronic acid is recommended in addition to assessing CEA and TPA concentration [12]. Assessment of some tumor markers (TPA, CEA, or oncoproteins) in easily accessible biological material (blood, serum, or urine) opens up new diagnostic possibilities for early detection of the risk of neoplastic disease under conditions of environmental and occupational exposure to carcinogenic chemicals. The introduction of biomarkers able to record the very early health effects produced by environmental carcinogenes make it possible to assess the risk of cancer incidence at a very early stage in its development. Of course, there are some limits to this assessment. It is known that exposure biomarkers—e.g., DNA or protein adducts (also call precancerogenic biomarkers)—occur more frequently under conditions of exposure to carcinogenes, and that the number of people in whom those adducts are detected is

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much greater than the number of people who may develop the neoplastic disease. Not every one of these people in whom DNA adducts have been detected will develop cancer. On the other hand, the failure to detect adducts does not mean that the person in question will not develop cancer. However, in many instances the likelihood of correct assessment of increased cancer risk on the basis of DNA or protein adduct determination can be augmented by the simultaneous determination of other biomarkers (for example, the presence of oncogenes) or their protein products (oncoproteins) in the cells or the biological fluids. A greater number of positive biomarker determination results shows significant increase in the risk of disease. However, the determination of a large number of biomarkers noticeably increases the cost of the research; this may be essential in epidemiological studies conducted on large populations. Therefore, before selecting such a biomarker set, the biomarkers should be carefully examined, and those which supply similar information, or those which are not related to the investigated pathologic process or harmful factor, should be eliminated. Biomarker-based biological monitoring is the most efficient measure capable of assessing the risk to an individual organism resulting from exposure to external harmful agents. According to the biomarker concept, environmental effects on the human organism can be assessed by the determination of exposure, health effect, and individual susceptibility, and enhanced analysis of the health risk in people exposed to environmental toxic agents has become possible. The assessments performed directly in the organism contribute to the elimination of uncertainties inherent in epidemiological studies.

REFERENCES 1. "Biomarkers and Risk Assessment: Concepts and Principles." Environmental Health Criteria 155. Geneva: World Health Organization, 1993. 2. Fowle, J. R. and Sexton, K. "EPA Priorities for Biologic Markers Research in Environmental Health." Environmental Health Prospectives, Vol. 98, 1992, pp. 235-241. 3. Gunn, P. M., Devra, L. and Perera, F. "Biological Markers in Environmental Epidemiology: Constrains and Opportunities," in Methods for Assessing Exposure of Human and Non-Human Biota, Chichester: John Wiley and Sons,Ltd, 1991, pp. 152-174. 4. Hanke, J., Indulski, J. A. and Lutz, W. "Biomarkers—A New Way of Evaluating the Effects of Environment on the Human Organism." Medycyna Pracy, Vol. 43, 1991, pp. 63-71 (in Polish). 5. US National Research Council. "Biological Markers in Environmental Health Research." Environmental Health Perspectives, Vol. 74, 1987, pp. 1-191. 6. Henderson, R. F. et al. "The Use of Biological Markers in Toxicology." Critical Reviews in Toxicology, Vol. 20, 1989, pp. 65-82.

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7. Gaylor, D. W., Kadluber, F. F. and Beland, F. A. "Application of Biomarkers to Risk Assessment." Environmental Health Perspectives, Vol. 98, 1992, pp. 139-141. 8. Indulski, J. A. and Lutz, W. "Biological Monitoring of Risk of Bladder Cancer in Persons Occupationally Exposed to Aromatic Amines." Polish Journal of Occupational Medicine and Environmental Health, Vol. 6, 1992, pp. 143-151. 9. Weinstein, B. J. "The Origin of Human Cancer: Molecular Mechanisms of Carcinogenesis and Their Implications for Cancer Prevention and Treatment." Twenty-seventh GAA Clowes Memorial Award Lecture. Cancer Research Vol. 48, 1988, pp. 4,135^,143. 10. Lutz, W. and Baranski, B. "The Role of Testing DNA and Protein Adducts in Cancer Risk Monitoring." Medycyna Pracy, Vol. 42, 1991, pp. 67-75 (in Polish). 11. Autrup, H. "Human Exposure to Genotoxic Carcinogens: Methods and Their Limitations." Journal of Cancer Research and Clinical Oncology, Vol. 117, 1991, pp. 6-12. 12. Pluygers, E. P. et al. "Biomarker Assessments in Asbestos Exposed Workers as Indicators for the Selective Prevention of Mesothelioma or Bronchogenic Carcinoma: Rationale and Practical Implementations (Parts I and II)." European Journal of Cancer Prevention, Vol. 1, 1991, pp. 57-68 and Vol. 2, 1922, 129-138. 13. Balmain, A. and Brown, K. "Oncogene Activation in Chemical Carcinogenesis." A^vflEfic^^" in Cancer Research, Vol. 51, 1988, pp. 147-182. 14. Brand-Rauf, P. W. "The Molecular Epidemiology of Oncoproteins." Scandinavian Journal of Work, Environment and Health, Vol. 18, 1992, pp. 4 6 ^ 9 . 15. Cooper, G. M. "Oncogenes as Markers for Early Detection of Cancer." Journal of Cellular Biochemistry, Suppl. 16G, 1922, pp. 131-136. 16. Niman, H. L. et al. "Anti-Peptide Antibodies Detect Oncogene-Related Proteins in Urine." Proceedings of the National Academy of Sciences, Vol. 82, 1985, pp. 7,924-7,928. 17. Brand-Rauf, P. W. et al. "Serum Oncogene Proteins in Foundry Workers." Journal of Social and Occupational Medicine, Vol. 40, 1990, pp. 11-14. 18. Brand-Rauf, P. W. and Niman, W. "Serum Screening for Oncogene Proteins in Workers exposed to PCBs." British Journal of Industrial Medicine, Vol. 45, 1988, pp. 689-695. 19. Brand-Rauf, P. W. "New Markers for Monitoring Occupational Cancer: The Example of Oncogene Proteins." Journal of Occupational Medicine, Vol. 30, 1988, pp. 399-404. 20. Travis, C. C. and Belefant, H. "Promotion as a Factor in Carcinogenesis." Toxicology Letters, Vol. 60, 1922, pp. 1-9. 21. Yamasaki, H. "Gap Junctional Intercellular Communication and Carcinogenesis." Carcinogenesis, Vol. 11, 1990, pp. 1,051-1,058.

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22. Trosko, J. E., Chang, C. and Medcalf, A. "Mechanisms of Tumor Promotion. Potential Role of Intermolecular Communications." Cancer Investigation, Vol. 1,1983, pp. 511-526. 23. Benchimol, S. et al. "Carcinoembryonic Antigen a Human Tumor Marker, Functions as an Intercellular Adhesion Molecule." Cell, Vol. 57, 1989, pp. 327-334. 24. Rutischauser, U. et al. "The Neural Cell Interactions." Science, Vol. 240, 1988, pp. 53-57. 25. Hosier, J. C. "Genes for Tumor Markers are Clustered with Cellular ProtoOncogenes on Human Chromosomes." Cancer Letters, Vol. 36,1987, pp. 235-245. 26. Herbeth, B. and Bargel, A. A. "A Study of Factors Influencing Plasma CEA Levels in an Unselected Population." Oncodevelopment and Biological Medicine, Vol. 1, 1980, pp. 191-198. 27. Pluygers, E. P. et al. "Tumor Markers for Cancer Detection." Cancer Detection and Prevention, Vol. 9, 1986, pp. 495-504 and 505-509. 28. Schilpkoter, H. W., Baginski, B. and Kramer, U. "The Carcinoembryonic Antigen (CEA) in Urban Populations: Epidemiological Studies." Zentralblatt fur Bakteriologie und Hygiene, Vol. 166, 1978, pp. 136-143. 29. Pluygers, E. P. et al. "Evaluation of the Cancer Risk Associated with Activities of the Chemical Industry in Production, Utilization and Environmental Pollution," in Occupational Health in the Chemical Industry. Copenhagen: Medichem and WHO, 1992, pp. 220-228. 30. Bernard, A. and Lauwerys, R. "Determination of Tumor Markers in Biological Fluids," in Indicators for Assessing Exposure and Biological Effects of Genotoxic Chemicals. A. Aitio et al. (Eds.), Brussels-Luxemburg: Office for Official Publications of the European Communities, 1988, pp. 153-169. 31. Anderson, H. A. et al. "CEA Levels in Workers Exposed to Vinyl Chloride Monomer," in The Abstract Book of the Medichem 4th International Conference, September 7-10, Haifa: 1976, pp. 47. 32. Jarvisallo, J. O. and Stemman, U. H. "Monitoring Human Exposure to Carcinogenic and Mutagenic Agents." IRAC Scientific Publications, Vol. 59, 1984, pp. 403-409. 33. Pluygers, E. P. et al. "Evaluating Carcinogenic Risk Among Workers in the Chemical Industry by Using Tumor Marker Assessments in Serum," in Occupational Health in the Chemical Industry. Medichem and WHO, Copenhagen, 1988, pp. 136-143. 34. Fischer, L. "TPA: Tracing Cancer in Serum and Tissue." International Clinical Products, Vol. 7/8, 1985, pp. 20-24. 35. Lutz, W. and Krajewska, B. "The Concentration of the Tissue Polipeptyde Antigen (TPA) and Carcinoembryonic Antigen (CEA) in the Serum of Workers Exposed to Aromatic Amines." Medycyna Pracy, Vol. 43,1992, pp. 297-301.

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36. Lutz, W., Indulski, J. A. and Krajewska, B. "Tissue Polypeptyde Antigen and Acetylation Phenotype," in Occupational Health in the Chemical Industry. Selected papers from the XX Medichem Congress, 6-9 October 1992, Copenhagen: World Health Organization Regional Office for Europe. 1993, pp. 127-131. 37. Kumar, S. et al. "Frequent Evaluation of Tissue Polipeptyde Antigen in the Sera of Workers Exposed to Bladder Carcinogens." International Journal of Cancer, Vol. 22, 1978, pp. 542-545.

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CHAPTER 4 SICK BUILDING SYNDROME Peter Dingle Environmental Science Murdoch University Murdoch Western AustraUa 6150

CONTENTS INTRODUCTION, 67 THE NATURE OF THE SYNDROME, 68 The Symptoms of SBS, 69 The Costs of SBS, 70 Causal Mechanisms, 70 ENVIRONMENTAL CONTAMINANTS ASSOCIATED WITH THE SYNDROME, 70 Indoor Air Pollution, 70 Physical Factors, 74 Biological Contaminants, 76 CAUSAL FACTORS AND SOURCES ASSOCIATED WITH THE SYNDROME, 79 Psychological Factors, 79 Psychosocial Phenomena, 80 The Building and Environmental Control Systems, 81 Office Materials, Equipment, and Furnishings, 82 Combination of Causes, 84 CURRENT RESEARCH, 85 SOLUTIONS TO SBS PROBLEMS, 85 CONCLUSIONS, 86 REFERENCES, 86 INTRODUCTION In a substantial number of large modem office buildings, office personnel have complained of a similar set of symptoms now commonly known as sick building syndrome (SBS). SBS was first reported as a problem in buildings more than thirty years ago in the U.S.A. and Scandinavia. The problem began to reach epidemic proportions when the oil crisis of the early seventies required Western countries to reconsider their use of energy [1]. The major strategy used to reduce energy consumption was to construct buildings more air tight. This was based on observations that buildings accounted for more than a third of America's fuel consumption [2] and with 75% of energy used on environmental control systems [3]. 67

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The syndrome is mostly associated with buildings used for non-industrial purposes, especially office buildings. It has been estimated that 30% of new and remodeled office buildings show signs of SBS and that 10-30% of occupants are affected [4]. While current evidence suggests that general SBS symptoms and complaints may cause no lasting damage to the long-term health of occupants, health complaints related to buildings can be a significant disruption to the people who experience them on a regular basis [5].

THE NATURE OF THE SYNDROME To date, the SBS has not been concisely defined and has been loosely applied to various situations. This has resulted in the lack of a systematic approach to studying the syndrome, which has caused some controversy and confusion. There is now a clear distinction between the syndrome and other building-related health problems such as "Building Related Illnesses" (BRI). This is based on the observation that a BRI is a clinically defined illness and can be diagnostically attributed to an environmental exposure. The most serious BRI is the occasionally fatal Legionnaires' Disease. Other well-known BRIs include Humidifier Fever and Pontiac Fever. In order to define the SBS, it is important to understand that it is not a clinically defined disorder but is a set of general symptoms with no known single underlying cause in all buildings [6]. Synonyms of the SBS have included "building illness syndrome," "ill buildings," "stuffy offices," and "tight office syndrome" [7]. The World Health Organization (WHO) and other researchers have defined SBS with the following observations: • An increase in the frequency of building occupant-reported complaints; • Acute non-specific symptoms; • Occurs in non-industrial environments; • No single causal substance or agent has been identified; and • The symptoms improve when the effected persons are away from the building [4, 8]. The WHO has also made a distinction between "temporary sick buildings," where onset is acute and usually declines within one year of occupancy, and "permanently sick buildings," where symptoms are non-specific and ongoing. The former are often caused by off-gassing of new fabrics, building materials, and furnishings, and can generally be treated [9]. The latter are invariably sealed, air-conditioned buildings designed to be energy-efficient [10]. The WHO has also suggested a more technical definition of SBS is when more than 20% of a building's occupants "often experience" a specific set of symptoms. However, while there is general agreement on the set of symptoms, what is defined as "often experienced" is not clear.

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The Symptoms of SBS The symptomology of SBS is varied, but five symptom complexes are regularly encountered and may occur singularly or in combination [8]. The primary symptoms of SBS include: • Mucous membrane irritation; • Neurotoxic symptoms; • Odor complaints; • Skin irritation; and • Asthma-like symptoms [11, 12, 13, 14]. These symptoms disappear or become considerably less pronounced minutes or hours after leaving the building [15]. However, some people do not get better until they have been away from the building for several days, or in some cases such as skin symptoms, several weeks [8, 15]. Table 1 shows a broader list of symptoms. Table 1 General glasses of symptoms Related to the sick building syndrome with examples of symptoms belonging to each glass

1. Sensory irritation in eyes, nose, and throat: • dryness • stinging, smarting, irritating sensation • hoarseness, changed voice 2. Skin irritations: • reddening of the skin • stinging, smarting, itching sensations • dry skin 3. Neurotoxic symptoms: • mental fatigue • reduced memory • lethargy, drowsiness • reduced power of concentration • headache • dizziness, intoxication • nausea • tiredness 4. Unspecific reactions: • running nose and eyes • asthma-like symptoms in non-asthmatic persons • respiratory sounds 5. Odor and taste complaints: • changed sensitivity • unpleasant smell or taste Source: M0lhave [12].

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The Costs of SBS Apart from the adverse health effects an individual may experience, SBS symptoms have been shown to impact on productivity and absenteeism [5]. SBS may also affect unofficial time off work, reduce overtime, increase staff turnover, and can make substantial demands on management to spend time on resolving problems [5]. In some extreme cases, buildings have been closed for a period while mitigation activities are carried out, with the potential to incur considerable costs. It is reasonable to assume that if workers are unhappy and irritated they are unlikely to be working at maximum capacity [16]. Some studies estimate that a reduction in efficiency by 20% may occur (self-assessed) [1]. Causal Mechanisms SBS symptoms are non-specific in character and have many potential causes [17], which are generally classified as physical, chemical, biological, or psychological [18]. Although a large number of causes of SBS have been hypothesized, only fragmentary evidence exists supporting any one hypothesis [5, 18]. The nature and time pattern of SBS symptoms suggests that it could result from a combination of allergic, irritant, and toxic reactions to environmental contamination [13]. One etiological hypothesis for SBS suggested by M0lhave and co-workers was developed from the observation that low-level exposure to volatile organic compounds can lead to symptoms similar to those typical of SBS [13, 19]. A plausible biologic hypothesis proposed by Bergland et al. [20] suggests that SBS symptoms may arise from multi-sensory adaptation to indoor air pollution. This is achieved by either exhaustive stimulation, or by a sensory deprivation of the variety of signal required to maintain normal sensory variation. A result may be sensory confusion, which becomes a strain on the particular system attempting to interpret the sensory signals. This hypothesis has been supported by cases where individuals no longer working in or visiting the original building concerned will continue to experience symptoms, or have an increased sensitivity to environmental exposures such as tobacco smoke, new paint odor, etc. [15]. This is also supported by the observation that SBS is predominantly stimulation of the olfactory and irritant receptors of the upper airways, and while the symptoms may not be severe, these receptors are peoples' warning systems. ENVIRONMENTAL CONTAMINANTS ASSOCIATED WITH THE SYNDROME Indoor Air Pollution Sources of indoor air pollutants include the occupants and their activities, building materials, microorganisms, and infiltration of contaminants from the exterior

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[21]. The indoor air quality of a building can be impacted by potentially hundreds of chemicals [22]. The concentration of an indoor air pollutant is a complex interaction between: • The source strength; • The location of the source; • The transportation and mixing of pollutants; • Indoor climatic factors; • Synergistic reactions with other pollutants; • Dilution with outside air; and • Pollutant removal mechanisms [22]. Major contaminants of indoor air are human bioeffluents, volatile organic compounds, formaldehyde, suspended particulate matter, environmental tobacco smoke, and outdoor air pollution. Human Bioeffluents Contaminants generated by the human body are described as bioeffluents and historically are a major indoor air quality (lAQ) concern of both odor and (dis)comfort. Carbon dioxide (CO2) is a bioeffluent produced by human beings as a byproduct of metabolism [19]. It is now well established that CO2 concentrations in the range of 600 ppm to 1,000 ppm and higher are associated with SBS [8, 19]. The concentrations normally observed in buildings are not associated with any symptoms, except the sensation of stale and stuffy air [8]. Other bioeffluents reported from building investigations include acetone, acetaldehyde, acetic acid, alkyl alcohol, amyl alcohol, butyric acid, diethyl ketone, athylacetate, ethyl alcohol, methyl alcohol, phenol, and toluene [23]. Volatile Organic Compounds (VOCs) Volatile organic compounds (VOCs) are one of the main pollutants inside sick new buildings. They are regarded as producing SBS effects even in low doses. Organic compounds include all chemicals containing carbon and hydrogen. VOCs are easily vaporized and are rapidly absorbed via the lungs, skin, and intestinal tract. It is generally accepted that VOCs include those organic compounds with a boiling point in the range of 50 to 250°C. Of the compounds in this range, over 900 compounds have been identified in indoor air. The number of VOCs present in any one building can vary considerably from as few as 20 to several hundred compounds [15]. VOCs are emitted from a wide variety of sources in buildings such as building materials, furnishings, consumer products, building maintenance materials, human bioeffluents, office equipment, tobac-

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CO smoke [8, 24], carpets, cleaning products, smoke, adhesives, caulking, paints, and solvents [25]. However, evidence of the role of VOCs in SBS has not been convincing [8]. This may be due in part to a lack of standard measuring methods. Different methods will yield different results and do not allow for the direct comparison of results from studies using different measurement techniques [15]. Formaldehyde Formaldehyde is a ubiquitous contaminant of indoor spaces [26] and is a known sensory irritant [27], an irritant of the respiratory tract [9, 28], an apparent neurotoxic substance [29], and may be responsible for allergic disorders including asthma [30]. However, its presence in indoor air is frequently attributed to mucous membrane irritation [19]. Major sources of formaldehyde in office and non-industrial buildings include: • Furniture made from pressed woods such as plywood, particle board, and medium density fiberboard (MDF) [26, 31]; • Storage cabinets, countertops, and workstations made from particle board; • Room office dividers with particle board or MDF cores; • Acid cured finishes on wood furniture [26]; • Office equipment and decomposition of VOCs [32]; • Urea formaldehyde foam insulation (UFFI); • A variety of products used for disinfection, cleaning, and painting [8, 26]; and • Fabrics, glues, and carbonless copy paper. Except in a few cases, formaldehyde concentrations reported in buildings have been too low to be responsible for SBS symptoms. Consequently, it has rarely been identified as a causal factor in problem buildings [8]. On the whole, it is unlikely that formaldehyde by itself is responsible for SBS symptoms, but it may be a contributor to SBS symptoms by potentiation of other factors [8]. Suspended Particulate Matter Airborne particulate matter is ubiquitous and exists in a wide range of particle sizes and chemical characteristics [33]. Dust in the indoor air consists of organic and inorganic particles, many of which can be classified as fibers [8]. The particulate size range of concern to human health is the range in aerodynamic diameters between 0.1 and 10 micrometers. This corresponds to the respirable suspended particulate matter (RSP) and the total suspended particulate matter (TSP) ranges [33]. Particles less than 3.0 mm in diameter (RSP) are the most biologically significant, as they can reach the thoracic or lower regions in the respiratory tract. Exposure to these particulates is suggested responsible for most of the adverse health effects of SBS [14, 26, 34].

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Typical sources of suspended particulate matter in buildings include: • Smoke, street dust, paper, duct insulation, water residue, tobacco smoke, carpets, heating, ventilation, air, and cooling (HVAC) filters; • Chemical disinfectants, corrosion or rust from humidification equipment; damaged insulation materials; and paper shredding; • Skin cells, dust mite feces, domestic animal dander on clothing, clothing fabrics, and biological activity [15]; and • Combustion by-products. Environmental Tobacco Smoke (ETS) The problems with environmental tobacco smoke (ETS), and sidestream smoke (SS) are extensively documented. This lead to the finding in the late eighties that the smoking of tobacco products indoors is the most important source of chemical pollution in indoor air [8, 35]. ETS is the mixture of smoke from the burning end of a cigarette, pipe, or cigar, and the smoke exhaled by a smoker [36]. The sidestream smoke of ETS is known to be responsible for mucous membrane irritation and has been found to be far more irritating than mainstream smoke [8]. Cigarettes contain a great number of chemicals, in the order of 3,800. Some of the more noxious chemicals include formaldehyde, carbon monoxide, nitrogen oxides, ammonia, nicotine, and an assortment of heavy metals and pesticides. Short-term studies have revealed that ETS can add significantly to indoor formaldehyde levels [32]. In buildings where smoking is allowed, environmental tobacco smoke (ETS) may account for 40% of the indoor pollution. Outdoor Air Because healthy indoor air quality requires the intake of outdoor air, and because of phenomena such as the thermal stack effect, pollution in the outdoor air is a potential problem. The most likely outdoor air pollutants are from adjacent roadways, parking lots, and loading bays. Indoor air pollution entering from outside may be from combustion by-products and may include carbon monoxide, carbon dioxide, nitrogen dioxide, sulphur dioxide, and VOCs, all of which can contribute to SBS. Oxides from combustion products are often respiratory irritants and can result in adverse health if long-term chronic exposure occurs. Indoor Surface Pollution There is growing evidence of an important role of surface dust or indoor surface pollution (ISP) in office buildings [14]. This is partly due to the observation that individuals create their own dust cloud when settled dust is stirred up during normal work activities and that concentrations in this dust cloud are significantly

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greater than ambient levels [5]. Exposure to settled dust can result in direct dermal contact, and possible transportation to eyes causing irritation, and disturbed or resuspended dust can result in potential respiratory, ocular, and dermal exposure [14]. The potential toxic or irritation effects of ISP may also be increased as a result of the adsorption of gases and vapors to particulates [5]. Initial studies by Skov et al. [37] found significant associations between floor dust levels and mucous membrane irritation symptoms. Intervention studies have found that steam-cleaning carpets and soft furnishings, wet wiping of hard surfaces, and high efficiency vacuum cleaning can significantly reduce symptoms, particularly when actions remove dust deposits from carpets and soft furnishings [5, 38, 39, 40]. Physical Factors Physical factors contributing to indoor environmental conditions with the potential to affect human health and comfort include temperature, relative humidity, air movement, ventilation, light intensity and flicker, noise, vibration, air ions, and electrostatic, electric, and magnetic phenomenon [41, 42]. Thermal Comfort Thermal comfort is described as a condition of mind, an expression of satisfaction [43], and is dependent on the level of work performed, the heat of metabolism, evaporative losses, radiant and convective heat transfers [19], individual physiology, and age [45]. Dissatisfaction with the thermal environment may be due to general bodily discomfort due to warm or cool ambient conditions, or to unwanted heating or cooling of a localized part of the body—local thermal discomfort. Unsatisfactory thermal conditions can act indirectly to produce irritable workers, which may increase the likelihood of SBS complaints arising. Temperature alone has been suggested as the most important indoor parameter affecting SBS. Thermal discomfort can also lead to synergistic effects with other parameters such as increased skin irritancy by particulates [44]. Relative Humidity There is no agreement on what constitutes the ideal range of relative humidity for indoor air [8]. An optimum range of relative humidity of 45-55% is now generally accepted, and is a much narrower range than previously thought [46]. A wider range may be acceptable for thermal comfort, but encourages bacterial, fungal, and viral growth. What is generally agreed upon is that raising humidity has a similar effect to raising the temperature [45]. Direct effects include discomfort at excessively high (above 70%) or low (below 20%) relative humidities [8, 45]. Humidities below 20% can evoke drying of mucous membranes or skin, and a particular dermatitis has been associated with

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dry, warm air and high rates of air movement [47, 48]. Indirect effects of humidity include phenomena such as significant increases in formaldehyde concentrations by a factor of 1% for every 1% rise in relative humidity. Lighting Some evidence exists that illumination could play a role in the SBS [49]. Artificial light only consists of some wavelengths in the spectrum, whereas natural light is a combination of all colors of the spectrum. Research has shown that people can suffer, to varying degrees, a syndrome known as seasonal affective disorder (SAD), which is a result of deprivation from full-spectrum lighting. Poor lighting conditions can cause eye strain and fatigue and appear to be associated with headache, dizziness, fatigue, nausea, and eye irritation [50, 51]. Excessive luminosity has been reported to be a major cause of complaints in some SBS investigations and has been suggested as a major causal factor in 20% of buildings with SBS [52]. Noise and Acoustic Conditions Acoustic conditions in a building depend on sound transmitted from the outside, from other rooms, the ventilation system, machines and persons in the room, and on structural conditions that provide attenuation or modification (structure-borne vibrations) [15]. However, few investigators have evaluated the significance of noise or vibrations for SBS [49]. Concern over the potential impact of noise is based on the notion that some sound vibrations have frequencies similar to the resonant frequencies of certain body parts, such as the eyes. Electromagnetic Radiation Offices full of electrical equipment are likely to have severely disturbed electromagnetic fields. In 1987, WHO endorsed a report that visual display unit (VDU) workers should not work within one meter of the rear or sides of other terminals unless they had been tested to emit only low levels of non-ionizing radiation (NIR). The dominant source of extremely low frequency (ELF) electromagnetic fields measured in buildings is now known to be all office equipment and not just the VDUs [53]. Research has shown that women who sit in front of a VDU for more than six hours a day are 40% more likely to have a miscarriage. Airborne Ions The indoor office environment has far more positive ions and far fewer negative ions than most outdoor environments. Electrical equipment, VDUs, synthetic fabric and carpets, and HVAC systems, which recirculate air through metal ducts, all contribute to ion loss. Some research has shown that negative ion generators can facili-

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tate removal of particulates from air by electrostatic precipitation. Godish [31] has concluded that the collective weight of the various studies indicates that air ions can have significant biological effects and that such effects can occur in humans. However, there is considerable debate occurring on the health impacts of air ions, and in particular their role in SBS. Biological Contaminants Biological contaminants come from many sources and include bacteria, molds, mildew, viruses, animal dander and cat saliva, house dust mites, cockroaches, pollen [36], yeasts, amoebae, and nematodes [54]. Some of the allergic reactions triggered by biological contaminants include hypersensitivity pneumonitis, allergic rhinitis, and some types of asthma [36]. A variety of biogenic contaminants have been suggested as risk factors for SBS symptoms regardless of atopic history. The major contaminants include: • Aerosols of both viable and nonviable organisms and their antigens; • Microbial products such as glucans, endotoxins, mycotoxins, and VOCs; and • Macro-molecular organic dust (MOD) found in particulate matter deposited on floor and indoor surfaces. Bioaerosols Biological aerosols are naturally occurring and have always been a factor in human health [55]; however, this does not mean they are benign. Water spray systems, humidifiers, filters packed with organic dust, high humidity, reduced ventilation, tighter buildings, and moisture damage to building materials all provide conditions for the growth of microorganisms. In particular, indoor air biological or microbial contamination is often the result of a fault in the building's HVAC system or maintenance procedures where microorganisms are provided with conditions to grow and proliferate [49, 56]. Airborne Infectious Disease Many of the typical symptoms for SBS are similar to those occurring in common airway infections [49]. Infectious illnesses such as influenza, measles, and chicken pox viruses are generally transmitted through the air [36] and close human contact. Many of these common airway infections may induce temporary bronchial hyperactivity [57], which could further act to make an individual more sensitive to irritants in the indoor air [49]. LaForce [58] and Brundage et al. [59] provide evidence that air conditioning systems may influence the spread of airborne infections.

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However, the pattern of SBS occurrence suggests that it most Ukely results from a combination of allergic, irritant, and toxic reactions to environmental contamination and not to infectious illness [14]. Bacteria While the concentrations of other biological contaminants in indoor air, such as mold spores, varies primarily as a function of outdoor concentrations, the concentration of bacteria in indoor air varies mainly with human occupancy [15]. Exposures to bacteria and microbial metabolites have rarely been studied as potential causal factors of SBS. Most cross-sectional epidemiological studies have failed to show any relationship between viable bacteria levels and SBS symptoms. Endotoxin Endotoxin is the name given to a class of biological molecules that have certain characteristic toxic effects. It is now recognized that the lipid portion of lipopolysaccharide (Lipid A) of Gram-negative bacteria is chemically distinct from all other lipids in biological membranes and is responsible for the molecules' characteristic toxicity [60]. Airborne endotoxin is ubiquitous in nature, as are the Gramnegative bacteria that produce endotoxins [61]. The most important type of reservoir for Gram-negative bacteria is recirculated water-based fluids that produce aerosols, such as the mechanism found in many humidifier systems [60]. Endotoxins are known to cause fever and malaise, changes in white blood cell counts, respiratory distress, shock, and even death when in the bloodstream in microgram quantities [60]. Endotoxins are also known to stimulate the immune system, which can have beneficial effects such as anti-tumor properties [62] and result in reduction of lung cancer risk [63, 64, 65]. Fungi Fungal spores are ubiquitous in outdoor air, but are considered a contaminant in indoor air [66]. However, very little baseline data exist on indoor air concentrations apart from case studies performed following occupant complaints or a diagnosis of illness due to environmental exposure [66]. Environmental factors that influence indoor fungus concentrations include outdoor air concentrations, type and rate of ventilation, and indoor moisture levels [67]. During the growing season, indoor fungus spore levels are typically 10-25% of outdoor levels [68]. Only in cases of extreme indoor contamination do indoor spore levels exceed outdoor levels [55]. A study on seven New York buildings by Morey and Jenkins [68] found indoor air levels exceeded outdoor levels by a factor of 1.5 to 13 during periods when the furniture and HVAC system were disturbed.

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Indoor environments exposed to contaminants such as quantities of bird or bat droppings are at contamination risk from strains of fungi such as Aspergillus fumigatus, Histoplasma capsulatum, and other fungi known to cause certain diseases. Exposure to airborne fungal spores, hyphal fragments, or metaboHtes is known to cause a variety of respiratory diseases. The full range of health effects due to fungal exposure include: • Allergic diseases including allergic rhinitis, asthma, and hypersensitivity pneumonitis; • Infectious diseases such as histoplasmosis, blastomycosis, and aspergillosis; • Acute toxicosis from exposure to mycotoxins; and • Cancer, also ascribed to exposure to mycotoxins [66]. Free-living Amoebae The impact of protozoa on human health in indoor environments is generally restricted to free-living amoebae. These can have direct effects as pathogens and allergens, or they can interact with bacteria and amplify bacterial pathogens [69]. Unlike bacterial contamination of reservoirs such as humidifier water, the aerosolization of living amoebae appears to pose a minimal threat. However, aerosolization of amoebic antigens may be harmful [69]. Of even greater potential impact on human health is the biological symbiosis between free-living amoebae and bacterial pathogens. It appears that free-living amoebae can harbor and or amplify known bacterial pathogens such as Mycobacterium, Listeria, and Legionella. This can result in the release of these bacteria and possible human exposure. However, the interactions between protozoa and potential human bacterial and viral pathogens and their impact on human health in the indoor environment are not fully understood. Allergens Potential allergens found in indoor environments include dust mite fecal wastes, mold spores, fungal hyphal fragments, and macromolecular organic dust (MOD). Exposure to allergens in the fecal wastes of dust mites appears to be the single most important cause of asthma and chronic allergic rhinitis in residential environments [70]. Allergens from domesticated animals can occur in buildings regardless of whether animals are kept on the premises. These allergens can be carried on clothing from places with domesticated animals, or may remain for many years after previous habitation by animals [15]. Surfaces of fabric-covered office panels can further act as reservoirs for cat dander allergen and fungal spores brought into the building by occupants [71].

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Volatile Organic Compounds as Bioejfluents Microorganisms such as bacteria, actinomycete, and fungi produce a variety of VOCs and semi-VOCs during metabolism [15]. VOC metabolites such as higher alcohols, ketones, and organic acids are responsible for odor problems associated with microbial growth. Exposure to these VOCs is suggested as a potential contributing factor to SBS symptoms, although only few studies have attempted to evaluate their potential health effects. CAUSAL FACTORS AND SOURCES ASSOCIATED WITH THE SYNDROME Psychological Factors There is litde direct evidence for the role of individual psychological factors in problem buildings [72]. The major role of psychosocial factors is most likely as modifiers of individuals' and organizations' responses to biological, chemical, and physical exposures in the office environment [72]. Some of the evidence from the research suggests some individuals have a predisposition to SBS [73]. It has also been found that the degree of control an individual has over the climatic conditions of an office affects the severity of reported complaints indirectly, via its effects on the workers' satisfaction with the prevailing environmental conditions and also their awareness of those conditions. Individual Sensitivity Individual sensitivity to exposures depends on factors such as a variety of personal characteristics and include: • Gender; •Age; • Marital status; • Atopic status; • A variety of psychosocial factors such as smoking, alcohol consumption, coffee consumption, regular exercise, and use of contact lenses; • Personal factors such as childhood exposures, residential factors, and industrial exposure to irritants [49]; • Skin type; • Previous exposures (sensitization); • Medication; and • Activity-related factors [6].

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Atopy Atopy is the genetic predisposition to allergic manifestation of exposure to common allergens such as dust mites, mold, pollen, and animal dander. Significant relationships have been shown between people with a history of atopy and the prevalence of SBS symptoms [6, 31]. Female Gender Female gender is generally associated with reporting higher levels of SBS symptoms [74, 75, 76, 78]. The differences can be as high as 3 to 1 with females consistently reporting higher rates of symptoms than males. These findings have been repeated by other researchers [73, 77]. PSYCHOSOCIAL PHENOMENA Psychosocial phenomena include mass psychogenic illness (MPI), job (dis)satisfaction, occupant density, satisfaction with the physical environment, and seasonal affective disorder (SAD). Job Satisfaction Physical discomfort, repetitive or boring work, under-utilization and career frustration, and poor relationships with co-workers or superiors are all factors that can add to job stress and result in reduced tolerance for substandard indoor air quality. Managers and professional or technical staff are also known to report fewer symptoms than clerical staff. Professional staff often have enhanced accommodation and have been identified as having a greater degree of control over their job with much greater ability to change their environmental conditions [56]. External Stressors Little research has been done on the effects of the domestic environment on the symptoms of SBS. External factors of life stress, such as home and family relations, are particularly important in influencing occupational stress [78]. One study found that smoking, high population-density flats, terraced houses, and building moisture or mold at home were related to a higher prevalence of headache or mucosal irritation [79]. Norback [49] found that childhood exposure to tobacco smoke, childhood and current urban residency, and preschool children at home were found to be related to SBS, with personal factors such as atopy, hyperactivity, nickel allergy, and infection-proneness also related to SBS.

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The Building and Environmental Control Systems Four key elements interact in a building to yield the conditions of the indoor environment. They are the building shell, the HVAC system and its condition, the outdoor environment, and the building's occupants and their activities [54]. Most modern buildings now have a mechanically controlled indoor environment that is provided by a central HVAC system. Environmental Control Systems Environmental control systems are generally considered the major causal factor contributing to occupant health complaints and dissatisfaction with indoor air quality. HVAC systems are designed to provide thermal comfort, to distribute outdoor air to occupants, to remove odors and contaminants through the use of exhaust fans or by dilution, and to control air pressure relationships between rooms [45]. However, these systems represent a considerable risk factor for building-related problems as they can serve to transmit contaminants from their source(s) to areas in a building where they may produce air quality problems. Outdoor Air Ventilation Rates Ventilation systems are of two basic types: naturally ventilated or mechanically controlled. Natural ventilation refers to the intentional displacement of air through openings such as doors and windows. Mechanical ventilation refers to the intentional use of air movers (fans) to bring in outdoor air or to exhaust indoor air [33]. The term ventilation efficiency is used to describe the ability of the ventilation system to distribute supply air and remove internally generated pollutants [45]. The effectiveness of the ventilation with outdoor air can be judged when comparing the elevation of carbon dioxide concentrations indoors to the average ambient outdoor concentration of approximately 325 ppm [19]. However, the focus on CO2 levels also disregards indoor air pollution emitted from sources other than human beings. The first attempt to regulate the indoor air of buildings was to develop a ventilation standard to control body odor and effluents. As a result of the energy conservation movement in the 1970s, the ventilation requirements in nonsmoking areas were reduced to minimum values [6]. In response to more recent interest in building hygiene, revised ventilation requirements have recommended minimum values for office spaces of 10 liters/sec/person by ASHRAE and 11 liters/sec/person by the Nordic Countries [6]. However, Fanger and co-workers proposed that in buildings with a large sensory pollution load (such as new or temporary sick buildings), the ventilation requirements may be as high as 50 liters/sec/person [43]. However, rarely has research investigated the ventilation requirements for providing and maintaining healthy indoor environments [6].

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Many outbreaks of SBS are "cured" by increasing the air exchange and the air flow. The main problem faced by health authorities regarding ventilation is that no one really knows what level of air exchange is necessary to stop SBS complaints from occurring [32]. Meeting current ventilation standards does not necessarily guarantee good indoor air quality and thereby reduce the symptoms of sick building syndrome. Inadequate Maintenance Problems with inadequate maintenance can result in poor operational procedures. These include: • Failure to keep air intakes clean; • Dirty filters not replaced or serviced adequately; • Fouled and contaminated heating and cooling coils; and • Disconnected components such as damper linkages, exhaust fans, and automatic controls [45]. A common problem of poor maintenance are fan motors with worn bearings, or fan belts that overheat and generate detectable odors [54]. Another typical example is the changing of HVAC filters while the air-handling unit fans remain operating. This can result in a concentrated release of particulates into the supply ductwork [54]. Maintenance of local fan coil units and other ventilation system components is also often difficult or impossible to achieve due to poor design and access [19]. Microbes, particulates, insects, rats, dead birds, animal feces, and many other unhygienic objects have been found in badly maintained HVAC systems [80]. Office Materials, Equipment, and Furnishings Investigations of complaint buildings and epidemiological studies have shown the health-affecting or contaminant-generating potential of carbonless copy paper (CCP), other papers, copying machines, VDUs and computer equipment. These products are considered an important source for VOCs, especially when products or materials are new [24]. Some furnishings, such as fabric-covered surfaces, can also act as a sink for VOCs and biological contaminants such as dust mites and molds [81]. Carbonless Copy Paper A variety of studies have implicated handling carbonless copy paper (CCP) or some component of CCP as a causal factor of skin and/or mucous membrane irritation [82], hoarseness, coughing, skin irritation, headache, fatigue [83], and contact dermatitis [84]. Controlled human exposures to the paper have also confirmed

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responses such as upper respiratory congestion, upper airway obstruction, contact urticaria, and acute systemic reactions. Ojfice Machinery Photocopying, the use of laser printers, and working with VDUs are all factors that influence the prevalence of SBS [15]. A major source of ozone, a known irritant, in indoor air is office machinery such as electrical equipment, computer terminals (or VDUs), laser printers, and dry-process photocopiers [81]. Wet-Process Photocopy Wet-process photocopy (WPC) machines have been reported to be a significant source of VOCs, and copier vapor has been reported to account for 90% of total VOC concentrations in a number of buildings [85]. This "older" type of copy process generally produces higher levels of VOCs than the dry-process type copiers. VOC emissions typically arise when a small amount of the solvent is released into the air each time a copy is made [81]. Kerr and Sauer [86] found that approximately 0.332 ml of solvent was released with each copy made. Dry-Process Photocopy In recent years, many office managers have replaced wet-process photocopiers with dry-process machines to reduce the emissions and servicing problems associated with toner and dispersant liquids [81]. However, emissions testing on dryprocess copiers has shown that this kind of equipment can be a significant indoor source of ozone [87, 88, 89, 90]. The dry-copy process typically requires 5 to 10 kilovolts to produce electrical charges and discharges of photoconductive material. This process has been reported as responsible for emissions of ozone [91], selenium, cadmium sulfide, zinc oxide, and organic polymers from the photo-conductive material, and carbon black particulate matter from toner powder in machine exhaust streams [92, 93]. It has been found that photocopying more than 25 sheets per week is a significant risk factor for mucous membrane symptoms [94]. Visual Display Units (VDU) Health and safety concerns associated with using VDUs in office work environments are controversial and represent a number of health concerns. There is general agreement that working with VDUs causes discomfort problems. A number of studies indicating that increasing hours of VDU usage are associated with higher SBS symptom reporting rates [95, 96, 97].

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Floor Coverings Floor coverings are a potentially significant risk factor for building-related health complaints and a source of indoor contaminants [49]. Several factors regarding floor coverings give rise to potentially significant effects on lAQ and human health comfort. These factors include: • The large quantities of materials that are not used; • The large surface-to-volume ratio of materials such as carpets; • The use of composite products that give them their physical and chemical properties; • They require physical or chemical fastening to substrates; and • They require periodic cleaning. Carpets are considered one of the major sources of VOCs indoors [98] and are a risk factor for increased symptom severity among asthmatics. Floor coverings can affect occupant health directly by releasing toxic contaminants into the indoor air, and can act indirecdy by releasing toxic substances from bonding agents, shampoos, and waxes, and by acting as a reservoir or a contaminant sink for VOCs and immunogenic macromolecular dust (MOD). Floor coverings are also known to act as a medium for microbiological contamination and growth. Combination of Causes SBS can no longer be dealt with in terms of simple dose-response relationships, as if it were a case of chemical exposure. A multi-factorial view of SBS is needed and may account for past failure to identify specific causal agents. The most likely underlying cause of SBS is a combination of environmental exposures with direct, indirect, and additive effects. For example, temperature added to humidity has not been shown to cause SBS directly [99], whereas increased temperature and low humidity has been shown to increase out-gassing of chemicals from indoor materials [100]. Osbom et al. [101] found that by raising indoor temperature from 23°C to 40°C, formaldehyde concentrations released from simulated urea formaldehyde foam insulation (UFFI) wall panels could be increased 13-fold. Similar results have been found with VOC emissions [100]. The synergistic and antagonistic relationships between contaminants and other factors are also important. It is now known that even when formaldehyde levels are extremely low, they can still be dangerous by reacting with one or more volatile organic compounds [102]. If activities such as smoking are allowed in buildings, then the entire indoor environment is changed by the many new toxins being introduced. Lercher et al. [41] found a strong correlation between low-level formaldehyde concentrations, unfavorable physical conditions, and SBS.

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CURRENT RESEARCH There are many gaps in the research and problems with design, collation, and interpretation of results. Hedge et al. [78] points out that none of the research from the USA has a theoretical framework, and neither does much of the European work [103]. Until recently, there has been no research on turnover intentions (the reasons people left their workplaces). Few studies have used multivariate statistical analysis, and none has surveyed a broad range of offices. The range of survey questionnaires used makes comparing data complex, and the inherent bias of self-administered surveys has left some researchers skeptical of others' findings. Many surveys have not used comparably designed "healthy" buildings as a baseline. SOLUTIONS TO SBS PROBLEMS Chemicals are ubiquitous in the workplace, and since the data on toxicological and sensory effects of VOCs are incomplete, overall reduction to exposure is the goal. This can be achieved through a variety of strategies including: • Selection of or replacement with low-emitting building materials and furnishings; • Continuous ventilation during and after installation to minimize the occurrence of temporary sick buildings; • A building bake-out method, increasing the temperature and ventilation for several days to speed up the off-gassing of chemicals; • Darkened glass or shading to minimize direct sun entry and thereby reduce episodic elevation of VOCs; • Use of non-toxic glues, pens, correction fluid, and other office materials; • Storing all chemicals, especially cleaning chemicals, in well-ventilated places with the lids secured; and • Dedicated outside exhaust fans near sources of indoor contaminants such as large office equipment. Air cleaning, filtration, and purification of contaminants from indoor air is now viable; however, technological limitations remain, such as the impracticality of removing organic chemicals [104]. Maintenance and repairs should also be done with the least disturbance to the building environment to avoid release of accumulated dust and fibers into the indoor air. Extensive work will require a ventilation strategy for the affected areas, otherwise the whole building may have to be temporarily closed to avoid occupant exposure to contaminants. Recent research is now showing the benefits of proper cleaning practices in reducing SBS. Studies by Raw [14] and Kemp and Dingle [34] have shown the positive benefits of dust mite reduction measures and high-performance cleaning practices applied to carpets and all other fabric-covered surfaces such as chairs and partitions. In particular, the research is showing that normal bag-type vacuum

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cleaners do not remove the smaller fraction of dust (RSP), which is the most biologically important. The appropriate cleaning of hard surfaces has also shown positive benefits in reducing SBS [39]. CONCLUSIONS Problems with indoor air quality arise from many sources in the office environment and contribute to many complaints concerning comfort, health, and well-being. These stressors interact in combination to produce more significant effects. The wide range of potential exposures in indoor environments is likely to cause a correspondingly wide variation in symptoms known as sick building syndrome (SBS). An ideal work environment has been suggested as one with no large changes in temperature or humidity and with no odors. However, ideal conditions have proven to be far more difficult to achieve than engineers and building designers would hope for. Satisfactory indoor air quality is of vital importance for health, comfort, work, and productivity, and is subsequently an important economic consideration. "Healthy" buildings also include comfort and welfare. Ensuring that people are safe at work may be an initial design goal, but the consideration of health should not end there. Once safety is ensured, avoidance of the potential allergenic, irritative, and toxic effects of indoor environments should also be a primary aim in the design, construction, and use of non-industrial buildings. Education as a solution to SBS is the most prudent path to follow. If people understand even the main causative factors of SBS, and are given the means of monitoring, controlling, and alleviating these factors, top-down action on management should not be necessary. The solutions to SBS also involve education on all the factors associated with the building process. REFERENCES 1. Sykes, J. M. "Sick Building Syndrome." Building Services Engineering Research and Technology, 10(1), pp. 1-11. 1989. 2. Spengler, J. D. and Sexton, K. "Indoor Pollution—A Public Perspective." Science, Vol. 221, No. 4605, pp. 9-16. 1983. 3. Turiel, I. et al. "The Effects of Reduced Ventilation on Indoor Air Quality in an Office Building." In: Atmospheric Environment, Vol. 17, No. 1, pp. 51-64. 1983. 4. WHO. Indoor Air Quality Research. EURO Reports and Studies, No. 103, 1986. 5. Raw, G. J. "Sick Building Syndrome." Building Research Establishment, September, EP230, BRE/109/1/3. 1993. 6. Sundell, J. "On the Association Between Building Ventilation Characteristics, Some Indoor Environmental Exposures, Some Allergic Manifestations and Subjective Symptom Reports."//PzJoorA/r. Supplement No. 2/94:9^2. 1994.

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7. Jaakkola, J. J. K., Heinonen, O. P., and Seppanen, O. "Mechanical Ventilation in Office Buildings and the Sick Building Syndrome; An Experimental and Epidemiological Study rindoor Air. No. 2/91:111-121. 1991. 8. Molina, C. et al. Sick Building Syndrome—A Practical Guide. COST 613 Report No. 4 Brussels/Luxembourg: Commission of the European Communities. 1989. 9. Whorton, M. D. et al. "Investigation and Work-up of Tight Building Syndrome." Journal of Occupational Medicine, Vol. 29, pp. 142-146. 1987. 10. Christie, B. Human Factors of Information Technology in the Office, John Wiley & Sons, New York, 1985. 11. Akimenko, W. et al. "The Sick Building Syndrome." In: Berglund, B., Berglund, U., Lindvall, T., and Sundell, J., Eds. Indoor Air. Vol. 6 (Evaluation and conclusions for health sciences and technology). Stockholm: Swedish Council for Building Research, Dl3:87-97. 1986. 12. M0lhave, L. "The Sick Building—A Subpopulation among Problem Buildings." In: Indoor Air '87, Proceedings of the Fourth International Conference on Indoor Air Quality and Climate 2. Berlin. (FP-469-473): Institute for Water, Soil and Air Hygiene. 1987. 13. Hodgson, M. J. et al. "Symptoms and Micro-Environmental Measures in Non-Problem Buildings." Journal of Occupational Medicine, Vol. 33, pp. 527-533. 1991. 14. Raw, G. J. et al. "Sick Building Syndrome: Cleanliness Is Next To Healthiness." Indoor Air. No. 3/93, pp. 237-245. 1993. 15. Kukkonen, E. et al. Indoor climate problems; investigation and remedial measures. Nordest Ventilation Group, Finland. NT TECHN REPORT 204. 1993. 16. Richards, A. "Office Sickness Syndrome." In: Occupational Safety and Health, 16(2): 20-22. 1986. 17. Fisk, W. J. et al. "The Californian Healthy Building Study, Phase 1: A Summary." Proceedings of Indoor Air '93. Vol. 1. 1993. 18. Wallace, L. A. The Sick Building Syndrome: A Review. United States Environmental Protection Agency. Washington, D.C. Report no. 88-110.6. 1988. 19. Kreiss, K. "The Epidemiology of Building Related Complaints and Illness." Occupational Medicine: State Of The Art Reviews. Vol. 4, No. 4: pp. 575-592. 1989. 20. Bergland, B. et al. "Characterisation of Indoor Air Quality and Sick Buildings." A5///M£ Trans. Vol. 90, pt. 1:1,045-1,055. 1984. 21. Pickering, C. A. C. "Links Between Indoor Air Exposures and the Presence of Symptoms: How Well Do We Know Them?" In: Indoor Air, an Integrated Approach. Gold Coast, Australia, p. 37. 1995. 22. Baechler, M. C. et al. "Sick Building Syndrome—Sources, Health Effects, Mitigation." Pollution Technical Review (No. 205), Noyes Data Corporation, USA. 1991.

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23. Wang, T. C. "A Study of Bioeffluents in a College Classroom." ASHRAE Trans. 81:32-44. 1975. 24. Girman, J. R. "Volatile Organic Compounds and Building Bake-out." Occupational Medicine State of Art Reviews 4(4), pp. 695-712. 1989. 25. Tuker, W. G. "Research Overview: Sources of Indoor Air Pollutants." In: Proceedings of lAQ '86: Managing Indoor Air for Health and Energy Conservation. Atlanta, ASHRAE, pp. 8-15. 1987. 26. Dingle, P. W. and Murray, F. "Indoor Air: An Australian Perspective." Indoor Environment. Vol. 2, pp. 217-220. 1993. 27. Kane, L. E. and Alarie, Y. Cited in Godish, T. (1995). Sick Buildings: Definition. Diagnosis and Mitigation. Lewis Publishers, CRC Press, Boca Raton, Florida. 1977. 28. WHO. Indoor Air Pollutants: Exposure and Health Effects. EURO reports and studies, 78:23-26. 1983. 29. Bach, B., M0lhave, L., and Pederson, F. "Human reaction during controlled exposure to low concentration of formaldehyde-performance tests." In: Indoor Air '87: Proceedings of the Fourth International Conference on Indoor Air Quality and Climate. Berlin. Institute for Water, Soil and Air Hygiene. 1987. 30. Hendrick, D. J., and Lane, D. J. "Occupational formalin asthma." Brit. J. Industr. Med. No. 34: 11-18. 1977. 31. Godish, T. Sick Buildings: Definition, Diagnosis and Mitigation. Lewis PubHshers, CRC Press, Boca Raton, Florida. 1995. 32. Gammage, R. B. and Gupta, K. C. "Formaldehyde." In: Indoor Air Quality. CRC Press, 5th ed., Boca Raton. 1987. 33. Yocom, J. E. and McCarthy, S. M. Methods for Measuring Indoor Air Quality: A Practical Guide. John Wiley, West Sussex, and McCarthy. 1991. 34. Kemp, P. C. and Dingle, P. W. "Dust Unkempt." Indoor Air: An Integrated Approach, International Workshop. Gold Coast, Australia. 1994. 35. Hodgson, M. J. et al. "The Sick Building Syndrome." Clin. Res. 37:314. 1989. 36. USEPA. United States Environmental Protection Agency Large Buildings Studies Integrated Protocol. Prepared for: Indoor Air Division of Office of Radiation and Indoor Air, Washington, D.C., and Atmospheric Research and Exposure Assessment Laboratory Office of Modeling, Monitoring Systems, and Quality Assurance. Research Triangle Park, NC 27711. Draft: June 1993. 37. Skov, P. et al. "Influence of Personal Characteristics, Job Related Factors and Psychosocial Factors on the Sick Building Syndrome." Scand. J. Work. Environ. Health. 15:2, pp. 6-95. 1989. 38. Kemp, P. Sick Building Syndrome: Diagnosis and Intervention Studies. Honours Thesis, Murdoch University. Perth, Western Australia. 1995. 39. Leinster, P. et al. "A Modular Longitudinal Approach to the Investigation of Sick Building Syndrome." Proceedings of the Fifth International Conference of Indoor Air Quality and Climate. Toronto. Vol. 1, pp. 292-297. 1990.

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40. Raw, G. J. et al. "A New Approach to the Investigation of Sick Building Syndrome." Proceedings of the CIBSE National Conference, London. 1991. 41.Lercher, P., Hortnagl, J. and Kofler, W. W. "Possible Combined Health Effects in White Collar Workers Caused by Complex Exposure." In: Indoor Air '87: Proceedings of the Fourth International Conference on Indoor Air Quality and Climate. Berlin. Institute for Water, Soil and Air Hygiene. 1987. 42. Rohles, F. H. and Woods, J. E. "Occupant Perception of the Work Environment." Proceedings of the Human Factors Society, 1987. 43. Fanger, P. O. "Introduction of the Olf and Decipol Units to Quantify Air Pollution Perceived by Humans Indoors and Outdoors." Energy and Build. 12, pp. 1-6. 1988. 44. Dingle, P. W. Personal Exposure to Formaldehyde. Doctoral Thesis, Murdoch University. Perth, Western Australia. 1995. 45. USEPA. Building Air Quality: A Guide for Building Owners and Facility Managers. United States Environmental Protection Agency (EPA/ 400/1-91/833) DHNS (NIOSH) Publication No. 91-114, December 1991. 46. Wallbank, C. "Indoor Air Quality: The Design Perspective." Paper presented at Building Owners and Managers Forum, February 1991. Perth, Western Australia. 1991. 47. Andersen, I. et al. "Human Response to 78-Hour Exposure to Dry Air." Arch. Environ. Health. 29:319-24. 1974. 48. Rycroft, R. J. G. and Smith, W. D. L. "Low Humidity Occupational Dermatoses." Cont. Derm. 6:488-92. 1985. 49. Norback, D. Environmental Exposures and Personal Factors Related to Sick Buildings Syndrome. Doctoral Thesis. Uppsala University, Sweden. 1990. 50. Plog, B. A. (Ed). Fundamentals of Industrial Hygiene. 3rd ed.. National Safety Council, Chicago. 1988. 51. Rask, D. R. et al. "Environmental Stressors and System Deficiencies Identified in Problem Office Buildings." In: 83rd Annual Meeting of the Air and Waste Management Association, Pittsburgh, PA. 1990. 52. Abbritti, G. et al. "Sick Building Syndrome: Prevalence in a New Air Conditioned Building." In: Fifth International Conference on Indoor Air Quality and Climate, Vol. 1, Toronto, pp. 513-518. 1990. 53. Sandstrom, M. et al. "The Office Illness Project in Northern Sweden—A Study of Offices with High and Low Prevalence of SBS: Electro-Magnetic Fields in our Indoor Environment." In: Proceedings of the Sixth International Conference on Indoor Air Quality and Climate. Vol. 1, pp. 303-307. 1991. 54. Bearg, D. W. Indoor Air Quality and HVAC Systems. Lewis Publishers, Boca Raton, 1993. 55. Burge, H. A. Bioaerosols. Lewis Publishers, Boca Raton, 1995. 56. Burge, H. A. et al. "Guidelines for Assessment and Sampling of Saprophytic Bioaerosols in the Indoor Environment." Appl. Ind. Hyg., Vol. 2, RlO-16. 1987.

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57. Empey, D. W. et al. "Mechanisms of Bronchial Hyperreactivity in Normal Subjects after Upper Respiratory Tract Infection." Am. Rev. Respir. Dis. 113:131-9. 1976. 58. LaForce, F. M. "Airborne Infections and Modern Building Technology." Environ. Int. 12:137-46. 1986. 59. Brundage, J. F. et al. "Building-Associated Risk of Febrile Acute Respiratory Diseases in Army Trainees." JAMA. 259:2,108-2,112. 1988. 60. Milton, D. K. "Endotoxin." In: Burge, H.A. Bioaerosols. Lewis Publishers, Boca Raton, 1995. 61. Andrews, J. H. and Hirano, S. S. Microbial Ecology of Leaves. Springer-Verlag. New York, 1992. 62. Engelhardt, et al. "Phase I Trial Intravenously Administered Endotoxin (Salmonella Abortus Equi) in Cancer Patients." Can. Res. 51(10):2524. 1991. 63. Enterline, P. E. et al. "Endotoxins, Cotton Dust, and Cancer." Lancet 2(8461):934. 1985. 64. Hodgson, J. T. and Jones, R. T. "Mortality of Workers in the British Cotton Industry in 1968-1984." Scan. J. Work Environ. Health. 16(2): 113. 1990. 65. Rylander, R. "Environmental Exposures with Decreased Risks for Lung Cancer?" M. J. Epidemiol. 19(1):567. 1990. 66. Levetin, E. "Fungi." In: Burge, H. A. Bioaerosols. Lewis Publishers, Boca Raton, 1995. 67. Burge, H. A. "Bioaerosols: Prevalence and Health Effects in the Indoor Environment." J. Allergy Clin. Immunol. 86:687. 1990. 68. Morey, P. R. and Jenkins, B. A. "What are Typical Concentrations of Fungi, Total Volatile Organic Compounds and Nitrogen Dioxide in an Office Environment?" In: lAQ '89. The Human Equation: Health and Comfort Proc. of ASHRAE. Geshwiler, M., Moran, M. and Montgomery, L. Eds. 1989. 69. Tyndall, R. L. and Vass, A. A. "The Potential Impact on Human Health from Free Living Amoebae in the Indoor Environment." Burge, H.A. Bioaerosols. Lewis Publishers, Boca Raton, 1995. 70. Andersen, I. and Korsgaard, J. "Asthma and the Indoor Environment— Assessment of Health Implications of High Indoor Humidity." In: Proceedings of the Third International Conference on Indoor Air Quality and Climate. Stockholm. Vol. 1, pp. 79-86. 1984. 71. Hung, L. L. et al. "Biocontamination on Fabric Modular Office Panels." In: Indoor Air: An Integrated Approach. Gold Coast, Australia, p. 50. 1995. 72. Baker, D. B. "Social and Organisational Factors in Office Building-Associated Illness." Occupational Medicine: State of the Art Reviews, Vol. 4, No. 4, pp. 607-624. 1989. 73. Skov, P. and Valbj0m, O. "The Sick Building Syndrome in the Office Environment." In: Indoor Air '87, Proceedings of the Fourth International Conference on Indoor Air Quality and Climate. Berlin. Institute for Water, Soil and Air Hygiene. 1987.

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74. Hedge, A. R. et al. "Psyhosocial Correlates of SBS." In: Proceedings of the Sixth International Conference on Indoor Air Quality and Climate. Helsinki. Vol. 1, pp. 345-350. 1993. 75. Lenvik, K. "Sick Building Syndrome Symptoms; Different Prevalences Between Males and Females." Environmental International. Vol. 8, pp. 11-17.1992. 76. Mendell, M. J. "Non-Specific Symptoms in Office Workers: A Review and Summary of the Epidemiological Literature." Indoor Air, Vol. 3, pp. 227-236. 1993. 77. Taylor, P. R. et al. "Illness in an Office Building with Limited Fresh Air Access." /. Environ. Health. 47:24-7. 1984. 78. Hedge, A. et al. "Work-Related Illness in Offices: A Proposed Model of the Sick Building Syndrome." Environ. Int., 15:143-158. 1989. 79. Valbjom, O. and Kousgard, N. "Headache and Mucous Membrane Irritation: An Epidemiological Study." In: Berglund, B., Lindvall, T., and Sundell, J. (Eds). Indoor Air, Vol. 2. Radon, Passive Smoking, Particulates and Housing Epidemiology. Stockholm: Swedish Council for Building Research, D17:249-54. 1984. 80. Robertson, A. and Burge, S. "Building Sickness—All in the Mind?" Occup. Health, pp. 78-81. March 1986. 81. Etkin, D. S. Office Furnishings, Equipment and lAQ: Health Impacts, Prevention and Mitigation. Cutter Information Corp. Arlington. 1992. 82. Morgan, M. S. and Camp, J. S. "Upper Respiratory Tract Irritation from Controlled Exposure to Vapour from Carbonless Copy Forms." Journal of Occupational Medicine, 28:41-49. 1986. 83. Marks, J. G. et al. "Contact Urticaria and Airway Obstruction with Carbonless Copy Paper." JAMA. 262:1,038-1,040. 1984. 84. Marks, J. G. "Allergic Contact Dermatitis from Carbonless Copy Paper." JAMA. 245:2331. 1981. 85. Tsuchiya, Y. and Stewart, J. B. "Volatile Organic Compounds in the Air of Canadian Buildings with Special Reference to Wet Process Photocopying Machines." Proceedings of Fifth International Conference on Indoor Air Quality and Climate. Vol. 2. Canada Mortgage and Housing Corporation, Ottawa, pp. 633-638; and Stewart, 1990. 86. Kerr, G. and Sauer, P. "Control Strategies for Liquid Process Photocopier Emissions." In: Indoor Air '90—The Fifth International Conference on Indoor Air Quality and Climate. Toronto, Canada. 3:759-764. 1990. 87. Sutton et al. "Predicting Ozone Concentrations in Residential Structures." ASHRAE Journal, pp. 21-26, Sept. 1976. 88. Allen, R. J. et al. "Characterisation of Potential Indoor Sources of Ozone." Am. Ind. Hyg. Assoc. J 39:456-466. 1978. 89. Selway, M. D. et al. "Ozone Production from Photocopying Machines." Am. Ind. Hyg. Assoc. J 41:455-459. 1980.

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90. Wadden, R. A. and Scheff, P. A. Indoor Air Pollution: Characterization, Prediction and Control New York, New York: John Wiley and Sons, p. 213. 1983. 91. Braun-Hansen, T. B. and Andersen, B. "Ozone and Other Air Pollutants from Photocopying Machines." Am. Ind. Hyg. Assoc. J. 47:659-665. 1986. 92. Markin, J. M. et al. "Elevation of Selenium Levels in Air by Xerography." Nature. 259:204-205. 1976. 93. Parent, R. A. "Elevation of Selenium Levels in Air by Xerography." Nature. 263:708. 1976. 94. Skov, P. et al. "Influence of Indoor Climate on the Sick Building Syndrome in an Office Environment." Scand. J. Work. Environ. Health. 16:363-71. 1990. 95. Zweers, T. et al. "Health and Indoor Climate Complaints of 7043 Office Workers in 61 Buildings in the Netherlands." Indoor Air. Vol. 2, pp. 127-136. 1992. 96. Nelson, C. J. et al. "EPA's Indoor Air Quality and Work Environment Survey: Relationships of Employee's Self-Reported Health Symptoms with Direct Indoor Air Quality Measurements." Proceedings of Healthy Buildings. Washington, D.C. pp. 22-32. Adanta: ASHRAE. 1991. 97. Wallach, C. "Video Display Health Hazard Safeguards." In: Proceedings of the Third International Conference on Indoor Air Quality and Climate. Stockholm. Vol. 3. 1991. 98. Levin, H. "Building Materials and Indoor Air Quality." Occupational Medicine: State of the Art Reviews. 4:667-93. 1989. 99. Morris, L. and Hawkins, L. "The Role of Stress in Sick Building Syndrome." In: Indoor Air '87: Proceedings of the Fourth International Conference on Indoor Air Quality and Climate. Berlin. Institute for Water, Soil and Air Hygiene. 1987. 100. Volkl, S., Gebfiigi, I. and Korte, F. "Emission of VOC from Coatings into Indoor Air." In: Indoor Air '90: Proceedings of the Fifth International Conference on Indoor Air Quality and Climate. Ottawa. Institute for Water, Soil and Air Hygiene. 1990. 101. Osbom, S. W. et al. "Urea-Formaldehyde Foam Insulation Study in Indoor Air Quality." CRC Press, 5th Edition, Boca Raton, 1987. 102. Berglund, B. and Lindvall, T. "Sensory Reactions to Sick Buildings." Eviron.Int. 12:147-159. 1986. 103. WHO. Indoor Air Pollutants: Exposure and Health Effects. EURO Reports and Studies, 78:23-26. 1983. 104. Hedge, A. R. et al. "Breathing Zone Filtration Effects on Indoor Air Quality and Sick Building Syndrome Complaints." In: Proceedings of lAQ '91: Healthy Buildings. ASHRAE. Atlanta, pp. 351-355. 1991.

CHAPTER 5 RESPIRATORY FINDINGS OF CONSTRUCTION WORKERS EXPOSED TO ASBESTOS DUST Isamu Ebihara The Institute of Science of Labour Kawasaki, Japan Mamoru Hirata Osaka Prefectural Institute of Public Health Osaka, Japan Naomi Hisanaga National Institute of Industrial Health Kawasaki, Japan Eiji Shibata Nagoya University School of Medicine Nagoya, Japan Kiyoshi Sakai Nagoya City Public Health Research Institute Nagoya, Japan

CONTENTS ASBESTOS UTILIZATION IN THE CONSTRUCTION INDUSTRY, 94 Asbestos-Containing Construction Materials, 94 Levels of Asbestos Exposure, 94 Delay of Measures of Occupational Safety and Health in the Construction Industry, 98 REGULATION OF ASBESTOS, 102 Asbestos Regulations in Different Countries, 102 ILO Convention No. 162 and Recommendation No. 172, 103 RESPIRATORY DISORDERS DUE TO ASBESTOS AMONG CONSTRUCTION WORKERS, 105 Asbestos Burden in Lung Tissues of Construction Workers, 105 Subjective Symptoms, 108 Pleural Plaques and Asbestosis Among Construction Workers, 108 Cancers of the Trachea, Bronchus, and Lung, 111 Malignant Mesothelioma, 113

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MEASURES AGAINST RESPIRATORY DISORDERS DUE TO ASBESTOS EXPOSURE, 118 Prevention of Asbestos Exposure, 118 Health Control, 120 REFERENCES, 123 ASBESTOS UTILIZATION IN THE CONSTRUCTION INDUSTRY Asbestos-containing Construction Materials Chrysotile and amphiboles (including crosidlite, amosite, and ansophylite) have traditionally been used in asbestos-containing construction materials. Today, however, due to regulations restricting their use, chrysotile is mainly used. The regulations include prohibition of crosidlite and often amosite, also. There is even a total ban of the utilization of asbestos in some European countries. Asbestos exposure from construction materials results from the use of cementreinforcement materials and insulatory materials against sound, heat, and fire (e.g., asbestos-containing materials are required by law for use as fireproof construction materials by the Building Standards Law in Japan). After asbestos control regulations came into effect, such materials began being replaced by man-made mineral fibers (MMMF), including fiberglass and rock wool, but asbestos continues to be used in cement-reinforcement and fireproofing materials, many of which are asbestos cement-made materials. In 1995, about 90% of asbestos consumption was accounted for by asbestos cement (A/C) products (asbestos slate, asbestos perlite, roofing tiles, etc.) [1]. For example, about 42,000 tons of asbestos was consumed as asbestos slate in Japan in 1994 (21% of total consumption of about 199,800 tons) [2]. Fireproof construction materials certified by the Ministry of Construction of Japan from 1989 to 1994 showed a decrease in the content of asbestos, which ranged from 2% to 20%. Also, the number of asbestos-containing materials certified by the Ministry of Construction of Japan has decreased: two types in 1994 and 1992, three in 1993, compared with 23 types in 1987, 16 in 1986, and 17 in 1988 (Table 1) [3]. Accordingly, the risk of asbestos exposure among construction workers in the 1990s should have been lower than in the 1980s. However, the total amount of asbestos utilization did not decrease, indicating that old types of asbestos-containing construction materials might still be in use with or without transformation. Levels of Asbestos Exposure Table 2 shows the available data of airborne asbestos concentrations associated with construction work [4-13]. In home construction, the asbestos exposure of workers ranged widely from 0.04 to 787 fibers/ml depending on the type of work and the ventilatory conditions. Indoor sawing with an electric circular saw is considered to be one of the most hazardous operations [6] (Figures 1 and 2).

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Table 1 Asbestos-containing fire-proof construction materials certified by the ministry of construction of Japan from 1989 to 1994, and asbestos contents Number of materials

Item

Fiber-mixed cement board Fiber-mixed calcium silicate board Fiber-mixed asbestos cement board Fiber-mixed slug cement board Fiber-mixed slug plaster board Fiber-mixed cement calcium silicate board Asbestos cement calcium silicate board Asbestos cement board Asbestos perlite slug plaster board Asbestos slate Fly ash mixed slug cement board Calcium silicate board

J.

Total

28

Range of content

4-5% 14-22.5 17 0.5 4.5 4.5 17 20.5 5 5 4.5 5

2

Source: Department of Construction Leading, Bureau of Housing, Ministry of Construction of Japan, Handbook of fireproof structure and materials, 1995 [3].

Table 2 Airborne concentrations of asbestos fibers in construction work

Reference

Application

Number of samples

Concentration (Mathematical mean, fibers/cm^) (range)

10 3 2 11 2 2 2 3 2

10.0(1.2-19.3) 8.6(3.5-19.8) 4.8 (0.7-8.8) 5.3(1.3-16.9) 2.3(2.1-2.5) 4.3(1.5-7.1) 47.2.(35.4-59.0) 5.8(0.5-13.1) 2.6(2.1-3.1)

1 1

41.4 26.4

Drywall construction

Fischbein et al. (1979) USA [4]

- pole sanding (3-5 ft.) background (8 ft.) same room background (25 ft.) adjacent room - hand sanding (3-5 ft.) background (8 ft.) same room background (15 ft.) adjacent room - dry mixing (3-5 ft.) background (10-20 ft.) same room background (16-35 ft.) adjacent room - sweeping floor (10-50 ft.) 15 minutes after sweeping 35 minutes after sweeping

(table continued on next page)

96

Health and Toxicology

Table 2 (Continued)

Reference

Number of samples

Application

Concentration (Mathematical mean, fibers/cm^) (range)

Drywall tapping process Verma& Middleton (1980) Canada [5]

- application - mixing (dry powder) - mixing (pre-mix) - mixing area (pre-mix) - hand sanding - pole sanding - sanding area - sweeping

10 3 7 7 22 52 10 10

3.2 (0.3-7.0) 15.1 (4.0-26.5)

4 3 8

214(125-787) 245 (103-630) 11.0(1.3-1319)

7 8

5.4(0.9-48.1) 2.0(0.3-14.1)

15 1 2

1.3(0.1-4.6) 12.1 0.08(0.04-0.12)

5

0.3(0.1-0.5)

12 8

6.67*1

3

1.10*1

2 2

0.19*1 3.07*1

22

8.5(0.1-24.3)

45

6.4(0.1-61.6)

0.9(0.4-1.3) 11.2(9.0-12.4) 2.4(1.2-3.2) 2.0(1.2-2.7) 11.5(2.1-24.2) 4.9(1.2-10.1)

Home construction work sitek Hisanaga etal. (1991) Japan [6]

- Sawing construction boards - 1.5-2 m from the above work - Screwing, drilling, or nailing (partly including sawing) - 1-10 m from the above work - Screwing, drilling, or nailing (not including sawing) - 1-4 m from the above work - Cutting and filing - 5-30 m from the work operating board - Finishing or cleaning (1 to 7 days after the work) Home construction work sitek

Sakai etal. (1993) Japan [7]

- Sawing - Screwing, drilling, nailing, cutting. or filing (partly including sawing) - Screwing, drilling, or nailing (not including sawing) - Cutting or filing (not including sawing) - Boarding

leY^

Insulation Balzer & Cooper (1968) USA [8]

- Prefabrication: precut and shaped materials using hands or power tools - Application: fitted, hammered, or curved and attached materials to surface by wiring or gluing

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

- Finishing: coating materials with asbestos-containing cements, resins, asbestos or cotton cloth, or petroleumbased sealers - Tearing out: removal of old or unusable materials in the process of insulating or reinsulating - Mixing: mixing mineral wool, asbestos, fibrous glass, and cements or glues - General: cleaning up of old insulation, transporting of materials (1 to 7 days after the work using board)

31

2.7 (0.1-24.4)

17

8.9 (0.2-26.3)

22

2.6 (0.2-10.7)

16

4.8 (0.1-22.9)

97

Renovation and demolition of asbestos-cement (A/C)-clad buildings

Brown (1987) Australia [9]

- Cleaning A/C roofing with water jets - Painting weathered A/C sheeting • by roller • by airless spray - Replacing weathered A/C roofing - Removing weathered A/C roofing - Demolishing A/C warehouses

2

0.08-0.10

3 6 27 20 40

0.12-0.22 0.01-0.14 0.03-0.27 <0.03-0.32 <0.03-l.l

Installing track light in the ceiling Sawyer (1977) USA [10]

- Sprayed with asbestos • electricians (4 ft. track light) • electricians ( 2 x 4 ft. lighting unit) • carpenters (4 ft. partition) - Removal of 8 x 12 ft. ceiling section • dry: no preparation • wet: untreated water • wet: amended water

6 5

7.7 (2.9)*2 1.1 (0.8)*2

4

3.1(1.1)*2

11 6 10

82.2 (24.7)*2 23.1 (4.9)*2 8.1(4.6)*2

Renovation and removal of sprayed asbestos

Paik et al. (1983) USA [11]

- Carpenter - Electrician - Sheet-metal worker -Painter

105 35 37 7

0.131 0.131 0.191 0.081

(3.46)*3 (3.23)*3 (4.05)*3 (2.38)*3

Dry and amended wet removal

Sawyer et al. (1985) USA [12]

- Dry removal: scraping dry friable material from structural surface - Wet removal: scraping wet friable material from structural surface following application of amended water - Bagging: initial cleanup and bagging of removed debris in a wet removal project

38.9(8.1-117.8) 226

1.1 (0.0-37.1) 3.9(0.0-8.1)

(table continued on next page)

98

Health and Toxicology Table 2 (Continued)

Reference

Application

Number of samples

Concentration (Mathematical mean, fibers/cm^) (range)

Spraying of thermal insulation Reitze etal. (1972) USA [13]

• man at nozzle (1) • man at nozzle (2) • man charging hopper - Spraying of fireproofing materials • man at nozzle (3) • man at nozzle (4) - Distance from nozzle man - 10 ft. - 15 ft. - 20 ft. - 35 ft. - 75 ft. 30 minutes after spray operation 60 minutes after spray operation

4 4 4

52.0 (24-85) 61.0(32-100) 10.8 (5-22)

5 2

35.2 (20^9) 96.8 (94.5-99.0)

2 1 2 1 1 4 5

70.5 (70-71) 17 51.8(37.6-66.0) 10 46 1.98(1.01^.22) 0.47 (0.26-0.76)

*^, Geometric mean; *^, mathematic standard deviation "^y geometric standard deviation Measuring method: Phase contrast optical microscopy; length-diameter ratio, >3:1; length, >5 jum; diameter, <3 pm (Balzer & Cooper [8] appliedlength-diameter ratio >3:1 and diameter <3.5 pm)

Airborne asbestos concentrations during renovation and demolition considerably vary depending on the type of construction materials (sprayed materials or fixed materials) and the method of application (dry or wet). Sprayed asbestos was extensively used as fireproof material, acoustic and thermal insulation, and decoration in the 1940s and 1970s. In the 1970s, the mean concentration during the spraying of asbestos for insulation ranged was as high as 10 fibers/ml [13]. These data show that construction workers may be exposed to high levels of asbestos fibers. However, the time-weighted average (TWA) exposure of construction workers is probably less than the mean concentrations during application because the majority of the workers only intermittently handle asbestos materials depending on their employment and the type of construction site. Delay of Measures of Occupational Safety and Health in the Construction Industry Outline of Construction Work and Occupational Health Many people work in the construction industry, including those self-employed, but statistical data do not distinguish between building construction and civil (or

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

99

Figure 1. Cutting asbestos-containing wallboard with an electric circular saw without local exhaust ventilation system. Airborne asbestos concentration during the work was more than 100 f/ml at the respiratory rate.

public) engineering work. Accordingly, our description of people in construction work includes civil engineering work. There were 8.118 million workers (about 6% of the economically active population of 128.54 million) in the United States of America in 1992, and 273,000 workers (about 6% of the economically active population of 4.46 million) in Sweden in 1992 engaged in construction work [14]. In Japan, 6.7 million workers (about 10% of total number of economically active population of 66 million), including 900,000 self-employed workers and 300,000 of their family members, are engaged in construction work from house-making to civil

100

Health and Toxicology

Figure 2. Fixing ceiling board to metal stud with electric screwdriver. Airborne asbestos concentration during the work was 8.8 f/ml at the respiratory zone.

engineering [15]. However, a large number of them are small-scale enterprises (Table 3), and workers are relatively older than those in other industries (Table 4) [16]. Adequate occupational health measures are often lacking in small-scale enterprises and among self-employed workers. Types of work included in construction are carpentry, floorlaying, roofing, siding and concrete work. (See Table 5) [17]. Since some types of workers frequently simultaneously worked in the same workplace, various types of workers are exposed to the same hazard. Insufficient Occupational Safety and Health Measures in the Construction Industry In the construction industry, occupational safety is given greater importance than occupational health because accidents are more frequent and more serious than occupational diseases. Health hazards in construction include respirable dust, such

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

101

Table 3 Scale structure of labor force of the Japanese construction industry

Scale (Number of employees)

Construction workers

All workers

1-4 5-29 30-99 more than 99

0.73 million (14.0) 2.51 (48.0) 0.85 (16.3) 1.12 (21.4)

3.93 million (7.6) 13.12 (25.2) 8.26 (13.9) 21.36 (41.1)

Total

6.45

66.66

(100.0%)

(100.0%)

Source: Japan Construction Safety and Health Association, Yearbook of safety and health in construction industry 1994,/?. 176 [16]. Table 4 Age structure of labor force of the Japanese construction industry Age class

Construction workers

15-29 years 30-44 45-54 Older than 55 years

1.27 million 2.12 1.59 1.43

Total

6.45

(19.8%) (33.1) (24.8) (22.3)

(100.0%)

All workers

15.45 million (24.0%) 20.68 (32.1) 14.82 (23.0) 13.92 (21.6) 66.66

(100.0%)

Source: Japan Construction Safety and Health Association, Yearbook of safety and health in construction industry 199A, p. 177 [16].

as asbestos and silica; liquids, gases, and fumes arising from the use of volatile organic solvents in paint, adhesives and preservatives, resins, strong acids and alkalis; noise; vibration; and radiation or artificial environments such as compressed air [18]. Occupational diseases due to those hazards in the construction industry, including civil engineering work, are more numerous than in other industries (acute poisoning due to organic solvents, 45% of all patients; pneumoconiosis, 33%; vibration syndrome, 45% of all patients in Japan, (Table 6) [19]). In Japan, abnormal findings in auditory examinations were recorded for 9.96% of the examined construction workers (7.87% of all examined workers), and abnormal findings were found in 14.2% of the examined construction workers (10.5% of all examined workers) [19]. Pneumoconiosis and vibration disease are mainly found in civil engineering workers. Asbestos exposure in civil engineering work are much smaller than that in building construction because there is hardly any need for fireproofing or sound and heat insulation materials. Construction workers handling asbestos-

102

Health and Toxicology Table 5 List of types of construction work General building contractors

Single-family houses Other residential buildings Operative builders Industrial buildings and warehouses Non-residential buildings Special trade contractors

Plumbing, heating, air conditioning Painting and paper hanging Electrical work Masonry and other stonework Plastering, drywall, insulation Terrazzo, tile, marble, and mosaic work Carpentry Floorlaying and other floor work Roofing, siding, and sheet metal work Concrete work Structural steel erection Glass and glazing work Excavation work Wrecking and demolition work Source: U. S. Department of Commerce Economics and Statistics Administration, Bureau of Census, Statistical abstract of the U.S. 114th edition, 1994, p. 725 [17].

containing materials, excluding those in civil engineering work, usually belong to small-scale enterprises or are self-employed, and health and safety measures are often inadequate (see Figure 3) [20]. REGULATION OF ASBESTOS Asbestos Regulations in Different Countries There are no special regulations regarding asbestos exposure of construction workers, except for demolition workers exposed to asbestos dust, although there is a total ban in some European countries. Various regulations are found around the world [21].

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

103

Table 6 Incidence of certified occupational diseases among construction workers and all workers in Japan

Disease Lumbago (1993) Pneumoconiosis (1993) Vibration syndrome (1993) Organic solvent poisoning (1984-1993)

All workers

Construction workers

Rate of construction/all workers

9,630 persons 1,025 405

1,661 persons 344 179

17.2% 33.6 44.2

486

221

45.9

Source: Japan Construction Safety and Health Association, Yearbook of safety and health in construction industry 1994, pp. 99-108 [19].

1. Total ban: Sweden, Denmark, Netherlands, Norway, and Germany. 2. Prohibition of amphiboles and spraying, and limitation of chrysotile (according to EEC directive, 83/477 in 1983, 87/217 in 1987, 91/382 and 91/659 in 1991): EU countries (France, Greece, Spain, UK and Portugal), Japan (amosite is also banned). Newly industrialized areas (NIES) including South Korea, Taiwan, Singapore, and Hong Kong are adopting similar regulations. Czech, Hungary, and former "socialist" countries follow same regulations. 3. Prohibition of import: Nigeria, Saudi Arabia, Sri Lanka. 4. USA: After the overturn of the U.S. Environmental Protection Agency's 1989 ban rule by the U.S. Court of Appeals on October, 18, 1991, prohibited products are as follows: corrugated paper, commercial paper, specialty paper, rollboard, flooring felt, and new uses of asbestos. ILO Convention No. 162 and Recommendation No. 172 The Convention and Recommendation described the principles of regulation of asbestos. The description by the Convention is as follows: technological control and work style to prevent asbestos exposure (§9), regulation of utilization of asbestos products (§9), replacement (§10), total or partial prohibition in some work processes (§10), prohibition of crocidolite (§11), prohibition of spraying (§12), establishment of exposure standards to evaluate work environment (§15), use of respiratory protective equipment in cases where reduction of asbestos exposure is not achieved (§15), responsibility of the employer (§16), regulations with respects of removing asbestos from buildings (§17), monitoring of the work environment and health of workers (§20). Additional items in the Recommendation are as follows: subjecting selfemployed workers to the regulations (§1), special attention to workers younger than

104

Health and Toxicology

Asbestos-related diseases Delay of occupational health countermeasure Difficulty in organizing—| occupational health activity

•Insufficient administrative assistance "Subcontract system

Self-employedworker Small-scale • establishment

CONSTRUCTION WORKER

-Obscure employeremployee relationship -Apprentice system

i:

Asbestos dust

Lack of risk assessment before introducing new technique

Asbestos-containing construction material

Noise, Vibration, Injury

Electric tool (Circular saw. Screwdriver, etc.) i

Demand of Low cost & High productivity Figure 3. Factors that contribute to asbestos exposure in the construction industry.

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

105

18 years (§1), information from employers to authoritative organization on asbestos control (sort, amount, work process, products, number of exposed workers, level of exposure, preventive and protective measures, information of health protection (§13), participation of exposed workers in the measures against exposure (§15), preservation of records of exposure (measured by time-weighted average) for 30 years (§30), contents of periodical health examination of exposed workers, and preservation of records of health examination and chest X-ray films for 30 years after removal from the work (§36). RESPIRATORY DISORDERS DUE TO ASBESTOS AMONG CONSTRUCTION WORKERS Asbestos Burden in Lung Tissues of Construction Workers Asbestos lung tissue burden of construction workers was reported to be almost 12-20 times higher than that of referent workers who were probably not exposed to asbestos [22, 23]. Kawami and Ebihara (1991) studied the asbestos fiber concentration in the lung tissues from 67 subjects obtained by autopsy or surgical operations [22]. The asbestos fiber burden was estimated by means of light microscopy (for ferruginous body) and scanning electron microscopy after lung tissues were digested by lowtemperature plasma ashing method. The fiber type was confirmed by the energy dispersive X-ray analysis system [24]. The asbestos fiber concentration of construction workers (group VI: 11 cases) were 12 times higher in median values than those of group IV, who were probably not exposed to asbestos [group I (housewives); group II (whitecollar workers); group III (farmers); group IV (park scavengers); group V (printers)] and were the same as shipyard welders (group VII) or longshoreman (group VIII) (Figure 4). In 91% (10/11) of the construction workers, the fiber concentration exceeded 1 million fibers per gram dry tissue, but in only 15% (3/20) of the referent group (group IV). It should be stressed that tremolite, anthophyllite, and actinolite were found in lung tissues of all 11 construction workers, and amosite was found in 82% (9/11) of the construction workers. The ferruginous body concentrations of construction workers were 56 times higher than that of referent groups (Figure 5). In a series of 65 surgically treated lung cancer patients, Karjalainen et al. reported the findings evaluated by scanning electron microscopy analyses of mineral fibers in lung tissue [23]. Lung tissue samples of 17 autopsied male office workers were analyzed as a referents. The fiber concentration in the lung tissue ranged from <0.1 to 15 million fibers per gram dry tissue in the nine lung cancer patients with a history of at least 10 years in construction occupations, including carpenters, electricians, sheet metal

106

Health and Toxicology

•• lO''

• •

• •• •• ••

10*

•

10* O

10^

A

10*

E ^ -

A

u

A

I

n m

IV V VI

vu

VI

m

iv

V

•

••

•

••

•

• •

••

o o

AA

A

I

o

A

A A A

•

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VI

•1

_.J VE IX X XI

housewives white-collar workers farmers park-scavengers printer, analyst, etc construction workers shipyard welders longshoremen

xn

XI

_J

_J VI

IX

X

XI

XII

:

lining manufactures asbestos sprayers asbestos textile workers asbestos cement workers talc workers, railway coach repairman (jobs with probable asbestos exposure)

(-)

or unknown A O : pl.pq. • : pl.pq. ( + )

Figure 4. Total asbestos fiber concentrations in 13 occupational groups (by scanning electron microscopy) Kawami and Ebihara [24].

workers, plumbers, and demolition and unspecified assistant housebuilding workers. On the other hand, the concentration ranged from <0.1 to 0.8 million fibers per gram dry tissue in the reference group. In 77% (7/9) of the lung cancer patients with a history of construction jobs, but in none of the referents, the fiber concentration exceeded 1 million fibers per gram dry tissue. Predominant fiber types found in the lung tissues of the patients of construction workers were anthophyllite, amosite, and crocidolite. Only a few chrysotile fibers were found.

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

107

]0*

F

• • •

10^ r L 10-

I

I—

I *

P I

I

*

i

M F I

j

k F

F 10*

*

I * I

• •' ••

I

r

10*

I

*

r

10'

**

° •

\

E

M

r '^ L

^

\

^

A O o \

o

I

I

I

*

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\

•

A

AAAA

I

*

I

\ ^ \

'^M^^''

h r

:

A

A

•o

n m i v v v i w Y i i x x x i x n x f l i housewives white-collar workers farmers park-scavengers printer, analyst, etc construction workers shipyard welders longshoremen

DC X JJ Xn Xffl

lining manufactures asbestos sprayers asbestos textile workers asbestos cement workers talc workers, railway coach repairman (jobs with probable asbestos exposure)

A O : pl.pq. ( —) or unknown : pl.pq. ( + )

Figure 5. Ferruginous body concentrations in 13 occupational groups (by light microscopy) Kawami and Ebihara [24].

Anttila et al. (1993) studied exposure to asbestos, pulmonary fibrosis, fiber count, and fiber size in the relation to the labor origin of lung cancer in 90 consecutive patients [25]. Among the 32 patients with a history of occupational exposure to asbestos, 22 were construction worlcers. The proportion of lower lobe tumors increased with the duration of exposure from 45% in those worlcing less than 15 years to 82% in those working 15 years or more in the construction trade, as compared with 25% in patients who were probably not exposed. The location of the tumor in the lower lobe was explained by the high number of total fibers (odds ratio

108

Health and Toxicology

[OR] = 9.0, CI = 2.3-34.6), of fiber 3 |im and longer (OR = 22.1, CI = 3.9-125), and fibers of anthophyllite (OR = 14.6, CI = 2.4-83.4) and crocidolite (OR = 7.0, CI = 1.2-41.2) when the effect of smoking and fibrosis was adjusted in the regression analysis. The location of the tumor did not correlate with fibrosis, pack-years, or the number of short (<3 |im) fibers. These findings suggest that the asbestos burden in the lung tissues of construction workers were enough to cause an excess of lung cancer and malignant mesotheliomas, although the occupational exposure to asbestos was intermittent or exposure levels were relatively low. Subjective Symptoms Some surveys on respiratory symptoms among construction workers exposed to asbestos have been conducted. The survey by Matsunaga et al. [26] showed that male construction workers who currently smoked and experienced frequent exposure to asbestos dust significantly complained more of shortness of breath, palpitation, sputum, and cough compared with male construction workers who currently smoked but were not exposed to asbestos and also compared with non-smoking male workers or those who had quit smoking (Table 7). This survey suggested that asbestos caused these complaints, but it might have been biased by low number of respondents (28.4%). Since these symptoms are generally observed among various kinds of dust work, it is not specific to asbestos exposure. Hisanaga et al. described similar conditions of construction workers exposed to asbestos (Table 8) [6]. Pleural Plaques and Asbestosis Among Construction Workers Asbestos-associated diseases are prevalent among workers employed in the construction trades. Workers in these trades are exposed to asbestos directly through their own work activities, and indirectly by inhaling airborne asbestos fibers generated by others working nearby on construction sites. Construction insulators are a high-risk group for asbestos-related lung disease. From a prevalence survey of pulmonary health parameters among current and retired construction insulators (50 years and older) from British Colombia, Canada [27], pleural abnormalities alone were found in 34% of active workers and 45% of non-active workers. Rates for parenchymal abnormalities for these same groups (ILO classification 1/0 or higher) were 17% and 20%. Sheet metal workers in the United States are employed in the fabrication and installation of heating, air conditioning, and ventilation ducts. Asbestos exposure occurred as sheet metal workers removed or distributed asbestos from ceilings or girders in order to install new ductwork. The extent of asbestos-related disease in this population of sheet metal workers is substantially higher than that generally found in unexposed populations. Baker et al. (1982) found 49 (49%) pleural thickening cases among 100 unselected individu-

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

109

Table 7 Rates of respiratory complaints for workers according to smoking habits

Symptom Palpitation

Shortness of breath Cough

Sputum

Prolonged cold symptoms

Class of exposure

Current smoker

Ex-smoker

Non-smoker

Frequent Moderate Rare No Frequent Moderate Rare No Frequent Moderate Rare No Frequent Moderate Rare No Frequent Moderate Rare No

34*** 23** 17 14 52*** 42** 31 30 59*** 55*** 41 39 75*** 50*** 49 44 3j*** 18 15 15

3g*** 26 17 23 46 39 38 35 55*** 43* 22 29 56*** 48 31 32 31** 24 14 15

29** 28** 12 12 4j*** 29 35 19 50*** 36* 26 23 5j*** 38* 26 22 27** 17 9 12

*, p<0.05; '^'^, p<0.01; ***, p<0.001; tested by chi square test between unexposed and exposed workers. Source: Matsunaga et al, A questionnaire survey on asbestos exposure and symptoms among construction workers, 1990 [26].

als who had worked for more than 10 years in their trades [28]. Baker et al. (1985) also found 152 workers (51%) had pleural abnormalities among 314 white male members of a local sheet metal workers union [29]. The prevalence of pleural abnormalities increased with duration of employment as a sheet metal worker, ranging from 6.3% in workers with 10 or fewer years in the trade to approximately 70% in individuals with more than 30 years of experience. Selikoff and Lilis also found the prevalence rate of pleural fibrosis in 47% among 1,330 sheet metal workers in the United States and Canada [30]. A total of 1,016 workers had been employed for at least 35 years in the industry, and the mean duration from onset of asbestos exposure was 39.5 years. The parenchymal changes (ILO classification 1/0 or higher) was present in 35%, and radiologic abnormalities increased as duration of exposure increased. Michaels et al. reviewed chest X-rays of 707 currently employed New York metropolitan area sheet metal workers and found that 10.9% of the workers had

110

Health and Toxicology

Table 8 Relationship between mean days of handling asbestos-containing materials per month in the previous year and prevalence of subjective respiratory symptoms

Days/months

0 0<-
Number

1,141 42 642 366 254 296 108 69

Palpitation

Shortness of breath

Cough

Sputum

Yes

Yes

Yes

Yes

17.3% 19.1 22.0 24.1 27.5 27.7 27.7 39.1

18.9% 19.0 23.2 29.5 28.7 30.0 28.7 39.1

27.7% 33.4 43.5 48.9 55.7 52.3 61.1 63.7

33.4% 42.9 50.2 55.5 59.9 57.1 62.0 63.8

Source: Hisanaga et al, Asbestos exposure among construction workers, 1991 [6]. parenchymal asbestosis (ILO classification 1/0 or higher) and 9.2% had pleural asbestosis [31]. There was a strong, statistically significant relationship between years in the trade and the prevalence of radiologic abnormalities. Ironworkers in construction sites who do not routinely use asbestos materials in their work may also be at risk for asbestos hazards. Fischbein et al. conducted a clinical survey on 869 ironworkers employed at a construction site in the New York metropolitan area and found that the prevalence rate of pleural abnormality was 38% and that of asbestosis was 7% [32]. Electricians may also receive substantial asbestos exposure, especially while performing renovation work. They frequently work in confined spaces with old asbestos insulation. To install or repair equipment, they must strip old asbestos from pipes, beams, and wires. In a cross-sectional study of 37 subjects of non-shipyard electricians, Hodgson et al. found the prevalence of asbestosis, defined as irregular opacities of at least 1/0 profusion, in 15% of overall and 25% after 20 years of service [33]. One person with 30 years of exposure had bilateral pleural disease. Plumbers and pipefitters in construction trade may receive "secondary" asbestos exposure. Sprince et al. evaluated plumbers and pipefitters employed in building construction and found pleural asbestosis in 26.1% and small irregular opacities of profusion 1/0 or greater in a group of 153 participants [34]. Other occupational groups in the construction industry have been exposed to asbestos directly or indirectly. Oksa et al. studied exposure to mineral dust among Finnish construction workers (N = 437) with the aid of questionnaire and a chest Xray examination of the lungs [35]. The results of the questionnaire showed that exposure to asbestos and to silica was found in every occupational group. Pipe and bitumen insulators, pipefitters, and carpenters were especially prominent in the

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

111

asbestos-exposed group. Pleural plaques and/or lung fibrosis (ILO 1/1) were found in 26% of the examined workers; the prevalence varied from 18 to 40% among the various occupational groups (Table 9). Ebihara and Kawami evaluated the chest radiographs of 5,712 male members of construction unions in Tokyo, Kanagawa, Chiba, and Saitama, Japan [36]. Pleural plaques were found in 47 (0.82%) of the workers. The prevalence of pleural abnormalities increased with age, ranging from 0% in younger than 35 years to 4.4% in age groups older than 65 years of age. The prevalence was found in 5.1% among insulators, in 4.8% among roofers, and varied 0% to 1.8% among other various occupational groups (Table 10). The prevalence rates of pleural plaques diagnosed by chest X-ray films seem to be rather low; however, pleural plaques were pathologically revealed in 19 cases among 20 consecutive autopsy cases of construction workers whose chest X-ray films showed no evidence of pleural plaques [36]. From these evidences, it should be noted that asbestos-related diseases among construction workers will be more prominent in the following decades in Japan. In fact, some cases with severe asbestosis were already compensated for disability every year in this trade. Figure 6 shows a severe asbestosis case found in a Japanese carpenter. Cancers of the Trachea, Bronchus, and Lung Epidemiological survey on cancers of the trachea, bronchus, and lung among construction workers is summarized in Table 11 [37-50]. Several epidemiological methods were employed in those surveys. Results are shown as proportional mortality ratio (6 reports), proportional cancer mortality ratio (1), standardized mortality ratio (3), relative standardized mortality ratio (1), standardized incidence ratio (2), odds ratio (2), and relative risk (4). Ratios of respiratory cancers were increased moderately in all the reports except one [47]. Construction workers can be exposed to multiple lung-carcinogenic substances (such as asbestos, diesel exhaust, welding fume and silica) which are common in construction sites [39]. Also, smoking habits are popular among construction workers. Consequently, the increased mortality or incidence from respiratory cancer would be partly explained by the exposure to those factors. It is impossible to distinguish the contribution of asbestos alone to the increase of respiratory cancers in the epidemiological surveys. It can be said, however, that contribution of asbestos would be considerable, because of the following reasons: (1) A number of construction workers have been exposed to asbestos for a long time, (2) Occurrences of malignant mesothelioma were observed simultaneously in the epidemiological surveys [37-42, 46, 47, 49], (3) Asbestos and smoking can affect synergistically the development of lung cancer, and (4) It has been suggested that lung cancer might be associated with asbestos even in the absence of radiologically apparent pulmonary fibrosis [51].

a a N

Table 9 Occurrence of small opacities and pleural plaques in 437 Finnish construction workers, 1987-1988 Workers with lung fibrosis

Occupational group

ILO 01- -010

Workers with small opacities ILO ILO 011-110 111-112

Workers with pleural plaques ILO s2/1

None

Uncertain

Definite2

2 ILO 111 and/or definite pleural plaques

I

2 (D

2

2 E. n ..

o_

Pipe insulators (N = 5 ) Bitumen insulators ( N = 15) Pipefi tters (N= 80) Carpenters ( N = 144) Female laborers (N = 27) Male laborers (N = 42) Cement workers (N = 30) Reinforcement workers (N = 25) Others (N = 67) Total (N = 437) Source: Oksa et al. [351.

0

(c1

1(20%)

2 (40%)

2 (40%)

-

5 (100%)

0 (0%)

0 (0%)

2 (40%)

7 (47%)

7 (47%)

1(6%)

-

10 (67%)

2 (13%)

3 (20%)

3 (20%)

41 (51%)

31 (39%)

7 (9%)

1 (1%)

45 (56%)

23 (29%)

12 (15%)

18 (23%)

61 (42%)

58 (40%)

25 (17%)

2 (1 %)

87 (60%)

30 (21%)

29 (20%)

47 (32%)

14 (52%)

9 (33%)

4 (15%)

-

19 (70%)

5 (19%)

3 (1 1%)

5 (19%)

13 (31%)

19 (45%)

7 (17%)

3 (7%)

25 (60%)

14 (33%)

3 (7%)

11 (26%)

9 (30%)

15 (50%)

5 (17%)

1(3%)

20(67%)

6 (20%)

4 (13%)

9 (30%)

16 (64%) 30 (45%) 192 (44%)

7 (28%) 27 (40%) 175 (40%)

2 (8%) 8 (12%) 61 (14%)

-

12 (48%) 41 (61%) 264 (60%)

9 (36%) 23 (34%) 112 (26%)

4 (16%) 3 (4%) 61 (14%)

6 (24%) 12 (18%) 113 (26%)

2 (3%) 9 (2%)

Y

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

113

Table 10 Pleural plaques in construction workers of Japanese metropolitan areas Occupational group

N

Workers with pleural plaques

3(5.1%) Insulator 59 Roofer 42 2 (4.8%) Demolition worker 220 4(1.8%) 3(1.3%) Pipefitter 236 4(1.1%) Electrician 353 2(1.0%) Tiler 192 20(1.0%) Carpenter 2,019 1 (0.8%) Stone mason 126 Ironworkers 256 2 (0.8%) Interior maker 143 1 (0.7%) Sheet metal worker 169 1 (0.6%) 354 Plasterer 2 (0.6%) Painter 362 2 (0.6%) Wood worker 0 (0.0%) 415 Designer 128 0 (0.0%) 124 0 (0.0%) Unknown 0 (0.0%) 514 Others Total 5712 47 (0.8%) Source: Ebihara, I. and Kawami, M. Asbestos exposure and occupational background. Evidence from asbestos fibre and ferruginous body concentrations in the lungs, 1991 [36]. The variety of trades listed in Table 11 should be assumed as high-risk job of asbestos-related cancers of the trachea, bronchus, and lung. Malignant Mesothelioma Robinson et al., reporting a proportional mortality ratio (PMR) analysis among construction workers 1984-1986 in the USA [40], described a significant increase of cancer of the pleura and peritoneum in plumbers (PMR = 327, 5 cases, p < 0.05) and in electricians (PMR = 331, 4 cases, < 0.01); cancer of the pleura in insulation workers (PMR = 13,486, 4 cases, p < 0.01); and cancer of the peritoneum in workers in general (PMR = 2,467, 2 cases, p < 0.01). In carpenters, cancer of the pleura and peritoneum showed a tendency to increase, but not significantly. The same tendencies were observed among white males (PMR = 141, 33 cases) and white males who died under age 65 (PMR = 131, 22 cases). These cancers may have included mesothelioma. Using the PMR method, Peto et al. analyzed mesothelioma death rates in England, Wales, and Scotland since 1968 [52]. They observed significantly more (text continued on page 118)

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Health and Toxicology

Figure 6. Severe asbestosis case found in a Japanese carpenter.

Table 11 Summary of epidemiological survey on cancers of the trachea, bronchus, and lung among construction workers Author1 Year'

Site of cance6

Epidemiological indicator

Adjustment for smoking

PMR4

1.18" 1.61" 1.22" 1.57""

no

T, B, L

PMR

1.60 (1.16-2.22)"

no

Laborer

T, B, L

PMR PCMR'

1.20 (1.14-1.27) 1.06 ( 1 .OO-1.12)

no no

Construction: White men Black men White women Black women Brickmason Carpenter Painter Plumber Insulation worker Operating engineer Elevator install & repair Structural metal worker Electrician Laborer Tile setter

T, B, L

PMR

1.14(1.11-1.16) 1.05 (0.98-1.12) 1.26 (1 .OO-1.57) 2.72 (1 .OO-5.93) 1.20 (1.06-1.37) 1.16 (1.1 1-1.21) 1.24 (1.14-1.34) 1.10 (1.01-1.20) 1.93 (1.57-2.29) 1.20 (1.09-1.3 1) 2.03 (1.05-3.55) 1.29 (1.07-1.54) 1.14 (1.05-1.23) 1.13 (1.06-1.21) 1.69 ( 1.15-2.40)

no

Country2

Job trade

Milham 1983

USA

Painter & Paperhanger Plasterer Plumber & Pipefitter Glazier & Roofer

T, B, L

Zoloth 1985 [38]

USA

Sheet metal worker

Stern 1995 1391

USA

Robinson 1995 [40]

USA

(table continued on next page)

9.

Table 11 (Continued) Author1 Year'

Job trade

Country2

Site of cancel3

Epidemiological indicator

Adjustment for smoking

25 s

Registrar General

Painter & Paperhanger Plasterer Plumber & Pipefitter Glazier & Roofer Laborer

UK

1986 [41]

T, B, L

PMR

1.23"" 1.38** 1.21"" 1.39** 1.22""

no

Y

UK

Construction Bricklayer Driver Foreman/Ganger Laborer Painter Plumber Roofer Scaffolder Terrazzo mosaic Woodworker

L

PMR

1.35 (1.29-1.40) 1.34 (1.12-1.51) 1.20 (1.02-1.37) I .24 (1.03-1.46) 1.49 (1.36-1.61) 1.44 (1.26-1.63) 1.58 (1.05-2.3 1 ) 1.72 (1.13-2.50) 1.58 (1.08-2.08) 1.77 (1.13-2.64) 1.16 (1.02-1.29)

n CF

USA

Laborer: White men Black men

T, L

SMR6 SMR

1.63 (1.39-1.91) 2.23 (1.67-2.92)

no no

L

SMR RSMR7

1.71** 1.69""

no

1995 [42]

~~~

Burkhart

~

1993 1431 Ng 1988 [44]

Hong Kong

Construction

--I

:5': 8

(P

~~~

Dong

Q

T, B, L L L

SMR SIR8 RR9

0.86 (0.79-0.95) 0.91 (0.83-1.00) 0.82 (0.60-1.12) 0.93 (0.66-1.31)

no no no yes

L

SIR

0.94 (0.88-1.01)

no

L

OR"'

2.18 (1.20-3.98)

Yes

Construction Road construction Unskilled worker

L

OR

1.8 (1.04-3.06) 3.7 (1.06-13.20) 2.7 (1.24-5.76)

Yes

B

RR

1.9 ( 1 .O-3.5)

no

RR

1.4 ( 1 .o-1.9) 1.7 ( 1 .O-2.9)

Yes

Engholm 1987 [45]

Sweden

Construction

Fletcher 1993 [46]

Sweden

Construction Workers including insulator, refrigeration, mechanic & plumber

Jockel 1992 [47]

Germany

Coggon 1984 [48]

UK

Construction

Coggon 1986 [49]

UK

Construction Laborer Workers including insulator, asbectos roofer and etc.

B

Building construction

L

~~

Yamaguchi 1992 1501

Japan

1.8 ( 1 .O-3.0) RR

1.95 (0.97-3.94)

yes

1) Year of publication, 2 ) C o u n t n surveyed, 3) T-Trachen, B-Bronchus, L-Lung, 4 ) Proportional mortality ratio, 5) Proportional cancer nzortulih ratio, 6) Standurdized mortality ratio, 7 ) Relutive stcindurdized mortality ratio, 8 ) Standardized incidence ratio, 9 ) Relative risk, 10) Odds ratio, I I ) 95% confidence interval, * pcO.05, ** p<0.01.

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Health and Toxicology

{text continued from page 113)

mesothelioma deaths in some jobs, including plumbers and gas fitters (PMR = 442.8, p < 0.001), carpenters (PMR = 365.7, p < 0.001), electricians (PMR = 290.5, p < 0.001), construction workers (PMR = 255.6, p < 0.001), plasterers (PMR = 202.8, p < 0.001), and painters and decorators (PMR = 131.0, p < 0.05). They concluded that asbestos exposure at work in construction and building maintenance could account for a large proportion of mesothelioma deaths. They warned that jobs related to asbestos removal may well increase the burden of future occupational asbestos diseases, and also that such workers should be aware of the risks and take appropriate precautions. Malker et al. reported 35 cases of peritoneal malignant mesothelioma based on the National Swedish Cancer Registry from 1961 to 1979 [53]. Thirteen cases were employed in the construction industry (Standardized Incidence Ratio (SIR) = 2.6, p < 0.01). As an occupational group, insulators had a very high risk of the disease (SIR = 163.9, 4 cases, p < 0.001). Mouri et al. analyzed 947 cases of malignant mesothelioma autopsied from 1974 to 1990 in Japan [54]. Among these, 49 cases were engaged in construction including 19 cases of electric and pipe work. In 1995, at least 3 cases of pleural mesotheliomas were found in the Construction Workers Union in the Japanese Metropolitan Area, which was organized by about 160,000 various construction workers. The three mesothelioma cases (2 in carpenters and 1 in an electrician) were certified as being of occupational origin. These findings showed the incidence of mesothelioma due to asbestos exposure among construction workers, which is expected to increase in the future [52]. Consequently, measures to prevent the incidence of mesothelioma and lung cancer or enable their early detection is needed among construction workers. MEASURES AGAINST RESPIRATORY DISORDERS DUE TO ASBESTOS EXPOSURE Prevention of Asbestos Exposure Preventive measures against asbestos exposure consist of substitution, work environment control, work control, and work education and training. Substitution: This is the most important measure preventing asbestos exposure. Not only manmade mineral fibers (e.g., rock wool, glass wool and ceramic fiber) but also natural non-asbestos fibers (e.g., wollastonite and sepiollite) are utilized as substitutes. It should be noted that harmful properties of those substitutes cannot be underestimated, even though they are assumed to be less harmful than asbestos.

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

119

Working Environment Control: First, minimizing asbestos dust generation is needed. As mentioned above, large amounts of dust containing asbestos can be generated by electric circular saws. It is desirable to cut boards containing asbestos based on a blueprint or pattern, at a factory with a complete ventilation system before forwarding the material to construction sites. Second, it is necessary to suppress asbestos dust emission. Local exhaust systems, vacuum cleaners, and electric circular saws equipped with dust-collecting bags should be utilized to keep clean worksites. Sweeping the floor with a broom should be done carefully (Figure 7). In case of renovation, demolition, and asbestos removal, the places with asbestos-containing materials must be identified before starting work. Sprinkling of water or solidification of fly able asbestos by resin should be done at those sites. Third, construction material wastes should be sealed in containers or bags. Fourth, airborne asbestos concentration should be determined to monitor the exposure of workers and air pollution around construction sites. Fifth, attention should be paid to asbestos exposure from mineral materials possibly including asbestos, such as serpentine rock, talc, and vermiculite [39, 55]. Work Control: First, workers should wear respiratory protective equipment during the work with asbestos exposure. Second, avoid taking food to worksites with asbestos exposure. Third, workers should be careful with asbestos pollution »of clothes and trousers during work, and change them before leaving construction sites (Figure 8).

Figure 7. Sweeping the floor with a broom. Emission of dust containing asbestos is remarkable. Airborne asbestos concentration during the work was 0.5 f/ml at the respiratory zone.

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Health and Toxicology

Figure 8. Trousers polluted by asbestos dust. It is important to avoid family contact with asbestos by changing clothes and trousers after the daily work.

Education and Training: The majority of construction workers are employed in small-scale enterprises or are self-employed. Work sites are temporary, therefore asbestos exposure is usually episodic in nature. Those factors make it difficult to implement measures mentioned above. Education and training is indispensable to get workers to protect themselves from asbestos exposure. Workers with a potential for bystander exposure from other workers' handling of asbestos should also be the subject of education and training. Health Control Health Examination In health examination, attention needs to be paid to the following. 1. Subjective symptoms including shortness of breath, palpitation, sputum, and cough are checked using various methods. To check dyspnea, the Hugh-Jones classification is a representative questionnaire [56].

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

121

2. Chest X-ray photographs are to be taken one or two times a year and classified mainly by ILO U/C International Classification of Radiographs of Pneumoconiosis [57]. According to the findings, the chest X-rays are divided into type 0 (no significant findings), type 1 (a few irregular-shaped shadows in both lungs), type 2 (many irregular-shaped shadows in both lungs), and type 3 (very many irregular-shaped shadows in both lungs). Lung cancer findings are also checked by X-ray film. Computerized tomography (CT scan) is also utilized to confirm the findings of pleural plaque and/or lung cancer. 3. In auscultation in physical examinations, fine crackles heard at the lower part of the bilateral lung are characteristic of and useful for identifying asbestos lung. 4. Lung function tests including vital capacity (VC), forced effective volumes (FEV), and V25/ HT are performed in some cases with significant asbestosis findings for the symptoms and chest X-ray film. Abnormal findings due to asbestos are fundamentally restrictive ones including reduction of VC and V25/HT. Since this test is often confounded by smoking, evaluation of the test must be done carefully. Workers with reduced lung function are subjected to blood gas analysis (measurement of partial pressure of oxygen [O2] and carbon dioxide [CO2] in the blood) and/or DLco test (diffusion capacity from the lung to the blood using carbon monoxide [CO]). 5. Cytology tests of the sputum are used for early detection of lung cancer. A worker suspected of lung cancer is examined by bronchial endoscopy with lung biopsy. The biopsied specimen is cytologically examined. 6. The examined workers are divided according to health and work control classification. Since the classification method varies in different countries, we show the classification of pneumoconiosis including asbestosis (Table 12) [58] and health disorders due to specified chemical substances including asbestos (Table 13) [58] in Japan as an example. Follow-up of Examined Workers The workers with evident chest X-ray film disorders due to asbestos must be advised to consult a medical doctor. Symptomatic therapy is done for asbestosis and cancer therapy including surgery for lung cancer. However, there is no effective treatment for workers with pleural plaque. Health control must be combined with technological measures to reduce exposure to asbestos. Occupational Health Education Occupational health education includes knowledge about asbestos-containing materials distinguished by the "a" mark for the materials including more than 5% asbestos, and a notice for careful handling so as not to provoke asbestos dust, and

122

Health and Toxicology Table 12 Management classification of pneumoconiosis due to asbestos in Japan

Management classification

Findings

Classification 1

No findings due to asbestos

Classification 2

Type 1 chest X-ray without evident abnormal lung function Type 2 chest X-ray without evident abnormal lung function

Classification 3A

Measures to be taken in occupational environment

No special occupational measures Measures to reduce exposure to dust Measures to reduce exposure to dust Recommendation to change work Instruction to change work

Type 3 or 4 (large shadows occupy more than one-third of the lung) chest X-ray without evident abnormal lung function Type 4 (large shadows occupy Classification 4 Treatment more than one-third of the lung) chest X-ray or Type 1, 2, 3 or 4 (large shadows occupy less than one-third of the lung) with evident abnormal lung function Source: Labour Standards Bureau, Ministry of Labour of Japan, General Guidebook on Industrial Health, 1994, p. 20-22 [58].

Classification 3B

Table 13 Management classification of health disorders due to specified chemicals including asbestos in Japan Management classification

Findings

Measures to occupation

No special occupational measures No finding due to asbestos Not applicable to C, but with Further medical examinations, findings due to asbestos restriction of work with exposure Classification C Suffering from disease due to Treatment and prohibition of asbestos work with exposure Source: Labour Standards Bureau, Ministry of Labour of Japan, General Guidebook on Industrial Health, 799-^, pp. 68-69 [59]. Classification A Classification B

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

123

the use of general exhaust vents, local exhaust vents, dust catchers, and respiratory protective equipment. Health education is important to stop smoking among workers exposed to asbestos in order to prevent lung cancer due to asbestos. This is because tobacco smoking and asbestos exposure synergistically interact to induce lung cancer. Epidemiological studies revealed that workers who smoke and are subjected to a high level of asbestos exposure are much more liable to suffer from lung cancer than those who do not smoke and are not exposed to asbestos [60, 61]. The issue is described in ILO Recommendation No. 172 (§43). REFERENCES 1. Asbestos International Association, Personal communication. 2. Japan Asbestos Association, Personal communication. 3. Department of Construction Leading, Bureau of Housing, Ministry of Construction of Japan, Handbook of fireproof structure and materials, Tokyo, Japan Construction Center, 1995 (in Japanese). 4. Fischbein, A. et al. "Drywall construction and asbestos exposure," Am Ind Hyg Assoc J, 1979, 40:402-407. 5. Verma, D. K. and Middleton, C. G. "Occupational exposure to asbestos in the drywall tapping process," Am Ind Hyg Assoc J, 1980, 41:264-269. 6. Hisanaga, N. et al. "Asbestos exposure among construction workers," Proceedings of 7th International Pneumoconiosis Conference, 1991, pp. 1,053-1,058. 7. Sakai, K. et al. "Airborne asbestos types, concentrations and size distribution at construction work sites," Wongphanich, M., et al. eds. Research Perspectives in Occupational Health Ergonomics in Asia and Other Countries, Bangkok, Mahidol University, 1993, pp. 344-350. 8. Balzer, J. L. and Cooper, W. C. "The work environment of insulating workers," Am Ind Hyg Assoc J, 1968, 29:222-227. 9. Brown, S. K. "Asbestos exposure during renovation and demolition of asbestos-cement clad buildings," Am Ind Hyg Assoc J, 1987, 48:478-486. 10. Sawyer, R. N. "Asbestos exposure in a Yale building: Analysis and resolution," £/iv/rt?n 7?^^, 1977, 13:146-169. 11. Paik, N. W. et al. "Worker exposure to asbestos during removal of sprayed material and renovation activity in buildings containing sprayed material," Am Ind Hyg Assoc J, 1983, 44:428-432. 12. Sawyer, R. N., Rohl, A. N., Langer, A. M. "Airborne fiber control in buildings during asbestos material removed by amended water methodology," Environ Res, 1985,36:46-55. 13. Reitze, W. B., Nicholson, W. J., Holaday, D. A., Selikoff, I. J. "Application of sprayed inorganic fiber containing asbestos: Occupational health hazards," Am Ind Hyg Assoc J, 1972,33:178-191.

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14. International Labour Office, International labour and economy yearbook in 1992, Geneva, ILO, 1993, pp. 270-338. 15. Bureau of Statistics, Management and Coordination Agency of Japan, Monthly Report of Labour Force Survey in June 1995, Tokyo, Bureau of Statistics, Management and Coordination Agency of Japan, 1995, pp. 10-11 (in Japanese). 16. Japan Construction Safety and Health Association, Yearbook of safety and health in construction industry 1994, Tokyo, Japan Construction Safety and Health Association, 1994, pp. 176-177 (in Japanese). 17. U.S. Department of Commerce Economics and Statistics Administration, Bureau of Census Statistical abstract of the U.S. 114th edition, 1994, p. 725. 18. International Labour Conference 73rd session. Safety and health in construction, 5th Item on the agenda, Geneva, ILO, 1987, p. 9. 19. Japan Construction Safety and Health Association, Yearbook of safety and health in construction industry 1994, Tokyo, Japan Construction Safety and Health Association, 1994, pp. 99-108 (in Japanese). 20. Hisanaga, N. et al. "Prevention of asbestos-related diseases in the small-scale construction industry." 21. Asbestos International Association, AIA information memorandum—Summary of main features of asbestos regulations, Paris, AIA, 1995, pp. 2-90. 22. Kawami, M. and Ebihara, I. "Asbestos exposure and occupational background. Evidence from asbestos fibre and ferruginous body concentrations in the lungs," J Science of Labour, 1991, 67(10):469-480 (in Japanese with English summary). 23. Karjalainen, A. et al. "Asbestos exposure among Finnish lung cancer patients: Occupational history and fiber concentration in lung tissue," Am J IndMed, 1993, 23(3):461-471, 1993. 24. Kawami, M. and Ebihara, I. "A study on a method to determine asbestos fiber and ferruginous body concentrations in the human lung tissue as indicators of asbestos exposure," / Science of Labour, 1990, 66(1): 1-23, (in Japanese with English summary). 25. Anttila, S. et al. "The lung cancer in the lower lobe is associated with pulmonary asbestos fiber count and fiber size," Environ Health Perspect, 1993, 101(2):166-170. 26. Matsunaga, I. et al. "A questionnaire survey on asbestos exposure and symptoms among construction workers," Jpn J Ind Health, 1990, 32:779 (in Japanese). 27. Kennedy, S. M. et al. "Lung function and chest radiograph abnormality among construction insulators," Am J Ind Med, 1991, 20:673-684. 28. Baker, E. L. et al. "Incremental value of oblique chest radiographs in the diagnosis of asbestos-induced pleural disease," Am J Ind Med, 1982, 3:17-22. 29. Baker, E. L. et al. "Respiratory illness in the construction trades: The significance of asbestos-associated pleural disease among sheet metal workers," J OccupMed, 1985, 27(7):483^89.

Respiratory Findings of Construction Workers Exposed to Asbestos Dust

125

30. Selikoff, I. J. and Lilis, R. "Radiological abnormalities among sheet metal workers in the construction industry in the United States and Canada: Relationship to asbestos exposure," Arch Environ Health, 1991, 46(l):30-36. 31. Michaels, D. et al. "Asbestos disease in sheet metal workers: Radiologic signs of asbestosis among active workers,'' Am J Ind Med, 1987, 12:595-603. 32. Fischbein, A. et al. "Respiratory findings among ironworkers: results from a clinical survey in the New York metropolitan area and identification of health hazards from asbestos in place at work," Brit J Indust Med, 1991, 48:404^11. 33. Hodgson, M. et al. "Asbestosis among electricians," / Occup Med, 1988, 30(8):638-640. 34. Sprince, N. L. et al. "Asbestos-related disease in plumbers and pipefitters employed in building construction," / Occup Med, 1985, 27(10):771-775. 35. Oksa, P. et al. "Parenchymal and pleural fibrosis in construction workers," AmJIndMed, 1992,21:561-567. 36. Ebihara, I. and Kawami, M. "A study on the incidence of pleural plaques among construction workers," / Occup Health, 1988, 30 (suppl):156. 37. Milham, S. J. "Occupational mortality in Washington state 1950-1979," DHHS (NIOSH) Publication No. 83-116, NIOSH, 1983, Cincinnati. 38. Zoloth, S. and Michales, D. "Asbestos disease in sheet metal workers: The results of a proportional mortality analysis," Am J Ind Med, 1985, 7(4):315-321. 39. Stern, F. et al. "Proportional mortality among construction labourers," Am J Ind Med, 1995, 27(4):485-509. 40. Robinson, C. et al. "Assessment of mortality in the construction industry in the United States, 19^4-19^6;'Amer J Ind Med, 1995,28:49-70. 41. Registrar General's Decennial Supplement for England and Wales, "1979-80, 1982-83 Occupational mortality, Office of Population Censuses and Surveys, Her Majesty's Stationery Office, 1986, London. 42. Dong, W. et al. "Mortality study of construction workers in the UK," Int J Epidem, 1995, 24(4):750-757. 43. Burkhart, G. et al. "Job tasks, potential exposure, and health risks of labourers employed in the construction industry," Am J Ind Med, 1993, 24(4):413-425. 44. Ng, T. P. "Occupational mortality in Hong Kong, 1979-1983," Int J Epidem, 1988, 17(1):105-110. 45. Engholm, G. et al. "Respiratory cancer incidences in Swedish construction workers exposed to mineral fibers and asbestos," Ann Occup Hyg, 1987, 31(4B):663-675. 46. Fletcher, A. C. et al. "The risk of lung cancer from asbestos among Swedish construction workers: Self-reported exposure and a job exposure matrix compared," Int J Epidem, 1993, 22 (Suppl 2):S29-S35.

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47. Jockel, K. H. et al. "Occupational and environmental hazards associated with lung cancer," IntJEpidem, 1992, 21(2):202-203. 48. Coggon, D. et al. "Use of job-exposure matrix in occupational analysis of lung and bladder cancer on the basis of death certificates," / Natl Cancer Inst, 1984, 72(l):61-65. 49. Coggon, D. et al. "A survey of cancer and occupation in young and middle aged men: Cancers of the respiratory tract," Brit J Ind Med, 1986, 43(5):332-338. 50. Yamaguchi, N. et al. "A case-control study on occupational lung cancer risks in industrialized cities of Japan," Jpn J Cancer, 1992, 83(2): 134-140. 51. Wilkinson, P. et al. "Is lung cancer associated with asbestos exposure when there are no small opacities on the chest radiograph?" Lancet, 1995, 345 (apr. 29): 1,074-1,078. 52. Peto, J. et al. "Continuing increase in mesothelioma mortality in Britain," Lancet, 1995, 345:535-539. 53. Malker, H. S. R. et al. "Peritoneal mesothehoma in the construction industry in Sweden," J Occup Med, 1987, 29:979-980. 54. Mouri, I. et al. "Occupational and regional distribution of malignant mesothelioma using database of Annuals of Pathological Autopsy Cases in Japan," JpnJPubl Health, 1993, 40(10):1,284 (in Japanese). 55. Hay ward, S. B. and Smith, G. B. "Asbestos concentration of vermiculite," Am J Pub Hlth, 1984, 74(5):519. 56. Hugh-Jones, P. "A simple standard exercise test and its use for measuring exertion dyspnoea," 5nY Me J / , 1952, 1 (Jan. 12):65-71. 57. International Labour Office, "International Classification of Radiographs of Pneumoconiosis," Geneva, ILO, 1981. 58. Labour Standards Bureau, Ministry of Labour of Japan, General handbook on industrial health, Tokyo, Japan Industrial Safety and Health Association, 1994, pp. 20-22. 59. Labour Standards Bureau, Ministry of Labour of Japan, General handbook on industrial health, Tokyo, Japan Industrial Safety and Health Association, 1994, pp. 68-69. 60. Hammond, Y. Y. et al. "Evaluation of dust exposure in asbestos cement manufacturing operators," Amer Ind Hyg Assoc J, 1979, 40:490-495. 61. Sellikoff, I. J. et al. "MortaHty effects of cigarette smoking among amosite asbestos factory workers," 7M/ Cancer Inst, 1980, 65:507-513.

CHAPTER 6 ASBESTOS EXPOSURE AND THE RISK OF LUNG CANCER IN URBAN POPULATIONS Anti Karjalainen and Anttila Sisko Finnish Institute of Occupational Health Topeliuksenkatu41 aA Helsinki, Finland

CONTENTS INTRODUCTION, 127 ASBESTOS MINERALS, 128 PRODUCTION AND USE OF ASBESTOS, 128 EXPOSURE TO ASBESTOS IN URBAN POPULATIONS, 129 HISTORY OF THE ASSOCIATION OF LUNG CANCER WITH ASBESTOS, 129 EPIDEMIOLOGY OF ASBESTOS-RELATED LUNG CANCER, 130 Dose-response, Latency Time, and Interaction with Smoking, 130 Effect of Asbestos Fiber Type and Size on Lung Cancer Risk, 130 Etiologic Fraction of Asbestos Exposure in Lung Cancer, 131 Asbestosis and the Risk of Lung Cancer, 131 Pleural Plaques as Indicators of Risk of Lung Cancer, 132 Site and Histological Type of Asbestos-related Lung Cancer, 132 REFERENCES, 132 INTRODUCTION Lung cancer is numerically the most important malignant disease in the world [1]. Tobacco smoke is by far the most important single cause of lung cancer, but several other factors—like arsenic, asbestos, beryllium, cadmium, chromates, mustard gas, nickel, polycyclic aromatic hydrocarbons, and radon daughters—are known to increase the risk of lung cancer [2, 3]. Asbestos has been widely used in buildings, construction materials, machines, equipment, transport vehicles, and consumer products. Exposure to asbestos is known to cause asbestosis, lung cancer, malignant mesothelioma (cancer deriving from the serosal lining of the pleural, pericardial, and peritoneal cavities), pleural plaques, diffuse pleural thickening, rounded atelectasis, and benign exudative pleurisy [4]. An excess of other neoplasms, like laryngeal and gastrointestinal cancers, have been observed in some exposed populations [5]. Lung cancer is numerically the most-important and mesothelioma the second most-important asbestosrelated malignancy. Most mesothelioma cases are related to past asbestos exposure [6]. The incidence of malignant mesothelioma is considered an indicator of past asbestos exposure in the population, and the number of all asbestos-related deaths 127

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in a population is usually two to three times the number of mesothelioma deaths in that population [7]. Despite the reduction of exposure in the 1980s and 1990s, the incidence of asbestos-associated diseases has continued to increase in most industrialized countries [7]. This increase is due to the long latent period of asbestos-associated diseases and the increased use of asbestos in the 1960s and early 1970s. The estimates based on national data of mesothelioma incidence indicate that the overall incidence rates for men appear to be stagnating in the United States and Finland [7, 8], but will continue to increase during the next 10 to 20 years in UK and Norway [9, 10]. ASBESTOS MINERALS Asbestos is a collective term for six naturally occurring crystalline silicate mineral fibers: chrysotile, amosite, crocidolite, anthophylHte, tremolite, and actinolite [11]. Chrysotile belongs to the serpentine group, and the other five to the amphibole group of minerals. Asbestos minerals are not defined entirely on a mineralogical basis, but rather on the basis of their common properties such as high tensile strength, flexibility, and chemical and physical durability. Their insulating, fireproofing, and reinforcement properties have made them widely exploited in industry. In studies of asbestos-related diseases, exposure doses are usually based on the fiber criteria used in the measurement of workplace air. These criteria require a length-to-width ratio over or equal to 3 and a width of less than 3 micrometers (jim) [12]. PRODUCTION AND USE OF ASBESTOS The industrial use of asbestos started in the late 1800s and became common within a few decades [13]. Chrysotile has been the main asbestos type, accounting for more than 95% of all asbestos used industrially [14]. Despite the rapid reduction in the use of asbestos in Western industrialized countries, production of asbestos has remained consistently high in the 1980s due to the increase of use in the Third World [15]. The former Soviet Union and Canada were the main producers of chrysotile asbestos in the late 1980s. Asbestos-containing products have been commonly used in construction materials, in machines, equipment, transport vehicles, and consumer products. There are ten major asbestos product categories [4, 7, 16]: 1. Asbestos cement products 2. Insulation and fireproofing 3. Friction materials 4. Asbestos paper products 5. Floor coverings 6. Asbestos-reinforced plastics

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7. Paints, coatings, adhesives, sealants 8. Packings and gaskets 9. Asbestos textiles 10. Miscellaneous (drilling muds, filters, etc.) The greatest amount of asbestos fibers has been used in the production of asbestos cement products such as construction sheets and pipes. Asbestos products also have been widely used in the manufacture of floor tilings, insulation and fireproofing materials, asbestos paper products, and friction materials. EXPOSURE TO ASBESTOS IN URBAN POPULATIONS The number of affected workers is not restricted to those exposed during the mining, milling, and manufacture of asbestos products, but also the many workers in shipyards, the construction industry, maintenance, and mechanic repair have been exposed. Furthermore, exposure has not concerned only those who have used asbestos products, but also others who worked in the vicinity of asbestos. It has been estimated that in the United States, 27.5 million workers were exposed to asbestos between 1940 and 1979 [17]. To avoid further exposures, stringent limits for the asbestos content of workplace air were adopted by most countries in the 1980s and 1990s. Many countries have also prohibited the use of most if not all asbestos-containing products. The International Labour Organization's convention has required since 1986 that dismanthng of asbestos be allowed only under Hcense and using special techniques [18]. National regulative actions conforming to these recommendations have been adopted especially in Western Europe. Apart from industrial exposure, much public attention has focused on the exposure of occupants of buildings containing asbestos materials. The average airborne asbestos levels in such buildings are, however, invariably very low during normal use [7, 19]. HISTORY OF THE ASSOCIATION OF LUNG CANCER WITH ASBESTOS The first reports suggesting a relationship between past exposure to asbestos and lung cancer were pubhshed in 1935 [20, 21]. In 1955, Doll reported a more than 10-fold excess of lung cancer deaths in a cohort of asbestos textile factory workers in Great Britain [22]. Since then, numerous scientific articles have been published on the issue, and in the mid-1960s the association between asbestos exposure and lung cancer was generally accepted [23, 24]. Thereafter, increasing interest has focused not only on the workers exposed in the production of asbestos and the manufacture of asbestos products, but also on the cancer risk among downstream users of asbestos products.

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EPIDEMIOLOGY OF ASBESTOS-RELATED LUNG CANCER Dose-response, Latency Time, and Interaction with Smoking Cohort studies of persons with occupational exposure to asbestos indicate that at high levels of exposure, the lung cancer risk is roughly proportional to the cumulative dose, but there is only limited data on the dose-response at low levels of occupational exposure [7]. The slope of the dose-response curve varies considerably according to the fiber type and industrial process [23, 25]. There is also some evidence of raised lung cancer rates among household contacts of asbestos workers, but due to the dominant confounding effect of cigarette smoking, any lung cancer cases attributable to non-occupational low exposures would be difficult to detect in epidemiological studies [26]. It is generally accepted that there is a long latency time between the onset of exposure and the occurrence of lung cancer. In the cohort of 17,800 North American insulators, no increase in the incidence was demonstrable until 10 to 15 years had elapsed from the onset of exposure. The major increase in lung cancer incidence occurred later than 20 years from the onset of exposure, and the highest excess in the incidence was observed at 30 to 40 years from the onset of exposure [27, 28]. Most lung cancers among asbestos-exposed workers occur in smokers or exsmokers, and a multiplicative model of interaction between smoking and exposure to asbestos has been introduced [29]. However, a variable pattern of interaction ranging from supramultiplicative to less than additive has been observed in other studies [30]. Cessation of smoking is expected to reduce the risk of lung cancer among asbestos-exposed smokers [29]. Effect of Asbestos Fiber Type and Size on Lung Cancer Risk The International Agency for Research on Cancer evaluated the studies of the carcinogenic effect of asbestos exposure and concluded that exposure to all types of asbestos fibers results in an increased risk of lung cancer [5]. The relative potencies of these fiber types in the causation of lung cancer are somewhat controversial, and the interpretation of results is difficult due to possible confounding effects of different industrial processes. In cohorts of asbestos miners and millers, the different amphibole asbestos types have produced higher risks of lung cancer than chrysotile [23, 31]. In other industrial applications of asbestos, especially in asbestos textile factories, high risks of lung cancer have been reported also in relation to exposure to chrysotile asbestos [32, 33, 34]. The increase in risk per a given exposure dose of chrysotile fibers has been up to 25 times greater in these studies as compared to that observed in chrysotile mining and milling. The reason for such differences remains debatable [35, 36]. It has been concluded that lung cancer is most closely associated with the numbers of fibers longer than 10 |Lim and thicker than 0.15 ^im [37].

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Etiologic Fraction of Asbestos Exposure in Lung Cancer Table 1 summarizes the studies that have estimated the etiologic fraction of past asbestos exposure in lung cancer among men. All the studies have been conducted in urban populations or highly industrialized geographical areas, where occupational exposure to asbestos has been common in the past. The asbestos-related proportion is lower in populations in which asbestos exposure has been less common (e.g., women and rural areas). In urban populations, various occupations in the construction industry seem to contribute most to the asbestos-related excess in lung cancer [38, 39, 40], although shipyard work and various industrial maintenance operations may also have a high contribution in certain areas [41, 42, 43]. Asbestosis and the Risk of Lung Cancer A question closely related to the dose-response relationship is whether there is a causal relationship between asbestos-related pulmonary fibrosis (asbestosis) and lung cancer, or whether asbestosis and the increased risk of lung cancer both are implications of past exposure to asbestos and thus usually, but not always, occur in the same individuals [45, 46]. As it is likely that there is a level of exposure below which fibrosis will not occur or will be insignificant [47], the question of the relationship between fibrosis and lung cancer is crucial from the point of view of preventive strategies and, of course, for the understanding of the carcinogenic mechanisms of asbestos. In addition, the question has obvious medico-legal implications that have polarized and probably distorted the debate [46]. Some epidemiological studies have suggested that the increased risk of lung cancer is confined only to those exposed individuals who have radiographic evidence of pulmonary fibrosis [48, 49], but recent studies have indicated that the risk is increased even in the absence of radiographic fibrosis [39] or histological fibrosis[38, 50, 51].

Table 1 Etiologic fraction of asbestos exposure in lung cancer among men in five international studies

Geographical area Telemark, Norway Trieste, Italy Helsinki, Finland Gothenburg, Sweden Glasgow and West of Scotland

Etiologic fraction

Reference

23% 20% 19% 16% 6%

41 42 38 43 44

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Pleural Plaques as Indicators of Risk of Lung Cancer As the occurrence of pleural plaques correlates strongly with past exposure to asbestos, it is obvious that within a given population individuals with pleural plaques have been more exposed on the average than those without plaques. This would also imply that they are at a higher risk of lung cancer and mesothelioma. Pleural plaques, on the other hand, may be caused by very low exposures, which probably imply only a low risk of lung cancer, if any. Some, but not all, studies have found indications of elevated risk of lung cancer among patients with pleural plaques. These studies and their limitations were reviewed recentiy with the conclusion that evidence of increased risk of lung cancer among carriers of pleural plaques is not convincing [52]. Since then, two studies have been pubhshed with some indication of an increased risk [53, 54], and one with no increase [55]. Finally it has been pointed out that epidemiological studies are not sensitive enough to provide a conclusive answer to the problem; unrealistically large population studies would be needed to observe the statistical relation between pleural plaques and lung cancer resulting from relatively low levels of environmental asbestos exposure [56]. Site and Histological Type of Asbestos-related Lung Cancer Several studies have demonstrated that the proportion of lung tumors arising from the lower lobes is increased in asbestos-exposed workers [38, 51, 57, 58, 60]. Yet some authors have found no difference in the lobar distribution pattern between asbestos-exposed and unexposed populations [62]. Some authors have found a preponderance of adenocarcinomas among asbestosexposed workers, but also contradictory results have been published [38, 62, 63, 65]. Both asbestos and tobacco smoke seem to be complex carcinogens that can affect more than one stage of lung carcinogenesis, and very limited information is available on the interaction between asbestos exposure and smoking in causing specific histological types of lung cancer [30].

REFERENCES 1. International Agency for Research on Cancer. Cancer: Causes, Occurrence and Control lARC Scientific Publications No. 100. lARC, Lyon, 1990. 2. Higginson, J., Muir C. S., Munoz, N. Human cancer: epidemiology and environmental causes. Cambridge University Press. Cambridge, 1992. 3. International Agency for Research on Cancer. I ARC Monographs on the evaluation of the carcinogenic risk of chemicals to humans. Vol. 58, "Beryllium, Cadmium, Mercury and exposures in the glass manufacturing industry." lARC, Lyon, 1993.

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4. Browne, K. "Asbestos-related disorders." In: Parkes, W. R. Occupational lung disorders. Third edition. Butterworth Heinemann, London, 1994:411-504. 5. International Agency for Research on Cancer. "Asbestos." Supplement No. 7. Overall evaluations of carcinogenicity: An updating of I ARC Monographs Volumes 1-42. lARC, Lyon, 1987. 6. McDonald, A. D., McDonald, J. C. "Epidemiology of malignant mesothelioma." In: Antman, K., Aisner, J. (eds.): Asbestos related malignancy. Boston, Grune & Stratton, 1987:31-56. 7. Health Effects Institute—Asbestos Research. "Health Implications of exposure to asbestos." In: Asbestos in public and commercial buildings: A literature review and synthesis of current knowledge. Health Effects Institute— Asbestos Research, Cambridge, 1991:6-1, pp. 6-104. 8. Karjalainen, A., Pukkala, E., Mattson, K., Tammilehto, L., Vainio, H. "Trends in mesothelioma incidence and occupational mesotheliomas in Finland 1960-95," Scand J Work Environ Health. 9. Peto, J., Hodgson, J. T., Matthews, F. E., Jones, J. R. "Continuing increase in mesothelioma mortality in Britain." Lancet, 1995; 345: pp. 535-539. 10. Mowe, G., Andersen, A., Osvoll, P. "Trends in mesothelioma incidence in "^ovv^diy:' Ann NY Acad Sci, 1991; 643: pp. 449-453. ll.Pooley, F. D. "Asbestos mineralogy." In: Antman, K., Aisner, J. (eds.). Asbestos related malignancy. Boston, Grune & Stratton, 1987: pp. 3-27. 12. WHO. "Asbestos and other natural mineral fibres." IPCS (International Programme on Chemical Safety). Environmental health criteria 53. WHO, Geneva, 1986. 13. Selikoff, I. J., Lee, D. H. K. Asbestos and Disease. Academic Press, New York, 1978. 14. Hines, W. "The third wave of asbestos disease: asbestos in place." The Workplace Health Fund, Washington, D.C., 1990. 15. Enqvist, J. "Asbestos market booms in Third World." Working Environment, 1992: pp. 22-25. Swedish Work Environment Association, Stockholm. 16. Oullette, R. P., Dilks, C. F., Thompson, W. C , Cheremisinoff, P. N. Asbestos Hazard Management. Technomic Publishing Company, Lancaster, Pennsylvania, U.S.A., 1987. 17. Nicholson, W. J., Perkel, G., Selikoff, I. J. "Occupational exposure to asbestos: Population at risk and projected mortality 1980-2030." Am J Ind Med, 1982; 3: pp. 259-311. 18. International Labor Organization. "Convention (No. 162) Concerning Safety in the Use of Asbestos," 1986. 19. Burdett, G. J., Jaffery, S., Rood, A. P. "Airborne asbestos fibre levels in buildings." In: Bignon, J., Peto, J., Saracci, R. (eds.). Non-occupational exposure to mineral fibres. lARC Scientific Publications No. 90. lARC, Lyon, 1989: pp. 277-290.

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20. Lynch, K. M., Smith, W. A. "Pulmonary asbestosis III: Carcinoma of the lung in asbestos silicosis." Am J Cancer, 1935; 24: pp. 56-64. 21. Gloyne, S. R. "Two cases of squamous carcinoma of the lung occurring in asbestosis." Tubercle, 1935; 17: pp. 5-10. 22. Doll, R. "Mortality from lung cancer in asbestos workers." Br J Ind Med, 1955; 12: pp. 81-86. 23. McDonald, A. D., McDonald, J. C. "Epidemiology of asbestos-related lung cancer." In: Antman, K., Aisner, J. (eds.). Asbestos related malignancy. Boston, Grune & Stratton, 1987: pp. 57-79. 24. Enterline, P. "Changing attitudes and opinions regarding asbestos and cancer l934-65r Am J Ind Med, 1991; 20: pp. 685-700. 25. Hughes, J. M. "Human evidence: lung cancer mortality risk from chrysotile exposure." Aww Occup Hyg, 1994; 38: pp. 415-416, 555-560. 26. Gardner, M. J., Saracci, R. "Effects on health of non-occupational exposure to airborne mineral fibres." In: Bignon, J., Peto, J., Saracci, R. (eds.). Nonoccupational exposure to mineral fibres. lARC Scientific Publications No. 90. lARC, Lyon, 1989: pp. 375-397. 27. Selikoff, I. J., Hammond, E. C , Seidman, H. "Latency of asbestos disease among insulation workers in the United States and Canada." Cancer, 1980; 46: pp. 2,736-2,740. 28. Selikoff, I. J., Seidman, H. "Asbestos-associated deaths among insulation workers in the United States and Canada 1967-1987." Ann NY Acad Sci, 1991; 643: pp. 1-14. 29. Hammond, E. C , Selikoff, I. J., Seidman, H. "Asbestos exposure, cigarette smoking, and death rates." Ann NY Acad Sci, 1979; 330: pp. 473-490. 30. Vainio, H., Boffetta, P. "Asbestos and smoking in the etiology of lung cancer: mechanisms of the combined effect." Scand J Work Environ Health, 1994; 20: pp. 235-242. 31. McDonald, J. C , Liddell, F. D., Dufresne, A., McDonald, A. D. "The 1891-1920 birth cohort of Quebec chrysotile miners and millers: mortality 1916-%^:'Br J Ind Med, 1993; 50: pp. 1,073-1,081. 32. Dement, J. M., Brown, D. P. "Lung cancer mortality among asbestos textile workers: a review and update." Ann Occupat Hyg, 1994; 38: pp. 412,525-532. 33. McDonald, A. D., Fry, J. S., Woolley, A. J., McDonald, J. C. "Dust exposure and mortality in an American chrysotile textile plant." Br J Ind Med, 1983; 40: pp. 361-367. 34. Peto, J., Doll, R., Hermon, C , Binns, W., Clayton, R., Goffe, T. "Relationship of mortality to measures of environmental asbestos pollution in an asbestos textile factory." Ann Occup Hyg, 1985; 29: pp. 305-355. 35. Dement, J. "Carcinogenicity of chrysotile asbestos: Evidence from cohort studies." Ann TVFAcaJ^d, 1991; 643: pp. 15-23. 36. Mossman, B.T. "Carcinogenesis and related cell and tissue response to asbestos: a review." Ann Occup Hyg, 1994; 38: pp. 423, 617-624.

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37. Lippmann, M. "Asbestos exposure indices." Environ Res, 1988; 46: pp. 86-106. 38. Karjalainen, A., Anttila, S., Vanhala, E., Vainio, H. "Asbestos exposure and the risk of lung cancer in a general urban population." Scand J Work Environ Health, 1994; 20: pp. 243-250. 39. Wilkinson, P., Hansell, D. M., Janssens, J., Rubens, M., Rudd, R. M., Newman Taylor, A., McDonald, C. "Is lung cancer associated with asbestos exposure when there are no small opacities on the chest radiograph?" Lancet, 1995; 345: pp. 1,074-1,078. 40. Muscat, J. E., Stellman, S. D., Wynder, E. L. "Insulation, asbestos, smoking habits, and lung cancer cell types.'' Am J Ind Med, 1995; 27: pp. 257-269. 41. Kjuus, H., Skjaerven, R., Langard, S., Lien, J. T., Aamodt, T. "A case-referent study of lung cancer, occupational exposures and smoking. II: Role of asbestos exposure". Scand J Work Environ Health, 1986; 12: pp. 203-209. 42. Bovenzi, M., Stanta, G., Antiga, G., Peruzzo, P., Cavallieri, F. "Occupational exposure and lung cancer risk in a coastal area of Northeastern Italy." Int Arch Occup Environ Health, 1993; 65: pp. 35-41. 43. Jarvholm, B., Larsson, S., Agberg, S., Oiling, S., Ryd, W., Toren, K. "Quantitative importance of asbestos as a cause of lung cancer in a Swedish industrial city: a case-referent study." EurResp J, 1993; 6: pp. 1,271-1,275. 44. De Vos Irvine, H., Lamont, D. W., Hole, D. J., Gillis, C. R. "Asbestos and lung cancer in Glasgow and the west of Scotland." BMJ, 1993; 306: pp. 1,503-1,506. 45. Browne, K. "Is asbestosis or asbestos the cause of increased risk of lung cancer in asbestos workers." Br J Ind Med, 1986; 43: pp. 145-149. 46. Cullen, M. R. "Controversies in asbestos-related lung cancer." In: Rosenstock, L. (ed.). Occupational Medicine: State of Arts Reviews. Volume 2, No. 2. Hanley & Belfus, Philadelphia, 1987: pp. 259-272. 47. Doll, R., Peto, J. "Effect in health of exposure to asbestos." Health and Safety Commission. Her Majesty's Stationery Office. London, 1985. 48. Sluis-Cremer, G. K., Beduizenhart, B. N. "Relation between asbestosis and bronchial carcinoma in amphibole asbestos miners." Br J Ind Med, 1989; 46: pp. 537-540. 49. Hughes, J. M., Weill, H. "Asbestosis as a precursor of asbestos related lung cancer: results of a prospective mortaUty study." Br J Ind Med, 1991; 48: pp. 229-233. 50. Karjalainen, A., Anttila, S., Heikkila, L., Kyyronen, P., Vainio, H. "Lobe of origin of lung cancer among asbestos-exposed patients with or without diffuse interstitial fibrosis." Scand J Work Environ Health, 1993; 19: pp. 102-107. 51. Anttila, S., Karjalainen, A., Taikina-aho, O., Kyyronen, P., Vainio, H. "Lung cancer in the lower lobe is associated with pulmonary asbestos fiber count and fiber size." Environ Health Perspect, 1993; 101: pp. 166-170.

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52. Weiss, W. "Asbestos-related pleural plaques and lung cancer." Chest, 1993; 103: pp. 1,854-1,859. 53. Hillerdal, G. "Pleural plaques and risk for bronchial carcinoma and mesothelioma: a prospective study." Chest, 1994; 105: pp. 144-150. 54. Rosier, J. A., Woitowitz, H. J., Lange, H. J., Ulm, K., Woitowitz, R. H., Rodelsperger, K. "Radiologic diagnosis of asbestosis and risk for asbestosrelated malignancy." Proceedings of the 8th International Conference on Occupational Lung Diseases. ILO and Czech Medical Society, Prague, 1993, Vol. 1: pp. 357-367. 55. Waage, H., Johnson, E., Hilt, B., Langard, S., "Asbestosis and pleural changes as risk factors for asbestos-induced lung cancer." Int J Occup Med Toxic 1994, 3: pp. 319-321. 56. Nurminen, M., Tossavainen, A. "Pleural plaques and lung cancer: Is there an association without asbestosis?" Scand J Work Environ Health, 1994; 21: pp. 62-64. 57. Hueper, W. C. Occupational and environmental cancers of the respiratory system. New York: Springer Verlag, 1966. 58. Kannerstein, M., Churg, J. "Pathology of carcinoma of the lung associated with asbestos exposure." Cancer, 1972; 30: pp. 14-21. 59. Whitwell, F., Newhouse, M. L., Bennett, D. R. "A study of the histological cell types of lung cancer in workers suffering from asbestosis in the United Kingdom." BrJIndMed, 1974; 31: pp. 298-303. 60. Ruffie, P., Hirsch, A., Marteau, D., Bignon, J., Chretien, J. "Etiological and histological study of 448 cases of lung cancer." Ann Med Intern, 1981; 132: pp. 1-15. 61. Auerbach, O., Garfinkel, L., Parks, V. R., Conston, A. S., Galdi, V. A., Joubert, L. "Histologic type of lung cancer and asbestos exposure." Cancer, 1984; 54: pp. 3,017-3,021. 62. Johansson, L., Albin, M., Jakobsson, K., Mikoczy, Z. "Histological type of lung carcinoma in asbestos cement workers and matched controls." Br J Ind Med, 1992; 49: pp. 626-630. 63. Martischnig, K. M., Newell, D. J., Barnsley, W. C , Cowan, W. K., Feinmann, E. L., Oliver, E. "Unsuspected exposure to asbestos and bronchogenic carcinoma." 5rMeJ 7, 1977; 1: 746-749. 64. Mollo, F., Pira, E., Piolatto, G., Belhs, D., Burlo, P., Anreozzi, A., Bontempi, S., Negri, E. "Lung adenocarcinoma and indicators of asbestos exposure." Int J Cancer, 1995; 60: pp. 289-293. 65. Churg, A. "Lung cancer cell type and asbestos exposure." JAMA, 1985; 253: pp. 2,984-2,985.

CHAPTER 7 EMPHYSEMA AND LUNG MINERAL CONTENT IN COALWORKERS J. Leigh, T. DriscoU, and B. Cole National Institute of Occupational Health and Safety National Occupational Health and Safety Commission GPO Box 58, Sydney NSW 2001 Australia CONTENTS DEFINITION OF EMPHYSEMA, 137 Morphology and Classification, 139 Centriacinar (Centrilobular) Emphysema, 140 Focal Emphysema, 141 Panacinar (Panlobular) Emphysema, 142 DEFINITION OF COALWORKERS' PNEUMOCONIOSIS, 143 QUANTITATIVE RELATION BETWEEN EMPHYSEMA AND LUNG COAL AND SILICA CONTENT, 144 Methods, 145 Tissue Preparation, 145 Emphysema and Pneumoconosis Measurement, 145 Analysis of Lungs for Coal and Silica Content, 150 Smoking History, 153 Lung Function Measurements, 153 Years at Face, 153 Statistical Methods, 156 Results, 156 Discussion, 161 REFERENCES, 165 The relationship between emphysema and coal and silica in the lungs of coalworkers has been a controversial issue for many years. It is only recently that the results of large-scale pathological studies, incorporating quantification of both emphysema and lung coal and silica content, have allowed a clear resolution of the question. DEFINITION OF EMPHYSEMA The definition of emphysema in anatomical terms requires a good knowledge of the microanatomy of the lung. The pleural surface of the lung is characterized by septa, which give it a mosaic appearance. The septa, which are composed of thin

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strands of collagenous tissue, contain veins and lymphatics. The latter are very clearly seen in the adult city-dweller's lung because of carbon deposition. These septa outline the secondary lobule described by Miller [1]. It would be convenient if they constituted the main respiratory unit of the lung—i.e., if they were all supplied by a terminal bronchiole (the end stage of the branching conducting airways) and were devoted in their entirety to gas exchange. Unfortunately, such is not the case. The connective tissue septa referred to above have a somewhat irregular and indefinite distribution within the depths of the lung parenchyma. A study of post-mortem bronchograms led Reid [2] to define a much more constant and satisfactory unit. She found a fairly sharp transition from a branching pattern at intervals of approximately 1 cm to one of intervals at 1 nmi; this occurred at the level of the terminal bronchiole. It was therefore proposed to describe that unit of lung tissue supplied by the terminal bronchiole as the acinus. The number of such units—i.e., the number of terminal bronchioles within secondary lobules—varies between three and five. The acinus is the true respiratory unit of the lung. Distal to the terminal bronchiole, the air passages (e.g., respiratory bronchioles and alveolar ducts) are concerned not only with conduction of gas to and from the most distal portions of the acinus but also, by virtue of alveoli present in their walls, with gas exchange. In 1958, a Ciba Guest Symposium [3] was arranged to consider "terminology, definition, and classification of chronic pulmonary emphysema and related conditions." As a result of their deliberations, a definition was formulated in anatomical terms as follows: Emphysema is a condition of the lung characterized by increase beyond the normal in the size of air spaces distal to the terminal bronchiole either from dilatation or from destruction of their walls. This, although an enormous advance, does not distinguish clearly between overinflation, a condition found for instance in asthma, in which the architecture of the lung parenchyma is intact, and the state of the lungs found in emphysema, where the architecture is plainly disturbed. To overcome this defect, Thurlbeck et al. [4] proposed a new and more precise definition. Pulmonary emphysema is defined as an abnormal permanent increase in size of the respiratory portion of the lung beyond (i.e., distal to) the terminal bronchiole, or portion of the lung known as acinus, accompanied by destructive changes. Destruction of a tissue by disease may be defined as the reduction of that tissue to a useless form or to nothingness. Destruction, occurring with respiratory airspace enlargement in emphysema, may be recognized by the naked eye, by low-power magnification of an inflation-fixed lung slice, or by light microscopic evaluation of thick (200 to 400 |Lim) or thin (4 to 6 jam) stained and mounted sections. At the subgross level, destruction is manifest by the disorder of the respiratory airspaces. At the microscopic level, disorder of the blood vessels, connective tissue network, and

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the other elements of respiratory airspace walls may be evident. Agreement was reached on the following definition: Destruction in emphysema is defined as nonuniformity in the pattern of respiratory airspace enlargement so that the orderly appearance of the acinus and its components is disturbed and may be lost [5]. This is now generally accepted. Both these definitions, by the use of words "increase in size," introduce a quantitative element into definition of the disease. Furthermore, they presuppose a knowledge of normal dimensions that is not altogether justified. It must, for instance, be remembered that although the normal alveolus has a mean "diameter" of 250 jam [6], there is considerable variation in alveolar size within the lung. Alveoli at the apex, for instance, have a much larger volume than those at the base [7]. However, many quantitative studies of the pathology of emphysema, which have employed the point-counting method on gross material, have taken any air space with a diameter of greater than 0.1 cm as being emphysematous [8]. Morphology and Classification Once the above definition of emphysema is accepted, it becomes apparent that the term covers not one disease but a group of disorders that show differing and distinct morphological features. The variety of appearances found in the lung has led to many attempts at classification [3]. The most logical is probably that proposed by Thurlbeck [9], which described emphysema according to the part of the acinus involved. Thus: (i) proximal acinar (centriacinar) emphysema includes disease of the respiratory bronchioles, (ii) panacinar emphysema is the term used when the entire acinus is affected, (iii) distal acinar emphysema is the name given to that state where alveolar ducts and sacs are selectively diseased, and (iv) irregular emphysema describes the condition when no selective portion of the acinus is predominantly affected. To a large extent, many of the classifications proposed represent a somewhat fruitless exercise for two main reasons. Firstly, even a superficial examination of fixed inflated lungs reveals that there is often, some would say usually, more than one type of disease present, so neat categorization of individual cases is not possible. Secondly, very few classifications have justified themselves from an etiological or functional viewpoint. This is not to say that certain well-recognized forms of disease do not have distinct etiologies (e.g., panacinar destructive emphysema and alantitrypsin deficiency). Because of these considerations, the main, well-recognized types of emphysema will be described, but it must be realized that, while "pure"

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forms of each condition may occur, more often than not more than one type of disease is present in any given lung. Centriacinar (Centrilobular) Emphysema Gross Appearances The characteristic feature is the presence of abnormal air spaces situated in the central portions of secondary lobules—hence the name—almost invariably surrounded by some normal lung parenchyma that intervenes between one abnormal space and the next. As is apparent from a study of the microanatomy, a more correct name for the disease would be centriacinar emphysema, and indeed there are often two or more emphysematous spaces within one secondary lobule. The upper zones are affected more frequendy than the lower zones [10-12]. This is particularly true of subclinical cases, i.e., those without any symptoms. In addition, Thurlbeck [12] has demonstrated that not only is the disease more common in the upper zones, but it is also more severe in that the abnormal spaces are larger and more numerous than in lower zones. The region of the emphysematous space is often the seat of carbon deposition, particularly in city dwellers. The relation of this to "focar'emphysema is considered below, but it may be stated here that dust is not an essential feature of the disease [13]. Macroscopic examination of the spaces frequently reveals that they are multilocular due to the presence of septa, which are, however, incomplete as there is free communication between the locules. Histology The nature of the emphysematous spaces was elicited by Leopold and Gough [14]. If serial sections are taken through a lobule affected by this condition, it will be found that the proximal airway supplying the space is either a terminal bronchiole or a first-order respiratory bronchiole. Furthermore, the parenchyma distal to the emphysematous space is composed of respiratory bronchioles of the third order or alveolar ducts. It follows from this that the centriacinar space is formed from diseased and largely destroyed respiratory bronchioles of the first or second order. Heppleston and Leopold [15] state that the spaces are composed of "groups of disrupted respiratory bronchioles." The word "disrupted" is best avoided, as it means forcible severance or breaking asunder and thus implies a mechanical or traumatic origin of the spaces when this is not intended. Indeed, Heppleston and Leopold were among the first to emphasize three important histological features in this disease that belie any traumatic etiology and will be referred to subsequently, namely: (i) The parent airway supplying the spaces is nearly always found to be the seat of inflammation, in that there is peribronchiolar polymorph or lymphocytic infiltration.

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(ii) Fragments of tissue—the septa seen on the gross specimen—are often the seat of chronic inflammation, (iii) The parent airway is often stenosed. Heppleston and Leopold speculate that the first and third features may result in rigidity of the parent proximal airway with resultant inability to dilate or elongate on inspiration, a concept of some considerable functional importance. Focal Emphysema This is a term that has been given to the disorder associated with simple pneumoconiosis of coal miners. It is not confined to these workers, as it occurs with varying degrees of severity in all those exposed to carbon dust inhalation and has been found in, among others, coal trimmers in docks [16], and in graphite and foundry workers [17]. The term is not a good one, as it implies disease occurring at any focus in the lung whereas in fact it is a disorder of respiratory bronchioles. The pathological anatomy of this condition was beautifully demonstrated by Heppleston [18, 19]. Gross Appearances The lung is very similar to that in centriacinar emphysema and indeed differs from it only in the degree of carbon pigmentation which is present. On opening the pleural cavities in a fully developed case, the lungs appear black. The cut surface of fixed inflated lung reveals numerous discrete foci of black pigmentation situated at the center of secondary lobules, more frequent and more intense in upper than lower parts of the lung, and associated with emphysematous spaces. Histology As Heppleston [18] so elegantly showed, the microscopic features of this condition are characteristic. The emphysematous spaces are formed by dilated respiratory bronchioles, and the very large quantity of carbon dust that is present accumulates primarily in alveoli in their walls. Fibrosis is minimal. There is, however, loss of smooth muscle in respiratory bronchioles. Heppleston and Leopold [15] emphasize that bronchiolitis, stenosis of supplying airways, and "disruption" of alveolar or bronchiolar walls are not essential features and may be absent in early stages of the disease. In fact, it is claimed that the condition is primarily one of dilatation of respiratory bronchioles associated with dust deposition. Is the Distinction between Focal and Centrilobular Emphysema Valid? It will be apparent from the above descriptions that there are remarkable similarities between the two conditions and the reader may well doubt whether any true

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distinction is justified. Heppleston and Leopold clearly think that these are two separate diseases. They base their view that focal emphysema is an entity in its own right on the following grounds: (i) Its distribution in the lung is more uniform than that of centrilobular emphysema, (ii) The airway supplying the emphysematous spaces is not inflamed or stenosed, (iii) There is a large quantity of dust present adjacent to the emphysematous space, and (iv) Initially there is no "disruption" of walls of respiratory bronchioles, but only dilatation. Clearly, these are formidable reasons if true, and they are reinforced by the authors' considerable experience in the South Wales coalfield. However, it is a view that has not gone unchallenged. Wyatt [20], using the whole lung papermounted section technique, considered the two conditions were not distinguishable, an opinion supported by Mitchell [21]. Furthermore, Duguid and Lambert [22] found that fibrosis was related to both dust deposits and emphysema. Ryder et al. [23] have shown that there is an excess of centriacinar emphysema in miners that is not associated with progressive massive fibrosis. The whole argument is extensively treated in Thurlbeck's excellent monograph [9], and he concludes on the basis of his own considerable experience and on evidence from the literature that coal miners have an excess of emphysema and that in mild cases this can be distinguished from centriacinar emphysema. In our view, the argument is merely a semantic one. The walls of the enlarged air spaces of focal emphysema show evidence of increased fenestrations, alterations in capillary density, and loss of elastic fibers representing a form of tissue destruction [24]. The lesion, therefore, differs from centriacinar emphysema only in that it is associated with the macular dust lesion. It is extremely difficult to be certain whether "disruption" and fibrosis are present in a lung that is the seat of very heavy carbon deposition, as the carbon deposition itself masks all other histological features. It would be an advantage if the term "focal emphysema" could be discontinued, as it would be simpler and more accurate and convenient to refer to all forms of emphysema where respiratory bronchioles are affected as centriacinar emphysema and to add whether or not they were associated with excess carbon dust deposition. Panacinar (Panlobular) Emphysema When the emphysematous process affects the acinus in a uniform manner, the term "panacinar destructive emphysema" is employed. The word "destructive" is inserted to distinguish this state from the overinflation of the acinus that occurs in asthma, where there is partially or wholly reversible airways obstruction, or in "compensatory" emphysema where there is permanent overdistension of the remaining lung following removal or obstructive collapse of another portion of lung.

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The term "panacinar emphysema" is to be preferred to "panlobular" for the simple reason that emphysema itself is defined in terms of the acinus. Gross Appearances Lungs that are the seat of severe panacinar destructive emphysema are enlarged and fail to collapse on opening the pleural cavities. The outstanding feature seen on the cut surface of the fixed inflated lung is a spectacular enlargement of air spaces within the acinus and lobule with obvious disorganization of architecture. Distribution of the process within the lung is somewhat variable but tends to affect lower rather than upper zones [12], though it may involve the entire lung. Sometimes the disease is localized to a few lobules, often situated along the anterior margins of an upper lobe. Minor degrees are difficult to recognize in gross material as they are hard to distinguish from overdistension not accompanied by dissolution of alveolar septa. A rough guide is that spaces greater than 0.1 cm in diameter, situated in the periphery of a secondary lobule or acinus, may be taken as abnormal, particularly in the lower zone of the lung. Histology The outstanding feature is total disorganization of acinar architecture. The affected lung is virtually devoid of any normal alveoli and replaced by large, thin-walled spaces of irregular shape and size. The size may be judged from the few remaining normal alveoli that can often be seen. Occasional terminal bronchioles can be made out within secondary lobules, but there is no evidence of orderly division of more distal airways. Indeed, respiratory bronchioles cannot be seen at all, and distinction between these structures, alveolar ducts, and sacs is lost. DEFINITION OF COALWORKERS' PNEUMOCONIOSIS The fundamental lesion of coalworkers' pneumoconiosis consists of a focal aggregation of coal mine dust-laden alveolar macrophages that accumulate in the first- and second-order respiratory bronchioles [25-27]. These cells become enmeshed by reticulin, elastin, and collagen fibers, leading to weakening dilation of the bronchiole wall. The collection of dust-laden macrophages is referred to as the coal dust macule and the dilated bronchioles as focal or centriacinar emphysema [18, 25-27]. Generally, the respiratory bronchioles lead to normal-size alveolar ducts and alveolar sacs that abut on a lobular septum. Panacinar emphysema is also observed in coal miners. Cigarette smoking can cause these lesions as well, making attribution difficult in the smoking miner with emphysema.

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Additionally, in some coalworkers exposed to coal mine dust with a higher percentage of silica, there may be fibronodular lesions as well [27]. The dust-laden activated alveolar macrophages in second-order terminal respiratory bronchioles release exaggerated amounts of oxidants. Increased oxidant release may injure alveolar and bronchial epithelial cells, providing access to interstitial connective tissue. An increased amount of antigenic elastase enzyme may be associated with the destruction of the elastin-containing connective tissue matrix and may attract neutrophils to potentiate the inflammatory response by oxidative reduction of antiprotease activity. Taken together, these cellular events in the local lung microenvironment lead to emphysema, a basic lesion of coalworkers' pneumoconiosis. QUANTITATIVE RELATION BETWEEN EMPHYSEMA AND LUNG COAL AND SILICA CONTENT* Well-designed large studies using post-mortem pathological material have shown that emphysema, especially centriacinar emphysema, is qualitatively related to coal dust content of lungs [28-30] and quantitatively related to pneumoconiosis severity, years at the coal face, and indices of obstructive lung disease obtained during life [31, 32]. Previous researchers have found an association between emphysema prevalence and lung coal content [28-30], but not between extent of emphysema and measured coal content. In a hospital-based case-control study, Cockcroft et al. [28] found an excess of emphysema in coalworkers compared to non-coalworkers, and an association between severity of emphysema and dust exposure. However, the dust exposure score was based on pathological assessment of lung specimens rather than on measured lung dust content or exposure measurements. Ruckley et al. [29, 30] showed a clear relationship between the presence of centriacinar emphysema and higher coal dust exposures (whether as inhaled dust or lung content of dust), but were unable to demonstrate a relationship between the amount of emphysema and coal dust exposure. They suggested that this may have been due to problems with lung inflation and tissue fixation or an indication that coal initiates the emphysema but other factors influence its progression. Recent studies of coalworkers have suggested that emphysema is relatively less likely with high lung silica content [30-33]. In contrast, emphysema has been found to be related to silica dust exposure in goldminers [34, 35]. Estimating the relationship between emphysema and coal dust exposure by evaluating pneumoconiosis pathologically is potentially biased because the coal dust lesions draw attention to the emphysema. This can be partly avoided by the use of radiological pneumoconiosis scores [32]. However, the radiological appearances may not be due to coal. The most objective exposure index, not subject to these biases, is actual lung coal content. *Based on Leigh et al. [66, 67].

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Underground coalworkers in the New South Wales, Australia, coal industry are exposed to high-rank bituminous coal (carbon dry ash free 82-90%). Silica content of the New South Wales coal seams is on average less than 2% [36]. Some workers would have been exposed to higher silica levels when working in shaft development and in other mines, predominandy in the United Kingdom. Some of these workers would have been exposed to much higher dust levels than those obtained today. The aim of this study was to determine the relationship between quantified emphysema and quantified lung content of coal dust and silica dust in coalworkers. Methods The data analyzed in this study came from workers in the New South Wales coal mining industry who died during the period July 1966 to January 1983. The New South Wales Joint Coal Board, with the assistance of relevant unions, has encouraged the conduct of post-mortem examinations on all deceased coalworkers since 1949. However, the proportion of coalworkers who underwent post-mortems is probably influenced by a number of factors, including perceived eligibility for compensation. The 264 subjects included in this study represent 70% of the 376 coalworkers (all male) who underwent post-mortem during the study period. Inadequacy of lung specimens was the main reason for exclusion of subjects. Other subjects who were excluded from the analyses had missing data. Subjects who underwent a post-mortem comprised about 20% of all coalworkers who died during the study period (based on the New South Wales annual male crude death rate of 9.6/1,000 in 1972 and a mean mining population of 14,000 over the study period). Tissue Preparation After removal from the body, both lungs were perfused immediately with 10% acetate formalin via vessels and bronchi under a pressure of 120 cm water without any vessels or airways tied off, until the lungs were inflated to a volume between functional residual capacity and total lung capacity. The lungs were stored in 10% formalin in plastic containers. Blocks for histology were taken from each lobe of each lung and from hilar lymph glands. In 35 subjects, Gough-Wentworth large lung sections [37] were made of the left lung, using a two centimeter-thick sagittal slice involving the hilar region. Emphysema and Pneumoconiosis Measurement Emphysema was quantified by one of two observers using a modification of the Heard method [38]. This was done on macroscopic examination of the surfaces of at least three cuts in the sagittal plane in each lung. Each cut was divided into three segments of approximately equal area. In each segment, the percentage of lung area involved as abnormal holes was measured and the mean for each of the three or

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more cuts calculated. Both fixed and Gough-Wentworth sections were used. Where both types of sections were available for the same subject, the emphysema estimations agreed closely. The mean for all the cuts from both lungs became the final percentage emphysema score. This was then coded 1-7 (1: nil; 2: 1-10%; 3: 11-20%; 4: 21-30%; 5: 31-40%; 6: 41-50%; 7: > 50%). There was good agreement shown between the two observers for separate readings of 400 lungs using this method [32]. Figures 1-8 illustrate different levels of emphysema. Classification of pneumoconiosis was made on the basis of macroscopic and microscopic examination of lung tissue and an examination of whole lung sections. Macules/nodules were classed as sparse (equivalent to ILO 1/0 or less), moderate (less than ILO 2/2), or profuse (ILO 2/2 or greater), and massive lesions documented. Distinction was made between coal only (macules), mixed coal/silica (nodules, palpable), and siHca only changes. One observer made all classifications. Pneumoconiosis classification was available for 259 (98%) subjects. Figures 9-12 illustrate typical histological appearances. (text continued on page 150)

Figure 1. Large lung sections from inflated post-mortem coalworkers' lungs showing normal lung (E score 1).

^~/-&&7^

Figure 2. <10% emphysema (E score 2).

Figure 3.10-20% emphysema (E score 3).

Figure 4. 20-30% emphysema (E score 4).

Figure 5. 30-40% emphysema (E score 5).

Figure 6. 40-50% emphysema (lifelong non-smoker) (E score 6).

Figure 7. 60% emphysema (E score 7).

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Health and Toxicology

Figure 8. Large lung section from coalworker showing classical silicosis with pigmentation <10% emphysema (E score 2).

(text continued from page 146)

Analysis of Lungs for Coal and Silica Content Specimens were freed of non-lung tissue, cut into approximately 2 cm cubes, homogenized in a blender, dried on shallow trays at 105°C in a laboratory oven, and ground in a mill to pass a 0.5 mm screen, giving an average particle size of 200 ^im. Lungs had been previously dissected at autopsy and no attempt was made to further separate lymphatic tissue. Tissue used for preparation of large lung sections was included. Powdered lung samples were hydrolyzed as follows using a modified version of the procedure of Guest [39]. Subsamples of the powdered lung (1 g) were hydrolyzed in duplicate in screw-capped polypropylene centrifuge tubes by heating with shaking for two hours at 60°C with concentrated hydrochloric acid (20 ml, 36%). The hydrolysis procedure was carried out three times. Each hydrolysis step was followed by dilution with ethanol (20-25 ml), separation of the solid residue by

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Figure 9. Coal macule and surrounding focal (centriacinar) emphysema (x 500).

Figure 10. Coal macules and surrounding focal (centracinar) emphysema (x 50).

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Figure 11. Silicotic nodules with coal pigmentation (x 50).

Figure 12. Mixed coal/silica nodule (x 500).

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centrifuging (30 min, 3,000 rpm), and careful siphoning off of the hydrolysis product (supernatant). The separated residue was repeatedly washed with ethanol and centrifuged as before, until the supernatant was clear (usually two to three washes). The residue in the centrifuge tube was transferred quantitatively to a pre-weighed platinum crucible using ethanol and toluene to transfer the slurried extract. Total lung dust, total coal, and total mineral matter (ash) were determined gravimetrically: the total lung dust after first drying the crucible contents at 110°C, the total mineral matter after ashing the crucible contents at 600°C overnight, and the total coal by calculating the difference between total lung dust and total mineral matter. Finally, siUca (a quartz) was determined in the ash by Fourier Transform Infrared Spectroscopy (FTIR) using the potassium bromide disc technique (1 mg ash per 250 mg disc), essentially using the method of Dodgson [40]. By ashing at 600°C, less interference in the quartz spectra was obtained at the expense of not being able to quantitatively determine kaolin and mica. The main results consider the total amount of coal and silica in the two lungs of each subject, standardized by predicted vital capacity (using height and age) [41]. For comparison purposes, results were also expressed as proportions of coal or silica by dry weight of lung, and as absolute amounts of coal and silica. Figures 13-16 show typical spectra, transformed spectra and calibration curves. Smoking History Data on smoking history were obtained from a standardized questionnaire given at Joint Coal Board routine clinical examinations held every two to three years. Smoking was expressed as ounces of tobacco smoked per week (1 ounce = 28 g), based on a lifetime average of tobacco intake. Lung Function Measurements Forced expiratory volume in one second (FEVj, body temperature, atmospheric pressure, and saturation) was measured using a Vitalograph spirometer with results recorded as the better of two satisfactory efforts. The measurements were performed at routine clinical examinations. Measurements were only accepted for the purposes of this study if taken within five years of death. Results were available for 183 (69%) subjects. Values were expressed as percentage predicted (FEVi%) [42]. Years at Face Information on years worked at the coal face was obtained from the detailed occupational histories taken at each routine Joint Coal Board clinical examination.

{text continued on page 156)

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600

900

700 800 Wavenumbers ( c m - 1 )

0.116, 0.224. 0.274, 0.474, 0.784, 0.991 m g A9950 q u a r t z s t a n d a r d s

Figure 13. Infrared absorption spectra for quartz standards.

1.2 r

C O

U.O

0.2

0.4

0.6

0.8

1.0

Quartz/mg Figure 14. Calibration lines for quartz absorption peaks at 779 and 798 c m - \

Emphysema and Lung Mineral Content in Coal workers

600

700 800 Wavenumbers (cm-1) Ashed lung pressed 20/5/92. No. 6261

155

900

Figure 15. Typical quartz spectrum for ashed coal miner's lung.

600 7321

700 800 Wavenumbers ( c m - l ) Res= 4 c m - l

900

0 6 / 0 5 / 9 2 11:21

Ashed lung pressed 2 0 / 5 / 9 2 . No.7321

Figure 16. Typical Fourier Transform of quartz spectrum for ashed coal miner's lung.

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{text continued from page 153)

Statistical Methods Calculations were performed using standard simple parametric and regression techniques as programmed in the Statistical Analysis System [43] with appropriate checking of residuals in regression models. Statistical significance was determined at the 5% level (two-sided). Results Frequency distributions, means, standard deviations, and quartile values for emphysema score, lung coal content, lung silica content, smoking amount, age at death, and FEVi% are given in Table 1. Age, smoking, and FEVi% had basically normal distributions, with similar medians and means. In contrast, the lung dust content variables and emphysema score were positively skewed. Excluded subjects had a similar mean emphysema score (3.1), age (67 years), smoking amount (3.1 oz/week), FEVi% (74% predicted), and years worked at the coal face (21) as subjects included in the analyses. Subjects without FEVi data had higher mean emphysema score (3.6 vs. 2.8), higher mean lung coal content (1.25 vs. 0.73 g/1), higher mean lung silica content (0.051 vs. 0.037 g/1), and were older (67 vs. 62 years), but had similar smoking amounts (2.9 vs. 3.2 oz/week) and years worked at the coal face (21 vs. 19) compared to subjects with FEVi data. The majority of subjects had minimal or mild pneumoconiotic changes, and there were only four subjects with silica-only changes (Table 2). Mean lung coal content

Table 1 Means, standard deviation, and percentiles of main variables Percentiles

N Emphysema score Adjusted lung coal content (g/1*) Absolute lung coal content (g**) Adjusted lung silica content (g/1*) Absolute lung silica content (g**) Tobacco smoked (oz per week) Age at death (years) FEVi%***

264 264 264 261 261 264 264 183

SD

25%

3.1

1.5

0.89 3.93 0.04 0.18

0.81 3.44 0.04 0.17

Mean

3.1

63.4 78.7

2.2

10.4 25.5

50%

75%

2

3

4

0.32 1.53 0.02 0.08

0.65 2.88 0.03 0.12

1.21 5.07 0.05 0.21

2

58 60

3

64 81

^Adjusted by predicted vital capacity ^^Absolute amount of lung coal or silica per subject *** Measured FEVj as a percentage of predicted (using height and age) [41]

4

71 98

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and mean lung silica content were highest in those subjects with a mixed pneumoconiosis, next highest in those subjects with primarily coal pneumoconiosis, and lowest in subjects with minimal changes. All differences were statistically significant (Table 3). When examined in the same model using logistic regression, lung fibrosis was highly significantly related to lung silica content (p < 0.005) and age (p < 0.0001), but not to lung coal content (p > 0.55). The distribution of emphysema score was positively skewed, suggesting that a log transformation might be appropriate when using regression techniques. However, results from the regression equations were similar with and without transformation. A slightly better fit was obtained when the log transformation of emphysema score was used. Residuals were appropriately distributed whether the emphysema score or its logarithm was used as the outcome variable. For clarity, only the analyses involving the untransformed emphysema score are presented. The correlation matrix for variables of interest is shown in Table 4. Emphysema score (E score) increased significantly with lung coal content, age, and amount of smoking (Equation 1). Equation 1 E score = -1.73 +0.22 (coal g/1) +0.07 (age years) +0.10 (smoking oz/week) (Adjusted R^ = 0.23)

t = 3.12 t = 2.05 t = 8.18 t = 2.50

p < 0.002 p < 0.05 p < 0.0001 p < 0.02

Table 2 Distribution of pneumoconiotic changes (frequency [n] and percent)

Pneumoconiosis severity Sparse n % Pneumoconiosis Type Coal Mixed Silica Sparse Total

123 (47) 123 (47)

Moderate n %

Profuse % n

72 29 4 105

10 11 21

(28) (11) (2) (40)

(4) (4) (8)

IVlassive n %

4 6 10

(2) (2) (4)

n

Total %

86 (33) 46 (18) 4 (2) 123 (47) 259 (100)*

^Subjects did not have data regarding pneumoconiotic change; errors in total percentages are due to rounding.

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Health and Toxicology Table 3 Mean coal and silica content by type of pneumoconoisis Lung content (g/l predicted vital capacity) Coal* Pneumoconiosis type

Sparse (n = 123) Coal (n = 86) (moderate, profuse, massive) Mixed (n = 46) (moderate , profuse, massive)

Silica**

Mean

SD

Mean

SD

0.53 1.15

(0.41) (0.84)

0.03 0.05

(0.02) (0.03)

1.41

(1.11)

0.07

(0.06)

^Mean lung coal content significantly different for each group (F = 31.8, p < 0.0001), using Duncan's multiple range test. ^^Mean lung silica content significantly different for each group (F = 30.6, p < 0.0001), using Duncan's multiple range test.

Table 4 Correlation matrix of main variables* (Pearson correlation coefficient (r)) Emphysema n=264 Emphysema (Score) Coal (g/l) Silica (g/l) Age (years) Smoking (oz/week) Face (years) FEVi%

1.00 0.20*** QJ9*** 0.46* 0.04 0.24* -0.44*

Coal n=264

Silica n=264

Age n=264

Smoking n=264

Face FEVi% n=264 n=183

1.00 0.54* 1.00 0.23** 0.26* 1.00 —0 15**** -0.25* -0.17*** 1.00 0.26* 0.45* -0 19*** 0.27* 1.00 0.05 -0.31* -0.02 -0.11 -0.01

1.00

*/7 < 0.0001 **/7 < 0.001 ***/? < 0.005 ****/? < 0.02

Lung silica content did not contribute significantly to the model. Addition of a coal-smoking interaction term improved the model slightly, with coal and smoking having a negative interaction (Equation 2). Note that the effect of lung coal content on E score must be obtained by using a combination of the coal-only and coal x smoking regression coefficients, as the effect of coal depends on the level of smoking.

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Equation 2

E score = -1.79 +0.62 (coal g/1) +0.06 (age years) +0.21 (smoking oz/week) -0.17 (coal X smoking) (Adjusted R^ = 0.25)

t = 3.28 t = 3.64 t = 7.76 t = 3.91 t = 2.99

p< p< p< p< p<

0.002 0.0003 0.0001 0.0001 0.004

Of the other potential interactions (coal x silica; coal x age; silica x age, silica x smoking; and age x smoking), none showed a significant effect. Similar results were obtained when the coal and silica contents were expressed in an unadjusted form (as absolute weight per subject) or as a proportion of the dry weight of lung (Table 5). When the 40 non-smokers were considered separately, the positive effect on emphysema score of lung coal content and age was striking, with these two variables accounting for about two-thirds of the variation in emphysema score (Equation 3). Lung silica content and all interaction terms did not contribute significantly to the model. The univariate association between lung coal content and emphysema for non-smokers is shown in Figure 17. Equation 3 E score = - 1 . 5 6 +0.78 (coal g/1) +0.06 (age years) (Adjusted R2 = 0.66)

t = 1.98 t = 5.43 t = 4.19

p < 0.06 p < 0.0001 p < 0.0002

Table 5 Regression parameters for various measures of lung coal content Measure of coal used in final regression equation

Parameter

coal age smoking coal X smoking adjusted R^

adjusted by vital capacity t value p > It!*

proportion of lung dry weight t value p > Itl*

absolute amount per subject t value p > it!*

3.64

0.0003

3.53

0.0005

3.79

0.0002

7.76 3.91 -2.99

0.0003 0.0001 0.004 0.25

7.86 3.28 -1.89

0.0001 0.002 0.06 0.26

7.87 4.02 -3.15

0.0001 0.0001 0.002 0.26

^probability of obtaining an absolute value oft as great or greater

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Emphysema score had a highly significant negative relationship with FEVi% for the 184 subjects who had available FEVi% measurements (Equation 4). Almost identical results were found when lung coal content was added to the regression model (P E score = -7.39, t = -6.73, p < 0.0001), with coal not making a significant contribution to the model (|3coal = 2.86, t = 1.00, p > 0.32). This relationship appeared to be similar for non-smokers, with the low numbers of non-smokers with available FEVi data (28) probably responsible for the borderline statistical significance of this relationship (Equation 5). Equation 4 FEVi%= 99.2 -7.24 (E score)

t = 28.22 t = 6.66

p < 0.0001 p < 0.0001

t= 10.24 t = 1.76

p<0.0001 p < 0.09

(R2 = 0.19)

Equation 5 FEVi%=

105.3 -7.21 (E score) (R2 = 0 . 1 1 )

Emphysema score was significantly higher in subjects with pneumoconiotic change (moderate, profuse, or massive) compared to subjects with minimal or no

Emphysema score

1

2

3

Lung coal content adjusted by vital capacity (g/l) Figure 17. Relationship between emphysema score and adjusted lung coal content in lifelong non-smokers: r = 0.81 p < 0.0001 (n = 40).

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pneumoconiotic changes (t = 4.90, p < 0.0001). This relationship was independent of lung coal and lung silica content. Mean FEVi% was higher, but not significandy so, in subjects with minimal or no pneumoconiotic change compared to those with definite pneumoconiotic change (82% vs. 76%; t = 1.40, p > 0.17). Discussion The results from this study show clearly that lung coal content is quantitatively related to the development of emphysema, taking into account age and smoking. This is true whether the effect of smoking is controlled using regression methods or by examining only non-smokers. The results indicate that a non-smoking miner with about the average lung coal content would have emphysema involving an extra 7 to 10% of the whole lung mass compared to a non-smoking person of the same age who was not exposed to coal. The influence of coal appears less marked in smokers, possibly due to a negative interaction between smoking and coal. Although other possible causes of emphysema in coalworkers, apart from coal and smoking, have been suggested [44], the very high R^ term in the regression equation for non-smokers strongly suggests that coal exposure and age are the main determinants of emphysema in non-smoking coalworkers. Biologically plausible mechanisms for a pathological effect of coal on lungs to produce emphysema are now well-documented [45, 46]. The analytical technique used in this study measures total coal and silica content of the lung, which includes both dust in the bronchi and in the parenchyma. This measurement is used as a proxy for the true measure of interest, parenchymal dust, as it is in the parenchyma that any pathological effects that might result in emphysema presumably occur. Therefore, any process (such as dust being trapped in the increased mucus produced in bronchitis) that might alter the proportion of total lung dust in the parenchyma could affect the observed relationship between total lung dust and emphysema. Unfortunately, it is not known how much variation in this proportion might occur due to pathological processes or differences between individuals, because it is too difficult to dissect out the bronchial tree from the parenchyma to allow separate measurements of dust content in each. However, known particle deposition distributions would suggest that the parenchymal component would be much larger than the bronchial component, except in very unusual circumstances. An alternative explanation for the observed coal-emphysema association might be that emphysema somehow impairs clearance of dust from the lungs. Greater emphysema would therefore be associated with dust in the lung because of decreased clearance rather than because the dust was causing the emphysema. The effect of emphysema on clearance is uncertain, and there is animal and human evidence of both decreased and increased clearance in emphysematous lungs [47^9].

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The strong positive association between age and emphysema found in this study is well-documented elsewhere [50]. An association between smoking and emphysema as found in this study has been well-documented in the general community [51], coal miners [52], and goldminers [34, 35]. The negative interaction between coal and smoking does not have a clear explanation. Perhaps it is due to the fact that smokers have more bronchitis [51], and that the increased mucus production traps more cqal in the bronchi before it reaches the lung parenchyma. Therefore, for a given amount of measured dust in the lung, more smoking may result in a smaller proportion of dust reaching the parenchyma and correspondingly less emphysema than would otherwise be expected. Alternatively, the negative interaction may be due to a form of competitive inhibition, with the individual effects of coal and smoking having a final common pathway in which the presence of one interferes with the effect of the other. This is plausible, as macrophage or polymorphonuclear leucocyte damage or activation, with subsequent release of elastase or other proteases, is a probable mechanism for the development of emphysema in both smokers [52] and coal miners [45, 46]. Finally, the observed relationship might simply be a result of statistical associations inherent in this data set rather than a reflection of a true biological phenomenon. Silica content in the lung was not found to be significantly associated with emphysema in this study. Previous investigators have found conflicting results. One group investigating British coalworkers found a negative effect of silica on emphysema prevalence in coalworkers [30]. This was true for lung silica content for subjects with progressive massive fibrosis; and for percentage silica of lung dust or percentage silica in lung for subjects with any fibrotic lung disease. They postulated that silica-induced fibrosis might make emphysematous change less likely. In goldminers exposed to "silica dust," the prevalence of centriacinar emphysema was found to be related to dust exposure level and to the presence of silicosis in one study [35]. In contrast, an earlier study of goldminers found a positive association between the presence of any emphysema and dust exposure levels, but no significant effect of silicosis [34]. It is possible that the relationship between emphysema and "silica dust" in these two studies was due to some other component of the dust apart from silica, although the dust is known to have had a high silica content [35]. A very recent study of lifelong non-smoking white South African goldminers found only low levels of emphysema, mainly panacinar, unrelated to dust exposure, pathological silicosis, or loss of respiratory function [53]. The lack of an emphysema-silica association in the present study may be due to relatively low silica exposures and correspondingly less silica-related fibrotic lung disease, even though there was a strong relationship between the presence of fibrotic lung disease (mixed coal-silica or silica-only disease) and lung content of silica. The subjects included in this study were mosdy recent miners who had a relatively low prevalence of fibrotic lung disease. Of the 259 subjects who had data available on lung histology, 19% had mixed coal-silica or silica-only nodules, with only 34% of these (6% of the total) showing profuse nodularity or massive fibrosis (ILO 2/2 or

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greater). In comparison, in a study of British coalworkers that showed a negative emphysema-siUca association, 79% of the subjects had some fibrotic lung disease and 33% were said to have progressive massive fibrosis [30]. A study of an earlier group of miners from the same mining regions as the subjects used in this study, but exposed to a higher concentration of respirable dust, had a much higher prevalence of fibrotic lung disease, with 48% having mixed nodules and 54% of these (26% of the total) having ILO 2/2 or greater [33]. In the latter group, there was a significantly lower prevalence of emphysema in those subjects with mixed nodules compared to those with coal-only macules. This difference was especially marked in those with profuse mixed nodules, supporting the suggestion that there may be a negative association between silica and development of emphysema. Unfortunately, the lungs of those subjects were not available for the mineral analysis used in this study. Lung silica content of subjects in this study was also lower than in other studies, both on an absolute and a percentage basis. This study showed a mean lung silica content of 0.11% of dry lung weight, compared to 0.35% in the above study of British coalworkers [30]. The comparable absolute amounts were 0.18 g compared with 0.30 g per subject. Percentage lung coal content could not be directly compared between the two studies because of differences in the data presentation, but the mean absolute amount of coal per subject found in this study (3.9 g) was lower than that found in the British study (4.9 g). Lung coal content was an order of magnitude higher than lung silica content in this study (mean coal g/1: mean silica g/1 = 22:1), similar to the British study, which found a corresponding ratio of about 16:1. Emphysema score had a highly significant negative relationship with FEVi% in this study. This result has been found previously in a larger group of coalworkers that included the subjects from this study [32] and in other studies of coalworkers [29, 30]. The emphysema-FEVi% relationship was independent of lung coal content, suggesting that the association was not a consequence of dust exposure independently causing emphysema and a decrease in FEVi%. The regression coefficient suggests that the emphysema found in coalworkers is of some functional relevance, with an increase of emphysema of 10% leading to a predicted decrease of FEVi% of about 7%. This same negative relationship between FEVi% and emphysema was suggested in the analysis, which included only non-smokers, but the small number of subjects with available FEVj measurements makes firm conclusions difficult. Recent evidence suggests that occupational exposure to coal dust alone might produce clinically important airways dysfunction [54-56], although this is not accepted by all researchers [57, 58]. In this study, as the severity of pneumoconiosis increased the severity of emphysema increased. This relationship appeared to be independent of lung dust content and suggests a direct pathological connection between pneumoconiosis and emphysema. FEVi% showed a non-significant decline with increased severity of pneumoconiosis. The lack of statistical significance of this relationship was possibly due to the limited number of subjects with available FEVi measurements.

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Lung content of coal and silica were both highly correlated with each other, as would be expected because the majority of coalworkers were exposed to mixed coal/silica dust. Lung contents of coal and silica were also both significantly correlated with years worked at the face, although the correlation coefficients of 0.26 and 0.27 suggest that years worked at the face gives a more qualitative estimate of the lung burden from dust exposure. Years at the face was highly significantly correlated with emphysema score, but once age was taken into account, years at the face no longer had a significant relationship with emphysema score, presumably because years at the face was acting as a proxy measure of age in the univariate correlations. A number of sources of bias are possible in a study of this type. However, for reasons outlined below, bias is in fact not thought to have been a significant problem. Most of the emphysema found in previous studies of coalworkers was reported to be of the centriacinar type [28, 29] with relatively little panacinar emphysema, and this was also the case in the present study. In advanced cases, centriacinar emphysema was seen to extend to involve whole lobules and to coalesce between lobules, ultimately involving the whole lung. In accordance with current thinking [59-61], we regarded the two types of emphysema as part of a spectrum and hence made no attempt to separately quantify panacinar emphysema. Tissue preparation methods were the same for all lung specimens used, and emphysema measurements were made by either of two observers using standard techniques, with a high degree of inter-observer agreement, blinded to the other variables of interest. Assessment of emphysema using macroscopic measurements has been criticized [59], but the technique is widely used [9] and, in this study, emphysema score had a highly significant negative relationship with FEVi%, as would be expected clinically. The measurements of overall emphysema are therefore believed to be appropriate and reasonably accurate. Dust analysis was performed using minor modifications of established techniques, with good agreement between duplicate samples, and analysis was done without knowledge of the emphysema measurements made on each specimen. Lung content of coal and silica can be regarded as the best quantitative estimate of exposure as, once clearance mechanisms are saturated, accumulation of dust in the lungs is proportional to further exposure [62]. Lung coal and silica contents were adjusted using predicted vital capacity, which was used as an estimate of lung volume, thereby providing a measure of the concentration of coal and silica in the lung. However, this adjustment introduces some (presumably non-differential) error. Expressing lung coal and silica content as a proportion of dry lung weight would appear to give an even better measurement of the concentration of dust in the lung. Unfortunately, this measurement can be criticized on the basis that more emphysematous lungs probably contain less total lung tissue. Thus, for a given amount of total lung dust, more emphysematous lungs would appear to have a higher concentration of dust per dry weight of lung, thereby potentially introducing a spurious positive relationship between dust content and

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amount of emphysema. Conversely, more fibrotic lungs might contain more lung tissue and so introduce an apparent negative relationship. Nevertheless, a number of authors have expressed lung dust content on a per dry weight of lung basis [30, 63, 64], and in this study analyses using this measurement for lung coal and silica content produced similar results to the main analyses performed using measures adjusted by predicted vital capacity. Using the absolute mass of coal or silica (unadjusted) does not give a measure of the concentration of dust in the lung as it does not take into account the fact that bigger lungs will contain more dust than smaller lungs. Therefore, this measure is likely to contain significant non-differential error, although analyses using these unadjusted measures gave similar results to the primary analysis. The 264 subjects included in this study are a subset of a continuous series of 1,086 post-mortem examinations on coalworkers in New South Wales from 1949 to 1987. Lung tissue obtained prior to 1966 was no longer available for mineral analysis. Subjects who had a post-mortem during the study period 1966-1983 but were not included in this study had similar values for the main known variables of interest compared to subjects who were included in the study. Since the main reason for exclusion of subjects at this stage was inadequate lung specimens, it is unlikely that significant bias was introduced because of this exclusion. It is likely that those miners who reached post-mortem had a higher prevalence of lung disease than the overall mining population of the same age during the study period. Post-mortem proven lung disease would be more likely to attract compensation for the families of the miners than lung disease that was not confirmed pathologically, so families of miners who suspected that they had lung disease are more likely to have agreed to the conduct of a post-mortem examination. However, there is no reason to believe that selection would have been influenced by emphysema severity and lung dust content in such a way as to artificially produce the results found in this study. In conclusion, the results from this study provide strong support for the hypothesis that emphysema in coalworkers is quantitatively and causally related to coal content in the lung and thus to exposure to coal in life. In this regard, it is of interest that emphysema has recently become a prescribed disease for underground coal miners in the United Kingdom [65]. The importance of age and smoking in emphysema severity is also confirmed. The results do not support a relationship between silica exposure and the development of emphysema. This review is based on the previously published papers [66, 67] and material in Reference [68]. REFERENCES 1. Miller, W. S. The Lung. 2nd edition. C. C. Thomas, Springfield, 1947. 2. Reid, L. "The secondary lobule in the adult human lung, with special reference to its appearance in bronchograms." Thorax, 1958; 13: pp. 110-115.

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3. Ciba Guest Symposium. "Terminology, definitions and classification of chronic pulmonary emphysema and related conditions." Thorax, 1959; 14: pp. 286-299. 4. Thurlbeck, W. M., Henderson, J. A., Fraser, R. G., Bates, D. V. "Chronic obstructive lung disease. A comparison between clinical, roentgenologic, functional and morphologic criteria in chronic bronchitis, emphysema, asthma, and bronchiectasis." Medicine, 1970; 49: pp. 81-145. 5. Snider, G. L., Kleinerman, J., Thurlbeck, W. M., Bengali, Z.H. "The definition of emphysema." Report of a National Heart, Lung, and Blood Institute, Division of Lung Diseases Workshop. Am Rev Resp Dis, 1985; 132: pp. 182-185. 6. Weibel, E. R. Morphometry of the Human Lung. Heidelberg: Springer. 1963. 7. Glazier, J. B., Hughes, J. M. B., Maloney, J. E., West, J. B. "Vertical gradient of alveolar size in lungs of dogs frozen intact." J Appl Physiol, 1967; 23: pp. 694-705. 8. Dunnill, M. S. "The contribution of morphology to the study of chronic obstructive lung disease." Am J Med, 1974; 57: pp. 506-519. 9. Thurlbeck, W.M. Chronic Airflow Obstruction in Lung Disease. Philadelphia: W. B. Saunders, 1976. 10. Heard, B. E. "Further observations on the pathology of pulmonary emphysema in chronic bronchitis." Thorax, 1959; 14: pp. 58-70. 11. Snider, G. L., Brody, J. S., Doctor, L. "Subclinical pulmonary emphysema. Incidence and anatomic patterns." Am Rev Resp Dis, 1962; 85: pp. 66-83. 12. Thurlbeck, W. M. "The incidence of pulmonary emphysema with observations on the relative incidence and spatial distribution of various types of emphysema." Am Rev Resp Dis, 1963; 87: pp. 206-215. 13. Gough, J. "The pathology of emphysema." Postgrad Med J, 1965; 41: pp. 392-400. 14. Leopold, J. G., Gough, J. "The centrilobular form of hypertrophic emphysema and its relation to chronic bronchitis." Thorax, 1957; 12: pp. 219-235. 15. Heppleston, A. G., Leopold, J. G. "Chronic pulmonary emphysema: anatomy and pathogenesis. Am J Med, 1961; 31: pp. 279-291. 16. Gough, J. "Pneumoconiosis in coal trimmers." J Path Bacteriol, 1940; 51: pp. 277-285. 17. Heppleston, A. G., Leopold, J. G. "Chronic pulmonary emphysema: anatomy and pathogenesis." Am 7 MeJ, 1961; 31: pp. 279-291. 18. Heppleston, A. G. "The pathological anatomy of simple pneumoconiosis in coal workers." 7 P<3^/i Bacteriol, 1953; 66: pp. 235-246. 19. Heppleston, A. G. "The pathogenesis of simple pneumokoniosis in coal workers." 7 Pfl//i Bacteriol, 1954; 67: pp. 51-63. 20. Wyatt, J. P. "Macrosection and injection studies of emphysema." Am Rev Resp Dis, 1959; 80: pp. 94-103.

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21. Mitchell, R. S. "Diffuse pulmonary emphysema and occupation." J Am Med Assn, 1962; 181: pp. 71-77. 22. Duguid, J. B., Lambert, M.W. "The pathogenesis of coal miner's pneumoconiosis." /Ptz^/z Bacteriol, 1964; 88: pp. 389-403. 23. Ryder, R., Lyons, J. P., Campbell, H., Gough, H. J. "Emphysema in coal workers' pneumoconiosis. Brit Med J, 1970; 3: pp. 481-487. 24. Heppleston, A. F. "The pathological recognition and pathogenesis of emphysema and fibrocystic disease of the lung with special reference to coal ^oxkersr Ann NY Acad Sci, 1972; 200: pp. 347-369. 25. Morgan, W. K. C , Lapp, N.L. "Respiratory diseases in coal miners." Am Rev RespDis, 1976; 113: pp. 531-559. 26. Green, F. H. Y., Laquer, W. A. "Coal workers' pneumoconiosis." Pathol Ann, 1980; 15: pp. 333-410. 27. Kleinerman, J., Green, F., Harley, R.A. et al. "Pathology standards for coal workers' ^ntwmoconio^is.'" Arch Pathol Lab Med, 1979; 103: pp. 375-432. 28. Cockcroft, A., Wagner, J. C , Ryder, R., Seal, R. M. E., Lyons, J. P., Andersson, N. "Post-mortem study of emphysema in coalworkers and non-coalworkers." Lancet, 1982; pp. 600-603. 29. Ruckley, V. A., Gauld, S. J., Chapman, J. S., et al. "Emphysema and dust exposure in a group of coal workers." Am Rev Respir Dis, 1984; 129: pp. 528-532. 30. Ruckley, V. A., Femie, J. M., Campbell, S. J., Cowie, H. A. "Causes of disability in coalminers; a clinico-pathological study of emphysema, airways obstruction and massive fibrosis." Edinburgh: Institute of Occupational Medicine, 1989. Report No. TM/89/05. 31. Leigh, J., Outhred, K. G., McKenzie, H. I., Wiles, A. N. "Multiple regression analysis of quantified aetiological, clinical and post-mortem pathological variables related to respiratory disease in coal workers." Ann Occ Hyg, 1982; 26: pp. 383-400. 32. Leigh, J., Outhred, K. G., McKenzie, H. I., Click, M., Wiles, A. N. "Quantified pathology of emphysema, pneumoconiosis, and chronic bronchitis in coal workers." BrJIndMed, 1983; 40: pp. 258-263. 33. Leigh, J. "Relationship between emphysema severity and histologic type of pneumoconiosis in coal workers." Eur Respir J, 1992; 5: p. 519s (Abst). 34. Becklake, M. R., Irwig, L., Kielkowski, D., Webster, L, De Beer, M., Landau, S. "The predictors of emphysema in South African gold miners." Am Rev Respir Dis, 1987; 135: pp. 1,234-1,241. 35. Hnizdo, E., Sluis-Cremer, G.K., Abramowitz, J.A. "Emphysema type in relation to silica dust exposure in South African gold miners." Am Rev Respir Dis, 1991; 143: pp. 1,241-1,247. 36. Click, M., Outhred, K. G., McKenzie, H. L "Pneumoconiosis and respiratory disorders of coal mine workers of New South Wales, Australia." Ann NY AcadSci, 1972; 200: pp. 316-341.

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37. Gough, J., Wentworth, J. D. "Thin sections of entire organs mounted on paper." In: Harrison, C.V., ed. Recent advances in pathology. 7th ed. London: Churchill Livingstone, 1960; p. 80. 38. Heard, B.E. Pathology of chronic bronchitis and emphysema. London: Churchill Livingstone, 1969; p. 12. 39. Guest, L. "The recovery of dust from formalin-fixed pneumoconiotic lungs: a comparison of the methods used at SMRE." Ann Occ Hyg, 1976; 19: pp. 37-47. 40. Dodgson, J., Whittaker, W. "The determination of quartz in respirable dust samples by infrared spectrophotometry-L The potassium bromide disc method." Ann Occ Hyg, 1973; 16: pp. 373-387. 41. Lazarus, R. "Lung-function reference values from Victorian power-industry workmen.''Med J Aust, 1982; 2: pp. 121-124. 42. Ferris, B.G., Anderson, D.O., Zickmantel, R. "Prediction values for screening tests of pulmonary function." Am Rev Respir Dis, 1965; 91: pp. 252-261. 43. SAS Institute. SAS System, version 6.03. Gary, NC: SAS Institute, 1987. 44. Kennedy, M. C. S. "Nitrous fumes and coalminers with emphysema." Ann Occup Hyg, 1972; 15: pp. 285-300. 45. Brown, G. M., Donaldson, K. "Inflammatory responses in lungs of rats inhaling coalmine dust: enhanced proteolysis of fibronectin by bronchoalveolar leukocytes." BrJIndMed, 1989; 46: pp. 866-872. 46. Rom, W. N. "Basic mechanisms leading to focal emphysema in coal workers' pneumoconiosis." Environ Res, 1990; 53: pp. 16-28. 47. Gross, P., Tuma, J., de Treville, R. T. P. "Emphysema and pneumoconiosis: a comparative quantitation of dust content of pneumoconiotic rodent lungs with and without emphysema." Arch Environ Health, 1971; 22: pp. 194-199. 48. Heppleston, A.G. "The pathologic recognition and pathogenesis of emphysema and fibrocystic disease of the lung with special reference to coal workers." Ann NY Acad Sci, 1972; 200: pp. 347-369. 49. Ferin, J. "Emphysema in rats and clearance of dust particles." In: Inhaled Particles III, part I, W. H. Walton, ed. Surrey: Unwin, 1970, pp. 283-292. 50. Ingram, R. H., Jr. "Chronic bronchitis, emphysema, and airways obstruction." In: Braunwald, E., Isselbacher, K. J., Petersdorf, R. G., Wilson, J. D., Martin, J. B., Fauci, A. S., eds. Harrison's Principles of Internal Medicine. 6th ed. New York: McGraw-Hill Inc., 1987; pp. 1,087-1,095. 51. Burrows, B. "An overview of obstructive lung diseases." Med Clin North Am, 1981; 65: pp. 455^71. 52. Cotes, J. E., Steel, J. "Chronic bronchitis and emphysema: roles of smoking, occupation and air pollution." In: Work-related lung disorders. Oxford: Blackwell Scientific Publications, 1990; pp. 373-386. 53. Hnizdo, E., Sluis-Cremer, G. K., Baskind, E., Murray, J. "Emphysema and airway obstruction in non-smoking South African gold miners with long exposure to silica dust." Occup Environ Med, 1994; 51: pp. 557-563.

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54. Becklake, M. R. "Occupational exposures: evidence for a causal association with chronic obstructive pulmonary disease." Am Rev Respir Dis, 1989; 40: pp. s85-s91. 55. Seaton, A. "Coal, emphysema, and compensation." Br J Ind Med, 1990; 47: pp. 433-435. 56. Oxman, A. D., Muir, D. C. F., Shannon, H.S., Stock, S. R., Hnizdo, E., Lange, H.J. "Occupational dust exposure and chronic obstructive pulmonary disease. A systematic overview of the evidence." Am Rev Respir Dis, 1993; 148: pp. 38-48. 57. Gee, J. B. L., Morgan, W. K. C. "Coalmining, emphysema, and compensation." Br J Ind Med, 1991; 48: pp. 70-72. 58. Morgan, W.K.C. "Coal mining, emphysema, and compensation revisited." Br J Ind Med, 1993; 50: pp. 1,051-1,053. 59. McLean, A., Warren, P. M., Gillooly, M., MacNee, W., Lamb, D. "Microscopic and macroscopic measurements of emphysema: relation to carbon monoxide gas transfer." Thorax, 1992; 47: pp. 144-149. 60. Gough, J. "The pathogenesis of emphysema." In: The lung. International Academy of Pathology Monograph No. 8., Baltimore: The Williams and Wilkins Company, 1967; pp. 109-133. 61. Gough, J., James, W. R. L., Wentworth, J. E. "A comparison of radiological and pathological changes in coalworkers' pneumoconiosis." J Faculty Radiol, 1950; 1: pp. 29-39. 62. Outhred, K. G. "The pneumoconioses." Proceedings, First Australian Pneumoconiosis Conference, Sydney, 1968: pp. 1-6. 63. Attfield, M. D., Vallyathan, V., Green, F. H. Y. "Radiographic appearances of small opacities and their correlation with pathology grading of macules, nodules, and dust burden in lungs." Ann Occ Hyg, 1994; 38 Supp 1: pp. 783-789. 64. Gibbs, A. R., Pooley, F. D., Griffiths, D. M., Mitha, R. "Silica and silicate pneumoconioses—a pathological and mineralogical study." Ann Occ Hyg, 1994; 38 Supp 1: pp. 851-856. 65. Industrial Injuries Advisory Council. "Chronic bronchitis and emphysema." Social Security Administration Act 1992. Department of Social Security: London, 1992. 66. Leigh, J., Driscoll, T. R., Cole, B. D., Beck, R. W., Hull, B. P., Yang, J. "Quantitative relation between emphysema and lung mineral content in coal workers." Occ Env Med, 1994; 51: pp. 400^07. 67. Leigh, J., Beck, R., Cole, B. "Emphysema severity, pneumoconiosis histopathology and lung coal and silica content in coal miners." J Occ Health Safety-ANZ, 1993; 9: pp. 137-146. 68. Dunnill, M. S. Pulmonary Pathology. Churchill Livingstone: Edinburgh, 1982.

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CHAPTER 8 SINONASAL CANCER AND WOOD DUST EXPOSURE A. Leclerc and D. Luce Unite 88 INSERM-HNSM 14 rue du Val d'Osne 94410 Saint-Maurice, France

CONTENTS INTRODUCTION, 171 SINONASAL CANCER, 172 WOOD DUST EXPOSURE, 172 RISK OF SINONASAL CANCER RELATED TO WOOD DUST EXPOSURE: EPIDEMIOLOGIC STUDIES, 174 Sinonasal Cancer and Woodworking in the United Kingdom, 175 CASE REPORTS FROM DIFFERENT COUNTRIES, 176 MORTALITY AND INCIDENCE OF SINONASAL CANCER IN WOODWORKERS, 179 CASE CONTROL STUDIES, 179 CARCINOGENIC EFFECTS OF WOOD DUST; SYNTHESIS FROM EPIDEMIOLOGIC STUDIES, 185 PREVENTION OF SINONASAL CANCER, 186 REFERENCES, 186 INTRODUCTION Many independent studies since 1965 have found an excess risk of sinonasal cancer among workers exposed to wood dust, with especially strong associations between adenocarcinoma, which is one of the histologic types of sinonasal cancer, and occupations such as cabinet makers [1]. The excess appears to be attributable to wood dust per se, rather than to other exposures in the workplace. However, the potential chemical and physical causative agents, which may differ qualitatively and quantitatively according to the species of wood and the working conditions, have not been precisely defined. This chapter includes some developments both on sinonasal cancer and on wood dust exposure. The largest part is devoted to results from epidemiologic studies, because most of the current knowledge on the carcinogenic effect of wood dust comes from epidemiology rather than from experimental studies.

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SINONASAL CANCER Sinonasal cancer (SNC), or cancer of the nasal cavities and paranasal sinuses, is a rare disease. The annual incidence rate adjusted for age and sex ranges from 0.3 to 1 case per 100,000 in most countries [2, 3]. The male/female ratio is about 2:1. Incidence rates are relatively low in North America, and higher in Japan and other East Asian countries [2]. Sinonasal cancer affects different sites: nasal cavity (coded C30 according to the tenth revision of the International Classification of Diseases [4]), and accessory sinuses (maxillary sinus, code C31.0; ethmoidal sinus, code C31.1; frontal sinus, code C31.2, and sphenoidal sinus, code C31.3). Cancers of the skin of the nose and of the nasal bone are not included. It must be noted also that the nasal cavity and paranasal sinuses do not include nasopharynx. Different histologic types must be distinguished, especially squamous cell carcinoma, which is the most frequent, and adenocarcinoma, because the magnitude of the association with wood dust differs according to the histologic type. However, in interpreting results from epidemiologic studies, one must be aware that a certain level of misclassification between histologic types cannot be avoided, especially in earlier studies. Even now, the certainty of histological type for a given subject depends on the source of the information. It is good if the source is a specialized hospital department. The reliability varies from one cancer registry to another. Histology is not necessarily indicated on the death certificate. The prognosis of sinonasal cancer is poor, especially if the treatment has been delayed. The reason for focusing on sinonasal cancer in studying the effect of wood dust exposure is that the associations between wood dust and other sites of cancer are unclear. Associations have been reported, especially for nasopharyngeal and laryngeal cancer. However, the results so far lack of consistency [1], although many studies have been performed, especially for the lung or other respiratory sites. It must be noted that wood dust has also non-cancerous and pre-cancerous effects on the upper respiratory tract. Both experimental and human studies indicate that inhalation of wood dust particles has chronic irritative effects. Squamous metaplasia and dysphasia are frequently found among woodworkers. These changes can potentially progress to nasal carcinoma [1, 5, 6]. WOOD DUST EXPOSURE Wood dust exposure occurs mainly as an occupational exposure in some industries and occupations. The partial list of exposed workers includes: loggers, sawmill workers, workers involved in the manufacture of plywood and other boards, wooden furniture workers and cabinet makers, workers in the manufacture of other wood products, and carpenters and joiners in the construction industry.

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The type and the level of exposure to wood dust can be described in several ways. A first important characteristic is the type of wood (hardwood, softwood, tropical wood) or the wood species. Hardwoods are deciduous trees such as oak, beech, lime, ash, birch, poplar, elm, or cherry trees. Softwood are coniferous trees, such as fir, spruce, or pine. Wood is also characterized by its moisture content, which depends both on the species and on the freshness of the wood. The concentration of airborne dust is generally measured in mg/m^ with standard methods [1]. The particle size is also an important parameter, because the deposition pattern in human upper and lower airways partly depends on particle size. Large particles (>10|Lim) are almost entirely deposited in the nose. Some authors consider that relatively few particles larger than 5jLim, which form most of the wood dust, pass into the lung, as far as the clearance mechanism of the nose is not impaired [6, 7]. However, up to 65% of the particles in some workplaces measure less than 5|Lim [1]. Globally, there is a lack of knowledge about depositing of wood dust in the lung and nasal sinuses according to the particle size [1]. The question of whether wood dust or dust from one or several woods contains carcinogens has been raised. There is no clear answer to this question. A very large number of chemical compounds are present in wood: cellulose, polyoses, lignin, terpene and terpenoids, fats, waxes, and phenolic compounds such as tannins, but none is a known carcinogen. However, wood may carry microorganisms (fungi and bacteria) capable of liberating carcinogens [7]. The type of exposure depends on the occupation: For example, loggers and sawmill workers are exposed to relatively large particles of fresh wood. The mean level of wood dust is generally lower than Img/m^ [1]. The species of wood depends on the country: In North America and in the Nordic countries, softwood is predominant. In some European countries, hardwood dominates. Sawmill workers may be exposed to chemical agents, such as chlorophenol, in addition to wood dust. In the manufacture of plywood and other boards, the mean level of exposure is often close to Img/m^. Many workers are exposed to formaldehyde in addition to wood dust. Wooden furniture manufacture and cabinet making involves a large number of woodworking processes, such as sawing, planing, chipping, sanding, and the use of different milling machines that produce wood dust. A wide variety of species are used, with a predominance of hardwood for high-quality furniture manufactured by cabinet makers, with some exceptions, for example in Asian countries. The concentration of airborne dust may be high especially if local exhaust ventilation is lacking or ineffective. Mean values higher than 5 mg/m^ are observed relatively often [1]. The mean size of the particles may be especially small, because the wood needs to be very dry (which produces smaller particles) and the sanding is finer for furniture than in other industries, for aesthetic reasons. In addition, there is some evidence that sanding of hardwood can generate smaller particles. Workers may be exposed to other substances such as formaldehyde, varnishes, paints, and glues.

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Health and Toxicology

Workers exposed to wood dust in the construction industry are mainly carpenters and joiners. The intensity of exposure depends on whether the work is done outdoors or not. If it is in shops, the situation is similar to that of furniture manufacture, except that the mean size of the particles may be larger, and the species may differ. Working on a construction site generally involves a lower level of exposure. For example, the level of exposure for carpenters is expected to be relatively low. However, some building trades involve dusty operations such as sanding a parquet before varnishing [1]. RISK OF SINONASAL CANCER RELATED TO WOOD DUST EXPOSURE: EPIDEMIOLOGIC STUDIES The first studies were case reports, which consist of a description of a series of cases of sinonasal cancer, with details on the disease and on the occupational exposure. The limitation of this type of study is that it does not provide an estimate of the relative risk (ratio of incidence or mortality among exposed subjects, compared to non-exposed). However, case reports on sinonasal cancer remain informative, both for historical reasons and because they often give more details on the jobs and the species of wood handled by the workers. In addition, a comparison of the occupations of the case subjects with the distribution of occupations in the general population has been made in some case reports. Descriptive studies based on systematic record and cohort studies of a group of exposed workers provide a comparison between exposed subjects and the general population (SMR or SIR, standardized mortality or incidence ratio, or comparison between the number of observed cases and an expected number). These studies differ according to the precision on histologic type and the precision on occupational exposure. A limitation in some studies is that occupation is occupation at death or at the time of diagnosis, which may not be the best information. In other studies, the number of observed and expected cases of sinonasal cancer is very small (close to zero) because sinonasal cancer is a rare disease. In case control studies, a group of cases presenting sinonasal cancer is compared to a control group (representing the general population or a population comparable to that of the cases) concerning the occupational history of cases and controls. The drawback of this approach is the need to estimate retrospectively the occupational exposure. Nevertheless, most of the recent knowledge on sinonasal cancer comes from case-control studies that include a retrospective assessment of the level of exposure to wood dust, in addition to a description of the occupations held in the past. Since providing results according to the level of exposure was not the usual approach until recently, the monograph from International Agency for Research on Cancer (lARC) in 1981 and its supplement in 1987 [8, 9] focused on industries and occupations in which exposure to wood dust can occur, but not on wood dust per se. Only the last monograph, issued in 1995 [1], addresses wood dust.

Sinonasal Cancer and Wood Dust Exposure

175

The studies described in this chapter represent only a small part of the studies on sinonasal cancer and wood dust exposure. For comprehensive reviews, the reader can refer to Ny lander [5] and to the I ARC 1995 monograph [1]. In the presentation of the studies, the quantification of the association between the disease and exposure is given for case-control studies as relative risk (RR), which is the risk among exposed subjects divided by the risk among the unexposed population. For descriptive or cohort studies, the measure (SMR, Standardized Mortality Ratio; SIR, Standardized Incidence Ratio; or a comparison between observed and expected number) is most often a comparison between exposed subjects and the general population, which comprises both exposed and unexposed subjects. If there is no association, the observed figure is close to one, irrespective of the measure used (SMR, SIR, or RR, comparison with the general population or with unexposed subjects). Sinonasal Cancer and Woodworking in the United Kingdom The studies from 1965-1972 in U.K. are especially important because the objective of many subsequent studies in different countries was to compare the situation with that observed in the U.K. In 1965, British otorhinolaryngologist Ronald Macbeth reported observations from one of his colleagues, Esme Hadfield, from High Wycombe, Buckinghamshire [10]. On a total of 17 male patients from High Wycombe with a malignant disease of the paranasal sinuses, no less than 15 were associated with the making of wooden chairs. Macbeth noted later that the tumors were all adenocarcinomas. In High Wycombe, there were several sites of furniture factories specialized in chair making. A wide variety of domestic and imported hardwoods were used. However, the percentage of woodworkers in the local male population did not exceed 23.5%. Following these observations, epidemiologic assistance was given by E.D. Acheson. A survey in the Oxford area, including High Wycombe, was planned. The survey included a total of 148 cases of nasal cancer from the period 1951-1965 [6, 11]. Cases were classified according to sex and histology. The results for men indicated a strong relationship between adenocarcinoma and present or past work in the furniture industry. Among the 33 cases of adenocarcinoma, 24 (73%) had been woodworkers, 22 of them (67%) in the furniture industry. Among the 65 remaining cases, the corresponding numbers were 5 (8%) and 3 (5%). For the subgroup of men economically active at the onset of their illness, diagnosed in 1956 or later, a detailed occupational history was obtained and could be compared with the findings of the 1961 census. It could be estimated that the risk of adenocarcinoma was similar among cabinet and chairmakers, and wood machinists, namely 0.7 ±0.2 per 1,000 per year during the decade 1956-1965—at least 500 times the risk in adult males in Southern England. The results suggested that there was no comparable

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risk in carpenters and joiners. Details on patients involved at some time in the wood trade were given, including the site of the tumor, histology, occupational history, and wood types (if known). Among the 16 patients for whom information was available, the wood types they had used before the second World War were oak (15/16), beech (11/16), and mahogany (11/16). Walnut was also frequently used. The list of cases included wood machinists, who did not use glues. Inquiries made about the use of insecticides and seasoning agents before machining the wood were negative. A national survey in England (excluding the Oxford area) was performed also by Acheson [12]. Cases of sinonasal cancer from cancer registries in England were collected (for the period 1961-1966 for most registries). The study included 107 cases of adenocarcinoma (80 men) and 110 other sinonasal cancer (85 men), when restricted to accepted cases, mainly on the basis of confirmation of the histologic classification. Thirty-three men with nasal adenocarcinoma had at some time worked as woodworkers; among them, 24 had worked in the furniture industry. The ratio observed cases/expected cases was 95 for furniture workers and 5 for other woodworkers (principally, carpenters and joiners) on the basis of the distribution of occupations at the 1961 census. Among nasal cancer cases other than adenocarcinoma, a significant excess of woodworkers was also observed. Details on the main types of wood dusts were known for a part of the woodworkers (both adenocarcinoma and other histologic types). The species most often indicated were oak (7 adenocarcinoma patients); mahogany (6 adenocarcinoma patients); and beech, birch, and walnut (5 adenocarcinoma patients for each of the species). Four patients (three adenocarcinomas, one squamous cell cancer) had used mainly softwoods. An average of 42.8 years between diagnosis of adenocarcinoma and first exposure to wood dust (latency) was found, combining material from the Oxford and national surveys [6]. CASE REPORTS FROM DIFFERENT COUNTRIES Following the results from U.K., many studies based on hospital cases were performed elsewhere (Table 1). In France, several independent studies were performed between 1969 and 1975. The conclusions were similar: In different regions in France, a large proportion of woodworkers were found among males with adenocarcinomas of ethmoidal sinuses [13-16]. The average latency was 40 years [13]. Woodworker cases were mainly cabinet makers [13, 14] or equally cabinet makers and joiners [16]. It must be noted that in France, it was frequent to work both as a joiner and a cabinet maker. The species used were various European hardwood and different tropical species, including mahogany. It was concluded that the risk was related to native woods [15]. This conclusion was also in accordance with comments from Hadfield in the U.K who noted that although the men themselves blamed the foreign woods

Table 1 Case Reports on Sinonasal Cancer and Wood Dust Exposure

Reference

Country

Sex

Histological Type

Exposed Cases/ Total Cases

Acheson [ 6, 1 1 ]

U.K. Oxford area

M

adenocarcinoma

24/33

Acheson [ 121

U.K. except Oxford area

M

adenocarcinoma

33/80

Luboinski and Marandas 161

France

M

adenocarcinoma, ethmoidal sinus

2 1143

19 joiners or cabinet makers

MF

adenocarcinoma

12/17 (men: 12/15)

10 cabinet and chair makers

10/140 (men: 10199) 77/85

2 cabinet and chair makers 55 joiners or cabinet makers joiner or cabinet maker various occupations, no cabinet maker various occupations, only one cabinet maker

Andersen [ 18, 191 Denmark

Kleinsasser [20]

Germany

M

other histological tY Pe adenocarcinoma

Engzel [21]

Sweden

M

adenocarcinoma

19/36

Ironside, Matthews [22]

Australia

M

adenocarcinoma

10/18

Voss (231

Norway

M

sinonasal cancer, not adenocarcinoma

1 1/44

Occupations

Species of wood

22 in the furniture industry 24 in the furniture industry

oak, beech, mahogany, walnut oak, mahogany, beech, birch, walnut; mainly softwoods for three cases European hardwood (oak, chesnut, wild cherry, walnut, beech, poplar); Tropical species, including mahogany primarily beech, oak, walnut: periodically mahogany, teak not given oak orland beech for all cases oak, birch, teak, mahogany native timbers

mainly softwood

A

-4 -4

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Health and Toxicology

because often they had acute irritating effects, these woods were of comparatively recent introduction, and native species were more likely to be implicated [17]. Andersen in Denmark presented results from a series of 157 patients treated for tumors of the nose and paranasal sinuses [18, 19]. The proportions of woodworkers were 12/17 among adenocarcinoma (12/15 if restricted to men), and 10/140 among other histologic types (10/99 if restricted to men). Ten out of the 12 woodworkers with adenocarcinoma were cabinet and chair makers. In the other group (non-adenocarcinoma) comprised of 10 woodworkers, only 2 were cabinet and chair makers. The author indicated that the risk of nasal adenocarcinoma among Danish woodworkers in the furniture industry could be estimated as 0.5/1000/year, which was comparable to that in England. The ratio 10/99 of woodworkers among male cases other than adenocarcinoma was significantly higher than expected from the number of woodworkers in relation to the total male population. This indicated that occupational exposure to wood dusts could be of importance for the development of nasal cancers other than adenocarcinomas. Several other studies in Europe, for example in Germany [20] and in Sweden [21], supported the fact that adenocarcinoma cases had handled mainly hardwoods, and that the average latency was close to 40 years. In Australia, Ironside and Matthews analyzed 19 cases of adenocarcinoma of the nose and paranasal sinuses from the Cancer Institute of Victoria [22], and compared them to the remaining 80 cases of cancer of the nose and paranasal sinuses from the same source. Among the 18 male cases of adenocarcinoma, ten could be classified as woodworkers in various occupations; none was a cabinet maker. The comparison with the distribution of the workforce in different occupations indicated a clear excess of woodworkers among adenocarcinomas. Native timbers were those most commonly used. The group of cases of other malignant tumors of the nose and paranasal sinuses included 55 males. Comparisons with the occupations in the general population did not indicate an excess of cases among woodworkers. Voss et al. [23] in Norway presented data from a group of 70 cases of sinonasal cancer. Among the 46 males, two had an adenocarcinoma. One of them was a woodworker. Among the 44 remaining males (not adenocarcinoma), 11 were woodworkers. The types of wood were pine and spruce (n = 9), spruce only (n = 1), and pine and lime (n = 1). The mean latency period was 44 years. The observed number of workers exposed to wood dust (n = 12) was significantly larger than the expected number of 4, calculated from the distribution of workers in the population. From the above studies it can be concluded that adenocarcinoma is strongly related to occupational exposure to hardwood, at least in Europe. A specific association between adenocarcinoma and the furniture industry has been observed in many European countries. The study from Ironside and Matthews [22] suggests that the coarse dust of a sawmill may be a hazard too. The study from Voss et al. [23] suggests an association between exposure to softwood and sinonasal cancer other than adenocarcinoma.

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179

MORTALITY AND INCIDENCE OF SINONASAL CANCER IN WOODWORKERS Selected studies based on systematic records and three cohort studies are summarized in Table 2. In Switzerland, Vader and Minder studied the mortality in 1980 and compared the mortality of furniture workers to that in the general population. A 6.6.-fold increase for death due to sinonasal cancer among furniture workers was found. For adenocarcinoma only, the corresponding figure was 230 [24, 25]. In Sweden, Malker et al. [26] studied the incidence of cancer in the period 1960-1979, according to the occupations at 1960 census. The SIR for woodworkers (in general) and all nasal cancer was only 1.3, not significantly different from 1. The SIR was higher when the association was restricted to adenocarcinoma and furniture workers. In cohorts of woodworkers, the number of cases of sinonasal cancer is generally very small, both for observed and expected numbers. However, two large cohorts of woodworkers show an association between the furniture industry and adenocarcinoma. In Sweden [27], in a cohort of 8,141 furniture workers, 14 cases of adenocarcinoma were observed (expected number 0.3; SIR = 44). No excess was observed for squamous cell carcinoma. In a cohort of 5,108 men from nine furniture factories in High Wycombe or the surrounding district in the United Kingdom [28], nine deaths from sinonasal cancer were observed. The ratio observed/expected was 8.1, significantly different from one. All nine deaths were due to adenocarcinoma, although this tumor usually represents less than 10% of nasal cancer death. The ratio observed/expected rose from 8.1 to 15.8 for "occupation very dusty," since all nine deaths were classified in this category, which comprised cabinet and chair makers, sanders, and wood machinists. In another cohort study of 36,622 workers in the furniture industry in the U.S.A., no excess of sinonasal cancer was observed [29, 30]. Only two nasal cancer deaths occurred among white men. The corresponding expected number was 1.8. CASE-CONTROL STUDIES A relatively large number of case-control studies on sinonasal cancer have been performed. The choice has been made here to present detailed results from a pooled reanalysis of twelve case-control studies [31]. Results from other studies, included or not in the reanalysis, are also presented. Demers [31] performed a pooled analysis of twelve case-control studies from seven countries of sinonasal cancer and exposure to wood. The geographical locations were: Shanghai, China [32]; Germany [33]; The Netherlands [34]; France [35]; Northern Sweden [36]; Siena, Verona, Vicenza, Italy [37]; Breschia, Italy [38]; Biella, Italy [39]; Vigevano, Italy [40]; North Carolina and Virginia, U.S.A. [41]; Los Angeles area, U.S.A. [42]; and Seattle area, U.S.A. [43, 44].

Table 2 Mortality and Incidence of Sinonasal Cancer in Wood Workers

I (D

Reference

Country

Method

Vader and Minder 124,251

Switzerland

Mortality according to the occupation at 1980 census, period 1980-1985.

Malker [26]

Sweden

Incidence of cancer according to the occupation at 1960 census, period 1960-1979.

Results

$ E

Gehardsson [27]

Sweden

Acheson [28]

U.K. (High Wycombe)

Miller [29,30]

U.S.A.

Record linkage between 1960 census and 1961-1979 cancer registry. Cohort study of 8,141 furniture workers. Cohort study of 5,108 men who worked in the Buckinghamshire furniture industry before 1968. Follow-up through 1982.

Cohort study of 36,622 furniture workers.

Furniture workers Sinonasal cancer SMR = 6.6 Adenocarcinoma SMR = 230 -Woodworkers (in general) and sinonasal cancer: SIR = 1.3 -Furniture industry adenocarcinoma: SIR= 17 squamous cell carcinoma: no association -Sawmill industry: no association with either squamous cell carcinoma or adenocarcinoma Squamous cell carcinoma: no excess risk Adenocarcinoma SIR=44 Sinonasal cancer: observed/expected = 8.1 Adenocarcinoma: observed/expected >80 Sinonasal cancer, occupation very dusty: observed/expected = 15.8 2 nasal cancer deaths (1.8 expected)

d

--I

:5:

0 0 a Y

Sinonasal Cancer and Wood Dust Exposure

181

There were 680 male cases, including 169 adenocarcinomas and 329 squamous cell cancers, 157 of other histology, and 25 of unknown histology. The number of female cases was 250 (26 adenocarcinomas, 101 squamous cell cancer, 105 of other histology, and 18 of unknown histology). They were compared to 2,349 male controls and 787 female controls, for work history and level of exposure to wood dust. Work history data were coded or recoded using the same classifications for occupational titles and industry titles. Seven categories were used: 1. Forestry workers 2. Logging workers 3. Pulp and paper workers 4. Sawmill workers 5. Furniture workers, including cabinet makers 6. Other wood product workers 7. Carpenter, joiner, or parquetry workers Jobs in wood-related occupations or industries were also classified as to their level of wood dust exposure using a job exposure matrix. Three categories were defined according to an estimation of the level of wood dust: less than Img/m^; 1-5 mg/m^; and more than 5mg/m^. Relative risk for sinonasal cancer was given by wood-related occupational category, separately for adenocarcinoma and squamous cell cancer. For adenocarcinomas among men, a high relative risk was associated with employment in wood-related jobs (RR = 13.5). The risk was higher among furniture workers (RR = 41.1). It was around 15 for sawmill workers and other wood product manufacture, and only 2.3 among carpenters, taking into account other high-risk occupations in the working life. The relative risk was lower than one for forestry, logging, and pulp and paper workers. No association was observed among men between squamous cell carcinoma and employment in all wood-related occupations (RR = 0.8). The number of exposed female cases was small. However, the relative risk was significantly different from 1 (RR = 4.6) for adenocarcinoma and all wood products, which was comprised of sawmills, furniture, and other wood products. Among men, the analysis by level of wood dust exposure indicated a clear relationship between the risk of adenocarcinoma and the level of exposure, with a RR of 45.5 for high exposure. The risk increased also with the duration of exposure. A significantly increased risk of squamous cell carcinoma in men was seen among those employed for 30 or more years in jobs with moderate or high levels of exposure to fresh wood, with a definition of fresh wood as employment in logging, pulp preparation, and sawmills (RR = 2.4).

{text continued on page 184)

w

Table 3 Case-control Studies on Sinonasal Cancer

5 Results Reference

Country

Sex, Cases/ Controls

Adenocarcinoma

Squamous cell Carcinoma

Reanalysis of 12 studies, seven countries

M 68012,349 F 2501787

RR = 13.5" (men) for all wood-related occupations increase with level and duration of exposure RR = 4.6* (female) for "all wood products"

RR = 0.8 (men) RR = 2.4" (men) restricted to 30 or more years in jobs with moderate or high level of exposure to freshwood

Leclerc [35]

France

M F 2071409

RR = 168" for hardwood exposure no additional effect of softwood exposure

RR = 2.5" restricted to first exposure to either hardwood or softwood before 1945

Hernberg [45]

Denmark, Finland, Sweden

M F 1671167

Sinonasal cancer RR = 2.0 Strong association for adenocarcinoma Sinonasal cancer RR = 3.3" RR = 2.5 for squamous cell carcinoma Sinonasal cancer RR = 12" Strong association for adenocarcinoma

Demers [31]

Elwood [46]

Canada

M 121 cases

RR = 2.5" for "sinonasal cancer" association for both histologic types

Comments

4

x. 0

$

(CI

Y

Hardwood only Softwood only Hardwood + softwood Predominantly softwood

Vaughan [43,>4]

I U.S.A.,

M F 271552

Not studied

RR = 3.1*, restricted to exposure 15 or more years before diagnosis

Seattle

Brinton [41]

U.S.A. Virginia, North Carolina

M F 1601290

Any wood-related job: RR = 3.7" Furniture industry RR = 5.7"

No excess risk

Fukuda [47]

Japan

M F 1691338

Not studied

RR = 2.9* (men) RR = 2 (women) maxillary sinus only

*Significantly different from 1.

Predominantly softwood

Softwood is more frequently used than hardwood in the Japanese furniture.

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Health and Toxicology

(text continued from page 181)

For adenocarcinoma, the results were consistent with the results of individual participating studies, but the magnitude of the excess risk was higher in most European studies than in studies from the U.S. or China. For squamous cell carcinomas, the number of exposed cases was smaller. The RRs for any wood exposure ranged from 2.7 to zero, according to the studies, with very small numbers of exposed cases in some studies. Some studies, included or not in the reanalysis, bring some additional information: In the French study (included in the reanalysis), hardwoods and softwoods were distinguished [35]. Among 82 cases of adenocarcinoma, 80 had been exposed to hardwood, alone or associated with another kind of wood. The two remaining cases had not been exposed to wood. For adenocarcinoma, two components of exposure to hardwood—duration and average level—contributed independently to the overall very elevated risk. Additional exposure to wood other than hardwood did not increase the risk. For squamous cell carcinoma, a significant increase in risk was observed among subjects whose first exposure to either hardwood or softwood occurred before 1945 (RR = 2.5). It was not possible to attribute this excess to hardwood or softwood, since the number of exposed cases was small and the two types of exposure were highly correlated. The results were consistent with results from the Italian studies included in the reanalysis and also with results from Hemberg in the Nordic countries. In this case-control study [45], not included in the reanalysis by Demers, a strong association between adenocarcinoma and hardwood dust only or hardwood plus softwood was observed. The RR associated with softwood dust alone was 3.3 (significandy different from 1) for all sinonasal cancers. It remained high (RR = 2.5) if only squamous cell carcinomas were considered. Two studies from North America suggest that exposure to softwood, or predominantly softwood, entails an increased risk of squamous cell carcinoma: In the first one, from Canada, an association for both histologic types was observed in a region where the wood dust exposure was mainly softwood [46]. The other one, included in the common reanalysis, was performed in Seattle area [43, 44]. A relative risk of 3.1, significantly different from 1, was observed for squamous cell carcinoma, restricted to exposure 15 or more years before diagnosis. Cases had been exposed mainly to dust from softwood. One of the studies included in the common reanalysis [41] was performed in another region in the U.S.A., Virginia and North Carolina. No excess was observed for squamous cell carcinoma. A significant association between adenocarcinoma and woodworking was observed. The relative risk was 3.7, increasing to 5.7 for furniture industry. These figures are lower than that in several European studies, given by Demers for any exposure to wood dust: 161 (France), 64 (Germany), and 12.6 (The Netherlands). However, in some of the European studies included in the common reanalysis, the RR were also low. The reasons for this difference might be differences in types of wood or dust conditions.

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In East Asian countries, the distribution of sites and histologic types of sinonasal cancer is different from the distribution in Europe and North America [3]. In Japan, squamous cell cancer of the maxillary sinus is predominant. In a case control study limited to this subgroup of sinonasal cancer, Fukuda [47] found a significant relative risk (RR = 2.9) for male woodworkers. Traditionally in Japan, softwood is more frequently used than hardwood in furniture making [48]. However, the species of softwood are not necessarily the same as in other countries where softwood is predominant. CARCINOGENIC EFFECTS OF WOOD DUST; SYNTHESIS FROM EPIDEMIOLOGIC STUDIES The results from epidemiologic studies clearly establish that exposure to hardwood dust entails an elevated risk of sinonasal cancer, with especially high relative risk for adenocarcinoma. The effects of exposure to softwood are less important; softwood exposure would be associated primarily with squamous cell carcinoma, with a long latency between first exposure and occurrence of the disease. However, the distinction between hardwood and softwood is purely empirical. The reason why the effects are different are unknown. Moreover, several kinds of wood, especially outside Europe and the U.S.A., cannot be classified as hardwood or softwood as they are usually defined. As a consequence, the present knowledge does not justify to limit to hardwood the carcinogenic effects of wood dust. The excess of risk appears to be attributable to wood dust per se, rather than to other exposures in the workplace, since the associations were observed in different periods, different countries and among different occupational groups. However, the causative carcinogenic agent(s) present in the wood dust have not been defined. The mechanisms of carcinogenicity in humans, including the role of lesions due to irritative agents on the occurrence of cancer, are largely unknown. Experimental studies bring little reliable information on the effects of wood dust. On the grounds of the above constatations, the conclusion of the lARC working group on the evaluation of carcinogenic risks to humans [1] was that there was "sufficient evidence in humans for the carcinogenicity of wood dust." The overall evaluation was that wood dust is carcinogenic to humans, with the following footnote: "This evaluation is based on the observation of a marked increase in the occurrence of cancers of the nasal cavities and paranasal sinuses among workers exposed predominandy to hardwood dusts." Occupational hazards due to wood dust exposure extends to leisure exposure entailing level of exposure comparable to that in a working environment. However, no studies were found on non-occupational exposure to wood dust by the working group at I ARC [1].

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PREVENTION OF SINONASAL CANCER Sinonasal cancer remains a health problem needing preventive measures for woodworkers, especially in some countries and in some conditions. Because the risk is related, at least partly, to the concentration of airborne wood dust, a reasonable objective for prevention is to reduce the concentration by improving exhaust ventilation. Several countries have set standards or guidelines for occupational exposure to wood dust. For example, in Canada, the limits for TWA (8-h timeweighted average) are 1 mg/m^ for hardwood and 5 mg/m^ for softwood. A description of regulations for selected countries can be found in Reference [1]. A medical surveillance of exposed workers, during and after the working life, must be encouraged, because chronic tissue damage may be considered as increasing the risk of development of sinonasal cancer. In several countries, sinonasal cancer is compensated as an occupational disease under certain conditions. It is not a negligible aspect, but efforts must be clearly put, first to prevention and secondly to early detection and treatment of nasal impairments among exposed workers.

REFERENCES 1. International Agency for Research on Cancer. Monographs on the evaluation of carcinogenic risks to humans, Vol. 62, "Wood dust and formaldehyde," 1995, Lyon. 2. "Cancer incidence in five continents." lARC Scientific Publication 120. lARC, Lyon, France, 1992. 3. Muir, C. S., Nectoux, J. "Descriptive epidemiology of malignant neoplasms of nose, nasal cavities, middle ear and accessory sinuses." Clin Otolaryngol, 1980, 5: pp. 195-211. 4. International Statistical Classification of diseases and related health problems, 10th revision. Vol. 1, WHO, 1992, Geneva. 5. Nylander, L. A. and Dement, J. M. "Carcinogenic effects of wood dust: review and discussion." Am J Ind Med, 1993, 24: pp. 619-647. 6. Acheson, E. D. "Nasal cancer in the furniture and boot and shoe manufacturing industries." Preventive Medicine, 1976, 5: pp. 195-315. 7. Medical Research Council. "The carcinogenicity and mutagenicity of wood dust." Scientific Report No. 1, 1982, Environmental Epidemiology Unit, Southampton. 8. International Agency for Research on Cancer. Monographs on the evaluation of carcinogenic risks of chemical to humans. Vol. 25, "Wood, leather and some related industries," 1981, Lyon. 9. International Agency for Research on Cancer. Monographs on the evaluation of carcinogenic risks to humans, Supp. 7, "Overall evaluation of carcinogenicity: an updating of lARC monographs," Vol. 1 to 42, 1987, Lyon.

Sinonasal Cancer and Wood Dust Exposure

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10. Macbeth, R. "Malignant disease of the paranasal sinuses." J Laryng, 1965, 79: pp. 592-612. 11. Acheson, E. D., Cowdell, R. H., Hadfield, E., Macbeth, R. G. "Nasal cancer in woodworkers in the furniture industry." Brit Med J, 1968, 2: pp. 587-596. 12. Acheson, E. D., Cowdell, R. H., Rang, E. "Carcinoma of the nasal cavity and sinuses in England and Wales." Brit J Industr Med, 1972, 29: pp. 21-30. 13. Gignoux, M., Bernard, P. "Tumeurs malignes de I'ethmoide chez les travailleurs du bois." Le Journal de Medecine de Lyon, 1969, 50: pp. 731-736. 14. Adenis, L., Vankemmel, B., Egret, G., Demaille, A. "Adenocarcinomes de I'ethmoide chez les ouvriers exposes aux poussieres de bois." Arch Mai Prof, 1972, 34: pp. 10-11,644-646. 15. Leroux-Robert, J. "Les cancers de I'ethmoide chez les travailleurs du bois." Cahiers d'ORL, 191 A, 9, 6: pp. 585-594. 16. Luboinski, B., Marandas, P. "Cancer de I'ethmoide: etiologie professionnelle." Arch Mai Prof, 1975, 36, 9: pp. 477-487. 17. Hadfield, E. H. "Adenocarcinoma of ethmoids in furniture workers." Ann Otol, 1971, 80: pp. 699-703. 18. Andersen, H. C , Solgaard, J., Andersen, I. "Nasal cancer and nasal mucustransport rates in woodworkers." Acrtz Otolaryngol, 1976, 82: pp. 263-265. 19. Andersen, H. C , Andersen, I., Solgaard, J. "Nasal cancers, symptoms and upper airway function in woodworkers." Br J Ind Med, 1911, 34: pp. 201-207. 20. Kleinsasser, O., Schroeder, H. G. "What's new in tumors of the nasal cavity? Adenocarcinomas arising after exposure to wood dust." Path Res Pract, 1989, 1984: pp. 554-558. 21. Engzel, U., Englund, A., Westerholm, P. "Nasal cancer associated with occupational exposure to organic dust." Acto Otolaryngol, 1978, 86: pp. 437^42. 22. Ironside, P., Matthews, J. "Adenocarcinoma of the nose and paranasal sinuses in woodworkers in the state of Victoria, Australia." Cancer, 1975, 36: pp. 1,115-1,121. 23. Voss, R., Stenersen, T., Oppedal, B. R., Boy sen, M. "Sinonsal cancer and exposure to softwood." Acto Otolaryngol, 1985, 99: pp. 172-178. 24. Vader, J. P., Minder, C. E. "Die Sterblichkeit an krebsen der nasen-und nasen-nebenhohlen bei schweizer schreinern." Schweiz Med Wschr, 1987, 117, 13: pp. 481-486. 25. Minder, C. E., Vader, J. P. "Sinonasal cancer and furniture workers: update and methodological points." Med Soc Prev, 1987, 32: pp. 228-229. 26. Malker, H.S.R., McLaughlin, J.K., Blott, W.J., Weiner, J. A., Malker, B. K., Ericcson, J. L. E., Stone, B. J. "Nasal cancer and occupation in Sweden, I96l-I919r Am J Ind Med, 1986, 9: pp. 477-485. 27. Gerhardsson, M. R., Norell, S. E., Kiviranta, H. J., Ahlbom, A. "Respiratory cancers in furniture workers." Br J Ind Med, 1985, 42: pp. 403-405.

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28. Acheson, E. D., Pippard, E. C , Winter, P. D. "Mortality of English furniture makers." Scand J Work Environ Health, 1984, 10: pp. 211-217. 29. Miller, B. A., Blair, A. E., Raynor, H. L., Stewart, P.A., Zahm, S. H. and Fraumeni, J. P., Jr. "Cancer and other mortality patterns among United States furniture workers." BrJIndMed, 1989, 46: pp. 508-515. 30. Miller, B. A., Blai, A., Raynor, H. L., Zahm, S. H. "Mortality among furniture workers: limitations in interpreting study findings when detailed work histories and exposure data are lacking." Scand J Work Environ Health, 1987, 13, 2: p. 167. 31.Demers, P. A., Kogevinas, M., Boffetta, P., Leclerc, A., Luce, D. et al. "Wood dust and sinonasal cancer: pooled reanalysis of twelve case-control studicsr Am J Ind Med, 1995, 28: pp. 151-166. 32. Zheng, W., Blot, W. J., Shu, X. O., Diamond, E. L., Gao, Y. T., Ji, B. T., Fraumeni, JF. "A population-based case-control study of cancers of the nasal cavity and paranasal sinuses in Shanghai." Int J Cancer, 1992, 52: pp. 557-561. 33. Bolm-Audorff, U., Vogel, C , Woitowitz, H .J. "Occupation and smoking as risk factors for nasal and naso-pharyngeal cancer." In Sakurai, H. et al. (eds). Occupational Epidemiology, New York, Elsevier Science Publications, 1990, pp. 71-74. 34. Hayes, R. B., Gerin, M., Raatgever, J. W., de Bruyn, A. "Wood-related occupations, wood dust exposure, and sinonasal cancer." Am J Epidemiol, 1986, 124: pp. 569-577. 35. Leclerc, A., Martinez Cortes, M., Gerin, M., Luce, D., Brugere, J. "Sinonasal cancer and wood dust exposure: results from a case-control study." Am J Epid, 1994, 140, 4: pp. 340-349. 36. Harden, L., Johansson, B., Axelson, O. "Epidemiological study of nasal and nasopharyngeal cancer and their relation to phenoxy acid or chlorophenol txposuvtr Am J Ind Med, 1982, 3: pp. 247-257. 37. Comba, P., Battista, G., Belh, S., de Capua, B., Merler, E., Orsi, D., Rodella, S., Vindigni, C , Axelson, O. "A case-control study of cancer of the nose and paranasal sinuses and occupational exposure." Am J Ind Med, 1992, 22: pp. 511-520. 38. Comba, P., Barbieri, P.G., Battista, G., Belli, S., Ponterio, F., Zanetti, D., Axelson, O. "Cancer of the nose and paranasal sinuses in the metal industry: A case control study." BrJIndMed, 1992, 49: pp. 193-196. 39. Magnani, C , Comba, P., Ferraris, F., Ivaldi, C , Meneghin, M., Terracini, B. "A case-control study of carcinomas of the nose and paranasal sinuses in the woolen textile manufacturing industry." Arch Environ Health, 1993, 48: pp. 94-97.

Sinonasal Cancer and Wood Dust Exposure

189

40. Merler, E., Baldasseroni, A., Laria, R., Faravelli, P., Agostini, R., Pisa, R., Berrino, F. "On the causal association between exposure to leather dust and nasal cancer: further evidence from a case-control study." Br J Ind Med, 1986, 43: pp. 91-95. 41. Brinton, L. A., Blot, W.J., Becker, J. A., Winn, D. M., Browder, J. P., Farmer, J. C , Fraumeni, J. F. "A case-control study of cancers of the nasal cavity and paranasal sinuses." Am J Epidemiol, 1984, 119: pp. 896-906. 42. Mack, W., Preston-Martin, S. "Case-control study of cancers of the nasal sinuses and nasopharynx among non-Asians in Los Angeles County (manuscript in preparation). 43. Vaughan, T. L. "Occupation and squamous cell cancers of the pharynx and sinonasal cavity." Am/MJM^J, 1989, 16: pp. 493-510. 44. Vaughan, T. L., Davis, S. "Wood dust exposure and squamous cell cancers of the upper respiratory tract. Am J Epidemiol, 133: pp. 560-564. 45. Hemberg, S., Westerholm, P., Schultz-Larsen, K. et al. "Nasal and sinonasal cancer. Connection with occupational exposures in Denmark, Finland and Sweden." ScandJ Work Environ Health, 1983, 9: pp. 315-326. 46. Elwood, J. M. "Wood exposure and smoking: association with cancer of the nasal cavity and paranasal sinuses in British Columbia." Can Med Assoc J, 1981, 124: pp. 1,573-1,577. 47. Fukuda, K., Shibata, A., Harada, K. "Squamous cell cancer of the maxillary sinus in Hokkaido, Japan: a case-control study." Br J Ind Med, 1987, 44: pp. 263-266. 48. Fukuda, K., Kojiro, M., Hirano, M., Hyams, V. J., Heffner, D. "Predominance of squamous cell carcinoma and rarity of adenocarcinoma of maxillary sinus among Japanese." The Kurume MedicalJournal, 1989, 36: pp. 1-6.

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CHAPTER 9 CHROMIUM ACCUMULATION OF CHROMATE WORKERS' BRONCHI Yuichi Ishikawa and Eiju Tsuchiya Department of Pathology, The Cancer Institute, Kami-ikebukuro, Toshima-ku, Tokyo 170, Japan

CONTENTS INTRODUCTION, 191 CHARACTERISTICS OF CHROMATE LUNG CANCER AND PULMONARY Cr BURDEN, 192 MATERIALS AND METHODS FOR STUDY OF Cr BRONCHIAL DISTRIBUTION, 194 Subjects, 194 Tissue Materials, 196 Calculation of Cr Concentration, 196 Neutron Irradiation, 197 BRONCHIAL BIFURCATIONS: "HOT SPOTS" OF CR ACCUMULATION, 197 DISCUSSION, 198 ACKNOWLEDGMENTS, 203 REFERENCES, 203 INTRODUCTION Lung cancer is the most frequent cause of cancer death in many industrialized countries, including the United States and Japan. Although its etiology is not fully understood, inhalation of environmental carcinogens is certainly one of the most important causes. Among the various kinds of pulmonary cancers, squamous cell and small cell carcinomas are most related to inhaled carcinogens. In fact, the incidences of these types of tumors are much greater in industrial than in rural areas, in smokers than in nonsmokers, and in underground miners than in the general population [1]. Thus, research findings support a strong relationship between bronchogenic carcinomas and inhaled carcinogens such as cigarette smoke, industrial emissions, mine dusts, and the radioactive gas radon [1,2]. One of the characteristic features of lung tumors induced by inhaled carcinogens is site specificity. Most lung carcinomas occurring in smokers originate in central rather than peripheral pulmonary regions. Further, there is evidence among smokers that epithelial lesions such as carcinomas, cell atypia, and loss of cilia are often found at or near bronchial bifurcations [3, 4]. To study the etiology of carcinomas caused by inhaled carcinogens and to estimate the risks of exposure, it is important to establish the relationship between cancer development and the pattern of their 191

192

Health and Toxicology

deposition in human bronchi. In particular, it is crucial to establish whether there is any preferential concentration at specific sites ("hot spots"). Several lines of evidence for preferential accumulation of inhalants in bronchi have been demonstrated: (i) enhanced deposition at bifurcations shown by computational modeling considering physical mechanisms [5-7], (ii) deposition experiments using hollow casts of airways and various types of inhalants including fibers [8-12], (iii) animal experiments featuring inhalation of radioactive [13, 14] and inorganic particles, including asbestos fibers [15, 16]. Particle size is crucial for distribution of inhalants in the lung. Schlesinger and Lippmann [12] showed in their comprehensive study using hollow casts that bronchial bifurcations are the site of enhanced accumulation from sub-|im particles (like cigarette smoke) to particles of more than 10 jum diameter. However, this approach is not able to estimate exact risk by a given inhalant to humans, because other mechanisms than those considered in hollow cast experiments can play a role in deposition in the human lung, and because human beings are long-lived and their airway physiology and pathology may be more complex than those of experimental animals. Hence, measurements using human organs/tissues are necessary. Nevertheless, very little information has been published on preferential sites of deposition in human bronchi. Little et al. [17] demonstrated high concentrations of ^^^Po at bronchial bifurcations in smokers by measurement of alpha-particle radioactivity. Although the half-life of ^^^^Po is only 138 days, continued exposure to cigarette smoke could result in persistently enhanced levels of this alpha emitter at specific sites. Still, the induction period for tumors of the respiratory tract is very long, generally in the order of several decades, and therefore information concerning the long-term exposure to inhaled particles is relevant to elucidating the underlying mechanisms that initiate tumor formation at hot spots. However, to the best of our knowledge, the availability of such information is very limited to date. To investigate the long-term accumulation of an inhaled carcinogen at specific sites of human bronchi, we chose a population of ex-chromate workers. Chromate workers have been established to be at high risk of developing respiratory tract carcinomas [18-26].

CHARACTERISTICS OF CHROMATE LUNG CANCER AND PULMONARY Cr BURDEN Since October 1975, we have followed up a population of 86 male ex-chromate workers by chest radiography, sputum cytology and bronchoscopic examinations. The subjects worked at a chromate manufacturing factory in Tokyo that produced chromate compounds from before World War II until August 1975. All were exposed to chromium (Cr) compounds, to a greater or lesser degree, including chro-

Chromium Accumulation of Chromate Workers' Bronchi

193

mate ore (FeOCr207), sodium chromate (Na2Cr04), and various dichromates (Na2Cr207, K2Cr207, CrOs). Our autopsy studies of this population demonstrated 21 carcinomas found in systemic organs of 12 cases: 16 lung, one maxillary, one esophagus, one common bile duct, and two gastric carcinomas [26], with latent periods of 27-53 years. Interestingly, a clearly positive "dose-response" relationship was seen between cancer occurrence and Cr pulmonary burden as shown in Figure 1. Although the Cr amount was not measured at bronchi but in peripheral tissues (bronchioles and alveoli), which are not preferential sites of chromate workers' lung cancers as detailed below, the obviously positive relationship does illustrate the importance of the Cr burden in the lung as an index of exposure. The histological types of the 16 lung carcinomas are indicated in Figure 2. Just like carcinomas observed among smokers, the prevalence of squamous cell carcinoma was significantly higher in exchromate workers (69%) than in sex-matched controls (32%) (p < 0.005). The distribution of lung tumors is also characteristic. When the 16 lung cancers were classified in origin as central (from main to segmental bronchi) or peripheral (subsegmental or more peripheral bronchi), 11 (69%) were central. These included an adenocarcinoma (Case 5 of Table 1) and a large cell carcinoma [26], both of which are usually seen in the periphery. An additional result of our autopsy studies was to reveal the eventual fates of precancerous lesions (dysplasias) that had been followed up by endoscopy. All five lesions detected were observed at the bronchial bifurcation, exactly in line with the

Cr concentration (^g/g (dry))

250

Multiple lung ca. (n=5)

Single lung ca. (n=2)

Other ca. (n=3)

No cancer (n=1)

Control (n=3)

Figure 1. Pulmonary Cr burdens measured in the peripheral lungs of chromate autopsy cases classified into four groups: (1) group with multiple lung cancers, (2) with a single lung cancer, (3) with non-pulmonary cancer(s), and (4) with no cancers. Ageand sex-matched controls were selected from non-chromate autopsies. For details, see ishikawa et al. [26].

194

Health and Toxicology

Exchromate workers (n=16)

6%

16%

IB ^^^ "'^ 1

AC Control (n=24,635)

-, 35%

,

SqCC

HIIIII|32%|H I 13% 1

0%

SCLC , / others , LCC

50%

10%

H

4%

100%

Figure 2. Comparison of histological types of lung cancers between ex-chromate workers (number of carcinomas = 16) and the control group (no. of cases = 24,635). AC, adenocarcinoma; SqCC, squamous cell carcinoma; SCLC, small cell lung carcinoma; LCC, large cell carcinoma; Others, other histological types.

finding in smokers that epithelial lesions such as dysplasias and squamous metaplasias occur at or near these bifurcations, like carcinomas. In summary, typical ex-chromate workers develop squamous cell carcinomas and dysplasias in the central part of lung—i.e., main to segmental bronchi, particularly at bifurcations—depending upon their Cr burden. Then, the question arises of the distribution of Cr within bronchi. Are there any deposition "hot spots" within bronchi even many years after cessation of exposure? We here show our results from Cr measurement in bronchi. MATERIALS AND METHODS FOR STUDY OF Cr BRONCHIAL DISTRIBUTION Subjects Seven consecutive autopsy cases were used for measurement of Cr. In addition, tissues from two cases in which lobectomy was performed following the diagnosis of lung tumors were available. Details of the cases are given in Table 1. In short, all the cases suffered from respiratory tract cancers with an average latency of 21 years. Case 2 was the only one who died from a cause other than cancer or cancer-related diseases. However, he developed a squamous cell carcinoma arising from the bifurcation of the main bronchus 2.5 years before his death which was cured by radiation therapy. Details of the tumors and a consideration of their relationship to chromate exposure have been described elsewhere [26]. From the nine cases, samples of tissue from 50 bifurcations and from neighboring sites were available for examination.

Table 1 Profile of ex-chromate workers' cases (male) examined Cancer Case no.

Reference no.

Age (y)

Exposure perioda(y)

Period from cessationb(y)

Smoking habitC

Lung Cr concentration

Site

Histologyd

Cause of death/operation

5 3

3. -

1

1

2 3 45 6 7 8 9 Average

S2524

69

19.3

14.3

0.5 x 52

468

S3141 S 3351 T28037 S3498

63 52 65 47

22.5 17.4 32 8.3

12.0 13.9 10 19.0

1 x 39 0.5 x 33 I1 xx456

138 28 n.e. n.e.

Lung Maxilla Epiphalynx Lung

SqCC SqCC SqCC AC

Radiation pneumonitis Cardiac infarction Cancer Cancer Cancer

S3583 S 3647 029571 029847

67 71 59 77 63.3

28.6 24.6 15.6 23.8 21.3

15.5 16.2 19.7 16.6 15.2

1 x33 0.5 x 36 1.5 x 41 1 x 47 32.7

84 15,800 40 76

Lung Lung Lung Lung

sqcc sqcc sqcc sqcc

Cancer Cancer Cancer Cancer

uPeriod of working in dusty environment. hPeriodfrom cessation of Cr exposure to death. cUnit: (pack (20 cigarettes) a day) x (years). dSmCC: small cell carcinoma, SqCC: squamous cell carcinoma, AC: adenocarcinoma.

Lung

SmCC

3 D

0 0

5

r 2

5' 3

0, 0

z

0

3

B

z

% 0

?

m

a3

0

3

196

Health and Toxicology

Tissue Materials

Tissue samples 2-5 mm thick and 2-20 mm^ in area, including non-cancerous epithelia and stromata as well as cartilage, were taken from the center and the edge of bifurcation ridges and from other regions (i.e., more central or more peripheral portions near the bifurcation) of each formalin-fixed lung (Figure 3). For the purpose of quality control, samples were taken in duplicate from the ridge edges of main bronchial bifurcations and adjacent sites of two cases in a pilot study, and the result was good agreement. Among the nine cases examined, there were some in whom tissues from bifurcations were used for other analyses or whose lung lobes had been surgically removed due to cancers and were not therefore available in totality for study. In the autopsy cases, invasion and/or metastasis of cancer were main reasons why systematic sampling could not be performed. All tissues were embedded in paraffin. The first section was cut for histology and morphometry, the following 20 to 100 consecutive sections were cut serially at 8 |im, piled up and double sealed with polyethylene for neutron irradiation, and the last section, again, was taken for histology and morphometry. Sectioning was performed by one technician always using the same microtome. Calculation of Cr Concentration The area of each tissue section excluding cartilage was measured using an image analyzer for both the first and the last sections. Exclusion of cartilage is reasonable because it is highly unlikely that the bronchial cartilage contains substantial amounts of Cr and because the fractional area of cartilage was not materially

More central part Bifurcation ridge

More peripheral part

Figure 3. Pictorial representation of definitions of the terms used and sampling sites from bronchial bifurcations, and a photograph of a typical specimen, 4.6 mm^ In area, taken from the ridge center of an intermediate trunk bifurcation to the middle lobar bronchus of Case 5. Bar, 1 mm.

Chromium Accumulation of Chromate Workers' Bronchi

197

different from specimen to specimen. Simultaneously, the lack of any primary cancer or invasion was confirmed histologically. Following quantification of Cr by activation analysis (see below), the concentration in the specimen was obtained by dividing the amount of Cr by the area of the tissue and the thickness of the irradiated sample. Using this method, we earlier successfully determined the concentration of thorium in samples from Thorotrast patients [27]. The ratio of Cr concentrations between the center of the bifurcation ridge and other portions near the bifurcation was also calculated. This ratio should indicate the degree of enhancement of Cr at the ridge center because our unpublished data showed systemic background concentrations of Cr in two cases to be in the order of 1% or less of pulmonary deposits, and consequendy were negligible in the context of this study. Comparison of the ratio with the generation of tracheobronchial branching allowed correlation coefficients to be calculated. Neutron Irradiation Concentrations of Cr in all samples were determined by neutron activation analysis at the Research Reactor Institute of Kyoto University, with a flux of 2.75 x 10^^ n cm~^ s~^ and irradiation times of up to 60 min. This leads to the production of ^^Cr by reaction of ^^Cr (n, 7) ^^Cr, where ^^Cr is one of the stable components of natural Cr with an abundance of about 4%. After a three-week wait for decay of short-lived nuclides, the radioactivity of ^'Cr (half-life 27.7 days) was measured with a cylindrical germanium detector of 76-mm diameter. The measurement duration was sufficient to give statistical uncertainties of 7% or less. Calibration was effected by dissolving accurately weighed pure chromate compounds for atomic absorption and counting. BRONCHIAL BIFURCATIONS: "HOT SPOTS" OF Cr ACCUMULATION The concentrations of Cr at the centers and the edges of bifurcation ridges and other portions near the bifurcations are listed in Table 2. The bifurcations where all three Cr values of ridge center (RC), ridge edge (RE) and other portions (Other) were available (n = 44) were classified into four categories in comparison with ridge values for other portions: (i) RC>RE>Other, (ii) RE>RC>Others, (iii) RC>Other or RE>Other, (iv) Other>RC and Other>RE.

198

Health and Toxicology

The results were: (i) 50%, (ii) 30%, (iii) 9%, and (iv)ll%. The most remarkable finding was the higher Cr concentrations at most (80%) of the bifurcations than at the other portions [(i) + (ii)]. Within the ridge of many bifurcations, concentrations at the centers were higher than at the edges. Whereas the concentrations were relatively constant in other portions, they varied considerably from site to site at the centers of bifurcation ridges (Table 2). This was particularly the case in more peripheral regions, where very high values were found almost exclusively at the ridge centers. In portions other than the centers of bifurcation ridges, Cr levels were generally higher in the peripheral than in the central bronchi in both lungs. Those at the trachea and tracheal bifurcations were the lowest, in agreement with the scarcity of cancers arising at these sites. Mean Cr concentration ratios between portions at ridge centers and at other portions for each generation of tracheobronchial branching, calculated from Table 2, are shown in Figure 4. For this purpose we propose the term "accumulation ratios at ridge centers." In all the generations of tracheobronchial branching from tracheal to segmental bifurcations, the mean accumulation ratios were more than 1. Interestingly, the mean accumulation ratios increased with generation of tracheobronchial branching, although the high values of 8.4 (n = 4) and 10.9 (n = 3) were largely due to two particularly high ratios of 25.4 (14.20/0.56) and 25.2 (38.54/1.53), respectively, both of which were found in Case 9. Using all the ratios (n = 44) from both sides of the lungs of all the cases, the correlation coefficient (R) regarding the accumulation ratios and branching generation was calculated to be significantly positive, at 0.06 < R < 0.58 (p < 0.05). DISCUSSION The present investigations revealed 80% of bifurcations to show higher concentrations of Cr at ridges than in other nearby portions. Furthermore, all mean accumulation ratios at ridge centers proved to be more than 1, with values increasing significantly with increasing generations of bronchial branching. These findings imply that the bifurcation ridges, and in particular the ridge centers, are hot spots of persisting Cr deposits even more than 10 years after cessation of exposure. This study thus produced solid evidence for the deposition hot spot concept of long-term retention. There are two main aspects, physical and pathophysiological, that should be taken into account in determining the factors responsible for formation of deposi-

Table 2 Cr concentrations of former chromate workers’ bronchi Tracheal Case

RC

RE

Main Other

Left lung and trachea 1

RE

Other

RC

RE

Segmental Other

RC

RE

Other 0

-

2 3

1.1

4 6

7

RC

Lobar

0.6

0.4

8 (table continued on next page)

1.7 2.6

-

0.8 2.2

0.2 0.7 1.9

0.2

4.4 1.3

1.8 2.0

2.7 1.1

0.4

13.4

6.4

1.1

4.7 3.0

1.1 7.4

1.o 2.9

12.3 0.4

3.9 0.8

zz.

0.7 0.4

1.0 8.0 3.8 8.4 14.4 14.6

1.4 4.3 3.1 4.0 4.1 6.5

1.9 2.6 1.4 1.4 2.6 3.3

2.1 2.2 2.3 3.3 1.5 4.2 9.2

3.4 1.8 2.6 4.8 1.6 2.8 4.5

1.1 1.3 1.8 1.9 1.3 1.1 2.2

C

3 D

9.2

5.8

1.8

0 0

C

3

E

$ !

2 s

3 a3

2 (D

z

% v?

m

a

3 0

z A

(0 (0

Table 2 (Continued) Cr concentrations of former chromate workers' bronchi Main Case

RC

RE

Intermediate Other

RC

RE

Other

Lobar RC

RE

pl

Segmental Other

RC

RE

Subsegmental

Other

RC

RE

Other

d

+ .!0

0 0

9

Right lung 1

1.2

1.2

1.9

2 4 5

2.2 1.6

2.0 1.1

0.8

6 7 9

1.1

0.3

0.4 4.6

0.4 2.3

0.5 1.2

0.8 0.04

1.2 1.2

0.7

-

1.1 2.4 2.2

1.8 2.3

1.1 1.9

8.0 3.7 12.6 0.6 1.9 1.5 6.7 6.4

3.9 0.5 3.4 0.2 3.0 5.7 -

2.3 3.1 3.3

18.0 12.3 14.7 2.5 1.6 1.7

4.9 3.9 1.9

2.5 2.1 9.7 0.7

1.9

14.2 16.5

0.6

1.9 4.2 38.5 7.8

0.8 1.5

-

-

1.0 1.0 1.5 0.9

Centers of bifurcation ridges (RC), the edges of bifurcation ridges (RE), and other (i.e., more central or more peripheral) parts near the bifurcation (Other). Measured by neutron activation analysis, at 15 years on average after cessation of exposure. Unit of Cr concentration is x l @"g/pm tissue thickness/mm2.

Chromium Accumulation of Chromate Workers' Bronchi

201

Mean Cr accumulation ratios at ridge centers of bifurcations 4

6

8

10

12

Tracheal bif. (n=1)

L Main br. bif. (n=7) L Lobar br. bif. (n=15) L. Seg. br. bif. (n=1)

R. Main. br. bif. (n=3) R. Interm. tr. bif. (n=5) R. Lobar, br. bif. (n=7) R. Seg. br. bif. (n=4) R. Sub-seg. br. bif. (n=3) Figure 4. Mean ratios of Cr concentrations between the ridge centers of bifurcations and other portions (mean Cr accumulation ratios) for each generation of bronchial branching. Note that all the mean Cr accumulation ratios are more than 1 and that the ratio increases with increasing generation of tracheobronchial branching. L., left; R., right; bif., bifurcation; br., bronchus or bronchial; Interm. tr., intermediate trunk; Seg., segmental; n, number of bifurcations available for calculation of the accumulation ratios.

tion hot spots at bifurcations. Physically, there are four ways in which solid particles can be deposited in the lung: sedimentation, inertial impaction, interception, and diffusion [28]. Among these, the former two are of primary importance in the enhanced deposition at bronchial bifurcations of the chromate workers because the other two may concern particles of irregular shape and very small particles. In fact, microscopic measurements in lung tissues obtained at autopsy showed that most of the chromate particles were 1-3 jim in diameter [29], with an approximately spherical shape. (Actual assessment of particle size distribution in the workplace is now impossible because the factory in which the workers were employed terminated production of Cr compounds in 1975.) Considering the size and shape of particles and airway generations of interest (main to subsegmental bronchi), inertial impaction and sedimentation may be chiefly responsible for the enhanced deposition in the workers' bronchi [28].

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Health and Toxicology

However, the results obtained by this approach should be interpreted in the light of physiological considerations, where possible, using animal models for example, because deposited particles are largely cleared by mucociliary streaming. It is known that the ciliary streaming of mucus and the lymphatic flow tend to slow down and stagnate around the bifurcation [14, 30]. Other factors that may facilitate augmented deposition are related to pathology in human beings. Prolonged exposure to toxic inhalants such as tobacco smoke causes loss of cilia and/or squamous metaplasia occurring preferentially at or near bifurcations. This would further delay the mucociliary removal and hence accelerate the formation of hot spots. Additionally, we should consider the question as to whether Cr sticks to bronchial tissues for more than 10 years or whether steady removal of Cr from alveolar regions is responsible for the enhanced concentration in bronchial bifurcations. If the former possibility is true, most of the Cr detected in bronchial tissues must be deposited in bronchial stroma rather than in epithelium, considering that cell renewal of the epithelium is estimated to be an order of 1 year or less for adults in our age range [31, 32]. On the other hand, the latter possibility would imply that most of the Cr measured is localized to the epithelium. Further fine topographical determination of Cr within bronchial tissue may answer this question. Another finding requiring discussion is the fact that higher enhancement was evident in the more peripheral bifurcations. This may be a clue to the question on how the deposition hot spots are formed, because one of the main causes is the stagnation of mucus stream at the bifurcations mentioned above. The mucus flow must carry considerable amounts of Cr compounds deposited in alveolar regions, which retain mainly particles with diameters of more than sub-|im and less than 4 [xm [33], comparable with typical chromate particles [29]. In other words, the fact that at the more peripheral bifurcations a more marked deposition was evident may imply that the primary mechanism of forming hot spots at bifurcations is the stagnation of mucus flow at such sites. Further, it is very interesting that the distribution of Cr is comparable with that of bronchial cancer which is known to arise most frequently at segmental bronchi, not at more central bronchi [3]. In this study we successfully examined up to subsegmental bifurcations, but could not analyze more peripheral bifurcations using the same methods because of their very small dimensions. Chemical forms may be crucial for metal carcinogenesis. As is well known, 6valence Cr is more harmful than the 3-valence form. However, actual measurements showed that most of the Cr deposited in pulmonary tissues was 3-valence like Cr203 [34]. We agree with Dr. T. Sano, a pioneer of occupational disease pathology in Japan, who proposed that 6-valence Cr is so strong an irritant that it can induce severe inflammation and subsequently is reduced to 3-valence, which can exist as an insoluble form and might persist to cause cancer [29]. It is known that carcinomas and atypical lesions preferentially occur at bronchial bifurcations in smokers [4]. This is also the case for ex-chromate workers [26]. Hence, the results of this study would indicate a direct relationship between the concentration of Cr deposits and neoplasia. Additionally, it should be noted that the

Chromium Accumulation of Chromate Workers' Bronchi

203

Cr concentrations at ridge edges were not found to be low. In fact, among the 44 bifurcations where the values at the ridge center, ridge edge, and another portion were available, 13 (30%) showed the highest level at the edge as mentioned above. Cigarette smoking may have been a factor in the induction of carcinomas in the bronchi of the population of ex-chromate workers used in this study. Actually, most of the cases were smokers with an average pack-year of 32.7 (Table 1). However, an epidemiological study of the same population of ex-chromate workers suggested that the exposure to Cr compounds played the main role in causing cancer development [20]. Summarizing briefly, the lung cancer morbidity rate for the ex-chromate workers with 9 years or more of exposure was 21.6 times higher than that for the general population. The mortality rate for lung cancers in non-chromate smokers, adjusted to the ex-chromate worker population by age of starting smoking and by number of cigarettes per day, is generally 3-4.7 times higher than for non-smokers in Japan. Unless cigarette smoking had an extraordinarily synergistic effect on cancer induction, or super-multiplicative synergism with Cr inhalation, we can therefore conclude that the Cr exposure was primarily responsible, always bearing in mind that there is a minor difference between morbidity and mortality. ACKNOWLEDGMENTS We wish to thank Drs. T. Kitagawa and H. Sugano, Cancer Institute, for their support; Drs. K. Nakagawa, Cancer Institute Hospital; T. Hirano, Hirano Kameido Himawari Clinic; and Prof. T. Tamai, Kyoto University, for collaboration. Y. I. is grateful to Professor Emeritus S. Hatakeyama for his encouragement. This study was supported financially by the Visiting Researchers' Program, Research Reactor Institute, Kyoto University, Osaka, Japan. REFERENCES 1. Kreyberg, L. Aetiology of lung cancer—a morphological, epidemiological and experimental analysis, Oslo: Oslo Universitets Forlaget, 1969, pp. 1-90. 2. National Academy of Sciences. Health Risks of Radon and Other Internally Deposited Alpha-emitters, BEIR IV Report, Washington, D.C.: National Academy Press, 1988, pp. 1-602. 3. Marsh, B. et al. "Bronchoscopic localization of radiologically occult cancer," in Recent Results in Cancer Research, Vol. 82, Early Detection and Localization of Lung Tumours in High Risk Groups. R. R. Band (Ed.), Heidelberg: Springer-Verlag, 1982. pp. 87-89. 4. Auerbach, O. et al. "Changes in bronchial epithelium in relation to cigarette smoking and in relation to lung cancer." New England Journal of Medicine, Vol.265, 1961, pp. 253-267.

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5. Hofmann, W. and Balashazy, I. "Particle deposition patterns within airway bifurcations—solution of the 3D Navier-Stokes equation." Radiation Protection Dosimetry, Vol. 38, 1991, pp. 57-63. 6. Kinsara, A. A. et al. "Computational flow and aerosol concentration profiles in lung bifurcations." Health Physics, Vol. 64, 1993, pp. 13-22. 7. Martell, E. A. "Alpha-radiation dose at bronchial bifurcations of smokers from indoor exposure to radon progeny." Proceedings of National Academy of Science of USA, Vol. 80, 1983, pp. 1,285-1,289. 8. Martonen, T. B. et al. "Cigarette smoke and lung cancer." Health Physics, Vol.52, 1987, pp. 213-217. 9. Ermala, P. and Holsti, L. R. "Distribution and absorption of tobacco tar in the organs of the respiratory tract." Cancer, Vol. 8, 1955, pp. 673-678. 10. Martin, D. and Jacobi, W. "Diffusion deposition of small-sized particles in the bronchial trees." Health Physics, Vol. 23, 1972, pp. 23-29. 11. Sussman, R. G. et al. "Asbestos fiber deposition in a human tracheobronchial cast. I. Experimental, and II. Empirical model." Inhalation Toxicology, Vol. 3, 1991, pp. 145-179. 12. Schlesinger, R. B. and Lippmann, M. "Selective particle deposition and bronchogenic carcinoma." Environmental Research, Vol. 15, 1978, pp. 424^31. 13. Gore, D. J. and Patrick, G. "The distribution and clearance of inhaled UO2 particles on the first bifurcation and trachea of rats." Physics of Medicine and Biology, Vol. 23, 1978, pp. 730-737. 14. Svartengren, M. et al. "Retention of particles on the first bifurcation and the trachea of rabbits." Bull Eur. Physiopathol Respir., Vol. 17, 1981, pp. 87-91. 15. Brody, A. R., et al. "Chrysotile asbestos inhalation in rats: deposition pattern and reaction of alveolar epithelium and pulmonary macrophages." American Review of Respiratory Disease, Vol. 124, 1981, pp. 670-679. 16. Brody, A. R. and Ore, M. W. "Deposition pattern of inorganic particles at the alveolar level in the lungs of rats and mice." American Review of Respiratory Disease, Vol. 128, 1984, pp. 724-729. 17. Little, J. B. et al. "Distribution of polonium-210 in pulmonary tissues of cigarette smokers." New England Journal of Medicine, Vol. 273, 1965, pp. 1,343-1,351. 18. Mancuso, T. F. and Hueper, W. C. "Occupational cancer and other health hazards in a chromate plant: a medical appraisal. I. Lung cancers in chromate workers." Industrial Medicine and Surgery, Vol. 20, 1951, pp. 358-363. 19. Abe, S. et al. "Chromate lung cancer with special reference to its cell type and relation to the manufacturing process." Cancer, Vol. 49, 1982, pp. 783-787. 20. Nakagawa, K. et al. "Surveillance study of a group of chromate workers— early detection and high incidence of lung cancer." (in Japanese) Japanese Journal of Lung Cancer, Vol. 24, 1984, pp. 301-310.

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205

21. Langard, S. and Norseth, T. "A cohort study of bronchial carcinomas in workers producing chromate pigments." British Journal of Industrial Medicine, Vol. 32, 1975, pp. 62-65. 22. Machle, W. and Gregorius, F. "Cancer of the respiratory system in the United States chromate-producing industry." Public Health Report, Vol. 63, 1948, pp. 1,114-1,127. 23. Newman, D. "A case of adeno-carcinoma of the left inferior turbinate body and perforation of the nasal septum in the person of a worker in chrome pigments." Glasgow MedicalJournal, Vol. 33, 1890, pp. 469-470. 24. Bidstrup, P. L. "Carcinoma of the lung in chromate workers." British Journal of Industrial Medicine, Vol. 8, 1951, pp. 302-305. 25. Baetjer, A. M. "Pulmonary carcinoma in chromate workers I. A review of the literature and report of cases." AMA Archives of Industrial Hygiene and Occupational Medicine, Vol. 2, 1950, pp. 487-504. 26. Ishikawa, Y. et al. "Characteristics of chromate workers' cancers, chromium lung deposition and precancerous bronchial lesions: an autopsy study." British Journal of Cancer, Vol. 70, 1994, pp. 160-166. 27. Ishikawa, Y. et al. "Quantitative determination of elements in paraffinembedded specimens by activation analysis—an example of Thorotrastinjected patients." (in Japanese) Journal of Clinical and Experimental Medicine (Igaku-no-ayumi), Vol. 141, 1987, pp. 959-960. 28. Parks, W. R. Occupational Lung Disorders, 2nd ed. London: Butterworth & Co., 1982, pp. 1-529. 29. Sano. T. "Occupational cancers in chromate workers." in Pneumoconiosis and Public by Dust in Japan, 2nd ed. (in Japanese) T. Sano (Ed), Kawasaki: Institute for Science for Labour, 1983, pp. 315-325. 30. Hilding, A. C. "Ciliary streaming in the bronchial tree and the time element in carcinogenesis." New England Journal of Medicine, Vol. 256, 1957, pp. 634-640. 31. Fawcett, D. W. A Textbook of Histology, Philadelphia: W. B. Saunders Co., 1986, p. 737. 32. Creagh, T., and Krausz, T. "13. The respiratory system, 13.1 Normal structure and function." in Oxford Textbook of Pathology, Vol. 2a, J. O'D. McGee et al. (Eds.), Oxford: Oxford University Press, 1992, pp. 943-947. 33. Lippmann, M. and Albert, R. E. "The effect of particle size on the regional deposition of inhaled aerosols in the human respiratory tract." American Industrial Hygiene Association Journal, Vol. 30, 1969, pp. 257-275. 34. Kudo, H. et al. "The concentration and X-ray microanalysis of chromium in the lung tissues of a chromate worker with multicentric lung cancer." (in Japanese) Japanese Journal of Lung Cancer, Vol. 19, 1979, pp. 385-392.

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CHAPTER 10 BENZENE TOXICOKINETICS IN HUMANS Frederic Yves Bois Indoor Environment Program Energy and Environment Division Lawrence Berkeley National Laboratory Berkeley, CA 94720

CONTENTS INTRODUCTION, ABSORPTION, DISTRIBUTION, ELIMINATION, BENZENE SECONDARY METABOLISM, MODELING OF BENZENE TOXICOKINETICS, REFERENCES, INTRODUCTION Benzene is an ubiquitous chemical, present in some natural foodstuffs and raw petroleum minerals, but mostly encountered in industry as a basis for plastic monomers and as a solvent [1]. It is also found in refined petroleum products such as gasoline. Benzene is volatile at usual temperatures and slightly miscible with water (0.8 parts by weight in 1,000 parts of water at 20°C). Overall, it has been estimated that 10 kg of benzene is lost per ton of benzene produced during transfer and storage, with approximately 94% lost as air emissions and 6% as water effluents [1]. It is therefore found in ambient air, water, and soil. For example, benzene has been detected in U.S. water supplies [2]. Concentrations between 0.3 to 2.4 ppm in the air near gas stations for benzene have been reported, compared to 0.017 ppb for rural areas [1]. The main exposure for smokers is from mainstream cigarette smoke: This represents 50% of the population exposure burden [3]. The highest exposure levels occur for workers exposed when using it as a solvent. Benzene is a known human carcinogen [4, 5], most likely through its metabolites. It has been long recognized that chronic exposure to benzene at high levels in the workplace can lead to bone marrow depression and aplastic anemia [6]. Benzene is also an etiologic agent of acute myelogenous leukemia and some of its variants [7]. Humans appear to be more susceptible to the leukemogenic potential of benzene than most animal species studied. Yet, most animals exposed to benzene develop bone marrow hypoplasia and cancers at various sites. At very high concentrations, unhkely to be encountered nowadays except in case of accident, it induces acute toxicity, particularly to the central nervous system [7]. Understanding the toxicokinetics of benzene in humans is important to achieve better monitoring of workers (through the choice of sensitive measurement endpoints and sampling 207

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times), and in risk assessment of low-level exposures (through a better characterization of effective exposure). ABSORPTION, DISTRIBUTION, ELIMINATION Data have been collected for the last hundred years on benzene toxicokinetics. Those experiments are usually summarized by a few characteristics (e.g., fraction absorbed, terminal half-life) whose interpretation requires some understanding of some basic concepts related to benzene absorption, distribution, and elimination. Absorption Benzene can enter the body by inhalation, ingestion, or dermal absorption. The respiratory route is believed to be the major source of human exposure to benzene from the workplace, gasoline vapors, automobile emissions, and tobacco smoke. Yet, the use of benzene for hand cleaning, by rotogravure workers for example, can lead to significant (but unquantified) exposures [1]. Skin absorption represents less than 5% of that absorbed through the lungs if exposure is only to the vapor phase [8]. There is apparently no human data on benzene absorption through ingestion, but it is likely that most if not all benzene ingested passes the intestinal wall because of its lipophilicity. The fraction absorbed by inhalation is difficult to measure and often to interpret because it is time and dose dependent, based on benzene blood concentration; therefore, penetration decreases when exposure length increases. At steady-state (achieved after about 60 hr of constant exposure, bur reasonably approximated after 10 hr), it can be measured by the difference between inhaled and exhaled air concentration and is the complement of the fraction metabolized. Yet, this fraction itself tends to decrease with the exposure concentration because benzene metabolism eventually saturates. Interindividual variability further complicate the picture. It should also be realized that in a closed-exposure setting, all benzene would eventually be absorbed and metabolized by the subjects. With all these caveats, at steady-state, for exposure levels of the order of 10 ppm or below, the fraction absorbed is about 50% [9, 10]. A potentially more useful quantity is the blood over air partition coefficient (i.e., the ratio of blood over air concentration at equilibrium). This partition coefficient, when measured in vitro, is close to 7 [8, 11, 12]. In vivo, values of about 14 (range 5-40) and 9.6 (SD 0.8) can be inferred from the data of Brugnone et al. [13], Pekari et al. [10], and Bois et al. [14] data, respectively. Lower in vitro values could be due to the effect of uninhibited metabolism, other losses, or inter-subject variability. Distribution Once in the body, benzene is dispersed mostly by blood through the various organs. It has a particular affinity, characteristic of apolar solvents, for fat tissues.

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209

where it accumulates. The affinity of benzene for the various tissues or organs can be measured by the organ-over-air-partition coefficients (Table 1). These coefficients can be combined to give tissue-to-tissue ratios: For example, the in vivo fatover-blood partition coefficient can be obtained as 183/9.6 = 19, indicating that at equilibrium the concentration of benzene in fat is about 20 times higher than its concentration in blood. Bone marrow, a target organ for toxicity, has a high partition coefficient and accumulates benzene more than most other tissues. It has been suggested that benzene could bind to blood proteins [15], but this claim is based on little evidence and has not been confirmed experimentally. Table 1 Benzene tissue-over-air-partition coefficients (mean ± SE)

in vitro [11] in vivo [10, 14]^^)

Liver

Bone marrow

Fat

Muscle

Viscera

425 ± 50 183 ±55

16 ±2.5 16 ±3.2

12±4 23 ± 4.5 — 9.7 ± 2.4 17 ±7.6 74 ±30

Blood 6.4 ± 0.6 9.6 ±0.8

('^^Population averages determined using a physiologically-based pharmacokinetic model.

It was noted experimentally that the contribution of smoking to blood and exhaled air benzene can be a confounder of occupational exposures, but this problem can be overcome if the subjects refrain from smoking on the day of sample collection, or if the collection takes place one hour after a high (>5 ppm) exposure [10]. Elimination Benzene is eliminated out of the body by exhalation and by metabolism. Very little benzene is found in the urine (less than 1%) [12]. Post exposure, benzene blood concentration-vs.-time curves are at least triphasic [16]. The terminal component of the concentration curve is controlled by the balance of exhalation, metabolism, and slow release from fat storage, processes which vary from individual to individual. Early studies give terminal half-Hves ranging from 16 hr to 75 hr [17-19]. It has been shown, however, that these studies lead to unreconcilable estimates of pharmacokinetic parameters [20]. The variability seen above could therefore be due to artifacts. Recently, the average terminal half-life was found to be 10 hr for 3 subjects in a controlled experiment [14]. Caution should be exercised in interpreting the results of some studies [16, 21, 22] with follow-up too short to observe the terminal phase. The population average of the fraction metabolized was estimated to be 57% ± 6% in a recent pharmacokinetic analysis of controlled exposure data [10, 14]. Benzene metabolism occurs in several organs, but mostly in the liver (at least for rodents [1, 7]), and is mediated by cytochrome P450 2E1 [7]. A single pathway.

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from benzene to benzene oxide is involved. Benzene oxide is itself further transformed (see secondary metabolism below). The high metabolic clearance of benzene points to the possibility of a perfusion limitation of benzene metabolism (particularly at exposure levels below 10 ppm) [14]. This would imply that benzene rate of metabolism is essentially controlled by its rate of delivery to the liver, and that it is linear for constant exposure levels below 10 ppm. The high hepatic metabolism of benzene would also induce a potential difference between the inhalation and the ingestion route. Upon ingestion, benzene is transported through the portal vein to the liver where P450 oxidizes it and only the unmetabolized fraction escapes to the general circulation. After inhalation, benzene is directly available to the other tissues. The impact of this "first pass" effect is unknown because all experimental data on humans are from inhalation exposures. Simultaneous treatment of animals with both benzene and toluene increases the excretion of unchanged benzene [1]. Toluene appears to inhibit benzene metabolism, thereby increasing its respiratory elimination. A report that this was not observed in humans [22] is inconclusive because the kinetics were not followed enough in time to make meaningful inference about altered metabolism. While the majority of the experimental evidence suggests that the liver is the major organ involved in benzene metabolism, the bone marrow is the target site for benzene toxicity. Hepatic metabolites are carried via the bloodstream to the bone marrow, where they may accumulate and undergo further transformations [7], but this has not been shown in humans. In situ bone marrow metabolism can also contribute to benzene toxicity [23]. These two possible mechanisms of bone marrow exposure to metabolites would lead to different patterns of metabolites accumulation, but no human data is available on this question. This complicates tremendously the identification of the actual active species responsible for bone marrow toxicity, and hence benzene risk assessments. BENZENE SECONDARY METABOLISM Benzene is actually not likely to be directly carcinogenic; rather, its metabolites are involved [1]. Most of the evidence relating to the reactions involved comes from animal experiments, but the same metabolites are found in humans, and it is reasonable to assume that similar pathways are followed. A brief summary is given here (Figure 1). A first step involves P450 oxidation of benzene, leading to benzene oxide. Three pathways then lead to mercapturic acids, ring-opened metabolites, or ring-hydroxylated metabolites. Premercapturic acid is formed by glutathione conjugation of benzene oxide. It leads to S-phenylmercapturic acid, which has been found in the urine of exposed workers and proposed as a biomarker of benzene exposure [24]. This pathway gives nontoxic metabolites but is quantitatively minor (representing less than 1% of all metabolites).

Benzene Toxicokinetics in Humans

H HOOC

0 =

I

H

I

C

I

0 =

C

I

H

COOH

H

H2C— O H — COOH

trans,trans-mucon\c acid

H OHO—C= C

I

H

211

t

I 0 =

I

I

S

benzene H C

OHO

I

N H — COCH3

P450

H

phenylmercapturic acid

frans, frans-m ucona Ide hyde epoxide hydrolase^

GSH benzene oxide

premercapturic acid

benzene-/ra/is-dihydrodioi

P450 phenol

OH

hydroquinone

catechol OH

I

A o

OH

1,2,4-benzenetrlol

o-benzoquinone

T o

p-benzoquinone Figure 1. IVIajor pathways of benzene metabolism in humans. Unlabeled arrows can represent several reactions.

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Ring opening of benzene oxide (which can be achieved by free radical reactions) leads to trans,trans-muconMthyde. Muconaldehyde is cytotoxic and could be involved in benzene toxicity. It is further oxidized in trans,trans-muconic acid, found in urine of benzene-exposed individuals. Muconic acid amounts to about 10% of all urinary benzene metabolites in humans. Several other minor muconic derivatives can also be formed, at least in vitro [7]. The major metabolites are ring-hydroxylated compounds. Phenol is formed by non-enzymatic rearrangement of benzene oxide. Direct oxidation of benzene by hydroxyl radicals could also be an important mechanism in micromolar concentrations (which is the range encountered in the body after occupational exposures) [7]. Phenol is detoxified by sulfo- and glucurono-conjugation. Phenol (free and conjugated) is the main metabolite found in urine (where it accounts for about 70% of total metabolites) [25]. Hydroquinone, catechol, and 1,2,4-benzenetriol are formed by further P450 mediated hydroxylation of phenol. Hydroquinone, catechol sulfate, and glucuronic acid conjugates can be detected in the urine of benzene-exposed individuals, where each represents about 10% of excreted metabolites. An alternative pathway for the formation of catechol involves metabolism of benzene oxide by epoxide hydrolase to benzene-rran^-dihydrodiol, converted to catechol by the action of dehydrogenase. Hydroquinone and catechol can be further oxidized into p- and o-benzoquinones. The hydroxylated metabolites are very likely to be involved in the toxicity of benzene [1, 7]. Little is known of the kinetics of these compounds in humans, and even less in the bone marrow. MODELING OF BENZENE TOXICOKINETICS Several models have been developed to describe the toxicokinetics of benzene in humans. Early work used classical pharmacokinetic models with three compartments to describe the various phases of benzene elimination from blood [16]. These models were only moderately helpful because they did not describe in any detail inhalation or metabolism and could not account for first-pass effects. They also did not provide insight on the fraction of benzene dose delivered to the bone marrow. For these reasons (and for the hypothetical possibility of extrapolating results from animals) physiologically based pharmacokinetic (PBPK) models have recently been developed [14, 20, 26-30]. A typical design for these models is given in Figure 2. The equations used to describe this type of models can be found in the literature [26, 30]. Problems remain with the calibration of those models. For example, Travis et al. [27], used visual fitting to model several sets of data [12, 17-19, 21, 31] and give no measures of uncertainty for the model parameters or predictions. Yet, many of these parameters, typically those controlling metabolism, are not known with precision. Proper statistical inference about their value is therefore necessary, while conserving the strong prior information conferred by their physiological definition. Bayesian statistics provide a natural way to merge a priori knowledge, gained by implementing a physiological model, with evidence from experimental data [32]. In

Benzene Toxicokinetics in H u m a n s

ALV ^ ^

213

Exhaled Air

r y^ VPR^'^Pba

^o 11 CD 1 o 1

WELL PERFUSED

>a>

Vwp, Pwp

c 1 1

^Fwp

1^

FAT

4:"

^

Vf,Pf

POORLY PERFUSED

^Fpp

Vpp, Ppp

^

BONE MARROW

^Fbm

\^

Vbm, Pbm

LIVER VI, PI VmaxI, Kmi

Vmaxbm, Kmbm

\ fKf -^

1

Fl

L^-J ^

9

1 METABOLITES

^

Ke, Fu

URINARY PHENOL OTHERS

*L_. 1-fp

Figure 2 . Schematic representation of a 5 - c o m p a r t m e n t physiological model used to simulate t h e distribution a n d m e t a b o l i s m of b e n z e n e in h u m a n s [14]. S y m b o l s are given in T a b l e 2.

addition, scientific interest generally resides in inference about benzene metabolism in humans—i.e., in a diverse population—rather than in any one individual studied in published experiments. It is therefore preferable to design a statistical model describing the relationships between individual and population physiological parameters to estimate population variability [33-35]. Once properly parameterized, PBPK models can be powerful simulation tools. Figures 3 and 4 show the fit obtained to data from one subject exposed for 4 hr to 1.7 ppm and 10 ppm benzene by inhalation, using the model depicted in Figure 2. Distributions for the parameter values of this model are given in Table 2. On the basis of this model, it was established that no significant dose-rate effect occur for benzene [20], that benzene primary metabolism is essentially linear below 10 ppm exposure levels, and that the fraction metabolized at those levels is close to 55% (Figure 5) [14, 27]. Many other endpoints of interest for benzene toxicology or risk

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Health and Toxicology

H 1 1 1 1 1 1 1 1—j 1 1 1 1 1 I I I I I I I I 1 I I I I I I I I I I I I I I I I t I I I

500

1000

500

1500

Time (min)

2500

Time (min)

Figure 3. Simulated and observed time course of tlie venous blood and exhaled air concentrations of benzene for subject 1 of Pekari et al. [10]. Exposures were to 1.7 ppm (A and C) and 10 ppm (B and D) for 4 hours. The data points are bracketed by ±2 estimated SDs. Results are similar for the other two subjects studied.

30H 25H

c o c o

o

"o c
0

I I I I I I I

500

1000

I

I

1500

I I I I I I I I I I I I I

2000

2500

3000

I

3500

Time (min)

Figure 4. Simulated and observed time course of urinary phenol concentration for subject 1 of Pekari et al. [10]. Exposure was to 10 ppm (30 |jg/L) for 4 hours, starting at time zero. The data points are bracketed by ±2 estimated SDs.

Benzene Toxicokinetics in Humans

215

Table 2 Population geometric means and standard deviations for the scaling coefficients of a physiologically based pharmacokinetic model of benzene distribution and metabolism in humans [14]. The means and SDs themselves are subject to uncertainty. Scaled parameter Ventilation over Perfusion Ratio (VPR) Blood flows per unit mass Well-perfused tissues (Fwp) Poorly-perfused tissues (Fpp)

Fat (Ffp

Bone Marrow (Fbm) Liver (Fl) Volumes Well-perfused tissues (Vwp) Poorly-perfused tissues (Vpp/^^ Fat (Vf)('^ Bone Marrow (Vbm) Liver (VI) Blood/air partition coefficient (Pba) Tissue/blood partition coefficients Well-perfused tissues (Pwp) Poorly-perfused tissues (Ppp)

Fatf/y;

Bone Marrow (Pbm) Liver (PI) Maximal rates of metabolism Liver (Vmaxl) Bone Marow (Vmaxbm) Michaelis-Menten coefficients Liver (Kml) Bone Marow (Kmbm) Endogenous metabolites formation rate (Kf) Metabolites excretion rate constant (Ke) Urine formation rate (Fu) Phenol fraction of excreted metabolites (fp)

Scaling function^^^

— SC X Vwp SC X Vpp SCxVfxO.92 SC X Vbm SCxVl SC X LM(^^

—

SC X Bw/0.92 SC X LM^"^^ SC X LM^"^)

—

Geometric mean

Geometric

1.7 ±0.20

1.14x^1.02

0.34 0.041 0.029 0.19 1.0

± 0.022 ± 0.0026 ± 0.0024 ±0.016 ±0.084

0.23 ±0.015 0.25 0.052 0.031 9.65

± 0.035 ± 0.004 ±0.0026 ± 0.77

SD^*')

1.05x^1.01 1.05x^1.01 1.05x-fl.01 1.05x^1.01 1.05x^1.01 1.05 x-^ 1.01 1.21 x-^ 1.02 1.05x^1.01 1.05 x-f 1.01 1.14x^1.01

— — — — —

1.0 ±0.18 1.7 ±0.20 19 ±2.3 7.67 ± 2.5 1.8 ±0.66

1.14 x-f 1.02 1.14x^1.01 1.14x^1.02 1.14x^1.02 1.14 x-f 1.02

SCxLM^^ SC X Vmaxl

5.8 ±2.4 0.16 ±0.054

1.41 x^ 1.05 1.41 x^l.04

Vmaxl/SC Vmaxbm/SC

0.60 ± 0.35 2.8 ±1.2

1.39x4-1.04 1.41 x-^ 1.05

—

0.014 ±0.0027

1.25x^1.04

— —

0.0016 ±0.0004 0.0025 ± 0.0003

L28 x-f 1.05 1.22x^1.02

—

0.78 ± 0.06

1.14x^1.01

^Scaled parameter =f(SC) where SC is the scaling coefficient (no scaling function means that no scaling is made). Units: weights in kg, flows in L/min, volumes in L, Vmax in mg benzene/min, Km in mg benzene, Kfin mg benzene/min; Ke in min-1, Fu in L/min. ^The transformation 1 -exp(SD) gives an approximate CV. ^The density of fat tissues is assumed to be 0.92. ^LM is the lean body mass (body mass, or weight, minus fat mass). ^This parameter was set at each Monte Carlo iteration so that the sum of the organ masses (plus skeleton, 17% ofLM) matched the imposed or sampled body weight.

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Health and Toxicology

1 llllp^

^ 30

40

50

I

60

I

70

I

80

90

Fraction Benzene Metabolized Per Day (%) Figure 5. Estimated population distribution of the fraction of benzene metabolized by humans. This fraction is independent of the exposure level (at least up to 10 ppm exposure). The histogram is based on 5,000 Markov chain Monte Carlo runs and represents both uncertainty and variability.

assessment can be simulated with such a model. Further advances on these questions will be made as data on metabolism in humans become available, particularly in the bone marrow. New biomarkers of exposure and effect are being developed and should help get insight on benzene leukemogenesis. Analysis of such biomarker data is an integral part of toxicokinetics. REFERENCES 1. Cooper, K. R. and Snyder, R. "Benzene metabolism (toxicokinetics and the molecular aspects of benzene toxicity)," in Benzene Carcinogenicity, M. Aksoy (Ed.), Boca Raton, Florida: CRC Press, 1988, pp. 33-58. 2. Lee, S. D. et al. "Assessment of benzene health effects in ambient water," in Carcinogenicity and Toxicity of Benzene, M. A. Mehlman (Ed.), Princeton, N.J.: Princeton Scientific Publishers, 1986, pp. Chap. 8, 91-125. 3. Wallace, L. A. "Major sources of benzene exposures," Environmental Health Perspectives, Vol. 82, 1989, pp. 165-169. 4. International Agency for Research on Cancer (lARC) Some Industrial Chemicals and Dye stuffs. Vol. 29. Lyon: International Agency for Research on Cancer, 1982, pp. 93-148, 391-398. 5. International Agency for Research on Cancer (lARC) Overall Evaluations of Carcinogenicity: An Updating of I ARC Monographs Volumes 1 to 42, Vol.

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Suppl. 7. Lyon: International Agency for Research on Cancer, 1987, pp. 120-122. 6. Snyder, R. and Kocsis, J. J. "Current concepts of chronic benzene toxicity," CRC Critical Reviews in Toxicology, Vol. 3, 1975, pp. 265-288. 7. Snyder, R. et al. "The toxicology of benzene," Environmental Health Perspectives, Vol. 82, 1993, pp. 31-35. 8. Sato, A. "Toxicokinetics of benzene, toluene and xylenes," in Environmental Carcinogens—Methods of Analysis and Exposure Measurement—Volume 10: Benzene and Alkylated Benzenes, I ARC Scientific Publication No. 85, L. Fishbein and I. K. O'neill (Eds.), Lyon: lARC, 1988, pp. 47-64. 9. Docter, H. J. and Zielhuis, R. L. "Phenol excretion as a measure of benzene exposure," A/2^a/5 of Occupational Hygiene, Vol. 10, 1967, pp. 317-326. 10. Pekari, K. et al. "Biological monitoring of occupational exposure to low levels of benzene," Scandinavian Journal of Work Environment and Health, Vol. 18, 1992, pp. 317-322. 11. Fiserova-Bergerova, V. et al. "Effects of biosolubility on pulmonary uptake and disposition of gases and vapors of lipophilic chemicals," Drug Metabolism Reviews, Vol. 15, 1984, pp. 1,033-1,070. 12. Srbova, J. et al. "Absorption and elimination of inhaled benzene in man," Archives of Industrial Hygiene and Occupational Medicine, Vol. 2, 1950, pp. 1-8. 13. Brugnone, F. et al. "Benzene in the blood and breath of normal people and occupationally exposed workers," American Journal of Industrial Medicine, Vol. 16, 1989, pp. 385-399. 14. Bois, F.Y. et al. "Population toxicokinetics of benzene," Environmental Health Perspectives, Proceedings of the "Benzene 95" Conference, Rutgers University, June 1995, in press. 15. Travis, C. C. and Bowers, J. C. "Protein binding of benzene under ambient exposure conditions," Toxicology and Industrial Health, Vol. 5, 1989, p. 1,989. 16. Sato, A. et al. "Pharmacokinetics of benzene and toluene," Internationales Archiv fur Arbeitsmedicine, Vol. 33, 1974, pp. 169-182. 17. Berlin, M. et al. "Breath concentration as an index of the health risk from benzene," Scandinavian Journal of Work and Environmental Health, Vol. 6, 1980, pp. 104-111. 18. Nomiyama, K. and Nomiyama, H. "Respiratory elimination of organic solvents in man," Internationales Archiv fur Arbeitsmedicine, Vol. 32, 1974, pp. 85-91. 19. Sherwood, R. J. "Benzene: the interpretation of monitoring results," Annals of Occupational Hygiene, Vol. 15, 1972, pp. 409-421. 20. Watanabe, K. et al. "Benzene toxicokinetics in humans—Bone marrow exposure to metabolites," Occupational and Environmental Medicine, Vol. 51, 1994, pp. 414-420.

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21. Sato, A. et al. "Kinetic studies on sex difference in susceptibility to chronic benzene intoxication—with special reference to body fat content," British Journal of Industrial Medicine, Vol. 32, 1975, pp. 321-328. 22. Sato, A. and Nakajima, T. "Dose-dependent metabolic interaction between benzene and toluene in vivo and in vitro,'' Toxicology and Applied Pharmacology, Vol. 48, 1979, pp. 249-256. 23. Subrahmanyam, V. V. et al. "Potential role of free radicals in benzeneinduced myelotoxicity and leukemia," Free Radical Biology and Medicine, Vol. 11, 1991, pp. 495-515. 24. Van Sittert, N. J. et al. "Application of the urinary S-phenylmercapturic acid test as a biomarker for low levels of exposure to benzene in industry," British Journal of Industrial Medicine, Vol. 50, 1993, pp. 460^69. 25. Hunter, C. G. and Blair, D. "Benzene—Pharmacokinetic studies in man," Annals of Occupational Hygiene, Vol. 15, 1972, pp. 193-201. 26. Woodruff, T. et al. "Design and analysis of a model of benzene toxicokinetics in mammals," in Proceedings of the IIth Annual International Conference of the IEEE Engineering in Medicine and Biology Society, Part 1/6. IEEE Publication No. 89CH2770-6, Y. Kim and F.A. Spelman (Eds.), NewYork: IEEE, 1989, pp. 254-255. 27. Travis, C. C. et al. "Pharmacokinetics of benzene," Toxicology and Applied Pharmacology, Vol. 102, 1990, pp. 400-420. 28. Medinsky, M. A. et al. "A toxicokinetic model for simulation of benzene metabolism," Experimental Pathology, Vol. 37, 1989, pp. 150-154. 29. Spear, R. C. et al. "Modeling benzene pharmacokinetics across three sets of animal data: parametric sensitivity and risk implications," Risk Analysis, Vol. 11, 1991, pp. 641-654. 30. Woodruff, T. et al. "Structure and parametrization of toxicokinetic models: their impact on model predictions," Risk Analysis, Vol. 12, 1992, pp. 189-201. 31. Nomiyama, K. and Nomiyama, H. "Respiratory retention, uptake and excretion of organic solvents in man," Internationales Archiv fur Arbeitsmedicine, Vol. 32, 1974, pp. 75-83. 32. Wakefield, J. C. et al. "Bayesian analysis of linear and non-linear population models using the Gibbs sampler," Applied Statistics—Journal of the Royal Statistical Society Series C, Vol. 43, 1994, pp. 201-221. 33. Racine-Poon, A. and Smith, A.F. "Population models," in Statistical Methodology in the Pharmaceutical Sciences, D.A. Berry (Ed.), New York: Marcel Dekker, Inc., 1990, pp. 139-162. 34. Wakefield, J. C. "The Bayesian analysis of population pharmacokinetic models," Journal of the American Statistical Society, 1994, pp. under revision. 35. Bois, F. Y. et al. "Population toxicokinetics of tetrachloroethylene," Archives of Toxicology, in press.

CHAPTER 11 FLUOROMETRY OF CARCINOGENIC POLYCYCLIC AROMATIC HYDROCARBONS IN BIOLOGICAL SYSTEMS Kuang-pang Li* and Ping Chiang Department of Chemistry, University of Massachusetts Lowell, Lowell MA 01854 Ruixia Song Technical Physics Department, Beijing University, People's Republic of China CONTENTS INTRODUCTION, 219 FUNDAMENTALS, 220 Nomenclature, 220 Transport and Metabolism, 221 Cytochrome P450, 224 MetabolitiesofBaP, 228 FLUORESCENCE OBSERVATIONS, 229 PAHs in Liposomes, 230 PAHs in Microsomes, 232 PAH in Living Cells, 234 The Rate Model, 235 CONCLUSION, 241 REFERENCES, 242 INTRODUCTION According to American Cancer Society, 1.1 million Americans were diagnosed with cancer (except for skin cancer) and 514,000 died from cancer in 1991 [1]. In 1992, about 146,000 Americans died of lung cancer alone. Ninety percent of these deaths were caused by cigarette smoking [2]. Smoking is responsible for about 30% of all cancer deaths in the United States, more than 155,000 each year [2]. Many researchers believe that deaths are caused by exposure to the incomplete combustion products in cigarette smoke, such as the polycyclic aromatic hydrocarbons (PAHs). PAHs belong to a wide class of fused-ring aromatics. They are found not only in the cigarette smoke but also in air, soil, water, and in the food chain, and have been considered as major environmental pollutants. Some of them, typified by benzo(a)pyrene (BaP), 3-methylcholanthrene (MC), and others, have been shown to induce tumor in animals and have also been implicated in human carcinogenesis. As a matter of fact, BaP was one of the first carcinogens identified. Partly because of '• To whom all correspondence should be addressed. 219

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this historical reason and partly because of its relatively high concentration in the environment—estimated at 2,000 tons of BaP per year being introduced into the air over the U.S. [3, 4]—investigations of the mode of action and metabolism of BaP have become almost a tradition. Use of BaP as a model carcinogen for environmental control purposes, on the other hand, has the following additional advantages: (1) Its invariable formation in the combustion of organic matter may serve as a useful indicator for industrial pollution. (2) Its wide spread provides useful information about its environmental and occupational carcinogenesis in man. (3) Its ready detection and quantitization by its fluorescence often makes the use of radioactive tracers not necessary in its pharmacological investigation. The number of papers referring BaP published each year has grown exponentially [5] in recent years, and most of them are in the biomedical field. It is, therefore, not the intention of this chapter to review past work in an exhaustive manner, nor to compile a large body of facts, figures, and references. Instead, we would like to emphasize the usefulness of fluorescence in the study of PAH carcinogenesis. It is hoped that, in conjunction with other observations, a comprehensive theory on the mechanism of PAH carcinogenesis may eventually be established. Some early findings about PAH activation will be first summarized to provide essential background knowledge for better comprehensibility. Observations of PAH fluorescence undertaken in various biological systems will be presented, and a kinetic model for BaP metabolism in the early stages will be proposed. FUNDAMENTALS Nomenclature BaP is a planar molecule with five benzene rings fused together as shown in the following structural formula:

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The naming and numbering are according to the rules of the International Union of Pure and Applied Chemistry (lUPAC) and are now commonly accepted. Usage of other nomenclature and numbering systems, such as benzo[a]pyrene, benzo[def]chrysene, 1,2-benzpyrene, 3,4-benzpyrene, etc., is therefore not recommended. The 4,5-bond (sometimes also the 11,12-bond) and the ClO-to-Cll area have been referred to as the "K-region" and the "bay region" of BaP, respectively. These terms are also avoided in this report. An extended discussion about the nomenclature of PAHs can be found elsewhere [6, 7]. Transport and Metabolism Ever since the carcinogenicity of BaP was recognized, its fate in animals has been widely investigated. Early studies [8, 9, 10] found that BaP transports through the body in the blood and lymph vessels when it is hypodermically introduced. Because BaP is very hydrophobic, this indicates that it must be carried by some components in the blood such as albumin [8] or other compounds [9, 10]. When intragastrically administered in rats, BaP is rapidly absorbed from the intestines. Four hours after administration, about 20% of the hydrocarbon can be found in the lymph glands, and the rest is presumably taken up directly into the bloodstream [11]. When BaP is inhaled either as finely divided powder or associated with particulates, it can be absorbed directly into the lung tissue or indirectly by the intestine and eventually distributed to all parts of the body [12]. When BaP reaches a cell, it is found first in the hydrophobic layer of the cellular membrane and later in the mitochondria and in lipid vacuoles within the cell [13]. Since foreign substances such as BaP can interfere with the normal functions of the membranes, the cell will evolve a system to convert them into hydrophilic products for easy excretion. This metabolism system is associated mainly with the endoplasmic reticulum in the cell. Most metabolites—e.g., phenols, ^ra^i^-dihydrodiols, and glutathione conjugates—are non-carcinogenic. So metabolism has been considered to be primarily a detoxication mechanism. However, some of the oxygenated metabolites, instead of being excreted, may covalently bind to the informational molecules of the cell—e.g., DNAs, RNAs, or proteins—leading to heritable modifications of the DNA genome, thus transforming a normal cell into a cancerous cell. These chemically reactive metabolites, referred to as the ultimate carcinogens, are believed to be electrophilic [14]. Their formation is under enzymatic control and is highly dependent on the structure of the parent compound and on the species, the strain, and certain environmental factors such as enzyme inducers or inhibitors. In most cases the induced changes would be expected to occur in the nuclear DNA, but mutations of mitochondrial DNA could also be observed [14]. Normally, carcinogens are classified into several types. The "direct-acting carcinogens," mostly synthetic compounds developed for research or antitumor drugs, do not require the participation of enzymes from the host organism to generate the key reactive intermediate, the ultimate carcinogen. Because of their high reactivity.

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they do not generally persist in the environment. On the other hand, chemicals that are active only after metabolic conversion by the host are labeled as "procarcinogens." These include most of the known chemical carcinogens existing in the environment. They impose the greatest potential cancer threat to man. The precursors from which carcinogens can be synthesized by the host may also be included in this class. Other agents such as croton oil exhibit little if any carcinogenicity themselves, but can significantly augment the effect of either direct-acting carcinogens or procarcinogens. These are referred to as "cocarcinogens." Tobacco tar and tobacco smoke contain many such promoting agents. Based on the above classification, BaP can be considered a procarcinogen. However, this was not realized until the late 1960s and early 1970s when studies showed that BaP could not mutate cells in culture and could not react well with molecular targets but could combine with DNA to a significant extent in the presence of a microsomal oxidative system [15, 16]. Studies also showed that benzoic hydrocarbons could undergo epoxidation with formation of arene oxides [17] and that many synthetic epoxides of certain PAHs, except BaP-4,5-oxide, were much more mutagenic and carcinogenic than their parent hydrocarbons [18]. These facts finally led to the disproof of the then very well known "K-region" theory of Pullman and others [19, 20], who hypothesized that metabolitic conversion of BaP into the 4,5-oxide, which then alkylated DNA in the cell, was the principle reaction responsible for the carcinogenicity of BaP. Later findings seemed to favor the metabolite BaP-7,8-diol9,10-epoxide to be the most likely ultimate carcinogen [21-23]. Mixed function oxidases

Epoxide hydrase

^>d0

OH) 0 PARENT HYDROCARBON

BENZO(A)PYRENE -7,8-EPOXIDE

BENZO(A)PYRENE

BENZO(A)PYRENE -7.8-DIOL

Mixed function oxidases

BENZO(A)PYRENE 7.8-DIOL-9,10-EPOXIDE ( ULTIMATE CARCINOGEN )

ULTIMATE CARCINOGEN BOUND TO GUANINE IN DNA

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The main rout of metbolitic activation of BaP and its site of binding to DNA is thought to be proceeded in the following manner [24]: The polycyclic compound is activated initially by a complex of enzymes associated with intracellular membranes, the cytochrome P450-associated mixed-function oxidases (MFO) also known as aryl hydrocarbon hydroxylase (AHH). The 7,8position double bond is opened to form an epoxide. The epoxide is a substrate for a number of enzymatic and non-enzymatic reactions. The important one for carcinogenicity involves soluble enzymes called epoxide hydrolases. The epoxide is converted into 7,8-diol, which is a good substrate for a second epoxidation of the 9,10position. The epoxide then binds to the 2-amino group of guanine in DNA, which is the major adduct found in BaP-induced carcinomas. The 7,8-oxide is very reactive and cannot be isolated from the metaboUte mixture of BaP. Its existence as an intermediate in BaP metabolism can be recognized only from its hydrolysis products. The two asymmetric carbon atoms in the epoxide give rise to two stereoisomers, namely the 7S,8R-oxide and the 7R,8S-oxide. When the synthetic racemic epoxide is hydrolyzed by incubation with rat liver microsomes, the 7S,8R-isomer will be converted stereospecifically to the 7S,8S-dihydrodiol, and the 7R,8S-oxide to the 7R,8R-dihydrodiol. The 7S-dihydrodiol appears to be the predominant product during the early stage of metabolism [25-27]. The dihydrodiol formed by metabolism of BaP is found to be purely 7R [5]. The 7,8-oxide intermediate must, therefore, be the pure 7R-isomer. The diol-epoxide, often referred to as benzo[a]pyrene-7,8-dihydrodiol-9,10oxide (BaPDE), has four asymmetric carbon atoms and can form eight stereoisomers.The four isomers with hydroxyl groups cis- to each other have not been observed as BaP metabolites. The four trans- isomer can have the epoxide ring above or below the molecular plane. They are informally named as 7R-anti-BdiF7,8-dihydrodiol-9,10-epoxide (or IR-anti-BsiFDE or (+)awr/BaPDE), IS-anti-BaF7,8-dihyroldiol-9,10-epoxide (or IS-anti-BaPDE, or (-)-anti-BsiFDE), IR-syn-BaF7,8-dihydroldiol-9,10-epoxide (or IR-syn-BaFDE, or (-)-^jn-BaPDE), and 7S-^y«-BaP-7,8-dihydroldiol-9,10-epoxide (or IS-syn-BaFDE, or (+)-syn-BaFDE), respectively. The prefixes (+) and (-) specify the rotation of polarized light to the right and to the left, respectively. The prefixes anti- and syn- indicate that the 7and 9- substituents are lying on opposite sides or on the same side of the molecular plane. Both mutagenicity tests (Ames test using tester strains of Salmonella typhimurium) and carcinogenicity tests (injection into newborn mice) show that the (+)-syn-enantiomer is the most potent mutagen and carcinogen, about 40-fold more active than the parent compound [28]. Other PAHs, e.g., methyl-cholanthrene, show similar results. These findings lead Jerina and Daly [29] to postulate the socalled "bay region theory" of PAH carcinogenesis. The hypothesis states that, for a compound to be highly mutagenic and carcinogenic, diol epoxides must be generated in the bay region formed by the polycyclic structures of the molecules. This hypothesis has been shown to be generally true. However, some carcinogenic hydrocarbons do not possess a bay region, so different structures will be required for their carcinogenicity.

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In spite of all the studies on metabolic activation of PAHs, there is still no formal proof that the above-mentioned reaction products detected in cells exposed to these agents are the ones actually involved in carcinogenesis. Besides, the 7,8-epoxide is not the only nor the most prominent metabolite of BaP. There are many other intermediates and products. Some may be in trace amounts and some may not have been detected. Yet they could be crucial. Because carcinogenesis is a multistage process, multiple actions of a carcinogen or actions of multiple carcinogens may be responsible for the production of a clinically detectable malignant neoplasm. Cytochrome P450 From the above discussion, BaP metabolism can be envisaged as proceeding in two phases, the conversion to oxygenated derivatives (epoxides, phenols, diols, diol-epoxides, tertrols, quinones, etc.) and the conjugation with sulfate, glucuronic acid or glutathione to give sufficiently hydrophilic products for easy excretion. The first phase of metabolism involves a mixed function oxidase comprised primarily of the membrane-bound cytochrome P450 and the NADPH-cytochrome P450 reductase. This is the primary step determining whether the substrate is being detoxicated or being activated to an intermediate, eventually leading to the ultimate carcinogen. The reductase is a flavoprotein consisting of a peptide bound to one molecule of flavin mononucleotide (FMN) and one molecule of flavin adenine dinucleotide (FAD). Its function is to transfer the two electrons needed for the oxygenation of the PAH molecule symbolized as RH in the following equation [30, 31]: RH + O2 + 2e- + 2H+ -^ ROH + H2O

(1)

These two electrons are introduced in sequential, one-electron steps [32, 33]. The electrons are accepted from NADPH, which is oxidized to NADP"^, by the FAD portion of the reductase and later transferred to P450 by the FMN [34, 35]. The pure reductase prepared by proteolytic cleavage from the endoplasmic reticulum shows no reduction activity toward P450 [36]. It is active only in the presence of surfactants such as detergents or phospholipids [37]. Topological studies show that the reductase (Mr ~ 78000) is anchored to the membrane via a small hydrophobic segment (Mr ~ 6000-10000) on the N-terminal portion of the protein [38, 39], whereas the hydrophilic portion, containing one molecule of FMN and FAD, protrudes outward from the membrane. Without the hydrophobic portion, the enzyme cannot reduce P450. Unlike the reductase, cytochrome P450 has many different forms (isozymes) [40, 41], depending upon the strains, the sources, and the method of induction. As purification techniques have greatly improved in recent years, new isozymes have been rapidly added to the list. Many have been cloned and amino acid sequence determined [42-50]. They are different proteins coded by different genes [5]. The capability of the mixed-function oxidase to metabolize a wide variety of endoge-

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nous substrates and many xenobiotics may be attributed to the multiplicity and inductibility of the P450 isozyme family. The active side of P450 is believed to be the iron-containing protoporphyrin IX prosthetic group:

When it is reduced with carbon monoxide, the enzyme exhibits an absorption maximum at 450 nm. This is where the trivial name P450 is derived. Because many of the individual P450s catalyze multiple reactions, the usual method of naming enzymes is inadequate for this group of heme proteins, and a systematic nomenclature has been devised based on structural homology [51, 52]. The system is still evolving as more P450 proteins are characterized. In general, it may be described as follows. Those P450 proteins with >40% sequence identity are included in the same family designated by an Arabic number, and those with greater than 55% identity are then included in the same subfamily designated by a capital letter. The individual genes are arbitrarily assigned numbers. For example, the major phenobarbital-inducible cytochrome in rabbit liver microsomes, originally called P450LM2, or form 2, is assigned to family 2 and subfamily B, and the gene and the enzyme are designated CYP2B4 and CYP2B4, respectively. The enzyme may also be called P450 2B4. The main advantage of the unified nomenclature is that structurally identical or highly similar P450s are easily recognizable, regardless of the source (species, tissue, or organelle), the inducer, or the catalytic activity examined. The microsomal P450s have not been crystallized. Therefore, knowledge on the active-site structure of these P450s can be obtained only indirectly, for example, by investigation of the similarities or dissimilarities in the amino acid sequences and effects of mutations, or by homology-mode building studies based on the crystal structure of P450 101 (a bacterial cytochrome P450 originally designated as cytochrome P450cam)- The existing knowledge on the membrane topology of these P450s, their possible role in the interaction with redox partners and their involvement in the orientation of substrates will be briefly summarized. Several proposed active-site models of individual cytochrome P450 will be presented.

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P450 isozymes from different sources or methods of induction are highly different in molecular structures. However, comparison of their primary amino acid sequences with that of P450 101 indicates there are two highly conserved regions, one in the heme binding site and the other at the central region of helix I, the oxygen binding site, which presumably constitutes part of the substrate binding site [53-55]. In all P450s, the fifth, axial heme ligand is believed to be a cysteinyl residue (alignment position 564 [54]). The Cys residue itself is part of a polypeptide chain that contains some other highly conserved and invariant residues such as Phe-557, Gly-560, and Gly-566. The three-dimensional structure surrounding this heme ligand Cys appears to be very similar in all P450s and may be related to the critical role of the Fe-S bond in the P450 catalytic mechanism. Moreover, the residue Arg at position 562 has been shown to interact with the heme proportionate in a manner similar to His-355 in P450 101. Hydrophobic residues of P450 1A2 such as Phe449, Leu-451, Gly-452, Ile-457, and Gly-458 located close to the axial thiolate ligand, on the other hand, are likely to hold the heme at the active site [56]. The central region between Gly-248 and Thr-252 of helix I in the P450 101 is distorted and widened to provide a pocket for molecular oxygen. The corresponding conserved domain around the Thr at alignment position 400 in the membranebound P450s also indicates a similar oxygen-binding pocket for all P450s. Some of the amino acid residues in this region, such as the Asn-391 to Phe-406 of P450 1A2, may constitute part of the substrate binding site and may influence the binding and orientation of the substrates [57]. The conservation of the three-dimensional structure around the prosthetic heme group and the oxygen-binding pocket in all P450s indicates similar requirements for oxygen binding and cleavage of the O-O bond. Substrate selectivity among P450 isozymes, therefore, must be attributed to the differences in architecture and nature of the substrate binding site. As a matter of fact, it has been demonstrated that substrates of the 3-MC-inducible cytochrome P450 (P450 1A1/1A2) are essentially planar molecules with large molecular dimensions, characterized by a large area/depth (A/D) ratio. Those of PB-inducible isozymes (P450 2B1/2B2) are rather bulky, nonplanar molecules, characterized by small A/D ratios. Accordingly, the binding site of P450 1 Al/1 A2 may contain aromatic amino acids that form a planar pocket to complement the aromatic rings of the substrate, whereas that of P450 2B1/2B2 should accommodate more hydrophilic amino acids capable of forming a hydrogen bond with the carbonyl and/or amine function of the substrates. Metabolism of polycyclic aromatic hydrocarbons is seen to occur more favorably by the 3MC-inducible isozymes, such as P450 lAl, than the PB-inducible ones. Membrane topology studies of the membrane-bound P450s have suggested that the P450s are anchored to the membrane by one [58-60] or two [53, 54, 59] transmembrane segments at the N-terminus. The helices C, D, G, I, K, and L of P450 101 are conserved in all membrane-bound P450s. The helices A, B, B', E, F, and H

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are not conserved, or are conserved only in some P450 families. Several B-structures of P450 101 are also present in the membrane-bound P450s [59]. The heme-containing portion of the microsomal P450s is exposed to the cytosolic side of the membrane. The prosthetic heme is suggested to be oriented with the substrate binding site facing the membrane, tilted with an angle different for different isozymes [61]. Two substrate-access channels are proposed to exist in P450s for their versatile capabilities of metabolizing hydrophobic and hydrophilic substrates. The hydrophobic substrates are thought to enter the active-site pocket from the membrane side, whereas hydrophilic substrates gain access from the cytosolic side [59]. Figure 4 summarizes the current knowledge on the three-dimensional topology of membrane-bound P450s in an illustrative way. The residues at the alignment positions 168, 392-398, and 448-468 constitute part of the substrate binding site, whereas positively charged residues at 137, 456, and 494 are involved in the electron transfer to the heme.

Q) Interaction with redox partners @ Substrate binding site

dU) Helix ^ ^ ^ P-sheet

Figure 4. Model for membrane-bound cytochrome P450. Helices C, D, G, I, J, K, and L, and B-sheets B3/B4 of P450 101 are conserved in the membrane-bound P450s.

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Topology of the active site of the membrane-bound cytochrome P450, e.g., cytochrome P450 lAl, for PAH metaboUsm can be illustrated with the following steric model proposed on the basis of the elegant work on the stereo-selective metabolism of BaP by Jerina, Levin, and co-workers [27, 62-65]. The model consists of a hydrophobic cleft asymmetrically disposed to the heme iron atom. BaP is depicted lying over a plane parallel to the porphyrin plane. The asymmetrical protein perimeter permits only one of two possible faces of the substrate to be oriented toward the active oxygen species at the iron in a fashion allowing epoxidation of the C9-C10 double bond:

To take into account the broad substrate specificity of the enzyme and the wide variety of positional isomeric metabolites, the binding pocket needs to be enlarged substantially to accommodate reorientation of the substrate molecule, or the angle of oxygen addition needs to be flexible. Topological information of the active site not only enhances our understanding of the mode of action of the enzyme in PAH carcinogenesis at the molecular level, but it also facilitates developments in the design of specific P450 inhibitors or drugs for therapeutic uses. Benefitted from the evolution of biotechnological methods for producing significant amounts of specific cytochrome P450 using recombinant systems, commercial applications of these P450 isozymes to pollution control, modification of undesirable food products, and synthesis of new pharmaceuticals have become very promising. The versatility of P450s for the fast removal of PAH from water samples has been tested, and efforts of using P450 for detoxication of industrial process waste eaters are being undertaken [66]. Metabolites of BaP Much of the fundamental work on BaP metabolism has used the hepatic microsomal fraction of rat treated with 3-methylcholanthrene before sacrifice. Other systems may have different metabolite profiles. Differences are usually quantitative, not qualitative.

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When BaP metabolites in rat or human liver microsomes are analyzed chromatographically with a methanol/water gradient on an ODS-permaphase column, dihydrodiols (9,10-, 4,5- and 7,8-, etc.) are eluted first, followed by the quinones (1,6-, 3,6-, and 6,12-, etc.) and phenols (9-OH, and 3-OH, etc.). The most prominent metabolite appears to be the 3-hydroxy-BaP [5, 67]. This metabolite fluoresces strongly (excited at 435 nm and emits at 522 and 475 nm) and has conventionally been used as an assay for the aryl hydrocarbon hydroxylase enzyme (AHH assay). It is thought to arise by rearrangement of the 2,3-oxide, not by free-radical hydroxylation [68]. When 3-hydroxy-BaP is subjected to further metabolism, the principal non-polar metabolite is BaP-3,6-dione [69-72]. Other metabolites such as 3-hydroxy-BaP [73], 3,5-dihydroxy-BaP [73], and 3,9-dihydroxy-BaP [74,75] are also produced. The 4,5-, 7,8-, and 9,10- dihydrodiols formed by metabolism are purely transand all have the R,R-stereochemistry. This is not a coincidence, but a consequence of the geometry of the active sites as mentioned previously. In view of the toxicity of these compounds, particularly the 7,8-dihydrodiol, it would be interesting to know what proportion of them are converted to the diol-epoxides. According to Keller et al. [76], most of the 7,8-diol is converted to diol-epoxide unless the BaP concentration is sufficiently high to saturate the oxygenase system, or BaP quinones accumulate and inhibit the enzyme. Under these conditions, the diol may be conjugated before it can be oxidized further. FLUORESCENCE OBSERVATIONS It is evident from the above summary that the most crucial step in PAH carcinogenesis/detoxication is the initial oxygenation. Addition of the oxygen atom at the critical position(s), e.g., 7- and 8- in BaP, would lead to metabolic activation of the PAH molecule, whereas at other positions it results in detoxication. In other words, the route to activation or detoxication is essentially determined by the configuration of the activated complex, P450-O-PAH. Because the active site, including the oxygen binding pocket, is highly conserved in all P450s for similar catalytic requirements, the isomeric differences in the activated complex must be attributed to the PAH molecular orientations in the substrate binding site. Knowledge and manipulation of the orientations will be of great value in the prevention of PAH carcinogenesis and in the environmental control of PAH contamination. Reports in this area have been very scarce. The following discussion will be based primarily on the preliminary attempt in the authors' laboratory. Because of its sensitivity to molecular structural and microenvironmental variations, fluorescence has been extensively used for molecular probing, including intramolecular distance measurements by energy transfer [77, 78], topographic determination by dynamic quenching [79], binding site location by lifetime measurements [80], just to name a few. Its high selectivity, fast response, and capability of detecting extremely low concentrations make it the unquestionable method of

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choice for monitoring the kinetics or steady-state of biochemical events in biological systems. Recent developments of instrumentation in ultrafast and site-selection spectroscopies open a doorway to the mechanistic studies of reactions in the subpicosecond range. These luminescence techniques will be very useful in deconvoluting the configurations of the activated complexes in PAH carcinogenesis. PAH metabolism is a very complicated process. Even with the simplest active reconstituted system composed of the P450, the reductase, and a phospholipid, the retrieval of the configurational information is still insurmountably difficult. Therefore, it is advisable to begin with a single component system, the phosopholipid bilayer, and work up to the complicated cell culture system. PAHs in Liposomes Phospholipids are amphiphatic molecules having a polar hydrophilic head group and two long hydrophobic hydrocarbon chains. When they are carefully dispersed in water, the fatty acid chains should be sequestered to the maximum extent feasible from contact with water, while the head group should be in direct contact with the aqueous phase in order to attain the lowest free energy state. As a result, bilayers or micellar aggregations would be formed. If conditions are right, uniformed spherical vesicles (tiny water chambers each encompassed by a single shell of bilayer membrane) can be prepared. The thickness of the bilayer with the fatty chains in a noncrystalline state is about 4.0-4.5 nm [81], and the diameter can be as small as 25.0 nm [82, 83]. Such phospholipid dispersions are often referred to as liposomes. PAHs (e.g., BaP, BeP and pyrene) are extremely hydrophobic. They form crystals and microcrystals when they are dispersed by sonication in water. The large crystals eventually settle out, whereas the invisible microcrystals stay suspended in solution. This can be demonstrated with the BaP emission spectra shown in Figure 6. Spectrum D and the insert spectrum are the emission of BaP (in different sensitivity scales) in low methanol/water solution, whereas spectrum A is that of BaP in pure methanol. Because BaP is completely dissolved in the methanol solution, the emission peaks at 405, 430, and 450 nm may be designated to the "dissolved" (or monomeric) BaP molecules. Judged by the intensities of these peaks, the concentration of the monomeric BaP in the low methanol/water solution is seen to be very insignificant. It is barely detectable in pure buffer solutions. The broad band at the longer wavelengths (480-590 nm) must be attributed to emission from the microcrystals. BaP molecules in these microcrystals are stacked very closely and orderly. An excited BaP molecule (BP*) may easily transfer its energy to a nearby groundstate BaP molecule (BP). They together emit as an entity designated as excimer (BP*BP). These bands will be referred to as "excimer bands" [84-89]. The excimer emission of BaP appears to be an overlap of two bands (the insert spectrum in Figure 6) whereas that of pyrene is bell-shaped [88]. BaP is an asymmetrical planar compound. It can be stacked either parallel or anti-parallel to each

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

231

8c5

440

490 Wavelength (nm)

540

590

Figure 6. Emission spectra of BaP. (A) BaP in pure methanol solution; (B) BaP in 40% Tri buffer/methanol; (C) BaP in 50% Tri buffer/methanol; (D) BaP in 60% Tri buffer/ methanol solution. Insert: same as spectrum D. The total amount of BaP is the same for all cases.

other in the microcrystalline state. Pyrene is symmetrical and can have only one excimeric arrangement. The emission band structure seems to reflect this difference in molecular orientations [87, 88]. Upon addition of liposomes to the BaP suspensions, it is seen that the excimer bands decrease and the monomer bands increase in magnitude with the increase of liposomal concentration [87]. In their study of particulate-facilitated transport of BaP, Lakowicz et al. [84, 85] indicated that binding of BaP microcrystals to the lipid vesicles was rapid and did not disrupt the vesicle integrity, and the rate of transport was not affected by the concentrations of either particulates or the vesicles. Li et al. [86, 90] monitored both BaP monomer emission at 405 nm and excimer emission at 490 nm and found the mass transfer to be a first-order process with a rate constant -0.36 hr~^ corresponding to an average half-life of 2.8 hr. Based on the hydrophobicity of the PAH molecules, one would expect that they would stay in the midplane of the bilayer, keeping a long distance from the aqueous phase. However, even in the presence of a large excess of liposomes, excimer BaP emission is still detectable [91, 92]. A similar phenomenon is observed in other PAHs, such as pyrene and BeP [87, 90]. Dynamic quenching studies using membrane-impermeable paramagnetic ion probes (e.g., Cu^"^, Mn^"^) clearly indicate two distinguishable sites of residence of BaP in the bilayer. One site is more easily

232

Health and Toxicology

accessible to the probing ion than the other [87, 91]. Because dynamic quenching is preferential, the more easily accessible site must be closer to the probing ion than the other site. That is, it must be close to the hydrophilic head group—e.g., in the vicinity of the glycerol backbone—for the ion to effectively quench the BaP fluorescence. The lateral movement of the BaP molecules residing in this site will be significantly restricted by the rigid structure of the carbon-carbon backbone. The BaP molecules cannot tumble as easily as those residing in the fatty acid chain region, i.e., the midplane of the bilayer. The restriction in molecular translation and tumbling greatly enhances the formation of excimers. The simplest liposomal system cannot oxidize PAHs, but its interaction with the PAHs in contact could be the most important pathway for the PAH molecules to enter our body. Transport of PAH from the site of entry to the target tissue sites is probably carried out by the plasma Hpoproteins. Smith and Doody [10] employed the excimer-to-monomer ratio, measured as the radiation intensity ratio (1520/1430)^ to study such transfer and found that the ratio was linearly dependent on BaP concentrations. Song and Li [87] used a similar technique to study the interactions of BaP and pyrene with hemoglobin. They found that the emission spectrum of BaP mixed with hemoglobin was quite different from that dispersed in buffer. The 490 nm band, which was the major excimer band in buffer, was now overshadowed by the 520 nm band. The spectrum gradually approached spectrum A in Figure 6 as its limit when aliquots of liposomes were added to the mixture. This seemed to indicate a weak binding to the heme and the preferential enhancement of the excimer emission at 520 nm would implicate certain steric selectivity. Measurements of the excimer-to-monomer ratios under O2 or CO2 saturation conditions showed a stronger Heme Fe-02-BaP binding than heme Fe-C02-BaP binding. Furthermore, the much slower decrease in the excimer-to-monomer ratio in the presence than in the absence of hemoglobin could be attributed to the dissociation of the heme-0BaP complex, not the mass transfer of BaP from microcrystals to the bilayer. PAHs in Microsomes Figure 7 illustrates the initial interaction of BaP with liver microsomes from maternal rats [91, 92]. The diluted liver microsome solution has little residual emission. Upon introduction of the microsome to the BaP dispersion, the monomer emission of BaP is rapidly enhanced. As reaction proceeds (Figure 8), the excimers emitting at 520 nm gradually dissipate whereas those emitting at 490 nm appear not to be affected. If the two different bands are emission from excimers with different configurations—i.e., parallel and anti-parallel oriented BP*BP—and if the access channel to the heme group of the P450 is capacious enough to allow only one BaP molecule to pass at a time, this would implicate a selective breakup of the excimers. The lifetimes of BaP in different environments have been measured by Li et al. [92] using a laboratory-constructed time-resolved fluorometer consisting of an N2 laser as the excitation source and a streak camera as the detector. The fluorescence

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

233

TOO

in

c >

395

445

495

Wavelength

545

595

(nm)

Figure 7. Emission spectra of BaP in rat liver microsomes, (a) Residual emission of microsomes (40 ML, protein content -25 mg/mL) in 2 mL-phosphate-buffered saline (PBS) solution; (b) BaP dispersed in PBS; and (c) BaP in microsome solution (40 [iL microsomes added to (b), spectrum taken at 30 s after mixing).

100

j^3

445

495

345

595

Wavelength (nm)

Figure 8. Microsomal residual emission corrected spectra of BaP. (a) 30 s after mixing; (b) 10 min after mixing; (c) 20 min after mixing; and (d) 30 min after mixing.

234

Health and Toxicology

decay curves were deconvoluted into multiple exponential terms, each specified with an independent half-life x. When BaP was dissolved in a methanol solution, only one half-life of 6.7 ns was observed, indicating one BaP species or one environment for BaP in the solution. In buffer suspensions, two half-lives of 8.6 and 11.1 ns were deconvoluted and could be assigned to the dissolved and microcrystalline BaP, respectively. The half-lives of BaP in liposomes were lipid-to-BaP ratio dependent. When the ratio was high, with apparently no microcrystals left suspending, the deconvoluted half-Hves (11.0 and 12.2 ns) were nearly identical. This could be attributed to the relatively fast exchange of BaP between the membrane midplane and the glycerol backbone. At low lipid-to-BaP ratios, the BaP in the membrane midplane could be easily distinguished from the microcrystalline BaP. Three half-lives—4.9, 10.3, and 10.7 ns—were observed. The last two half-lives could be assigned to BaP near the carbon backbone and in the microcrystals, respectively, because of their similarities in molecular orderliness. In the presence of microsomes, however, the BaP half-lives varied with reaction time, from x's ~ 3.1 and 10.9 ns at 1.5 min, to 8.0, 10.1, and 12.7 ns at 8.5 min and 10.7, 11.4, and 12.4 ns at 22 min after mixing. As the authors pointed out, these assignments were only speculative because of the non-optimized conditions of instrumentation. The implication of the experiment, however, was very interesting. If the differences in half-lives really reflect differences in the configuration of the P450-O-BaP activated complexes, with better monitoring devices and by the introduction of proper inhibitors it would be possible to metabolize BaP to non-toxic products only. Fast detoxication of PAH-containing industrial waste would be realistic as mass production of P450 becomes commercially available. PAH in Living Cells Many different culture systems have been used for studying cell transformation induced by chemical exposures. In some cases, near normal, diploid cells can be converted with reasonable efficiency into cells that can grow into invasive tumors if they are put back into a suitable host. Such transformed cells are referred to as tumorigenic, and the transformation process can be studied as a model for chemical carcinogenesis in vivo. BaP and other potent PAHs have been conclusively demonstrated to be capable of inducing transformation and eventually tumorigenic potential in epithelial cells such as skin keratinocytes, salivary gland duct cells, urinary bladder cells, etc., from some rodents, treated with the carcinogen in primary cultures [24]. The frequency of transformation depends on a variety of factors and on the species. Primary cultures of human and avian cells rarely transform spontaneously. The transformation is a multistage process and usually takes a relatively long period of time. However, sometimes just for a single short exposure, cells may emerge to grow as tumors in an appropriate animal host. This indicates the vital importance of the initial cell-carcinogen interaction in chemical carcinogenesis. Observations of the

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

235

initial contact of BaP and BeP with liver and kidney cells from different sources [90] will be summarized below, and a theoretical rate model [93] will be presented in the next section. Shown in Figure 9 are emission spectra of BaP measured about 30 s after mixing with different amounts of mouse liver (a), pig kidney (b), monkey kidney (c), and human cancer cell (d), respectively. In these experiments, an excess of BaP is intentionally employed so that the variations in the excimer bands can be clearly observed. Because measurements are performed shordy after mixing, insufficient time for BaP metabolism, spectral variations may, therefore, be attributed to BaP transfer from the microcrystalline state to the plasma membrane and to its distribution within the membrane. As in liposomes, the monomeric emission is enhanced, more significantly in the normal liver cells than in liver cancer cells or kidney cells. This may reflect different cross-membrane distribution for different types of cells. On the other hand, the decrease in excimer emission is essentially the same for all types of cells, indicating similar mass transfer of BaP from microcrystals to the cell membrane. When BaP is allowed to interact with the cells for a certain period of time, metabolism starts to take place (Figure 10). The excimer emission is seen to dissipate continuously as reaction proceeds, whereas the monomer emission decreases with different patterns for different types of cells. The invariant rate of mass transfer (rate constant k ~ 0.36 hr~^) with cell types indicates that metabolism does not occur direcdy to the microcrystalline BaP. Therefore, by monitoring the rate of change in the concentration of the membrane-bound BaP, i.e., variation in the monomer emission, the initial rate of metabolism could be estimated. Chiang et al. [93] estimated the rate to be fastest in mouse liver cells and slowest in pig kidney cells. The Rate Model Based on the results of the aforementioned observations, a theoretical rate model was proposed [93] for PAH metabolism in the initial stage, i.e., phase I metabolism. The essential features of the model can be depicted in the simplified sketch shown in Figure 11. The model is better applied to reconstituted systems and microsomal systems, but may be used as the first approximation for living cells. Let mi, m2, and m3 be the concentrations (or number densities) of BaP in the microcrystals, on the membrane surface, and in the membrane midplane, and the rate constants of the mass transfer, the intra-membrane transport, and the binding to the heme designated as k|, k2, k3, and k4 respectively. The initial and boundary conditions can be specified as follows: At time t = 0 t = oo,

m^ = mo (mo is the amount of BaP added) m2 = m3 = 0 mj = m2 = m3 = 0

236

Health and Toxicology

120

A

&

90

8

60

o

/^^ D

//^^

D

o

=3

E

30

0 390

,

440

490

540

590

Wavelength (nm)

180

-

A

///y^^''^

D

^

1 120 o

g

90

1

6d 30

- D

//v — ^

0 390

A

440

490 Wavelength (nm)

540

590

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

237

150

A

120

>.

v'^'"^ B

1 9"

A

/

>y^

y'''''!^--''''***'*^^^.

\

o

i

60

l-i

o 30 -jirvv ^"V'^A u •

390

^^^

440

490

540

590

Wavelength (nm)

Figure 9. Emission spectra of BaP measured at 30 s after mixing with increasing amounts of cells, (a) BaP in buffer with 0 |jL (A), 10 |jL (B), 20 pL (C), and 30 |JL (D) of mouse liver cells; (b) BaP in buffer with 0 |JL (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of pig kidney cells; (c) BaP in buffer with 0 ML (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of monkey kidney cells; (d) BaP in buffer with 0 pL (A), 10 |JL (B), 20 |JL (C), and 30 ML (D) of human cancer cells. Cell counts are approximately 100/|JL. The rate equations can be written as: drnj dt dni2

"dT dt

= -kimi

(2)

= kiiTii - (k2 + k4) m2 + k3m3

(3)

= k2m2 - k3m3

(4)

Solution of these equations yield: i^2 A -kt T^ bt f /C ,\sinat — ^ = Ae ^^+Be^^Jcosat+ —+ b kimo [ \B / a

(5)

s-b (s - b)^ + a^ -k(t-u)U^-ku^g^bu

•+ •

C/B + b

(s - b)^ + a '

/ C , \ sin au cos au + — + b IB

) a

.du

(6)

120

a

390

440

490

S90

540

Waveloopb (om)

Wavsloapb bn)

n

b

d

80 x

'z

60

H 9

40

8 20

0'

J

390

uo

490 Wwclcngtb (om)

540

590

I 390

uo

490

540

S90

Wavelength (nm)

Figure 10. Emission spectra of BaP measured at different incubation times in the presence of (a) mouse liver cells, (b) pig kidney cells, (c) monkey kidney cells, and (d) human liver cells. Incubation times: (A) 0 hr, (B) 1 hr, (C) 2 hr, and (D) 3 hr.

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons C^^FQI^ J]l'

239

heme group of P-450

Iic4

membrane

BaP

microcrystals BaP(m) m3 membrane

Figure 11. Simplified sketch of the theoretical model.

where:

hz^^_

A

(7)

kik2 + kik3 + kik4 - kj - k3k4

^'-^'

B

,

(8)

kik2 + kik3 + kik4 - kj - k3k4

Q^

^3 (^2 + k3 - ki) kik2 + kik3 + kik4 - kj^ ~ k3k4

^^^mn^

(10)

and: a2 = k3k4-b2

(11)

The rate constant k^ can be obtained from the rate of dissolution of BaP microcrystals [90] or that of BaP uptake by the membrane [86]. Rate constants k2 and k3 can be obtained, at least in principle, from kinetic measurements of BaP distribution in phospholipid bilayers, but have not been reported. Because of this, the rate constant of metabolism (or more accurately, the rate constant of association with P450) k4 can only be estimated indirectly.

240

Health and Toxicology

From the structure of the phospholipid bilayer, it is conceivable that k2 should have a larger value than k3 and both of them should be greater than, or at least comparable to, k4. This is because the inward diffusion of BaP is a transport process from an orderly environment, the glycerol backbone, to a much more random domain, the membrane midplane. It is also because the membrane midplane is more hydrophobic than the membrane surface, making the inward diffusion of BaP energetically more favorable than the reversed transport process. Based on the assumption that k4 is rate determining, one would expect that the midplane BaP concentration m3 would rise, pass through a maximum, and eventually die away as BaP microcrystals completely disappear. The incubation time needed for m3 to approach zero level, of course, depends on the value of k4. The larger k4 is, the shorter the incubation will be. A similation of the k4 effect on m3 is given in Figure 12 and temporal emission profiles of BaP in different cell cultures is given in Figure 13. Comparison of these profiles with the similated ones reveals that the mouse liver cells have the largest and pig kidney the smallest k4.

0.25

2: 0.15

0.05

Time (hour)

Figure 12. Effect of k4 on m^ of BaP is similated using Equation 5. The value of k4 used for similation is (A) 0.04 hr^; (B) 0.4 hr^; (C) 1.0 hr^ and (D) 5.0 hr\ respectively.

Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons

0.6

-

/

0.5

A

0.4

/

0.3

/

-

^ ^

^-^

•

1

B

/ A

c

"/

oJ

-

-0.1 -0.2

^

/

0.2 0.1

-

/

241

1

^^ „.

-v"^^^ ^ s ; ; ; ,

1 ,

J

^^

1 24

TIME (hour) Figure 13. Corrected temporal profiles of BaP monomer emission. BaP emission at zero incubation time was used as background for correction. BaP in (A) human liver cancer cells, (B) monkey kidney cells, (C) mouse liver cells, and (D) pig kidney cells.

CONCLUSION PAH carcinogenesis is a long, multiple-stage process. The crucial step leading to the ultimate carcinogen is conceived to be the metabolic activation of the hydrocarbon by the enzyme cytochrome P450 to form an arene oxide at a critical position, e.g., in the bay region. Oxygenation at other positions most likely gives rise to nontoxic metabolites.The substrate binding site of the enzyme is capacious enough to accommodate some molecular reorientations but restrictive enough to permit stereoselectivity. In other words, the heme-0-BaP complex could have only a few configurations. If these configurations were stable enough to permit characterization and differentiation, biochemical modifications to inhibit activation or to enhance detoxication could be designed. Many P450 isozymes have been expressed and cloned. Production of large quantities of isoforms with specific structures for commercial or industrial uses in environmental control is not out of reach. Analytical methods capable of monitoring and differentiating the configurations of the heme-substrate complexes can be very helpful in the construction and production of these specific isoforms. Among these methods, fluorescence, particularly the time-resolved technique, is probably the most appropriate one and should be further explored.

242

Health and Toxicology

REFERENCES 1. Cancer facts and figures. 1991, American Cancer Society. 2. Cancer facts and figures. 1992, American Cancer Society. 3. Particulate Polycyclic Organic Matter (1972): National Academy of Sciences, Washington, D.C. 4. Heidelberger, C. "Studies on the Mechanisms of Carcinogenesis by Polycyclic Aromatic Hydrocarbons and Their Derivatives" in Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism, and Carcinogensis, R.I. Freudenthal and P.W. Jones (Eds), Raven Press, New York, 1976, pp. 1-8. 5. Osborne, M. R. and Crosby, N. T. Benzopyrenes, London, Cambridge University Press, 1987. 6. Jacob, J. and Grimmer, G. lARC monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans 3, Lyon: International Agency for Research on Cancer, 1977, pp. 15-30. 7. Loening, K. L. and Merritt, J. E. "Some Aids for Naming PAH and their Heterocyclic Analogs," in Polycyclic Aromatic Hydrocarbons, M. Cooke and A.J. Dennis (Eds), Columbus, Ohio: Battelle Press, 1983, pp. 819-843. 8. Ma, J. K. H., Fu, P. P., and Luzzi, L. A. "Protein Binding of Benzo[a]anthracene and BaP," 7 P/zarm Sci 66, 1977, pp. 209-213. 9. McKenzie, M., McLemore, T., Rankin, P., Martin, R. P., Wray, N., Cantrell, E. and Busbee, D. "Human Plasma Component that Binds BaP," Cancer, 42, 1978, pp. 2,733-2,737. 10. Smith, L. C. and Doody, M. C. "Kinetecs of Benzo[a]pyrene transfer between Human Plasma Lipoproteins," in Polycyclic Aromatic Hydrocarbons: Chemical Analysis and Biological Fates, M. Cooke and A. J. Dennis (Eds), Columbus, Ohio: Battelle Press, 1981, pp. 615-624. 11. Rees, E.D., Mandelstrom, P., Lowry, J. Q., and Lipscomb, H. "A Study on the Mechanism of Intestinal Absorption of BaP," Biochem Biophys Acta, 225, 1971, pp. 96-107. 12. Mitchell, C. E. "Distribution and Retention of BaP in Rats After Inhalation," Toxicology, 28, 1982, pp. 65-73. 13. Wade, C. G., Baker, D. E., and Bartholomew, J. C. "Selective Fluorescence Quenching of BaP and a Mutagenic Diolepoxide Derivative in Mouse Cells," Biochemistry, 17, 1978. pp. 4,332^,337. 14. Miller, E. C , and Miller, J. A. "Biochemical Mechanisms of Chemical Carcinogenesis" in The Molecular Biology of Cancer, H. Busch (Eds.), New York: Academic Press, 1974, Chapter X, pp. 377^02. 15. Gelboin, H. V. "A Microsome-dependent binding of Benzo[a]pyrene to DNA," Cancer Res. 29, 1969, pp. 1,272-1,276. 16. Grover, P. L. and Sims, P. "Enzyme-catalysed Reaction of Polycyclic Hydrocarbons with Deoxyribonucleic Acid and Protein in vitro,'' Biochem 7 110, 1968, pp. 159-160.

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17. Daly, J. "Enzymatic Oxidation at Carbon," in Concepts in Chemical Pharmacology, Part II, B.B. Brodie and J.R. Gillette (Eds.), New York, Springer, 1971, pp. 285-311. 18. Heidelberger, C. "Current Trends in Chemical Carcinogenesis," Fed. Pro. 32, 1973, pp. 2,154-2,161. 19. Pullman, A. and Pullman, B. "Electronic Structure and Carcinogenic Activity of Aromatic Molecules: New Developments," Adv Cancer Res, 3, 1955, pp. 117-169. 20. Baird, W. M., Harvey, R. G., and Brookes, P. "Comparison of the Cellular DNA-bound Products of BaP with the Products Formed by Reaction of BaP-4,5- oxide with DNA," Cancer Res, 35, 1975, pp. 54-57. 21. Huberman, E., Sachs, L., Yang, S. K., and Gelboin, H. V. "Identification of Mutagenic Metabolites of Benzo(a)pyrene in Mammalian Cells," Proc Natl AcadSci U.S.A. 73, 1976, pp. 607ff. 22. Weinstein, I. B., Jeffrey, A. M., Jennette, K. W., Blobstein, S. H., Harvey, R.G., Harris, C , Antrup, H., Kassai, H., and Nakanishi, K. "Benzo(a)pyrene Diol Eposides as Intermediates in Nucleic Acid Binding in vitro and in vivo,'' Science, 193, 1976, pp. 592ff. 23. Jerina, D. M., Yagi, H., Hernandez, O., Dansette, P. M., Wood, A. W., Levin, W., Chang, R. L., Wislocki, P. G., and Conney, A. H. "Synthesis and Biological Activity of Potential Benzo(a)pyrene Metabolites," in Carcinogenesis—A Comprehensive Survey, Vol. 1, R. Fredenthal and P. W. Jones (Eds.), New York, Raven Press, 1976, pp. 91-113. 24. Wigley, C. "Chemical Carcinogenesis and Precancer," in Introduction to the Cellular and Molecular Biology of Cancer, L.M. Franks and N.M. Teich (Eds.), New York, Oxford University Press, 1986, pp. 131-153. 25. Yang, S. K., McCourt, D. W., Leutz, J. C , and Gelboin, H. V. "BaPDE: Mechanism of Enzymatic Formation and Optically Active Intermediates," Science, 196, 1977, pp. 1,199-1,201. 26. Yang, S. K., Roller, P. P., and Gelboin, H. V. "Enzymatic Mechanism of BaP Conversion to Phenols and Diols and an Improved HPLC Separation of BaP Den\a.iivcs,'' Biochemistry, 16, 1977, pp. 3,680-3,687. 27. Levin, W., Buening, M. K., Wood, A.W., Chang, R. L., Kedzierski, B., Thakker, D. R., Boyd, D. R., Gadaginamath, G. S., Armstrong, R. N., Yagi, H., Karle, J. M., Slaga, T. J., Jerina, D. M. and Conney, A. H. "An Enanatiomeric Interaction in the Metabolism and Tumorigenicity of (+) and (-) BaP-7,8-oxide," 7^/0/ Chem, 255, 1980, pp. 9,067-9,074. 28. Conney, A. H. "Induction of Microsomal Enzymes by Foreign Chemicals and Carcinogenesis by Polycyclic Aromatic Hydrocarbons," Cancer Res 42, 1982,pp.4,875ff. 29. Jerina D. M. and Daly, J. W. "Oxidation at Carbon," in Drug Metabolism— from Microbe to Man. D. V. Parke D. V. and Smith, R. C. (Eds), London, Taylor and Francis, 1977, pp. 13-32.

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30. Mason, H. S., Fowlks, W. L, and Peterson, E. "Oxygen Transfer and Electron Transport by the Pnenolase Complex," J Am Chem Soc, 1955, 77, pp. 2,914-2,915. 31. Haysishi, O., Katagiri, M., and Rothberg, S. "Mechanism of the Pyrocatechase Reaction," J Am Chem Soc, 1955, 77, pp. 5,450-5,451. 32. Peterson, J. A., White, R. E., Yasukochi, Y., Coomes, M. L., O'Keefe, D. H., Ebel, R. E., Masters, B. S. S., Ballou, D. P., and Coon, M. J. "Evidence that Purified Cytochrome P450LM is a One Electron Acceptor," J Biol Chem 1976, 251, pp. 4,010-4,016. 33. Peterson, J. A. "Camphor Binding by Pseudomonas putida Cytochrome P450," Arch Biochem Biophys 19071, 144, pp. 678-693. 34. Vermilion, J. L. and Coon, M. J. "Identifaction of the High and Low Potential Flavins of Liver Microsomal NADPH-Cytochrome P450 Reductase," J Biol Chem 1978, 253, pp. 8,812-8,819. 35. Peterson, J. A. and Prough, R. A. "Cytochrome P450 Reductase and Cytochrome b5 in Cytochrome P450 Catalysis," in Cytochrome P450: Structure, Mechanism, and Biochemistry, P. R. Ortiz de Montellano (Eds.), New York, Plenum Press, 1986, pp. 89-117. 36. Coon, M. J., Strobel, H. W., and Boyer, R. F. "On the Mechanism of Hydroxylation Reactions Catalyzed by Cytochrome P450," Drug Metab Dis/?05 1973, 1, pp. 92-97. 37. Lu, A. Y. H., Levin, W., and Kunzman, R. "Reconstituted Liver Microsomal System that Hydroxylates Drugs, other Foreign Compounds and Endogenous Substrates, VIL Stimulation of Benzphetamine N-demethlation by Lipid and Detergent," Bioch Biophys Res Commun, 1974, 60, pp. 266-272. 38. Gum, J. R. and Strobel, H. W. "Isolation of the Membrane-binding Peptide of NADPH-cytochrome c (cytochrome P450) reductase: Characterization of the Peptide and its Role in the Interaction of Reductase with Cytochrome ?A5^r JBiol Chem 1979, 254, pp. 4,177-4,185. 39. Black, S. D., French, J. S., WilHams, C. H., Jr., and Coon, M. J. "Role of a Hydrophobic Polypeptide in the N-terminal Region of NADPH-cytochrome P450 Reductase in Complex Formation with P450^^," Biochem Biophys Res Commun 1979, 91, pp. 1,528-1,535. 40. Lu, A. Y. H. and West, S. B. "Multiplicity of Mammalian Microsomal Cytochromes P450," Pharmacol Rev 1980, 31, pp. 277-291. 41. Johnson, E. F. "Multiple forms of Cytochrome P450: Criteria and Significance," in Reviews in Biochemical Toxicology, E. Hodgson, J.R. Bend and R. M. Philpot (Eds.), Amsterdam, North Holland, Elsivier, 1977, pp. 1-26. 42. Ueng, Y. F., Ueng, T. H. "Induction and Purification of Cytochrome P450 lAl from 3-Methylcholanthrene-treated Tilapia, Oreochomis Niloticus X Oreochromis Aureus,'' Arch Biochem Biophys 1995, 322(2), pp. 347-389.

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43. Romkes, M., Faletto, M. B., Blaisdell, J. A., Raucy, J. L., Goldstein, J. A. "Cloning and Expression of Complimentary DNAs for Multiple Members of the Human Cytochrome P450 IIC Subfamily," Biochemistry, 1993, 32, p. l,390ff. 44. Sun, B. Y., Fukuhara, M. Takanaka, A. "Characterization of Benzo(a)pyrene Metabolism and Related Cytochrome P450 Isozymes in Syrian Hamster Livers," J Toxicol Environ Health, 1995, 46(1), pp. 47-56. 45. Zeldin, D. C , DuBois, R. N., Falck, J. R., Capdevila, J. H. "Molecular Cloning, Expression and Characterization of an Endogenous Human Cytochrome P450 Arachidonic Acid Epoxygenase Isoform," Arch Biochem Biophys 1995, 322(1), pp. 76-86. 46. Sakamoto, K., Kirita, S. Baba, T., Nakamura, Y., Yamazoe, Y., Kato, R., Takanaka, A., Matsubara, T. "A New Cytochrome P450 Form Belonging to the CYP2D in Dog Liver Microsomes: Purification, cDNA Cloning and Enzyme Charaterization," Arc/z Biochem Biophys 1995, 319(2), pp. 372-401. 47. Imaoka, S., Hiroi, T., Tamura, Y., Yamazaki, H., Shimada, T., Komori, M., Degawa, M., Funae, Y. "Mutagenic Activation of 3-Methoxy-4Aminoazobenzene by Mouse Renal Cytochrome P450 CYP4B1: Cloning and Characterization of Mouse CYP4B1," Arch Biochem Biophys 1995, 321(1), pp. 255-262. 48. Richardson, T. H., Jung, F., Griffin, K. J., Wester, M., Raucy, J. L., Kemper, B., Bornheim, L. M., Hassett, C , Omiecinski, C. J., Johnson, E. F. "A Universal Approach to the Expression of Human and Rabbit Cytochrome P450s of the 2C Subfamily in Escherichia coli/' Arch Biochem Biophys 1995, 323(1), pp. 87-96. 49. Waxman, D. J., Azaroff, L. "Phenobarbital Induction of Cytochrome P450 Gene Expression," Biochem J 1992, 281, pp. 577-592. 50. Porter, T. D., Coon, M. J. "Cytochrome P450: Multiplicity of Isoforms, Substrates, and Catalytic and Regulartory Mechanisms," J Biol Chem 1991, 266(21), pp. 13,469-13,472. 51. Nebert, D. W., Nelson, D. R., Coon, M. J., Estabrook, R. W., Feyereisen, R., Fujii-Kuriyama, Y., Gonzalez, F. J., Guengerich, F. P., Gunsalus, L C , Johnson, E. F., Loper, J. C , Sato, R., Waterman, M. R., Waxman, D. J. "The P450 Superfamily: Update on New Sequences, Gene Mapping, and Recommended Nomenclature," DA^A Cell Biol. 1991, 10, pp. 1-14. 52. Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R W., Feyereisen, R., Gonzalez, F. J., Coon, M. J., Gunsalus, L C , Gotoh, O., Okuda, K., and Nebert, D. W. "The P450 Superfamily: Update on New Sequences, Gene Mapping, Accession Numbers, Early Trivial Names of Enzymes, and Nomenclature," DA^A Cell Biol. 1993, 12, pp. 1-51.

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53. Nelson, D. R. and Strobel, H. W. "On the Membrane Toplogy of Vertebrate Cytochrome P450 Proteins," 7 B/o/ Chem, 1988, 263, pp. 6,038-6,050. 54. Nelson, D. R. and Strobel, H. W. "Secondary Structure Prediction of 52 Membrane-Bound Cytochrome P450 Shows a Strong Structural Similarity to P450cam » Biochemistry, 1989, 28, pp. 656-660 55. Koymans, L., Donne-op den Kelder, G. M., Koppele, J.M., and Vermeulen, N. P.E. "Cytochromes P450: Their Active-site Structure and Mechanism of Oxidation," Drug Metab Rev. 1993, 25, pp. 325-387. 56. Shimizu, T., Hirano, K., Takahashi, M., Hatano, M., and Fujii-Kuriyama, Y. "Site-Directed Mutageneses of Rat Liver Cytochrome P450s: Axial Ligand and Heme Incorporation," Biochemistry, 1988, 27, pp. 4,138^,141. 57. Shimizu, T., Sadeque, A. J. M., Hatano, M., and Fujii-Kuriyama, Y. "Bindings of Axial Ligands to Cytochrome P450^ Mutants: A Difference Absorption Spectral Study," Biochim Biophys Acta, 1989, 995, pp. 116-121. 58. Tretiakov, V. E., Degtyarenko, K. N., Urarov, V. Y., and Archakov, A. I. "Secondary Structure and Membrane Topology of Cytochrome P450," Arch Biochem Biophys, 1989, 275, pp. 429-439. 59. Brown, C. A. and Black, S. D. "Membrane Topology of Mammalian Cytochrome P450 from Liver Endoplasmic Reticulum," J Biol Chem,l9S9, 264, pp. 4,442^,449. 60. Vergeres, G., Winterhalter, K. H., and Richter, C. "Localization of the N-terminal Methionine of Rat Liver Cytochrome P450 in the Lumen of the Endoplasmic Reticulum," Biochim Biophys Acta, 1991, 1063, pp. 235-241. 61. Ingelman-Sundberg M. "Cytochrome P450 Organization and Membrane Interactions," in Cytochrome P450: Structure, Mechanism and Biochemistry, P.R. Ortiz de Montellano (Eds.), New York, Plenum Press, 1986, pp. 119-160. 62. Thakker, D. R., Yage, H., Akagi, H., Koreeda, M., Lu, A. Y. H., Levin, W., Wood, A. W., Conney, A. H., and Jerina, D. M. "Metabolism of Benzo[a]pyrene. VI. Stereoselective Metabolism of Benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol Epoxides," Chem Biol Interact 1977, 16, pp. 281-300. 63. Amstrong, R. N., Levin, W., Ryan, D. E., Thomas, P. E., Mah, H. D., and Jerina, D.M. "Stereoselectivity of Rat Liver Cytochrome P450c in Formation of Benzo[a]pyrene 4,5-oxide," Biochem Biophys Res Commun 1981, 100, pp. 1,077-1,084. 64. Miwa, G. T. and Lu, A. Y. H. "The Topology of the Mammalian Cytochrome P450 Active Site" in Cytochrome P450: Structure, Mechanism and Biochemistry, P.R. Ortiz de Montellano (Eds.), New York, Plenum Press, 1986, pp. 77-88. 65. Yagi, H. and Jerina, D. M. "Absolute Configuration of the trans-9,10dihydrodiol Metabolite of the Carcinogen Benzo[a]pyrene," J Am Chem Soc, 1982, 104, pp. 4,026-4,027.

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66. Kulisch, G. P. and Vilker, V. L. "Application of Pseudomanas putida PpG 786 Containing P450 Cytochrome Monooxygenase for Removal of Trace Naphthalene Concentrations," Biotechnol Prog 1991, 7, pp. 93-98. 67. Selkirk, J. K., Yang, S. K., and Gelboin, H. V. "Analysis of Benzo[a]pyrene Metabolism in Human Liver and Lymphocytes and Kinetic Analysis of Benzo[a]pyrene in Rat Liver Microsomes," in Carcinogenesis—A Comprehensive Survey, Vol. 1, R. Fredenthal and P.W. Jones (Eds.), New York, Raven Press, 1976, pp. 153-169. 68. Selkirk, J. K. "Comparison of epoxide and free-radical Mechanisms for Activation of BaP by Sprague-Dawley Rat Liver Microsomes," J Natl Cancer Inst. 1980,64,771-774. 69. Capdevila, J., Estabrook, R. W., and Prough, R. A. "The Microsomal Metabolism of BaP Phenols," Biochem Biophys Res Commun, 1978, 82, pp. 518-525. 70. Jernstrom, B., Orrenius, S., Undeman, O., Graslund, A., and Ehrenberg, A. "Fluorescence Study of DNA-binding Metabolites of BaP Formed in Hepatocytes Isolated from 3-Methylcholanthrene Treated Rats," Cancer Res, 1978, 38, pp. 2,600-2,607 71. Wiebel, F.J. "Metabolism of Monohydroxy-BaPs by Rat Liver Microsomes and Mammalian Cells in Culture," Arch Biochem Biophys, 1975, 168, pp. 609-621. 72. Yang, S. K., Selkirk, J. K., Plotkin, E. V., and Gelboin, H. V. "Kinetic Analysis of the Metabolism of BaP to Phenols, Dihydrodiols and Quinones by HPLC Compared to Analysis by Aryl Hydrocarbon Hydroxylase Assay, and the Effect of Enzyme Induction," Cancer Res, 1975, 35, pp. 3,642-3,650. 73. Ribeiro, O., Kirkby, C. A., Hirom, P. C , and Millburn, P. "Secondary Metabolites of BaP: 3-Hydroxy-rraw5'-7,8-dihydro-7,8-dihydroxy-BaP, a Biliary Metabolite of 3-Hydroxy-BaP in Rat," Carcinogenesis, 1985, 6. pp. 1,507-1,511. 74. Capdevila, J. Estabrook, R. W., and Prough, R. A. "The existence of BaP3,6-Quinone Reductase in Rat Liver Microsomal Fractions," Biochem Biophys Res Commun, 1978, 83, pp. 1,291-1,298. 75. Prough, R. A., Saeki, Y., and Capdevila, J. "The Metabolism of BaP Phenols by Rat Liver Microsomal Fractions," Arch Biochem Biophys, 1981, 212, pp. 136-146. 76. Keller, G. M., Turner, C. R., and Jefcoate, C. R. "Kinetic Determinants of BaP Metabolism to Dihydrodiol Epoxides by 3-methylcholanthrene Induced Rat Liver Microsomes," Mol Pharmacol 1982, 22, pp. 451^58. 77. Amir, D. and Haas, E. "Estimation of Intramolecular Distance Distributions in Bovine Pancreatic Trypsin Inhibitor by Site-Specific Labeling and Nonradiative Excitation Energy-Transfer Measurements," Biochemistry, 1987, 26, pp. 2,162-2,175.

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78. Kunz, B. C , Rehorek, M., Hauser, H., Winterhalter, K. H., and Richter, C. "Decreased Lipid Order Induced by Microsomal Cytochrome P450 and NADPH-cytochrome P450 Reductase in Model Membranes: Fluorescence and Electron Spin Resonance Studies," Biochemistry, 1985, 24, 2,889-2,895. 79. Markello, T., Zlotnick, A., Everett, J., Tennyson, J., and Holloway, P. W. "Determination of the Topography of Cytochrome b5 in Lipid Vesicles by Fluorescence Quenching," Biochemistry, 1985, 24, 2,895-2,901. 80. Meyerhoffer, S. M. and McGown, L. B. "Microenvironments of Fluorescence Probe in Sodium Taurocholate and Sodium Taurodexoycholate Bile Salt Media," Anal Chem, 1991, 63, pp. 2,082-2,086. 81. Stoeckenius, W. "Some Electron Microscopical Observations on Liquid-crystalline Phase in Lipid-water Systems," J Cell Biol, 1962, 12, pp. 221-229. 82. Bystrov, V. F., Dubrovina, N. L, Barsukov, L. L, and Bergelson, L. D. "NMR Differentiation of the Internal and External Phospholipid Membrane Surfaces Using Paramagnetic Mn"^^ and Eu"^^ Ions," Chem Phys Lipids, 1971, 6, pp. 343-350. 83. Levine, Y. K., Lee, A. G., Birdsall, N. J. M., Metcalfe, J. C , and Robinson, J.D. "The Interaction of Paramagnetic Ions and Spin Labels with Lecithin Bilayers," Biochim Biophys Acta, 1973, 291, pp. 592-607. 84. Lakowicz, J. R. and Bevan, D. R. "Effects of Asbestos, Iron Oxide, Silica, and Carbon Black on the Microsomal Availability of Benzo(a)pyrene," Biochemistry, 1979, 18, pp. 5,170-5,176. 85. Lakowicz, J. R., Bevan, D. R., and Riemer, S. C. "Transport of a Carcinogen, Benzo(a)pyrene, from Particulates to Lipid Bilayers: A Model for the Fate of Particle-Adsorbed Polynuclear Aromatic Hydrocarbons which are Retained in the Lungs," Biochim Biophys Acta, 1980, 629, pp. 243-258. 86. Li, K. P., Li, Y. Y., and Boley, L. "Liposomal Uptake of Microcrystalline Benzo[a]pyrene Studied with Synchronous Fluorescence," Biochem Biophys Res Commun. 1983, 112, pp. 1,069-1,076. 87. Song, R. and Li, K. P. "Fluorometry of Carcinogenic Polycyclic Aromatic Hydrocarbons in Biological Systems," Applied Spectrosc, 1993, 47, pp. 1,604-1,608. 88. Song, R. and Li, K. P. "Fluorescence and Raman Spectrometry of Polycyclic Aromatic Hydrocarbons in Biological Systems," Polycyclic Aromatic Compounds, 1994, 5, pp. 249-258. 89. Li, K. P. "Fluorescence Studies of Binding and Transport of Carcinogenic Polynuclear Aromatic Hydrocarbons across Vesicular Bilayers," in Polynuclear Aromatic Hydrocarbons: Chemical Analysis and Biological Fate, M. Cooke and A. J. Dennis (Eds.), Columbus, Ohio, Battelle Press, 1981, pp. 593-602.

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90. Chiang, P., Li, K. P., and Hseu, T. M. "Spectrochemical Behavior of Carcinogenic Polynuclear Aromatic Hydrocarbons (PAHs) in Biological Systems: I. Steady-state Fluorometry of BaP and BeP in Living Cells," Applied Spectrosc, 1996, 50, pp. 1, 345-351. 91. Li, K. P., Click, M. R., IndraHngham, R., and Winefordner, J. D. "Steadystate and Time-resolved Fluorimetry of Benzo(a)pyrene in Liposomes and Microsomes," Spectrochim Acta, 1989, 45A pp. 471-477. 92. Li, K. P., Click, M. R., IndraHngham, R., and Winefordner, J. D. "Timeresolved Fluorimetry of Benzo(a)pyrene in Liposomes and Microsomes," Proceedings Internat. Conf. Laser '88, 1989, pp. 580-585. 93. Chiang, P., Li, K. P., and Hseu, T. M. "Spectrochemical Behavior of Carcinogenic Polynuclear Aromatic Hydrocarbons (PAHs) in Biological Systems: IL A Theoretical Rate Model for BaP Metabolism in Living Cells," Applied Spectrosc, 1996, 50, pp. 1, 352-351, 359.

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CHAPTER 12 OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS DURING PAINT STRIPPING AND PAINTING Marc Charretton Caisse Regionale d'Assurance Maladie Rhone-Alpes Laboratoire de Chimie du Val Rosay F-69370 St Didier au Mont d'Or, France Raymond Vincent Institut National de Recherche et de Securite Service Evaluation et Prevention du Risque Chimique BP 27, F-54501 Vandoeuvre Cedex, France CONTENTS INTRODUCTION, 252 ASSESSMENT OF OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS, 252 Evaluation of Exposure by Atmospheric Sampling, 253 Exposure Evaluation by Biological Sampling, 255 PAINT STRIPPING, 256 COMPOSITION OF CHEMICAL STRIPPING AGENTS, 258 TOXICITY OF DICHLOROMETHANE, 259 Metabolism, 259 Acute Toxicity, 260 Chronic Toxicity, 261 LIMIT VALUES FOR OCCUPATIONAL EXPOSURE TO DICHLOROMETHANE, 262 LEVEL OF EXPOSURE TO DICHLOROMETHANE DURING STRIPPING OPERATIONS, 262 CURRENT TRENDS IN PAINT-STRIPPING TECHNIQUES, 264 GENERAL OVERVIEW OF PAINTS, 265 THE CONSTITUENTS OF PAINTS, 269 Binders, 269 Pigments, 271 Extenders, 271 Additives, 271 Solvents, 272 TOXICITY OF SOLVENTS FOUND IN PAINT, 272 Pathways by w^hich Solvents can Enter into the Organism, 272 Effects of Solvents on the Organism, 273

251

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DIFFERENT AREAS IN WHICH PAINTS ARE USED, 275 METHODS OF APPLICATION OF LIQUID PAINTS, 275 Application by Pressurized Spraying of Paint, 276 Application by Electrostatic Pulverization, 279 Application by Dipping in Paint Baths, 280 Other Processes for Application of Liquid Paints, 281 SECTORS OF USE AND APPLICATIONS OF PAINT, 283 Industry, 283 OCCUPATIONAL EXPOSURE TO PAINT SOLVENTS, 283 Epidemiological Studies, 283 EVOLUTION OF PAINT FORMULATIONS, 285 REFERENCES, 300 INTRODUCTION The use of paint dates back to the beginning of civilization. Employed throughout prehistory, paints were used to create ritual decorations, and to embellish monuments and dwellings. Since then, their use has expanded to include a protective role: to prevent damage to wood and construction materials, and to stop the corrosion of metals. Due to relatively recent chemical developments and the appearance of polymers, the manufacturing of paint has lost the empirical character that dominated it for millennia. In our industrial society, the production of objects made from different materials (wood, plastics, metals, concrete) is closely associated with the application of paint—an application that both protects and beautifies the object, be it an automobile, an airplane, furniture, a boat, or a building, etc. The majority of paints, varnishes and paint strippers (with the exception of coating powders) contains solvents. These volatile components allow the paint to be easily spread, or in the case of stripping agents, to participate in the destruction of the paint film. The use of paints and stripping agents thus leads to the emission of organic solvent vapors, to which different categories of people are exposed during the course of their occupational, or domestic, activities. ASSESSMENT OF OCCUPATIONAL EXPOSURE TO ORGANIC SOLVENTS Two methods are generally used to evaluate the degree of exposure of workers to organic solvents:

Occupational Exposure to Organic Solvents

253

1. the measurement of the concentration of pollutants in the atmosphere at the workplace; and 2. the measurement of the quantity of a toxic substance absorbed by the worker, accomplished by evaluating the levels of pollutant or metabolites in the biological fluids of the individual: urine, blood, exhaled air. Evaluation of Exposure by Atmospheric Sampling [1,2,3] This approach usually consists of two steps in industrial hygiene: 1. sampling of air polluted by organic solvents; and 2. determination of concentration of pollutants in the air. This type of approach can only be used to evaluate exposure caused by the inhalation of polluted air. Sampling In the classical approach, sampling is usually carried out with a sampling tube that contains some type of absorbent material that traps the solvent vapors. This tube, placed in a support, is attached to a battery-powered individual sampling pump (Figure 1) that assures a regular and constant air flow rate. The support is placed near the respiratory tract of the individual through the sampling period (Figure 2). The most commonly used sampling tubes contain activated charcoal for

Figure 1. Sampling pump and tubes.

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Figure 2. Worker equipped with a sampling device.

non-polar solvents (e.g., hydrocarbons), and silica gel for polar components (e.g., alcohols). Porous polymers (e.g., TENAX, CHROMOSORB, etc.) containing variable quantities of absorbent material (on the order of tens to hundreds of mg) can also be used. The choice of which tube to use thus depends on the polarity of the solvent in question, its concentration (assumed to be low, high), sampling flow rate (on order of tens to hundreds of ml/min), and the sensitivity of the method of analysis for the pollutant(s) being sought. Sampling badges, or passive samplers that do not need a pump, can also be used. In this case, the absorbent material is contained in a pouch, covered by a grid that regulates the rate at which air diffuses across the absorbent material. The solvent vapors are trapped on the absorbent material in the same manner as for the sampling tubes (Figure 3) [4]. The workers wear the sampling device in question for periods representative of the exposure related to their activity (on the order of a few minutes to several hours). At the end of the sampling period, using a known flow rate (usually measured at the

Occupational Exposure to Organic Solvents

255

Figure 3. Different sampling badges.

beginning and end of the sampling period), the volume of air sampled can also be calculated. The solvent vapors present in the sampled air are trapped in the tube and their respective concentrations quantified using different analytical methods. Analysis The first step of the analysis consists of desorbing the vapors trapped in the sampling tube or badge. The absorbent material is transferred to a glass flask to which 1 or 2 ml of solvent (carbon disulfide normally) are added. The vapors are desorbed by being solubihzed in the added solvent. It is the analysis of this desorption solution that will yield the amount of pollutants trapped in the sampler. This can be used in turn to estimate the atmospheric concentration using the estimate of the volume of air that was sampled. The analysis of the desorbed solution constitutes the second step of the analysis. The most widely used analytical technique is gas chromatography, often coupled with mass spectrometry to identify and quantify the pollutants. The results are generally expressed in terms of mg of pollutant per cubic metre (mg/m^), or in parts per million (ppm). The industrial hygienist then compares the results to the current occupational limit values (TLV-TWA, TLV-STEL, PEL, etc.) to evaluate the exposure to which the worker(s) was (were) exposed. Exposure Evaluation by Biological Sampling [5] The second method is based on the measurement of the concentration of the pollutant itself, or of its metabolites in a blood or urine sample taken at the beginning or end of the exposure period. It is also possible to measure the concentration of pollutant in the air exhaled by the individual at the end of the work shift. This method allows one to evaluate respiratory and cutaneous exposures, and, in certain cases, the amount of pollutant ingested by the subject. Additionally, this method

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also accounts for the level of physical activity since, in the case of heavy physical effort, respiration is accelerated and the quantity of polluted air inhaled increases. Given the kinetics of the elimination of toxins by the organism, the moment at which the biological samples are taken is very important in order to be able to evaluate the exposure levels of the individual with any precision. A wide range of analytical techniques are available to measure pollutant concentrations, and the one chosen will depend on the nature of pollutant or metabolite to be measured. Once the concentration of pollutant has been evaluated, it is compared to the value of the Biological Exposure Indices (BEI) proposed by different organizations such as the ACGIH. The ACGIH currently proposes BEI values for approximately sixty substances [6]. The BEI values for conmion substances used in paints and in stripping agents are listed in Table 1. PAINT STRIPPING It is necessary to periodically apply a new coat of paint or varnish to trains, airplanes, boats, outdoor furniture, and parts of buildings (e.g., doors, wooden or metal shutters and blinds) among other objects. In order to guarantee a maximum level of protection and aesthetic quality, it is necessary to remove the old layers of paint from the surfaces to be treated. The elimination of old paint can be accomplished using different techniques: • abrasion of paint film by sanding, milling, or projection of particles (sandblasting); • burning and abrasion; • stripping with laser light (used in restoring art treasures); or • chemical strippers. Table 1 BEI Values (ACGIH) recommended for certain organic solvents

Substance Acetone Methyl alcohol (Methanol) Ethylbenzene Ethylglycol Ethylglycol acetate Methylethylketone Methylisobutylketone Xylenes

Parameter

Fluid

BEI

Sampling time*

Acetone Methanol Formic acid Mondelic acid 2-ethoxyacetic acid

Urine Urine Urine Urine Urine

100 mg/1 15mg/l 80 mg/g creatinine 1,500 mg/g creatinine 100 mg/g creatinine

A A I D D

Methylethylketone Methylisobutylketone Methylhippuric acids

Urine Urine Urine

2 mg/1 2 mg/1 1,500 mg/g creatinine

A A A

Sampling time: A = end of shift; D = end of shift at week's end; I = before last shift of work week.

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257

Chemical stripping agents are often used when it is necessary to protect the mechanical properties of the materials in question, for example in the case of airplanes, or in instances where mechanical stripping would aesthetically damage the surface of the object, e.g., furniture. Additionally, chemical stripping, when performed with a liquid product, can be applied to all parts of the object, even those where it would be difficult to strip the paint by abrasion or scraping. Two principal techniques are typically used for the stripping of painted surfaces. The technique to be used is generally chosen as a function of the size of the object to be stripped:

Figure 4. Hand stripping of furniture dipped in a stripping agent (Courtesy of Ets COMONT).

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Figure 5. Industrial stripping of an airplane by spraying (Courtesy of Didier TOULORGE).

• soaking, widely used for the stripping of furniture, shutters, doors, etc. (Figure 4). • direct application of stripping agent with a brush or spray gun, followed by elimination of the paint by sanding or scraping. This type of procedure is generally used for large objects such as airplanes (Figure 5). Because chemical stripping agents always contain organic solvents, these types of operations lead to exposure to solvent vapors of the individuals performing the work. The intensity of the exposure will depend on the size of the surfaces to be treated, the premises, the procedures used, etc. COMPOSITION OF CHEMICAL STRIPPING AGENTS A chemical stripping agent is generally composed of: • a caustic product, the primary function of which is to soften the paint film. Potassium and sodium hydroxides are the most widely used products for the stripping of paint applied to wood and ferrous metals. For other metals, such as aluminum, copper, and tin and their alloys, phenol or organic acids are generally used to strip paint in order to avoid corrosion associated with the formation of metal hydroxides in the presence of sodium or potassium hydroxide.

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259

• organic solvents to permeate the paint film. The action of these solvents, sometimes coupled with that of a caustic agent, also softens the paint film and causes it to become detached from the surface. Methylene chloride, or dichloromethane (CH2CI2), is by far the solvent most widely used in paint strippers—in fact this is the principal application of dichloromethane [7]. Methanol, or methyl alcohol, is also often present in these formulations. A study on the composition of paint strippers [8] showed that 67.9% of the 215 formulations studied contained dichloromethane at an average concentration of 73% (standard deviation ±12%), 45.6% contained methanol (average concentration 11%, standard deviation ±4%), and 19.5% contained phenol (average concentration 13%, standard deviation ±7%). Methylene chloride is widely used in this type of formulation because it is such a good solvent, but also because it is not flammable [7]. • thickeners such as waxes or methyl cellulose. (The methanol present in paint strippers swells the methyl cellulose.) The thickeners form a barrier at the surface of the stripper that limits the evaporation of the solvents and guarantees that they produce their maximum effect on the paint surface. • surfactants that improve the wetting capacity of the solution and increase the speed at which the stripper works at ambient temperature. Paint strippers are sold in different containers depending on their end use: in small bottles for domestic use, or in barrels or tanks for industrial applications. Given the composition of paint strippers, occupational exposures to organic solvents resulting from these products is generally related to exposure to dichloromethane. TOXICITY OF DICHLOROMETHANE Metabolism The biotransformation of dichloromethane can follow one of two paths, both of which lead to the production of electrophile reactants [9, 10, 11]. The first pathway, dependent on cytochrome P450, leads to the production of a carbon monoxide (CO) endogen that causes an increase in the levels of carboxyhemoglobin. The second path follows the conjugation of dichloromethane with glutathion (GSH), and leads to the formation of formaldehyde. The two different pathways are shown in Figure 6. In subjects that have been intensively exposed to dichloromethane vapors at levels high enough to make them lose consciousness, carboxyhaemoglobin levels can be as high as 50% [12]. Given this phenomenon of hypoxia, those suffering from cardiac problems can be considered to be particularly at risk if they inhale strong doses of dichloromethane vapors [13]. The second pathway of biotransformation that leads to the formation of formaldehyde could explain the mutagenic character of dichloromethane that has been

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Health and Toxicology

I CYTP-450'~|

H

o

O

II

C

H - O - C - CL

C^rLj v^Lo

NADPH I CYTOSOL + GSH

I

CL

|

\

:C = 0

H CL

\ HCL

HCL + GSH

h^HCL GS - CH2 - CL

D

^

^HCL GS -'CH2 - OH

//

G-S-C I "^H ^

CH2O + GSH

H

NAD+

>.^°

o

v&

CO9

GS-C

\ i

O

// GS-C \

o H

HCOOH + GSH GSH

CO,

CO,

Figure 6. Metabolism of dichloromethane as described by Andersen et al. [11].

observed by several authors [14-16]. Given the polymorphism of the enzyme involved in the formation of the formaldehyde (glutathion-S-transferase Theta; GSH), the actual quantity of formaldehyde produced is highly dependent on the level of GSH activity—an activity that can vary greatiy from one individual to the next. For this reason, certain subjects have a higher risk than others of genetic damage. Acute Toxicity Animal Studies The LD50 for oral administration in rats is approximately 3,000 mg/kg [17], and for inhalation, the LC50 is 52,000 mg/mV6h [18]. For mice, the LC50 is 49,100 mg/m3/6h [19]. Numerous studies carried out on different species of animals (rats, mice, guinea pigs, rabbits, dogs, and monkeys) have demonstrated that dichloromethane acts on the liver, kidneys, heart, and central nervous system after only short exposures (on the order of a few minutes to several hours) at atmospheric concentrations varying

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from 3,600 mg/m^ (1,000 ppm) to 60,000 mg/m^ (approximately 17,000 ppm). Damage to the central nervous system is reflected by a decrease in overall activity levels, narcosis, and perturbation of the sleeping patterns [13]. Human Studies The exposure of humans to atmospheric concentrations of 3,600 mg/m^ (1,000 ppm) leads to the appearance of anesthetic effects. After 5 minutes of exposure to concentrations of up to 8,000 mg/m^ (2,300 ppm), symptoms including dizziness, and irritation of the eyes and of the respiratory tract can appear [20]. After two hours of exposure to atmospheric concentrations of 700 mg/m^ (200 ppm) neurobehavioral changes were observed, including problems of alertness and the alteration of overall performance levels in surveillance/pursuit tests [21]. The narcotic effect of its vapors can lead to a deep coma [12], or even death [22-25], whereas cutaneous contact with dichloromethane lasting on the order of a few minutes can cause severe bums [26, 27]. Chronic Toxicity Animal Studies Rats and mice are the studied animals that are the most sensitive to this substance. An increase in dose-dependent levels of mammary benign tumors (in both males and females) was noted in groups of rats exposed to 0, 500, and 3,500 ppm for 5 hours a day, 6 days a week for 2 years. Sarcomas in the region of the salivary glands were also observed, but only in males—this effect was statistically insignificant in females [28]. These results were confirmed by another study where groups of rats and mice were exposed in much the same conditions of frequency and duration, but to atmospheric concentrations of 1,000, 2,000, and 4,000 ppm. Other than the appearance of tumors in the mammary and salivary glands, the authors also demonstrated a dosedependent increase in the frequency of pulmonary (males and females) and hepatic (in males only, statistically insignificant in females) cancers [29]. Human Studies There are relatively few epidemiological studies on the risks associated with prolonged exposure to dichloromethane [30-32]. None of those studies available was able to demonstrate a particular cause (pulmonary and liver cancer, cardiac problems) of an increase in mortality rates associated with a occupational exposure to dichloromethane. A long-term study from 1964 to 1984 of a cohort of 1,013 workers exposed to an average atmospheric concentration of 26 ppm of dichloromethane per day showed

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that there was an insignificant increase in the number of deaths due to pancreatic cancer—8 observed cases against 4.2 expected cases [32]. The prolongation of this cohort study until 1988 did not lead to the confirmation of this observation [33]. More recently, an examination of a group of workers exposed to average atmospheric concentrations on the order of 475 ppm for at least ten years did not reveal any significant cardiac or hepatic damage, nor any damage to the central nervous system [34]. The International Centre for Cancer Research (CIRC) [35] has classified dichloromethane in the category 2B: a substance suspected to have a possible carcinogenic effect on humans. Additionally, when discussing the toxicity of dichloromethane, it should be pointed out that the thermal degradation of this substance leads to the production of phosgene gas, a highly toxic compound that causes severe lesions to the pulmonary system. This phenomenon can arise when paint strippers are used near a source of heat such as a gas-burning radiator [36]. LIMIT VALUES FOR OCCUPATIONAL EXPOSURE TO DICHLOROMETHANE The 8-hour exposure value in the United States (TLV-TWA: Threshold limit value, time-weighted average) reconmiended by the American Conference of Governmental Industrial Hygienists (ACGIH) is currently 50 ppm (174 mg/m^). The ACGIH has also classified this substance in the category A2: substance suspected of having a carcinogenic effect in humans [6]. In 1993, the Occupational Safety and Health Administration (OSHA) reduced the Permissible Exposure Limit (PEL) from 500 ppm to 25 ppm [37]. In France, the Ministry of Labour recommends an average limiting exposure value (AEV) over eight hours of 50 ppm. The short-term limiting exposure value (LEV) for periods no longer than 15 minutes has been fixed at 100 ppm. Dichloromethane is classified in category C3: carcinogenic effects observed on animals [38]. In Germany, the 8-hour exposure value (the MAK value) has been fixed at 100 ppm, and dichloromethane has been classified in category IIIB: substance suspected of having a carcinogenic potential. The values of biological indicators of exposure to dichloromethane in Germany are a level of carboxyhaemoglobin of 5%, or 1 mg dichloromethane/1 in a blood sample taken at the end of the working day [39]. LEVEL OF EXPOSURE TO DICHLOROMETHANE DURING STRIPPING OPERATIONS Anundi et al. [40] studied the exposure of workers to organic solvents during the elimination of graffiti from subway stations. The exposure levels of dichloromethane varied from 18 to 1,188 mg/m^ (5-330 ppm) over the course of the workday, but varied from 6 to 5,315 mg/m^ (1.7 to 1,476 ppm) for short periods. The same authors also measured the atmospheric concentration of other sol-

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vents contained in the stripping agents, but found that exposure to these substances, as well as their atmospheric concentration levels, were relatively low. They detected the presence of some glycol ethers, including dipropyleneglycolmonomethylether (DPGME), propyleneglycolmonobutylether (PGBE), and nmethyl-2-pyrolidone (nMP). The trimethylbenzenes present in the stripping agents were not detected in the air of the workplace. The concentrations of the different glycol ethers ranged from 3.4 to 11.8 mg/m^, and exposure to nMP was detected at 9.9 mg/m^, but only in one single sample. McCammon et al. [41] evaluated exposure of 12 workers from 5 different workshops involved in furniture stripping to dichloromethane with both atmospheric and biological samples. The atmospheric concentrations measured over the course of the workday varied from 54 to 1,317 mg/m^ (15-366 ppm). At the end of the workday, the concentration of dichloromethane in the air exhaled by the workers varied from 2.3 to 167 ppm, and the concentrations of carboxyhemoglobin varied from 2.1 to 13.5%. The concentration of dichloromethane in these same blood samples varied from 0.1 to 8.8 ppm (approximately 0.1 to 8.8 mg/1). The exposure values varied from one workshop to the next, and depended to a great extent on the procedures employed by the workers and on the means of protection provided (i.e., ventilation). The authors also point out that the concentrations of dichloromethane measured during the course of the study are probably the lowest observed during the year because their study was carried out during the summertime when all the doors of the different workshops were wide open, thereby offering the maximum level of ventilation possible. Hall et al. [42] also studied exposure to dichloromethane in a furniture stripping workshop to aid in the conception of a ventilation system designed to reduce exposure levels. Before modification, exposure levels varied from 230 to 2,200 ppm (828-7,920 mg/m^). After the ventilation system was installed, the exposure varied from 6 to 93 ppm (21.6-334.8 mg/m^). Hall and Rumach [43] gave questionnaires to workers from 21 furniture stripping workshops to determine how dichloromethane was used, the ventilation systems employed, methods of individual protection, and the physiological effects noticed by the individuals in question during the stripping operations. Half of the workers questioned stated that they suffered nausea, headaches, and dizziness during this type of work. Given the descriptions of symptoms provided by the workers, the authors deduced that the exposure level in these workshops was between 500 and 5,000 ppm for periods varying from a few minutes to several hours. Shusterman et al. [44] presented a case study of a worker in a furniture stripping shop exposed to between 100 and 200 ppm of dichloromethane, and whose carboxyhemoglobin level attained 10.4%. The authors also point out the lack of efficiency of the protective respiratory equipment using activated cartridges; the authors state that the worker reported being able to detect solvent odors while wearing the mask.

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This phenomenon was studied experimentally by Moyer and Peterson [45]. Their results demonstrate that the duration of the trapping efficiency of an activated carbon cartridge varied as a function of the relative humidity and dichloromethane concentration, and was relatively short—approximately 30 minutes in the case of a dichloromethane concentration of 50 ppm at a relative humidity of 80%. This lack of dichloromethane capture efficiency in the cartridges of respiratory masks is linked to the physical characteristics of this solvent, especially the low molecular weight and low boiling point (high vapor pressure). Vincent et al. [8] measured individual exposure levels to dichloromethane in a door, shutter, and furniture stripping workshop, and found that they varied from 258 to 659 ppm (929-2,373 mg/m^). Concentration levels in the same workshop were measured by ambient sampling, and were between 69 and 683 ppm (250-2,460 mg/m^). In order to reduce exposure to dichloromethane in furniture stripping workshops, NIOSH published a good practices guide that suggests the use of appropriate and efficient ventilation systems [46]. The most crucial problem associated with such stripping operations concerns the treatment of large objects, for which it is difficult to install a ventilation system. This is especially true in the case of airplanes. When Vincent et al. [47] measured the exposure to dichloromethane and to phenol during the stripping of a Boeing B747 aircraft, they found that the total surface to be treated was on the order of 2,500 m^, and required the participation of a dozen workers for approximately 72 hours. The exposure of workers performing the actual paint stripping was on the order of 83 to 525 ppm (299-1,889 mg/m^), an exposure that corresponds to a daily level of between 24 to 344 ppm (86-1,240 mg/m^). Those workers who did not directly handle the paint stripper, but performed other tasks in the workshop, were exposed to concentrations between 88 and 212 ppm (317-762 mg/m^) during the stripping work carried out by the other workers. This corresponds to an 8-hour exposure of 27-48 ppm (97.2-174.6 mg/m^). Atmospheric phenol concentrations—phenol being present in the stripper—varied from 1.1 to 5.3 mg/m^ (0.3-1.4 ppm), which represents levels below the exposure levels generally recommended for this product (TLV-TWA, AGCIH = 5 ppm or 19 mg/m^). CURRENT TRENDS IN PAINT-STRIPPING TECHNIQUES Given the supposedly carcinogenic nature of dichloromethane, the decreasing limiting occupational exposure values, new laws on the emissions of VOCs in the atmosphere, the difficulty of installing efficient ventilation systems while at the same time respecting the environment, and the lack of efficient protective respiratory devices, industrial groups are looking for substitute chemical stripping processes that do not rely on the use of dichloromethane. Two routes are currently being

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explored: Replace the dichloromethane by other solvents thought to be less toxic, or use mechanical stripping procedures based on the projection of different materials. In strippers for domestic use, dichloromethane has been replaced by different mixtures of ketones, alcohols, esters, etc. The principal problem associated with the implementation of these new stripping agents is higher cost, lower efficiency, and especially problems of flammability when used in confined spaces. Furthermore, the use of hot caustic sodium hydroxide solutions for the stripping of furniture is not possible because this product attacks wood. In the area of aeronautics, the International Air Transport Association (lATA) has created a working group to study different alternatives to paint strippers based on dichloromethane and/or phenol [48]. Several different processes are being considered. Also being considered are acidic or basic strippers containing benzylic alcohol. This product has a low toxicity, a relatively high boiling point (200 °C), and it acts 10 to 20 times more rapidly than dichloromethane-based stripping agents. Paint stripping through the projection of particles of ice, carbon ice, starch, and water under high pressure can also be envisaged. The use of a pulsed laser beam also seems to be a possible means for stripping airplanes, railway cars, etc. The principal obstacles to the use of such new processes is also their higher cost as compared to that of chemical processes using dichloromethane. The operating principal of a process using the projection of ice particles that can be used to eliminate exposure to toxic substances is illustrated in Figures 7 and 8. The efforts undertaken by different companies to commercialize new stripping processes should lead to a considerable reduction in the use of chemical stripping agents over the course of the next few years, and at the same time reduce occupational exposure to organic solvents. GENERAL OVERVIEW OF PAINTS A paint is a fluid preparation (liquid, paste, or solid) that can be spread in thin layers on all types of materials (called subjectiles), which, after a hardening period (referred to as drying), forms a resistant, adhesive thin coating or layer (film or sheet) that plays both a decorative and/or protective role. If the film is opaque (white, or colored), it is classified as a paint. It is the presence of pigments in the paint that gives it the ability to cover or mask the support on which it is applied. A varnish corresponds to a paint without pigment. The development of synthetic macromolecules has led to the creation of a wide diversity of products—a development that makes the task of formulating paints more complex, but that also enriches the possibilities of new formulations, better adapted to a given end use. Constraints related to hygiene and safety in the workplace and in the environment have also contributed to the evolution of the makeup of paints. The manufacturer of a paint must currently consider the following parameters when developing a new product:

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Figure 7. Schematic diagram of the operating principle of a process using the projection of ice particles to strip paint (Courtesy of LINDE-CRYOCLIN, France).

• end use of the paint—whether it is an adherent, an anti-corrosive agent, an intermediate layer, or for finishing an object. • the nature of the support onto which it will be applied—whether this be wood, concrete, metal, aluminum, or a plastic material. • the desired properties of the paint—its hardness, flexibility, weather resistance, resistance to chemical agents, etc. • form in which the paint is sold. The different forms in which paint is found are summarized in Table 2. • is the chemical reaction necessary. Generally begun during the hardening phase of a paint, if the reaction is to occur at ambient temperature, it is necessary to

Figure 8. Stripping of a painted surface using the projection of ice particles to strip paint (Courtesy of LINDE-CRYOCLIN, France).

Table 2 Fornfis in which paints are found Presentation

Solid Aqueous Liquid • solution • emulsion Organic Liquid • solution • emulsion

Common name

Coating powders

Containers

1

Waterbome paints Water-thinnable paints

lor 2 lor 2

Solvent-based paints High solids paints Plastisols Organosols

lor 2 lor 2 1 1

mix the reactive constituents only at the instant of application. In this case, the paint is generally delivered in two separate containers. It can be seen from Table 2 that practically all types of liquid paints (aqueous or organic) can be conditioned in two separate containers, depending on their reactivity. • method of curing—summarized in Table 3.

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Health and Toxicology Table 3 Paint curing methods

Drying category

Physical curing

Chemical curing

Physico-chemical curing

Type of drying

Solvent evaporation Coalescence Fusion Oxidation Polycondensation Polyaddition Polymerization Each type of chemical curing method can be preceded by one of the three types of physical curing methods

Example of binding agents used

Acrylics, chlorinated rubbers, nitrocellulose Acrylics, vinyls Polyesters, acrylics Alkyds Alkyd-melamines Polyurethanes Unsaturated polyesters See above-mentioned binding agents.

Whenever a paint cures only by the simple evaporation of solvents, no chemical transformation of a binder occurs during the drying stage. The paint film therefore remains sensitive to solvents even when it is dry. In this case, the binders used are thermoplastic. Coalescence is the mechanism by which film forming occurs in emulsion paints when paint is applied. The evaporation of water causes the spreading, coming together, and finally the fusion of the emulsion droplets. It should be remembered that after the drying process, the film is insensitive to water. If a chemical reaction occurs in the film at the same time as the coalescence process, the film can equally become resistant to the majority of solvents (physico-chemical drying). In the opposite case, it can remain sensitive to solvents that dissolve the binder present in the paint. Fusion is the mechanism by which coating powders, or plastisols and organosols, form films. In the case of emulsion paints, if a chemical reaction occurs at the same time as fusion, the film becomes insensitive to attack by the majority of solvents. The binders used in paints that dry via a chemical process (or a physico-chemical process) are generally thermoset resin. During the drying process they undergo an irreversible transformation. This transformation can also cause the paint film to become insoluble in the majority of solvents. The chemical process can occur at ambient temperature (2 component paints), via the addition of a catalyst (oxidation catalysts: siccatives or acid catalysts), or be caused by radiation (e.g., infrared, ultraviolet, or beta-rays). The principal methods for applying paints, as well as their capacity for solvent emission (+-I-++ = high emissions, + = weak emission), are summarized in Table 4.

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Table 4 Methods for applying paint

Category

Pulverization

Dipping Pouring Other processes

Nature

Pneumatic Airless Airmix HVLP Purely Electrostatic Assisted Electrostatic Conventional processes Electrodeposition Flowcoating Rolling machines Sray curtains Coil coating Can coating Brush, roller

Estimated level of emission of solvents

++++ + +++ ++ + ++ +++ + +++ ++ + + ++ +

The rheological properties of paints must be adapted to the way in which they are used, and the.manufacturer must account for this in the formulation of the paints. Other than the above-mentioned parameters, the manufacturer must also consider the toxicity of the substances used in the paints in order to satisfy the health and safety requirements for the workplace and environmental legislation, during both the manufacturing and use phases. The importance of the list of requirements shows that paints are highly technical products, and the technological evolutions in this area have led to the creation of highly specific formulations; each paint is formulated for a specific end use. THE CONSTITUENTS OF PAINTS The constituents necessary for paint formulation are listed in Table 5. The column corresponding to coating powders is mentioned for the sake of completeness, but this category of paint does not produce any solvent vapors and will therefore not be discussed. Binders The binder is the essential ingredient of a paint. It forms bonds between all of the components and assures that the paint film adheres to the surface to which it is applied. The principal characteristics of a paint depend on the binder, and paint is

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Health and Toxicology Table 5 Composition of paints

Water-based paints Constituents Binders Pigments Extenders Solvents -Organic -Water -Coalescing agents -Cosolvents Additives -Conservation agents -Neutralizing amines

Coating powders

Waterborne Solventpaints based paints

Water-thinnable paints

+ + +

+ + +

+ + +

+ + +

+ -

+ + -

+ + + +

+ + + + -

(+) presence (-) absence

frequently named after which principal binder it contains. A paint can have one binder, or a combination of many different ones. The "principal binder" is the one whose chemical and physical characteristics define the general functional properties of the paint. The "complimentary binders" are those added to bring particular properties to the finished product. Binders are made up of macromolecules of natural (e.g., cellophane), artificial (nitro-cellulose, chlorinated rubbers), or synthetic origin (acrylics, epoxides, polyesters, polyurethanes, etc.). The principal characteristics of a binder include: • form—liquid or solid; • chemical nature—organic (natural and synthetic), organo-mineral (silicones), or mineral (silicates); • reactivity—non-reactive binders such as cellulose or rubber derivatives, reactive binders such as acryUcs, polyesters or epoxides, etc.; • molecular weight—low (oils), or medium to high (synthetic binders); • form in which they are used—solution, dispersion, powder; • solubility—in solvents or water (waterborne binders). It should be noted that the type of solvent used in a paint is dictated by the solubility-related properties of the binder that it contains.

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Pigments As previously mentioned, pigments provide the opacity and color of the paint film. In addition, they can also have an impact on certain other properties such as corrosion resistance, hardness, impermeability, etc. Pigments are pulverized solids of submicron-sized particles, either white or colored, mineral or organic, and insoluble in the dispersion medium. Mineral pigments are metal derivatives. With the exception of titanium oxide (the white pigment found in the majority of paints), mineral-based pigments are being used less and less because of the danger of toxicity that they can pose (e.g., lead or zinc chromates). Mineral pigments provide high levels of opacity and have average tinting capacities. There is a wide variety of formulas for organic pigments, but the most commonly used are phthalocyanine, azoic, and quinacridone derivatives. Organic pigments offer poor degrees of opacity, but very high powers of coloration. Extenders Originally, extenders were added to paints to lower production costs. However, experience has shown that this practice can have an impact on the mechanical, chemical or rheological properties of the paint. Extenders are generally pulverized soHds, usually white, mineral-based, and insoluble in the dispersion medium of the paint. The most widely used include calcium carbonate, barium sulphate, and silica derivatives. Extenders have poor powers of opacity. Additives Additives can bring different properties throughout the life cycle of a paint: •Fabrication: aid in the dispersion of pulverizing agents, anti-foaming agents (surfactants); • Storage: "anti-skin forming" agents (cetoxime derivatives), anti-sedimentation agents, rheological agents; • Film formation: spreading agents (surfactants), agents that render the film matte (silica), anti-corrosion agents (nitrites), anti-bacterial agents or fungicides; • Curing: accelerating agents (siccatives, acids), coalescing agents (glycol derivatives). Additives are introduced at low concentrations (<5%) in paints, and can be either organic or mineral products.

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Solvents

Solvents are what allow the paint to be applied to the subjectile. They do not remain in the film once it has been applied and dried (with the exception of reactive solvents in epoxide paints, and unsaturated acrylic monomers used in coatings cured with ultraviolet radiation, or styrene in unsaturated polyesters). Given the wide diversity of binders used in paints, practically all categories of chemical products are encountered as paint solvents. The most widely used solvents are presented as a function of their chemical categories in Table 6. TOXICITY OF SOLVENTS FOUND IN PAINT [49-57] Pathways by which Solvents can Enter into the Organism Solvents can enter the human body via: The Respiratory Tract: Any solvent vapors inhaled pass into the organism through the lungs and circulatory system. They accumulate in body tissues having high lipid contents (nerves, brain, bone marrow, adipal tissues, liver, kidneys). Cells can be damaged either by the solvents themselves or by their decomposition products. Cutaneous Pathways: Because solvents are lipophillic, they can be absorbed cutaneously. It would seem that this is the most important means of absorption for certain solvents (e.g., low-molecular-weight glycol ethers). The Digestive Tract: This is more rare than the others, but if ingestion occurs, it can lead to diarrhea and gastric problems. Table 6 Main types of solvents used In paint Chemical category

Aromatic hydrocarbons Aliphatic hydrocarbons Alcohols Esters Ketones Chlorinated hydrocarbons Glycol or propylene glycol ethers

Name

Toluene, xylenes, naphtha solvents White spirit Ethanol, butanol, isopropyl alcohol, etc. Ethyl acetate, butyl acetate, ethylglycol acetate, etc. Methylethylketone, methylisobutylketone, cyclohexanone, isophorone, etc. Methylene chloride, chlorinated paraffins, etc. Butylglycol, butyldiglycol, etc. Methoxypropyleneglycol, ethoxypropyleneglycol, acetates

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R- 0 - CH2 - CH2 - OH Alkoxyethanol Alcohol dehydrogenase

NADH R- 0 - CH2 - C - H Alkoxyacetaldehyd 0

Aldehyde dehydrogenase

NADH

R - 0 - CH2 - C - OH Alkoxyacetic acid 0

Figure 9. Metabolism of ethyleneglycol ethers.

Effects of Solvents on the Organism The effects that solvents can have on the body depend on both the concentration and the duration of the exposure. Even though the relationship between the dose and effects of acute exposures to solvents are relatively well-established, the same cannot be said for the effects of long-term occupational exposures to low concentrations. Divergent opinions are often found in the literature on this subject. Such disagreement could be explained by: • Differences between the methods used to evaluate the effects of solvents on the human organism. Such effects can be studied using biochemical, chemical, or epidemiological techniques. It appears evident that direct comparison of results obtained using these different techniques is not always possible. • Difficulties in obtaining precise information on occupational exposure to solvents. Exposure to a range of solvents is also quite frequent in industrial environments, and this type of multiple exposure makes it difficult to interpret any observed toxicological effects of a single solvent. • Non-specific reactions for chemical intoxications (with a few exceptions) caused by exposure to different solvents. The development of an illness is an insidious process, and there is also the problem of an important inter-individual variation of symptoms. Acute Toxicity The symptoms of an acute solvent-induced intoxication include nausea, dizziness, drowsiness, headaches, loss of consciousness, and narcotic effects associated with perturbations of the central nervous system.

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Chronic Toxicity Certain studies have demonstrated neuro-psychological effects that persist in individuals who were chronically exposed to organic solvents outside exposure periods. These symptoms are often labeled "psycho-organic syndrome." The basic symptoms include accumulated fatigue, memory loss, difficulties with concentration, and loss of motivation associated with free time and work-related activities. As mentioned above, the existence of this syndrome has not been universally recognized. Effects on the Skin Solvents can act on the skin in two ways: • They can dissolve the natural fatty layer of the skin, leading to cracking. Microorganisms and dust particles can then more easily penetrate the organism and cause infections. • They can act directly on the skin and mucous membranes to cause irritations (dermatitis, conjunctivitis). Specific Effects The Hematological Effects of organic solvents are dominated by benzeneinduced myelotoxicity. Benzene has not been used in paints for a number of years now; however, industrial solvents can contain trace impurities of this substance. French legislation limits the maximum amount of benzene allowed in industrial solvents to 0.1% w/w. Glycol Ethers (Co-solvents in Waterborne Paints) can provoke serious hemopathies, including aplasia (methyl and ethyl glycol) and hemolysis (butylglycol). It is suspected that styrene and toluene also present hemopathic dangers. Glycol Ethers and their Acetate Derivatives can also have adverse effects on the reproductive system. Ethylglycol and methylglycol are teratogens that can cause malformations, and butylglycol can lead to testicular atrophication, embryotoxicities, foetotoxicities, but not malformations. Toluene is classified in the same group as glycol ethers in Germany as far as reproductive effects are concerned. According to the I.A.R.C Classification, some solvents are classified in the following groups for carcinogenic and mutagenic effects: • Group 2B for styrene and carbon tetrachloride, • Group 3 for toluene, xylenes, cyclohexane, and isopropyl alcohol.

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Metabolism of Solvents The measurement of urinary levels of hippuric acid can be used to evaluate exposure to toluene. Based on a study of 303 workers exposed to solvents, Ukai et al. [57] concluded that the relationship between toluene exposure and the urinary excretion of hippuric acid is not modified by the presence of methylethylketone, nor that of isopropyl alcohol. Langman [56] points out that the measurement of the levels of hippuric acid in the urine is a better indicator of exposure to xylenes than the measurement of exposure by using air sampling. The ACGIH proposes a value for the Biological Exposure Indice (BEI) of 2.0 g MHA per liter of urine for xylenes. Ninety-five percent of the xylenes absorbed by the body is metabolized in the liver to yield methylhippuric acid. Seventy to 80% of the metabolites are excreted in the urine within the 24 hours that follow the exposure. The principal metabolic pathways for glycol ethers are shown in Figure 9. DIFFERENT AREAS IN WHICH PAINTS ARE USED [58-63] The large number and diversity of paints presents the possibility of numerous classifications (by type of curing, form of product, chemical nature, sector of use, etc.). It would seem that one of the more simple classifications would be by sector of use, because there are usually only six well-defined areas: • Building; • Industrial—auto bodies, furniture, household appliances, agricultural and public works machinery, aeronautics, coil coating, can coating; • Anti-corrosion; • Marine; • Automobile repair; and • Domestic use. METHODS OF APPLICATION OF LIQUID PAINTS There are several industrial processes that can be used for the application of liquid paints. The choice of which process to use is dictated by: • The characteristics required of the coating—adhesive undercoating, anti-corrosion, or surface finishing (e.g., there are different requirements for the finishing coats on automobile bodies and on heavy machinery); • The characteristics of the paint to be applied—its final form (viscous or fluid, powder), how it dries (physical curing, chemical curing); • The characteristics of the object to be painted—nature of constitutive material, its form, size, etc. • Speed of production (requires automation of painting process in certain cases);

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• Reduction of risk of exposure of person applying paint to the emission of dangerous substances (industrial health and safety), or the release of same into the environment; • The cost of the application procedure (energy costs, raw material and labor costs, etc.). Economic factors are often the principal basis on which the choice of a process is made. Because of this, the evolution of the technology in the area of the application of paint tends to favor processes having a low energy cost, that can be automated, and have high yields. Currently, the application of paint through electrodeposition, coil coating, and electrostatic pulverization correspond the most closely to such requirements. Application by Pressurized Spraying of Paint The principle of this technique resides in breaking the (liquid) paint down to the form of the smallest possible droplets, and dispersing these droplets on the surface to be covered. Originally, the only method for pulverizing paint was pneumatic. Small droplets were obtained through the generation of mechanical forces that resulted from the impingement of two jets: one jet of paint, the other of air. Economic (pneumatic pulverization has a relatively low yield) and ecological (this process leads to the formation of a mist of pollutants that is harmful in both the workplace and the environment) constraints pushed people to develop processes that better satisfy these considerations. New pressurized processes include airless pulverization (paint under pressure), mixed pulverization (combination of airless and pneumatic pulverization), and low pressure pulverization (HVLP = High Volume Low Pressure). Pneumatic Pulverization The mixing of air and paint that causes the droplet formation takes place in an annular space in the head of a spray gun, into which the compressed air and the concentric paint jet are fed. Other jets of air in the head regulate the form of the jet of pulverized droplets at the same time as they participate in the formation of the droplets themselves. The pressure drop between the compressed air and the outlet entrains the paint jet at high speed towards the subjectile. It is this difference of speed between the jets of compressed air and that of the paint that leads to the formation of fine particles that are carried away in the jet leaving the head of the spray gun. The pressure (2 to 5 bars) causes the paint to be vaporized into droplets that are on the order of 10 to 100 microns in diameter (the diameter being a function of the viscosity of the paint, paint flow rate and air pressure). The paint can be fed to the spray gun under the action of gravity, by aspiration, or from a pressured reservoir. Pneumatic pulverization requires an air flow rate of around 15 to 25 m^/h (at a

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pressure of 2 to 5 bars). The maximum paint flow rate is 500 cmVmin. The thickness of the paint layer produced by this method is on the order of 10 to 30 microns, and depends on the characteristics of the paint. Pneumatic pulverization produces paint films of excellent quality. However, because it requires that the paint be highly diluted, this process has a relatively low yield of approximately 40% (application yield defined as ratio of the mass of paint deposed on the surface divided by the mass of paint pulverized). Furthermore, the generation of paint fogs or mists makes this process highly polluting. Pneumatic pulverization can be used to apply the majority of liquid paints. It has been widely employed in a large number of industrial sectors, but the low yields and risks that it poses have significantly restrained its use, despite the excellent quality of the film it generates. It is still used in auto body work (applied to metallic bases using robots) and automobile repair (painting of auto bodies, manual application—see Figure 10), and in the furniture industry.

Figure 10. Application of paint by pulverization under pressure (high pressure air) (Courtesy of SOVAB-RENAULT).

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Health and Toxicology

Airless Pulverization Paint put under high pressure by a pump (100 to 300 bars) vaporizes as it leaves the nozzle of the spray gun at ambient pressure. However, the droplets formed in this manner are larger than those obtained using pneumatic pulverization. Airless pulverization can be used to apply up to 20 liters of paint per minute, and the diameter of the nozzles employed in this process vary between 300 and 900 microns. The major advantage of this technique is that it can be used to apply highly viscous paints with little to no dilution. Much less paint mist is generated with this process than with pneumatic pulverization, and the yield is somewhat higher—estimated to be on the order of 60 to 70%. The formation of larger droplets leads to the creation of a paint film with a less satisfying visual aspect than that obtained with pneumatic pulverization, and the paint films are always thicker than 30 microns. Airless pulverization can be used to apply all types of liquid paints, and is well adapted for applications on large surfaces that do not require extremely high finishing quality. It is used in the building industry, naval constructions, railway construction, for heavy machinery (e.g., cranes), and for tractor-trailer trucks. Airmix Pulverization This is a hybrid of the airless and pneumatic pulverization processes. In the airmix process, paint is put under a lower pressure (20 to 40 bars) than with the airless process, and additional low-pressure (0.5 to 2 bars) air jets are situated at the outlet of the nozzle in order to improve the pulverization of the paint and the homogeneity of the resulting jet. This process has a yield of 40 to 60%, and offers a combination of less fog formation (than the pneumatic process) and finer application (than the airless process). This process is found in more or less the same types of applications as mentioned for the airless process above. Low Pressure Pulverization (HVLP) Pulverization is obtained in the HVLP process by combining the paint with a high volume of air at low pressure (0.7 bars at maximum with respect to 2 to 5 bars in the other processes). This lower pressure enables one to obtain better control of the pulverization and to reduce the amount of paint that rebounds off the surface, thereby giving a higher yield than the pneumatic process. The paint flow rate is on the order of 150 to 200 cm^/min, and the air flow rate is 300 to 500 m^/h (compared to 15 to 20 m^/h for pneumatic pulverization). The yield can be as high as 85%. As is the case with pneumatic pulverization, the paint can be fed by aspiration, under the influence of gravity, or from a pressurized reservoir. The quality of the film obtained with this process is equivalent to that obtained via pneumatic pulverization, with film thicknesses of the same order of magnitude

Occupational Exposure to Organic Solvents

279

(10 to 30 microns). Given the fact that it can be used to apply highly viscous paints and that it offers a good yield, this process (the principle of which is not recent) might progressively replace the pneumatic pulverization process. Application by Electrostatic Pulverization In this type of process, pulverization is induced either uniquely through the effect of electrostatic forces applied to the paint ("pure" electrostatic), or through the use of one of the processes described earlier, plus a simultaneous or subsequent electrostatic charge being put on the paint ("assisted" electrostatic). In the pure electrostatic process, a thin film of paint is placed on a disk with tapered edges that is subjected to a high voltage. Once on the disk, the paint is subjected to a very strong force field. The paint film is then dispersed on the surface to be covered, first as thin filaments, then as charged droplets. Only moderately viscous paints with electrical conductivities between 5.0 x 10"^ to 5.0 x 10"^ S/cm can be applied with this process. The pulverization can be further improved with centrifugal forces if the disk is rotated. The diameter of the disk is on the order of 400-700 mm, with a rotational speed of 3,000 rpm. It is also possible to use a rotating bowl instead of a disk (diameter 70-250 mm, rotational speed 1,500 rpm). These purely electrostatic methods of pulverization are little used because they do not provide an acceptable application on all shapes of pieces. For example, Faraday cages are formed in hollow pieces, and because electrostatic forces no longer have any effect in such formation, the paint is not deposed on the surface. Assisted electrostatic methods are more practical because pulverization occurs with mechanical forces. The application of an electric field is simply a means of transporting the paint to the grounded surface to be treated. Pulverization can be obtained by: • Bowls, 30-80 mm in diameter turning at speeds of 15,000-40,000 rpm under a voltage of 50-120 kV; • Disks with diameters of 150-250 mm, turning at speeds of up to 30,000 rpm under voltages in the range 20-250 kV; • Processes with air (pneumatic, airless, airmix, HVLP) in which a charge is placed on the paint by means of needle electrodes with voltages of 50-100 kV that are placed directly in the paint jet. Such processes can have yields on the order of 75-80% because the paint droplets are attracted by the surface to be painted, with the possibility of painting the reverse side of the (grounded) object. The individual applying the paint must be equipped with conductive boots and gloves. All types of paint (including powders) can be applied using these electrostatic pulverization procedures if their conductivity lies within the range given above (there are certain additives that can be used to adjust the conductivity of paint). These electrostatic processes are often

280

Health and Toxicology

encountered in the household appliance, automobile body, agricultural machinery, metallic furniture, machine tool, and metal-forming sectors. Application by Dipping in Paint Baths Application by dipping is one of the simplest and oldest methods of coating. In addition to the classical methods of dipping pieces in solvent-based or waterbome paints, electrodeposition is also widely used in large-scale industrial production. Conventional Dipping In the conventional dipping process, the objects to be painted are plunged into a paint bath, then withdrawn. The liquid paint sticks to the surface and dries. In the rapid dipping process, the objects are withdrawn from the bath much more quickly than it takes for the paint to drain off of it, and there is difference in the thickness of the paint layers at the top and bottom of the piece. The paint used in this process should be relatively fluid, and the piece to painted should have a simple, regular shape with no sharp angles. The finish obtained with this process is very elementary. In the slow dipping process, the piece is withdrawn at a speed less than or equal to the speed at which the paint drains off, and it thus requires a mechanized installation. The paints used in this process are very viscous, and solvent vapors present above the bath can improve the surface tension of the film. It is possible to obtain very good finishes in one or two passages. Heating radiators, gear box casings, and door and window frames are examples of objects painted with the conventional immersion process. Electrodeposition Paints used in the electrodeposition process contain waterbome binders having organic solvent contents of less than 5%. With this method of application, the object to be painted serves as an electrode onto which a maro-ion is deposited due to a continuous current that passes between the object and the tank of the paint bath (the object to be painted must therefore be a conductor of electricity). In anaphoresis, the object to be painted is the anode, and in cataphoresis, it is the cathode. Anaphoresis has been gradually replaced by cataphoresis because a better penetration of paint into the subjectile (coating of inaccessible parts) and improved corrosion resistance are obtained with the latter. The binders used in cataphoresis should lead to the formation of positive maro-ions so that the paint is deposited on the negatively charged cathode (i.e., object to be painted) when an electric current is passed through the system. These binders are generally epoxides or acrylics with end groups composed of saturated

Occupational Exposure to Organic Solvents

281

amines. The neutralization of this type of end group with organic acids (e.g., acetic acid) leads to the formation of quaternary anmionium derivatives: R

I

(R-N-H+ + RCOO-) R that render the binder soluble in a mixture of water and organic solvents called cosolvents, which are often glycol or propylene glycol derivatives. When a current is passed through the bath, the paint is deposited on the object to be painted so long as the latter is not totally covered (so long as it remains possible to pass a current). The binder loses its charge upon contact with the subjectile and becomes insoluble in the dispersion medium. Upon being removed from the bath, the painted object is abundantly rinsed with water and then placed in an oven in order to allow the coat of paint deposited on the surface to definitively harden. Cataphoresis requires a very high initial investment. In addition to the electrophoretic dipping bath, it is necessary to install an ultrafiltration system in order to continuously maintain the quality of the paint bath. It is also necessary to have continuous current generator capable of delivering voltages of 200 to 400 V at intensities of 25 to 2,000 amperes. In addition, the pieces to be painted must undergo surface treatment in order to ensure that the entire surface is exposed in the same manner and to obtain a good level of adhesion of the deposited coating. Because of this, this type of process is used only on production lines that permit the automation of the procedure that includes: • One or more surface treatment baths; • The cataphoretic immersion bath (with ultrafiltration system); • Ovens for the hardening of the cataphoretic coating (140-180 °C). Automobile and bus bodies, certain automobile accessories, and several different types of industrial accessories are coated in this process, shown in Figure 11. Other Processes for the Application of Liquid Paints Brushes and rollers: These methods of application are used only in small-scale applications, home handiwork, and in the building industry. Rolling machines: The pieces to be painted are passed between rollers that coat their surfaces. There is a marked difference between the direct system in which the roller and object are moving in the same direction (layer deposited on the order of a few microns), and the indirect system in which the roller and the object are moving in opposite direction (the paint layers can be on the order of 100 microns). These processes are used in particular for the painting of wood panels (doors, planks).

282

Health and Toxicology

Figure 11. Production line for application of anti-corrosion undercoating by cataphoresis (Courtesy of SOVAB-RENAULT).

Flow-coating: The piece is coated by the aspersion of paint. The excess paint is recovered and recycled. This process can only be used to paint surfaces having simple shapes, and provides at best an average finish (not useful for the application of a brilliant finish). It is used for coating cranes and metallic doors. Curtain machines: The elements to be painted pass under a continuous curtain of paint. The paint is pumped into a reservoir having an adjustable slit at its base. As is the case with flow coating, the excess paint is recovered and recycled. This process provides a good surface finish, and is used essentially in the woodworking industry (panels, doors, planks) because it can only be used to paint planar surfaces. Coil coating: This process can be used to paint metal coiled around a spool. One process line, the metal is unwound from the spool, undergoes a surface treatment (degreasing and adhesive treatments), is covered using rollers (e.g., same type as inking rollers), is cooked in an oven at 230-250°C for approximately 30 seconds.

Occupational Exposure to Organic Solvents

283

and is rewound onto a spool in its initial form. The metal wires can be treated at speeds of up to 150 m/min. This process can only be used for flat metals. It provides an attractive finish with good resistance characteristics. It is used for metallic siding and roofing materials, caravans, and the bodies of small household appliances such as microwave ovens. Can coating: This is a process for painting metallic packaging materials (e.g., soft drink containers). Techniques employed can include sheet-to-sheet strip varnishing, pneumatic-electrostatic pulverization (inside the containers), and roller machines for decorations. SECTORS OF USE AND APPLICATIONS OF PAINT [49; 58-63] The methods of application of paint in different industrial sectors, the different types of paint used in these same sectors, as well as the types of solvents encountered are listed in the following tables. Industry The different activities included in this sector are: • building sector (Tables 7 and 8); • automobile bodies (automobile construction) (Table 9); • automobile repair (Table 10); • appliances, metallic furniture (Table 11); • aeronautics (Table 12); • furnishing (Table 13); • agricultural and heavy equipment (Table 14); • coil coating (Table 15); • anti-corrosion coatings (Table 16); • Naval construction (Tables 17 and 18). OCCUPATIONAL EXPOSURE TO PAINT SOLVENTS Epidemiological Studies Based on the epidemiological studies on industrial exposure to solvents presented in Table 19, it can be stated that atmospheric exposure to organic solvents during the stages of the application and fabrication of paints appears to be rather moderate. Aromatic hydrocarbons (especially toluene and xylenes) are frequently mentioned as being present in paints. The percutaneous absorption of solvents is not systematically taken into account even though it has an important impact on biological results when present.

284

Health and Toxicology Table 7 Building sector—exterior paints

Support Brick, plaster, concrete, stone

Nature of binders

Solvents encountered

Normal method of application

Silicates

Water

Acrylic and vinyl-acrylic emulsions "Pliolite"—styrene co-acrylics Silicones Two-component

Water + coalescing agents (glycol derivatives) Aromatic hydrocarbons. toluene, xylenes Water Esters, ketones, dilution by aromatic hydrocarbons

Brush, rollers Pulverization: pneumatic, airless, airmix

Wood

High oil content alkyds Acrylic emulsions Alkyd-based stains

White spirit Water + glycol derivatives White spirit

Brush, rollers Pulverization: pneumatic, airless

Ferrous metals

Modified alkyd-based primers

Aromatic hydrocarbons, toluene, xylenes Esters, ketones White spirit

Brush Pulverization: pneumatic, airless

Bi-component-based primers High oil content alkyd-based finishing paints Non-ferrous metals

Chlorinated rubber-based primers PVC copolymer-based primers High oil content alkyd-based finishing paints

Plastic (PVC panels/siding)

Chlorinated rubber-based primers Chlorinated polyethylenebased primers High oil content alkyd-based finishing paints

Esters, ketones, aromatic hydrocarbons, chlorinated hydrocarbons Ketones, esters, chlorinated hydrocarbons

Brush Pulverization: pneumatic

White spirit Esters, ketones, aromatic hydrocarbons, chlorinated hydrocarbons Ketones, chlorinated hydrocarbons White spirit

Brush Pulverization: pneumatic

The perfection of methods for the development of job-exposure matrices should lead to the better evaluation of the exposure of workers to organic solvents during the course of their occupational activities. Results obtained from the measurement of the levels of the metabolites of certain solvents confirms the interest of this type of test in the evaluation of occupational exposures because they can account for absorption by both inhalation and cutaneous contact. The long-term effects of exposure to solvents are often difficult to demonstrate because of the presence of confounding factors (age, lifestyle, general health), but generally concern the central and peripheral nervous systems. The use of aqueous-phase paints can lead to a significant improvement in the quality of the atmos-

Occupational Exposure to Organic Solvents

285

Table 8 Building sector—interior paints

Support Walls and Ceilings: Plaster Floors: Concrete

Nature of binders Acrylic or vinyl-acrylic emulsion High oil content alkyds

White spirit

Epoxy with no solvent

Glycidyl-based solvents

High solids loading epoxies Acrylics

Esters, ketones Esters, ketones, toluene, xylenes Water + cosolvents Water + coalescing agents Esters, ketones, dilution with aromatic hydrocarbons

Hydrosoluble epoxies Acrylic emulsions Monocomponent polyurethanes Wood

Bi-component polyurethanes Hydrosoluble acrylics

Wood and metal panelling/siding

Solvents encountered Water + cosolvents

Normal method of application Brush, rollers pneumatic Pulverization

Brush, rollers Pneumatic pulverization. HVLP

Esters, ketones dilution with aromatic-hydrocarbons Water + cosolvents (glycol derivatives)

High oil content alkyds

White spirit

Acrylic emulsions

Water -i- cosolvents (glycol derivatives)

Brush pneumatic pulverization

phere in the workplace, even if certain risks of exposure to some of the constituents of the paints are not totally eliminated (especially cosolvents and coalescing agents). EVOLUTION OF PAINT FORMULATIONS [80-84] At the current time, environmental and health and safety constraints appear to be the principal motivations in the modification of paint formulations. These constraints primarily target a reduction in the use of organic solvents: Insofar as occupational health and safety is concerned, the limiting exposure values of organic solvents are rapidly decreasing because of an ever-increasing understanding of their effects on the organism obtained from epidemiological studies. Legislation enacted to protect the environment is based on the best technology currently available, selected on the basis of the following aspects: • choice of products leading to use of the lowest possible quantities of solvents; • choice of application technique leading to the lowest possible emission of organic solvents; • purification of wastes by development of techniques for treatment of volatile organic compounds—incineration, catalytic incineration, biological treatment, adsorption, absorption, condensation.

286

Health and Toxicology Table 9 Automobile bodies

Nature of film applied

Nature of binders

Solvents encountered

Normal method of application

Anti-corrosion undercoating

Hydrosoluble epoxies

Water + cosolvents

Electrodeposition: cataphoresis

Intermediate layers (fillers)

Polyester-urethanes in a solvent phase Polyester—melamine in aqueous phase Polyester-urethane in aqueous phase

Esters, ketones, toluene. xylenes Water + cosolvents (glycol derivatives) Water + cosolvents (glycol derivatives)

Electrostatic with bowls rotating at high speeds

Non-metallic finishes:

Acrylics and urethanes in solvent phase

Esters, ketones, aromatic hydrocarbons

Electrostatic with bowls rotating at high speeds

Metallic finishes:

Acrylics and urethanes in solvent phase

Esters, ketones, aromatic hydrocarbons

Electrostatic with bowls rotating at high speeds

Base coat

Acrylics and urethanes in aqueous phase

Water + cosolvents (glycol derivatives)

Electrostatic with bowls rotating at high speeds (1 layer)

Alkyds—melamines

Esters, ketones, toluene. xylenes

Pneumatic (1 layer)

Acrylics, urethanes

Esters, ketones, toluene. xylenes

Electrostatic with bowls rotating at high speeds

Clear coat

Table 10 Automobile repair Solvents encountered

Normal method of application

Solvent-based acryUcs

Esters, ketones, aromatic hydrocarbons

Pneumatic pulverization, HVLP

Solvent-based acrylics and urethanes Combination Thermoset acryUcs in aqueous phase + Acrylic urethane in solvent phase

Esters, ketones, aromatic hydrocarbons Water + coalescent agents

Nature of film applied

Nature of binders

Under coat

Finished coat

Esters, ketones, aromatic hydrocarbons

Pneumatic pulverization. HVLP

Occupational Exposure to Organic Solvents

287

Table 11 Home appliances, metallic furniture Nature of film applied

Nature of binders

Finishing coat

Finishes for components of home appliances

Finishes for small appliances (ovens, microwave ovens, etc.)

Solvents encountered

Normal method of application

Thermoset acrylics

Esters, ketones, toluene, xylenes

Electrostatic: disks or bowls turning at high speeds

Hydrosoluble epoxies

Water + cosolvents

Electrodeposition

Acrylics urethanes

Esters, ketones, toluene, xylenes

Electrostatic

Polyesters, urethanes

Esters, ketones, aromatic hydrocarbons

Coil coating

Table 12 Aeronautics Nature of film applied

Nature of binders

Solvents encountered

Normal method of application

Base coat primer

Wash primer

Isopropyl alcohol, butyl alcohol

Pneumatic pulverization,

Anti-corrosion

Bi-components epoxyamine coatings Epoxy-amino amids

Esters, ketones, aromatic hydrocarbons Esters, ketones, aromatic hydrocarbons Esters, ketones, aromatic hydrocarbons

HVLP, electrostatic

Epoxy-urethane Finishing

Bi-component acrylicurethanes

Esters, ketones, aromatic hydrocarbons

It is clear that the constraints push the development of paint formulations in two principal directions: • reduction in the use of organic solvents in the formulation itself; and • reduction of waste generated during the application step. As far as the reduction of the use of organic solvents in paints is concerned, paint formulators tend to be taking the following directions:

288

Health and Toxicology Table 13 Furnishing

Nature of film applied

Undercoats and finishing coats

Nature of binders

Solvents encountered

Solvent-based nitro cellulose

Alcohols, esters, ketones thinnable in aromatic hydrocarbons

Acrylics, urethanes

Esters, ketones, aromatic hydrocarbons

Unsaturated polyesters

Styrene

Acrylics curing with UV (or electron beam)

Unsaturated acrylic monomers

Normal method of application

Pneumatic pulverization, HVLP electrostatic Dipping

Unsaturated polyesters that Styrene can be reticulated with UV(or electron beam)

Table 14 Agricultural and heavy machinery Nature of film applied

Nature of binders

Solvents encountered

Normal method of application

Cranes

Solvent-based, air-drying White spirit, aromatic alkyds (high oil content) hydrocarbons

Flow coating Airless

Agricultural machinery, heavy machinery

Medium oil content alkyds-melamines

Airless

Esters, ketones, aromatic hydrocarbons

• increasing the solids loading of the paint. This is associated with the perfection of new application procedures for these products having special theological properties. • the use of water. Development of applications of waterborne and waterthinnable paints in new industrial sectors. It should be noted that in certain sectors, the introduction of this type of paint is being slowed down due to the reluctance of certain users to change processes, and especially due to the high investment costs needed to change over from traditional formulations to waterbased formulations, etc.

Occupational Exposure to Organic Solvents

289

Table 15 Coil coating Nature of film applied

Nature of binders

Solvents encountered

Epoxy-based primers rich Esters, ketones, aromatic Steel, galvanized steel, hydrocarbons m zmc aluminum Esters, ketones, aromatic Acrylic primers hydrocarbons Polyester primers Esters, ketones, aromatic hydrocarbons Acrylic finishes Esters, ketones, aromatic hydrocarbons Polyester finishes Esters, ketones, aromatic hydrocarbons PVDF Isophrone, (polyvinylidene dimethylphthalate fluoride) finishes Silicon modified Esters, ketones, aromatic polyester finishes hydrocarbons

Normal method of application

Coil coating

Table 16 Anti-corrosive Coatings Nature of film applied

Nature of binders

High oil content alkyds Bridges (roads. railways). Electric pylons. Chlorinated rubbers Storage reservoirs. Chemical plants, Pipelines, PVC (plastisols. Containers organosols)

Solvents encountered

White spirit, aromatic hydrocarbons Aromatic hydrocarbons, chlorinated compounds. esters, ketones Dioctyl and dibutylphthalates, ketones, esters Epoxy resins (hardened Aromatic hydrocarbons with amino amid resins, (toluene, xylenes) with or amine adducts) esters, ketones and alcohols (butanol) Ethyl silicates, rich in Ethanol, isopropanol. zmc ketones, esters

Normal method of application Brush Rollers Pulverization: pneumatic, airless

290

Health and Toxicology Table 17 Naval construction (Pre-treatment of steel)

Support Steel

Nature of binders Wash primer Hardened epoxy primers with amino amid resins, or with amine adducts, or with polyamides Ethyl silicates with low zinc contents

Solvents encountered Isopropyl alcohol, isobutyl alcohol Aromatic hydrocarbons, esters, ketones, alcohols

Normal method of application Automated pneumatic pulverization

• the use of reactive diluents that participate in the filmification of the paint. Such reactive diluents are akeady being used in acrylic coatings that harden under ultraviolet light, unsaturated polyesters, and coatings that are reticulated by radiation. • the use of coating powders. Insofar as waste reduction is concerned, the following developments should be noted: • the development of cataphoretic processes; • the development of coil-coating applications; • the development of application by electrostatic pulverization and low pressure HVLP; and • the use of supercritical carbon dioxide instead of the solvents used for the dilution of paints at the moment of application. Research in the field of paints has really taken a large step in the direction of the protection of the environment and the improvement of conditions in the workplace. Even if these improvements were only undertaken following legislative changes (workplace, environment), these efforts should be stressed. By taking such a leap, the paint industry has shown its maturity and real will to integrate itself with our environment.

Occupational Exposure to Organic Solvents

291

Table 18 Naval construction (structural aspects) Support

Nature of binders

Superstructures Medium oil content alkyds Medium oil content alkyds Epoxy primers Acrylics urethanes (polyurethanes) Hulls, bridges

Polyvinyl acetochlorides

Chlorinated rubbers Epoxy primers Acryhcs, urethanes (polyurethanes) Brai epoxy Reservoirs

Anti-fouling

Ethyl silicate rich in zinc

Solvents encountered

Normal method of application

Esters, ketones, aromatic hydrocarbons White spirit, aromatic hydrocarbons Aromatic hydrocarbons, esters, ketones, alcohols, (butanol) Esters, ketones, aromatic hydrocarbons

Brush, rollers Pneumatic pulverization, HVLP

Esters, ketones, chlorinated solvents, dilution with aromatic hydrocarbons Aromatic hydrocarbons, chlorinated compounds, esters, ketones Aromatic hydrocarbons, esters, ketones, alcohols Esters, ketones, aromatic hydrocarbons Esters, ketones, aromatic hydrocarbons

Pneumatic pulverization, airless

Ethanol, isopropanol, ketones, esters Aromatic hydrocarbons, esters, ketones

Epoxy primers hardened with amine adducts or polyamides or isocyanates Acryliques urethanes Aromatic hydrocarbons, (polyurethanes) esters, ketones

Pneumatic pulverization, airless

Acrylic copolymers (triaryl or trialkyl-tin acrylic esters)

Pneumatic pulverization

Esters, ketones, aromatic hydrocarbons

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Table 19 Epidemiologicalstudies on industrial exposure to solvents

Authors

Activity

No. subjects

Nature of solvents, level of exposure

Studied effects, tests, results, conclusions

4 groups for a total of 41 1 subjects

Hydrocarbons, mineral oils, toluene, xylenes, butanol Exposure < VME

Mechanism of action of possible effects of solvents on the kidneys. Circulating laminin antibodies, serum laminin, soluble E. selectin. The two groups exposed to mineral oils and solvents gave different test results.

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Stevenson et al. [64]

Paint Production

Foo S.C. et al. [65]

Paint Production

Glass D.C. et al. [66].

2 Paint Production

Ukai H. [57]

21 exposed 21 controls

303 exposed 135 controls

Mixture of paint solvents Exposure < 0.09 VME for each solvent, and toxicity index between 0.03 and 0.24

Neuro-behavioral effects. Tests: digit span, grooved pegboard, digit symbol, Z-score. Test results are lower at statistically significant level for exposed with respect to control subjects.

Creation of job-exposure matrix based on: - results of atmospheric samplings - estimations of exposure levels - historical data on work stations

Trials for retrospective estimation of exposure to solvents in two paint manufacturing plants.

Mixture of solvents Toluene: g.m = 18 ppm MEK: g.m = 16 pprn IPA: g.m = 7 pprn Ethyl acetate: mg = 9 ppm

Monitoring of toluene exposure with badges, biological analyses, analysis of urinary hippuric acid, hematological studies, analysis of biological serum to monitor liver function, and follow-up

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Rees D. et al. [68]

Colvin M. et al. [69]

Paint Production

Paint Production

Paint Production

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68

89 exposed

Toxicity index most often less than one, with a maximum value of 8 (g.m = geometric mean)

questionnaire on subjective evaluation of symptoms. Hematology and hepatic functions do not show significant differences. Objectives symptoms essentially include local imtations and depression of nervous system. Only toluene toxicity demonstrated, with no visible synergetic effect of other solvents.

Mixture of solvents Exposure less than 0.72 TLV-TWA

Objective of study was to look at validity of a battery of 17 tests used to estimate the neuro-psychological effects of solvents.

Mixture of organic solvents at low concentrations. Exposure of long duration for most subjects.

Estimation of exposure based on measurements taken by company.

Study of hepatic effects of alcohol consumption combined with exposure to solvents. Examination of hepatic enzyme activity levels: activity of gamma glutamyl transferase and aminotransferase aspartate. Enzymatic activity levels higher in those exposed, but conclude that there are no visible effects on liver (excessive alcohol consumption is an important factor.) Study of neuro-behavioral effects caused by chronic exposure to solvents. Test: World Health Organization

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Table 19 Epidemiologicalstudies on industrial exposure to solvents

Authors

Activity

No. subjects

Nature of solvents, level of exposure

Cumulative exposure index established for homogeneous group of exposed workers. Exposure < VME

Ruijten MW Paints et al. [70] in naval construction

Wieslander Paints in G . et al. [71] building industry

415 exposed 18 controls

Studied effects, tests, results, conclusions

Neurobe-havioral Core Test Battery, NES-2 computerized battery plus 4 South African tests. Cumulative exposure index linked to speed of visual-motor tracking, that indicates possible precocious neurotoxic effects.

Mixture of solvents and xylenes

Study of the neuro-behavioral effects of long term exposure to xylenes and mixtures of solvents. Tests: questionnaire on observed symptoms, measurement of peripheral sensory, motor nerve parameters and computerized performance tests. Neuro-physiological parameters can be linked to exposure indices. Behavioral tests reveal problems in visuomotor performance of exposed subjects.

Mixture of paint solvents in aqueous phase, white spirit, toluene, ethyleneglycol and propyleneglycol derivatives

Study of the effect of exposure to aqueous phase paints in respiratory functions using questionnaires. Test: questionnaire on observed symptoms sent to exposed workers (self-filled questionnaire).

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The study concluded that there was a cumulative risks of damage to the respiratory function of painters working on buildings, especially when they used solvent-based paints. Wieslander Paints in G. et al. [72] building industry

Kishi R. et al. [73]

Appliers of industrial paints

255 exposed 302 controls

81 exposed

Low exposure to solvent vapors

Mixture of paint solvents at low concentrations

Study of the effects of water-based paints on the skin and eyes. Self-questionnaire on symptoms observed. Water-based paints give better working environment than do solvent-based paints. Very few effects on skin and eyes described. Less discomfort and irritation with waterbased paints. Study of neuro-behavioral effects of chronic exposure to organic solvents. Self-questionnaire on symptoms felt by workers, and neuro-psychological examinations. Study showed that: Certain behavioral tests are well-adapted to the detection of the effects of an exposure to solvents at low concentrations (e.g., Santa Ana co-ordination tests and Benton visual retention test turned out to be significantly useful for the detection of exposure to toluene).

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Table 19 Epidemiologicalstudies on industrial exposure to solvents

Authors

No. subjects

Activity

Nature of solvents, level of exposure

Studied effects, tests, results, conclusions

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However, no valid model was observed in terms of describing the effects of solvents on the neuro-behavioral function corresponding to type I problems of the central nervous system (classification established by the WHO). Triebig G., Appliers of 168 exposed Lang C. [74] building and 8 1 controls industrial paints

Daniel1 W. et al. [75]

Paints used in automobile repair

92 exposed : - 39 highly -

exposed 24 moderately

Mixture of paint solvents at concentrations inferior to threshold limit values

Using of brain imaging techniques to evaluate effects of exposure to solvents. Techniques used: X-ray computed tomography (CAT). It was not possible to establish a correlation between neuro-psychological tests, exposure to solvents and CAT scans. It was concluded that long-term exposure to solvents (at levels less that the TLV) does not cause any accumulated atrophication of the brain (when corrections are made for age, alcohol use, and other personal factors in the subjects studied). Study of the effects of solvents using neuro-psychological performance tests and self-filled questionnaires on symptoms observed.

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Paints in automobile body works and printers' shops

exposed 29 slightly exposed. 24 controls

113 in automobile body works work and printing, 81 controls

Application 105 exposed of paint 58 controls by pulverization

(table continued on next page)

It was observed that the exposed subjects reported more symptoms than did control group. The highly exposed subjects performed significantly more poorly on tests of visual perception and memorization, but no differences were noted for visuo-motor performances, depressive states, or motor control. Toluene, xylenes, MEK and mixtures of solvents. Exposure level: degree of toxicity between 0.10 and 2.29.

Toluene, xylenes, trimethylbenzenes, aliphatic hydrocarbons (heptane), ethyl and butyl acetate. Level of exposure: - None of solvents exceeded TLV, - In certain cases the toxicity index as high as 2 or 3. Duration of exposure: 10 to 44 years.

Neuro-behavioral effects caused by exposure to solvents. Of the 7 tests used, only 4 revealed evidence of differences between exposed and control subjects: simple retention time, Santa Ana dexterity, digit symbol, and Benton visual test. Study of neuro-toxicity of solvents on individuals applying paints by pulverization. Estimation of solvent concentrations in the body at time of study by biological sampling did not reveal evidence of exposure to solvents. Only the dosing of methylhippuric acid gave higher results in exposed subjects than in controls (80 mg/l vs. <20 mg/l)-this is a sign of exposure to xylenes. Estimation of past exposure by questionnaire on duration of exposure, quantity of solvent involved, ventilation and effects on health.

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Table 19 Epidemiologicalstudies on industrial exposure to solvents

Authors

Triebig G . et al. [79]

Activity

No. subjects

Application 105 exposed of paint by 58 controls pulverization

Nature of solvents, level of exposure

Toluene, xylenes, trimethylbenzenes, aliphatic hydrocarbons (heptane), ethyl and butyl acetate. Level of exposure: - None of solvents exceeded TLV, - In certain cases the toxicity index as high as 2 or 3. Duration of exposure: 10 to 44 years.

Studied effects, tests, results, conclusions

After examination of possible confounding factors, only 83 of the exposed subjects and 42 of the controls were kept for study analysis. Conclusions of study: *No damage to central or peripheral nervous system. *Depression, loss of interest and lack of concentration more prevalent in exposed subjects than controls. *The psychological and performance tests did not reveal any statistically significant differences (after adjustment). This suggests that there is only a very slight risk of mental dysfunction due to exposure to solvents. *A relationship was established between the subjective complaints on healthrelated problems and long-term exposure; however, the effects of age cannot be totally discounted.

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Toluene: a.m = 4 ppm Xylenes: a.m = 0.9 ppm Exposure level estimated to be 8.4% of TWA defined by the ACGIH. Exposure index varied from 0 to 0.62. - 49 exposed workers had had no cutaneous exposure to the solvents - 33 had had slight or accidental contact, - 5 had had moderate to high cutaneous exposures. (a.m = arithmetic mean)

Study of the absorption of organic solvents by inhalation and cutaneous contact. Levels of urinary methylhippuric acids were rather low with respect to the BE1 defined by the ACGIH (median value of 2% for range of values from 0 to 12% of the BEI). However, some of these values are strongly correlated with atmospheric concentrations of xylenes and cutaneous contact with this solvent. It appears that a cutaneous contact with xylenes lasting for 15 minutes is equivalent to a daylong atmospheric exposure at the work station. The study concluded that atmospheric sampling alone significantly underestimates the danger of solvent exposure if cutaneous contact is present.

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REFERENCES 1. Soule, R. D. "Industrial Hygiene Sampling and Analysis." In: Patty's Industrial Hygiene and Toxicology, Vol. IB, Clayton G. D. and Clayton F. E. (Eds.), New York, John Wiley & Sons, 1994, pp. 73-135. 2. Institut de Recherche en Sante et en Securite du Travail au Quebec. "Guide d'echantillonnage des contaminants de I'air en milieu du travail." IRSST, Montreal, 1994. 3. Lynch, J. "Industrial Hygiene." In: Kirk Othmer Encyclopaedia of Chemical Technology, Vol. 14, 4th ed.. New York, John Wiley & Sons, 1994, pp. 199-219. 4. Mulik, J. D. and Lewis, R. G. "Recent Developments in Passive Sampling Devices." In: Advances in Air Sampling, ACGIH, Lewis Publishers, Chelsea, 1988, pp. 117-131. 5. Roach, S. A. "Alternative Ways of Monitoring Occupational Exposure." In: Exposure Assessment of Epidemiology and Hazard Control, Rappaport, S. M. and Smith, T. J. (Eds.), Lewis Pubhshers, Chelsea, 1991, pp. 3-20. 6. American Conference of Governmental Industrial Hygienists. "1994-1995 Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices." Cincinnati, 1994. 7. Holbrook, M. T. "Methylene chloride." In: Kirk Othmer Encyclopaedia of Chemical Technology, Vol. 5, New York, John Wiley & Sons, 1993, pp. 1,041-1,050. 8. Vincent, R. et al. "Occupational Exposure to Methylene chloride," INRSCahiers de Notes Documentaires, 1994, No. 155, pp. 157-167. 9. Kubik, V. et al. "Metabolism of Dihalomethanes to Carbon monoxide. I. In vivo Studies." Drug Metabolism Disposition, 191 A, No. 2, pp. 53-57. 10. Ahmed, A. and Anders, M. "Metabolism of Dichloromethanes to Formaldehyde and Inorganic Halide." Biochemistry Pharmacology, 1978, No. 27, pp. 2,021-2,025. 11. Andersen, M. E. et al. "Physiologically Based Pharmacokinetics and the Risk Assessment Process for Methylene Chloride." Toxicology and Applied Pharmacology, 1987, No. 87, pp. 185-205. 12. Fagin, J. et al. "Carbon Monoxide Poisoning Secondary to Inhaling Methylene Chloride." British MedicalJoumal, 1980, No. 281, p. 1,461. 13. World Health Organization (WHO) "Methylene chloride," Environment Health Criteria No. 32, 1986, 63 pages. 14. Jongen, W. M. F. "The Effect of Glutathione Conjugaison and Microsomal Oxydation on the Mutagenicity of Dichloromethane in Salmonella Thyphimurium." Mutation Research, 1982, Vol. 95, pp. 183-189. 15. Nestmann, E. et al. "Mutagenicity of Paint Removers Containing Dichloromethane." Cancer Letters, 1981, No. 1, pp. 295-302.

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301

16. Hallier, E. et al. "Metabolism of Dichloromethane (Methylene Chloride) to Formaldehyde in Human Erythrocytes: Influence of Polymorphism of Glutathione Transferase Theta (GST. Tl-1)." Archives of Toxicology, 1994, No. 68, pp. 423-427. 17. Kimura, E. T. et al. "Acute Toxicity and Limits of Solvent Residue for Sixteen Organic Solvents." Toxicology and Applied Pharmacology, 1971, No. 19, pp. 699-704. 18. Bonnet, P. et al. "Determination de la concentration letale 50 des principaux hydrocarbures aliphatiques chlores chez le rat." Archives des maladies professionnelles, 1978, No. 41, pp. 317-321. 19. Gradiski, D. et al. "Toxicite aigiie comparee par inhalation des principaux hydrocarbures chlores." Archives des Maladies Professionnelles, 1978, No. 39, pp. 249-257. 20. Torkelson, T. R. "Halogenated Aliphatic Hydrocarbons—Methylene Chloride." In: Patty's Industrial Hygiene and Toxicology, Vol. 2E, 4th ed., Clayton, D. C and Clayton, F. E. (Eds), New York, John Wiley & Sons, 1994, pp. 4,034-4,045. 21. Putz, V. R. et al. "A Comparative Study of the Effect of Carbon monoxide and Methylene chloride on Human Performance." Journal of Environmental Pathology and Toxicology, 1979, No. 5, pp. 97-112. 22. Winek, C. L. et al. "Accidental Methylene chloride Fatality." Forensic Science International, 1985, No. 18, pp. 165-168. 23. Logemann, E. and Van der Smissen, G. "Intoxikation mit einem Dichlormethan-haltigen Abbeizmittel." Arch. KriminoL, 1991, No. 108, pp. 159-166. 24. Shinomiya, T. and Shinomiya, K. "A Case of Poisoning by Methylene chloride during the Spraying of a New Cargo Vessel." Acta Med. Leg. Soc, 1985, No. 35, pp. 135-156. 25. Leikin, J. B. et al. "Methylene chloride: Report of Five Exposures and Two Deaths." American Journal of Emergency Medicine, 1990, No. 8, pp. 534-541. 26. Wells, G. G. and Waldron, H. A. "Methylene chloride Bums." British Journal of Industrial Medicine, 1984, No. 41, p. 420. 27. Weber, M. et al. "Intoxication aigiie par le chlorure de methylene et methanol par voie percutanee." Archives des maladies professionnelles, 1990, No. 51, pp. 103-106. 28. Burek, J. D. et al. "Methylene chloride: A Two-year Inhalation Toxicity and Oncogenicity in Rats and Hamsters." Fundamental and Applied Toxicology, 1984, No. 4, pp. 30-^7. 29. National Toxicology Program. "Toxicology and Carcinogenesis Studies of Dichloromethane in F3441 N Rats and B6C3F1 Mice (Inhalation Studies)." Washington, U.S. Department of Health and Human Services, 1986, NTPTR 306, NIH Pub. No. 862562.

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30. Friedlander, B. R. et al. "Epidemiologic Investigation of Employees Chronically Exposed to Methylene chloride. Mortality Analysis." Journal of Occupational Medicine, 1978, No. 20, pp. 657-666. 31. Ott, M. G. et al. "Health Evaluation of Employees Occupationally Exposed to Methylene chloride." Scandinavian Journal of Work, Environment and Health, 1983, No. l,pp. 1-38. 32. Hearne, F. T. et al. "Methylene chloride Mortality Study: Dose-response Characterization and Animal Model Comparison." Journal of Occupational Medicine, 1987, No. 3, pp. 217-228. 33. Hearne, F. T. et al. "Absence of Adverse Mortality Effects in Workers Exposed to Methylene chloride: An Update." Journal of Occupational Medicine, 1990, 32 (3), pp. 234-240. 34. Soden, K. J. "An Evaluation of Chronic Methylene chloride Exposure." Journal of Occupational Medicine, 1993, 35 (3), pp. 282-286. 35.1 ARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon, CIRC/IARC, 1987, suppl. 7, pp. 194-95. 36. Snyder, R. W. et al. "Pulmonary Toxicity Following Exposure to Methylene chloride and its Combustion Product, Phosgene." Chest, 1992, No. 101, pp. 860-861. 37. OSHA Proposed Standard on Methylene chloride (56 FR 57082-83). 38. INRS "French Limit Values for Occupational Exposure to Chemicals." INRS-Cahiers de Notes Documentaires, 1995, No. 153, pp. 557-574. 39. Deutsche Forschungemeinschaft "List of MAK and BAT Values 1994." New York, VCH Publishers, 1994, Report No. 30, p. 40, p. 138. 40. Anundi, H. et al. "High Exposures to Organic Solvents among Graffiti Removers." International Archives of Occupational and Environmental Health, 1993, No. 65, pp. 247-251. 41.McCammon, E. S. et al. "Exposure of Workers Engaged in Furniture Stripping to Methylene chloride as Determined by Environmental and Biological Monitoring." Applied Occupational Environmental Hygiene, 1991, No. 5, pp. 371-379. 42. Hall, R. M. et al. "Control of Methylene chloride—furniture stripping dip tank." Applied Occupational Environmental Hygiene, 1995, No. 3, pp. 188-195. 43. Hall, A. M. and Rumack, B. H. "Methylene Chloride in Furniture Stripping Shops: Ventilation and Respirator Use Practices." Journal of Occupational Medicine, 1990, 30 (1), pp. 33-37. 44. Shusterman, D. et al. "Methylene chloride Intoxication in a Furniture Refinisher. A Comparison of Exposures Estimates Utilizing Workplace Air Sampling and Carboxyhemoglobin Measurements." Journal of Occupational Medicine, 1990, 30 (5), pp. 451^54.

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45. Meyer, E. S. and Peterson, J. A. "Organic Vapor (ov) Respirator Cartridge and Canister Testing against Methylene chloride." Applied Occupational Environmental Hygiene, 1993, No. 6, pp. 553-563. 46. National Institute for Occupational Safety and Health (NIOSH). "Questions and Answers. Methylene chloride Control in Furniture Stripping." DHHS (NIOSH), 1993, Pub. No. 93-133. 47. Vincent, R. et al. "Occupational Exposure to Organic Solvents during Paint Stripping and Painting Operations in the Aeronautical Industry." International Archives of Occupational and Environmental Health, 1994, No. 65, pp. 377-380. 48. Powles, R. and Hans, H. "Paint Stripping with Non-toxic Chemicals." SURF AIR X: 10th International Conference on Surface Treatments in the Aeronautical and Aerospace Industries, 8-10 June 1994, Cannes (France). Proceedings. 49. Stoye, D. "Paints, Coatings and Solvents." Weinheim: VCH Publishers 1993, pp. 201-211, pp. 241-264. 50. Cicolella, A. "Les ethers de glycol. Etat actuel des connaissances. Perspectives de recherche." INRS-Cahiers de Notes Documentaires, 1992, No. 148, pp. 359-378. 51. Cavigneaux, A. "De la toxicite des peintures." Surfaces, 1991, No. 185, pp. 27-31. 52. David, M. "Les effets pathogenes des peintures," Biennale prevention des risques professionnels.—CRAM Dijon, 4 November 1995, Besangon (France). 53. Juntunen, J. "Neurotoxic Syndromes and Occupational Exposure to Solvents," Environmental Research, 1993, 60 (1), pp. 98-111. 54. de Ceaurriz, J. "Les solvants organiques et le systeme nerveux"—Conference Internationale Copenhague, 2--4 May 1990—INRS-Cahiers de Notes Documentaires, 1990, No. 140, pp. 733-735. 55. Matheson D. "Solvents, Industrial." Encyclopedia of Occupational Health and Safety, Vol. 2, 3rd ed., 1991, pp. 2,085-2,088. 56. Langman, J. M. "Xylene: its Toxicity, Measurement of Exposure Levels, Absorption, Metabolism and Clearance." Pathology, 1994, 26 (3), pp. 301-309. 57. Uka'i, H. et al. "Occupational Exposure to Solvent Mixtures: Effects on Health and Metabolism." Occupational and Environmental Medicine, 1994, 51 (8), pp. 523-529. 58. Remontet, P. "Peintures industrielles liquides." Biennale prevention des risques professionnels—CRAM Dijon, 11 April 1995, Besangon (France). 59. De Vilbiss-Ransburg, "La pulverization basse pression." Galvano-Organo Traitements de Surface, No. 637, 1993, pp. 667-670. 60. Desfilhes, P H. "Basse pression, le brouillard s'eclaircit." Plastiques modernes et elastomeres, 1994, pp. 61-62.

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61. Saatwerber, D. and Wuppertal, H. "Protection anticorrosion par les peintures industrielles." Galvano-Organo—Traitements de surface, No. 619, 1991, pp. 893-900. 62. National Safety Council. "Electrostatic Paint Spraying and Detearing." Data Sheet 1-468, rev. 91, pp. 1-5. 63. Catherin, J.Y. "Peintures industrielles 1'antipollution moteur du developpemenV' Industries et techniques, 1991, pp. 131-144. 64. Stevenson, A. et al. "Biochemical Markers of Basement Membrane Disturbances and Occupational Exposure to Hydrocarbon and Mixed Solvents." Quarterly Journal of Medicine, 88 (1), 1995, pp. 23-28. 65. Foo, S. C. et al. "Chronic Neurobehavioural Effects in Paint Formulators Exposed to Solvents and Noise." Annals Academy Medecine Singapore, 23 (5), 1994, pp. 650-654. 66. Glass, D.C. et al. "Retrospective Assessment of Solvent Exposure in Paint Manufacturing"—Occupational and Environmental Medicine, 1994, 51 (9), pp. 617-625. 67. Nell, V. et al. "Neuropsychological Assessment of Organic Solvents Effects in South Africa: Test Selection, Adaptation, Scoring and Validation Issues." Environmental Research, 1993, 63 (2), pp. 301-318. 68. Rees, D. et al. "Solvent Exposure, Alcohol Consumption and Liver Injury in Workers Manufacturing Paint." Work Environmental Health, 1993, 19 (4), pp. 236-244. 69. Colvin, M. et al. "A Cross-sectional Survey of Neurobehavioral Effects of Chronic Solvent Exposure on Workers in a Paint Manufacturing Plant." Environmental Research, 1993, 63 (1), pp. 122-132. 70. Ruitjen, M W. et al. "Neurobehavioral Effects of Long-term Exposure to Xylenes and Mixed Organic Solvents in Shipyard Spray Painters." Neurotoxicology, 1994, 15 (3), pp. 613-620. 71. Wieslander, G. et al. "Occupational Exposure to Water-based Paints and Self-Reported Asthma, Lower Airway Symptoms, Bronchial Hyperresponsiveness and Lung Function." International Archives of Occupational and Environmental Health, 1994, 66 (4), pp. 261-267. 72. Wieslander, G. et al. "Occupational Exposure to Water-based Paint and Symptoms from the Skin and Eyes." Occupational and Environmental Medicine, 1994, 51 (3), pp. 181-186. 73. Kishi, R. et al. "Neurobehavioral Effects of Chronic Occupational Exposure to Organic Solvents among Japanese Industrial Painters." Environmental Research, 1993, 62 (2), pp. 303-313. 74. Triebig, G. and Lang, C. "Brain Imaging Techniques Applied to Chronically Solvent Exposed Workers: Current Results and Chemical Evaluation." Environmental Research, 1993, 61 (2), pp. 239-250.

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75. Daniell, W. et al. "Neuropsychological Performance and Solvent Exposure among Car Body Repair Shop Workers." British Journal of Industrial Medicine, 1993, 50 (4), pp. 368-377. 76. Lee, S. H. "A Study on the Neurobehavioral Effects of Occupational Exposure to Organic Solvents in Korean Workers." Environmental Research, 1993, 60 (2), pp. 227-232. 77. Triebig, G. et al. "Neurotoxicity of Solvent Mixtures in Spray Painters. I. Study Design, Workplace Exposure, and Questionnaire." International Archives of Occupational and Environmental Health, 1992, 64 (5), pp. 353-359. 78. Triebig, G. et al. "Neurotoxicity of Solvent Mixtures in Spray Painters. II. Neurologic, Psychiatric, Psychological, and Neuroradiologic Findings." International Archives of Occupational and Environmental Health, 1992, 64 (5), pp. 361-372. 79. Daniell, W. et al. "The Contributions to Solvent Uptake by Skin and Inhalating Exposure." American Industrial Hygiene Association Journal, 1992, 53 (2), pp. 124-129. 80. Laout, J. C. and Joly, M. "L'Industrie des peintures face aux defis des annees 2000." Galvano-Organo—Traitements de surface. No. 647, 1994, pp. 497-499. 81. Joly, M. "Les peintures: composition, mise en oeuvre." Biennale prevention des risques professionnels—CRAM Dijon, 4 November 1995, Besan^on (France). 82. Duesco, N. "Cabines de peinture et environnement: la reduction des emissions de COV." G2i\v^.no-OrgMvo-Traitements de surface. No. 647, 1994, pp. 503-507. 83. Clin, I. "Les peintures ecologiques de I'an 2000." Galvano-Organo-rraiYements de surface, No. 647, 1994, p. 467. 84. Moudden, B. "CO2 supercritique, une solution aux emissions de COV." Plastiques modernes et elastomeres, 1993, pp. 58-60,

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CHAPTER 13 OCCUPATIONAL EXPOSURE TO METALLIC COBALT G. Mosconi, M. Bads, and P. Leghissa Department of Occupational Medicine Ospedali Riuniti Bergamo Bergamo, Italy C. Sala Department of Safety and Hygiene Chemical Division USL 16 Lecco, Italy CONTENTS INTRODUCTION, 307 THE FIRST STUDY, 309 THE SECOND STUDY, 314 CONCLUSION, 322 REFERENCES, 324 INTRODUCTION Cobalt is an essential oligoelement for many human beings. It enters in the composition of vitamin B12. For the general population, food and beverages represent the main source of cobalt exposure. Traces of cobalt are also present in cement and various household products. Cobalt is produced primarily as a by-product of the mining and processing of copper and nickel ores, which usually contain <1% cobalt. Cobalt refineries manufacture the metal as cathodes or powders, which are mainly used for the production of various alloys and cobalt compounds. The cobalt alloys include: (a) the strong and corrosion-resistant superalloys, (b) the magnetic alloys, (c) the high-strength steels, (d) the electrodeposited alloys, (e) the special alloys used for surgical implants and (f) the hard-metal alloys (tungstencarbide, cemented carbide, or Widia) that are used for the production of tips of saws, cutters, drill belts, and other devices that must be resistant to heat and wear. Such objects often consist of a hard metal cemented to the body of the tool. Cobalt powder is also used in the production of

307

308

Health and Toxicology

diamond abrasive or other tools used for the grinding and sharpening of tools, for stone cutting, and for polishing diamonds. In hard metal alloys and diamond abrasive, the Co acts as a binder for the different powder components. Except in the production of cobalt powders, the uses of these products involve exposure not only to cobalt but also to other substances such as tungsten carbide, iron and other metals and diamond that may modulate the biological reactivity of cobalt, which results as the main cause of disease for occupationally exposed workers. Toxic effects have been reported following inhalation of cobalt-containing dusts. Absorption through the skin can occur, but is low. In workers exposed to pure cobalt metal powder, cobalt salts, and cobalt-containing dusts, the two main target organs are the skin and the respiratory tract. Table 1 summarizes the clinical findings in relation to exposure to Co and its salts. Contact dermatitis, bronchial asthma, and irritative forms of the respiratory apparatus are well-established diseases in relation to Co exposure. However, Co exposure alone should be an essential but not sufficient condition for developing lymphocytic alveohtis and interstitial lung fibrosis hard metal disease (HMD) [30]. The concomitant presence of other metals in airborne dusts (e.g., W, Ta, Mo, Ni, Cr, V), lubricating oils, or diamond powder should increase the probability of the disease. Concerning pulmonary disease, with the exclusion of dose-dependent irritative forms, a role of Co in inducing individual hypersusceptibility is highly probable [12]. The analysis of the bronchoalveolar lavage (BAL) from subjects affected by lymphocytic alveolitis and interstitial lung fibrosis supports this theory as indicative of an immunological response both in diseased and asymptomatic individuals [18, 37]. However, it is not possible at present to establish if a single pathogenetic mechanism is responsible for the clinical findings described or if the latter should be considered as distinct pathogenetic entities. The type of exposure (e.g., wet or dry, and multielement exposure) would probably influence the clinical evolution of the lung disease. Table 1 Health effects of cobalt exposure

Target organ Skin Lung

Heart

Disease Allergic contact dermatitis Allergic bronchial asthma Interstitial fibrosis^ Lymphocytic alveolitis^ (hypersensitivity pneumonitis) Acute or chronic irritation forms Cancer Myocardiopathy^

^Probably associated with other hard metal compounds. ^2B lARC classification on experimental basis and soluble Co salts.

Role of Co Definite Definite Definite Definite Very probable Possible^ Probable

Occupational Exposure to Metallic Cobalt

309

Possible cardiotoxic actions of Co in hard metal exposure have also been hypothesized on the basis of an initial right ventricular impairment, more evident in interstitial lung fibrosis, which is ascribed to an initial pulmonary hypertension with a possible direct toxic action of Co on the myocardium [16, 66]. Studies are urgendy required to establish if a myocardiopathy (and its pathogenetic mechanisms) can be induced in those who work with hard metals. The possible carcinogenicity induced by Co in humans is not well documented and is mainly based on evidence from animals [81]. However, statistically significant increases in lung cancer deaths in hard metal workers have been reported with some suggestive evidence of an interaction between tobacco consumption and hard metal dust [30]. In addition, some experimental data support a possible genotoxic role for soluble Co salts [82]. The main clinical pictures involve the respiratory apparatus, in particular asthma and HMD, although not for their prevalence, which is rather low, but for the certain seriousness of their cases (that can be lethal). Therefore, a periodic preventive onthe-spot surveillance is required as well as a regular check-up of exposed workers, which makes it necessary to take a census of all the workers at risk. This is what we tried to do in our 1991 survey. THE FIRST STUDY A number of papers describing cases of HMD [1-7, 10-13, 22, 23], or reporting the results of sanitary surveys carried out on exposed workers [14, 17-19, 24], are reported in the medical literature. However, no survey concerns all possible working activities where operators could be potentially exposed to hard metal dusts. We have therefore decided to carry out a survey in our province, aimed at estimating the number of workers at risk of developing respiratory disease due to the inhalation of metallic cobalt. The study was carried out after having observed 11 cases of differently exposed workers affected by hard metal disease and who worked in different production areas (Table 2). The Province of Bergamo is situated in Lombardia, Northern Italy (Figure 1). Its main activities are: the metal and mechanical, textile, building, chemical, wood and furniture industries, which employ about 82% of the labor force. The following are the main industrial activities of the Province of Bergamo and the numbers (in thousands) which they employ: metal and mechanical, 63; textile, 41; building, 35; chemical, 18; wood and furniture, 10; total, 167. The total employees in industrial activities in the Province of Bergamo: 203,000. In this context, a group of 45 firms, where hard metal exposure was certain or extremely probable (group A), and a group of 2,039 firms at potential risk of exposure (group B) was identified. The related data are reported in Tables 3 and 4. The presence of factories producing sintered hard metal tools was identified. Onthe-spot inspections were carried out in 304 factories to verify the existence of work

310

Health and Toxicology Table 2 Cases of "hard metal disease" diagnosed in the department of occupational disease in Bergamo Hospital since 1984

Cases

Job

Industrial activity

1

Mold-filler

2

Mold-filler

3

Mold-filler

4

Grinder and welder Grinder Grinder Grinder Grinder and paper Grinder

Diamond abrasive production Diamond abrasive production Diamond abrasive production Diamond abrasive production Grinding Grinding Grinding

5 6^ 7a 8 9 10^ IP

Printing Small metal parts production Grinder Foundry Dental Dental skeletal technician prosthesis prod.

Duration of exposure

CoA (mg/m^)

Daily exposure

3

0.4-0.6

8h

3

0.4-0.6

8h

8

0.4-0.9

8h

0.04-0.06 0.005 (0.005)^ (0.005)^

8h 8h 8h 8h

0.0007

20min

30 30

0.008-0.02 —

4h 8h

30

—

30 min-1 h

6 10 20 14 5

^Exposed. ^Similar exposition to 5th case.

techniques at risk. This number included the 45 factories with "certain" or "very probable" risk, and about 10% of those selected as being "potentially" at risk. Further information was also gathered during the inspections regarding the number of exposed workers, their jobs, their daily exposures and the year in which the activity at risk had started. Two hundred and fifty-nine airborne samples were analyzed. Airborne dust samples were collected in all plants at different stages of the manufacturing process. Both personal samples from fixed locations in the work area ("static") for "total dust" were collected at a constant flow rate of 3.7 1/min using a cone-shaped filter holder (air speed at orifice, 1.2 m/s) on cellulose esther membrane filters (diameter 37 nmi, pore size 0.8 m). Single sampling periods ranged from 2 to 4 h. In any department, personal samples were collected by a PERSPEC [6] aerosol spectrometer for a realistic evaluation of size distribution of respirable, tracheobronchial and extrathoracic fractions of airborne dusts. Cobalt content was determined in atmospheric and settled dust samples. The sampled dusts were submitted to acid digestion ("aqua regia"). Also, 600 urine samples were taken for analysis at the end of the shift and at the end of the week, in order to evaluate the degree of exposure to cobalt. The concentration of cobalt in airborne and biological

Occupational Exposure to Metallic Cobalt

\ ) (

D

BERGAMO ^ A ^

Milano

/

\

Venezia

^ ^ ^ ^

311

V °

Torino

Bologna \ V San Marino

Pisa

\

FIrenze

J

\

\

Roma

Napoli

Cagliari Messina

^ ^ • ^

Catania (

Figure 1. Map of Italy and location of the Bergamo Province.

samples, here reported, were determined by atomic absorption spectrometer [16-20] in accordance with the procedures indicated by NIOSH [21]. The results of the on-the-spot inspections were carried out in the factories where professional cobalt exposure was certain or very likely. The following activities were involved: diamond abrasive production (n = 6), grinding (n = 23), tool production (n = 14) and hard metal alloy filHng (n = 2). From a total of 45 firms, we ascer-

312

Health and Toxicology Table 3 Industrial activities with certain or quite probable hard metal exposure and number of people exposed

Industrial activity^

Number of factories

Number of inspections

Number of factories at risk Employees Exposed

Diamond abrasive production 6 6 6 62 113 Grinding 23 23 23 209 76 Tool production 14 14 6 182 35 Hard metal alloy filling (Stellite) 2 2 2 12 9 Total 45 45 37 516 182 ^There is an absence of factories producing sintered hard metal tools in Bergamo Province. Table 4 Industrial activities with possible hard metal exposure and number of people exposed

Industrial activity Metal turning and small metal parts production Mold production Mechanical construction of industrial machines Foundry Wood turning Sawmill and furniture factory Printer Dental skeletal prosthesis production Total

Number of inspections

Number of factories at risk

322 113

52 19

18 9

540 92

39 52

493 86 151

30 7 21

17 6 9

3,366 2,610 409

35 30 19

598 269

51 33

6 3

657 989

6 3

7 2,039

5 218

5 73

63 8,727

37 221

Number of factories

Employees

Exposed

tained 37 cases of working methods at risk with 182 exposed subjects out of a total of 516 workers. The results of the inspections in the factories where the risk was possible, but not certain, are reported in Table 5. Here, the industrial activities selected are very different and they include: metal turning and small metal part production (n = 322), mold production (n = 113), mechanical construction of industrial machines (n =

Occupational Exposure to Metallic Cobalt

313

493), foundries (n = 86), wood turning (n = 151), sawmills and furniture factories (n = 598), printing (n = 269) and dental skeletal prosthesis production (n = 7). A total of 218 on-the-spot inspections (i.e., 10.7% of the factories comprised in group B) were carried out in the above industries and showed that in 73 factories with 8,727 employees, 221 workers were exposed. The latter, added to the 182 workers of the factories included in group A, gave a total of 403 confirmed exposed workers. The exposed group includes diamond abrasive operators, mold fillers, sinters, grinders, and mechanics, grinders or sharpeners of hard metal tools, stellite welders and founders, grinders of paper cutting blades, and hard metal form or surface grinders (Table 4). The survey (Table 6) documents a significant cobalt exposure, especially for the operators working in diamond abrasive production, and in particular in mold-filling and sintering units, where the environmental limits are regularly exceeded. In sharpening, tool production, and hard metal alloy filling, exposure is much more restrained. The concentrations determined in the factories of group B (identified as "Other" in Table 6) overevaluate the actual exposure, since they refer to measurements

Table 5 Jobs involving hard metal exposure in selected industrial activities Industrial activity

Diamond abrasive production

Grinding Tool production Hard metal alloy filling Metal turning and small metal parts production Wood turning Sawmill and furniture factory Printing Foundry Mold production Dental skeletal prosthesis production

Job

Mold-filler Sinter Grinder Mechanic Hard metal tools grinder Hard metal tools grinder Hard metal form grinder "Stellite" welder Hard metal tools grinder Hard metal tools grinder Hard metal surface grinder Hard metal tools grinder Hard metal tools grinder Grinder of paper cutting blades Hard metal tools grinder Hard metal tools grinder Hard metal form grinder Hard metal surface grinder "Stellite" founder Skeletal prosthesis grinder

314

Health and Toxicology

taken during the performance of the specific activity at risk. In these productive situations, the activity at risk often lasts < 1 h/day, although this can vary notably from one factory to another (Table 7). Biological monitoring results confirm data from environmental samples (Table 8). Figure 2 illustrates the year in which the activity at risk started (working/ machining or production of hard metals), for all the factories under examination. The result confirms what had already been known by the entrepreneurs themselves, namely, that the technological transformation occurring in the past 20 years has involved a notable increase in the use of hard metal-based materials and of Widia tools, especially in the metallurgical and mechanical industries. This probably corresponded to an increase in the number of exposed workers in the years between the end of the 1970s and the beginning of the 1980s. THE SECOND STUDY In our second work, the aim was to estimate the recent occupational exposure to cobalt in comparison with the data available in the literature. Exposed groups included hard-metal and diamond grinding tool producers, grinders and tool sharpeners (Table 9). Three groups of factories were examined: (1) two producing hard metal; (2) seven producing diamond grinding tools; (3) 11 grinding, machining or sharpening hard metal tools.

Table 6 Occupational cobalt exposure: results of 259 air samples

CoA fixed sampling (mg/m^

CoA personal sampling (mg/m^

Industrial activity

Median

Range

Median

Range

Diamond abrasive production Mold-filling Sintering Grinding Mechanical-working Grinding Tool production Hard metal alloy filling Other^

0.2200 0.1015 0.0220 0.0200 0.0050 0.0060 0.002 0.00265

0.0470-0.960 0.0320-0.2400 0.0150-0.0450 0.0120-0.0440 0.0025-0.094 0.0050-0.047 0.0008-0.003 0.0023-0.015

0.3820 0.309 0.2300 0.0400 0.0093 0.017 0.005 0.05

0.076-2.600 0.2380-0.4130 0.0820-0.6900 0.0071-0.0650 0.0015-0.178 0.004-0.028 0.001-0.107 0.01-0.29

^Cobalt exposure was determined during the process of grinding tools.

315

Occupational Exposure to Metallic Cobalt Table? Daily exposure in different jobs and industrial activities^

Median (min/die)

Range (min/die)

Mold-filler Sinter Grinder Mechanic Hard metal tools grinder Hard metal tools grinder Hard metal form grinder "StelUte" welder Hard metal tools grinder

480 480 480 480 480 480 480 480 30

— — — — — — — —

Hard metal tools grinder Hard metal surface grinder

30 30

30-300 30-300

Hard metal tools grinder Hard ruetal tools grinder Grinder of paper cutting blades Hard metal tools grinder Hard metal tools grinder Hard metal form grinder Hard metal surface grinder

15 75

10-30 10-240

40 30 30 30 30

30-420 30-240 10-45 10-45 10-45

60

—

360

—

Industrial activity

Job

Diamond abrasive production

Grinding Tool production Hard metal alloy filling Metal turning and small metal parts production Wood turning Sawmill and furniture factory Printing Foundry Mold production Dental schelectikal prosthesis production

"StelHte" founder Skeletal prosthesis grinder

^Mean duration of exposure.

Health and environmental surveillance in these factories was carried out by Departments of Occupational Medicine or Safety and Hygiene in some provinces of North Italy. The processes analyzed are as follows: Hard metal production. The stages in hard metal production are: I Weighing WC + (Tie + NbC + TaC) + Co + paraffin + solvent II Mixing, milling, and sieving of powders III Size enlargement by agglomeration

316

Health and Toxicology Table 8 Cobalt exposure: results of biological monitoring of 314 people exposed

CoU (Mg/i) Industrial activity

Median

Mean

Range

320 167.5 61 50 15 11.5 5 1"

587 193 151 67.2 31.5 19.4 4.8 2.85

39-2,100 102-390 34-520 14-165 0.8-730 0.8-100 0.8-18 0.8-72

Diamond abrasive production Mold-filling Sintering Grinding Mechanical-working Grinding Tool production Hard metal alloy filling Other ^Similar to reference value.

16 T 14 12 10

84

4+

i

n•

n

on

1—1- i,Ul. 0-P ' 1 — P ^ i — ' I — I — 1 — 1 — I — 1 — I P-4 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 85 87 88 89 90 91

Figure 2. Startup year for the at-risk activity in the factories seen in 1991

317

Occupational Exposure to Metallic Cobalt Table 9 Expected exposure in "hard metal" and diamond grinding tools manufacturing processes

Intensity of exposure

Frequency of occurrence

Manual work

Weighing Mixing-miUing Sieving Size enlargement Pressing Forming of presintered pieces Presintering Final sintering Grinding, sharpening of hard metal pieces

Discontinuous Continuous Discontinuous Discontinuous Continuous

High Low High High High

Continuous Continuous Continuous

High Slow Slow

Continuous

High

Mixing Filling and pressing "Finishing" Sintering cycle Mold recovery

At intervals Continuous Discontinuous At intervals At intervals

High High High Low High

Operation

Hard Metal High

Moderate

Grinding Tools High Moderate

IV Pressing into molds V Presintering in a bell shaped oven (500-700°C) VI Forming of presintered pieces VII Final sintering in inert atmosphere or in vacuum (1,350°, 1,500°C) VIII Grinding-sharpening of hard metal pieces. The most common cobalt content is 10%. Diamond grinding tools manufacturing. Summarized as follows: I Mixing of raw material powders: copper or brass and other additives II Manual or automatic filling in mold and pressing III Sintering cycle in electric oven IV Sharpening, grinding, polishing, brightening ("finishing")

318

Health and Toxicology

The most common cobalt content is 90%. Typical exposure can be estimated as shown in Table 1. Grinding processes. The occupational exposure for hard metal grinding in small workshops or factories is related to the presence and efficiency of local aspiration plants. The results proved that personal and "static" samples of airborne cobalt-containing dust and other exposure parameters before and after use of local aspiration plants showed very different cobalt exposure levels (see Tables 10-13). A substantial lowering of contamination was obtained for all stages of the hard metal manufacturing process; significant results were also obtained in grinding operations with efficient local aspiration. However, it is very difficult to control the degree of cobalt exposure in major stages of diamond tools manufacturing. Recent data are summarized in Table 14 for four manufacturing plants after a second improvement of local ventilation.

Table 10 Average atmospheric concentrations of cobalt (mg/m^) in medium-sized hard metal manufacturing plant before and after technical control Personal samples Values

Mixing

Pressing

Forming

Sharpening

Before technical control

Geometric mean Geometric standard deviation Range Number of samples

0.245

0.082

0.772

0.010

2.51 0.096-0.967 6

3.41 0.040-0.514 7

3.09 0.181-5.27 10

2.21 0.004-0.070 15

0.050

0.029

0.082

2.21 0.012-0.127 9

1.48 0.023-0.050 4

2.39 0.015-0.19 18

After technical control

Geometric mean Geometric standard deviation Range Number of samples

Occupational Exposure to Metallic Cobalt

319

Table 11 Average atmospheric concentrations of cobalt (mg/m^) in a small hard metal manufacturing plant Personal samples

Values

Without local aspiration

With inefficient local aspiration

With efficient local aspiration

Geometric mean Geometric standard deviation Range Number of samples

0.428 1.88 0.139-0.978 21

0.198 1.47 0.090-0.380 6

0.014 1.45 0.006-0.040 7

Table 12 Average concentrations of cobalt (mg/m^) in five diamond grinding tools small plants before and after technical control Personal samples

Values

Weighing and pressing

Static samples

Filling

Sintering^

Near workplaces^

7.46

2.04

0.62

0.717

2.42 2.05-21.87 8

2.41 0.418-9.04 16

2.28 0.277-2.714 8

2.61 0.106-3.93 11

0.78

0.61

0.45

0.15

3.38 0.080-2.4 8

2.61 0.060-1.480 14

1.78 0.147-1.171 13

1.99 0.024-0.300 12

Before technical control

Geometric mean Geometric standard deviation Range Number of samples After technical control Geometric mean Geometric standard deviation Range Number of samples

^to obtain a measurement of the general contamination in workroom. ^to obtain a measure of contamination in the immediate vicinity of a process.

At the sintering stage, high cobalt exposure is due to particular operations such as: recovery of sintered pieces, holders and graphite supports, graphite dust removal, and workplace cleaning, as shown in Table 15.

320

Health and Toxicology Table 13 Average concentrations of cobalt (mg/m^ in 11 small grinding plants for hard metal tools Samples from workplace without local aspiration

Values

Geometric mean Geometric standard deviation Range Number of samples

Samples from workplace with local aspiration

Personal

Static

Personal

Static

0.091

0.040

0.009

0.012

3.27 0.005-1.338 51

3.16 0.005-0.110 52

3.07 0.001-0.038 55

.94 0.004-0.058 47

Table 14 Average atmospheric concentrations of cobalt (mg/m^) after recent improvement of local ventilation Weighing Values

Personal

0.751 Geometric mean Geometric standard deviation 1.46 Number of samples 7

Filling and pressing

Sintering

Sharpening and grinding

Polishing

Personal

Static

Personal

Static

Personal

Static

Personal

Static

0.303

0.118

0.248

0.092

0.039

0.021

0.205

0.023

3.14 61

2.36 6

2.21 26

1.86 16

1.51 28

1.68 13

3.11 10

1.47 11

The average concentration of cobalt in settled dusts in places rarely cleaned (sampled at a greater distance than 1.5 m above the floor) is very near to the concentration of cobalt in suspended dusts found in hard metal and diamond tools manufacture. The higher content of cobalt in settled dusts can cause a higher secondary pollution of workroom air (Table 16). In a workroom used for the hard metal sintering process, the distribution of dust and cobalt concentrations in different job conditions has been studied. The concentration profile was uniform as shown in Table 17. The samples obtained for size distribution using PERSPEC showed: (a) The respirable fraction of cobalt is similar in hard metal and diamond grinding tools sintering processes; (b) The extrathoracic and tracheobronchial fractions are very different (Table 18).

Occupational Exposure to Metallic Cobalt

321

Table 15 Average atmospheric concentrations of cobalt (mg/m^) in sintering-related operations and sintering alone Sintering, holders and supports recovery, graphite removal, cleaning Values

Geometric mean Geometric standard deviation Number of samples

Sintering

Personal

Static

Personal

Static

0.358 1.47 14

0.138 1.78 13

0.049 1.52 9

0.041 1.50 6

Table 16 Percentage of cobalt in settled and in suspended particulate matter

Diamond g rinding tools

1Hard metal Co (%) Average value Standard deviation Number of samples

Settled

Suspended

Settled

Suspended

10.1 ±1.4 6

9.4 ±2.6 8

65.1 ±7.4 4

62.7 ±13.7 11

Table 17 From the breathing zone to ceiling concentration of dust and cobalt

Sampling height above floor (m)

Workroom and job condition

Dust (mg/m^)

Co (mg/m^)

Co (%)

4.2 2.8 1.4 4.2 2.8 1.4 4.2 2.8 1.4

Open window

1.07 1.27 1.20 2.72 3.00 2.73 0.99 1.01 1.03

0.050 0.058 0.047 0.100 0.108 0.108 0.047 0.047 0.044

4.66 4.56 4.06 3.66 3.62 3.94 4.71 4.70 4.26

Normal job Closed windows Normal job Closed windows Low-rate job

322

Health and Toxicology Table 18 Average cobalt content in conventional fraction (%)

Conventional fraction

Hard metal manufacturing

Diamond tools manufacturing

Respirable Tracheobronchial Extrathoracic

22.1 40.0 37.9

22.8 52.9 26.3

CONCLUSION

In a literature review of 250 works of the past 20 years, we found several studies providing a description of the activities at risk due to inhalation of metallic Co dusts. Many also report the results of on-the-spot or biological surveys that often document how the TLVs are exceeded for those workers professionally exposed to Co. A lower number of studies describes cases of HMD or Co asthma which, considering the gravity of illness, corresponds to the main important clinical issues. These findings are direct proof of the degree of risk related to the working activity. The results confirm what we highlighted during our studies, as previously illustrated. Table 19 provides an abstract of this literature review and indicates, next to the production area involved, the number of HMD and Co asthma cases described (a total of 89), as well as a short evaluation of the "level" of exposure to metallic Cobalt dusts (high, medium, low). Hard metal and diamond abrasive production and machining (grinding and sharpening) prove to be the operations more at risk. These activities are also those involving an exposure to the highest airborne concentration of Cobalt [15, 21, 24-28, 50, 63-65, 70, 73]. The airborne limits of ACGIH (0.05 mg/mc) are frequently exceeded, especially in the weighing, mixing, milling, sievening of hard metal production, as well as in the grinding and sharpening of hard metal tools. Seventy-nine out of 89 cases do actually come from the production areas. Besides being the most diffused ones, these operations also involve the exposure, at the same time, to other metals and in particular to WC, and to refrigerating oils. This strengthens Lauweris' and Lison's theses of an adjuvant action of other agents in the determinism of respiratory diseases. The exposure proves significant also in the welding and coating with hard metal, as well as in the diamond polishing. Surprising is the number of respiratory disease cases recorded among the operators working in skeletal prostheses production. Actually, although the exposure level is almost always within the TLV [ACGIH 1994-1995], there is a high case record of HMD and asthma. In this case as well, the type and methods of exposure can play an important part in the development of the disease. However, the risks in cobalt mining and in chemical manufacturing with cobalt powder handling prove to be limited.

Occupational Exposure to Metallic Cobalt

323

Table 19 Working activities at risk, cases of HIVID and co bronchial asthma, short evaluation of the occupational exposure levels to co: results of a review of the past 20 years

Industrial activity Hard metal production and diamond Grinding tools manufacturing Grinding and sharpening process Welding and coating with hard metal (stellite) Dental prostheses production Diamond polishing Cobalt mining and Co ores refining Chemical manufacturing with Co powder handling

Cases HMD and Co asthma

Level of exposure

Reference 2, 5, 7, 9, 21-22, 24-27, 31,35 38,40-41,46-47,49-51, 57,62-65,70-71,73-74

29

HIGH

_

_

46

MEDIUM-HIGH

3

MEDIUM-LOW

6 4

LOW MEDIUM-LOW

10, 23, 32, 40, 56, 68-69 4, 19, 34, 58

1

MEDIUM-LOW

4 3 ^ 4 , 77-79

0

LOW

75

38, 40, 45, 48, 62, 76 1,3,17,23,32,36,38, 40^1,47,54,56,59,62, 70-72

These studies evidence an unexpected wide diffusion of cobalt exposure. A number of productive areas are involved, and many workers are professionally exposed to different levels of hard metal dusts, often exceeding the TLV for cobalt. The "hard metal" occupational risk has greatly increased in recent years and can be controlled, but it is not easy. This environmental situation requires an improvement in exposure control strategies to obtain better performance from special local ventilation to remove dusts from the workers' breathing zone, and to achieve better manufacturing process layout. The aim of control also requires: (1) periodic assessment of cobalt exposure by environmental and biological monitoring; (2) separation of single stage of process; (3) cleaning of work places and departments [62]. It is necessary to discuss exposure conditions in terms of both quality and quantity. This is important, not only to schedule efficient clean air operations—needed now more than ever—but also because we strongly suspect that the operations

324

Health and Toxicology

themselves could play an important part in the development of the disease. Furthermore, according to our case record and considering that even low exposures could determine the disease, surveillance programs on exposed workers and former workers should be developed. We now have at our disposal, methods for an effective reduction and control of disease; however, we believe the risk of hard metal disease (HMD) to be considerably underestimated, considering its rate of incidence and the seriousness it bears on the clinical picture. Further investigations seem urgently needed for a better identification of workers at risk of cobalt exposure.

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64. Scansetti, G. et al. "Absorption and excretion of cobalt in the hard metal industry." Sci. Total Environ., Vol. 150, No. 1-3, 1994, pp. 141-144. 65. Seghizzi, P. et al. "Cobalt myocardiopathy. A critical review of literature." ScL Total Environ., Vol. 150, No. 1-2, 1994, pp. 105-109. 66. Sesana, G. et al. "Cobalt exposure in wet grinding of hard metal tools for wood manufacture." Sci. Total Environ., Vol. 150, No. 1-3, 1994, pp. 117-119. 67. Sherson, D. et al. "A dental technician with pulmonary fibrosis: a case of chromium-cobalt alloy pneumoconiosis?" Eur. Resp. J., No. 3, 1990, pp. 1,227-1,229. 68. Sherson, D. et al. "Small opacities among dental laboratory technicians in Copenhagen." Brit. J. Ind. Med., No. 45, 1988, pp. 320-324. 69. Shirakawa, T. et al. "Occupational asthma from cobalt sensitivity in workers exposed to hard metal dust." Chest, No. 95/1, Jan. 1989, pp. 29-37. 70. Shirakawa, T. et al. "Combined effect of smoking habits and occupational exposure to hard metal on total IgE antibodies." Chest, No. 101/6, 1992, pp. 1,569-1,576. 71. Sjogren, I. et al. "Hard metal lung disease: importance of cobali in coolants." Thorax, No. 35, 1980, pp. 653-659. 72. Sprince, N. L. et al. "Cobalt exposure and lung disease in tungsten carbide production." Am. Rev. Respir. Dis., No. 138, 1988, pp. 1,220-1,226. 73. Stebbins, A. I. et al. "Cobalt exposure in a carbide tip grinding process." Am. Ind. Hyg. Assoc. J., No. 53, 1992, pp. 186-192. 74. Swennen, B. et al. "Epidemiological survey of workers exposed to cobalt oxides, cobalt salts, and cobalt metal." Brit. J. Ind. Med., No. 50, 1993, pp. 835-842. 75. Tabatowski, K. et al. "Giant cell interstitial pneumonia in a hard metal work." Acta Cytologica, Vol. 32, No. 2, March-April 1988. 76. Van Cutsem, E. J. et al. "Combined asthma and alveolitis induced by cobalt in a diamond polisher." Eur. J. Respir. Dis., No. 70, 1987, pp. 54-61. 77. Van den Eeckhout, A. V. et al. "La patologie pulmonaire due au cobalt et aux metaux durs." Rev. Mai. Respir., No. 6 (3), 1989, pp. 201-207. 78. Van den Oever, R. et al. "Exposure of diamond polishers to cobalt." Ann. Occup. Hyg., Vol. 34, No. 6, 1990, pp. 609-614. 79. Zanelli, R. et al. "Uncommon evolution of fibrosis alveolitis in a hard metal grinder exposed to cobalt dusts." Sci. Total Environ., Vol. 150, No. 1-3, 1994, pp. 225-229. 80. Nordberg, G., "Assessment of risks in occupational cobalt exposures." Sci. Total Environ., Vol. 150, No. 1-3, 1994, pp. 201-207. 81. Binderup, M. L. et al., "Genotoxicity testing of cobalt compounds used in the Danish porcelain industry." Sci. Total Environ., Vol. 150, No. 1-3, 1994, p. 217.

CHAPTER 14 CLIMATE AND CHRONIC RESPIRATORY DISEASE Paul Beggs and Peter Curson Climatic Impacts Centre, School of Earth Sciences Macquarie University New South Wales 2109, Australia

CONTENTS ABSTRACT, 329 INTRODUCTION, 330 THE PAST, 331 CLIMATE AND CHRONIC RESPIRATORY DISEASE, 331 Direct Impacts of Climate, 332 Indirect Impacts of Climate, 334 DWELLING CHARACTERISTICS, DAMP HOUSING, MOLD GROWTH AND ASTHMA, 341 SYNERGISTIC EFFECTS, 342 EPIDEMICS, 342 THE FUTURE: CLIMATE CHANGE AND CHRONIC RESPIRATORY DISEASE, 343 Air Quality (Irritants), 344 Allergens, 345 Climate, 345 Infections, 345 TECHNOLOGICAL WARNING SYSTEMS, 346 ENVIRONMENTAL CONTROL, 346 CONCLUSIONS, 347 REFERENCES, 347 ABSTRACT This chapter examines the relationship between climate and chronic respiratory disease (CRD), with particular reference to asthma. The chapter pays specific attention to the direct and indirect effects of weather and climate on such diseases, as well as the interplay between a number of biophysical and socioeconomic factors, and the role played by the outdoor and indoor environments. In particular, it discusses the relationship between climate, weather, allergens, and air pollution and chronic respiratory diseases. It also argues that climate change will greatly influence the prevalence and distribution of many chronic respiratory diseases. Finally, it outlines some of the ways in which the indoor environment can be controlled to produce a less conducive environment for harmful triggers of CRD. 329

330

Health and Toxicology

INTRODUCTION Chronic respiratory diseases such as chronic bronchitis, emphysema, and asthma remain important public health issues in many countries of the world. Morbidity and mortality rates in many places are not only very high but have increased substantially over recent years. The prevalence of such diseases would seem to be closely linked to human activity and the biophysical environment, particularly climate. In many places, an unacceptable proportion of deaths from chronic respiratory diseases is untimely or premature. Mortality alone, however, tends to disguise the real impact of such diseases. Many respiratory diseases such as asthma and emphysema do not prematurely kill, but slowly undermine, debilitate, and severely downgrade the lifestyle and quality of life of the sufferer. Today, chronic respiratory disease remains one of the most important causes of chronic illness in developed countries and is responsible for a substantial proportion of absenteeism from work and school as well as for considerable human suffering. As such, both the economic and social costs of CRDs in many places are enormous. Among the chronic respiratory diseases, asthma is the most prevalent throughout the world. The disease is a chronic inflanmiatory disorder of the airways in which many cells play a role, including mast cells and eosinophils. In susceptible individuals, this inflammation causes symptoms usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with intervention, and causes an associated increase in airway responsiveness to a variety of stimuli [1]. Basically, asthma refers to a condition in which there are rapid changes in the diameter of the airways of the lung, accompanied by one or more of the following symptoms: wheeze, shortness of breath, constriction in the chest, cough (sometimes productive), and nighttime symptoms. Asthma is a multifactorial disease triggered by a wide variety of stimuli including allergens such as house dust mites, pollens, and molds; air pollutants such as ozone, nitrogen dioxide, sulfur dioxide, and particulates; as well as certain climatic and weather phenomena such as rapid temperature changes and cold dry air. Asthma has a differential impact on demographic groups in most countries. For example, the prevalence of asthma is higher in children than in adults, although more elderly than young people die from this disease. Further, the proportion of males to females suffering from the disease varies with age. Most studies of children have found that more boys than girls suffer from asthma, and some have found asthma to be more severe in boys than in girls. In adults there is a less striking preponderance of males, with some surveys reporting an equal sex distribution, while others report a preponderance of females. There is also evidence that CRDs are higher among the more disadvantaged sectors of industrial societies. In Australia, for example, McMichael [2] has found that males in the lowest socioeconomic category had 2.8 times the rate of bronchitis, emphyse-

Climate and Chronic Respiratory Disease

331

ma, and asthma compared with those in the highest group. There are many other groups in society that are particularly vulnerable to the effects of climate [3,4, 5]. THE PAST The concept of an association between climate and CRD is not new and is embedded in the traditional folklore of many civilizations. For example, since the time asthma was first recognized as a disease, physicians have appreciated the importance of the local biophysical environment as a source of triggers. A relationship between prevaiUng winds and asthma was first advanced in the Hippocratic writings of the fourth and fifth centuries B.C. [6], while Van Helmont in the seventeenth century identified dusts, climate, and weather as triggers of the disease [7]. Indeed, if one looks at the most notorious episodes of air pollution this century, it is clear that temperature inversions are intimately associated with many outbreaks of CRD. Perhaps the most infamous of these, the London pea soup smogs of 1952, caused thousands of excess deaths and involved the trapping of sulphur dioxide and particulates close to the ground by temperature inversions. Many of these excess deaths resulted from respiratory illness, with 10 times more patients dying from bronchitis as compared to the usual bronchitis death rate [8]. Similar episodes have occurred in the Meuse Valley, Belgium (1930), in Donora, Pennsylvania (1948), Poza Rica, Mexico (1950), and in New York (1966). As with the London episode, all such events occurred during winter and involved the trapping of pollutants by temperature inversions. CLIMATE AND CHRONIC RESPIRATORY DISEASE There have been many attempts to clarify the etiology and pathogenesis of CRDs as well as to try and understand the role of climatic factors in the chain of events leading to disease. It would seem clear that climate and weather are inseparably linked to many CRDs, although the precise mechanisms involved still remain illusive. Climate would seem to affect the human respiratory system in a variety of ways. In the first place, there are broad seasonal effects that represent the combined effect of climatic stimuli on basic physiological and biochemical processes, as well as broad climate-inspired changes in the biophysical environment. Secondly, although the mechanisms remain poorly understood, there may be direct climate effects on respiratory sensitivity such as those associated with rapid falls in temperature, low temperatures, and cyclonic and frontal activity. Generally, the effects of climate on CRDs can be divided into those which are direct and those which are indirect. For example, a number of climate variables play a direct and/or indirect role in the process leading to an attack of asthma (Figure 1). Temperature and humidity can directly stimulate the airways to produce symptoms. These parameters, along with wind and rainfall, also indirectly affect asthma via their effects on allergens and air pollutants.

332

Health and Toxicology

Climate / Weather (Rainfall, Temperature, Humidity, Wind)

Indirect

Direct

Indirect

Aeroallergens (e.g. pollen, mould spores, house dust mite)

Air Quality (e.g. ozone, nitrogen dioxide, sulphur dioxide, particulates)

Susceptible Populations

Asthma Figure 1. Schematic illustration of the direct and indirect links between climate and asthma.

Direct Impacts of Climate Temperature and Humidity It is possible that asthmatics may differ physiologically and in their biochemistry from non-asthmatics. In particular, there would seem to be differences in allergic status, blood pressure, adrenal gland function, thermo-regulatory efficiency, and bronchial contraction between the two groups [9]. With respect to their hypothalmic thermo-regulatory mechanism, asthmatics would appear to function less efficiently with a different re-warming curve and a longer period of adjustment to rapid falls in temperature. Consequently, a number of studies have shown that a sudden fall in temperature could directly precipitate attacks of asthma [8]. Bates and Sizto [10] found highly significant associations between excess total and respiratory hospital admissions and temperature in southern Ontario, Canada, although not for asthma admissions alone. In a later study [11], however, they found asthma admissions were correlated with temperature on the same day and also with temperature one and two

Climate and Chronic Respiratory Disease

333

days before. Ponka [12] found the number of admissions for asthma in Helsinki, Finland, increased during cold weather, although not for children. Although asthma is often thought of as being an "allergic" disease, there are other triggers of mast cell activation besides allergic (IgE-dependent) mechanisms [13]. Research by Lee et al. [14, 15], for example, has indicated that mast cell mediator release also occurs in response to non-immunological stimuli such as respiratory heat exchange via exercise. It has been suggested that increased airway resistance induced by cold air in asthmatic persons is the result of a stimulation of upper airways receptors, causing reflex bronchoconstriction [8]. Bronchoconstriction can also be induced by heat loss from airway mucosa, and this mechanism has been considered responsible for exercise-induced asthma. Haines and Fuchs [16] stated that "the study of meteorological factors in human disease is complicated by a number of factors, including the interaction of different variables such as temperature and humidity," and the varying response by different sub-groups according to local micro-environment. Bar-Or et al. [17], in their study of the effects of dry and humid air on exerciseinduced asthma in children and preadolescents, concluded that such asthma was more likely in dry air than in humid air, possibly due to heat loss at the airway mucosa caused by evaporation. Derrick [18] suggested that the respiratory system might be direcdy irritated by cold air, while Wells et al. [19] were of the opinion that cold air induced bronchospasm in susceptible individuals. Derrick [18] correctly states that "it is difficult to separate the effects of dry air and of cold air on the respiratory mucosa." In explanation, he continues, "cold air will also have a drying effect, as it must become unsaturated when warmed to 37°C. Dry air will also have a cooling effect, as moisture evaporates from the mucosa." It would seem probable, therefore, that increases in asthma might result directly from irritation by dry air. The lower the water content of the air, the greater the cooling of the airway mucosa [8]. On the other hand, there are a number of studies, such as that by Lopez and Salvaggio [8], which argue that high humidity is detrimental to asthmatics. A possible mechanism for this, proposed by Swartz [20], is that with high humidity there is decreased evaporation followed by an increase in respiratory rate, decreased blood carbon dioxide, and a tendency to develop alkalosis, causing symptoms in predisposed patients. It has also been suggested that increased stress on the thermo-regulatory system can result in a higher frequency of asthma attacks. Barometric Pressure Atmospheric pressure has also been shown to have an affect on CRD, particularly asthma [21], and such effects have been reviewed by Lopez and Salvaggio [8]. Several studies have associated increases in asthma symptoms with rapid pressure decreases [22, 23]. Khot et al. [24] found an association between low barometric pressure and child asthma admissions in Brighton. Girsh et al. [25], on the other

334

Health and Toxicology

hand, found an increased incidence of asthma associated with periods of high pressure. Relationships have also been found between asthma and synoptic weather systems. For example, Amolt et al. [26] found asthma crises in Rosario City, Argentina, were more common in circumstances of cyclonic atmospheric circulation. Indirect Impacts of Climate In some ways, the indirect relationships between climate and chronic respiratory disease are more important than the direct relationships discussed above. There are numerous indirect relationships between climate and chronic respiratory disease. Winds affect the composition of the atmosphere, and the production of some air pollutants such as ozone and aeroallergens such as mold/fungal spores, pollens, and that from house dust mites are intimately related to certain climate and weather conditions. It is these air pollutants and aeroallergens that have a well-documented effect on CRDs. Seasonality Some studies have investigated the effect of seasonal influences and variations on asthma (Table 1). For example. Derrick [28] found asthma attendances in Brisbane, Australia, peaked in autumn and, to a lesser extent, in spring. In Australia there is a maximum of bronchitis in winter. Indeed, respiratory disease mortality in Australia shows a strong seasonal distribution with a peak in winter (Figures 2 and 3). Table 1 Some recognized seasonal effects on chronic respiratory diseases Season

Condition

Possible mechanism

Winter

colder temperatures increased viral exposure

Spring

acute exacerbations of chronic obstructive airways disease/ chronic bronchitis/emphysema asthma asthma

Summer

asthma

Autumn

chronic obstructive airways disease/emphysema asthma

After Ayres [27].

viral-induced episodes spring rains/thunderstorms pollen release agricultural chemicals pollen release/thunderstorms hot still weather/air pollution ozone hot still weather/photochemical smog colder temperature/pollen release (e.g., Parietaria)

J

J

A

Month Figure 2. Seasonal distribution of respiratory disease mortality, Australia, 1988-91. (From unpublished Australian Bureau of Statistics data.)

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Figure 3. Seasonal distribution of respiratory disease mortality, New South Wales, 1970-90. (From unpublished Australian Bureau of Statistics data.)

336

Health and Toxicology

Similarly, respiratory disease deaths, such as those from chronic bronchitis, rise markedly during the colder winter months in Britain [29]. Seasonal influences [30, 31, 32] include gradual changes in temperature, and radiation period and intensity. Such changes regulate the growth of allergen-producing organisms, such as the house dust mite, molds, and plants. Wind Wind, a collective term for characteristics of air flow, including air speed, direction, and turbulence, has a marked influence over the composition of the atmosphere. It plays a major role in the dispersion and distribution of air pollution. Low wind velocity leads to an accumulation of pollutants, whereas high velocity and turbulence tend to disperse particulates and decrease their concentration [8]. Temperature inversions suppress vertical mixing and thus reduce the dispersion of pollutants. Outdoors, wind can trigger the release of pollen grains from the anthers of flowers and the distribution of mold/fungal spores. Onshore winds in coastal areas reduce the concentration of airborne allergens such as spores and pollen [33, 34], where as offshore winds may lead to increases in airborne allergen concentrations [33]. Winds such as katabatic (downslope or drainage) and anabatic (upslope) breezes are topographically generated. Land and sea breezes result from thermal circulation systems generated by contrasting thermal responses of land and water. Radiative heating of surfaces produces convective updrafts and mixing. Indoors, air movements can be generated by human activity. These air movements are the mechanism by which allergenic particles such as house dust mite feces and mold/fungal spores can be lifted to the level of the mouth or nose. Air Pollution The respiratory sensitivity of people suffering from diseases like asthma and emphysema makes them especially sensitive to episodes of atmospheric pollution. Ozone, sulphur dioxide, and nitrogen dioxide, as well as heavy loads of particulate matter, increase airway resistance and have an irritant effect on mucous membranes. All these elements are common byproducts of living in modem cities. The production of ozone (O3), a major constituent of photochemical smog, requires NOx (NOx = nitric oxide + nitrogen dioxide) and reactive organic compounds as precursors, and high temperature and ultraviolet radiation intensity for its formation [35]. Temperature inversions are perhaps the other major link between climate, air pollution, and CRD. In many places there is a strong seasonal character to air pollution episodes. Summer-type episodes often involve photochemical smog, while winter-type episodes more typically involve sulphur dioxide and suspended particulate matter [36]. Both have been associated with CRD [37]. Indeed, Bates and Sizto [11] have found respiratory admissions in Southern Ontario to be correlated with ozone, SO4, and SO2. They refer to this as the Acid Summer Haze Effect.

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337

Mold and Fungal Spores Many people suffering from chronic respiratory diseases are atopic with some degree of allergic status. Plants and animals provide the greatest store of allergens. Pollens, mold/fungal spores, and house dust mite feces are the allergen groups of most importance. CRDs such as asthma, which are affected by mold and fungal spores, are affected by climate indirectly via its effects on the growth of molds and the liberation of mold fungal spores. Moisture is required for mold growth, although requirements of different species vary widely [38]. Maximum growth for most fungi occurs at a relative humidity of 95-100% and growth declines or ceases in humidity of 80-85%, although a few fungi will grow at a relative humidity as low as 65% [39]. Reed [40] commented that mold growth would not occur unless the humidity consistendy exceeded 70%. The ability to grow on a given substrate is partially determined by the moisture content of the substrate. The liberation of mold/fungal spores into the air is also very much related to the humidity of the atmosphere. While the production of allergens may depend on moist conditions, their liberation into the air may be favored by dry air. For example, spores from certain of the Fungi Imperfecti (Deuteromycetes), Cladosporium and Alternaria, are shed into dry winds [8, 41]. Other spores, however, such as those from Ascomycetes and lower Basidiomycetes classes, are discharged during periods of high humidity (rainfall and dampness). As well as being sensitive to air humidity and substrate moisture levels, the growth of molds is responsive to temperature. All fungi have minimum temperatures below which they will not grow and maximum temperatures above which they are inactivated. They achieve their best growth at what are known as optimum temperatures [42, 43]. These temperatures can vary according to the stage of life of the fungus. Different molds are suited to different temperature regimes, so much so that they have been grouped accordingly: mesophilic, thermophilic, psychrophilic, and thermotolerant fungi growing in moderate, high, low, and wide temperature ranges, respectively. Various fungi can grow over a wide range of temperatures, though most (known as mesophilic fungi) grow at moderate temperatures within the range of 10-40°C [38], with optimal growth generally occurring between 15-40°C. Thermophilic fungi grow at higher temperatures with an optimum for growth of about 40°C, tolerating a maximum up to about 60°C and a minimum of 20°C [43]. Other fungi known as psychrophiles (e.g., certain Cladosporium species) grow at low temperatures, to a minimum of-5°C [42] with a maximum of 20°C and an optimum of 0-17°C [43, 44]. By contrast, thermotolerant fungi (e.g., certain Aspergillus species) tolerate a wide temperature range [41]. Aspergillus fumigatus, for example, grows between 12-55°C. The production or germination of spores often requires more specific conditions than does vegetative growth. Particles of any type of airborne allergen over 2|im in diameter are liable to be washed out of the atmosphere by rain [18, 28, 45]. Rain therefore decreases the atmospheric concentration of many types of spores, although some like Ascospores

338

Health and Toxicology

are increased [8]. Experimental work conducted by Hirst and Stedman [46] has shown that large transient increases in the concentration of some dry airborne spores can be due to rapid air movement in advance of radially spreading raindrop splashes and vibration. High wind velocity has also produced decreased atmospheric concentrations of some spores such as Altemaria, Cladosporium, and Botrytis species, and some Basidiospores [8]. Plants and Pollen In a similar way, CRDs of an allergic nature are indirectly affected by climate via its effects on plant growth and airborne pollen concentrations (Figure 4). The growth and distribution of some significant asthma-triggering plants, such as Parietaria judaica, is partially determined by temperature and rainfall levels [47]. Pollen release is also closely related to daily fluctuations in temperature, and occurs predominantly in the morning. Flowering, and thus the production of pollen, of many plants is responsive to the relative amounts of light and dark sensed by the plant. This phenomenon is known as photoperiodism. Ragweed, for example, is a short-day plant, flowering when the light period is shorter than a critical length. There is evidence to suggest that the initial cause of asthma, or at least sensitization, may be closely related to climate through seasonal variation in flowering and pollen production. Exposure to high allergen levels in the first year of life may lead to the development of asthma. More specifically, the timing and type of the first high allergen exposure may be critical. For example, Bjorksten et al. [48] and Bjorksten [49] found an increased risk of immediate hypersensitivity to birch pollen in patients who were born in the months immediately prior to the birch flowering season. Humidity also effects the concentration of pollens in the atmosphere. Lopez and Salvaggio [8] comment that pollen is shed into dry winds. Bass and Bass [47] comment that pollen release from P. judaica is closely related to daily fluctuations in humidity, and occurs predominantly in the morning. Pollen release into the air would seem to be triggered by a fall in relative humidity [45]. Conversely, high humidity can lead to the accumulation of moisture on airborne particles and accelerate their deposition. Rain also decreases the atmospheric concentration of pollen [8]. Much of the wind-borne pollen produced globally is lost by fallout at or within several meters of its source [50]. For example, probably no more than 5% to 10% of short ragweed pollen achieves appreciable dispersion. House Dust Mite At the broad level, climate exerts a considerable influence on the levels of house dust mite allergen and mites (Figure 4). Lower levels of house dust mite allergen have been found at high altitudes compared to sea level [51], and inland locations tend to have fewer mites than locations on the coast [52, 53, 54]. However,

Climate and Chronic Respiratory Disease

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Figure 4. Scanning electron microscope photograph of a female house dust mite (Dermatophagoides pteronyssinus), upper right; mite feces, upper left; mite egg, lower left; and rye grass pollen, lower right. (Courtesy of Dr. Euan Tovey, University of Sydney.)

Tovey [54] has noted that "while regions with similar climates have similar mean levels of mite allergens, the level between houses in the same region may cover at least a hundredfold range ... for reasons which are not yet characterized." A wide range of allergen concentrations and mite numbers have been determined, ranging from negligible in locations such as Alice Springs in central Australia [54] and Briangon in the French Alps [51], to high on the east coast of Australia [54, 55] and Sao Paulo, Brazil [56]. It has become clear in recent times that the house dust mite and the allergen it produces is a major factor in asthma in many locations around the world. As such, considerable research has been conducted on its life history, and it has been determined that it has quite specific microclimatic requirements. Some of this research has focused on domestic design as a means of controlling house dust mite numbers and therefore improve asthma severity. For example, house dust mites such as Dermatophagoides farinae and D. pteronyssinus are very sensitive to the temperature and humidity of their micro-environment, thriving at moderate temperatures and high humidities. Tovey [54], who has examined mite allergen levels in Australia,

340

Health and Toxicology

suggests that patterns of mite abundance are controlled by regional humidity. A relative humidity of 70-80% (at 25 °C) would seem optimal for the development of a population of house dust mites [57]. Specifically, it has been determined that the optimum conditions for mite development and reproduction are 25 °C and 75% relative humidity. Humidity greatly influences fecal production in mites. A reduction of humidity from 85% to 65% reduces feeding (and therefore production of allergen-containing feces) by more than tenfold, so although mites may not be killed by minor reductions in humidity, allergen production is severely curtailed. At a relative humidity of 60% or lower, the population stops growing and dies out. It has been found that very few house dust mites can develop if the internal relative humidity is less than 45% at an indoor temperature of 20-22°C [58]. House dust mite populations and allergen levels have been surveyed at many locations around the world (Table 2). Three dwelling sites have been studied: the subject's bed, bedroom floor, and the living-room floor. Of these three sites, beds have usually been found to contain the greatest numbers of mites or quantity of allergen, though a few studies have found higher numbers on the bedroom floor or living-room floor (e.g., Korsgaard [59] and Tovey [54], in Wellington NZ). This may reflect the microclimate and food supply generated by humans as they sleep. It has been demonstrated that in some locations such as Sydney, Australia, and South Manchester, United Kingdom, there is little fluctuation in the quantity of house dust mite allergen with season of the year [60, 61]. Studies at other locations such as in the United States of America [31, 62] have indicated seasonal variation in house dust mite allergen quantities and house dust mite body counts, which are related to seasonal variation in humidity. Table 2 House dust mite allergen quantities for selected locations Site* Reference

Location

Charpin et al. [51]

Brian^on, France Martigues (near Marseille), France Sydney, Australia Sao Paulo, Brazil

Green et al. [55] Naspitz et al. [56] Tovey [54]

Alice Springs, Australia

Sample (n)

Bed

Bedroom floor

Living-room floor

—

—

0.36 9 to 11 years old (211)

15.8

random (72-74)

22.42(18.3, 38.86 (95% c.i.** 31.8, 47.5) 27.5) 38.4 >4

13.68 (10.7, 17.6) >4

negligible

negligible

moderate to severe asthmatic and positive sldn test (20) general

negligible

* Quantities are geometric means expressed as ng{ofmite allergen Der p I)/g of fine dust. **Confidence interval.

Climate and Chronic Respiratory Disease

341

Viruses The seasonality of upper and lower respiratory tract infections, and their common association with exacerbations of asthma, chronic bronchitis, and emphysema, suggests that viral infections are both related to the climate and can provoke respiratory diseases [63, 64]. Derrick [18] has proposed a mechanism whereby an asthma attack is brought about by a sudden cold temperature change first inducing a respiratory infection. He cites Andrewes' [65] suggestion that cold temperatures might activate latent viruses, and that rhinoviruses are very prevalent among the cold infections associated with the first cold weather of autumn. It is likely that very low humidity can cause irritation of mucous membranes and may predispose the susceptible to respiratory tract infections. DWELLING CHARACTERISTICS, DAMP HOUSING, MOLD GROWTH, AND ASTHMA It has recendy become clear that certain characteristics of the home environment, such as the presence of damp, mold, and house dust mites, may be of the utmost importance in some CRDs like asthma. Older buildings, for example, can have problems of rising damp resulting from the disintegration of damp-proof building materials. The activities of humans in buildings can also gready effect the indoor climate. Activities involving clothes driers, showers, gas heaters, and cookers can inject large volumes of moisture into the air. Burr et al. [66], in their survey of respiratory disease in a South Wales town, found an association between those with a history of wheezing with breathlessness, and reported dampness and use of open coal fires throughout the year. Strachan and Elton [67], in a study of the relationship between respiratory morbidity in children and the home environment in Edinburgh, Scotland, found parental reports of wheeze, nocturnal cough and school absence owing to chest trouble were significantly more common among children from damp or moldy housing. They also found an association between coal fires and nocturnal cough. Brunekreef et al. [68], in a study of children living in the United States, also found a strong and consistent association between measures of home dampness and respiratory symptoms. However, only a weak relationship was found between home dampness and pulmonary function. Similar results have been found in a more recent North American study [69] where the odds ratio between symptoms and dampness was 1.62 (95% c.i. 1.48 to 1.78). Burr et al. [70, 71] found asthmatics reported mold on the inside walls of their homes in Cardiff more than twice as often as controls (Table 3). Piatt et al. [72], in a study of the relation between damp and mold growth and symptomatic ill health in the United Kingdom, found that both adults and children living in damp and moldy dwellings had a greater prevalence of respiratory symptoms such as breathlessness and wheeze compared with those living in dry dwellings. Although other studies have found such an association (e.g., Strachan [73]), it is possible that the simple awareness of dampness or mold in the home might be a determinant of parental reporting of respiratory symptoms.

342

Health and Toxicology Table 3 Studies of reported and measured prevalence of mold in dwellings

Reported mold (%)

Reference

Location

Sample (n)

Brunekreef et al. [68]

Kingston, TN Steubenville, OH Watertown, MA St. Louis, MO Topeka, KS Portage, WI

eight- to 12-yr-old children (4,625)

38.1 27.9 20.9 26.9 33.0 35.4

Burr et al. [70] &Burretal. [71]

Cardiff

asthmatics (72) controls (72)

26.0 12.5

Piatt et al. [72]

Glasgow, Edinburgh and London

households (597)

Strachan & Elton

Edinburgh

children (165)

Actual mold

45.9%

21.0

m Finally, it has recently become clear that there are close relationships between damp housing and the prevalence of molds and house dust mite populations. A recent study in the Netherlands found home dampness was associated with increased sensitization to dust mite and molds [74]. SYNERGISTIC EFFECTS One of the major challenges that faces modem society is the need to understand the complex synergistic interactions involved in CRD. Experimental studies by Bethel et al. [75] and Sheppard et al. [76] suggest how one synergy operates. This study found that cold dry air potentiated S02-induced bronchoconstriction, and that exposure to 0.1 ppm SO2 potentiated the effect of cold and dry air. Further, Linn et al. [77] found that warm humid air ameliorated the response to SO2. EPIDEMICS The circumstances surrounding epidemics of respiratory diseases like asthma have been the focus of many investigations. Many asthma epidemics have been associated with thunderstorms, which are characterized by rapid temperature drops, wind speed and direction changes, and heavy rainfall. The actual mechanisms involved in such epidemics have often been difficult to unravel. A number of studies have identified a sudden increase in airborne mold spores, as well as the characteris-

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tic atmospheric changes, as the cause of the outbreak [78]. Recently, however, more complex mechanisms have been proposed, particularly that sulphuric acid particulates could be transported to the surface from high altitude by thunderstorms [79]. It would also seem that the rainfall associated with thunderstorms could trigger the release of allergenic starch grains from pollens [80, 81]. The passage of cold fronts, involving rapid temperature decreases, wind speed and direction changes, and pressure changes, have also been shown to be associated with asthma attacks [9, 23, 82]. In some circumstances, epidemics of asthma may result from a combination of meteorological and aerobiological factors. Asthma epidemics in Barcelona, for example, seem to have originated from plumes of soybean particles being produced when beans were unloaded into silos from ships in the harbor on days with high pressure and little wind, the city lying downwind on every occasion in which the air movement occurred. Although there is still much we do not know about the impacts of the atmospheric environment on CRDs, at least one attempt has been made recently to integrate the sum of knowledge gained through the numerous and varied studies conducted to date [83]. Given the apparently complex interrelated factors involved, these authors have constructed a schematic model showing the linkages and relationships between the human and biophysical environments and the asthmatic person. Such models are beneficial as they can clarify and enhance our understanding of complex disease processes. THE FUTURE: CLIMATE CHANGE AND CHRONIC RESPIRATORY DISEASE In the context of global warming, the relationships and interactions between climate and CRD assume a new significance. Any deterioration of the status of chronic respiratory diseases associated with future climate change will undoubtedly have significant social and economic consequences. To date, reviews of climate change and health with respect to asthma have largely considered the impacts of atmospheric triggers such as urban air pollution and, to a lesser extent, allergens and the direct impacts of climate and meteorological phenomena. There are a number of problems and difficulties associated with gauging the impacts of global climate change on asthma. These include difficulties in predicting regional and local patterns of climate change and in assessing human and architectural influences on asthma triggers, as well as the problems associated with clarifying the effects of individual factors in a multifactorial disease [84]. Further, Longstreth [85] recognized that there are such complexities involved in even assessing the role current weather plays in human disease that one could only speculate at the effects of global warming on human health. Climate change is generally expected to lead to increases in morbidity and mortality from respiratory diseases like asthma. Many of the effects of climate change on respiratory diseases will be indirect, or secondary, as well as direct, or primary

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[16, 86, 87]. As will be discussed below, climate change will affect asthma indirectly through its impacts on atmospheric irritants and allergens and infections, and directly through its influence on the climate (Figure 5). Air Quality (Irritants) Longstreth [85] recently suggested that "respiratory diseases brought about by increased air pollution," such as asthma, would be "one of the largest public health issues" associated with global warming and stratospheric ozone depletion. Many studies have discussed the effects of atmospheric pollutants on asthma and how their occurrence may vary with climate change. An acceleration of photochemical reaction rates among the precursors of photochemical smog would be associated with increased temperatures and ultraviolet-B (UV-B) radiation [87-92]. Thus, high levels of O3 would be achieved earlier in the day and maintained longer more frequently [85, 93]. Such increases in ozone would be significant, as this irritant has been linked to sunmier peaks in the incidence of asthma [16].

Figure 5 Change in January air temperature over the globe associated with an equivalent duubling uf uarbuH diUAide. Frum the Austrcilicin Buruciu uf Muttijurulugy Research Centre's General Circulation Model. (Courtesy of Dr. Bryant MoAvaney and Ann-Maree Hansen.)

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Future emissions of NO2 and SO2 will rely primarily on human decisions and actions, largely being dependent on trends in fossil fuel consumption and, to a lesser extent, on the balance between the need for heating and cooling [91]. For example, increased winter temperature would lessen the demand for energy production for heating and thus would reduce acid aerosol exposure and reduce asthma morbidity [89]. Regional-scale (synoptic) conditions such as high pressure systems (anticyclones) and local-scale conditions such as katabatic flows and sea breezes play a large role in air pollution production. Changes in the occurrence of such regional and local conditions will influence urban air quality [92]. Allergens There is a clear relationship between many allergen-producing organisms such as plants, mold, and house dust mites, and climatic factors such as humidity, temperature, rainfall, and sunshine. Increased levels of pollens and other biologic agents causing allergic respiratory system reactions in humans may be expected to result from higher temperatures coupled with elevated humidity. Last and Guidotti [94] and Last [95] have suggested there would be significant changes in the distribution of vegetation, including many allergens. Similarly, Longstreth [85] has suggested that "changes in the prevalence or intensity of asthma . . . episodes in affected individuals" could come about through likely "quantitative and/or qualitative changes in the airborne concentration of allergens" such as mold spores and pollens. Climate Wind plays a major role in the transport of pollens, dust and other irritants [96]. Curson [97] has suggested "drier winters and changed wind movements and cyclonic activity" may lead to asthma increases in Australia. Waves of extreme heat and cold impose stress on asthmatic individuals [98]. Ewan et al. [92, 99] indicated "heat stress combined with high humidity act as a trigger to attacks." Hennessy [100] has estimated the change in frequency of extreme cold events in southeast Australia (winter days when temperature falls below 0°C) associated with a 3°C warming. It is clear that such events may become far less frequent. It is likely that acute asthma symptoms triggered by sudden exposure to cold air will be reduced if such climate change occurs in this region. Similarly, Langford and Bentham [29] have suggested that there would be fewer winter deaths from respiratory disease such as chronic bronchitis associated with the warming predicted to occur with climate change. Infections Ewan et al. [92] have stated that "infections such as the common cold and influenza" could increase in southeast Australia and decrease in southwest Aus-

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tralia due to predicted increases and decreases in rainfall, respectively. Such infections are commonly associated with asthma symptoms. These potential increases are significant because peaks in hospital admissions for asthma in autumn/winter have been related to viral infections [16, 101]. In the case of asthma, each asthmatic is unique in his sensitivity to, and the composition of, factors that provoke acute symptoms. As such, it is difficult to generalize about the overall impacts that climate change will have on the status of the disease. Asthmatics rarely, if ever, react or are reactive to only one trigger. While there is increasing evidence that triggers act synergistically or in combination, such complex mechanisms are not fully understood. Indeed, Goldstein and Reed [86], in their commentary on global atmospheric change and research needs in environmental health sciences, recognized the need to better understand both the individual and multiple effects of indoor and outdoor irritants and allergens on people with chronic respiratory diseases such as asthma. It is possible that a wide range of factors will be suited to a variety of environmental conditions. Just as the conditions close to the coast favor the house dust mite while those inland favor grass pollen production (reflected in the status of respective allergies present in the two populations), different molds grow over very different temperature ranges. As such, no matter what the change in climate, there may still be molds that can grow in that new climate. TECHNOLOGICAL WARNING SYSTEMS Because of the knowledge base that has been built with regard to the relationships between climate and weather and the occurrence of air pollutants and allergens, local and regional authorities are able to issue warnings and advise the public in advance of potentially threatening events. Warnings of elevated levels of air pollution are provided in many urban centers. Such warnings require an intimate knowledge of the local and regional setting for which the forecast is to be made. Factors that are often taken into consideration include the forecast wind direction and speed, the existence of temperature inversion, the day of the week, and whether rain is expected. As such, it is usually the local environmental protection authority and meteorological bureau that are best placed to issue such warnings. The need for warnings of elevated pollen and mold spore levels is perhaps more wideranging because it is not only urban areas that are affected by such events. Such warning systems have not been as well developed as those for air pollution, though the potential benefits could be equal to or greater than those derived from air pollution warnings. ENVIRONMENTAL CONTROL In addition to making forecasts of potentially harmful levels of air pollution and airborne allergens, attempts have been made to determine housing characteristics

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that are best suited to the prevention and management of CRDs. Recently, adequate ventilation and dehumidification have been targeted as essential factors in the control of the house dust mite [102, 103]. In cold climates, ventilation systems have been designed to exchange cold and dry outdoor air for warm and moist indoor air. In order to further reduce the humidity of indoor air, and thus house dust mite populations, a heat exchange system warms the outdoor air (which is to replace the indoor air), thus reducing its relative humidity. Indoor climate can also be controlled by using moisture absorbers, extractor fans in rooms where large amounts of moisture are generated such as bathrooms, and by using flues on gas appliances to prevent moisture from gas combustion being added to the indoor atmosphere. Older dwellings also often have structural defects such as leaking roofs and deteriorated damp-proof materials. The renovation of such dwellings can greatly improve the indoor microclimate that is so important for house dust mites and molds. The control, prevention, and treatment of mold growth in dwellings have been well documented [38]. These, too, focus on moisture control and ventilation. CONCLUSIONS There is no doubt that climate is related to chronic respiratory disease in many ways. Climate and weather have been linked to major outbreaks of chronic respiratory disease, seasonal variations of such disease, and more subtle instantaneous effects on the respiratory system. This chapter has viewed the relationship between climate and chronic respiratory disease by first looking at the direct effects of climate and weather such as the well-documented effects of cold dry air on asthmatics, and second by examining the diversity of indirect effects. The latter include the effects of climate and weather on potent stimuli such as air pollutants, and allergens, including mold spores, pollens, and house dust mite. It has also been shown that both the outdoor and indoor environments are important, with some stimuli such as house dust mites exclusively occurring in the latter. Perhaps the major challenge is to understand the complex synergistic relationships between many environmental agents and chronic respiratory disease. Such relationships may prove to be the dominant mechanisms producing such disease. Though our understanding of chronic respiratory disease is far from complete, we must look to the future and see what it holds for these significant diseases. While climate change may in some ways be detrimental to diseases such as asthma, our growing understanding of the relationships between the natural biophysical environment and human disease enables us to predict changes in and improve our environment. REFERENCES 1. National Institute of Health (NIH). International Consensus Report on Diagnosis and Management of Asthma. U.S. Department of Health and Human Services, NIH publication 92-3091, 1992.

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ation," in Climate Change, The IPCC Impacts Assessment. W. J. McG. Tegart, G. W. Sheldon, and D. C. Griffiths (Eds.), Canberra: Australian Government Publishing Service, 1990. 91. World Health Organization. Potential Health Effects of Climate Change. Geneva: World Health Organization, 1990. 92. Ewan, C., Bryant, E., and Calvert D. (Eds.). Health Implications of Long Term Climatic Change. Canberra: Australian Government Publishing Service, 1991. 93. Robinson, P. "The Effects of Climate Change," in Global Climate Change Linkages: Acid Rain, Air Quality, and Stratospheric Ozone. J. C. White (Ed.), New York: Elsevier Science PubUshing Co., Inc., 1989, pp. 23-41. 94. Last, J., and Guidotti, T. L. "Implications For Human Health of Global Ecological Changes." Public Health Reviews, Vol. 18, No. 1, 1990/91, pp. 49-67. 95. Last, J. M. "Global Change: Ozone Depletion, Greenhouse Warming, and Public Health." Annual Review of Public Health, Vol. 14, 1993, pp. 115-136. 96. Davis, D. L., Miller, V., and Reisa, J. J. "Potential PubUc Health Consequences of Global Climate Change," in Preparing for Climate Change. Rockville, Maryland U.S.A.: Climate Institute, Government Institutes, Inc., 1988, pp. 366-376. 97. Curson, P. "Human Health in the Greenhouse Era," in Planning for the Greenhouse, a Conference Designed for Planners, Engineers, Health Surveyors and Resource Managers. K. McCracken and R. Blong (Eds.), July 17-18, 1989, Macquarie University, Sydney, pp. 140-150. 98. Longstreth, J. "Overview of the Potential Effects of Climate Change on Human Health," in Coping with Climate Change. J.C. Topping (Ed.). Proceedings of the 2nd North American Conference on Preparing for Climate Change. December 1988. pp. 142-146. 99. Ewan, C. et al. "Potential Health Effects of Greenhouse Effect and Ozone Layer Depletion in Australia." The Medical Journal of Australia, Vol. 154, No. 8, 15 April 1991, pp. 554-559. 100. Hennessy, K. J. "Regional Climate Scenario for South-Eastern Australia in 2030," in Agriculture and Greenhouse in South Eastern Australia. I. E. Galbally and P. Holper (Eds.). CSIRO Division of Atmospheric Research. 1992. 101. Haines, A. "Global Warming and Health." British Medical Journal, Vol. 302, No. 6778, 23 March,1991, pp. 669-670. 102. Emenius, G. et al. "Mechanical Ventilation Protects Against High Humidity and Mite Growth in Dwellings." All. and Clin. Immunol. News, Vol. 2 supp., 1994, p. 470. 103. Korsgaard, J. "Ventilation and House Dust Mites." Der Allergiker, Vol. 1, 1988, pp. 16-18.

CHAPTER 15 EFFECTS OF ACID PRECIPITATION ON THE ENVIRONMENT AND ON HUMAN HEALTH Lars Gerhardsson and Staffan Skerfving Department of Environmental and Occupational Medicine Lund University Hospital S-221 85 Lund, Sweden Agneta Oskarsson Department of Food Hygiene Swedish University of Agricultural Sciences S-750 07 Uppsala, Sweden CONTENTS INTRODUCTION, 355 AIR, 356 WATER AND SOIL, 357 Calcium, 358 Aluminum, 358 Cadmium, 359 Copper, 359 Lead, 359 Magnesium, 360 Mercury, 360 Metal Exposure in Farmers, 360 CONCLUSIONS, 361 REFERENCES, 362 INTRODUCTION Environmental acid precipitation is a global problem that may cause a number of adverse health effects in man. These effects can be of both primary and secondary nature. Primary effects in humans are mainly confined to the respiratory tract and are caused by inhalational exposure. These effects most often result in impairment of pulmonary function, and symptoms such as cough, dyspnea, and increased mucus secretions. Secondary effects are caused by increased solubilization and mobilization of toxic metals—such as aluminum, cadmium, copper, lead, and mercury—in lakes and waters, leading to groundwater pollution and uptake in plants [1]. Through the animal and vegetable food chain, ingestion of polluted sources, e.g., fish and vegetables, will increase the intake of toxic metals in man, which may lead to health implications [2]. For some elements, such as the essential element selenium, acid 355

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precipitation results in decreased mobility and decreased levels taken up in crops. The greatest problems are found in regions with a combination of high fallout of acid precipitation and a low buffering capacity of bedrock, water, and soil. AIR The atmospheric acidity consists of both gaseous and particulate phases, which are mainly formed from the oxidation of sulfur oxides (SOx, e.g., SO2), nitrogen oxides (NOx, e.g., NO2), and hydrocarbons from combustion sources [3]. The SO2 gas passes a series of photo-oxidation reactions in the atmosphere, leading to formation of sulfate particles. The principal forms are sulfuric acid (H2SO4) and ammonium sulfate [(NH4)2S04]. They are characterized by relatively long residence time and long-range atmospheric transport. Laryngeal and tracheobronchial deposition is expected to be greater when breathing H2SO4 as fog droplets as compared with humid haze particles. On the other hand, deposition of acid in the alveolar region seems to be greater via humid haze particles than via fog droplets [4]. Various NOx compounds are formed in combustion processes and emissions from motor vehicles. The principal component is NO2, which is hydrolyzed to HNO2 (nitrous acid) and HNO3 (nitric acid), occurring mainly in fog or cloud droplets. Most of the HNO3 is expected to be deposited in the nose and upper part of the respiratory tract when breathing either haze or fog. In the former case, the acid is inhaled and rapidly removed as a gas, whereas in the latter case it may be efficiently removed in large droplets by the extrathoracic airways [4]. Also, through photo-oxidation processes, NOx contributes to the formation of ozone (O3), which is implicated as a main human health hazard in photochemical pollution. Acid aerosols in ambient air are in the respirable size and will reach the alveolar region of the lung [3]. In animal experiments, acid aerosols have caused airway narrowing, changes in particle lung clearance, and changes in response to inflammation [3]. In humans, a relation between increased acid aerosol levels and total mortality has been reported [5]. Other adverse health effects connected to exposure to acid aerosols include increased hospital admissions due to mainly respiratory symptoms [6]. Exposure to acid aerosols has been associated with increased prevalence of bronchitis symptoms in children [7]. Concomitant exposure to acid aerosols and ozone have affected lung function in normal children exercising outdoors [3]. For example, decrements of forced expiratory volume (FEVl) and peak expiratory flow (PEP) have been associated with air pollution episodes [8]. In the latter study, a larger decrement was indicated for children with a positive response to metacholine challenge compared to their nonresponsive counterparts. In experimental studies in humans [9, 10], asthmatics show statistically significant dose-related increases in respiratory symptoms, as well as dose-related decrements in forced expiratory performance when exposed to sulfuric acid aerosols. An association between summer hospital admissions for respiratory diseases in Southern

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Ontario and daily concentrations of particularly SO4 has been reported [6]. Multiple regression analysis showed that SO4 accounted for about 3% of the variance. Pollution episodes are often worse in winter, when high concentrations of pollutants may be trapped in the bottom few hundred meters of the atmosphere [11]. High concentrations may lead to severe adverse health effects. One of the worst disasters took place in London in 1952 in which coal smoke containing sulfuric acid was implicated as the causing agent. It is estimated that more than 4,000 deaths, primarily in elderly people, occurred in London during that single episode [12]. During the 1950s and early 1960s, deaths connected to chronic bronchitis were higher in London than in other, less-polluted areas in the U.K. and considerably higher than in other countries in northern Europe [12]. Later investigations showed that the excess mortality in London was more closely associated with blackness of sampled particles (BS) than with SO2. However, BS is mainly a measure of the carbon content of the aerosol, and is thus unlikely as a causal factor. It seems more probable that another aerosol compound, e.g., the free hydrogen ion (H"^), was the causal factor [12]. Increases have been observed in the total number of deaths (8%), hospital admissions (15%), and ambulance use (30%) for respiratory and cardiovascular illnesses in subjects living in a polluted area during the January 1985 air-pollution event in western Europe, compared to a before-and-after interval, and with a control area [13]. However, the effects could not be attributed to acidic aerosols as such [14]. The observation of an association between fine particulate air pollution and health outcomes in regions where little aerosol acidity has been measured suggests that particulate acidity alone may not be the primary component causing fine particulate air pollution toxicity [15]. Thus, respirable particulate air pollution is probably an important contributing factor to respiratory disease (e.g., symptoms and decreased lung function), increased hospitalizations, and other health care visits for respiratory and cardiovascular disease [16, 17]. There are two important defense systems against inhalation of acid aerosols. One is the conversion of acid to the ammonium salts by respiratory ammonia, and the other is buffering by the mucous lining of the airways [18], especially by its glycoproteins. The mucous increases in viscosity, leading to decreased clearance, as well as increased airway resistance and reduced pulmonary gas exchange [19, 20]. Further, after saturating the buffering capacity, the underlying tissues will receive an increased dose of acid. WATER AND SOIL Environmental pollution increases the exposure to metals in two ways. They can either be deposited from the atmosphere or mobilized from soil and water [21]. The solubilized metals will cycle through the ecosystems. The primary exposure to man is mainly through food and water, and adverse health effects may occur if the intake exceeds critical levels.

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Rainwater contains a variety of chemicals, including both strong and weaker acids. As strong acids such as sulfuric and nitric acids contribute up to 95% or more of the total acidity, the pH of rainwater can be used as an index of the acidity. Calcium Present acid rainfalls in the range pH 4.0-4.5 in many European countries will have a great impact on aquatic and terrestrial systems [22]. Generally, about 90% of the rain will pass through one or more soil layers before reaching a stream that feeds a lake. Water flowing over chalk and limestone will normally be completely neutralized. In more acidic soils, however, a cation exchange process may take place. In this process, hydrogen ions from the acid rain will be exchanged by soilderived elements, e.g., calcium. This gradually decreases the acidity of the water passing through the soil, and in parallel increases the calcium concentration in the water. In contrast, in soils naturally poor in exchangeable calcium, the soil may become progressively acidified, leading to increased leakage of, e.g., aluminum instead of calcium into the water [11]. The bedrock in, for example, Norway and Sweden has a low buffering capacity, which makes these countries more susceptible to acid downfalls. Afforestation may also worsen the effects of soil acidification. Growing trees take up calcium and other basic elements from the soil. The harvesting of timber removes this calcium, which otherwise in a natural ecosystem would be recycled [11]. Adequate concentrations of calcium seems to be as important for fish survival as is low acidity. The calcium/acidity ratio has been suggested as an index for fish survival as high values are associated with well-stocked lakes and low values with fishless lakes. High concentrations of calcium also seem to counteract harmful effects of toxic aluminum compounds as shown by experimental studies [11]. Liming of lakes has been practiced in Sweden for several years, showing good results. Usually, limestone has been added to the water itself. It would, however, probably be more efficient to lime the lake catchment instead [11]. Then it would be possible not only to increase the calcium supply to the soil, but also to prevent the release of aluminum into water. Aluminum Aluminum is an ubiquitous metal constituting about 8% of the earth's crust. It has generally been considered relatively non-toxic to humans. However, some studies have reported an increased incidence of Alzheimer's disease in regions with elevated aluminum concentrations in drinking water. Headwater streams exhibit episodic declines in pH and increases in dissolved aluminum leaching from soils during rainfall and snowmelt events. The elevated aluminum levels in source streams can directly affect the quality of domestic tap-

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water. Acid precipitation runoff episodes, thus, may give significant decreases in tapwater pH and increases in tapwater aluminum [23]. Cadmium Important exposure sources for the general population are diet and tobacco smoke. In many regions, the margin between present exposure levels and adverse health effects is small or non-existent. This is exemplified by the Cadmibel study in Belgium, where approximately 10% of the general population had an internal dose of cadmium sufficient to cause slight renal dysfunction [24]. The cadmium uptake in crops is considerably raised by acid precipitation, leading to increased human dietary intake. The long biological half-time (10-30 years) in the kidneys, the main storage pool in the body, means that virtually all absorbed cadmium is retained. More than two thirds of the increased concentrations of cadmium in winter wheat in Sweden seem to originate from increased soil concentrations. In this context, alterations of soil pH is probably of less importance. In the southern part of Sweden, cadmium concentrations in the grain of winter wheat close to or exceeding the maximum permissible level of 100 |Lig Cd/kg, as suggested by FAOAVHO, is often found. Thus, not much could be gained by Uming, because wheat is grown on the best soils with a near optimal pH [25]. Copper Copper is an essential element for man with a daily need of 1-1.2 mg in adults (0.3 mg in children up to one year of age). The safety margin between beneficial intake of copper and potentially harmful effects seems to be small [26]. The concentration of copper in drinking water originates from the piping system. Acidic water increases the corrosion processes and elevates the copper concentration. However, after flushing, the levels diminish rapidly. In a Swedish study of 600 children aged 9-21 months (Pettersson and Rasmusson, personal communication), most children had a daily intake of copper from water below 0.5 mg. Approximately 25% had an intake between 0.5-1 mg/day, and 10% exceeded 1 mg/day. Thus, copper in drinking water accounts for more than the daily need, for most children in this study. Lead Acid precipitation will increase the lead solubility, mobilizing lead from soils, especially in poorly buffered systems. The decreased pH will also solubiHze lead from pipes and lead soldered joints in cisterns. Acid deposition from the environment will also accelerate the weathering of lead-painted surfaces.

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Magnesium During the past few decades, there have been suspicions that acid deposition may severely damage growing trees. This issue attracted serious concern in the early 1980s when suddenly and unexpectedly a rapid decline of Norway spruce began in southern Germany. Recent studies suggest that soil magnesium deficiency is a main reason for this type of forest damage, probably due to a reduced magnesium supply [11]. Mercury Inorganic mercury is transformed to methylmercury in water. The methylation mostly takes place on sediments in fresh and ocean waters, but also in fresh and sea waters, and the process is enhanced by acidity. Through the food chain, the methylmercury concentration is increasing, giving levels exceeding 1.2 mg/kg in e.g. shark, swordfish, pike, and bass. Through this mechanism, long-term and high consumption of fish from contaminated waters will increase the risk of toxic effects on the central nervous system [21], as shown from the epidemic in Minamata. Historic discharges in Sweden have the greatest impact on present mercury concentrations in pike, followed by water acidity and airborne exposure of mercury emissions from other countries. The latter are of greatest importance in southern Sweden. A program has been implied, testing various remedial measures such as lake and wetland liming, potash and selenium treatment, and intensive fishing [27]. After proper liming, a 30% reduction of mercury concentrations in small perch have been observed in 2 years. The levels in 1-kg pikes can probably be reduced by 15^0% after another 2 years. No discernible differences between various sorts of lime were noted with respect to the reduction of mercury concentrations in small perch [25]. Intensive fishing caused a reduction of the biomass in the lake by about 25%, and reduced the mercury levels in 1-kg perch by 20-30% after 2 years. It is expected that the effect of intensive fishing would last about 6-8 years. When using selenium treatment, the dosing is very important, as the reproduction of perch was shown to decrease considerably. Also, other species of animals and plants may be sensitive to increased selenium levels in the water. On a long-term basis, however, the best solution is to minimize the mercury contamination of air, soil and water [25]. Metal Exposure in Farmers A Swedish study included 237 farmers with a high consumption of locally produced food and water from their private wells, showed different grades of acidity (range of pH 4.7-8.6). A significant correlation between the pH and the metal concentration in drinking water was observed [1]. The relation was negative for aluminum (median 0.07 jimol/L), cadmium (0.44 nmol/L), copper (0.24 jimol/L)

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and lead (1.9 nmol/L), and positive for calcium (0.62 mmol/L) and magnesium (0.21 mmol/L). However, no relation was observed between metal uptake in the subjects, as indicated by concentrations in blood and urine on the one hand, and pH in drinking water on the other (used as an acidity index). Other sources of metal exposure had a stronger impact on the concentrations in blood and urine than the acid precipitation had. Thus, the concentration of mercury in blood was related to fish consumption, cadmium and lead in blood to smoking, aluminum in urine to intake of antacids, lead in blood to rifle shooting and hunting, and mercury in blood to hunting [1]. In this study, the acid precipitation had an effect on the metal concentrations in drinking water, but not on the body retention of those elements. In another study in Scandinavia [28], concentrations of cadmium, nickel, zinc, and selenium were studied in spring wheat, potatoes, and carrots from commercial farming fields and correlated to pH and different soil factors. The selenium levels in potatoes decreased with decreasing pH. However, with decreasing pH in soil, there was an increasing concentration of cadmium, nickel, and zinc in potatoes and carrots, while in wheat only nickel and zinc levels were raised. In a multiple regression analysis, a combination of pH in soil, organic matter content, and cadmium concentration in soil explained 25, 68 and 41% of the total variation in cadmium concentration in potatoes, carrots, and spring wheat, respectively. Preliminary findings indicate that liming with CaC03 will lead to a decreased uptake of nickel and zinc in spring wheat and potatoes. The effect on cadmium, however, seems to be more complex, probably due to a competition between cadmium and calcium on the soil particles. Approximately 75% of the total dietary intake of cadmium originates from cereals, potatoes, and vegetables. It is thus probable that acid deposition causing decreases in pH of arable soils in combination with other acidifying processes in agriculture, e.g., fertilization and harvest of biomass in connection with insufficient liming, will lead to an increased cadmium intake via food as well as altered dietary exposure of other elements. CONCLUSIONS Air pollution with SOx and NOx causes exposure to acid pollutants and may result in irritant effects in the respiratory tract of man, causing symptoms such as cough and dyspnea. Impaired pulmonary functions have been reported at such exposures. Environmental acidification is a global problem that increases levels of toxic metals in water and food. Of these metals, aluminum, cadmium, lead, and methylmercury are of particular concern. Acid precipitation may increase the concentrations of aluminum, cadmium, copper, and lead in drinking water. On the other hand, levels of calcium and magnesium seem to be positively related to the pH. Furthermore, acid rain can cause an accumulation of methylmercury in fish in rivers and lakes.

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In parallel, acid precipitation may lead to increased metal concentrations in plants and vegetable foods, although, as shown for cadmium, the soil concentration seems to be more important. Accordingly, acid precipitation may have an impact on the exposure/uptake of metals. For several elements, e.g., cadmium, lead and mercury, there is a clear connection between environmental exposure and measured concentrations in biological indicator media such as blood and urine. The safety margin between current exposure and critical effect levels in humans is small for many toxic metals in some geographical areas. Moreover, in other regions around the world, these critical effect levels are indeed exceeded, causing effects on the health status of the population. One example is cadmium, which has been observed to affect the kidney function in a minor portion of the general population. For exposure to lead and methylmercury, the fetus has an increased susceptibility, and developmental effects in infants and children may occur at various exposure levels in pregnant women living in several regions. Up to now, no firm evidence linking acid precipitation to metal-related adverse health effects in man has been presented. However, there is a small or non-existent margin between the present intake of some metals, on the one hand, and toxic exposures, on the other. Further, for some essential metals, acidification may decrease the intake. This makes it necessary to follow the ongoing acid precipitation with greatest concern. To deal with this task, a global approach has to be adopted. It is important that accurate steps are taken on both national and international levels to enforce legislation that protects everyone. REFERENCES 1. Bensryd, I. et al. "Effect of acid precipitation pn retention and excretion of elements in man." Sci. Total Environ., Vol. 145, 1994, pp. 81-102. 2. NAPAP, National Acid Precipitation Assessment Program. "Indirect health effects associated with acidic deposition. National Acid Precipitation Assessment Program. Acid deposition: state of science and technology." NAPAP Report 23, Government Printing Office, Washington, D.C., 1990, p. 173. 3. American Thoracic Society. "Health effects of atmospheric acids and their precursors. Report of the ATS workshop on the health effects of atmospheric acids and their precursors." Am. Rev. Respir. Dis., Vol. 144,1991, pp. 464-467. 4. Larson, T. V. "The influence of chemical and physical forms of ambient air acids on airway doses." Environ. Health Perspect., Vol 79, 1989, pp. 7-13. 5. Thurston, G. D. et al. "Reexamination of London, England, mortality in relation to exposure to acidic aerosols during 1963-1972 winters." Environ. Health Perspect., Vol. 79, 1989, pp. 73-82. 6. Bates, D. V. and Sizto, R. "The Ontario air pollution study: identification of the causative agent." Environ. Health Perspect., Vol. 79, 1989, pp. 69-72.

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7. Speizer, F. E. "Studies of acid aerosols in six cities and in a new multicity investigation: design issues." Environ. Health Perspect., Vol. 79, 1989, pp. 61-67. 8. Raizenne, M. E. et al. "Acute lung function responses to ambient acid aerosol exposures in children." Environ. Health Perspect., Vol. 79, 1989, pp. 179-185. 9. Hackney, J. D., Linn, W. S. and Avol, E. L. "Acid fog: effects on respiratory function and symptoms in healthy and asthmatic volunteers." Environ. Health Perspect., Vol. 79, 1989, pp. 159-162. 10. Koenig, J. Q., Covert, D. S. and Pierson, W. E. "Effects of inhalation of acidic compounds on pulmonary function in allergic adolescent subjects." Environ. Health Perspect., Vol. 79, 1989, pp. 173-178. 11. Crane, A. J. "Acid Rain." The Journal of the Royal Society of Health, Vol. 110, No. 3, 1990, pp. 77-80. 12. Lippmann, M. "Background on health effects of acid aerosols." Environ. Health Perspect., Vol. 79, 1989, pp. 3-6. 13. Wichmann, H. E. et al. "Health effects during a smog episode in West Germany in 1985." Environ. Health Perspect., Vol. 79, 1989, pp. 89-99. 14. Shy, C. M. "Review, discussion, and summary of epidemiological studies." Environ. Health Perspect., Vol. 79, 1989, pp. 187-190. 15. Brauer, M. et al. "Measurement of acidic aerosol species in eastern Europe: implications for air pollution epidemiology." Environ. Health Perspect., Vol. 103, 1995, pp. 482^88. 16. Pope, C. A. Ill, Bates, D. V., and Raizenne, M. E. "Health effects of particulate air pollution: time for reassessment?" Environ. Health Perspect., Vol. 103, 1995, pp. 472-480. 17. Schwartz, J. "Air pollution and hospital admissions for respiratory disease." Epidemiology, Vol. 7, 1996, pp. 20-28. 18. Folinsbee, L. J. "Human health effects of exposure to airborne acid." Environ. Health Perspect., Vol. 79, 1989, pp. 195-199. 19. Holma, B. "Effects of inhaled acids on airway mucus and its consequences for health." Environ. Health Perspect., Vol. 79, 1989, pp. 109-113. 20. Graham, J. A. "Review, discussion, and summary: toxicology." Environ. Health Perspect., Vol. 79, 1989, pp.191-194. 21. Goyer, R. A. et al. "Potential human health effects of acid rain: report of a workshop." Environ. Health Perspect., Vol. 60, 1985, pp. 355-368. 22. Oskarsson, A. "Ground-water acidification and health effects," in Water and Public Health, A. M. B. Golding, N. Noah and R. Stanwell-Smith (Eds.), Great Britain: Smith-Gordon, 1994, pp. 195-203. 23. Sharpe, W. E. and DeWalle, D. R. "The effects of acid precipitation runoff episodes on reservoir and tapwater quality in an Appalachian mountain water supply." Environ. Health Perspect., Vol. 89, 1990, pp. 153-158. 24. Buchet, J. P. et al. "Renal effects of cadmium body burden of the general population." The Lancet, Vol. 336, 1990, pp. 699-702.

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25. Gerhardsson, L., Oskarsson, A., and Skerfving, S. "Acid precipitation— effects on trace elements and human health." Sci. Total Environ., Vol. 153, 1994, pp. 237-245. 26. Pettersson, R. and Sandstrom, B. "Copper," in Risk Evaluation of Essential Trace Elements, Essential versus toxic levels of intake, A. Oskarsson (Ed.), Copenhagen, Denmark: Nord Series, Nordic Council of Ministers, 1995, pp. 149-167. 27. Hakansson, L. "Measures to reduce mercury in fish." Water Air Soil Pollut., Vol.55, 1991, pp. 193-216. 28. Obom, I., Jansson, G. and Johnsson, L. "Acidification of arable land—a field study on the influence of soil pH on trace element uptake in spring wheat, potatoes and carrots." Abstract No. 445 at "Acid Rain '95," Gothenburg, Sweden, 1995. To be published in Water Air Soil Pollut.

CHAPTER 16 TOXIC EFFECT OF TANNERY EFFLUENT ON THE BIOCHEMICAL CONSTITUENTS IN ORGANISMS G. Varadaraj and M. A. Subramanian P.G. & Research Department of Zoology Chikkaiah Naicker College Tamil Nadu, India CONTENTS INTRODUCTION, 365 OVERALL EFFECTS OF TANNERY EFFLUENT (TE), 366 EFFECTS OF TE ON BIOCHEMISTRY OF ORGANISMS, 367 In Plants, 367 In Animals, 368 REFERENCES, 372 INTRODUCTION The tannery industry is growing in diverse proportions at cottage-, medium-, and large-scale sectors, particularly in developing countries. It provides employment opportunities to several thousand people and earns considerable sums via foreign exchange, thereby contributing to the economy. Unfortunately, this industry is linked with water pollution. The quantity of water used is voluminous in the tannery unit; it discharges 3,000-3,200 liters of water per 100 kg of skins and hides processed. The spent water of the tanning industry is invariably let off as effluent, becoming either stagnant or, in most cases, confluent with the nearby water bodies. Thus, pollution due to tannery wastes and its control has gained international importance. Hides and skins are the principal raw materials in the tannery industry. Common salt, lime, sodium sulfate, ammonium sulfate, ammonium chloride, enzymatic products, sulfuric acid, sodium carbonate, dyes, and sulfated vegetable oils are the chemicals used in the industry. Small tannery units may use vegetable tanning materials like wattle bark extract, pungum oil, myrobalam, and kodukai, whereas big tanneries perform chrome tanning. The inherent nature of the tanning process leads to the discharge of wastewater having foul smell, highly putrescible matter and toxic materials. Generally, the effluent consists of both organic matter such as fatty acids, protein, other soluble tannin, and inorganic matter like chlorides, trivalent chromium, nitrates, phosphorus, sulfides, sulfates, etc.

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OVERALL EFFECTS OF TANNERY EFFLUENT (TE) Considerable work has been carried out on the characterization of tannery wastes (Guruprasada Rao and Nandakumar, 1981; Manivasakam, 1987; Subramanian et al., 1988; Kadam, 1995). Available literature clearly indicates that TE contains a high concentration of total solids, high biochemical oxygen demand (BOD) and chemical oxygen demand (COD), chlorides, sulfides, chromium, oil, grease, tannin and lignin, and low dissolved oxygen (DO) (Table 1). The TE when discharged into water courses adversely affects aquatic life and thereby poses a threat to the aquatic ecosystem (Eye and Lawrence, 1971). Sastry and Madhavakrisnan (1984) have reported that TE affects physical, chemical and biological characteristics of water, and depletes DO from the water body. On land, tannery wastes are found to cause damage to the soil quality, crop pattern, vegetation, and yield (Miakhan and Raman, 1972; Guruprasada Rao and Nandakumar, 1981). Harihara Iyer et al. (1957), Yusuff and Ismail (1984) and Varadaraj and Subramanian (1995a) have noticed the seepage of effluents from tannery units into the surrounding irrigation as well as drinking wells. Moreover, dermatitis, bronchitis, acute pharyngitis and other respiratory diseases, and acid burns are associated with tannery workers (Backyavathy et al., 1986).

Table 1 Physicochemical characteristics of tannery effluent and tolerance limit (ISi, 1981)

Characteristics

Tannery effluent

Tolerance limit (ISI)

Color Odor Temperature pH Total solids BOD COD DO Chlorides Sulfides Sodium Chromium Oil and grease Tannin and lignin

Brown or black Disagreeable, foul 29°C-31°C 8-10 1,250-23,920 96-1,200 116-27,810 0.84-1.10 298-6,700 10-84.7 74 32-381 12-16 50-90

Without practicable color Without unpleasant odor Within 40°C 5.5-9.0 2,950 30 250 — 1,000 2 — 2 10 —

Values in mgA except temperature andpH.

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EFFECTS OF TE ON BIOCHEMISTRY OF ORGANISMS In Plants On Seed Germination The first process in the growth of a plant is germination, a resumption of metaboHc activity by the seed involving dehydration, utilization of nutrient reserves, and gradual development of biosynthetic systems, enabling the young plant to assume an autotrophic existence. All plants store proteins as reserve food in their seeds. Mobilization of these stored proteins and their rapid translocation from the cotyledons to the growing axes involves proteolytic activity. During germination, insoluble carbohydrates like starch are first hydrolyzed into maltose in the presence of amylase. Maltose is then converted into glucose, which is transported to the growing embryonic axes. TE has been reported to diminish the reducing and non-reducing sugar levels as well as proteolytic and amylolytic activity levels in cotyledons and embryonic axes of Vigna radiata (Figure 1) (Varadaraj and Subramanian, 1995b). Saxena et al. (1986) also have documented restricted growth of radicals and plumules and suppression of seed germination with raw TE in Vigna mungo, Cicer arietinum, and Pisum sativum. The alteration in seed germination could be due to the increased salinity condition of TE as noticed by Narale et al. (1969) in Oryza sativa, Gaur and Tomar (1975) in Helianthus annus, Ramana and Ramarao (1978) in Rhaphanus sativus, and Varma and Poonia (1979) in Pennisetum typhoides. This is further strengthened by the investigation of Ogra and Baijal (1982), who have shown decreased activity of amylase and acid protease in Sorghum sp. during seedling growth in increased salinity. On Chlorophyll Content In green plants, chlorophyll is involved in the formation of organic substances from simple inorganic ones. A marked decrease in the chlorophyll content has been noticed in the shoots of Cicer arietinum in concentrations of 25, 50, 75, and 100% TE (39.40%, 41.84%, 43.56%, and 58.58%, respectively) (Guruprasada Rao and Nandakumar, 1983). Similar decrement in the chlorophyll content has been recorded by Kadam (1995) in the leaves of C arietinum under the influence of 75% and 100% TE (34.25% and 25.30%), whereas the chlorophyll content has been shown to increase (13.10%) in 25% concentration of TE. This reduction in the chlorophyll content in plants could be due to the effect of chromium present in the effluent as envisaged by Arvind Bharati et al. (1979).

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50 Concentration (%)

Figure 1. Effect of TE on sugar and activity levels of enzymes in y. radiata during germination on 5th day. (Varadaraj and Subramanian, 1995b) PC—Protease activity in cotyledon AC—Amylase activity in cotyledon PE—Protease activity in embryonic axis AE—^Amylase activity in embryonic axis NSC—Non sugar in cotyledons RSC—Reducing sugar In cotyledons NSE—Non reducing sugar in embryonic axis RSE—Reducing sugar in embryonic axis

In Animals Invertebrates Varadaraj et al. (1994) have reported decreased levels of body constituents like total free amino acids, total proteins, total free sugars, glycogen, and total lipids in various tissues of tannery effluent-treated Pila globosa (Table 2). They have shown that the effect of the effluent on the biochemical constituents of tissues is dosedependent. The influence of TE and its ingredients on the succinic dehydrogenase (SDH) activity level in the hepatopancreas of Pila globosa has been investigated by Guruprasada Rao and Nandakumar (1982) (Table 3). Total haemolymph proteins, free amino acids, and free sugars of the freshwater prawn, Macrobrachium idella, exhibit marked diminution (6.94-32.22%, 8.16-36.90%, and 10.12-41.21%, respectively) when exposed to different sublethal

Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms

369

Table 2 Dose-dependent changes in biochemical constituents in the tissues of P. globosa exposed to tannery effluent Ranges of % decrease over control Biochemical constituent

Total free amino acids Total proteins Total free sugars Glycogen Total lipids

Liver

Gill

Mantle

Foot

11.81-37.00 43.33-66.67 43.04-58.06 6.94-38.38 6.25-31.25

14.01-42.92 17.65-47.04 28.12-46.87 16.75-62.50 9.09-27.27

14.28-61.90 4.78-28.57 4.08-44.89 7.14-21.42 13.63-45.45

7.14-21.43 4.00-42.85 8.10-59.45 6.67-20.00 11.76-47.05

Varadaraj et al (1994).

Table 3 Effect of different media on SDH activity levels in the hepatopancreas oipila globosa after 10-day exposure Medium

Tannery effluent Potassium dichromate Tannin Sodium chloride

Concentration

% change in SDH activity

10% 2.5 ppm 20ppm 1,000 ppm

-16.43 (P< 0.005) +20.75 (P < 0.05) -19.76 (P< 0.05) -31.16 (P< 0.05)

- indicates % decrease over control. + indicates % increase over control. Guruprasada Rao and Nandakumar (1982).

concentrations of TE (5, 10, 15, 20, and 25%) for a period of 15 days (Subramanian and Varadaraj, 1993). After exposure to 25% tannery effluent for 15 days, the concentrations of total haemolymph proteins, free amino acids and free sugars in the larvae of dragonfly Macromia cingulata exhibit a reduction of about 20% (Varadaraj et al., 1993). Subramanian and Varadaraj (1994) have reported decreased total free amino acids in haemolymph (1.60% to 20.35%), cuticle (9.96% to 26.97%), fatbody (17.90% to 33.46%), and nervous tissue (2.90% to 21.16%) in the larvae of dragonfly Pantala flavescens under sublethal concentrations of TE (1.2, 1.4, 1.6, 1.8, and l.Wc). The effect of TE leading to mortality in the larvae of P. flavescens is found to be dose- and duration-dependent (Subramanian, 1994). In these animals, the tannery effluent reduces total proteins, total free sugars, glycogen, and total lipids, and

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increases lactic acid and Ach content in vital tissues (Table 4). Activity levels of various enzymes such as protease, LDH, acid and alkaline phosphatases, and lipase increase in the tissues under the influence of sublethal concentrations of TE (1.2, 1.4, 1.6, 1.8, and 2.0%). At the same time, all the tissues exhibit a decreased rate of SDH and acetylcholine esterase (AchE) activities (Table 5).

Table 4 Ranges of percent changes of various biochemical constituents in the tissues of larvae of P. flavescens under sublethal concentrations of tannery effluent

Biochemical constituents/tissue

Haemolymph

Cuticle

Fat body

Nervous tissue

Total proteins* Total free sugars* Glycogen* Total lipids* Lactic acid** Ach*

20.52-52.33 19.57-52.72 — 6.37^3.58 8.04-24.90 9.26-50.00

17.32-53.15 20.00-51.33 23.08-69.23 5.33-50.30 14.28-61.90 13.04-95.65

26.03-67.64 24.48-56.51 10.69-66.63 12.13-36.38 2.41-54.25 25.00-187.50

31.50-65.35 18.92-70.27 9.41-59.22 16.67-47.22 1.38-61.29 13.13-71.05

* Ranges of % decrease over control. ** Ranges of% increase over control. Subramanian, 1994.

Table 5 Ranges of percent changes in the activity levels of enzymes in the tissues of larvae of P. flavescens under sublethal concentrations of tannery effluent

Activity levels of enzymes/tissue Protease** SDH* LDH** Acid phosphatase** Alkaline phosphatase** Lipase** AchE*

Haemolymph

Cuticle

Fat body

Nervous tissue

22.22-66.67 17.11-64.33 21.42-67.00

22.22-100.00 24.88-52.07 15.68-54.90

25.71-97.14 15.85-78.05 13.21-75.47

16.67-83.33 6.72-39.08 8.16-49.00

5.36-35.50

6.45-37.10

13.13-48.67

14.29-71.43

5.56-37.04 7.41-34.57 8.57-60.00

5.94-27.72 4.59-39.08 12.24-53.06

12.87-39.11 6.18-29.45 11.43-60.00

11.76-39.22 30.00-60.00 16.39-70.49

* Ranges of % decrease over control. ** Ranges of% increase over control. Subramanian, 1994.

Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms

371

Vertebrates A sublethal concentration of TE (0.2, 0.4, 0.6, 0.8, and 1.0%) is found to cause decreased levels of biochemical constituents in various tissues of the fingerlings of Cyprinus carpio (Varadaraj and Subramanian, 1995c). The percent decrease of total free amino acids ranges between 20.37-28.89, 17.70-48.03, 4.00-38.82, and 25.20-55.20 in gills, intestine, liver, and muscle, respectively; the values for total proteins are 0.46-14.16, 7.53-24.92, 0.52-21.28, and 2.72-15.40; for total sugars 7.78-54.86, 2.29-27.12, 19.14-33.33, and 9.43-40.57; for glycogen content 11.11-55.56, 10.53-36.84, 8.03-57.28, and 12.24-46.26; and for total lipids 16.84-37.76, 1.49-30.21, 0.49-24.27, and 3.72-23.09. Contamination of chromium compounds of TE is shown to cause a high degree of pollution in the waters of irrigation reservoirs (Nandakumar, 1990). Backyavathy and Nandakumar (1992) have recorded pronounced decrease in the activity levels of dehydrogenases, adenosine triphosphatase (ATPase) and AchE in various tissues of albino rats under chromium contaminated irrigation well water (Figure 2). The increase in percent inhibition of the activity levels of dehydrogenases could affect the oxidative metaboUc activity of citric acid, glycolytic pathways, and energy production. The impairment of oxidative phosphorylation due to chromate through inhibition of dehydrogenases and cytochrome system of mitochondria has also been reported by Sigova (1974). Decrement of the activity level of ATPase in the tissues could possibly impair oxidative phosphorylation from ADP to ATP (Berwin et al., 1977; Luciana et al., 1979; Marino et al., 1980). The chromium has been reported to deplete the activity level of AchE disrupting neural transmission in rats (Burmistrov, 1978) and in frogs (Zefirov et al., 1980). The perusal of literature clearly indicates that the toxicants in TE inflict stress on animals. To combat the stress, they have to allocate excessive energy. As a result, they are forced to utilize metabolic reserves, particularly glycogen. The TE causes a shift in the metabolism toward the anaerobic side, leading to the accumulation of lactic acid and the reduction of glycogen reserves and free sugars. At a point of time during stress, metabolism shifts to the utilization of lipid stores as well as tissue proteins, leading to the decrease of these biochemical constituents as well. These results explain the overall decrease in the biochemical parameters in animals, the increase in the levels of lactic acid and LDH activity, and the decrease in SDH activity. The change in the levels of Ach and AchE activity suggests spontaneous and inhibited nervous activity, which would cause fatigue and breakdown of the nervous system. These biochemical alterations would cause the death of animals under TE stress. Subramanian (1994) has recorded Juvenile Hormone Analogue (JHA) mimetic property of TE on dragonfly larvae, and therefore the effluent may be further investigated for application in the control of insects.

372

Health and Toxicology

20

10020

B

10020

K

10020

L

ioo"

M

iiir

Mi -t-.U: '20 100 20

10020

B

K

SDH

10020'

L

20

10020

B

M

10020

K

10020

L

100

M

GDH

Concentration

20

10020

10020

B

MDH

100*

MtVc

:Mni

K

(%)

n-ni:

iod2o

L

AT Paso

100

M

rir i_ 20

B

T 1C020

r 10020

K

TJ|.!.i.>.M-i10020 100

L

M

AchE

C o n c e n t r a t i o n (%)

Figure 2, Enzyme activity levels in tissues of rat under chromium contaminated water. (Backyavathy and Nandakumar, 1992) B—Brain; K—Kldney; L—Liver; M—Muscle; SDH—Succinic dehydrogenase GDH—Guanine dehydrogenase LDH—Lactic dehydrogenase MDH—Malate dehydrogenase ATPase—^Adenosine triphosphatase AchE—^Acetylcholine esterase

REFERENCES Arvind Bharathi, Saxena, R. P., and Pandy, G. N. 1979. "Physiological imbalances due to hexavalent chromium in freshwater algae." Ind. J. Env. Hlth., 21: 243. Backyavathy, D. M., and Nandakumar, N. V. 1992. "Effects of industrial chromium on rat dehydrogenases, ATPase and AchE." Poll. Res., 11 (3): 139-150.

Toxic Effect of Tannery Effluent on the Biochemical Constituents in Organisms

373

Backyavathy, D. M., Nandakumar, N. V., and Bhaskar, 1986. "Epidemiological studies on tannery industrial workers exposed to occupational environment." Poll Res., 5 (2): 73-78. Berwin, Yip., Rudolph, P., and Frederick, B. 1977. "Effect of chromium ATP on the association of the reacting forms of yeast hexokinase." J. Biol. Chem., 252 (6): 1,844-1,846. Burmistrov, N. V., Sanitari, Sb. Tr. Nil., and Virieny. 1978. "Determination of the quantitative dose time dependence for hexavalent chromium as a pollutant of wastes of a circulating water supply." Gruz., 14:12-1 A. Eye, J. D., and Lawrence, L. 1971. "Treatment of waste from sole leather tannery." /. Wat. Pollut. Cont. Feb., 43: 2,291-2,303. Gaur, B. L., and Tomar, D. S. 1975. "Effect of salinity on germination of sunflower (Helianthus annuus L.) varieties." Sci. and Culture, 41: 429-430. Guruprasada Rao, M., and Nandakumar, N. V. 1981. "Physicochemical characteristics of tannery effluent-contaminant irrigation reservoir." Ind. J. Env. Hlth., 23.-239-241. Guruprasada Rao, M., and Nandakumar, N. V. 1982. "The tannery industrial effluent effect on succinate dehydrogenase activity pattern in a freshwater snail, Pila globosa." Proc. Indian Acad. Sci. (Anim. Sci.)., 91: 427-481. Guruprasada Rao, M., and Nandakumar, N. V. 1983. "Impact of tannery effluent on seed germinability and chlorophyll content in Cicer arietinum L." Poll. Res., 2 (1): 33-36. Harihara Iyer, C. R., Rajagopalan, R., and Pillay, S. C. 1957. Curr. Sci., 10: 187. ISI 1981. Indian Standards Institution, Indian standard tolerance limits for industrial effluents discharged into inland surface water. IS-2490 (Part I). Kadam, S. D. 1995. "A study on the effect of tannery effluent on the growth of Cicer arietinum." J. of Env. and Poll., 2 (2): 59-62. Luciani, S. Dal., Rebellta, A. M., and Levis, A. G. 1979. "Effect of chromium compounds on plasma membrane magnesium ATPase activity of B cells." Chem. Biol Interest., 27(1): 56-69. Manivasakam, N. 1987. Industrial effluents—origin, characteristics, effects, analysis, and treatment, Sakthi Publications, Coimbatore, TN., India. Marino, D., and Cheland, W. W. 1980. "Investigation of substrate specificity and reaction mechanism of several kinases using chromium (III) adenosine 5' triphosphate." ^/oc/z^m/^^ry, 19(7): 1,506-1,515. Miakhan, M., and Raman, A. 1973. Proc. Symp. on Envi. Pollution, NEERI, Nagpur, India. Nandakumar, N. V. 1990. "Tannery and chromate industrial effluent effect on soil, animals and plants." In: Soil Pollution and Soil Organism. (Ed. P. C. Mishra). Ashish PubHshing House, New Delhi, India, 81-105. Narale, R. P., Subramanyam, T. K., and Mukherjee, R. K. 1969. "Influence of salinity on germination, vegetable growth and grain yield of rice (Oryza sativa var. Dulas)." Agraw. /., 61: 341-344.

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Ogra, R. K., and Baijal, B. D. 1982. "Physiological studies on the effect of salinity on Sorghum. I. Changes in alpha-amylase and acid protease during seedling growth." Ind. J. PL Physiol, 25: 133-140. Ramana, K. V. R., and Ramarao, V. S. 1978. "Physiological studies on the influence of salinity and alkalinity. Changes in growth, respiration, carbohydrates and fats during seedling growth of radish (Raphanus sativus L)." Ind. J. PI Physiol, 21: 93-105. Sastry, C. A., and Madhavakrishnan, W. 1984. "Pollution problems in leather industries of India." Dept. of Environment, Govt, of India Public, 34-35. Saxena, R. M., Keral, P. F., Yadav, R. S., and Bhatnagar, A. K. 1986. "Impact of tannery effluents on some pulse crops." Indian J. Environ. Hlth., 28 (4): 345-348. Sigova, N. V. 1974. "Mechanism and means of rendering the toxic effects of chromium." Yopu. Exsp. Klin. Ter. Profil Prom. InasikatsL 110-115. Subramanian, M. A. 1994. "Studies on the impact of tannery effluent on ecophysiology and biochemistry of the nymphs of dragonfly, Pantala flavescens (Fabricius) (Libellulidae: Anisoptera)." Ph.D. Thesis, Bharathiar University, Coimbatore, TN., India. Subramanian, M. A., Viswanathan, S., and Varadaraj, G. 1988. "A report on the physicochemical parameters of the sewage and industrial effluents in and around Erode." The Indian Zool, 12 (1 & 2): 185-187. Subramanian, M. A., and Varadaraj, G. 1993. "The impact of tannery effluent on the biochemical constituents in the haemolymph of the freshwater prawn, Macrobrachium idella Heller," J. Environ. Biol, 14 (3): 255-259. Subramanian, M. A., and Varadaraj, G. 1994. "Impact of tannery effluent on tissue free amino acids in the larvae of dragonfly Pantala flavescens (Fabricius)." Fraseria, 1 (1): 19-21. Varadaraj, G., Subramanian, M. A., and Jayasuriya, S. 1993. "Sublethal effects of industrial effluents on the biochemical constituents of the haemolymph in the larvae of Macromia cingulata Rambur (Anisoptera: Corduliidae)." Odonatologica, 22 (1): 89-92. Varadaraj, G., Jayasuriya, S., and Subramanian, M. A. 1994. "Toxic effect of tannery effluent on the biochemical constituents in different tissues of Pila globosa." Environment & Ecology, 12 (2): 303-307. Varadaraj, G., and Subramanian, M. A. 1995a. "Studies on groundwater contamination at the vicinity of tanneries near Erode." (Unpublished). Varadaraj, G., and Subramanian, M. A. 1995b. "A study on the physicochemical properties of tannery effluent and its effect on the germination of Vigna radiata (L) Wilezek." (UnpubHshed.) Varadaraj, G., and Subramanian, M. A. 1995c. "Studies on food utilization, oxygen consumption and biochemistry in the fingerlings of common carp Cyprinus carpio on exposure to tannery effluent." (Unpublished.)

Toxic Effect of Tannery Effluent on the Biochemical Constituents In Organisms

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Varma, S. R., and Poonia, S. R. 1979. "Germination and early seedling growth of pearl millet, Pennisetum typhoidis (L) as affected by salinity." Ind. J. PI. Physiol, 22 (2): 144-146. Yusuff, R., and Ismail, S. A. 1984. "Contamination of drinking water in wells by tannery effluents at Pammal (Pallavaram, Madras)." IV Annual Conference of STOX India, P.O. Institute of Basic Medical Sciences, University of Madras, Madras, India, Abs. No. C18. Zefirov, A. L., Poletaer, G. I., and Guselikova, G. G. 1980. "Effect of chromium ions on neuromuscular synapse function." Rifolizile., 25 (1): 115-119.

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CHAPTER 17 BLADDER CANCER AND WATER DISINFECTION METHODS Michael A. McGeehin Centers for Disease Control Atlanta, GA 30341-3724 John S. Reif Colorado State University Boulder, CO

CONTENTS INTRODUCTION, 377 WATER CHEMISTRY, 378 Organic Compounds in Source Waters, 378 Disinfection Processes, 378 CARCINOGENICITY OF DISINFECTION BY-PRODUCTS, 381 Trihalomethanes, 381 Haloacetonitriles, 381 Chlorophenols, 382 Summary, 382 BLADDER CANCER EPIDEMIOLOGY, 382 Bladder Cancer Risk Factors, 382 REFERENCES, 390 INTRODUCTION Since early in this century, the chlorination of drinking water has been considered one of the most significant advances in public health by preventing the waterborne transmission of infectious disease. In 1974, it was discovered that chlorine combines with organic materials in water to form halogenated compounds [1]. Some of these compounds are possible human carcinogens [2]. Since this discovery, a number of epidemiologic studies have been conducted to investigate the possible association between cancer and exposure to chlorinated drinking water. Based on the results of early investigations, more recent studies have focused on bladder cancer as the health outcome of interest. Cancer of the urinary bladder accounted for 4.7% of all newly diagnosed cancers in the United States between 1983 and 1987 [3]. Approximately 50,000 cases of bladder cancer occurred in the United States in 1995, almost three-fourths in men [4]. Bladder cancer incidence has been increasing among white males at approximately one percent per year since 1973 and at a slightly lower rate among white females [3]. Although the incidence rate among blacks is lower than among whites, 377

378

Health and Toxicology

it has been increasing faster among members of this racial group [3]. Bladder cancer will claim approximately 11,200 lives nationally in 1995 [4]. Certain risk factors for bladder cancer, such as smoking and occupation, have been studied extensively. The results of numerous investigations have indicated that cigarette smoking increases the risk of bladder cancer approximately two-fold [5]. Investigators conducting a nationwide case-control study of bladder cancer concluded that a moderately increased risk was found for several occupations, but that only 21% of bladder cancer incidence could be attributed to occupational exposure [6]. After accounting for the contribution of smoking and occupation, however, a large portion of bladder cancer cases have no known cause. One of the possible etiologic factors under investigation is exposure to chlorinated water. WATER CHEMISTRY The interest in possible adverse health effects associated with chlorinated drinking water centers on the by-products produced by reactions of chlorine with certain organic compounds present in raw water. Rook's [1] discoveries that chloroform and other trihalogenated methanes (THMs) are formed during the chlorination of drinking water first focused attention on this subject. In subsequent investigations, Trehy and Bieber [7, 8] identified dihaloacetonitriles (DHANs) as other by-products of the chlorination disinfection process with potential implications for human health. The results of these studies caused an acceleration in the development of analytical procedures to isolate, identify, and quantify microorganic drinking water contaminants. Organic Compounds in Source Waters The production of hazardous by-products during the chlorination of drinking water depends on the concentration of organic material in the source water [9]. The majority of organic material in natural waters comes from natural sources through decay of vegetation and animal tissues, photosynthetic by-products, animal excretion, and release of organic matter by plankton and aquatic microphytes [9]. These natural humic substances constitute most of the carbon found in typical surface waters [10, 11]. Disinfection Processes In addition to the natural humic material found in source waters, the production of potentially hazardous by-products during drinking water treatment requires the presence of free chlorine used in the disinfection process. When free chlorine combines with organic carbon precursors, trihalomethanes (THMs) are formed [1, 12]. The four THMs most commonly found in potable water are chloroform, bro-

Bladder Cancer and Water Disinfection Methods

379

modichloromethane, dibromochloromethane, and bromoform [13]. A fifth, dichloroiodomethane, is found less frequently [14]. The concentration of THMs found in treated water is heavily dependent on the concentration of humic material, reaction time, pH, temperature, chlorine dose, and residual chlorine [14-16]. Chlorination remains the most common water disinfection method used in the United States. Alternative methods, such as chloramination, are becoming more prevalent. The basic chemistry of these processes will be reviewed, along with the potential for formation of toxic by-products. Chlorination Chlorination has been the dominant method for disinfecting drinking water for more than 70 years. Chlorine is employed as a disinfectant principally because of its potent germicidal activity, but it is also unique in its ability to maintain a sustained, residual disinfecting action during distribution of the water after treatment. When chlorine gas is dissolved in water, the following chemical reactions occur [17]: CI2 + H2 = HOCl + H^ + CI-

(1)

HOCl = H^ + OCl-

(2)

At the pH normally found in water treatment. Equation 1 proceeds rapidly to the right with complete conversion to dissociated hypochloric acid within a few tenths of a second [17]. Hypochlorous acid (HOCl) is a weak acid and ionizes according to Equation 2. During chlorination, the relative amount of HOCl and OCl (-) (hypochlorite ion), together termed "free chlorine," is a function of the pH of the water [18]. HOCl, a more effective biocide than OCl (-), dissociates into OCl (-) between a pH of 7.0 and 8.0, the range in which most drinking water undergoes treatment [18]. Natural waters have a "chlorine demand" produced by inorganic molecules, organic molecules, and microbiota reacting with and consuming free chlorine ^19]. Addition of chlorine beyond the chlorine demand "breakpoint" produces a freechlorine residual, which forms the basis for determining required amounts of disinfectant [18]. The "breakpoint reaction" involves the reaction of HOCl with ammonia (NH3) to form products known collectively as chloramines [20]. The following reactions are classically used to characterize the formation of chloramines [21]: NH3 + HOCl = NH2 CI + H2O

(3)

NH2 CI + HOCl = NHCI2 + H2O

(4)

NHCI2 + HOCl = NCI3 + H2O

(5)

380

Health and Toxicology

The product in Equation 3 is monochloramine, followed by dichloramine in Equation 4 and trichloramine in Equation 5. Although these chloramines are not as reactive nor as biocidal as hypochloric acid, they are more stable and provide persistent disinfectant activity throughout the distribution system [17]. The chlorination of water supplies involves two steps in the disinfection process. The first is primary disinfection where an agent of sufficient biocidal strength is used to kill any pathogen [17]. HOCl is the only agent formed in the chlorination process with the potency against all forms of pathogenic organisms to fulfill the requirements as a primary disinfectant. The second step is residual disinfection, which involves the maintenance of a small concentration of germicide throughout the distribution system to preserve the integrity of the water supply. Both chloramine and HOCl are suitable for this purpose [17]. Chloramination Chloramination is becoming more widely used as a drinking water disinfection process, primarily because it limits the concentration of trihalomethanes produced when compared to chlorination [22, 23]. Monochloramine produces chlorine substitution into humic material to yield an organic halogen that can be measured using the total organic halogen (TOX) method [24, 25]. The quantity of TOX produced by monochloramine is only 5% to 50% of that produced by an equivalent amount of free chlorine, but the concentration of monochloramine is higher because it is a less-effective disinfectant [25]. In practice, chloramination has been applied in three different ways. Each of these methods results in a water product of different bacteriological and chemical quality [26]. In the first method, marginal chlorination is practiced when chlorine is added to water containing ammonia to generate monochloramine as the primary disinfectant. However, as discussed above, monochloramine is a much poorer disinfectant than chlorine [27]. Marginal chlorination, therefore, may not provide the necessary biocidal activity to prevent bacterial growth in the system [26]. Much greater biocidal activity can be generated if sufficient chlorine is added beyond that needed to remove ammonia from the source water, producing a freechlorine residual [18]. Ammonia is then added to the water after a contact time sufficient to achieve primary disinfection. This process achieves maximum disinfection, but it also produces chlorinated by-products [18]. The third method of chloramination utilizes a solution of preformed monochloramine as both the primary and residual disinfectant [18]. Although the bactericidal quality of this method may be better than marginal chlorination, the poorer disinfection capacity of monochloramines may still pose a problem [28-30]. The reactions between chlorine and ammonia which result in the formation of monochloramine and dichloramine were represented in Equations 3 and 4. The difference in the methodologies is essentially the length of time during which the "free chlorine" is allowed to react with the waterbome pathogens prior to the addition of

Bladder Cancer and Water Disinfection Methods

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ammonia. Because "free chlorine" has much greater biocidal activity compared to chloramines, this "free chlorine" reaction period greatly influences the disinfection efficacy of the process. This period, however, is also the time during which the chlorine reacts with organic compounds to form THMs and other potentially harmful halogenated products. CARCINOGENICITY OF DISINFECTION BY-PRODUCTS The reaction of free chlorine with naturally occurring organic substances produces thousands of organic compounds [31]. Approximately 90% of these compounds have yet to be identified and are present in very low concentrations [32]. The remaining 10% have been identified as volatile organic compounds (VOCs), of which trihalomethanes are the major constituent [32]. Meier et al. [33] investigated the mutagenicity of the mixture of compounds resulting from chlorination of surface water and found that more than 90% of the total mutagenicity was attributed to unidentified compounds. Because of the lack of appropriate toxicological information for large numbers of the by-products of water disinfection, this review will concentrate on three categories of volatile organic compounds, with particular emphasis on the trihalomethanes. Trihalomethanes The trihalomethanes are the most widely recognized by-products of the chlorination of drinking water. The most common forms include chloroform, dichlorobromomethane, dibromochloromethane, and bromoform [13]. Of the THMs, the toxicological effects of chloroform have been characterized most completely [34]. The evidence that trihalomethanes are carcinogenic is primarily based on data from bioassays of chloroform. It is clear that chloroform can produce renal tumors in one strain of rats and one strain of mice [35, 36]. When given in an oil-based vehicle, which adds significantly to the lipid intake of the subject, chloroform induced liver tumors in one strain of mice [37]. These results suggest that chloroform may be carcinogenic in humans [18]. In a 2-year rodent bioassay, dibromochloromethane was shown to increase the incidence of liver carcinomas, but not renal tumors, in male mice [38]. Haloacetonitriles The haloacetonitriles that have been identified in chlorinated drinking water have been the dihalogenated chlorinated and brominated derivatives [39]. The investigations into the carcinogenicity of this class of compounds have been limited to four compounds: dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromochloroacetonitrile (BCAN), and dibromoacetonitrile (DBAN). All four of these

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Health and Toxicology

compounds have been shown to be powerful mutagens [2]. However, evidence of carcinogenicity of these chemicals in experimental animals is very limited. Although the haloacetonitriles are the first group of by-products of chlorination other than THMs to show both mutagenic and carcinogenic properties [34], this class of compounds does not seem to represent a serious carcinogenic hazard to humans. There is no evidence available that clearly demonstrates the haloacetonitriles' ability to initiate cancer by an oral route of administration [34]. Chlorophenols Small quantities of chlorinated phenol derivatives result from the chlorination of drinking water [34]. The principal products formed from the reaction of chlorine with humic acid in source water include 4-chlorophenol, 2,4-dichlorophenol, and 2,4,6-trichlorophenol [40]. Carcinogenic testing has been done only for 2,4,6-trichlorophenol. High-dose rodent bioassays have shown a dose-related increase in leukemias and lymphomas in male rats and an increase in liver carcinomas in male and female mice [41]. The National Research Council has recommended that further toxicological data be developed for this class of compounds [18]. Summary The lack of certainty concerning disinfection by-products from water chlorination makes it difficult to estimate the overall carcinogenic hazard to humans. However, it is not necessary for each of the chemicals in a mixture of compounds to be identified and assayed before epidemiologic studies investigate the effects of the mixture on human health. There is sufficient toxicological evidence to suspect that by-products of drinking water chlorination may be carcinogenic in humans. Wellconducted epidemiological studies will assist in assessing the human cancer risk that these substances may represent, while the sciences of chemistry and toxicology attempt to assay the individual compounds and their possible interactions. BLADDER CANCER EPIDEMIOLOGY Bladder Cancer Risk Factors Smoking Much of the increase in bladder cancer incidence may be attributable to changing cigarette smoking habits [42]. Analysis of Connecticut incidence rates for bladder cancer shows a close correspondence of trends for bladder cancer with trends for cigarette smoking by birth cohort [43]. Although the increases are not as steep as those for lung cancer, the points trends are essentially the same in each gender [43].

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The association between tobacco smoking and bladder cancer has been thoroughly investigated. A positive association has been found consistendy in descriptive, case-control, and cohort studies. Ecological studies have found positive correlations of incidence [44] and mortality [45] with cigarette consumption. Cohort analyses of incidence in the United States and Denmark [46] and mortality in England [46-48] showed an association with tobacco consumption in those countries. The epidemiological evidence strongly suggests that cigarette smoking is a risk factor for bladder cancer [49]. Although dose-response was not evaluated in the early investigations, in those studies in which the quantity of cigarettes or duration of smoking was characterized, a dose-response relationship was demonstrated [50, 51]. In general, ex-smokers have elevated risks compared to non-smokers, but the increases are lower than those of current smokers [51]. Cessation had to have been seven to fifteen years before the risk was reduced [49]. The results of these studies suggest that the latency period for bladder cancer with respect to smoking may be as long as 20 years [5]. Occupation Although the association between bladder cancer risk and occupation is one of the most investigated areas in occupational cancer epidemiology, the results remain unclear. Certain occupations have been consistently associated with increased risk; e.g., printers, dye workers, rubber workers [6]. However, numerous studies during the past 30 years have suggested approximately 40 occupations that may have an increased risk for bladder cancer. Most of these studies have been hampered by small numbers of exposed subjects and results that were not statistically significant. It is difficult to compare the various studies' outcomes because each used a different method to classify occupation and different analytical methods were employed. Artificial Sweeteners Collectively, epidemiologic studies have shown no increased risk of bladder cancer in relation to intake of artificial sweeteners. The one case-control study that did show an elevated risk for males showed a protective effect for females [52]. It appears that if the use of artificial sweeteners represents any risk of bladder cancer in humans, it is negligible. Coffee Drinking Epidemiologic investigations of coffee drinking and bladder cancer have produced mixed results. Most of the epidemiological studies have shown slight to moderate increased risk for coffee drinkers with the risks being inconsistent for various subgroups [53, 54]. While it appears that coffee may not play a causal role in the

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etiology of bladder cancer, there is sufficient evidence to suggest that coffee should be investigated in any study of an association of bladder cancer and risk factors. Hair Dye There is presently no epidemiologic evidence of an association between bladder cancer and hair dye use. This possible risk factor, however, has not been extensively investigated. Water Disinfection Epidemiologic studies investigating the association between exposure to chlorinated surface water and risk of bladder cancer have had mixed results. The early investigations were ecologic studies using various measurements of water quality as surrogate exposure variables. In a review of these early studies. Cantor [55] reported that eight of the eleven ecologic studies showed positive and statistically significant associations between bladder cancer rates and some measure of chlorinated water contamination for white males. Case-control and cohort studies have also been conducted to investigate the association between bladder cancer and drinking water disinfection. Although the results of these studies were not completely uniform, those studies that used an interview to assess exposure and controlled for possible confounders all found an association between bladder cancer and drinking water chlorination. Most epidemiologic studies investigating an association between drinking water disinfection and bladder cancer have had important methodologic weaknesses [56]. The most serious of these is the lack of historical environmental measurements to assess individual past exposures to water contaminants. Ecologic Studies Early studies utilized an ecological design and failed to show a consistent relationship between chlorinated drinking water consumption and deaths from bladder cancer [57-60]. Cantor et al. [57] investigated the association of cancer mortality with trihalomethane levels by analyzing cancer mortality rates by county. Age-standardized cancer mortality rates by sites and sex in whites for the years 1968-1971 were adjusted for socioeconomic, industrial, and demographic factors. The analysis included counties with more than 50% of the population served by the sampled water supply. Positive correlations with THM levels measured by the Environmental Protection Agency were found for bladder and brain cancers among both males and females [57]. Non-Hodgkin's lymphoma and kidney cancer rates were associated with THM levels in males only. Bladder cancer mortality rates in both sexes showed the strongest correlation with THM exposure after controlling for differ-

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ences in ethnic group, urbanization, region of the U.S., and industrialization of the county. This study provided the first evidence of a possible association between THM levels and surface water chlorination with bladder cancer. Hogan et al. [61] examined the association between chloroform levels in drinking water and cancer mortality data from 1950 to 1969 for all counties in the 48 contiguous states of the United States plus the District of Columbia. The researchers used 20-year average annual, age-adjusted, sex- and race-specific rates for 16 cancer sites. The analysis was restricted to whites only. County estimates from the 1960 census of family income, years of education, and percent of the population which was non-white were included in a regression model to attempt to control for socioeconomic factors. The investigators found associations between potable water chloroform levels and rates of cancer of the large intestine, rectum, and bladder for both sexes. Urinary tract cancer mortality was studied in Houston, Texas, populations exposed to heavily chlorinated and lightly chlorinated drinking water [58]. In 1954, construction of a reservoir changed the type of drinking water supplied to many Houston residents from ground water to surface water. Mortality rates were studied by gender, race, and age cohorts for the period 1940 to 1970. Deaths due to cancer of the urinary tract were compared to mortality rates from respiratory cancer, emphysema, and homicide. Census tracts were classified according to duration of use of surface water. By the 1970s, approximately 20 years following the change to surface water, white females showed an increase in urinary tract cancer mortality rates [58]. A similar effect was not seen among males, nor among the non-white males or females. The investigators had difficulty in classifying exposure because the water supply was changed for each of the study areas gradually. Further, the study could not control for smoking or occupational exposure. The follow-up period may not have been long enough for bladder cancer to become manifest because the maximum induction period allowed was less than 20 years. These ecological studies used existing data bases and evaluated the mortality experience of geographically defined population groups. Exposure at the level of the individual was not evaluated. Case-control Studies—Death Certificates Case-control studies conducted prior to 1980 relied on data obtained from death certificates [61-64]. Exposure to water in each of these studies was classified based on the individual's residence as listed on the death certificate. Excess urinary tract and gastrointestinal cancer deaths were investigated for an association with chlorinated water use in a case-control study of seven New York counties [61]. The study populations included 3,446 deaths due to these cancers from 1968 to 1970 and 3,444 individually matched control deaths from other causes for the same period. Exposure to surface water or ground water and chlorinated or nonchlorinated water was determined based on the residence listed on the death certificate. The

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researchers attempted to control for occupation by using the occupation listed on the death certificate for a number of randomly chosen cases and controls. The odds ratio for bladder cancer for residence in a county served by chlorinated versus nonchlorinated water was 1.69 for both genders combined (p ^ .025) [61]. The main limitation of this study was that exposure was based entirely on the residence listed on the death certificate, not on a lifetime residential history. An analysis of data from the National Bladder Cancer Study has shown that determining exposure by death certificate residence depresses the odds ratio estimates compared to lifetime residential history [65]. The study was also not able to control for smoking status. A death-certificate-based case-control study design was used to investigate the association of various cancers with water supply among white women in Wisconsin [62]. Cases included deaths for various cancer sites between 1972-1977 in the 28 study counties of eastern Wisconsin. Non-cancer deaths were matched to each case on gender, race, year of death, county of residence, and age. Exposure was assigned on the basis of the water facility that served the population of the town, city, or village listed on the death certificate as the usual place of residence. More detailed information on water quality was obtained through a questionnaire mailed to each water utility superintendent. Logistic regression analysis was used to determine odds ratios for site-specific cancer deaths for high, medium, and low chlorine levels as compared to unchlorinated water exposure. There were no significantly elevated odds ratios for any chlorine dose category in an analysis of 460 bladder cancer deaths. However, classifying lifetime chlorination exposure by the residence listed on the death certificate may have resulted in misclassification of exposure in the Wisconsin study. Exposure misclassification in this study may have been nondifferential, which could have decreased the odds ratios [66]. An investigation in Massachusetts examined the relationship between site-specific cancers and drinking water disinfection methods [64]. Death certificates for approximately 50,000 fatal cancers were compared to those for approximately 215,000 controls who died from cardiovascular, cerebrovascular, pulmonary disease, or lymphatic cancer. Exposure was classified according to residence at death, and the water systems were categorized as chlorinated or chloraminated. After adjusting for age, a moderate increase in risk in exposure to chlorinated water systems was found among bladder cancer deaths compared to controls who died from lymphatic cancer (O.R. = 1.7; 95% CI = 1.3-2.2). No association was found for chlorinated water exposure among bladder cancer deaths when compared to all controls combined (O.R. = 1.1; 95% CI = 0.97-1.14) [64]. None of the death-certificate-based case-control studies utilized interviews to assist in an accurate determination of exposure. The researchers were unable to estimate duration or quantity of exposure. Control for smoking and other potential confounders was generally not possible, and control for possible occupational exposure was limited to county-wide census estimates. Most importantly, malignancies with good long-term survival, such as bladder cancer, may be under-represented in studies based on death certificate data.

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Cantor has suggested that case-control studies based solely on death certificate information may underestimate the true exposure to carcinogenic by-products of the chlorination process [56]. In an analysis of Iowa data, investigators also found an underestimation of exposure to chlorinated water for bladder cancer cases based on death certificate residence compared to interviewing for lifetime exposure [65]. Case-control Studies—Interview-based More recent studies have improved on the limitations of the earlier analyses. Investigators analyzed data from the National Bladder Cancer Study, a large casecontrol study (2,982 cases and 5,782 controls) conducted in 10 geographic areas of the U.S. [67]. Exposure was determined by interview and based on lifetime residential information, which was combined with water utility data. Logistic regression analysis was used to control for possible confounding by age, smoking, gender, employment in a high-risk occupation, and geographical area. Duration and frequency of exposure were examined by stratification. The analysis was limited to white respondents (2,805 cases and 5,258 controls). The results showed that estimates of bladder cancer risk increased with total tap water consumption among respondents using chlorinated water sources. Study participants using nonchlorinated ground water did not show an increased risk for bladder cancer with tap water consumption. Increasing odds ratios for increasing duration of exposures to chlorinated water was found among women (p < .02) and nonsmokers of both sexes reporting higher-than-median tap water consumption (p = .01) (Table 1). For nonsmokers with an exposure duration of at least 60 years, the odds ratio was 3.1 (95% CI = 1.3-7.3). For female bladder cancer cases with an exposure of at least 60 years, the odds ratio was 3.2 (95% CI = 1.2-8.7) [67]. In a case-control study in Massachusetts, surrogates for 614 individuals who died of bladder cancer and 1,074 individuals who died of other causes were interviewed Table 1 Estimated risk of bladder cancer for nonsmokers with duration of residence with chlorinated surface water for respondents reporting tap water consumption above the median [67]*

# of years

Odds ratio

95% CI

0 1-19 20-39 40-59 >59

1.0 1.1 1.9 2.0 3.1

0.5-2.4 1.0-3.7 1.0-4.1 1.3-7.3

"^Adjusted for age, smoking habit, high-risk occupation, population size of usual residence, reporting center, and sex.

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about residential and smoking information [68]. Exposure was defined according to duration of residence in communities using either chlorination or chloramination disinfection. Individuals were classified as having "usual exposure" to chlorinated by-products if they spent more than 50% of their lives in communities using chlorinated drinking water and "usual nonexposure" if they spent more than 50% of their lives in communities using chloraminated drinking water (Table 2). An odds ratio of 1.6 (95% CI = 1.2-2.1) was found for bladder cancer among individuals who resided only in communities supplied with chlorinated drinking water compared to those who resided only in communities served with chloraminated drinking water. Participants classified as "usual exposure" had an estimated risk of bladder cancer of 1.4 (95% CI = 1.1-1.8) compared to the "usual nonexposure" group. An historical cohort study was conducted on 31,000 persons in Washington County, Maryland [69]. Three cohorts, each distinguished by a different degree of exposure to chlorinated water, were examined for site-specific cancer incidence rates. Rates for cancer of the bladder in men and cancer of the liver in women were nearly two-fold higher in the group exposed to chlorinated water than in the nonchlorinated cohort. Neither rate elevation was statistically significant. Morris and his colleagues used meta-analytic methods to pool the results of epidemiologic studies and examine the association between chlorination of drinking water and cancer [70]. Relative risk estimates from pertinent case-control and cohort studies were abstracted and combined. The pooled relative risk estimate for consumption of chlorinated water and bladder cancer was 1.2 (95% CI = 1.09-1.34). A population-based case-control study of bladder cancer and drinking water disinfection methods was conducted during 1990-1991 in Colorado [71]. Surface water in Colorado has historically been disinfected with chlorination in some municipalities and chloramination in others. A total of 327 histologically verified bladder cancer cases were frequency matched by age and sex to 261 other-cancer controls. Subjects were interviewed about residential and water source histories. Table 2 Estimated risk of bladder cancer mortality in massachusetts associated with drinking water disinfection process [68]* Exposure duration

Lifetime: chlorine vs. chloramination Usual exposure: chlorine vs. chloramination

OR

95% CI

1.6

1.2-2.1

1.4

1.1-1.8

"^Adjusted for age, sex, cigarette pack-years, residence in community with high risk occupation.

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This information was linked to data from water utility and Colorado Department of Health records to create a drinking water exposure profile. After adjustment for cigarette smoking, tap water and coffee consumption, and medical history factors by logistic regression, years of exposure to chlorinated surface water were significantly associated with risk for bladder cancer (p = 0.0007). The odds ratio for bladder cancer increased for longer durations of exposure to a level of 1.8 (95% CI = 1.1-2.9) for more than 30 years of exposure to chlorinated surface water compared with no exposure (Table 3). The increased bladder cancer risk was similar for males and females, and for nonsmokers and smokers. In summary, epidemiologic studies of the association between water disinfection and bladder cancer have been of four general types—ecological, case-control mortality, case-control incident, and cohort studies. Each of the studies used a different measure of exposure, and each looked at a different period of exposure. Only three studies were able to control for smoking. Only two studies controlled for high-risk occupational exposure and included interviews with the cases to determine lifetime exposure at the individual level. Despite differences in study design, eight of the epidemiologic investigations reviewed above showed some association between bladder cancer and chlorinated water. Similar findings from independently conducted studies in different populations and geographical locations by independent investigators strengthens the plausibility of the association [72]. All four of the studies that used interviews for lifetime exposure assessment found an association between bladder cancer and chlorinated drinking water. Although the epidemiologic evidence of the association between bladder cancer and chlorination of drinking water certainly does not warrant abandoning chlorination as a water disinfection method, efforts should be made to reduce chlorine concentration and levels of disinfection by-products by protecting water sources and using water filtration for all but the most pristine water sources.

Table 3 Estimated risk of bladder cancer for lifetinfie duration of exposure to chlorinated water in Colorado [71]*

# of years

OR

95% CI

0 1-10 11-20 21-30 >30

1.0 0.7 1.4 1.5 1.8

0.4-1.3 0.8-2.5 0.8-2.9 1.1-2.9

"^Adjusted for sex, age, smoking, tap water intake, family history of bladder cancer, and medical history of bladder infection.

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REFERENCES 1. Rook, J. J. "Formation of haloforms during chlorination of natural waters." V^ater Treat Exam, 1974; 23: 234. 2. Bull, R. J., Meier, J. R., Robinson, M., Ringhand, H. P., Laurie, R. D., and Stober, J. A. "Evaluation of mutagenic and carcinogenic properties of brominated and chlorinated acetonitriles: By-products of chlorination." Fund Appl Toxicol 1985; 5:' 1,065-1,074. 3. NCI (National Cancer Institute). "Cancer statistics review," 1973-1987. NIH 90-2789. Bethesda: 1990. 4. ACS (American Cancer Society). "Cancer facts and figures—1990." Atlanta, Georgia: American Cancer Society, 1990. 5. Matanowski, G. M., and Elliot, E. A. "Bladder cancer epidemiology," Epidemiol Rev, 1981; 3: 203-229. 6. Silverman, D. T., Hoover, R. N., Albert, S., and Graff, K. M. "Occupation and cancer of the lower urinary tract in Detroit." JNCI, 1983; 70: 237-245. 7. Trehy, M., and Bieber, T. Proc. Am. Water Works Assn., 1980 Annual Conference, American Water Works Assn., Denver, Colorado, 1980. 8. Trehy, M., and Bieber, T. In: Jolley, R. L., et al., eds. Water Chlorination Environmental Health Impacts. Vol. 4. Ann Arbor, Michigan: Ann Arbor Science Publications, 1983. 9. Maier, W. J., McConnel, H. L., and Conroy, L. E. A Survey of Organic Constituents in Natural and Fresh Waters. Springfield, Virginia.: National Technical Information Service, 1974. 10. Thurman, E. M. Organic Geochemistry of Natural Waters. Boston: Martinus Nijhoff/Dr. W. Junk PubHshers, 1985. 11. Thurman, E. M. "Dissolved organic compounds in natural waters." In: Ram, N. M, Calabrese, E. J., Christman, R. P., eds. Organic Carcinogens in Drinking Water. New York: John Wiley and Sons, 1986: 55-92. 12. Bellar, T. A., Lichtenberg, J. J., and Kroner, R. C. "The occurrence of organohalides in chlorinated drinking water." / Am Water Works Assoc, 1974; 66: 703. 13. Symons, J. M., Bellar, T. A., Carswell, J. K. "National organics reconnaissance survey for halogenated organics." J Am Water Works Assoc, 1975; 67: 634-647. 14. Rook, J. J. "Haloforms in drinking water." J Am Water Works Assoc, 1976; 68: 168-172. 15. Stevens, A. A., Slocum, C. J., Seeger, D. R., and Robeck, G. G. "Trihalomethane formation in drinking water." / Am Water Works Assoc, 1976; 68: 615.

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16. Kajino, M., and Yagi, M. "Formation of trihalomethanes during chlorination and determination of halogenated hydrocarbons in drinking water." In: Afghan, B. K., Mackay, D., eds. Hydrocarbons and Halogenated Hydrocarbons in the Aquatic Environment. New York: Plenum, 1980: 491. 17. Morris, J. C. "Aqueous chlorine in the treatment of water supplies. In: Ram, N. M., Calabrese, E. J., and Christman, R. F., eds. Organic Carcinogens in Drinking Water. New York: John Wiley & Sons, 1986: 33-54. 18. NRC (National Research Council). "Drinking water and health." Vol. 7. Washington, D.C.: National Academy Press, 1987. 19. Morris, J. C. "Reaction dynamics in water chlorination." In: Jolley, R. L., Bull, R. J., Davis, W. P., Katz, S. K., Roberts, M. H., and Jacobs, V. A. Water Chlorination Chemistry, Environmental Impact and Health Effects. Vol. 5. Chelsea, Michigan: Lewis, 1985: 701-711. 20. Morris, J. C. "Kinetics of reactions between aqueous chlorine and nitrogenous compounds." In: Faust, S. D., and Hunter, J. V., eds. Principles and Applications of Water Chemistry. New York: John Wiley & Sons, 1967: 23-53. 21. Weil, I., and Morris, J. C. "Kinetic studies on the chloramines." J Am Chem Soc, 1949; 71: 1,664-1,670. 22. Fleischacker, S. J., and Randtke, S. J. "Formation of organic chlorine in public water supplies." J Am Water Works Assoc, 1983; 75: 132-138. 23. Johnson, J. D., and Jensen, J. N. "THM and TOX formation: routes, rates, and precursors." J Am Water Works Assoc, 1986; 78: 156-162. 24. Jensen, J. N., St. Aubin, J. J., Christman, R. F., and Johnson, J. D. "Characterization of the reaction between monochloramine and isolated aquatic fulvic acid." In: Jolley, R. L., Bull, R. J., Davis, W. P., Katz, S., Roberts, M. H., and Jacobs, V. A., eds. Water Chlorination: Chemistry, Environmental Impact, and Health Effects. Vol. 5. Chelsea, Michigan: Lewis, 1985. 25. EPA (U.S. Environmental Protection Agency). "National interim primary drinking water regulations; control of trihalomethanes in drinking water." Fed. Regist., 44: 68,624-68,707, 1980. 26. Arber, R. M., Speed, A., and Scully, F. "Significant findings related to formation of chlorinated organics in the presence of chloramines." In: Jolley, R. L., Bull, R. J., Davis, W. P., Katz, S., Roberts, M. H., and Jacobs, V. A., eds. Water Chlorination: Chemistry, Environmental Impact and Health Effects. Vol. 5. Chelsea, Michigan: Lewis, 1985. 27. Feng, T. H. "Behavior of organic chloramines in disinfection." J Water Pollut Control Fed, 1966; 38: 614-628. 28. Marks, H. C , and Strandskov, F. B. "Halogens and their mode of action." Ann N YAcad Sci, 1950; 53: 163-171.

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29. Wolfe, R. L., Ward, N. R., and Olson, B. H. "Inorganic chloramines as drinking water disinfectants: a review." J Am Water Works Assoc, 1984; 76: 74-88. 30. Wolfe, R. L., Ward, N. R., and Olson, B. H. "Interference in the bactericidal properties of inorganic chloramines by organic nitrogen compounds." Environ Sci Technol 1985; 19: 1,191-1,198. 31. Loper, J. C , Lang, D. R., and Schoeny, R. S. "Residue organic mixtures from drinking water show in vitro mutagenic and transforming activity." / Toxicol Environ Health, 1978; 4: 919-938. 32. NRC (National Research Council). "Drinking water and health." Vol 1. Washington, D.C.: National Academy Press, 1977. 33. Meier, J. R., Ringhand, H. P., Coleman, W. E., Munch, J. W., Streicher, R. P., Kaylor, W. H., and Schenck, K. M. "Identification of mutagenic compounds formed during chlorination of humic acid." Mutat Res, 1985; 157:111-122. 34. Bull, R. J. "Carcinogenic hazards associated with the chlorination of drinking water." In: Organic Carcinogens in Drinking Water: Detection, Treatment, and Risk Assessment. New York: John Wiley & Sons, 1986. 35. NCI (National Cancer Institute). "Report on the carcinogenic bioassay of chloroform." Bethesda: National Cancer Institute, 1976. 36. Jorgenson, T. A., Meierhenry, E. F., Rushbrook, C. J., Bull, R. J., and Robinson, M. "Carcinogenicity of chloroform in drinking water to male OsbomeMendel rats and female B6C3F1 mice." FundAppl Toxicol, 1985; 5: 760-769. 37. NTP (National Toxicology Program). "Toxicology and carcinogenesis studies of chlorodibromomethane in F344/N rats and B6C3F1 mice." Research Triangle Park: National Toxicology Program, 1985. 38. Bull, R. J., Brown, J. M., Meirhenry, E. F., Jorgenson, T. A., Robinson, M., and Stober, J. A. "Enhancement of the hepatotoxicity of chloroform in female B6C3F1 mice by com oil: Implications for chloroform carcinogenesis." Environ Health Perspect, 1986; 69: 49-58. 39. Trehy, M., and Bieber, T. "Detection, identification and quantitative analysis of dihaloacetonitriles in chlorinated natural waters." In: Keith, L. H., ed. Advances in the Identification and Analysis of Organic Pollutants in Water. Vol. 2. Ann Arbor, Michigan: Ann Arbor Science, 1981. 40. Rockwell, A. L., and Larson, R. A. "Aqueous chlorination of some phenolic acids." In: Jolley, R. I., et al. Water Chlorination: Chemistry, Environmental Impacts and Health Effects. Vol. 5. Chelsea, Michigan: Lewis Publ., 1984. 41. NCI (National Cancer Institute). "Bioassay of 2,4,6-trichlorophenol for possible carcinogenicity." Bethesda, Maryland: National Cancer Institute, 1979. 42. Devesea, S. S., and Silverman, D. T. "Cancer incidence and mortality trends in the U.S." JNCI, 1978; 60: 545-571. 43. Roush, G., Halford, T., Schefmura, M., and White, C. Cancer Risks and Incidence Trends. Washington, D.C.: Hemisphere Publ., 1985.

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44. Morrison, A. S. "Geographic and time trends of coffee imports and bladder cmcQrr Eur J Cancer, 1978; 14: 51-54. 45. Fraumeni, J. F. "Cigarette smoking and cancers of the urinary tract: geographic variation in the United States." JNCI, 1968; 41: 1,205-1,211. 46. Hoover, R., and Cole, P. "Population trends in cigarette smoking and bladder cmctxr Am J Epidemiol, 1971; 94: 409-418. 47. Armstrong, B., and Doll, R. "Bladder cancer mortality in England and Wales in relation to cigarette smoking and saccharin consumption." Br J Prev Soc Med, 1974; 28: 233-240. 48. Stevenson, R. G., and Moolgavkar, S. H. "Estimation of relative risk from vital data: smoking and cancers of the lung and bladder." JNCI, 1979; 63: 1,351-1,357. 49. Wynder, E. L., and Stellman, S. D. "Artificial sweetener use and bladder cancer: a case-control study." Science 1980; 207: 1,214-1,216. 50. Wynder, E. L., and Goldsmith, R. "The epidemiology of bladder cancer—a second look." Cancer, 1977; 40: 1,246-1,268. 51. Howe, G. R., Burch, J. D., and Miller, A. B. "Artificial sweeteners and human bladder cancer." Lancet, 1977; 2: 578-581. 52. Howe, G. R., Burch, J. D., and Miller, A. B. "Tobacco use, occupation, coffee, various nutrients, and bladder cancer." JNCI, 1980; 64: 701-713. 53. Marrett, L. D., Walter, S. D., and Meigs, J. M. "Coffee drinking and bladder cancer in Connecticut." Am 7 £p/J^m/6>/, 1983; 117: 113-127. 54. Hartge, P., Hoover, R., West, D. W., and Lyon, J. L. "Coffee drinking and the risk of bladder cancer. 7A^C/,1983; 70: 1,021-1,026. 55. Cantor, K. P., and McCabe, L. J. "Epidemiologic studies on the health effects of waterbome carcinogens." In: Proceedings, 1978 annual conference. Denver: Am. Water Works Assoc, 1979. 56. Cantor, K. P. "Epidemiological evidence of carcinogenicity of chlorinated organics in drinking water." Environ Health Perspect, 1982; 46: 187-195. 57. Cantor, K. P., Hoover, R., Mason, T. J., and McCabe, L. J. "Associations of cancer mortality with halomethanes in drinking water." JNCI, 1978; 61:979-985. 58. Cech, I., Holguin, A. H., Littell, A. S., Henry, J. P., and O'Connell, J. "Health significance of chlorination byproducts in drinking water: The Houston experience." M / £/7/J^m/o/, 1987; 16: 198-207. 59. Carlo, G. L., and Mettlin, C. J. "Cancer incidence and trihalomethane concentrations in a public drinking water system." Am J Public Health, 1980; 70: 523-525. 60. Alvanja, M., Goldstein, I., and Susser, M. "Case control study of gastrointestinal and urinary cancer mortality and drinking water chlorination." In: Jolley, R. J., Gorchev, H., and Hamilton, D. H., Jr., eds. Water Chlorination: Environmental Impact and Health Effects. Vol. 1. Ann Arbor: Ann Arbor Science Publ. 1978: 395-409.

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61. Hogan, M. D., Chi, P. Y., Hoel, D. G., and Mitchell, T. B. "Association between chloroform levels in finished drinking water supplies and various site-specific cancer mortality rates." / Environ Pathol Toxicol, 1979; 3: 873-887. 62. Young, T. B., Kanarek, M. S., and Tsiatis, A. A. "Epidemiologic study of drinking water chlorination and Wisconsin female cancer mortality." JNCI, 1981; 67: 1,191-1,198. 63. Gottlieb, M. S., Carr, J. K., and Clarkson, J. R. "Drinking water and cancer in Louisiana: A retrospective mortality study." Am J Epidemiol, 1982; 116:652-667. 64. Zierler, S., Danley, R., and Feingold, L. "Type of disinfectant in drinking water and patterns of mortality in Massachusetts." Environ Health Perspect, 1986; 69: 275-279. 65. Lynch, C. F., Woolson, R. F., O'Gorman, T., and Cantor, K. P. "Chlorinated drinking water and bladder cancer: Effects of misclassification of risk estimates." Arch Environ Health, 1989; 44: 252-259. 66. Breslow, N. E., and Day, N. E. Statistical Methods in Cancer Research, Volume 1—The Analysis of Case-control Studies. Lyon: International Agency for Research on Cancer, 1980. 67. Cantor, K.P., Hoover, R., Hartge, P., Mason, T. J., and Silverman, D. T. "Bladder cancer, drinking water source, and tap water consumption: a casecontrol study." 7A^C/, 1987; 79: 1,269-1,279. 68. Zierler, S., Feingold, L., Danley, R. A., and Craun, G. "Bladder cancer in Massachusetts related to chlorinated and chloraminated drinking water: A case-control study." Arch Env Health, 1988; 43: 195-200. 69. Wilkins, J. R., and Comstock, G. W. "Source of drinking water at home and site-specific cancer incidence in Washington County, Maryland." Am J Epidemiol, 1981; 114: 178-190. 70. Morris, R. D., Audet, A. M., Angelillo, I. F., Chalmers, T. C , and Mosteller, F. "Chlorination and cancer: a meta-analysis." Am J Pub Health, 1992; 82: 955-963. 71. McGeehin, M. A., Reif, J. S., Becker, J. C , and Mangione, E. J. "Case-control study of bladder cancer and water disinfection methods." Am J Epidem, 1993;137:492-501. 72. Cantor, K. P., Kanarek, M. S., and Young, T. B. "Epidemiologic approaches to the assessment of carcinogens in drinking water." In: Ram, N. M., Calabrese, E. J., and Christman, R. F., eds. Organic carcinogens in drinking water. New York: John Wiley and Sons, 1986.

CHAPTER 18 METAL-INDUCED DEVELOPMENTAL TOXICITY IN MAMMALS Jose L. Domingo School of Medicine Rovira i Virgili University 43201 Reus, Spain

CONTENTS SUMMARY, 395 DEVELOPMENTAL TOXICITY OF ENVIRONMENTAL TOXIC ELEMENTS, 396 Arsenic, 396 Cadmium, 397 Lead, 398 Mercury, 399 Uranium, 400 Vanadium, 401 DEVELOPMENTAL TOXICITY OF INDUSTRIAL METALS, 402 Chromium, 403 Cobalt, 403 Manganese, 403 Zinc, 404 DEVELOPMENTAL TOXICITY OF THERAPEUTIC METALS, 405 Aluminum, 405 Gallium, 405 Lithium, 406 REFERENCES, 406 SUMMARY Metals are widely distributed in nature. Elements such as arsenic, cadmium, lead, and mercury are well-known environmental pollutants that have profound effects upon embryonic and fetal development, whereas other metals such as uranium or vanadium are developmental toxicants. Although several metals such as chromium, cobalt, manganese, and zinc are considered essential for mammals, these elements may also represent a serious health hazard as a result of high environmental concentrations derived from a number of industrial activities. On the other hand, metals such as aluminum, gallium, and lithium are examples of current therapeutic elements that also possess potential embryotoxicity and fetotoxicity. The developmental toxicity in mammals of various elements is herein reviewed.

395

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DEVELOPMENTAL TOXICITY OF ENVIRONMENTAL TOXIC ELEMENTS Toxicities are summarized in Table 1 and are detailed in the text following. Arsenic Although exposure to arsenic (As) compounds may occur from sources such as herbicides, insecticides, rodenticides, paint pigments, and wood preservatives [1], environmental arsenic exposure has received attention primarily because of disease resulting from ingestion of food or water containing arsenic [2]. Most of environmental inorganic arsenic is present as arsenate (As^"^), but arsenite (As^"^) which is more toxic than arsenate, is also found in some cases [3]. Abundant evidence exists for the developmental toxicity of arsenic in the hamster, mouse, and rat [4, 5]. Arsenate teratogenicity has been demonstrated in golden hamsters [6] and rats [7] when given at high doses. In those species, malformations consisted mainly of exencephaly, eye defects, renal agenesis, and gonadal agenesis. Fetal anomalies resulting from intraperitoneal (ip) arsenate treatment of pregnant mice on one of gestation days 6-12, or from oral administration on one of gestation days 7-15, have also been reported [8-10]. It was found that oral dosing with arsenate had less effect on the conceptus than did treatment by ip injection, suggesting that fetal arsenic uptake is more rapid and extensive following ip than oral maternal exposure, with significantly greater peak levels attained [9, 10]. It was suggested

Table 1 Developmental toxicity in mammals of a number of environmental toxic elements: A summary of results

Element Arsenic Arsenate Arsenite Cadmium Lead Mercury Inorganic Methylmercury Uranium (uranyl ion) Vanadium Vanadyl Vanadate

Species

Route

Developmental effects

References

hamsters, mice, rats hamsters, mice, rats hamsters, mice. rats hamsters, rats

oral, ip oral, ip iv, diet, sc iv, ip

Teratogenicity Embryolethality, teratogenicity Embryotoxicity, teratogenicity Embryotoxicity, teratogenicity

[4-10] [4, 5, 12, 14] [22-32] [43, 44]

rats hamsters, cats, rats, mice mice

iv oral, ip

Teratogenicity Embryolethality, teratogenicity

[63] [52-59]

oral, sc

Embryolethality, fetotoxicity (including teratogenicity)

[69-75]

mice

oral

hamsters, rats, mice

oral, ip, iv

Embryolethality, fetotoxicity (including teratogenicity) Embryolethality, fetotoxicity (including teratogenicity)

[78] [79-84]

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that the primary effect of arsenate might be to prevent the neural folds from approaching close enough for fusion to occur during critical development periods [11]. With regard to arsenite, the first report that it might adversely affect the conceptus was the study by Hood [12], which found that arsenite treatment in mice resulted in high rates of fetal deaths, and tended to decrease fetal weights when given at 10 or 12 mg/kg on one of days 7-12 of gestation. Exencephaly, micrognathia, open eye, tail defects, and skeletal anomalies of the ribs and vertebrae were the most common defects associated with arsenite treatment. More recently, the effects of orally and ip administered arsenic were compared in the mouse [1] and hamster [13], with oral dosing being significantly less effective. In pregnant mice, trivalent arsenic administered ip or by gavage on gestation day 18 was readily transferred via the placenta and entered the near-term fetuses [14]. Arsenic uptake was significantly greater following maternal ip vs. oral dosing, whereas methylation of the administered arsenic occurs to a considerable extent regardless of treatment made and is presumed to decrease the toxic effect [14]. Embryotoxic effects of arsenite and arsenate on the development of mouse embryos during early organogenesis were also found using the whole embryo culture technique [15]. Moreover, at least one case of human neonatal death has also been ascribed to maternal ingestion of arsenic [16]. With regard to the prevention of arsenic-induced developmental toxicity, subcutaneous (sc) treatment with 2,3-dimercaptopropanol (BAL) diminished the incidence of arsenate-induced gross malformations and growth retardation in mice [17], although sc administration of this chelator was not able to alleviate the embryotoxicity and teratogenicity of arsenite [18, 19]. However, 2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane-l-sulfonate (DMPS) were effective in the prevention of both arsenite- and arsenate-induced developmental toxicity in mice [19-21]. Cadmium In animal models, cadmium (Cd) has been found to be a developmental toxin that can induce defects in the development of the lung, brain, testes, eye, and palate, as well as intrauterine growth retardation and fetal death [22-25]. In rats, a single intravenous (iv) injection of 1.25 mg Cd^'^/kg between days 9 and 15 of gestation resulted in a high incidence of hydrocephalus together with other malformations in the fetuses, while 1.35 mg Cd^"^/kg killed all the embryos. The no-teratogenic-observed-effect level (NOEL) was 1.1 mg Cd^"^/kg [26]. Other threshold doses for embryotoxicity of Cd chloride in rats were 1,000 ppm in the diet and 10 mg/kg by oral gavage [27]. Fetal death in rats was produced by sc injection of CdCl2 (40 jimoles/kg) on day 18 of pregnancy. The high incidence of these deaths (74.9%) following maternal exposure to Cd would not be solely explained by the direct effect of the metal on the fetus, because the fetal toxicity may also be the result of some extra-fetal mechanisms such as maternal toxicity or the observed

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placental necrosis [24]. Hydrocephalus was the most frequent abnormality when Cd was given between days 8 and 12 of gestation. Other malformations in rats included eye defects, gastroschiasis and umbilical hernia, and forelimb ectrodactyly [26, 28]. The placenta is another target organ for Cd toxicity [25]. In hamsters, the types of malformations depend upon the stage of organogenesis during which Cd exposure occurs, but consisted primarily of craniofacial and limb bud defects when the metal was injected iv [29, 30]. Growth retardation and malformations were also observed in mice [31, 32]. In general terms, the adverse effects of Cd are produced despite limited embryonic and fetal accumulation of the metal. A number of studies showed that the placenta serves as a barrier to the free movement of Cd^"^ to the fetus. It was suggested that many of the teratogenic effects of Cd^"^ might result from damage to the placenta and the disruption of the delivery of nutrients to the fetus [33-35]. The oral administration of alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) (50 mg/kg) protected hamsters against cadmium-induced malformations and embryonic death following a teratogenic dose of cadmium sulfate (2 mg/kg, iv, day 8 of gestation) [36]. The MFA protective mechanism against cadmium-induced teratogenesis in the hamster was mediated by the chelation of the cadmium ion rather than by metallothionein synthesis. A similar conclusion was obtained in a subsequent study, when the relative ability of four dithiocarbamate agents (DTC) to influence the developmental toxicity of parenteral cadmium in the hamster was examined [37]. Lead Early reports indicated that high doses of lead (Pb) could induce abortions in humans [38]. While Pb-induced malformations have been demonstrated in birds, fish and rodents [39], there is no evidence that Pb produces gross malformations in humans [40]. Although it would be possible that fetuses aborted by high-dose exposure to Pb were malformed, there is no evidence supporting this suggestion. The possibility that a number of adverse maternal health conditions can affect the transfer of Pb to the fetus and/or the retention of Pb by the mother or the fetus has received little systematic study [40]. In experimental investigations, negative findings were obtained in studies on Pb acetate for teratogenicity in rats and mice given oral doses of 3.9-390 mg Pb/kg/day during organogenesis [41], whereas Jacquet et al. [42] established that Pb added to the diet of pregnant mice from day 1 of gestation provoked a marked reduction in fertility as well as a retarded growth in utero. Studies by McClain and Becker [43] showed embryotoxic and fetotoxic effects of Pb in rats. The toxic effects depended on the day of gestation that Pb was administered. On day 9, teratogenic effects were observed with few resorptions, while Pb given on day 16 of pregnancy resulted in hydrocephalus and central nervous system hemorrhage. These effects were only observed when Pb was given iv. Very large doses of Pb given orally to rats produced few effects, whereas the ip injection of Pb on individ-

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ual days of gestation produced up to 100% resorptions, although much fewer and nonspecific malformations were detected [43]. In hamsters, malformations induced by iv administration of Pb were primarily localized within the developing sacral and tail vertebrae, and were characterized by varying degrees of tail malformations ranging from stunting to complete absence of the tail [44]. In mice, ip injection of Pb acetate increased the postimplantation mortality and the rate of skeletal anomalies among the fetuses. These anomalies were restricted to the anterior part of the axial skeleton and consisted essentially of the fusion of two or more cervical vertebrae [45]. Cohen and Roe [46] reviewed 23 teratogenicity studies of Pb acetate or nitrate given orally to rats and mice. No evidence of teratogenic effects was found. The route of administration of Pb appears to play an important role in the specificity of malformations provoked by this metal. Although significant quantities of Pb are transferred into the fetus, the placenta might limit the passage of Pb, because large maternal-fetal concentration gradients exist [43]. An assessment of the teratogenicity and fetal toxicity of a 1:1 molar ratio of lead to four chelating agents—ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), iminodiacetic acid (IDA), and penicillamine (PEN)—was made in rats at three stages of gestation. The iv administration of lead nitrate with the chelating agents resulted in less embryotoxic and fetotoxic effects than those produced by lead nitrate alone. EDTA produced the greatest reduction, with PEN and NTA intermediate, and IDA resulting in the least overall protective effects [47]. Mercury Agriculture, consumption of fossil fuels, and industry are the main sources of pollution by mercury (Hg), which is discharged into the environment as metallic mercury, as inorganic mercury compounds, or as organic compounds. Inorganic mercury can be transformed into methylmercury by action of microorganisms, particularly in sediments, and this biotransformation in aquatic food chains is recognized as a serious environmental hazard [48]. Methylmercury is known as the main causative agent of Minamata disease, one of the most visible tragedies caused by methylmercury pollution. This was the first known incident in history where the natural bioaccumulation of a toxin (methylmercury) in the aquatic environment killed human beings who ate fish accumulating the contaminant. More than 50 people died, whereas over 700 people around Minamata Bay (Japan) were permanently paralyzed. A high rate of congenital abnormalities was also observed. They included high palates, auditory canal defects, syndactylia, congenital retinochoroidal colobamata, congenital heart abnormalities, congenital pupil malformations, microcornea, microcephalia, hydrocephaha, archynoid cysts, and so forth [49]. Although the placenta may represent a certain barrier to Hg, transfer of alkyl Hg is possible, being more important than that of aryl or inorganic Hg [48]. Methylmercury has been recognized to cause neurologic disturbances and death in

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human adults and teratological anomalies in newborn infants [50, 51]. Methylmercury has been reported to be embryotoxic and teratogenic in golden hamsters [52, 53], cats [54], rats [53, 55, 56], and mice [55, 57-59]. A high incidence of resorptions and dead fetuses was observed, and cleft palate was the most frequent malformation. Generalized edema, brain lesions, wavy ribs, assymetrical stemebrae, and decreased ossification of parietal and occipital bones were other malformations and developmental variations seen in animals exposed to methylmercury [55, 58, 60]. The inorganic mercury detected in the fetal liver of hamsters after maternal exposure to methylmercury would probably be due to demethylation of methylmercury in the dam and transplacental transfer of inorganic mercury [61]. Mercuric mercury (Hg^"^) not only produces a range of toxic manifestations in adult male and non-gravid female animals [62], but is also fetotoxic, and at lower dose levels may affect embryonic development and cause fetal malformations in pregnant rodents [39]. Holt and Webb [63] suggested that the fetal abnormalities observed in rats given Hg^"^ iv resulted not from any direct action of Hg on the conceptus, but either from the effects of the metal on the transport of essential metabolites from the mother, or the maternal kidney disfunction. In mice exposed to low, non-toxic levels of methylmercury, the deposition of mercury in offspring due to transplacental transport was quantitatively more important than later deposition due to intestinal absorption of mercury from mother's milk [64]. Exposure of guinea pigs to inorganic mercury or methylmercury via breast milk resulted in significant accumulation of mercury in the offspring, the pattern being dependent on the chemical form of mercury given to the dams [65]. The chelating agents DMSA [58], DMPS [66], and 2-mercaptopropionylglycine [67] were effective in alleviating methylmercury-induced teratogenesis in mice, whereas the administration of penicillamine to pregnant rats poisoned with methylmercury prevented the production of morphological changes in the fetal brain [68]. In contrast, the methylmercury-induced teratogenicity was not influenced by the administration of trisodium nitriloacetate (NTA) [56], while the incidence of malformations produced by methylmercury was increased by treatment with N-acetylcysteine (NAC) [57]. Uranium The best known use of uranium (U) is as a fuel in nuclear power reactors and nuclear weapons. The increasing role of this metal in nuclear industry results in an increase in occupational exposure to uranium and puts the general population at risk for chronic exposure to low levels of uranium either by inhalation or by dietary intake. Although uranium can exist in the oxidation states of +3, +4, +5, or +6, in solution the uranyl ion (U02^"^) is the most stable species and the form in which this element is present in the mammalian body [69]. In recent years, it has been demonstrated that uranium is a developmental toxicant when given orally or sc to mice [4, 69]. Teratogenic effects of U were found in

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mice when uranyl acetate dihydrate (UAD) was given by gavage at 5, 10, 25, and 50 mg/kg/day on gestational days 6-15 [70]. Fetal toxicity consisted primarily of reduced fetal body weight and body length as well as an increased incidence of malformations (cleft palate, bipartite sternebrae) and developmental variations (reduced ossification and unossified skeletal variations), while in contrast, there was no evidence of embryolethality at any dosage level. In a subsequent study, UAD was given orally to pregnant mice at daily doses of 0.05, 0.5, 5, and 50 mg/kg from day 13 of pregnancy until weaning of the litters on day 21 post-birth. Although U exposure had no significant effect on sex ratio, mean litter size, pup body weight, or pup body length throughout lactation, significant decreases in the mean litter size on postnatal day 21 and in viability and lactation indices were observed at the 50 mg/kg/day dose level [71]. No adverse changes on fertility of mice were found when UAD was tested for its effects on reproduction, gestation, and postnatal survival. Mature male mice were orally treated with UAD (5, 10, and 25 mg/kg/day) for 60 days prior to mating with mature virgin female mice treated orally for 14 days prior to mating with UAD at the same doses as those of males. Significant increases in the number of dead young per litter were seen at birth and at day 4 of lactation in the 25 mg/kg/day group, whereas the growth of the offspring was always significantly lower for the U-exposed animals [72]. The effects of multiple maternal sc injections of UAD (0.5, 1, and 2 mg/kg/day) from day 6 to 15 of gestation were also evaluated in mice [73]. Although not doserelated, embryotoxicity occurred in all U-exposed groups. Fetal body weight was significantly decreased at 1 and 2 mg/kg/day, whereas the number of total internal and skeletal defects showed dose-dependent increases at 0.5, 1, and 2 mg/kg/day. Most morphological defects were developmental variations, while malformations (cleft palate) were only detected at 1 and 2 mg/kg/day. Most of these anomalies were not resulting from maternal toxicity [74], and they were probably the primary effects of the developmental toxicity of U. Investigation of whether the day of exposure would modify the embryo/fetal toxicity of U in mice showed that gestational day 10 would be the most sensitive time for U-induced developmental toxicity [75]. When the ability of Tiron, an effective chelator in acute uranium poisoning, to protect the developing mouse fetus against uranium-induced developmental toxicity was assessed, the results offered only modest encouragement with regard to its possible therapeutic potential for pregnant women exposed to this heavy metal [76]. Vanadium Vanadium (V) occurs at relatively high concentrations in crude oils and coals. Combustion of these fuels constitutes the major source of vanadium emissions into the atmosphere. Other possible sources of vanadium contamination are effluent discharges from titanium and uranium processing plants. Consequently, in the non-

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Health and Toxicology

industrial community, vanadium exposure in humans arises primarily from its environmental presence. Vanadate (V^"^) is the most stable form of vanadium at neutral pH in the presence of oxygen. However, reducing environments readily convert vanadate to vanadyl (V^"^). Although vanadate is more toxic than vanadyl, oxidation states IV and V interconvert easily under physiological conditions. It has been demonstrated that both vanadate and vanadyl cross the placental barrier and reach the mammalian conceptus [77, 78]. Vanadate has been found to cause developmental toxicity in pregnant hamsters, rats, and mice, whereas vanadyl exposure causes embryotoxic and teratogenic effects in mice. The ip exposure to ammonium vanadate (0.47, 1.88, 3.75 mg/kg/day) of pregnant Syrian golden hamsters from gestation days 5 to 10 resulted in micrognathia, supernumerary ribs, and alterations in stemebral ossification, while embryolethality was not observed at any dosage level [79]. Also, the iv administration of 0.15 ml of V2O5 1 mM injected in mice on day 8 of pregnancy caused a low degree of skeletal ossification without increasing the number of nonviable implants [80]. On the other hand, the oral administration of vanadyl sulfate pentahydrate (37.5, 75, 150 mg/kg/day) to pregnant mice during organogenesis caused maternal toxicity, embryofetotoxicity, and teratogenicity (cleft palate, micrognathia) at all dose levels tested [78], while the NOEL for maternal toxicity and for developmental toxicity were, respectively, 7.5 and 15 mg/kg/day of sodium orthovanadate when this compound was given by gavage to mice on gestational days 6-15 at doses of 7.5, 15, and 30 mg/kg/day [81]. A significant increase in the number of resorptions and dead fetuses was also observed when sodium metavanadate (NaV03) was administered orally to rats (20 mg/kg/day) during organogenesis [82]. Fetotoxicity (decreases in fetal body weight) and teratogenicity (increases in the incidence of cleft palate) were also found when NaV03 was injected ip to Swiss mice during organogenesis [83], with gestational day 12 being the most sensitive time for NaV03-induced developmental toxicity in mice [84]. The chelating agent sodium 4,5-dihydroxybenzene-l,3-disulfonate (Tiron) has been reported to ameliorate the vanadate-induced developmental toxicity in mice [85]. There was a significant decrease in the number of resorbed fetuses, an increase in the mean fetal weight, and a reduction in the incidence of the skeletal variations (reduced ossification of supraoccipital, parietal, metacarpals, and metatarsals) caused by vanadate. DEVELOPMENTAL TOXICITY OF INDUSTRIAL METALS Toxicities are summarized in Table 2 and are detailed in the text following.

Metal-Induced Developmental Toxicity in Mammals

403

Table 2 Developmental toxicity in mammals of various industrial metals: a summary of results Element Chromium Cobalt Manganese Zinc

Species

Route

Developmental effects

References

hamsters, mice hamsters, rats mice rats, sheep

oral, ip oral sc diet

Embryotoxicity, teratogenicity No remarkable embryo/fetal effects Embryotoxicity, fetotoxicity Embryotoxicity

[86-88] [90, 93] [98, 99] [103, 104]

Chromium The major toxic action of chromium (Cr) is due to its hexavalent form, which acts as a strong oxidant. Chromium (VI) crosses the placenta in humans, as evidenced by elevated concentrations in the newborns. In hamsters, chromium trioxide (Cr03) produced malformations in 64% of the pregnant animals receiving a parenteral dose of 15 mg/kg [86]. However, no malformations or developmental variations were observed in mice treated ip on day 9 of gestation with 15 mg Cr03/kg [57]. Embryonic and fetal developmental changes in mice receiving chromium (VI) in drinking water from conception to day 19 of gestation were also found [87], while gestational weight gain of dams, fetal weight and crown-rump length were decreased when female mice received 500 or 750 ppm chromium (VI) as potassium dichromate in drinking water on days 14 to 19 of pregnancy [88]. Cobalt Although cobalt (Co) is an essential trace element, excess dietary cobalt produces toxic effects in mammals [89]. Early teratogenesis tests of Co salts were negative in hamsters [90], whereas Co (II) chloride caused cleft palate and delayed ossification in mouse embryos [91]. When C0CI2 was administered in the drinking water of male rats for 60 days prior to mating, and female rats for 14 days [92], by gavage (25, 50 and 100 mg/kg/day) on days 6 to 15 of gestation [93], or from the 14th day of gestation through 21st day of lactation [94], no embryotoxic or fetotoxic effects were observed at doses as high as 100 mg/kg/day. However, cobalt affected the period of late gestation as well as the postnatal survival and development of the pups [92, 94]. Manganese IVIanganese (JMn) is also an essential nutrient. However, an excess of this element in an individual's environment can produce devastating results. For select popula-

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tions of humans, in addition to CNS pathologies, Mn toxicity (acute and chronic) can influence lung, liver, kidney, pancreas, and reproductive function [95]. Developmental toxicity of Mn may also be a serious health hazard for pregnant women living in high-exposure environments. Experimental studies have shown that although administration of Mn during pregnancy did not result in structural malformations in rats, mice, and hamsters, ip exposure during the organogenic period in hamsters caused embryolethality [90, 96, 97]. Unlike the fetus, it has been reported that the neonate seems to be particularly vulnerable to high levels of manganese [97]. Recent investigations also showed embryotoxic and fetotoxic effects in mice following sc exposure to 2, 4, 8, and 16 mg/kg/day of manganese chloride tetrahydrate from gestation day 6 through 15. Embryotoxicity was evidenced by significant increases in the number of late resorptions as well as in the percentage of postimplantation loss. Fetotoxicity consisted primarily of reduced fetal body weight and an increased incidence of skeletal defects. No teratogenic effects were seen in any of the groups [98]. Although the mouse conceptus would be adversely affected by parenteral exposure to Mn in any of the gestational days, mice would be more susceptible to Mn-induced embryo/fetal toxicity on days 9 and 10 of gestation [99]. Zinc Zinc (Zn) is a common metal in the human environment and constitutes an important trace element intervening in many biological processes [100]. Postulated mechanisms for zinc deficiency-induced abnormalities include: reductions in protein and/or nucleic acid synthesis, abnormal microtubule polymerization, free radical damage, altered gene expression, and altered cell cycles with subsequent distortion in morphogenesis [101, 102]. Little is known on the potential developmental toxicity of Zn to human subjects, but it was reported that of four women dosed orally with 300 mg Zn/day during pregnancy, three gave birth prematurely and the child of a fourth was stillbom [103]. Zinc concentrations of up to 5,000 mg/kg in the diet of pregnant rats induced hypocuprosis in the fetuses and caused a high incidence of stillbirths and fetal resorptions [104], toxic effects which were also observed in pregnant sheep receiving 750 mg Zn/kg diet [103]. In mice, ip Zn administration (20 mg/kg) on days 8, 9, 10, or 11 of gestation produced a delay in development and some abnormalities in fetal ossifications [105]. However, iv administration of Zn sulfate to pregnant golden hamsters at 2 mg/kg on day 8 of gestation did not reveal a pattern of malformations in the embryos [90], whereas a slight increase in the frequency of hydrocephalus in rat embryos was found following excess Zn in the maternal diet [106].

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Table 3 Developmental toxicity in mammals of some therapeutic metals: A summary of results Element/ compound

Species

Route

Developmental effects

References

Aluminum hydroxide nitrate Gallium nitrate Lithium

rats, mice rats mice rats, mice

oral oral ip oral

No embryo/fetal effects Fetotoxicity Embryotoxicity, fetotoxicity Fetotoxicity

[108, 109] [112] [115] [117, 120]

DEVELOPMENTAL TOXICITY OF THERAPEUTIC METALS Toxicities are summarized in Table 3 and are detailed in the text following. Aluminum It is well established that aluminum (Al) is a developmental toxicant when administered parenterally [107]. However, until recently there was little concern about embryo/fetal consequences of aluminum ingestion because bioavailability was considered low. The importance of the route of exposure and the chemical form of the aluminum compound on the developmental toxicity of this element are now well established. Although no evidence of maternal and embryo/fetal toxicity was observed when high doses of aluminum hydroxide were given orally to pregnant rats and mice during organogenesis [108, 109], signs of maternal and developmental toxicity were found in mice when aluminum hydroxide was given concurrently with citric [110] or lactic acids [HI]. In contrast to those studies, oral administration of Al nitrate nonahydrate (13, 26, and 52 mg Al/kg/day) to pregnant rats on days 6-14 of gestation resulted in decreased fetal body weight and increased incidence and types of external, visceral and skeletal abnormalities in all the Al-treated groups [112]. On the other hand, studies in rabbits have shown that aluminum-induced behavioral toxicity is greater in adult and aged animals than in young adults [107, 113]. Maternal dietary exposure to excess Al during gestation and lactation, which did not produce maternal toxicity, would be capable of causing permanent neurobehavioral deficits in weanling mice and rats [113]. Gallium There are very few data concerning the developmental toxicity of gallium (Ga). When this element was injected iv to pregnant hamsters as Ga sulfate at 40 mg/kg

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on day 8 of gestation, only three embryos from Ga-treated mothers showed mild malformations consisting of one limb bud abnormality, one case of spina bifida, and one mild exencephaly [114]. In a subsequent study, maternal toxicity was observed in mice given ip gallium nitrate at 12.5, 25, 50 and 100 mg/kg/day on days 6, 8, 10, 12, and 14 of gestation. Embryo/fetal toxicity was also evidenced at all doses by a decrease in the number of viable implants, a reduction in fetal weight, and an increase in the number of skeletal variations (delayed ossification of parietal and occipital, dorsal hyperkiphosis, wavy ribs), whereas no significant teratogenic effects were observed. The NOEL for both maternal and developmental toxicity of Ga nitrate was < 12.5 mg/kg [115]. Lithium Lithium (Li) carbonate is used in the treatment of manic depressive psychosis and other psychiatric disorders. Hence, the knowledge of the teratogenic potential of this element has been important. Although lithium-related congenital abnormalities in rats were reported by Gralla and Mcllhenny [116], a number of anomalies were found by other investigators after administration of Li salts [117-119]. Reductions in number and weight of the litter, increase in the number of resorptions, wavy ribs, short and deformed bones of the limbs, or an increased incidence of incomplete ossifications of sternebrae, thoracic vertebrae, phalanges, and metatarsal and metacarpal bones were the most remarkable effects caused by Li when given orally to rats [117, 120]. Exencephaly, fused ribs, and defective vertebra were the morphologic defects found in mice after oral administration of Li carbonate during pregnancy [118]. There is also sufficient evidence that lithium, when administered for therapy, causes developmental toxicity in offspring of humans who have major affective disorders. Data from registries, prospective studies, and case histories of women who used lithium therapy during their pregnancies indicate that lithium causes developmental toxicity in some offspring [121, 122]. However, recent epidemiological data indicate that the teratogenic risks of first-trimester lithium exposure is lower than those previously suggested [123]. REFERENCES 1. Baxley, M. N. et al. "Prenatal toxicity of orally administered sodium arsenate in mice." Bulletin of Environmental Contamination and Toxicology, 26,1981,749-756. 2. Franzblau, A., and Lilis, R. "Acute arsenic intoxication from environmental arsenic exposure." Archives of Environmental Health, 44, 1989, 385-390. 3. Vahter, M. "Biotransformation of trivalent and pentavalent arsenic in mice and rats." Environmental Research, 25, 1981, 286-293.

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4. Domingo, J. L. "Metal-induced developmental toxicity in mammals: A xtViQ^'' Journal of Toxicology and Environmental Health, 42, 1994, 123-141. 5. Golub, M. S. "Maternal toxicity and the identification of inorganic arsenic as a developmental toxicant." Reproductive Toxicology, 8, 1994, 283-295. 6. Ferm, V. H., and Carpenter, S. J. "Malformations induced by sodium diT^tndiit'' Journal of Reproduction and Fertility, 17, 1968, 199-201. 7. Beaudoin, A. R. "Teratogenicity of sodium arsenate in rats." Teratology, 10, 1974, 153-158. 8. Hood, R. D., and Bishop, S. L. "Teratogenic effects of sodium arsenate in micQ'' Archives of Environmental Health, 24, 1972, 62-65. 9. Hood, R. D. et al. "Prenatal effects of oral versus intraperitoneal sodium arsenate in mice." Journal of Environmental Pathology and Toxicology, 1, 1978, 857-864. 10. Hood, R. D. et al. "Distribution, metabolism, and fetal uptake of pentavalent arsenic in pregnant mice following oral or intraperitoneal administration." Teratology, 35, 1987, 19-25. 11. Morrissey, R. E., and Mottet, N. K. "Arsenic-induced exencephaly in the mouse and associated lesions during neurulation." Teratology, 28, 1983, 399-^11. 12. Hood, R. D. "Effects of sodium arsenite on fetal development." Bulletin of Environmental Contamination and Toxicology, 7, 1972, 216-222. 13. Hood, R. D., and Harrison, W. P. "Effects of prenatal arsenite exposure in the hamster." Bulletin of Environmental Contamination and Toxicology, 29, 1982,671-678. 14. Hood, R. D. et al. "Uptake, distribution, and metabolism of trivalent arsenic in the pregnant mouse." Journal of Toxicology and Environmental Health, 25, 1988,423-434. 15. Chaineau, E. et al. "Embryotoxic effects of sodium arsenite and sodium arsenate on mouse embryos in culture." Teratology, 41, 1990, 105-112. 16. Lugo, G. et al. "Acute maternal arsenic intoxication with neonatal death." American Journal of Diseases in Childhood, 117, 1969, 328-330. 17. Hood, R. D., and Pike, C. T. "BAL alleviation of arsenate-induced teratogenesis in mice." Teratology, 6, 1972, 235-238. 18. Hood, R. D., and Vedel, G. C. "Evaluation of the effect of BAL (2,3-dimercaptopropanol) on arsenite-induced teratogenesis in mice." Toxicology and Applied Pharmacology, 73, 1984, 1-7. 19. Domingo, J. L. et al. "Amelioration by BAL (2,3-dimercapto-l-propanol) and DMPS (sodium 2,3-dimercapto-l-propanesulfonic acid) of arsenite developmental toxicity in mice." Ecotoxicology and Environmental Safety, 23,1992,274-281. 20. Bosque, M. A. et al. "Effects of m^5o-2,3-dimercaptosuccinic acid (DMSA) on the teratogenicity of sodium arsenate in mice." Bulletin of Environmental Contamination and Toxicology, 47, 1991, 682-688.

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21. Domingo, J. L. et al. "m^50-2,3-dimercaptosuccinic acid and prevention of arsenite embryotoxicity and teratogenicity in the mouse." Fundamental and Applied Toxicology, 17, 1991, 314-320. 22. Ahokas, R. A. et al. "Cadmium-induced fetal growth retardation: Protective effect of excess dietary zinc." American Journal of Obstetrics and Gynecology, 136,1980,216-221. 23. Holt, D., and Webb, M. "Teratogenicity of ionic cadmium in the Wistar rat." Archives of Toxicology, 59, 1987, 443^47. 24. Levin, A. A., and Miller, R. K. "Fetal toxicity of cadmium in the rat: Maternal vs. fetal injections." Teratology, 22, 1980, 1-5. 25. Parizek, J. "Vascular changes at sites of oestrogen biosynthesis produced by parenteral injection of cadmium salts: the destruction of placenta by cadmium salts." Journal of Reproduction and Fertility, 7, 1964, 263-265. 26. Samarawickrama, G. P., and Webb, M. "Acute effects of cadmium on the pregnant rat and embryo-fetal development." Environmental Health Perspectives, 28, 1979, 245-249. 27. Machemer, L., and Lorke, D. "Embryotoxic effect of cadmium on rats upon oral administration." Toxicology and Applied Pharmacology, 58,1981,438^143. 28. Samarawickrama, G. P., and Webb, M. "The acute toxicity and teratogenicity of cadmium in the pregnant rat." Journal ofApplied Toxicology, 1,1981,264-269. 29. Perm, V. H. "Developmental malformations induced by cadmium. A study of timed injections during embryogenesis." Biology of the Neonate, 19, 1971, 101-107. 30. Gale, T. P., and Homer, J. A. "The effect of cadmium on the development of the facial prominences: Surface area measurements on day 10-8 A.M. hamster embryos." Teratology, 36, 1987, 379-387. 31. Layton, W. M., and Layton, M. W. "Cadmium induced limb defects in mice: Strain associated differences in sensitivity." Teratology, 19, 1979, 229-236. 32. Webster, W. S. "Cadmium-induced fetal growth retardation in the mouse." Archives of Environmental Health, 33, 1978, 3 6 ^ 2 . 33. Goyer, R. A. "Transplacental transfer of cadmium and fetal effects." Fundamental and Applied Toxicology, 16, 1991,22-23. 34. Levin, A .A., and Miller, R. K. "Fetal toxicity of cadmium in the rat: decreased uteroplacental blood flow." Toxicology and Applied Pharmacology, 5S,19SI, 291-306. 35. MahaUk, M. P. et al. "Teratogenic effects and distribution of cadmium (Cd^"^) administered via osmotic minipumps to gravid CF-1 mice." Toxicology Letters, 76, 1995, 195-202. 36. Perm, V. H., and Hanlon, D. P. "Inhibition of cadmium teratogenesis by a mercaptoacryhc acid (MPA)." Experientia, 43, 1987, 208-210. 37. Hatori, A. et al. "Dithiocarbamates and prevention of cadmium teratogenesis in the hamster." Teratology, 42, 1990, 243-251.

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38. Rom, W. N. "Effects of lead on the female and reproduction: a review." Mount Sinai Journal of Medicine, 43, 1976, 542-552. 39. Chang, L. W. et al. "Prenatal and neonatal toxicology and pathology of heavy metals''Advances in Pharmacology and Chemotherapy, 17, 1980, 195-231. 40. Ernhart, C. B. "A critical review of low-level prenatal lead exposure in the human: 1. Effects on the fetus and newborn." Reproductive Toxicology 6, 1992, 9-19. 41. Kennedy, G. L. et al. "Teratogenic evaluation of lead compounds in mice and rats." Food and Cosmetic Toxicology, 13, 1975, 629-632. 42. Jacquet, P. "Embryonic death in mouse due to lead exposure." Experientia, 31, 1975, 1,312-1,313. 43. McClain, R. M., and Becker, B. A. "Teratogenicity, fetal toxicity, and placental transfer of lead nitrate in rats." Toxicology and Applied Pharmacology, 31, 1975,72-82. 44. Perm, V. H., and Carpenter, S. J. "Developmental malformations resulting from the administration of lead salts." Experimental and Molecular Pathology, 7, 1967, 208-213. 45. Jacquet, P., and Gerber, G. B. "Teratogenic effects of lead in the mouse." Biomedicine, 30, 1979, 223-229. 46. Cohen, A. J., and Roe, F. J. C. "Review of lead toxicology relevant to the safety assessment of lead acetate as a hair colouring." Food and Chemical Toxicology, 29, 1991, 485-507. 47. McClain, R. M., and Siekierka, J. J. "The effects of various chelating agents on the teratogenicity of lead nitrate in rats." Toxicology and Applied Pharmacology, 31, 1975,434-442. 48. Leonard, A. et al. "Mutagenicity and teratogenicity of mercury compounds." Mutation Research, 114, 1983, 1-18. 49. Harada, M. "Congenital Minamata disease: Intrauterine methylmercury poisoning," in Teratogen Update: Environmentally Induced Birth Defect Risks, New York: Alan R. Liss, 1986, pp. 123-126. 50. Koos, B. J., and Longo, L. D. "Mercury toxicity in the pregnant woman, fetus and newborn infant." American Journal of Obstetrics and Gynecology, 126, 1976, 390-409. 51. Matsumoto, H. et al. "Fetal Minamata disease. A neuropathological study of two cases of intrauterine intoxication by a methylmercury compound." Journal of Neuropathology and Experimental Neurology, 24, 1965, 563-574. 52. Harris, S. B. et al. "Embryotoxicity of methyl mercuric chloride in golden hamsters." Teratology, 6, 1972, 139-142. 53. Hoskins, B. B., and Hupp, E. W. "Methylmercury effects in rat, hamster and squirrel monkey." Environmental Research, 15, 1978, 5-19. 54. Khera, K. S. "Teratogenic effects of methyl mercury in the cat: Note on the use of this species as a model for teratogenicity studies." Teratology, 8, 1973, 293-304.

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55. Fuyuta, M. et al. "Embryotoxic effects of methylmercuric chloride administered to mice and rats during organogenesis." Teratology, 18, 1978, 353-366. 56. Nolen, G. A. et al. "Effects of trisodium nitrilotriacetate on cadmium and methyl mercury toxicity and teratogenicity in rats." Toxicology and Applied Pharmacology, 23, 1972, 222-237. 57. Endo, A., and Watanabe, T. "Analysis of protective activity of N-acetylcysteine against teratogenicity of heavy metals." Reproductive Toxicology, 2, 1988, 141-144. 58. Sanchez, D. J. et al. "Effects of m^5o-2,3-dimercaptosuccinic acid (DMSA) on methyl mercury-induced teratogenesis in mice." Ecotoxicology and Environmental Safety, 26, 1993, 33-39. 59. Su, M. Q., and Okita, G. T. "Embryocidal and teratogenic effects of methylmercury in mice." Toxicology and Applied Pharmacology, 38,1976, 207-216. 60. Spyker, J. M., and Smithberg, M. "Effects of methylmercury on prenatal development in mice." Teratology, 5, 1972, 181-190. 61. Dock, L. et al. "Demethylation and placental transfer of methylmercury in the pregnant hamster." Toxicology, 94, 1994, 131-142. 62. Chang, L. W. "Pathological Effects of Mercury Poisoning," in The Biogeochemistry of Mercury in the Environment, J. O. Nriagu (Ed.), New York: Elsevier, 1979,519-580. 63. Holt, D., and Webb, M. "The toxicity and teratogenicity of mercuric mercury in the pregnant rat." Archives of Toxicology, 58, 1986, 243-248. 64. Nielsen, J. B. and Andersen, O. "A comparison of the lactational and transplacental deposition of mercury in offspring from methylmercury-exposed mice. Effect of seleno-L-methionine." Toxicology Letters, 76,1995,165-171. 65. Yoshida, M. et al. "Milk-transfer and tissue uptake of mercury in suckling offspring after exposure of lactating maternal guinea pigs to inorganic or mQihyXmidvcmyr Archives of Toxicology, 68, 1994, 174-178. 66. Gomez, M. et al. "Evaluation of the protective activity of BAL (2,3-dimercaptopropanol) and DMPS (sodium 2,3-dimercaptopropane-l-sulfonate) on methylmercury-induced developmental toxicity in mice." Archives of Environmental Contamination and Toxicology, 26, 1994, 64-68. 67. Fujimoto, T. et al. "Prevention by tiopronin (2-mercaptopropionylglycine) of methylmercuric chloride-induced teratogenic and fetotoxic effects in mice." Teratology, 20, 1979, 297-302. 68. Matsumoto, H. et al. "Preventive effect of penicillamine on the brain defect of fetal rat poisoned transplacentally with methyl mercury." Life Sciences, 6, 1967, 2,321-2,326. 69. Domingo, J. L. "Chemical toxicity of uranium." Toxicology and Ecotoxicology News, 2,1995,lA-1%. 70. Domingo, J. L. et al. "The developmental toxicity of uranium in mice." Toxicology, 55, 1989, 143-152.

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71. Domingo, J. L. et al. "Evaluation of the perinatal and postnatal effects of uranium in mice upon oral administration." Archives of Environmental Health, 44, 1989, 395-398. 72. Patemain, J. L. et al. "The effects of uranium on reproduction, gestation, and postnatal survival in mice." Ecotoxicology and Environmental Safety, 17, 1989, 291-296. 73. Bosque, M. A. et al. "Embryotoxicity and teratogenicity of uranium in mice following subcutaneous administration of uranyl acetate." Biological Trace Element Research, 36, 1993, 109-118. 74. Khera, K. S. "Maternal toxicity—A possible factor in fetal malformations in mice." Teratology, 29, 1984, 411-416. 75. Bosque, M. A. et al. "Embryofetotoxicity of uranium in mice: Variability with the day of administration." Revista de Toxicologia, 9, 1992, 107-110. 76. Bosque, M. A. et al. "Effectiveness of sodium 4,5-dihydroxybenzene-l,3disulfonate (Tiron) in protecting against uranium-induced developmental toxicity in mice." Toxicology, 79, 1993, 149-156. 77. Edel, J., and Sabbioni, E. "Vanadium transport across placenta and milk of rats to the fetus and newborn." Biological Trace Element Research, 22, 1988, 265-275. 78. Patemain, J. L. et al. "Developmental toxicity of vanadium in mice after oral adminisiraXion.'' Journal of Applied Toxicology, 10, 1990, 181-186. 79. Carlton, B. D. et al. "Assessment of teratogenicity of ammonium vanadate using Syrian golden hamsters." Environmental Research, 29, 1982, 256-262. 80. Wide, M. "Effect of short-term exposure to five industrial metals on the embryonic and fetal development on the mouse." Environmental Research, 33, 1984, 47-53. 81. Sanchez, D. J. et al. "Developmental toxicity evaluation of orthovanadate in the mouse." Biological Trace Element Research, 30, 1991, 219-226. 82. Patemain, J. L. et al. "Embryotoxic effects of sodium metavanadate administered to rats during organogenesis." Revista Espanola de Fisiologia, 43, 1987, 223-228. 83. Gomez, M. et al. "Embryotoxic and teratogenic effects of intraperitoneally administered metavanadate in mice." Journal of Toxicology and Environmental Health, 37, 1992, 47-56. 84. Bosque, M. A. et al. "Variability in the embryotoxicity and fetotoxicity of vanadate with the day of exposure." Veterinary and Human Toxicology, 35, 1993, 1-3. 85. Domingo, J. L. et al. "Prevention by Tiron (sodium 4,5-dihydroxybenzene1,3-disulfonate) of vanadate-induced developmental toxicity in mice." Teratology, 48, 1993, 133-138. 86. Hemminki, K. "Occupational chemicals tested for teratogenicity." International Archives of Occupational and Environmental Health, Al, 1980, 191-207.

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87. Trivedi, B. et al. "Embryotoxicity and fetotoxicity of orally administered hexavalent chromium in mice." Reproductive Toxicology, 3, 1989, 275-278. 88. Junaid, M. et al. "Chromium fetotoxicity in mice during late pregnancy." Veterinary and Human Toxicology, 37, 1995, 320-323. 89. Domingo, J. L. "Cobalt in the environment and its toxicological implications." Reviews of Environmental Contamination and Toxicology, 108, 1989, 105-132. 90. Perm, V. H. "The teratogenic effects of metals on mammalian embryos." Advances in Teratology, 6, 1972, 51-75. 91. Kasirsky, G. et al. "Inhibition of cortisone-induced cleft palate in mice by cobaltous chloride." Journal of Pharmaceutical Sciences, 56, 1967, 1,330-1,332. 92. Paternain, J. L. et al. "Estudios en ratas de los efectos producidos por la administracion oral de C0CI2 sobre la fertilidad, gestacion, parto y lactancia." Revista de Toxicologia, 2, 1985, 93-103. 93. Paternain, J. L. et al. "Developmental toxicity of cobalt in the rat." Journal of Toxicology and Environmental Health, 24, 1988, 193-200. 94. Domingo, J. L. et al. "Effects of cobalt on postnatal development and late gestation in rats upon oral administration." Revista Espanola de Fisiologia, 41,1985,293-298. 95. Keen, C. L., and Zidenberg-Cherr, S. "Manganese," in Present Knowledge in Nutrition, M. L. Brown (Ed.), Washington D.C.: ILSI, Nutrition Foundation, 1990, 279-286. 96. Laskey, J. W. et al. "Effects of chronic manganese (manganese oxide) exposure on selected reproductive parameters in rats." Journal of Toxicology and Environmental Health, 9, 1982, 677-687. 97. Webster, W. S., and Valois, A. O. "Reproductive toxicology of manganese in rodents, including exposure during the postnatal period." Neurotoxicology, 8, 1987, 437-444. 98. Sanchez, D. J. et al. "Maternal and developmental toxicity of manganese in the mouse." Toxicology Letters, 69, 1993, 45-52. 99. Colomina, M. T. et al. "Effect of the day of exposure on the developmental toxicity of manganese in mice." Veterinary and Human Toxicology, 38, 1996, 7-9. 100. Leonard, A. et al. "Mutagenicity, carcinogenicity and teratogenicity of zincr Mutation Research, 168, 1986, 343-353. 101. Keen, C. L., and Hurley, L. S. "Zinc and reproduction: Effects of deficiency on fetal and postnatal development," in Zinc in Human Biology, C. F. Mills (Ed.), New York: Springer-Verlag, 1989, 183-220. 102. Peters, J. M. et al. "Influence of short-term maternal zinc deficiency on the in vitro development of preimplantation mouse embryos." Proceedings of the Society for Experimental Biology and Medicine, 198, 1991, 561-568.

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103. Campbell, J. K., and Mills, C. F. "The toxicity of zinc to pregnant sheep." Environmental Researchy 20, 1979, 1-13. 104. Ketchenson, M. R. et al. "Relationship of maternal dietary zinc during gestation and lactation to development and zinc, iron and copper content of the postnatal rat." Journal of Nutrition, 98, 1969, 303-311. 105. Chang, C. H. et al. "Teratogenicity of zinc chloride, 1,10-phenanthroUne, and a zinc-l,10-phenanthroUne complex in mice." Journal of Pharmaceutical Sciences, 66, 1977, 1,755-1,758. 106. O'Dell, B. L. "Trace elements in embryonic development." Federation Proceedings, 27, 1968, 199-204. 107. Domingo, J. L. "Reproductive and developmental toxicity of aluminum: A review." Neurotoxicology and Teratology, 17, 1995, 515-521. 108. Domingo, J. L. et al. "Lack of teratogenicity of aluminum hydroxide in mice." Life Sciences, 45, 1989, 243-247. 109. Gomez, M. et al. "Evaluation of the maternal and developmental toxicity of aluminum from high doses of aluminum hydroxide in rats." Veterinary and Human Toxicology, 32, 1990, 545-548. 110. Gomez, M. et al. "Developmental toxicity evaluation of oral aluminum in rats: Influence of citrate." Neurotoxicology and Teratology, 13, 1991, 323-328. 111. Colomina, M. T. et al. "Concurrent ingestion of lactate and aluminum can result in developmental toxicity in mice." Research Communications in Chemical Pathology and Pharmacology, 11, 1992, 95-106. 112. Paternain, J. L. et al. "Embryotoxic and teratogenic effects of aluminum nitrate in rats upon oral administration." Teratology, 38, 1988, 253-257. 113. Golub, M. A., and Domingo, J. L. "What we know and what we need to know about developmental aluminum toxicity." Journal of Toxicology and Environmental Health, 48, 1996, 585-597. 114. Perm, V. H., and Carpenter, S. J. "Teratogenic and embryopathic effects of indium, gallium and germanium." Toxicology and Applied Pharmacology, 16, 1970, 166-170. 115. Gomez, M. et al. "Developmental toxicity evaluation of gallium nitrate in xmctr Archives of Toxicology, 66, 1992, 188-192. 116. Gralla, E. J., and Mcllhenny, H. M. "Studies in pregnant rats, rabbits and monkeys with lithium carbonate." Toxicology and Applied Pharmacology, 21,1972,428-433. 117. Marathe, M. R., and Thomas, G. P. "Embryotoxicity and teratogenicity of lithium carbonate in Wistar rat." Toxicology Letters, 34, 1986, 115-120. 118. Smithberg, M., and Dixit, P. K. "Teratogenic effects of lithium in mice." Teratology, 26, 1982, 239-246. 119. Szabo, K. T. "Teratogenic effect of lithium carbonate in the fetal mouse." Nature, 225, 1970, 73-75.

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120. Hoberman, A. L. et al. "Developmental toxicity study of orally administered lithium hypochlorite in rats." Journal of the American College of Toxicology, 9, 1990, 367-379. 121. Weiner, M. L. "Overview of Lithium Toxicology," in Lithium in Biology and Medicine, G. N. Schrauzer (Ed.), New York: VCH, 1991, 83-99. 122. Moore, J. A. et al. "An assessment of lithium using the lEHR evaluative process for assessing human developmental and reproductive toxicity of 2igtni%r Reproductive Toxicology, 9, 1995, 175-210. 123. Cohen, L. S. et al. "A reevaluation of risk of in utero exposure to lithium." The Journal of the American Medical Association, 111, 1994, 146-150.

CHAPTER 19 GENETIC EVALUATION OF PESTICIDES IN DIFFERENT SHORT-TERM TESTS Patrizia Hrelia Department of Pharmacology University of Bologna Bologna, Italy

CONTENTS INTRODUCTION, 415 THE ASSESSMENT OF GENOTOXICITY OF PESTICIDES, 416 MUTAGENICITY TESTING OF PESTICIDES: EXPERIENCE WITH OLD COMPOUNDS, 417 NEW APPROACHES TO THE STUDY OF PESTICIDES, 422 Integration of Cytogenetic Assays with Biochemical and Metabolism Studies, 422 Potential to Detect Cytogenetic Damage by Using Molecular Cytogenetic Techniques, 427 CONCLUSIONS, 427 REFERENCES, 428 INTRODUCTION The high level of agricultural productivity that has been achieved during the last half-century is due, at least in part, to the availability of a wide range of pesticides, e.g., fungicides, herbicides, insecticides and other plant protection products. Moreover, pesticides are used domestically in wood preservation or as household insecticides. A principal goal in pesticide research and development is identifying the specificity of harmful action of an agent toward life forms. However, it has to be recognized that many pesticides may be toxic to organisms other than those they are intended to control, and as such may constitute a potential health hazard to livestock, domestic animals, wildlife, and man. The impact of pesticides on the health of agricultural workers and consumers is still largely unknown, even if pesticides are extensively studied from a toxicological point of view. Continued attention is focused on pesticides applied to crops or soil because of the large amount of these chemicals currently in use, the extent of field worker exposure and the possibility of direct entry of pesticides into the food chain. Clinical poisoning by residues seems to be extremely rare, and acute poisoning seems likely to be a very uncommon occurrence.

415

416

Health and Toxicology

Cancer and the induction of hereditary damage represent the stochastic effects of agricultural chemicals and are regarded as the principal risk to health. Such effects are of special concern because of the generally irreversible nature of the processes and the long latency associated with their manifestation. Damage to germ cells may result in a genetic mutation expressed in a later generation; if pesticides induce lesions in critical genes of somatic cells, the result may be cancer induction in the exposed individual. Pesticide induced lesions in cells may be manifest at any level of organization of the genetic material, i.e., the chromosome, the gene, or the primary DNA level. Chromosome aberrations and gene mutations are both part of a complex and overlapping spectrum of genetic events, but have different genetic consequences and are induced in different proportions by different chemical agents. In some cancers, oncogenes are commonly activated by chromosome aberrations, whereas in others gene mutations are responsible [1]. Clear examples are now available of each of these mutational changes in different tumors, and these provide critical support for the somatic mutations theory of carcinogenesis [2, 3, 4]. These observations support the use of mutagenicity assays in the evaluation of carcinogenic risks of chemicals to humans and in the elucidation of mechanisms of action of known carcinogens. THE ASSESSMENT OF GENOTOXICITY OF PESTICIDES The use of short-term tests for mutagenicity is an important tool for identification of chemicals with the potential of posing a threat to human health. A large number of genotoxicity tests are presently available, and in order to assess adequately any expression of genotoxicity, a simplified systematic approach to the selection of these tests is required. In the early 1970s, Bruce Ames popularized that all mutagens were carcinogens [5, 6], but this optimistic relation between genotoxicity and carcinogenicity was clearly demonstrated to be incorrect. It was shown that mutagenicity is not always synonymous with carcinogenicity, and that in vitro conditions do not adequately mimic the metabolism and physiology of animal models or human exposure [7]. The activity of genotoxic carcinogens in the Ames test and in other tests for genotoxicity provide supports for discouraging reliance solely on the Salmonella assay. There is a genetic specificity of mutagenic action [8], and the induction of damage by chemicals can be specific or preferential for one or other genetic endpoints. As mentioned before, two basic categories of endpoints, gene mutations and chromosome alterations are believed responsible for the induction of somatic (including carcinogenics) as well as heritable mutations leading to genetic disorders in offspring. For a newly developed chemical, such as a pesticide, or for a hitherto unstudied "existing" chemical, the process of ascertaining its significant primary mutagenicity and potential genotoxic carcinogenicity usually involves the conduct of, at most, four or five genetic toxicity assays [9]. Because the reliability of risk evaluation

Genetic Evaluation of Pesticides in Different Short-Term Tests

417

mainly depends on the quality of testing, adherence in test protocols to the current state of the art is obligatory. In Europe, EEC has recently been revising its recommendations for design of studies [10]. The protocols usually to be followed are those of OECD [11, 12]. In the United States, the USEPA developed a set of guidelines [13, 14]. The harmonization of testing methods will ensure mutual acceptance of data in the international community. MUTAGENICITY TESTING OF PESTICIDES: EXPERIENCE WITH OLD COMPOUNDS In this very problematic field, regulatory authorities have to be concerned with the evaluation of safety of so called "old" compounds. Many of these compounds were not subjected to systematic toxicological investigations, and therefore risk assessment has to be made on the basis of incomplete data. Mutagenicity data are part of the weight-of-evidence approach for classifying potential human carcinogens [15]. The basis of the approach is to evaluate a pesticide on the basis of results from a range of functionally distinct in vitro assays (e.g., gene mutations, chromosome mutations, DNA damage), and then to assess individual activities in in vivo assays with the same genetic endpoint. Moreover, most inadequate information is published, and the evidence that does exist is mostly based on non-mammalian bioassays. In several studies, technical-grade chemicals were used, and therefore it is possible that the reported genotoxic activity may be related to the presence of contaminants. The observation of positive results in vivo is regarded as defining a potential hazard, while activity that is evident only in vitro is regarded with circumspection and may even be completely ignored [16, 17, 8]. Negative and dubiously positive results from insufficiently performed tests usually require reinvestigation. Results of such tests could only be considered if they are clearly positive. In recent studies, we investigated the genetic safety of a series of pesticides with a relevant economic importance and widespread agricultural use: cyanazine, cyhexatin, dicamba, and DNOC (see Table 1 for chemical structure). Insufficiently performed or poorly documented tests covering in vitro and in vivo toxicological assays were submitted, and therefore further tests were required to enable risk/benefit decision. As an example, the oncogenic potentials of cyanazine and dicamba were negative in a feeding study on rats and mice [18, 19], but no data are available on the carcinogenic potential of DNOC. Cyhexatin did not cause increases in tumor incidence in rats and mice [20, 21]. Nevertheless, criticisms exist about experimental protocols of these bioassays. Scanty and often inconclusive and conflicting results are available in the open literature on genetic activity (Table 2). Most of the data come from mutation assays with bacteria and yeasts, but very few results on induced chromosomal damage are available. We concentrated our efforts on genetic endpoints evaluated in the whole

418

Health and Toxicology Table 1 Chemical structure of pesticides tested

Chemical

Cyanazine (H)

CAS No.

21725-46-2

Structure CH

1

^W N

N

NH-CH,CHj

Cyhexatin (A)

13121-70-S

Dicamba (H)

1918-00-9

DNOC (I)

534-52-1

lO-l

SnOH

COOH

OH 02N^X,CH,

¥ NO2

Fenarimol (F)

60168-88-9

Vinclozolin (F)

50471-44-8

Metalaxyl (F)

57837-19-1

^

^

I ^N

y\J^zH=cH,

Cl

32809-16-8

23564-05-8

H, herbicide; A, acaricide; /, insecticide; F, fungicide

CHC0.OCH3

t r'"' 0

-f"'

^ "^H N >=/ V- ^ c - ,

Ct

Thiophanate-methyl (F)

1

CH3OCHJC0 CHj

Procymidone (F)

OH

fY

0

NHCS.NHCO.OCH

K. Jl NHCS.NHCO.OCH

419

Genetic Evaluation of Pesticides in Different Short-Term Tests Table 2 Summary of available results on the genotoxicity of pesticides tested Compound

Test organism

Genetic effect

Cyanazine

S. typhimurium

Gene mutations with mammalian activation Gene mutations with plant activation Gene mutations Chromosome aberrations

-

[24, 26] [24]

+

[25]

Cytological effects

+

[27]

S. cerevisiae Human peripheral lymphocytes in vitro Viciafaba and tradescandia

Result

Reference

[23] +

Cyhexatin

S. typhimurium

Gene mutations

-

[23]

Dicamba

S. typhimurium S. typhimurium

Gene mutations Gene mutations with plant activations

-

[28, 29]

+

-

[24] [24]

+ + +

[24] [29, 30] [29, 30]

-

[29] [28, 29]

Maize S. cerevisiae Bacillus subtilis rec A Escherichia coli pol A Drosophila melanogaster Human lung fibroblasts Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat liver in vivo DNOC

Drosophila melanogaster Proteus mirabilis Hela cells Mouse in vivo Human peripheral lymphocytes in vitro

Gene mutations with mammalian activation DNA damage DNA damage Sex-linked recessive lethal mutations UDS SCE UDS DNA strand breaks Sex-linked recessive lethal mutations DNA damage UDS DNA strand breaks Chromosome aberrations Chromosome aberrations

[31] -H -h

+ + +

+ +

[31] [31] [35] [36] [37] [37] [38] [38]

420

Health and Toxicology

animal and in in vitro systems that utilize human cells. Chromosomal aberrations are direct indicators of genetic damage and serve as excellent markers to assess the genotoxic and clastogenic potential of pesticides. Description of each bioassay system, procurement of pesticides, purity, and qualitative aspects of pesticide data were reported previously [22]. Either the lowest effective dose or the highest dose tested that produced no effect was recorded for each test agent and bioassay system. For each negative result, the highest dose studied was termed the highest ineffective dose (HID). Similarly, for positive results, the lowest effective dose (LED) was recorded. The doses or concentrations for all in vitro tests are expressed as pig/ml, and these for in vivo tests were reported in mg/Kg body weight (Table 3). Cynazine was uniformly negative in three tests for genotoxicity: unscheduled DNA synthesis (UDS) and sister chromatid exchanges (SCE) in cultured human

Table 3 Results on the genotoxicity of pesticides tested

Compound (purity >99%) Cyanazine

Cyhexatin

Test organism Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo

Dicamba

Mouse in vivo

DNOC

S. typhimurium TA98, TAIOO, TA97, TA102 Human peripheral lymphocytes in vitro Human peripheral lymphocytes in vitro Rat in vivo

Genetic effect

Results^ Metabolic activation with without

Dose*' LED/HID

UDS

100

SCE

100

Chromosome aberrations UDS

224 50

SCE

25

Chromosome aberrations Chromosome aberrations Reverse mutations

160 832 1,000

UDS

100

SCE

50

Chromosome aberrations

15

"+, positive; -, negative ^In vitro tests, \ig/ml; in vivo tests, mg/Kg b.w. LED: lowest effective dose; HID: highest ineffective dose.

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421

lymphocytes, and in vivo cytogenic assay in rat bone marrow. These results are in agreement with reported data on microorganisms [23, 24]. The reported clastogenic response in human lymphocytes based only on a doubling of break frequency at one dose level [25] is regarded with circumspection. Altogether this evidence establishes that cyanazine did not exert genotoxic activity in experimental systems. Nevertheless, clear evidence of a specificity of mutagenic action which depends upon plant activation was obtained in three studies [24, 26, 27]. Cyhexatin was reported to be non-mutagenic in Salmonella [23]. Negative results on the induction of UDS in human lymphocytes and of chromosome damage in rats endorse that the observed induction of SCEs (Table 3) cannot be regarded as providing evidence of mutagenicity for cyhexatin. An extensive number of in vitro data sets exist for dicamba. Conflicting results were obtained in microbial assay systems with mammalian or plant activation [28, 29, 24]. Dicamba was found positive in Bacillus subtilis rec A and in E. coli pol A assay [29, 30], in human lymphocytes in vitro by means of SCE stimulation and UDS activity [31], but not in human lung fibroblasts [28, 29]. Studies in rats have resulted in the production of DNA-damaging activity in the liver [31], but not of clastogenic activity in the bone marrow (Table 3). This discrepancy may imply that mutagenic species produced in the whole animal do not reach the bone marrow in sufficient concentration, if at all. Interaction of dicamba with germ cell DNA was excluded by Waters et al. [29] in Drosophila melanogaster sex-Hnked recessive lethal mutation assay. The genotoxic effects of DNOC in different experimental systems are outlined in Table 2. Clearly, the data from both in vitro systems and rodents establish that DNOC is genotoxic. In particular, the significant activity exhibited in vivo (Table 3) may be of potentially significant health concern. Shelby [32] and Shelby and Zeiger [33] have noted that the majority of human carcinogens are clastogenic to the rodent bone marrow, almost irrespective of the tissue in which these carcinogens induce cancer in rodents and man. The previously reported DNA-damaging activity in the liver of treated rats [34] provides strong confirmation of this potential risk. Interaction of DNOC with germ cell DNA was suggested by Muller and Haberzettl [35], who demonstrated sex-linked recessive mutations in Drosophila melanogaster, while no sperm abnormalities have been found following subchronic toxicity testing in mice [39]. Nevertheless, the identification of DNOC as a potential human mutagen requires other studies to verify an interaction with mammalian germ cells. Clearly, the data from experimental animals and cells in vitro estabhsh that pesticides tested may exert genotoxic activity as judged by the methodologies usually used to make these evaluations. Although the quantitative significance of these responses has not been determined, results on genotoxic activity provide additional information that may contribute to the design of legislative and regulatory instruments to minimize significant risk to humans from pesticides. Research into the etiology of cancer has provided us with a better understanding of the processes and agents that are crucial for the induction of tumors. This knowl-

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edge provides support for the idea that minimization of exposure to carcinogens is a prudent objective and is consistent with actions taken to safeguard public health. If one accepts that pesticides may contribute to the develop of human cancer, a logical question is how tumor induction occurs and what the mechanisms of carcinogenicity and toxicity are. NEW APPROACHES TO THE STUDY OF PESTICIDES Integration of Cytogenetic Assays with Biochemical and Metabolism Studies As stated before, carcinogenesis in human and animals is a complex, multistage process [4]. Tumors are known to show a range of genetic disturbances, such as chromosomal aberrations, aneuploidy, and gene amplification, which may not be detected using only the Salmonella assay. As suggested by Ashby [8], definition of the genotoxicological character has to be done avoiding to conduct intrinsically insensitive or repetitive assays that add no new positive information, or those that have an insecure basis. Development of assays with a cytogenetic endpoint, either in vitro or in vivo, will leave essentially no genotoxins undetected. However, it is increasingly clear that the primary biological effect of many carcinogenic chemicals involves events other than direct DNA reactivity in many experimental rodent models as well as human cancers. At this time, it is predominantly the nongenotoxic carcinogens that present the greatest problems in detection and risk assessment [40]. Chemicals that act to promote the probability that an initiated cell will progress to malignancy may be important as risk determinants [41, 42]. Recently we proposed a new approach which combines cytogenetic data with a comprehensive examination of the mechanisms of toxicity down to the metabolic level as a possible surrogate measure for promotional and cocarcinogenic activity [43]. This approach permits one to study the pathogenic mechanisms of cellular and tissue lesions at various stages of carcinogenesis (initiation and promotion). Many carcinogens can alter cytochrome P450 enzyme expression (induction or inhibition), leading to metabolic differences that may be related to variations in kinetic factors and in tissue dosimetry [42]. Moreover, the promoting properties of cyt. P450 enzyme inducers (e.g., barbiturates, haliphatic halogenated hydrocarbons, alcohols, dioxins) are acknowledged [44, 45]. We focused our attention to fungicides because these agents have been stated to contribute nearly 60% of all estimated oncogenic risk from pesticides [46]. Our recent studies on fenarimol, vinclozolin, metalaxyl, and procymidone (see Table 1 for the chemical structures) demonstrate a means of identifying potential carcinogenic pesticides that can act through genotoxic and nongenotoxic mechanisms of carcinogenesis. Fenarimol is not carcinogenic in mice [47], and results on vinclozolin are doubtful [48]. Metalaxyl did not exert oncogenic effects in rodents [49], whereas procymidone was found to increase the incidence of testicular interstitial cell tumors [50]. The evidence in the literature on genotoxic effects of the

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fungicides is conflicting and incomplete. In most cases, data consist of summary of results from mandatory toxicological assays submitted for registration and cited in USEPA or FAO-WHO reports (Table 4). Fungicides were assessed with two different cytogenetic assays, namely, frequency of chromosome aberrations in human peripheral lymphocytes and an assessment of the micronuclei frequency in mouse bone marrow. Description of each bioassay system and qualitative aspects of results were reported previously [56, 57].

Table 4 Sunnmary of available results on the genotoxicity of fungicides tested Compound

Test organism

Genetic effect

Fenarimol

S. typhimurium E. coli Mouse lymphoma cells Primary rat hepatocytes Mouse bone marrow

Gene mutations Gene mutations Gene mutations UDS Micronuclei (two repeated doses) Chromosome aberrations Dominant lethals Mitotic non-disjunction Gene mutations Gene mutations Gene mutations SCE Dominant lethals Mitotic recombination Mitotic non-disjunction Hemoglobin adducts Gene mutations Gene mutations Gene mutations Gene mutations Chromosome aberrations Host mediated assay with S. typhimurium Chromosome aberrations SCE UDS UDS Mitotic crossover Mitotic gene conversion Reverse mutations UDS Gene mutations Dominant lethals

Vinclozolin

Procymidone

Metalaxyl

Chinese Hamster in vivo Rat in vivo A. nidulans S. typhimurium S. pombe S. typhimurium CHO cells Mouse in vivo A. nidulans Rat in vivo S. typhimurium E. coli B. subtilis Mammalian cells CHO-Kl cells Mouse in vivo Mouse in vivo Mouse embryo cells Rat hepathocytes Epithelian human cells S. cerevisiae Primary rat hepatocytes Mouse lymphoma cells Mouse in vivo

Result

Reference

-

[47]

+ + + + + + — -

[51] [52] [48] [53, 54] [55] [50]

[49]

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Treatment of human lymphocytes in vitro with different doses of fenarimol, vinclozolin, and procymidone (1-100 ^igA), both in the presence and in the absence of exogenous microsomal activation (S9 fraction) did not induce any significant increase in chromosome aberrations (Figure 1). Dose-dependent enhancements in chromosomal damage, not associated with mitotic inhibition or cell death, were shown in metalaxyl-treated (300-1,000 fxg/l) lymphocytes. On the other hand, in in vivo experiments metalaxyl (75 to 300 mg/Kg b.w.), as well as procymidone (97.5 to 390 mg/Kg b.w.), had no effects on the frequency of micronuclei, detected in murine polychromatic erythrocytes (Figure 2). Significant increases in the incidence of micronucleated erythrocytes were found in fenarimol (75 to 300 mg/Kg b.w.) and vinclozolin-treated mice (312.5 to 1,250 mg/Kg b.w.). The discordant results from the in vitro and in vivo cytogenetic assays for the fungicides tested suggest that metalaxyl has an intrinsic genotoxic activity which is

Figure 1. Incidence of structural chromosome aberrations induced in cultured human peripheral blood lymphocytes treated with fungicides. Treatment dose: fenarimol, procymidone and vinclozolin, 100 pg/ml; metalaxyl, 1,000 pg/ml. • , without metabolic activation; 0 with metabolic activation. * p < 0.05, ** p < 0.01 with respect to controls pc^ test).

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Figure 2. Frequency of polychromatic erythrocytes with micronuclel (MN) In mice treated with fungicides. Data Indicate the percentage mean ± S.E. valued on 1,000 erythrocytes after an intraperitoneal dose of fenarlmol (300 mg/Kg), metalaxyl (300 mg/Kg), procymidone (390 mg/Kg), vinclozolin (1,250 mg/Kg). * p < 0.05, ** p < 0.01 with respect to controls (X^ test).

expressed only in vitro; metabolism in the whole animal provides detoxified products which do not react with DNA moieties in bone marrow erythrocytes. Bioavailability data in blood of metalaxyl [49] provide sufficient evidence of bone marrow exposure. Fenarimol and vinclozolin are clastogenic in vivo but non in vitro. This behavior may reflect a deficiency of the activating fraction in vitro, or effects detected may be due to interactions with other cellular structures than chromosomes resulting in mitotic anomalies. Procymidone was confirmed to be nongenotoxic; therefore, the induction of Leyding cell tumors evaluated in chronic and oncogenic studies [50] may be a species-specific nongenotoxic mechanism. Biochemical analysis, performed on purified microsomes from different organs of treated mice [58], indicated a toxic, co-toxic, co-carcinogenic, and "promoting" activity of fungicides tested through the induction of P450 function. Results shown in Figure 3, as activity profiles in liver of treated animals, pointed out a selective

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1.000

pNFI (CYP 2E1) PROD (CYP 2B1) APND (CYP 3A) ECOD (CYPs)

Figure 3. Expression of carcinogen metabolizing enzymes in liver microsomes from fungicide treated mice. Intraperitoneal doses for 3 days: fenarlmol, 150 mg/Kg; metalaxyl, 200 mg/Kg; procymidone, 800 mg/Kg; vinclozolin, 750 mg/Kg.

increase of the expression of CYP 2B1 by fenarimol, CYP 2E1 by vinclozolin, and CYP 3A by metalaxyl, whereas procymidone was a non-selective substrate, inducing all the considered isoforms. These inductive capabilities were confirmed at a molecular level. There are two major reasons why it is important to establish whether or not a pesticide is an enzyme inducer of inhibitor. First, to determine if the pesticide is likely to cause chemical interactions. Secondly, to evaluate the possible toxicological consequences of perturbation of drug-metabolizing enzymes. A combined study in which critical genetic endpoints have been correlated with alterations in oxidative metabolism, and with urine metabolites, was carried out in mice treated with fenarimol and trichloroethylene as a probe carcinogen [59]. In this time-course study, we found that the enhanced effect of fenarimol is correlated with a significant induction of cyt. P4502B1 family of isozymes and the increased presence of activated trichloroethylene metabolites in vivo. Observed significant potentiation of clastogenic effects of trichloroethylene (about two-fold increase, p < 0.01) by pretreatment (1 h) with fenarimol was paralleled by a 20% increase in excretion of trichloethanol. However, concomitant exposure to trichloroethylene

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and fenarimol decreased both the metabolism (by 51%) and the genetic effects of trichloroethylene (by 38%, p < 0.05), probably because of inactivation of the microsomal enzymes critical for its metabolism; both trichloroethylene and fenarimol are substrates of the same P450 enzymes. Potential to Detect Cytogenetic Damage by Using Molecular Cytogenetic Techniques Structural and numerical chromosome aberrations are associated with a range of clinical genetic disorders and diseases, such as spontaneous abortions, mental retardation, congenital malformations, and cancer [60, 61]. These same events are important in tumor initiation and progression [62, 63]. It is therefore important to develop methods for cytogenetic analysis that are applicable to both laboratory animals and humans. Recent advances in molecular biology led to facilitated analysis of cytogenetic abnormalities. One approach uses fluorescence in situ hybridization (FISH) with centromeric DNA probes to detect increased frequencies of hyperploidy in human lymphocytes that have been exposed to chemicals in vitro [64]. Initial studies performed on Thiophanate-methyl (see Table 1 for chemical structure), which has a well-documented aneugenic activity in lower eukaryotes, indicate that the fungicide (0.3 to 30 [ig/ml) induced dose-dependent increases in structural chromosome aberrations (up to eleven-fold, p < 0.01) and micronucleus frequency (up to two-fold, p < 0.01) in human lymphocytes in vitro [65]. Thiophanate-methyl was relatively more active in the induction of centromere-negative than centromere-positive micronuclei (73% vs. 27%). The two types of micronuclei were present in similar proportion in cells treated with bleomycin, used as positive control, indicating a relatively stronger clastogenic activity of the fungicide. CONCLUSIONS Evidence for genotoxicity of pesticides in different short-term tests has been and still is viewed as a useful diagnostic signal for potential human genetic diseases and cancer. Genotoxicity testing will continue to play an increasingly important role in distinguishing genotoxic and nongenotoxic compounds, which are treated differently in risk assessment models. Currendy, the Salmonella typhimurium test is the most widely used short-term test for the screening of mutagens and carcinogens. However, it may be only poorly correlated to carcinogenesis [66]. Because each test system detects some genotoxins than other might not, a battery of tests is liable to attain a greater number of hits and possibly a higher sensitivity. The vast majority of genotoxic pesticides will be alerted to by a consideration of chemical structure coupled to an assessment of mutagenic activity in the Salmonella assay and in a cytogenetic assay, either in vitro or in vivo, or perhaps both [8]. Evidence was pre-

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sented to illustrate the challenge to scientists to evaluate the genetic safety of pesticides and improve the standard of genotoxicity assays despite their limitations. Although it is possible to prioritize cancer risk using only genetic data, additional information is needed for quantitative risk evaluation. The prediction of potential mutagenic and/or carcinogenic pesticides is mainly toxicological. One most challenging and useful next step forward for the development of testing strategies is to consider biochemical techniques or metabolism studies that can identify and characterize the cellular mechanisms capable of initiating and promoting the carcinogenic response. The addition of toxicokinetic studies with relevant carcinogens offers a pointer to investigate interactive effects between agents to which humans are exposed. Moreover, new techniques (e.g., molecular cytogenetic) provide a means to increase further the information obtained from routine studies and to answer questions that could not be answered before.

REFERENCES 1. Land, H., Parada, L. F., and Weinberg, R. A. "Cellular oncogens and multistep carcinogenesis." Science, 22, 1983, 771-778. 2. Miller, J. A. "Carcinogenesis by chemicals: an overview. G.H.A. Clowes Memorial Lecture." Cancer Res., 30, 1970, 559-575. 3. Miller, J. A., and Miller, E. C. "The mutagenicity of chemical carcinogens: correlations, problems and interpretations," in Chemical Mutagens, Vol. 1, A. HoUaender (Ed.), Plenum Press, 1976, pp. 83-119. 4. International Agency for Research on Cancer. "Mechanisms of Carcinogenesis in risk identification. A consensus report of an I ARC monograph working group." lARC Technical Report 91/002, Lyon: WHO, 1991. 5. Ames, B. N. et al. "Carcinogens are mutagens: A simple test system combining liver homogenates for activation and bacteria for detection." Proc. Natl Acad. Sci. (USA), 70, 1973, 2,281-2,285. 6. McCann, J. et al. "Detection of carcinogens as mutagens in the Salmonella/ microsome test." Part 1. Assay of 300 chemicals. Proc. Natl. Acad. Sci. (USA), 72, 1975,5,135-5,139. 7. Douglas, G. R., Blakly, D. H., and Clayson, D. B. "Genotoxicity tests as predictors of carcinogens: an analysis," ICPEMC Working Paper No. 5. Mutation Res., 196, 1988, 83-93. 8. Ashby, J. "Use of short-term tests in determining the genotoxicity or nongenotoxicity of chemicals," in Mechanisms of carcinogenesis in risk identification, H. Vainio, P. N. Magee, D. B. McGregor, and A. J. McMichael (Eds.), Lyon: International Agency for Research on Cancer, lARC, 1992, pp. 135-164. 9. Health Protection Branch Mutagenicity Guidelines "The assessment of mutagenicity." Environ. Mol. Mutagen., 21, 1993, 15-37.

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10. EEC, Annex V to EEC Directive 79/831/EEC, Part B "Methods for the Determination of Toxicity." Official Journal of the European Communities, L251, Vol. 27, 19 September 1984, K.H. Narjes, EEC, Brussels. 11. OECD Guidelines for Testing of Chemicals. "Genetic Toxicology" No. 471^74. Organisation for Economic Co-operation and Development, Paris, 26 May 1983. 12. OECD GuideUne for Testing of Chemicals. No. 475-478, 4 April 1984. 13. USEPA Part 798 "Health Effects Testing Guidelines," Subpart F-Genetic Toxicology. Fed. Reg., 50, 39,435-39,458, 1985. 14. USEPA "Guidelines for mutagenicity risk assessment." Fed Reg., 51, 34,006-34,012, 1986. 15. Auletta, A. E., Dearfleld, K. L., and Cimino, M. C. "Mutagenicity test scheme and guidelines: U.S. EPA Office of Pollution Prevention and Toxics and Office of Pesticide Programs." Environ. Mol Mutagen., 21, 1993, 38-^5. 16. Bridges, B. "Evaluation of mutagenicity and carcinogenicity using a three tier system." Mutation Res., 41, 1976, 71-72. 17. Ashby, J. "The prospect for a simplified and internationally harmonized approach to the detection of possible human carcinogens and mutagens." Mutagenesis, 1, 1986, 3-16. 18. U.S. EPA "Dicamba," in: Tox. Chem., No. 295, 1985. 19. U.S. EPA "Cyanazine," in: Tox. Chem., No. 188 C "Bladex," July 26, 1985, pp. 1-7. 20. FAO "Cyhexatin." Pesticide residues in food: Evaluations 1981, Food and Agriculture Organization of the United Nations, Rome, 1982, pp. 75-77. 21. OMS "Cyhexatin." Organisation des Nations Unies pour 1'Alimentation et I'Agriculture. Evaluation de quelques residues de pesticides dans les deurces alimentarires, 191Q, Organisation Mondiale de Sante, Rome, 1972, pp. 599-622. 22. Hrelia, P. et al. "Genetic safety evaluation of pesticides in different shortterm tests." Mutation Res., 321, 1994, 219-228. 23. Moriya, M. et al. "Further mutagenicity studies on pesticides in bacterial reversion assay systems." Mutation Res., 116, 1983, 185-216. 24. Plewa, M. J. et al. "An evaluation of the genotoxic properties of herbicides following plant and animal activation." Mutation Res., 136, 1984, 233-245. 25. Roloff, B., Belluk, D., and Meisner, L. "Cytogenetic effects of cyanazine and metachlor on human lymphocytes exposed in vitro.'' Mutation Res., 281, 1992, 295-298. 26. Means, J. C , Michael, J. P., and Gentile, J. M. "Assessment of the mutagenicity of fractions from s-triazine-treated Zea mays.'' Mutation Res., 197, 1988, 325-336. 27. Ahmed, M., and Grant, W. F. "Cytological effects of the pesticides phosdrim and bladex on Tradescandia and Vicia faba.'' Can. J. Genet. CytoL, 14, 1972, 157-165.

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28. Poole, D. C , Simmon, V. F., and Newell, G. W. "m vitro mutagenic activity of fourteen pesticides." Toxicol Appl. Pharmacol, 46, 1977, 196. 29. Waters, M. D. et al. "An overview of short term tests for the mutagenic and carcinogenic potential of pesticides." /. Environ. ScL Health B, 15,1980, 867-906. 30. Leifer, Z. et al. "An evaluation of tests using DNA repair-deficient bacteria for predicting genotoxicity and carcinogenicity. A report of the U.S. EPA's Gene-Tox Program." Mutation Res., 87, 1981, 211-297. 31. Perocco, P. et al. "Evaluation of genotoxic effects of the herbicide Dicamba using in vivo and in vitro test system." Environ. Mol Mutagen., 15, 1990, 131-135. 32. Shelby, M. D. "The genetic toxicity of human carcinogens and its implications." Mwraton/?^5., 204, 1988, 3-17. 33. Shelby, M. D., and Zeiger, E. "Activity of human carcinogens in the Salmonella and rodent bone marrow cytogenetic tests." Mutation Res., 234, 1990,257-261. 34. Grilli, S. et al. "m vivo unwinding fluorometric assay as evidence of the damage induced by Fenarimol and DNOC in rat liver." J. Toxicol Environ. Health, 34, 1991, 4S3-492. 35. Muller, J., and Haberzettl, R. "Mutagenicity of DNOC in Drosophila melanogaster." Arch. Toxicol, Suppl. 4, 1980, 59-61. 36. Adler, B. "Repair-defective mutants of Proteus mirabilis as a prescreening system for the detection of potential carcinogens." Biol Zbl, 95, 1976,463^69. 37. Amadori, D., Zoli, W., and Ravaioli, A. "Rischi da pesticidi: Effetti tossici, mutageni, teratogeni e cancerogeni," 1st. Oncol. Romagnolo-USL 38, 31-32; 48-50, Forli, 1982. 38. Inventario Nazionale delle Sostanze Chimiche "Scheda n. 50-07-0198-001 DNOC," Istituto Superiore di Sanita, Roma, 1988. 39. Quinto, I. et al. "Effect of DNOC, Ferbam and Imidam exposure on mouse sperm morphology." Mutation Res., 224, 1989, 405^08. 40. Butterworth, B. E. "Consideration of both genotoxic and non genotoxic mechanisms in predicting carcinogenic potential." Mutation Res., 239,1990,117-132. 41. Leonard, I. B. et al. "Comparison of hepatic carcinogen initiation-promotion system." Carcinogenesis, 3, 1992, 851-853. 42. Guengerich, F. P. "Metabolic activation of carcinogens." Pharmacol Ther., 54, 1992, 17-61. 43. Cantell-Forti, G., Paolini, M., and Hrelia, P. "Multiple end point procedure to evaluate risk from pesticides." Environ. Hlth. Perspect., 101 (suppl. 3), 1993, 15-20. 44. Diwan, A. B. et al. "P450 enzyme induction by 5-ethyl-5-phenilhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoter phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats." Cancer Res., 48, 1988, 2,492-2,497.

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45. Schulte-Hermann, R. "Tumor promoting in the liver." Arch. Toxicol, 57, 1985, 147-158. 46. National Research Council "Regulating pesticides in food." Washington, D.C.: National Academy Press, 1987. 47. U.S. EPA. Pesticide Fact Sheet "Fenarimol." Tox. Chem. N. 207AA, June 17, 1985. 48. U.S. EPA. Pesticide Fact Sheet "Vinclozolin." Tox. Chem. N. 323C, Sept. 7, 1983; April 23, 1986; April 24, 1987. 49. FAOAVHO "Pesticides residues in food—Metalaxyl." Joint Meeting on Pesticide Residues, Rome, 1982, pp. 259-270. 50. FAOAVHO "Pesticide residues in food—Procymidone." Eval. II-ToxicoL, 100(2), 1989, 161-181. 51. Bellicampi, D. et al. "Membrane-damaging agents cause mitotic nondisjunction in Aspergillus nidulans." Mutat. Res., 79, 1980, 169-172. 52. Chiesara, E. et al. "Detection of mutagenicity of Vinclozolin and its epoxide intermediate." Arc/z. Toxicol, suppl. 5, 1982, 345-348. 53. Georgopoulos, S. G., Sarris, M., and Ziogas, B. N. "Mitotic instability in Aspergillus nidulans caused by the fungicides iprodione, procymidone and vinclozolin." Pest. Scl, 10, 1979, 389-392. 54. Vallini, G., Pera, A., and de Bertoldi, M. "Genotoxic effects of some agricultural pesticides in vitro tested with Aspergillus nidulans." Environ. Poll, 30, 1983, 39-58. 55. Sabbioni, G., and Neumann, H.G. "Biomonitoring of arylamines: hemoglobin adducts of urea and carbammate pesticides." Carcinogenesis, 11, 1990, 111-115. 56. Hrelia, P. et al. "A battery of biomarkers for detecting carcinogenic risk from fungicides." Clin. Chem., 40 (7), 1994, 1,460-1,462. 57. Hrelia, P. et al. "Genetic and biochemical markers as predictors of carcinogenic effects of pesticides." Pharmacol Toxicol, 76 (suppl. 1), 1995, 18 (Abs.). 58. Paolini, M. et al. "Wide spectrum detection of precarcinogens by simultaneous superinduction of multiple forms of cytochrome P450 isoenzymes." Carcinogenesis, 12, 1991, 759-766. 59. Hrelia, P. et al. "Interactive effects between trichloroethylene and pesticides at metabolic and genetic level in mice." Environ. Hlth. Perspect. 102 (suppl. 9), 1994, 65-68. 60. Oshimura, M., and Barrett, J. C. "Chemically induced aneuploidy in mammalian cells: Mechanisms and biological significance in cancer." Environ. Mutagen., 8, 1986, 129-159. 61. Hecht, F., and Hecht, B. K. "Aneuploidy in humans: Dimensions, demography, and dangers of abnormal numbers of chromosomes," in: Aneuploidy, Part A: Incidence and Etiology, B. K. Vig and A. A. Sandherg (Eds.), New York: Alan R. Liss, 1987, pp. 9-49.

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62. Nowell, P. C. "Origins of human leukemia: An overview," in: Origins of Human Cancer, J. Brugge, T. Curran, E. Harlow, and F. McCormick (Eds.), Plainview, NY: Cold Spring Harbor Laboratory Press, 1991, pp. 513-520. 63. Knudson, A. "Genetic events in human carcinogenesis," in: Origins of Human Cancer, J. Brugge, T. Curran, E. Harlow, and F. McCormick (Eds.), Plainview, NY: Cold Spring Harbor Laboratory Press, 1991, pp. 17-25. 64. Becker, P., Scherthanh, H., and Zanki, H. "Use of centromere-specific DNA probe (p82H9) in non isotopic in situ hybridization for classification of micronuclei." Genes Chromosome Cancer, 2, 1990, 59-62. 65. Hrelia, P. et al. "A cytogenetic approach to the study of genotoxic effects of fungicides: an in vitro study in lymphocyte cultures with thiophanate methyl" AJLA, 24,1996,597-601. 66. Tennant, R. W. et al. "Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays." Sciences, 236, 1987, 933-944.

CHAPTER 20 PESTICIDE RESIDUES IN FOOD Peter Dingle Adrian Strahco and Peter Franklin Murdoch University Perth, Western AustraUa 6150 CONTENTS INTRODUCTION, 433 HISTORY, 434 RESIDUES AND FACTORS AFFECTING RESIDUE LEVELS, 435 PESTICIDE REGULATION IN THE DEVELOPED AND DEVELOPING WORLD, 437 TOXICOLOGICAL TESTING OF PESTICIDES, 438 Limitations, 440 ESTABLISHING STANDARDS, 441 MONITORING OF RESIDUES, 442 INTERNATIONAL TRADE AND PESTICIDE RESIDUES, 443 RESIDUE LEVELS, 444 HEALTH EFFECTS, 445 Acute Poisonings, 446 REFERENCES, 447 INTRODUCTION Pesticides are used to control insect pests, weeds, rodents, and diseases on food and other crops. They have also been used to control insect-borne diseases in humans (e.g., control of malaria in the tropics). The major groups of pesticides include insecticides, fungicides, herbicides, and rodenticides. The use of pesticides has developed principally because such chemicals are capable of destroying certain animal species perceived as economic threats. In no instance, however, has it been shown that these chemicals are only toxic to target species. It is well known that most pesticides are toxic to a wide range of other species, including, in many cases, humans. In spite of such knowledge, the use of pesticides remains a global pursuit, particularly in the agricultural industry, where the reduction of losses to pests through the use of pesticides has been the principal benefit. According to Melifrondes and Kashouli-Kouppari (1994) the benefits of pesticides are that their use "enhances and stabilizes crop yield, protects the nutritional integrity of food, facilitates storage to assure year round supplies, and provides for attractive and appealing food products" [1]. One of the major benefits of pesticides has been the reduction in food losses caused by insect pests, crop diseases, and 433

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weeds. It has been estimated that on a world scale there would be 30% more food, cotton and other crops if it were not for losses caused by insects, weeds, and disease [2]. International losses in agricultural production as a result of pest attack have been estimated at $74.9 U.S. billion annually [3]. One of the legacies of pesticide use, however, has been the adverse affect that pesticides can have on human health. The main source of non-occupational exposure to pesticides is through the diet [4, 5]. For example, it has been estimated that greater than 90% of DDT in human tissue came from food [4]. However, considering food pesticide residues in isolation may give a false sense of safety to the public, who may in fact conceive that food pesticide residues are the only significant source of contamination. Studies such as the Non-Occupational Pesticide Exposure Study (NOPES) carried out by the U.S. Environmental Protection Agency concluded that for "14 of the 25 pesticides tested, food appears to be the major contributor to total exposure, whereas air appears to be the dominant contributor for six of the other eleven compounds" [6]. Although a wide variety of pesticides finds its way into our food, debate has raged over whether pesticide residues in food can affect our health and what, if any, levels are safe. However, consumer surveys have shown that the majority of people consider pesticide residues in food to be a major concern. Consumers worldwide are increasingly concerned about the chemical residues in the food they eat and expect safeguards to ensure that chemical residues do not pose a health risk. This chapter will examine the history of pesticide use, factors that affect pesticide residue levels in food, regulations and standards, and exposure to pesticide residues and the associated health risks. HISTORY Pesticides have been used throughout history; however, it was not until the insecticidal potential of chlorinated organic compounds was first discovered during the Second World War, with the use of dichlorodiphenyl trichloroethane (DDT) and hexachlorocyclohexane (HCH) that pesticide use in agriculture proliferated [7, 8]. For example, in 1945 there were 15 chemicals (60 formulations) registered in the U.K., but by 1975 the number had grown to 200 (more than 800 formulations) [9]. In the U.S., agricultural pesticide use more than doubled from 150 million kg in 1964 to 400 million kg in 1980 [7]. World sales of agrochemicals amounted to $950 U.S. million in 1964; however, international sales of pesticides alone was $2.32 U.S. billion in 1971 [3]. In 1991, 2.53 million tons of pesticides, approximately $20 billion worth, was used worldwide [1]. The United States uses approximately one-fifth of this with an annual cost of $4.1 billion [10], and of the pesticides used in the United States, 69% are herbicides, 19% are insecticides, and 12% are fungicides [10].

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Equally, the concern about the ecological consequences and health effects of pesticide use has increased regulations relating to their use. For example, due to its known persistence and possible carcinogenicity, DDT was banned in Sweden in 1970 and the U.S. in 1973 [11]. Although its use was severely restricted, it was not totally banned in the U.K. and Australia until the late 1980s. Now, the use of 20 or more individual pesticides of major economic importance have been banned or restricted in one or more countries [12]. More recently, there seems to be an effort in many industrialized countries to reduce the reliance on pesticides in agriculture. Some Western European countries now have policies to reduce pesticide use over the next decade. These countries include The Netherlands, Sweden, Denmark, and Germany [13, 14]. In other countries, such as the U.K., it is felt that pesticide approval regulations, as well as more effective application techniques and the increased use of products that are more biologically active at lower dosage rates, will decrease pesticide use without the need to impose arbitrary reduction targets [15]. Although pesticide use has seemed to stabilize or even decrease in developed countries in recent years, world consumption is still growing. Indeed it has been estimated that pesticide use will increase at an annual rate of 12.5% [2]. The reason for this is the increasing amount of pesticides that are being applied in developing countries [2]. This includes substances that have been banned or severely restricted in developed countries. For example, organochlorines, which are banned in most developed countries, are being used at ever-increasing rates in Asian tropical agroecosystems and in southern Pacific islands [16]. RESIDUES AND FACTORS AFFECTING RESIDUE LEVELS A residue can be defined as something that is left behind. "A pesticide residue is any substance or mixture of substances in food for humans or animals resulting from the use of a pesticide and includes any specified derivatives, such as degradation products, metabolites, reaction products and impurities, which are considered to be of toxicological significance" [17]. Residues will originate from the normal use of agricultural chemicals, misuse of chemicals, and unintended exposure or environmental contamination. Normal use may result in residues, particularly if the chemical is applied to protect the produce on its way to the consumer. Chemicals are applied with the expectation that they will break down or disperse over time, and that the produce will have little or no residue on it by the time it gets to market. Misuse of chemicals includes: failure to observe the withholding period, the application of too much of the chemical, or the use of the chemical on a commodity for which there is not registered utility [18]. The most common form of incorrect use is the failure to have the correct withholding period between application of the pesticide and harvesting [18]. Cases also exist where illegal pesticides or excessive concentrations of legal pesticides have left residues that are a health risk [19, 20].

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The other sources of residues are the unintended exposures of crops or animals to pesticides, either prescribed or illegal. Environmental contamination may be the result of past pesticide use, and some has remained due to its persistence and nonbiodegradability, such as in the case of the organochlorines, or pesticide drift from nearby applications. Apart from residues acquired during growing, some pesticides are applied after harvesting has occurred. They are applied during storage and transport to ensure that they last. Fungicides are common to prevent mold and other storage diseases, and pests. For example, potatoes, leafy vegetables, and grains are treated [18]. A fungicide is added to a wax, and together they help keep the fruits' and vegetables' appearance intact and prevent mold. There was concern that the fungicide, not the wax, could have some health effects as a result of increasing the residue level present [22]. Waxes are used primarily to make foods look more attractive to the consumer and therefore increase produce marketability. A number of fungicides that are added to waxes have been shown to cause health problems [23]. Foods that are typically waxed include apples, citrus fruits, and tomatoes. A number of factors affect the residue levels in produce at harvest. Hayes [24] and Precheur et al. [25] noted the following as contributing factors: a) Dosage and number of applications—several small doses led to more residue than did one large dose. b) Time from application to harvest—shorter intervals between last dose and harvest resulted in elevated residues [25]. Reduction in residues involves two factors: 1) loss of pesticide through means, and 2) relative loss of residue through plant growth. c) Nature of the compound—where some compounds break down faster than others. d) Character of the formulation—dusts give rise to lower residues than liquid formulations. If the plant surface is wet, then the reverse is true. e) Character of the plant—waxy plants retain less residue than hairy plants. f) Migration from the soil—depended on soil type and crop, where sandy soils retain the most pesticide. Carrots were found to absorb more pesticide than any other crop. g) Weather—rain soon after application decreased residues. h) Volatilization—when some chemicals evaporated due to temperature, higher residues resulted, i) Method of harvest—handling during harvest decreased residues considerably, j) Possible intentional or unintentional post-harvest application—grains may have several applications during transportation, k) Method of storage—under different storage conditions, residues may or may not decrease to safe levels [24]. Even when taking all these factors into account, uncertainty still remains as to resultant residue levels.

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Many factors also affect residue levels between harvest and consumption. Eilrich (1991) traced vegetables treated with the fungicide chlorothalonil from picking to packing houses, grocery stores, and restaurants [26]. The residue levels fell steadily between test points. During processing of foods, most residues will decrease greatly while others may undergo as much as a ten-fold concentration, as in the processing of tomatoes [25]. The amount of reduction or concentration depends upon the properties of the individual pesticide, the food on which it is present, and the types of processing steps [27]. The National Food Processors Association (NFPA) in the U.S.A. showed that washing and peeling removed 99% of carbaryl and malathion residues from tomatoes. Benomyl residues were reduced 83% by washing and 98% by subsequent processing into tomato puree and sauce [28]. Processing may also lead to the formation of breakdown products that may be toxic. In the case of the ethylenebisdithiocarbamate (EBDC) fungicides such as maneb, zineb, mancozeb, and metiram, ethylenethieourea (ETU) is occasionally detected as a breakdown product. Toxicology studies have shown that ETU possesses carcinogenic, mutagenic, goiterogenic, and teratogenic properties [27]. In the U.S. there was much public concern when supplies of apple juice were recalled from the market after discovery that they were contaminated with unsymmetrical dimethylhydrazine (UDMH), a breakdown product from the pesticide daminozide (Alar). PESTICIDE REGULATION IN THE DEVELOPED AND DEVELOPING WORLD Most developed countries have registration requirements and regulatory systems for pesticides [11]. In the U.S., for example, all pesticides must be registered with the U.S. EPA, which also sets tolerances (the maximum amount of residue that is permitted in or on a food) [29]. The U.S. Department of Agriculture is responsible for setting tolerances for meat, poultry, and certain egg products, while the Food and Drug Administration (FDA) is charged with monitoring and enforcing residue levels [29]. In the European Community, each of the individual member states is able to have its own pesticide registrations and set individual tolerances; however, most legislate to take into account a European Community Directive which seeks to harmonize registration requirements throughout the Community [30]. This directive provides for a list of "Active Substances Authorized for Incorporation in Plant Protection Products"; however, authorization of products containing these active substances is at a member state level [30]. In most industrialized countries, for a pesticide to be registered it must comply with certain criteria regarding its effectiveness in controlling pests and diseases, persistence, toxicity, and residue tolerance [14]. Before approving the use of an individual pesticide, regulatory bodies may require the manufacturer to provide data from standard animal toxicity tests and from field studies [31].

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In contrast to the developed world, many developing countries do not have pesticide regulations. Indeed, 50% of developing countries have no legislation for pesticide availability and use (the proportion in Africa was 76%) [32]. As a case in point, over the past 10-15 years in Nigeria there has been a great upsurge in pesticide use; however, apart from a recent publication of guidelines and standards for environmental pollution control from the Federal Environmental Protection Authority, there is no legislation or control of pesticide use [33]. Where there is some form of regulation, it may well be inadequate or poorly implemented [34]. The situation in Third World countries is exacerbated by a lack of resources and expertise to monitor use, a growing population that needs feeding, and a lack of political will to deal with the situation [35]. A number of UN and other international agencies have collaborated in the development of national policies on pesticides. There are about 50 international organizations, approximately 15 of them within the UN system, with some involvement in pesticides [12]. A good review of the international structure is provided by Ekstrom and Akerblom (1990) [12]. Following is a brief outline of some of the areas the UN has contributed to the regulation of pesticides: • Food and Agriculture Organization (FAO), in cooperation with WHO, UNEP and others, has developed a code of conduct on the distribution and use of pesticides, outlining the potential hazards and appropriate actions for their prevention. It also defines the responsibilities of national regulatory bodies. • FAO has published guidelines for improved pesticide management including registration and control. • World Health Organization (WHO) has conducted a literature review of the public health impact of pesticides used in agriculture. • Data sheets about pesticides, where the information is available, are published jointly by FAO and WHO. • FAO and WHO have recommended restrictions on the availability of pesticide formulations, and the UN has published a list of pesticides that have been banned, withdrawn, or severely restricted by governments. The FAO and WHO through the Codex Alimenterius Commission recommend maximum residue levels (MRLs) for pesticides in foods. In most developed countries, the basic principles for assessing pesticides and setting tolerances follow those set by the joint committee of the FAO and WHO. TOXICOLOGICAL TESTING OF PESTICIDES Toxicological testing is done to assess acute and chronic toxic effects of pesticides. These include the potential to promote or initiate tumors, exhibit neural toxicity, alter genetic material, affect fetal development, affect reproductive capacity, or give rise to chronic effects such as allergies or environmental sensitivities [20].

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The cost of these toxicological and field trials is high, approximately $20 million per chemical. Acute toxicity is that which is due to short periods of exposure to high concentrations of the toxin, usually less than 96 hours. These tests have largely evolved from the concerns of the groups of people most at risk, such as through occupational exposure. Given the ubiquity of chemicals in our total environment, though, they are increasingly seen as a tool for investigating community concerns. The most common techniques used to measure acute toxicity determine the amount of a toxic substance required to kill 50 percent of target organisms in a given time. This is referred to as the LD50 and is a measure of the concentration of toxic substances ingested or absorbed through the skin. In contrast, the LC50 is a measure of the concentration of a toxic substance in the ambient environment of the organism, such as in the air or water. This technique is particularly relevant and more realistic for air pollutants that may be inhaled, but is more difficult to assess and more expensive to determine than the LD50. Subacute toxicity studies assess the toxic effects of substances at sublethal concentrations. Subacute toxicity is most commonly tested on rodents by route of intended exposure to three or four different concentrations. A high dose, just below the acute toxicity levels, is usually selected to highlight any subacute affects, and a very low dose is selected to identify the level at which no toxic effect is noticeable, referred to as the no observable effect level (NOEL). These levels are usually set in the testing protocols established by regulatory agencies such as the U.S. EPA. The remaining concentrations tested are intermediate to these two levels. Subacute tests are usually carried out over 90 days or for 10 to 15% of an organism's normal lifespan. Observations on test animals are usually numerous and diverse, and include changes in body weight, diet consumption, organ dysfunction, metabolic, physiological and somatic changes, and obvious behavioral changes. Chronic toxicity tests are designed to investigate the long-term effects of exposure to subacute concentrations of toxic substances and typically explore the genotoxic, mutagenic, carcinogenic, and/or teratogenic potential of the substance. Classical chronic toxicology studies consist of animal bioassays using surrogate species. Carcinogenic bioassays are usually performed on medium-sized small mammals, such as rats. During the course of the study, some of the experimental animals are sacrificed and pathological examinations are used to determine whether tumors have developed. The remaining animals are sacrificed for the same purpose at the end of the study. Teratogenic bioassays are designed to assess malformations induced during development from conception to birth. Teratogens are most effective during the first trimester of pregnancy, the period of organogenesis, so most studies expose the pregnant animals during this period. As a result of the high financial and animal costs and time (very important), there has been much interest shown in developing short-term tests (STT). The first attempt to identify carcinogens using STT was using a Esherichia coli mutagenesis

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assay. However, these earliest studies failed to take into account that most mutagens require metabolic activation before they become active genotoxins, thus few mutagens were identified. In the 1970s, liver extracts were first used to metabolize promutagens to mutagens. Limitations From the above it is clear that the tools used by toxicologists are important in assessing how toxic substances adversely affect living organisms. However, it is also apparent that these tools have inherent limitations that need to be considered when assessing the toxicology of a substance. Clearly there is a large gap between our present levels of understanding in toxicology and the knowledge needed to make confident judgments about chemicals in our environment. The limitations of our understanding must be recognized and taken into account so we do not develop a false sense of our knowledge. It must also be recognized that total understanding is not necessary for action to be taken. Some of the limitations include the fact that the toxicological tests carried out do not consider the possible synergies between chemicals, such as the ability of nicotine to enhance the ability of certain organophosphate pesticides to inhibit the enzyme acetylclolinesterase, causing chronic weakness, blurred vision, and in extreme cases respiratory failure [37]. Toxicological studies often only consider the effects of the active ingredient, even though toxic effects many be attributable to the so-called "inert" ingredients [38]. In addition, there are concerns that toxicological calculations do not adequately consider the different dietary habitats and susceptibility of particular groups in the population, such as infants [39]. Only about one-third of the active ingredients used in agricultural chemicals have even been assessed by the Codex Committees [40]. The toxicological effects of many chemicals in widespread use are unknown. A number of older chemicals that were previously thought to be safe, based on overseas assessment, were found to be carcinogenic in animals [20]. These include Caftan, Captafol, Folpet, a major metabolite of Chlordimeform, and alachlor. At present, as much as 80% of the registered chemicals have not been fully tested for health effects on humans, to say nothing of the environmental effects [41]. Despite the lengthy process to introduce chemicals into the market, there is no guarantee that a chemical is entirely safe for use. An example of this is where fraud occurred when a major independent laboratory. International Bio Test of Illinois, was found to have faked tests. As the cost of the tests is so high, the regulatory bodies cannot afford to duplicate many of the tests, so it is uncertain how often this fraud takes place. The problems of such fraud would be carried through when other companies developing new formulations use the fraudulent data as a referential standard. While the testing may be carried out to the best possible standard, there still remain many adverse health effects that the tests fail to acknowledge, especially in regard to hypersensitivity. At present the basis for hypersensitivity is not fully

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understood. Hypersensitivity includes such ailments as asthma, migraines, arthritis, and skin rashes. Nor is it fully understood how the chemicals affect such things as emotions, intelligence, memory, and other complex neural functions and thus the general quality of life [31]. It is hard to establish a causal link between increased exposure levels of chemicals and allergies, as no long-term research on chronic effects of pesticides on humans has been undertaken [42]. ESTABLISHING STANDARDS After completion of the toxicological tests, the data are used to develop standards. The no observable adverse effect level (NOAEL) is the highest dose at which there are no observable effects in the most sensitive test species in the most sensitive tests [31, 43]. From this the acceptable daily intake (ADI) is set. The ADI represents the amount of a chemical that you can consume every day over an entire lifetime "without appreciable risk" [31]. The ADI is usually set at one-hundredth the NOAEL of the most sensitive animal studied. The ADI is expressed in milligrams of the chemical, as it appears in the food, per kilogram of body weight (mg/kg) per day. In order to arrive at an ADI, the following information is desirable: • The chemical properties of the residue and its derivatives due to metabolization in the plant/animal tissues. • The toxicity of the chemicals forming the residues based on data from acute, long-term and short-term toxicity studies (usually in animals), and knowledge of metabolism, mechanism of action, and carcinogenicity of residue chemicals when consumed. • An adequate knowledge of the effects of the chemicals in people. The maximum residue limit (MRL) is defined to be the maximum concentration of a residue that is recommended to be legally permitted, or recognized as acceptable in, or on, that food at any stage of harvesting, transport, marketing, or preparation, up to the final consumption. Firstly, the MRL is set with regard to the ADI of the chemical concerned. The MRL is set so that the ADI is unlikely to be exceeded. This is done through assessment of the population's diet and also exposure through non-food sources. Secondly, field trials are conducted to determine the levels of a chemical and its metabolites expected to be present when the chemical is used according to officially sanctioned directions or good agricultural practice (GAP). Residue levels can vary depending on soil, climatic, and other factors even when GAP is used. MRLs need to accommodate countries where climatic conditions, pests, crop, and farming practices can cause GAP to vary considerably. Thus it is not appropriate for countries simply to adopt standards from other countries because differing agricultural conditions require different practices. For instance, hot dry conditions in Australia

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require high insecticide use, whereas cool damp conditions in Europe require use of fungicides. Trade and poUtical considerations also come into the setting of MRLs. ADIs for pesticides are reconmiended by the WHO/FAO Joint Meeting on Pesticide Residues (JMPR). International MRLs are set by FAO/WHO through the Codex Committees on Pesticide Residues (Codex). MONITORING OF RESIDUES A critical component of dietary pesticide risk assessment is the estimation of the levels of pesticides that are likely to be in foods [29]. In practice, the major approach to estimation of pesticide residues involves monitoring a sample of foods, selected to be representative of the normal diet of the population, and extrapolating this information to the broader food market. Monitoring programs also aim to detect illegal use of pesticides. Because it is not practical to analyze all commodities for all possible residues, the analytical program is based on risk profiles. Commodity-residue combinations of highest risk are included in most surveys. The following factors are taken into account in assessing risk profiles: • Toxicology of residue • Incentive for misuse • Persistence • Previous survey results • Trade considerations • Cost and availability of analytical method. The Food and Drug Administration (FDA) of the U.S. conducts an annual monitoring program, the Total Diet Study or Market Basket Survey. It is designed to estimate the total dietary intake of pesticide residues of 8 age/sex groups ranging from infants to senior citizens [45]. Each basket within the FDA study contains 234 foods compared to a maximum of 54 foods in the Market Basket Survey. These foods were purchased four times within the year to account for a greater variety of foods purchased under different climatic conditions. In total, the FDA study analyzes 19,798 foods for a total of 270 pesticides [45]. The FDA survey could not be considered as conclusive as not all the pesticides or foods used in America are tested, but in comparison to the other countries, it is more comprehensive. The FDA (1989) reported that approximately 40% of the foods monitored had detectable residues and that approximately 3% exceeded the tolerance levels with similar results being reported in other years [45]. While many countries conduct market basket surveys, they generally have a number of limitations and at best can only be used as a guide.

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For example, the surveys are limited because: • A small number of pesticides is questioned. • No account is taken of those pesticides not permitted to be used on certain crops but that may still be freely available to growers. • In many cases the environmental conversion products or metabolites are not sought. In addition, not all breakdown products or even contaminants of some pesticides are known. It is therefore not possible to test for these chemicals, let alone identify how toxic they may be. • The cumulative total or toxic equivalents of all pesticides present within a sample is also often not accounted for within the test procedures. Two or more of a particular group of pesticides in a sample must not exceed the MRL allowable for a single substance. • Only a limited number of foods can be monitored for pesticides. In Australia, a maximum of only 21 samples of a particular food were purchased. As little as 7 samples were collected for some foods. • Samples analyzed may be composites and, consequently, the values of the residues only represent average values, not the values that may be present within individuals. It is likely then that individual samples of foods with residue levels above the maximum allowable levels will be diluted by samples within the composite for which little to no residue levels exist. This fact alone renders the results of the residue monitoring of little use. INTERNATIONAL TRADE AND PESTICIDE RESIDUES Different registration requirements and regulatory systems for pesticides among countries has meant that compounds that may be banned in one country can still be used in another [11]. Countries may also differ in national tolerance levels for pesticides. The Codex Committee on Pesticide Residues (CCPR) was established through the Codex Alimenterius Commission to try to achieve international harmonization of national tolerances of pesticide residues in food [46] to protect public health and facilitate international trade. In 1989, approximately 160 pesticides were subject to Codex tolerances but very few countries adopted these unless they already complied with existing national levels [46]. Countries include the monitoring of imported foods in their sampling surveys. The U.S. puts a large emphasis on the monitoring of imported food with 60% of sampled food being imports [46]. Imported foods in the U.S. are subject to the same tolerance levels as domestically produced goods, and most violations occur through the discovery of pesticide residues of compounds not registered in the U.S. [46]. Foods that violate U.S. laws can be refused entry into the U.S..

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The General Agreement on Tariffs and Trade (GATT) proposes that Codex standards be used for international trade and to remove non-tariff barriers [13], and under new changes all restrictions, even those based on health, will no longer be possible. The new proposals for GATT would set worldwide standards for acceptable pesticide residue levels called harmonization. Under harmonization, 42% of current U.S. food residue standards would be changed to allow higher levels of pesticide residue. The changes in the EC and Australia are anticipated to be slightly less. The allowed food residues of pesticides such as the organochlorines, no longer used in agriculture in the U.S., EC and Australia, would be higher than present levels [13]. This can increase the limits for residues in food in some countries as the U.S. and EC often prescribe tighter controls on pesticide residues than does Codex [13]. The harmonization of health, safety, and environmental standards could mean, in practice, the lowest standards of the most poorly regulated member state could be adopted. RESIDUE LEVELS Human exposure to a wide variety of pesticides in foods is extensive. Since concerns were first raised about the environmental and health effects of organic pesticides, a number of have been banned or their use restricted in various countries [12]. Of these, the organochlorines have been one of the most tightly controlled. The bans on a number of the organochlorine pesticides, including DDT, heptachlor, aldrin, and dieldrin, have been reflected in the results of many monitoring surveys with marked decreases in residue levels since restrictions on their use [47, 48]. Leoni et al. (1995) reported that total dietary intake of chlorinated pesticides in the U.S. has dropped from about 100% of ADI in the early 1970s to 10% by the mid-1980s [49]. Despite these decreases in residue levels, detectable levels of organochlorine pesticides are still found in a high number of tested samples. Trotter and Dickenson (1993) found that 49.4% of milk supplies monitored throughout the U.S. had pesticide residues, and p,p'-DDE and dieldrin accounted for 84.4% of these [50]. In Spain, DDT was found in 83% of lamb samples, although at moderate levels [48]. There is also concern that pesticide residues in developing nations are likely to be higher than those experienced in the developed countries. For example, Dogheim et al. (1990) reported that most milk samples in Egypt had high residue levels of 15 pesticides included in the investigation [51]. Butter in Argentina contained detectable levels of Lindane in 93% of samples, heptachlor in 78%, aldrin in 55%, and DDT and dieldrin in 30% [52]. Kashyup et al. (1994) reported that the total dietary intake of DDT for the average Indian vegetarian adult was 19.24 |Lig/day with 50% of this being contributed by fatty foods [5]. Organophosphorous pesticides are used extensively as grain protectants and have therefore been found in a number of grains and grain products. Organophosphate pesticides are broken down rapidly, and this probably accounts for the low residue

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levels reported from most [53]. Mean intakes of organophosphate (OP) pesticides reported from a number of different countries are generally below 1% of the respective ADIs [47]. In Italy, the sum of all the percentages of ADIs of nearly all organophosphates ingested was about 20 [49]. Smart (1987), in a review of OP residues in foods from EC countries, reported that only a small proportion of fruit and vegetables contain levels that exceeded FAOAVHO MRLs or EC Maximum Limits [53]. Violations of the recommended levels were generally below 1. It would seem that pesticide residues reported from most surveys are sufficiently below MRLs for the respective compounds. In the 1994 U.S. Market Basket Survey, only 3.4% of the samples violated U.S. levels [29]. However a high number of samples (in some cases 100%) still have detectable levels of pesticide residues. Much of the concern over residue levels, particularly for consumers, is whether current levels are safe rather than whether they are under the legal tolerance level [54]. Toxic impHcations of pesticide residues in humans are not completely understood, and they may be unsafe even at low daily intake [48]. HEALTH EFFECTS The possible effects of pesticides include acute or chronic poisonings, altered immune responses, allergic reactions, effects on the nervous system, and mutagenic, teratogenic, or oncogenic effects. Although acute poisoning is of great concern, it is fairly rare and easily detectable as the acute toxicity of many pesticides to humans is well documented. Less is known about the health effects of chronic exposure to pesticides at much lower doses, with findings in relation to such exposure often being debated. Whether or not an exposure of twenty to thirty years will cause cancer or nerve damage, for example, is a very real concern. Various studies have linked chronic pesticide exposure with oncogenetic, mutagenetic, fetotoxic, and teratogenic activity, as well as damage to primary metabolic body organs (kidneys and liver) and central nervous system degradation. It is difficult to detect the cause of some delayed effects or subtle effects. This is due to the fact that there are many possible causative agents to which an individual is exposed, and the amount of exposure is likely to be unknown. The International Agency for Research on Cancer reported sufficient evidence on carcinogenicity for eighteen pesticides and limited evidence on carcinogencity on another 15 pesticides based on animal studies [56]. Animal studies have shown the tumorigenic effects of organochlorine and dithiocarbamate pesticides, while the long-term effects of a range of other agricultural chemicals are unknown [3]. Many other chronic health effects are also being associated with pesticide use. The risk associated with pesticide residues in foods depends on the dosage of the chemical, the time of exposure, and the susceptibility of the individual human. The National Academy of Science (1987) estimates that nearly 60% of the carcinogenic risk from pesticides in foods comes from fungicides, 27% from herbicides, and

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13% from insecticides [58]. In addition, approximately 20% of the current cancer risk is associated with the consumption of processed foods; within this, fungicides account for approximately 75% of this risk. Linuron, a herbicide, accounts for more than 98% of the risk associated with herbicides, while the insecticides chlordimeform and pyrethrin account for more than 95% of the estimated dietary risk from insecticides [58]. The National Academy of Science (1987) also suggested 15 foods are associated with approximately 80% of the dietary cancer risks from pesticide residues. These include tomatoes, beef, potatoes, oranges, and lettuce. Tomatoes pose the highest estimated risk with a dietary carcinogenic risk as high as 15% of the total risk attributed to all pesticide residues as a result of the high use of fungicides [58]. The National Academy of Science (1987) concluded that the estimated additive cancer risk for the American public averages at no greater than 1 x 10"^ [58]. Archibald and Winter (1989) suggest a lower estimated cancer risk than the National Academy of Science [58]. However, Gots (1992) reports that it is impossible to directly relate any increase in cancers directly to pesticide residues [60]. The role of trace amounts of pesticide residues at the parts per million and parts per billion level are not understood and will continue to be debated. Some researchers suggest that the health risks associated from natural chemicals in foods are greater than those from pesticide residues in food [61, 62]. They claim that specific constituents of common fruits such as apples, bananas, grapefruit, orange juice, peaches, pineapples, and vegetables such as broccoli, cauliflower, cabbage, and celery are more toxic than chemical pesticide residues [64]. The extent of risk to human health associated with naturally occurring toxicants remains a scientifically contentious matter. There is an almost complete lack of data on the effects on human populations of long-term ingestion of natural toxicants in foods. At present, there is no firm evidence to demonstrate a link between common foods and any chronic human illness [64], and considerably less evidence supporting this than there is for the evidence that pesticide residues in food cause long-term health effects. Acute Poisonings There have been very few reported cases of acute pesticide poisoning from residues on food. In Singapore during December 1988, 110 people were treated for gastroenteritis after ingestion of a green leafy vegetable, known as gai-lan, that was contaminated with the organophosphates methamidophos and profenofos, and also dithiocarbamate. Based on the gastrointestinal systems of the patients and the depressed serum cholinesterase levels detected, methamidophos was assumed to be mainly responsible for the poisoning. Methamidophos is highly hazardous and has an LD50 in rats of 30 mg/kg body weight and its no-effect level (NEL) for cholinesterase activity in rates is 2 ppm. Profenophos is moderately hazardous with an LD50 of 350 mg/kg body weight. Its NEL is 0.38 ppm. In this case, human poisoning occurred after ingestion of approximately 0.56 mg of insecticide [65].

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In another case, California during July 1985 experienced the largest known outbreak of illness due to pesticide-contaminated food in the U.S.A. when the pesticide Aldicarb was illegally applied to watermelons. In that case, 1,370 illnesses were reported after the consumption of watermelons contaminated with the carbamate pesticide. Although no deaths occurred, some people were severely ill with possible life-threatening conditions: brachycardia, and hypotension. In addition, two stillbirths were reported in women who were pregnant at the time of their poisoning. Symptoms included vomiting, abdominal pain, blurred vision, loss of consciousness, seizures, and muscle twitching [19]. These cases illustrate the problems of assuring the safe use of potent pesticides. When inadvertent or illegal uses of a pesticide occur, there is no method of protecting the public from exposure. Mild symptoms are likely to be mistaken for those of unspecified gastroenteritis and the cause not investigated [19, 65]. The major problem in this area is establishing a scientifically proven link between pesticide exposure and the physical symptoms. Only where there has been relatively large-scale poisoning, requiring hospitalization of a large number of people, has a pesticide link been diagnosed. Lower-level exposures are unlikely to be detected and traced to pesticide exposure. It could be said that the vast majority of pesticide residue levels in food are relatively safe and are within legal limits. It is also true to say that some levels will continue to decrease, as people become aware of the adverse effects of certain pesticides and decline the usage of those chemicals. However, the effect of these low levels of pesticides is still poorly understood and may, for all we know, be contributing to long-term chronic health effects. REFERENCES 1. Melifrondes, I. D., and Kashouli-Kouppari, A. (1994). "Pesticide use and regulation in Cyprus." Reviews of Environmental Contamination and Toxicology, 134: 91-1,103. 2. British Medical Association (1990). Pesticides, Chemicals and Health. Report of the Board of Science and Education, October 1990. 3. Spynu, E. I. (1989). "Predicting pesticide residues to reduce crop contamination." Reviews of Environmental Contamination and Toxicology, 109: 89-107. 4. Barthel (1993). Pesticides and Cancer Risk, Z. Erkrank Atm-Org., 161: pp. 257-265. 5. Kashyap, R., Iyer, L. R., and Singh, M. (1994). "Evaluation of daily dietary intake of dichloro-diphenyl-trichloroethane (DDT) and benzene hexachloride (BHC) in India." Archives of Environmental Health, 49: 63-66. 6. United States Environmental Protection Agency (1990). Pesticide Exposure Study (NOPES), U.S. Environmental Protection Agency, Research Triangle Park, N.C.

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7. Lichtenberg, E., and Zilberman, D. (1986). "Problems of pesticide regulation: Health and environment versus food and fibre." In: Phipps, T. T., Crosson, P. R., and Price, K. A. (Eds). Agriculture and the Environment. Resources for the Future, Washington, D.C. pp. 123-145. 8. Hassall, K.A. (1990). The Biochemistry and Uses of Pesticides, 2nd edn, Macmillan Press Ltd, London. 9. Sly, J. M. (1977). "Changes in the use of pesticides since 1945." In: Perring, F. H., and Mellanby, K. (Eds.). Ecological Effects of Pesticides. Academic Press, London. 10. Pimental, D., McLaughlin, L., Zepp, A., Lakitan, B., Kraus, T., Kleinman, P., Vancini, P., Roach, W., Graap, E., Keeton, W., and Selig, G. (1991). "Environmental and economic impacts of reducing U.S. agricultural pesticide use." Handbook on Pest Management in Agriculture. Boca Raton, Florida: CRC Press, pp. 679-718. 11. Costa, L. G. (1986). "Toxicology of pesticides: A brief history." In: Costa, L. G., Galli, C. L., and Murphy, S. D. (Eds). Toxicology of Pesticides: Experimental, Clinical and Regulatory Perspectives. Springer-Verlag, Berlin, pp. 1-10. 12. Ekstrom, G., and Akerblom, M. (1990). "Pesticide management in food and water safety: International contributions and National approaches." Reviews of Environmental Contamination and Toxicology, 114: 23-55. 13. Beaumont, P. (1993). "GATT and pesticides in food." Chemistry and Industry, p. 424. 14. Jansma, J. E., van Keulen, H., and Zadoks, J. C. (1993). "Crop protection in the year 2000: a comparison of current policies towards agrochemical usage in four Western European countries." Crop Protection, 12: 483-489. 15. Lawson, H. M. (1994). "Changes in pesticide usage in the United Kingdom— policies, results and long term implications." Weed Technology, 8: 360-365. 16. Iwata, H., Tanabe, S., Sakai, N., Nishimura, A., and Tatsukawa, R. (1994). "Geographical distribution of persistent organochlorines in air, water and sediments from Asia and Oceania, and their implications for global redistribution from lower latitudes." fnv/ronm^nra/Pt^/to/ow, 85: 15-33. 17. Guidelines on Residue Trials (1981) Department of Primary Industry. Australian Government Publishing, Canberra. 18. BRR (1989). Report on National Residue Survey: 1987 and 1988 Results, Australian Government Publishing Service, Canberra. 19. Goldman L. R., Smith, D. F., Neutra, R. R., Saunders, L. D., Pond, E. M., Stratton, J., Walker, K., Jackson, R., and Kizer, K. (1990). "Pesticide food poisoning from contaminated watermelons in California 1985." Archives of Environmental Health, 45: 229-236. 20. AustraHan Science and Technology Council (ASTEC) (1989). Health, Politics and Trade. Controlling Chemical Residues in Agricultural Products. AGPS, Canberra.

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21. Americans For Safe Food (AFSF) (1988). The Wax Cover Up—What Consumers Aren 't Being Told about Pesticides in Fresh Produce. Center for Science in the Public Interest, Washington, D.C., U.S.A. 22. Americans For Safe Foods (1990). The Wax Cover Up. What Consumers Aren't Told about Pesticides on Fresh Produce. Washington, D.C. 23. Hayes, W. J. (1975). Toxicology of Pesticides. Waverley Press: Baltimore. 24. Precheur, R. J., Bennet, M. A., Ridel, R. M., Wiese, W. L., and Dudet, J. (1992). "Management of Fungicide Residues on Processing Tomatoes." Plant Diseases. 76: 700-702. 25. Eilrich, G. L. (1991). "Tracking the fate of residues from farm gate to the table." In: Tweedy, B. G., Dishburger, H. J., Ballantine, L. G., and McCarthy, J. (eds). "Pesticide Residues and Good Safety: a Harvest of Viewpoints." Am^ncaw Chemists' Society, Washington, D.C, pp. 202-212. 26. Winter, C. K. (1992). "Dietary pesticide risk assessment." Reviews of Environmental Contamination and Toxicology, 127: 23-67. 27. Elkins, E. R. (1989). "Effect of commercial processing on pesticide residues in selected fruit and vegetables." Journal of the Association of Official Analytical Chemists, 72: 533-535. 28. Food and Drug Administration (1994). Pesticide Program. Residue Monitoring 1994. 29. Chapman, P. J., and Mason, R. D. (1993). "British and European Community regulations and registration requirements for non-pesticidal co-formulants in pesticides and for adjuvants." Pesticide Science, 37: 167-171. 30. World Health Organization (WHO) (1990). "Principals for the Toxicological Assessment of Pesticide Residues in Food." (Publications of the World Health Organization: Finland.) 31. Loevinsohn, M. E. (1993). "Improving the pesticide regulation in the Third World: The role of the independent hazard auditor." Environmental Management, 17: 705-712. 32. Mbagwu, I. G., and Ita, E. O. (1994). "Pesticide use in the sub-humid zones of Nigeria: Implications for conservation of aquatic resources." Environmental Conservation, 21: 214-219. 33. Bajet, C. M., and Tejada, A. W. (1995). "Pesticide residues in the Philippines: an analytical perspective." Trends in analytical chemistry, 14: 430^34. 34. Pelfrene, A. F. (1986). "Pesticide use exposure and regulation in developed and developing countries." In. Costa, L. G., Galli, C. L., and Murphy, S. D. (Eds). Toxicology of Pesticides: Experimental, Clinical and Regulatory Perspectives. Springer-Verlag, Berlin, pp. 253-262. 35. Hayes, W. J. (1982). Pesticides Studied in Man. Baltimore, Williams and Wilkins, 1982. 36. Hallenbeck, W. H., and Cunningham-Burns, K. M. (1985). Pesticides and Human Health. (Springer-Verlag: New York.)

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37. Blair, D. (1989). "Uncertainties in Pesticide Risk Estimation and Consumer Concern." Nutrition Today, November/December 1989, pp. 13-19. 38. Martin, R. (1989). Health, Politics, Trade: Controlling Chemical Residues in Agricultural Products. Australian Government Publishing Service, Canberra. 39. Jerry, M. (1989) "Don't Downplay the Risk of Pesticides." Pest Control. 57 (10): 55. 40. The Parliament of the Commonwealth of Australia (POCA) (1990). Report of the Senate Select Committee on the Agricultural and Veterinary Chemicals in Australia. (Commonwealth government Printer: Canberra.) 41. Bureau of Rural Resources (BRR) (1992). Report on the National Residue Survey 1989-1990 Results (AGPS: Canberra). 42. Food and Drug Administration (1989). Residues in Food 1989. Food and Drug Administration Pesticide Program, Washington, D.C. 43. Wessel, J. R., and Yess, N. J. (1991). "Pesticide residues in foods imported into the United States." Reviews of Environmental Contamination and Toxicology, 120: 83-104. 44. Galal-Gorchev, H. (1991). "Dietary intake of pesticide residues, cadmium, mercury, and lead." Food Additives and Contaminants, 8: 793-806. 45. Herrera, A., Arino, A. A., Conchello, M. P., Lazaro, R., Bayarri, S., and Perez, C. (1994). "Organochlorine pesticide residues in Spanish meat of different species." Journal of Food Protection, 57: 441-444. 46. Leoni, V., Caricchia, A. M., Cremisini, C , Chiavarini, S., Fabiani, L., Morabito, R., Rodolico, S., and Vitali, M. (1995). "Levels of pesticide residues in food—evaluation of data from total diet studies in Italy." International Journal of Environmental Analytical Chemistry, 58: 411-422. 47. Trotter, W. J., and Dickerson, R. (1993). "Pesticide residues in composited milk collected through the United States pasteurised milk network." Journal of AOAC International, 76: 1,220-1,225. 48. Dogheim, S., Nasr, E., Almaz, M., and El-Tohamy, M. (1990). "Pesticide residues in milk and fish samples collected from two Egyptian govemorato." Jour. Assoc. Off. Anal. Chem., 73: 19-21. 49. Lenardon, A., Maitre de Hevia, M. I., and Enrique de Carbone, S. (1994). "Organochlorine pesticides in Argentinian butter." The Science of the Total Environment, 144: 273-277. 50. Smart, N. (1987). "Organophosphorous pesticide residues in fruits and vegetables in the United Kingdom and some other countries of the European Community since 1976." Reviews of Environmental Contamination and Toxicology, 98: 99-147. 51.Gianessi, L. P., and Greene, C. R. (1988). Vegetables and Specialties. Situation and Outlook Report. United States Department of Agriculture, pp. 27-41. 52. WHO/UNED (1989). Public health impact of pesticides used in agriculture. Geneva: World Health Organization/United Nations Environment Program.

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53. National Academy of Sciences (1987), Regulating Pesticides in Food, The Delaine Paradox, Washington, D.C., National Academy Press, 272 pp. 54. Gots, R. E. (1992). Toxic Risks: Science, Regulation and Perception. Lewis Publishers: Florida. 55. Ames, B. N., Magaw, R., Gold, L. S. (1987). "Ranking possible carcinogenic hazards." Science, 236: 271-280. 56. Ames, B. N., and Gold, L. S. (1989). "Pesticides, risks and applesauce." Science, 244: 755-757. 57. Culliney, T. W., Pimentel, D., Pimentel, M. H. (1992). "Pesticides and Natural Toxicants in Foods, Agriculture, Ecosystems and Environment," 41:297-320. 58. Goh, K. T., Yew, F. S., Ong, K. H., Tan, I. K. (1990). "Acute organophosphorus food poisoning caused by contaminated green leafy vegetables." Archives of Environmental Health, 45: 180-184.

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CHAPTER 21 BIOLOGICAL MONITORING BY MEANS OF URINARY SAMPLES AND PROBLEMS CONCERNING CONCENTRATION-DILUTION OF SPOT URINE Andrea Trevisan Istituto di Medicina del Lavoro Universita di Padova Padova, Italy

CONTENTS INTRODUCTION, 453 UNADJUSTED URINE, 454 ADJUSTMENT FOR OSMOLALITY, 454 ADJUSTMENT FOR SPECIFIC GRAVITY, 455 ADJUSTMENT FOR CREATININE, 456 OTHER TYPES OF ADJUSTMENTS, 456 CONCLUDING REMARKS, 457 REFERENCES, 458 INTRODUCTION Urinary sampling is a method utilized to detect occupational xenobiotic exposure and to determine damages of the nephron caused by diseases, drugs, poisons, or industrial substances. Commonly, 24-hour collection of urine is of clinical use, but this type of collection is subjected to practical and scientific problems during biological monitoring in an industrial environment. The practical restriction is that a careful collection is often difficult even during hospitalization, and much more so on the job or during leisure time. The scientific restriction is that metabolites are frequently excreted during and immediately after the end of exposure to xenobiotics; thus, prolonged collection period intervals could cause a dilution of the samples with urine not containing the xenobiotic and therefore underestimate its true value. For these reasons, long time collection is not reliable during biological monitoring. Other timed collections (4, 8, 12 hours) show similar problems; in addition, a too-short period could cause problems related to an overestimation of the values. Another common difficulty is the precise control of the time-related volume. The type of urine collection that appears to be most suitable to monitor work exposure and possible effects of industrial substances on the kidney is urine spot sampling. Obviously, this method presents the problem of concentration-dilution of urine, and four practical solutions were suggested:

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1) unadjusted 2) adjustment 3) adjustment 4) adjustment

urine; for osmolality; for specific gravity; for creatinine concentration excretion.

These, in addition to other types of adjustment requiring a short period of collection, are reviewed in this chapter. UNADJUSTED URINE Some authors support unadjustment of the urinary values during biological monitoring, justifying that a rough concentration factor, such as specific gravity, had been multiplied by a precise analytical measurement [1]. Other following studies [2-4], regarding creatinine adjustment also, agree against urine adjustment for concentration-dilution factors. The main argumentation supporting this choice is that no significant difference was observed among correlation between spot samples unadjusted, adjusted for specific gravity or creatinine, and values measured in 24-hour collected urine [3, 5]; thus, no practical advantage is offered by adjusted values in biological monitoring. ADJUSTMENT FOR OSMOLALITY Osmolality measures one of the colligative properties of water and depends on the number of particles of solutes in solution. Its unit of measure is mOsmoles for kg of water (mOsm/kg H2O): 1 mOsm/kg H2O means 6 x 10^^ particles of solutes are in one kg of water (1/1,000 of Avogadro's number). Sodium, the main extracellular cation, along with accompanying anions are the determinants of osmolality. In normal subjects, the value of osmolality in plasma is between 285-295 mOsm/kg H2O, with slight interindividual differences. On the contrary, depending on water consumption, urine osmolality shows high inter- and intraindividual differences, with values that range from less than 100 mOsm/kg H2O (high diluted urine), to urine that is four times the concentration of plasma with values higher than 1,000 mOsm/kg H2O (very concentrated urine). The maximum urine concentration capacity of the kidney is about 1,200 mOsm/kg H2O; this value is obtained when water consumption is limited to 0.5 1/day. Therefore, osmolality is a good parameter by which to measure concentration-dilution of urine, but, as with specific gravity (detailed in the next section), it is dependent on sodium and other solutes. Diseases that cause a variation of solute excretion, water restriction, or water loading, or an increase of sweat during work may modify osmolality. The use of osmolality adjustment is rare in biological monitoring and is used only for comparison with other methods of adjustment. In healthy subjects, an

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almost perfect correlation between adjusted values for osmolality and for specific gravity was found [6]; this was not unexpected in that urinary excretion did not contain abnormal concentrations of proteins or sugar. The osmolality is measured by the freezing point depression method with an osmometer. ADJUSTMENT FOR SPECIFIC GRAVITY Specific gravity (relative density or density number) of a substance is the result of a ratio of its density to the density of a reference substance under conditions that must be stated for both. Commonly, the reference substance for liquids and sohds is water. Sodium chloride, urea, sulfate, and phosphate [7] are the main contributors to specific gravity. Urea contributes about 20%, and phosphate and sulfate account for another 20%. Wide variations of urinary specific gravity were observed in a healthy population (coefficient of variation ranging from 5.8% to 13%) submitted to 24-hour and consecutive urine collections [8]. According to these observations, a reference value for specific gravity is difficult to define. In the United Kingdom, a mean value of the standard population is 1.016, whereas in the United States this value is 1.024 [9]. These authors [9] observed that the urine's solid content is roughly proportional to the specific gravity of 1.024. In an Italian population of 333 subjects, the average specific gravity, using a 24-hour collection period, was 1.024, whereas in 1,079 spot specimens this value was 1.027 [10]. These differences agree with differences in salt, protein, and fluid intake and ambient temperature. As with osmolality, specific gravity may be influenced by dysfunction of kidney tubules with impairment of sodium, phosphate, and protein reabsorption. In these subjects, correction for specific gravity during biological monitoring seems inadequate. This is true particularly for workers exposed to substances that are toxic to the renal tubules. A standard specific gravity is needed before this type of adjustment can be made. Commonly, a reference specific gravity of 1.024 is often used, but this, or other choices such as 1.016, must always be specifically expressed with the results. According to Elkins et al. [11] the correction is made as follows: unadjusted value x 0.024 ^ Unit of measure (^ig, mg, g or others)/liter (L) specific gravity of sample - 1.000 adjusted for a specific gravity standard of 1.024

It is common practice to reject over-diluted (specific gravity lower than 1.010) or over-concentrated (specific gravity higher than 1.030) urine samples. Recent research [10] supports a range in spot specimens between 1.010 and 1.035 because in 1,079 spot samples analyzed by the Laboratory of Industrial Toxicology of the Institute of Occupational Health of Padua University Medical School

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(Italy), a high percentage (almost 40%) of urine samples showed specific gravity values higher than 1.030. If the acceptable range is increased to 1.035, only 6% of the values exceed the upper limit. In addition, specific gravity of 1.035 corresponds to a creatinine concentration of about 3 g/1, the suggested upper limit for this urinary parameter (detailed in the next section). Commonly, specific gravity is measured by means of a difractometer. ADJUSTMENT FOR CREATININE Folin was the first [12] to report that creatinine output during a 24-hour period was constant. On the contrary, following studies showed that daily excretion may change significantly not only in different subjects but in the same subject from day to day [13]. Creatinine excretion is related to body muscle mass and muscular activity; creatinine output is also affected by ingestion of cooked meats, but there are fewer dietary factors that affect excretion of creatinine compared to specific gravity. In 24-hour specimens, an average creatinine concentration of 1.25 g/1 was found, and the relationship with specific gravity of 1.024, calculated from the correlation equation, was 0.88 g/1 [10]. The choice of creatinine was related to its high concentration in urine and to the hypothesis that it was filtered by the glomerulus and not reabsorbed or secreted by the tubule. This conviction was corrected by the observation that a small portion of creatinine is added to the urine by active tubular secretion. Measurement of creatinine is based on Jaffe's alkaline-picrate reaction. Usually, unadjusted results are related to g (or nmioles) of creatinine; one gram of creatinine corresponds to 8.8 nmioles (molecular weight (WB) of creatinine is 113.07). Biological Threshold Limit Values (BTLVs) of substances and metabolites in urine are conventionally expressed adjusted for creatinine, as indicated in ACGIH publications [14] and by Lauwerys and Hoet [15]. As for specific gravity, urine with a creatinine concentration lower than 0.5 g/1 (4.4 mmoles/1) or higher than 3 g/1 (26.5 mmoles/1) should be discarded and the collection repeated. More than 95% of spot samples collected in our laboratory [10] show a creatinine concentration within this range. Furthermore, following observations [16] show that adjustment for creatinine is justified in a wide range of concentrations (too diluted or concentrated urine). OTHER TYPES OF ADJUSTMENTS Following early observation by Araki [17] that some urinary indices were influenced by urinary volume, an equation was introduced to eliminate the effects of urinary volume on urinary concentration [18]. This equation is as follows: U(V-i) = UixVl'

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where:

U(v=i) = concentration adjusted to one ml urinary volume per minute; Ui = urinary concentration of the substance examined; Vi = urinary volume (ml/min); b = slope of regression coefficient of the relationship between urinary volume and urinary concentrations in each subject. This adjustment needs a timed collection of urine and a preliminary evaluation of "b" values for the measured substances, but the urinary volume-adjustment concentration is applicable to virtually all urinary substances within a wide variation in urinary volume [19]. In addition, concentration adjusted to urinary flow rate is independent by definition of urinary flow; therefore, this adjustment is applicable for highly diluted and highly concentrated urine samples without repetition of urine collection [20]. More recently, according to observations that creatinine elimination is not constant, adjustment for creatinine related to urinary flow was introduced [21]. The estimated slope (b) of the log-log dependence of creatinine excretion rate on urinary flow is 0.67 ± 0.07 standard error of the mean (SEM), and this value was used as an exponent for urinary volume to correct the measured concentrations to a standardized volume. Obviously, this type of adjustment required a timed, although short (60-90 minutes), collection. The following formula defines the urinary flow adjusted creatinine ratio: mg substance

1000 x substance concentration ^ x flow ^ ^^^

g creatinine

creatinine concentration ^^

Customarily, units are chosen as milligrams of substance per gram of creatinine (which requires multiplying the concentration ratio by 1,000). Substance concentrationM is the crude substance concentration (in mg/dl), and creatinine concentratiouM the measured creatinine concentration (in mg/dl). Flow^i^^ is urinary flow (subscript M designates the measured volume) in ml/min, and superscript 0.67 is slope (b). CONCLUDING REMARKS Discussions about urinary values of xenobiotic substances measured in biological monitoring may often be academic. Omitting long-time collection for the known reasons, short-time collections are also difficult to obtain during or immediately after the work shift. Urinary volume adjustment or urinary flow-adjusted creatinine ratio are also difficult to apply, requiring timed collection and appHcation of complicated mathematical calculations.

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Health and Toxicology Table 1 Intra-individual coefficients of variation of urinary values

Within day Between day

Creatinine

Specific gravity

Osmolality

Unadjusted

22.4 15^6

32.2 22^0

36.5 —

47.3 37.3

Results are drawn from [22].

A spot urine sample offers the only practical alternative in biological monitoring, and we agree with Pryde [22] that these are unacceptable without some adjustment for short-term concentration-dilution effects because of the wide variation in urine output from hour to hour. Creatinine correction reduces uncertainty of a spot urinary concentration, reflecting accurately the true substance excretion in an individual subject. Although the majority of researchers [4, 5, 16] do not find great differences in the relationship between values of unadjusted spot samples, samples adjusted for creatinine or specific gravity and 24-hour samples, adjustment for creatinine, and to a lesser extent for specific gravity, reduces scatter of the results and intraindividual coefficients of variation [23], as summarized in Table 1. Moreover, to evaluate individual exposure, unadjusted urine appears inadequate because in occupational medicine the aim is the safety of the workers and the reduction of the exposure within acceptable limits. Thus, a single unadjusted value, influenced by concentration-dilution of urine, could be underestimated or overestimated, and could cause failure of prevention. In conclusion, adjustment of urinary values in biological monitoring is needed for the explained reasons. Adjustment for creatinine appears more satisfactory in that it yields results in subjects with altered renal function, a situation that instead influences specific gravity and osmolality. Furthermore, adjustment for creatinine appears to offer less intraindividual variations. REFERENCES 1. R. J. Graul and R. L. Stanley, "Specific gravity adjustment of urine analysis results," Am. Ind. Hyg. Assoc. J. 43, 863 (1982). 2. C. N. Ong, B. L. Lee, S. C. Foo, H. Y. Ong, and L. H. Chua, "Specific gravity adjustment for urinary analysis of 6-aminolevulinic acid," Am. Ind. Hyg. A^^oc.y. 4(5, BIO (1985). 3. A. Berlin, L. Alessio, G. Sesana, A. Dell'Orto, and I. Ghezzi, "Problems concerning usefulness of adjustment of urinary cadmium for creatinine and specific gravity," Int. Arch. Occup. Environ. Health 55, 107 (1985).

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4. A. DeirOrto, A. Berlin, F. Toffoletto, B. Losito, and L. Alessio, "Creatinine and specific gravity adjustment of ALA in urinary spot samples: Is there any need?," Am. Ind. Hyg. Assoc. J. 48, A331 (1987). 5. L. Alessio, A. Berlin, A. Dell'Orto, F. Toffoletto, and I. Ghezzi, "Reliability of urinary creatinine as a parameter used to adjust values of urinary biological indicators," Int. Arch. Occup. Environ. Health 55, 99 (1985). 6. T. E. Barber and G. Wallis, "Correction of urinary mercury concentration by specific gravity, osmolality, and creatinine," J. Occup. Med. 28, 354 (1986). 7. J. W. Price, M. Miller, and J. M. Hayman, "The relation of specific gravity to composition and total solids in normal human urine," J. Clin. Invest. 19, 537 (1940). 8. H. Buchwald, "The expression of urine analysis results-observations on the use of a specific gravity correction," An/i. Occup. Hyg. 7, 125 (1964). 9. L. Levine and J. P. Fahy, "Evaluation of urinary lead determinations. I. The significance of the specific gravity," J. Ind. Hyg. Toxicol. 27, 217 (1945). 10. A. Trevisan, "Concentration adjustment of spot samples in analysis of urinary xenobiotic metabolites," Am. /. Ind. Med. 17, 637 (1990). 11. H. B. Elkins, L. D. Pagnotto, and H. L. Smith, "Concentration adjustments in urinalysis," Am. Ind. Hyg. Assoc. J. 35, 559 (1974). 12. O. Folin, "Approximately complete analyses of thirty 'normal' urines," Am. J. Physiol. 13, 45 (1905). 13. G. Curtis and M. Fogel, "Creatinine excretion: Diurnal variation and variability of whole and part day measures," Psychosom. Med. 32, 337 (1970). 14. Threshold Limit Values and Biological Exposure Indices. ACGIH 1994-95. 15. R. R. Lauwerys and P. Hoet, Industrial Chemical Exposure. Guidelines for Biological Monitoring, 2nd ed., Lewis Publishers, 1993. 16. A. Trevisan, G. Nicoletto, S. Maso, G. Grandesso, A. Odynets, and L. Secondin, "Biological monitoring of cadmium exposure: Reliability of spot urine samples," Int. Arch. Occup. Environ. Health 65, 373 (1994). 17. S. Araki, "The effects of water restriction and water loading on urinary excretion of lead, 6-aminolevulinic acid and coproporphyrin," Br. J. Ind. M^J. 55, 312(1978). 18. S. Araki, "Effects of urinary volume on urinary concentrations of lead, 6aminolevulinic acid, coproporphyrin, creatinine, and total solutes," Br. J. Ind. Med. 37, 50 (1980). 19. S. Araki, H. Aono, and K. Murata, "Adjustment of urinary concentration to urinary volume in relation to erythrocyte and plasma concentrations: An evaluation of urinary heavy metals and organic substances," Arch. Environ. Health 41, 171(1986). 20. S. Araki, F. Sata, and K. Murata, "Adjustment for urinary flow rate: an improved approach to biological monitoring," Int. Arch. Occup. Environ. Health 62, 471(1990).

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21. G. N. Greenberg and R. J. Levine, "Urinary creatinine excretion is not stable: A new method for assessing urinary toxic substance concentrations," /. Occup. Med. 31, 832 (1989). 22. D. E. Pryde, "Is it appropriate to adjust the results of urine analyses for concentration-dilution effects?," International Workshop on Biological Indicators of Cadmium Exposure: Diagnostic and Analytical Reliability, CEC-IUPAC, Luxembourg, 7-9 July 1982. 23. H. J. Mason and I. M. Calder, "The correction of urinary mercury concentrations in untimed, random urine samples," Occup. Environ. Health 51, 287 (1994).

CHAPTER 22 CAUSES OF ARTIFACTS IN SORPTION STUDIES WITH TRACE ELEMENTS Lorenzino Giusti School of the Environment, Sunderland University, Benedict Building, St. George's Square, Sunderland, SR2 7BW, UK CONTENTS INTRODUCTION, 461 SORPTION STUDIES: THEORETICAL BACKGROUND, 462 ARTIFACTS IN SORPTION STUDIES, 466 Artifacts caused by contamination, 466 Artifacts caused by separation techniques and type of sorbent material, 466 Artifacts associated with the use of voltammetric methods, 473 Artifacts caused by the use of radioactive tracers, 474 Artifacts caused by incorrect data manipulation, 475 SUMMARY, 481 REFERENCES, 485 INTRODUCTION The interactions between solid phases and trace elements dissolved in a liquid phase have been the object of investigation by scientists working in very diverse disciplines. Examples of fields in which sorption plays an important role include the following: the leaching of elements from the lithosphere and their transport and deposition in aquatic environments, pedogenetic processes, the transfer of nutrients from soil to vegetation, and more in general the uptake of substances by living organisms; transmembrane ion fluxes in patients with a number of physical and mental disorders. Environmental chemists, ecotoxicologists, and water engineers need to investigate the partitioning of trace elements between solution and particulate material as this will affect their bioavailability, toxicity, and the methods to be used in the treatment of water intended for domestic and industrial uses. Nuclear scientists are interested in the sorption mechanisms taking place between radionuclides and soil particles, sediments, suspended material in aquatic environments, living cells, humic substances, clays, and materials used to encapsulate nuclear waste. Waste disposal engineers have to ensure the efficient sequestration by natural or synthetic sorbents of potentially toxic pollutants that may contaminate groundwater. The reliability of the data obtained from sorption experiments and related computer simulations must be carefully assessed in order to rule out the possibility that

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some of the results may be attributed to experimental artifacts. Some of these problems have been described in the literature [1-5]. The purpose of this chapter is to describe the main sources of these and to review the methods used for their detection and correction. SORPTION STUDIES: THEORETICAL BACKGROUND The aim of this section is to describe the basic principles relating to the interactions between particulate material and trace elements, and to summarize the terminology commonly used in sorption studies. Sorption processes such as adsorption, absorption, ion exchange, and surface precipitation are interactions between solutes (sorbates) and sorbing phases (sorbents). Figure 1 illustrates the main categories of sorption. The term adsorption refers to the reactions of a solute with surface functional groups of sorbing phases (sorbents) such as living or dead organic matter; humic and fulvic acids; aluminosilicates (especially clays); iron-, manganese-, and aluminium oxyhydroxides; and sulphides. In this process, the solute (sorbate) accumulates on or near the surface of the sorbent. Depending on the prevailing type of attractive forces [6], adsorption can be physical (involving polar molecules), chemical (chemisorption, arising from hydrogen and covalent bonds), and electrostatic (due to ion-ion and ion-dipole forces). The term absorption is used to describe the actual penetration of the solute into the sorbent. Ion exchange involves the replacement of an ion in a solid phase by another ion present in the solute. When the solute concentration near the surface of the sorbent is relatively high, the solute forms a precipitate onto the surface, i.e., a new solid phase; in this case the interaction between the different phases is called precipitation. The uptake of metal ions and other elements or compounds by living cells can be achieved in a number of ways depending on the level of specialization of the cell itself [7]. The terminology used by biochemists and clinical pharmacologists to describe the possible interactions of metals with living tissue is thus more complex. The term ligand refers to any substance which binds to a specific binding site, and the term binding site is normally used for any molecule of the cell membrane which interacts with the ligand. The behavior of trace elements in biogeochemical cycles depends on two main factors: (i) the chemical species in which they are present in the system, and (ii) the interactions of trace metals with dissolved complex formers and with particulate matter. Although metal complexation by organic compounds and inorganic ligands in natural water keeps a significant fraction of metals in solution [8-14], a number of studies has indicated that an important fraction of trace elements is associated with suspended particles and sediments [15-17]. According to Martin and Meybeck [18], more than 95% of heavy metals in freshwater is transported in the form of

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463

(a)

Solid particlej

ABSORPTION

(b)

Figure 1. Simplified models representing three sorption mechanisms: (a) adsorption, in which the solute (Pb) reacts with the surface of the solid particle (sorbent); (b) absorption, characterized by the penetration of the solute Into the particle; and (c) ion exchange, which results in the replacement, both on the surface and inside the sorbent phase, of one ion (Zn in this case) by another (Pb).

particulate material. In aqueous environments, the distribution of metals between natural water and suspended particles (inorganic and organic) depends mostly on i) the affinity of metals for solute ligands and solid particles, ii) particle coagulation and sedimentation rates, iii) the seasonal composition of settling particles, iv) pH conditions, and v) the ratio of metal concentration to suspended particle concentration. Fe- and Mn-oxides are known to be important scavengers in lake water, sea water, and soils [19-21]. Organic matter, in the form of phytoplankton and humic substances, is a major carrier of metal ions [22-25]. Carbonates and aluminosilicates appear to be less efficient in removing metals from surface water [26].

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Suspended and settling particles tend to agglomerate and form heterogeneous particles. In natural water, inorganic particles are normally coated with humic substances and other organic material [27, 28]. As there is no completely successful method for the determination of the metal fraction bound to each type of carrier phase, model substances (especially hydrous metal oxides) have been used in many laboratories to mimic components of the natural particles [29-33]. Useful information concerning the adsorption/desorption mechanisms of trace metals have been obtained from these experiments. The fewer studies carried out with natural particles [19, 22, 24, 34-37] have confirmed that the sorption models developed for artificial phases may be applicable to more complex natural mixtures of particles. However, longer metal equilibration times have been reported in natural settings than in experiments with pure metal oxides [37], the difference being ascribed to biological activity or considered to be a result of the use of more realistic particle concentration. Also, the metal binding capacity of cell surfaces has been found to be orders of magnitude larger than for metal oxides [22, 24]. The effect of the simultaneous presence of natural concentrations of mineral surfaces and humic substances has only rarely been investigated [36, 38, 39] despite the fact that humic-metal interactions may modify the pH dependency and kinetics of the metal adsorption. Many adsorption studies failed to take into account particle residence times. The reported conclusions may thus be valid only for systems where the particle residence time is long with respect to reaction half-life. Normally, adsorption of trace metals onto particulates increases from nearly 0% to almost 100% as the pH increases. By plotting the adsorption data as a function of pH, a typical S-shaped curve is obtained, usually referred to as the adsorption (or binding) curve. The pH range over which absorption increases from undetectable levels to almost 100% is called the "adsorption edge." The position of the adsorption edge on the pH scale is an indication of the degree of affinity of the metal for the binding sites on the surface of the particulate phase(s). Figure 2 summarizes the typical adsorption trends reported for some trace metals. The following generalizations can be drawn from the available information: (i) metal binding to particles increases with increasing pH, (ii) increased adsorption (at a given pH) is observed with increasing particle concentration, reaction time, and temperature, (iii) decreased adsorption occurs with increasing metal concentrations, and increasing competing cations. The latter decrease the available binding sites. Some anions (e.g., sulphate) appear to promote adsorption of metals [30]. In experiments with synthetic and natural samples (sorbents and sorbates), metal desorption processes can be detected, especially in media of relatively high pH.

Figure 2. Diagrams showing the general sorption trends of metals onto sorbent phases. The percent sorption onto a constant amount of particulate material increases with increasing pH (all diagrams), decreases with increasing metal concentration (a), and competing cations (b), and increases at higher temperature (c), longer reaction time (d). Assuming other parameters are constant, increasing particle concentration has the effect of increasing fractional sorption of the metal in solution (e).

466

Health and Toxicology

ARTIFACTS IN SORPTION STUDIES Artifacts Caused by Contamination Contamination may affect both accuracy and precision of analytical results. It may occur during sampling, transport, storage, sample preparation, or during analysis in the laboratory. Sampling methods, sampling equipment, containers, labware, or dust may introduce contamination in trace and ultra-trace analysis. The addition of stabilizing reagents and buffers can also be responsible for significant levels of contamination. Routine quality controls and interlaboratory calibrations are a must to minimize potential errors. Proper selection and maintenance of sampling equipment and labware, clean-air facilities, and routine blank determinations (partialmethod and total-method blanks) are likely to result in a considerable reduction and correction of bias associated with contamination. In trace-element adsorption studies, particular care is necessary when physical separation of sorbent phases from the incubating medium is required. Contamination at this stage and in the subsequent steps before analysis is quite common given the small amounts of analyte normally involved and the contaminants that can be released by filtration apparatus. In addition to causing changes to the physico-chemical speciation of the trace elements, preconcentration procedures on natural samples are likely to result in high blanks. With the exception of cases of extremely polluted environments, the concentration of trace elements of environmental samples is usually quite low (often smaller than 10"^ to 10"^ mol). Similarly, synthetic solutions containing extremely low concentrations of metals are required in simulated sorption studies designed to mimic environmental conditions. Contamination and adsorption during storage and separation procedures must thus be minimized. Useful reviews of recommended techniques have been published [40-42]. Many workers avoided the problems associated with contamination by setting up experimental conditions in which the solute under investigation and/or the sorbent phase(s) were present in high or very high amounts. Consequently, contamination from lab apparatus and sorption onto containers and filters became unimportant. The main shortcoming of the data gathered from these experiments is their limited applicability to natural settings. Artifacts Caused by Separation Techniques and Type of Sorbent Material Filtration and Ultrafiltration Filtration and ultrafiltration (i.e., filtration with membranes of nominal pore size smaller than 15 nm) will inevitably affect the concentration and speciation of trace metals in filtered solutions. Important fractions of trace elements are normally associated with particulate and colloidal material so that the size discrimination of filters and ultrafilters will vary depending on pore size and material used. Filter membranes with different nominal pore size will give filtrate residues containing different amounts, size fractions, and variety of sorbing particles. Clogging of

Causes of Artifacts in Sorption Studies with Trace Elements

467

filters arid ultrafilters may cause adsorption of trace metals onto the material retained by the membranes [43]. Large fractions of the trace metals adsorbed onto suspended particles in aquatic systems are likely to be associated to colloidal-sized particles (< l|Lim) as a result of their large surface area. As colloids can pass through the pores (0.45|im, 0.2|Lim, 0.1 ^im) of filter membranes conventionally used to separate the "dissolved" fractions of trace metals, binding experiments that make use of filtration may be affected by large errors, unless a strict experimental protocol including a number of control and blank determinations is followed [4]. Values of partition coefficients (K^, see below) have been found to decrease with increasingly high concentration of suspended soHds [44, 45]. One of the possible explanations of this trend is improper phase separation with membrane filters (e.g., increasing concentrations of colloidal particles passing through the filter pores that are accounted for as "dissolved" fraction). The lapse of time between collection of natural samples and execution of an experiment must be taken into consideration because coagulation of colloids into larger particles is bound to cause errors of size fractionation of the particles in suspension and thus bias the final partitioning of trace metals between "dissolved" and "particulate" fractions. An example of this problem is summarized in Table 1, which shows the variations in Fe concentration in natural water samples collected from the anoxic hypolimnion of a lake. These variations are a direct result of the filtration/centrifugation of the water, of the oxidation of the samples (Fe^"^ -^ Fe^"^) and a function of the time lag between sampling and analysis. The term "colloidal pumping" [46] describes the gradual accumulation of trace metals into increasingly larger particles forming as a result of aggregation of colloids in natural aquatic environments. Artifacts caused by changes of chemical/physical properties of natural particles prior to filtration can be minimized by carrying out the filtration immediately after Table 1 Changes in Fe concentration (MQ/L) in anoxic water samples from Esthwaite Water, Lake District, Cumbria, UK (n = 3) Sample treatment

Not filtered/Not centrifuged Filtered (0.45^im)/Not centrifuged Not filtered/Not centrifuged Filtered (0.45|Lim)/Not centrifuged Not filtered/Centrifuged Not filtered/Centrifuged Not filtered/Centrifuged Not filtered/Centrifuged

Fe concentration {\ig/L)

311.5 267.4 89.2 85.5 190.4 171.1 149.7 60.8

(5.7) (3.2) (4.3) (0.6) (1.2) (1.7) (2.5) (1.7)

Time of analysis

2h 2h 2d 2d 2h Id 2d 28d

after after after after after after after after

collection collection collection collection collection centrifugation centrifugation centrifugation

468

Health and Toxicology

sampling. During filtration, a relatively low flow rate and stirring may reduce the problems of particle coagulation and sorption by the filters [47]. Coagulation and settling of particles during sorption experiments are also important problems that can be partially offset by gentle stirring and sonication. Filtration can thus cause a number of artifacts resulting in apparent increased or decreased metal concentrations. Figure 3 shows the concentration of Al detected in samples of surface water filtered with different cellulose membranes. In this case dialysis was more effective, though more time-consuming, in removing colloidal and larger particles. In general, the smaller the nominal pore size of the filter membrane the smaller the Al concentration in the "dissolved" fraction. Samples from sites 7 and 8 did not follow this trend, probably due to their high concentration in humic substances and other organic material. Living or dead cells in suspension may break during filtration, especially when excessive positive or negative pressure is applied, and release a number of elements and organic compounds that affect the behavior of trace metals. Filters, ultrafilters, and dialysis membranes should normally be decontaminated with diluted nitric acid and later rinsed in distilled deionized water. In order to prevent losses of trace elements, apparatus and filters may also need to be preconditioned with solutions such as NaN03 or Ca(N03)2. Figure 4 shows a reduction in Pb losses at pH 6.3 when a 0.005 M NaN03 solution is used instead of Milli-Q water.

300

z o p

<

200

• NOT FILTERED H FILTERED (0.45 m) 0 FILTERED (0.2 /7m) ID DIALYSED (MWCO = 1000)

Q:

IZ

LLi

U

z o u

100

SAMPLE NUMBER Figure 3. Typical Al concentrations detected in "dissolved" fractions obtained using different filters for water samples collected at eight sites of a typical moorland stream in Galloway (southwest Scotland, UK). Twelve water samples were collected at each site: three were not filtered, six were filtered with Millipore cellulose filters (three with pore size of 0.45pm, three with pore size of 0.2Mm), and the remaining three samples were dialyzed for 24h with Spectra/Por 7 cellulosic membrane (MWCO = 1,000).

Causes of Artifacts in Sorption Studies with Trace Elements

469

40 -<5

NaNOS (n = 3)

30

=1.

(b)

MQ(n = 3)

20 + -0

CL

^—

lOi pH = 6.3 0

20

40

60

80

100

120

TIME (min) 40

30

(a)

lfrh-+-4^ MQ(n = 3)

n a.

NaN03 (n = 3)

20

10 pH = 4.0 20

40

60

80

100

120

TIME (min) Figure 4. Comparison of metal (Pb) losses to container walls and filters in two different media (Milli-Q water and 0.005M NaNOa) over a period of 2h at pH 4.0 (a) and pH 6.3 (b). Metal losses were lower in the NaNOs solution. Lab apparatus was previously rinsed with either Milli-Q water or 0.005M NaNOa (n = number of experiments).

Trace metal adsorptive losses to Nuclepore polycarbonate filters were found to be negligible [48] or, when significant, usually lower than those caused by cellulose triacetate filters. These findings apply to synthetic solutions and samples of fresh water. Polycarbonate filters, however, adsorb humic substances, and mercury from sea water [49, 50]. Teflon (PTFE) filters have been recommended by some authors [50, 51]. Florence [52] found no Cu, Pb, Cd, or Zn losses during the filtration of natural waters with an all-glass Millipore apparatus. However, adsorptive losses of metals onto glass filtration apparatus were reported for synthetic solutions [53] and

470

Health and Toxicology

for sea water [54, 55]. DeMora and Harrison [41] reviewed the problems associated with the use of physical separation techniques in speciation studies. A study carried out by Lalande et al. [56] has shown that it is not always necessary to filter samples of natural water after collection. No significant differences were observed for Ca, Mg, K, NO3, SO4, F and monomeric Al. In their experiments, the concentration of total Al decreased with decreasing pore size of filter media. While most laboratories filter their samples on a routine basis, others have decided to resort to filtering only when the water is not clear [57-59]. Dialysis Even though not always very efficient, this technique has been used successfully in the separation of dissolved material from colloidal particles [60]. Unfortunately, dialysis techniques require relatively long equilibration times of at least 24 hours [61, 62], whereas studies of adsorption kinetics require measurements at considerably shorter time intervals (seconds or minutes). In addition, the negative charges present on the surface of the dialysis membrane do not allow all ions and molecules to diffuse through the membrane at the same rate [63]. Nonspecific binding of ligands to the dialysis membrane is a possible source of artifacts and can be corrected for with routine blank determinations. Centrifugation Centrifugation allows the separation of particulate material of different size and density by selecting appropriate rotation rates and times. Continuous flow centrifugation over a period of 12-24 hours at rotor speed in the range of 15,000-40,000 rpm can result in the efficient removal of particles and organic complexes with diameters significantly lower than those observed in conventional filtration techniques. This method is normally used to recover humic substances from natural water [64, 65]. Unfortunately, sorption experiments carried out with the residue collected with this method may be affected by changes of the physical and chemical properties of the recovered material as high rotation rates tend to cause particle agglomeration and compaction (Figure 5). Centrifugation of unstable natural solutions poses additional problems. For example, the separation of suspended particles in water samples collected in anoxic environments (e.g., groundwater, the hypoUmnion of lakes, soilwater from peat bogs) is quite difficult as oxidation of the water samples will cause neoformation of particles such as Fe oxyhydroxides. An example of this process is shown in Table 1. Centrifugation is routinely used by biologists and pharmacologists to separate free ligands from ligands bound to biological tissue. The main disadvantage of this technique is the incomplete separation of the free ligand trapped between the particles. This is an important drawback especially when very low concentrations of trace element and radioactive tracers are used in the experiments.

0

E 8 v)

r

; i '

5

...

b

2

; Figure 5. Amounts of (a) Fe, (b) Cu, (c) Pb, and (d) Zn released in solution (500 mi of Milli-Q or 0.005M NaN03)after 7d by identical amounts (25mg/L) of particulate material recovered either by centrifugation (Beckman J2-21, rotor speed 15,000 rpm) or after natural particle settling. Two samples of lOOL of water each were collected from the hypolimnion of Esthwaite Water, Cumbria, UK. Solids obtained by centrifugation were morphologically different (i.e., more scaly and compact) than those obtained by undisturbed settling in the plastic containers. Higher concentrations of Fe, Cu, Pb, and Zn were found in all solutions containing centrifuged particles. The data from five replicates were used to calculate the standard deviation.

f

472

Health and Toxicology

Field-Flow Fractionation (FFF) This separation technique is similar to Hquid chromatography. Samples suspended in carrier solutions are separated in ribbon-shaped channels of about 0.2 mm thickness. Centrifugation creates a gravitational field that separates the particles according to their size and density. The size of the particle fractions is determined by means of a UV detector. Colloids, biological material, and polymers have been successfully characterized by this technique [66-69]. Important applications of this technique are (i) the separation of particles in the size range of 0.05-100 ^im from natural water, (ii) the determination of the trace metal concentrations associated with different colloidal size fractions, and (iii) the determination of the molecular weight of fulvic and humic acids. The use of this technique has been quite limited thus far, and it will take some time before possible artifacts are reported. The main disadvantage of FFF in sorption studies is the need of very sensitive equipment and ultraclean laboratories capable to cope with the very small mass of the samples separated. Chromatographic Techniques Metal-organic interactions in marine organisms, in natural waters, and in sewage effluents have been carried out by gel filtration chromatography [70, 71]. Details of GFC are given in a number of publications [41, 72-76]. Porous polymeric beads are packed into chromatographic columns. Size fractionation of the phases present in the samples depend on their diffusion rate through the interstitial cavities of the beads. Two main categories of gel filtration chromatography procedures can be used to detect the amount of binding of a ligand for surface sites: (i) samples containing ligands and sorbant material already equilibrated are passed through a preconditioned resin, and, at the elution stage, free ligands are separated from those bound to particles or molecules; and (ii) the resin is preconditioned with an elution buffer containing a radioactive ligand. After applying the sample to the resin, elution is carried out with more buffer containing the same radioligand. Therefore, different levels of radioactivity in the eluant allow calculation of the amount of sorption. The main advantage of GFC is that it provides information of metal concentrations over a continuous size spectrum as opposed to the discrete size ranges typical of filtration and ultrafiltration [41]. However, during the elution step, trace elements become very diluted. Consequently, high blanks may be found, thus limiting

Causes of Artifacts in Sorption Studies with Trace Elements

473

the use of this technique to samples with relatively high concentrations of the species of interest [77]. An additional drawback is the possibility of metal binding to the resin bed of the columns, thus reducing the technique recovery efficiency and possibly causing cross-contamination errors. Adsorption artifacts and dissociation of metal complexes can be prevented or reduced by appropriate selection of pH, ionic strength, temperature, and composition of the eluant [78]. In receptor-ligand binding studies, it is important that the separation technique used be sufficiently quick to avoid the problem of dissociation of the receptor-ligand complexes. Dissociation of the bound ligand during column elution is thus a main shortcoming of this technique. Approximate upper limits for separation times can be calculated for various equilibrium binding constants [79]. Ion exchange resins have been used extensively in the separation of "resin-reactive" ("labile") from "resin-unreactive" ("non-labile") metal fractions [62, 80-82]. Unfortunately, the same complex or chelate can contribute to any of these two fractions depending on factors such as type of resin pretreatment and type of eluant. Partial dissolution or disaggregation of colloidal-sized resin particles may cause an apparendy reduced binding of trace elements to the resin and, as a result, low values of partition coefficients [5]. This artifact can be minimized by miniaturizing the experiment in order to reduce the amount of resin used in each test. Sequential Leaching An alternative approach to the use of individual artificial phases for adsorption experiments has been to carry out selective and sequential leaching on natural, heterogeneous particle assemblages in order to establish indirectly the extent of metal binding to operationally defined phases [15, 83, 84]. Belzile and Tessier [85] and Tessier et al. [86, 87] calculated the sorption of many trace elements on iron oxyhydroxides from lake sediments by leaching the sediments with a reagent that selectively dissolved the iron oxyhydroxides. Some workers [2, 88, 89] have questioned the accuracy of the data obtained with these techniques, as sequential leaching causes many artifacts. The main problems are due to the fact that the reagents normally used to dissolve specific phases present in natural sediments can in fact dissolve or leach other sediment fractions and their associated trace elements. Also, there is no guarantee that the trace elements extracted by the dissolution of one solid phase will not be readsorbed by the remaining particles. Artifacts Associated with the Use of Voltammetric Methods Electrochemical techniques can be used to distinguish between dissolved and particulate concentrations of trace elements without recourse to physical separation of the two phases. A number of options are available, depending on the nature of the samples under investigation and on the concentration range of trace metals.

474

Health and Toxicology

Ion-selective electrodes have been used extensively in water analysis [42, 90, 91], but the range of determinands that can be analyzed for is relatively Umited. Unfortunately, ion-selective electrodes are not sufficiently sensitive to measure the low concentrations of trace elements typically found in environmental samples. Voltammetric techniques (anodic stripping voltammetry, differential pulse polarography, differential pulse anodic stripping voltammetry, cathodic stripping voltammetry) offer increased sensitivity and lower detection limits. Anodic stripping voltammetry (ASV) and other voltammetric techniques have become an important tool in trace metal analysis and speciation and adsorption studies [22, 24, 92-95]. Some authors [3, 96], however, have pointed out some of the problems associated with the use of these techniques on systems with low ionic strength (1-10 mM), low concentrations of trace metals, and in the presence of organic complex formers. Davison et al. [3] reported a suppression in the ASV signal for low concentrations (5 x 10"^ M) of Pb, Cd, and Zn as the pH was increased or as the ionic strength was decreased. Adsorption onto the surfaces of the cell assembly was suggested as a possible cause of these trends. Diaz-Cruz et al. [96] provided evidence of adsorption onto cell components made of different material (glass, polyfluoroethylene. Teflon, polymethacrylate, nylon, and polystyrene) and found glass and polystyrene to be the least adsorbing materials. These problems indicate that ASV cannot be easily applied to real water samples and that more research is needed in this field to reduce or avoid possible artifacts. Whenever experiments on natural samples are carried out, routine measurements of blanks are necessary in order to reduce potential errors to an acceptable level. Formation of intermetallic compounds (other than mercury amalgam) within the electrode can be an important cause of interference resulting in reduced peak currents and a new peak for the intermetallic compound. Also, elements having similar plating potentials in a given medium may cause substantial errors. These and other sources of bias are described in a number of publications [97, 98] that recommend appropriate calibration procedures. Artifacts Associated with the Use of Radioactive Tracers Radioactive tracers are often used for studying metal ion binding to inorganic particles and biological material [35, 37, 99-101]. This technique involves the incubation of the radioisotope with the particulate phase in suitable medium (natural or artificial solutions). Once equilibrium is obtained, the particulate phase is separated (usually by filtration or centrifugation), and the amount of radioisotope sorbed onto it is measured by gamma spectrometry. Radiochemical techniques have the advantage of involving relatively simple handling protocols and, given the good sensitivity offered by existing analytical methods, they allow the use of very small amounts of trace metals and particulate material. Experimental conditions can closely mimic typical environmental conditions. However, a number of drawbacks are associated with radiochemical methods. Sorptive losses to container walls (if centrifugation is used to separate the incubation solution from the particulate phase)

Causes of Artifacts in Sorption Studies with Trace Elements

475

and to the filters (in case filtration is used) can be a problem. Also, some unbound radioisotope may end up trapped in the interstices between the separated particulate material, leading to an overestimation of the amount of metal actually bound. This problem can be overcome by running, in addition to the normal set of samples, a number of samples incubated with a metal ion or other substance known to have high affinity for the available binding sites. Sorptive losses can thus be calculated from the difference between the total amount of adsorbed metal in samples not containing the competing substance and the amount adsorbed in the presence of it. Partition coefficients determined in the laboratory with radiotracers are often lower than those obtained from natural samples, probably a result of the difference between the residence time of suspended material in natural water and the equilibration time (usually shorter) set in laboratory experiments [102]. Also, unrealistic concentrations of particles are often suspended in the solutions used in the laboratory. In studies with biological material, the binding site is called a "receptor," i.e., a molecule that chemically interacts with the ligand (and the radioligand used as a tracer) present in the medium. Radioligands chosen for laboratory experiments have high affinity for the receptor studied. Also, they are chosen for their high specific radioactivity to compensate for the low density of receptors normally present on biological tissues. Tritium (^H) and ^^P are two of the most commonly used isotopes in the preparation of radioligands. Evidence of significant cell death in control cultures containing the beta emitter radiotracer ^^P has been reported in the literature [103]. If a significant reduction in biological activity is induced by radioactive tracers, the results obtained may be affected by significant errors. It is thus necessary to compare the number of cells in the samples treated with the tracer to that of control cultures. Additional difficulties arise when working with microorganisms. In particular, their growth rates in different experimental conditions must be taken into account in binding measurements. The number of cells must be counted in sample aliquots so that the sorption data is expressed in mass of trace element sorbed per unit mass of living organisms. Variations in oxygen concentration in the samples under investigation affect the growth of microorganisms and may thus be a considerable source of error. In order to avoid these difficulties, many laboratories prefer to use dead cells. This means that the observed sorption trends cannot be readily extrapolated to environmental conditions. Artifacts Caused by Incorrect Data Manipulation Relationship Between Trace Element Concentration in Solution and Adsorption Patterns Adsorption isotherms. The partitioning of a solute between the solvent and the sorbent phase can be expressed with the ratio of their relative concentrations, also called distribution coefficient (K^) or partition coefficient (Kp):

476

Health and Toxicology

Amount of trace element sorbed m the solid phase

K(j =

S =— Amount of trace element dissolved in the aqueous phase D

(1)

Plots of the dissolved concentration (D) of an element against its adsorbed concentration (S) are called adsorption isotherms. In the simplest case, the amount of solute adsorbed (q) by the sorbent can be directly proportional to the concentration of solute in solution, i.e. q = Kd-[M]eq

(2)

where q is the concentration of adsorbed substance, K^ is the distribution coefficient, and [M]eq is the concentration of the substance under investigation that is still in solution at equilibrium. This adsorption pattern is represented graphically by a linear isotherm. The slope of the linear isotherm (i.e., the K^) will vary depending on the experimental conditions, as described earlier. This type of isotherm usually describes adsorption at low solute concentrations or for sorbent phases of low sorption capacity. Any interpretation or modeling of such data can only be limited to the range of metal concentrations investigated. Any extrapolation beyond the calibration range may be totally meaningless. These linear trends may become non-linear at higher solute concentrations. Adsorption isotherms are often parabolic (Freundlich isotherms) or hyperbolic (Langmuir isotherms) as shown in Figure 6. Langmuir isotherm. This relationship is based on the following assumptions: 1. The adsorption energy of each ion or molecule is constant independently of the amount bound onto the surface; 2. Only one layer of solute can be adsorbed onto the surface of the sorbent; 3. Adsorption occurs at specific sites; and 4. There is no interaction between adsorbed substances. The Langmuir expression is of the type q =

qmax-b-[M] l + b-[M]eq

where q^ax b q [M]eq

(3)

is the amount of solute sorbed when the monolayer is complete, is a coefficient related to the adsorption energy, is the concentration of solute sorbed, and is the adsorbent concentration remaining in solution at equilibrium.

Causes of Artifacts in Sorption Studies with Trace Elements

477

SORPTION ISOTHERMS

D Figure 6. Types of sorption isotherms: (a) linear, (b) parabolic, and (c) hyperbolic. The dissolved concentration of a given trace element is represented by D, whereas the amount sorbed in the solid phase is represented by S. Usually, a linear isotherm describes sorption at very low solute concentrations and is often not applicable at relatively high solute concentration. The value of a K^ determination is thus quite limited as it may change at different concentrations and at different conditions of pH, ionic strength, temperature, etc.

The Langmuir equation can also be derived by applying the mass law. The adsorption of a free metal ion in solution onto binding sites of an available solid surface can be expressed as a reversible equilibrium reaction. If S = surface site, M = substance in solution, and SM = substance bound onto surface sites, then

[M] + [S] -*-

K.ads

[SM]

Kads = [SM]/[S] [M]

(4) (5)

where K^ds is the adsorption constant for the substance M. The Langmuir equation can also be written in the form q = qmax[M]/(l/Kads + [M])

(6)

which, multiplying both numerator and denominator by Kajs becomes q = qmax{Kads[M]/(l+Kads[M]))

(7)

478

Health and Toxicology

This equation can be modified to include cases where more solutes can bind to the same sites, or instances where different solutes can bind to different adsorbent phases, or to sites of different affinities [104]. For example, if the metals Cu and Pb are competing for all the available binding sites but each metal has a different affinity for these sites, the following equations can be used: q = qmax [Cu] Kcu/ (1 + Kcu[Cu] + Kpb[Pb]) q = qmax [Pb] Kpb/ (1 + Kcu[Cu] + Kpb[Pb]). Each of these equations can be fitted separately into a regression program assuming a different q^ax for each metal, or can be used together in the same non-linear regression fit assuming an identical q^ax for Cu and Pb. The amount of solute (adsorbent) bound to particulate material increases in hyperbolic fashion and approaches q^ax as the unbound concentration of adsorbent increases. Half of the available binding sites will be occupied (q = Yi qmax) at a solute concentration [M]eq = 1/b. Therefore, it is possible to derive the parameters qmax and b by (i) determining the amount q bound at different solute concentrations, and (ii) fitting experimental data to Equation 3. The Freundlich isotherm has been used quite extensively to describe sorption on heterogeneous surfaces. It can be written as: q = K-[M]%

(8)

where K is an equilibrium constant indicative of the adsorption strength, and n shows the sorption intensity. A linear form of this equation is used to derive both coefficients: logq = logK + nlog[M]eq

(9)

If log q is plotted against log [M]eq the slope of the resulting straight line will give n, and the intercept on the ordinate will give q. Very often the analysis of binding experiments is based on different methods of data linearization, the most popular of which is the Scatchard plot [105]. This is a graph in which the concentration of adsorbed substance (S) is plotted on the x-axis, and the ratio of adsorbed substance over its concentration in solution (S/D) is plotted on the y-axis (Figure 7). In experiments with biological material, it was established that experimental data obtained at equilibrium can be interpreted using a Scatchard plot only when the free ligand concentration is in the range O.IK^-IO K^ [106]. A Scatchard plot can be a reliable method for measuring the number of surface sites and binding constants only if two additional conditions are met. Firstly, it must

S/D

0.6

(b)

J Scatchard plots

0.5 «

t

o •

0.4

S/D

0.3

J \

Data set 2 1 Data set 1

0 '^

0.2

^ 0.1-j

1

^

\^

0.0 -1—•—r—'—r

0

20 40 60 80 100 120 140 160 180 200

D (/ig/L)

20 0 •

(c)

Data set 2 Data set 1 0

o

12 H

s (Aig/L)

o

J

o o o

1

^ •

r-'-,

10

100

1000

Figure 7. (a) Scatchard plot of data (set 1) obtained from the sorption of a trace metal onto particulate matter. By fitting the data points to a straight line it is possible to determine the maximum number of binding sites (qmax) and the Kd on the basis of the slope and intercept on the abscissa, respectively, (b) The Scatchard plot of an additional set of data (set 2) obtained in a replicate experiment using higher dissolved metal concentrations (D) gives very different values of Qmax and Kd- Figure 7(c) shows that no inflection point is reached with data set 1 on a semilogarlthmic binding plot. A larger number of experiments is required to make sure that data points represent sorption at saturation levels of metal in solution. (D = metal concentration in solution; S = metal concentration sorbed in the solid phase.)

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Health and Toxicology

have been established by other binding measurements that there is only one type of surface sites. If every binding site is identical and its affinity for a specific metal is the same, then a Scatchard diagram will be linear and its intercept on the x-axis will represent the maximum number of surface sites. Secondly, the concentration of solute used in the experiment must be sufficiently high to saturate all the available surface sites. If the diagram on the Scatchard plot is a straight line, then a semilogarithmic plot (in which the bound metal is plotted against the concentration of free metal on a logarithmic scale) will be an S-shaped curve. Assuming all the surface sites are identical, the amount of metal bound at the inflection point of the curve will be 50% of the metal concentration at the saturation point. Therefore, only when the free metal concentration is on the plateau above the inflection point is the second condition met and the intercept on the Scatchard plot meaningful [107]. An example of incorrect application of a Scatchard diagram is shown in Figure 7. Non-linear plots can sometimes be explained with a decreasing binding affinity of the particles with increasing metal concentration. This means that an ion or a ligand binds first to surface sites of very high affinity and later to those of more limited affinity. Other possible explanations include losses of solute(s) and, in the presence of more than one solute, competition for the available binding sites becoming gradually dominant. Different equilibrium constants will be obtained at increasing metal concentrations. It is therefore impossible to determine adsorption parameters from curved Scatchard plots. Experimental data may alternatively be fitted using a non-linear regression analysis based on Langmuir's or Freundlich's equations. The maximum number of adsorption sites and the equilibrium dissociation constant (K^), which is equivalent to the inverse of the adsorption constant (1/Kads) for a specific metal (M), can be calculated by means of computer iterations. This non-linear analysis circumvents the problem of linearization and solves the regression problem directly without transforming the data. For better accuracy, the variance of each point can also be taken into account by using weighted least squares. The application of the Langmuir or Freundlich isotherms to experimental data on adsorption of trace metals onto soils, sediments, and natural particles suspended in aquatic environments may not be justified for a number of reasons: (i) the adsorption energy of solutes onto the very heterogeneous assemblages of natural particles may be quite different, (ii) there may be interactions between different sorbed substances, (iii) multilayer formation cannot be ruled out, and (iv) once bound, solutes may diffuse inside the sorbing particles and become internalized. This means that one cannot rely solely on the fitting of experimental data to a Langmuir or a Freundlich equation (or others available) to conclude that adsorption is the

Causes of Artifacts in Sorption Studies with Trace Elements

481

mechanism involved. Quite often, a minor change in the experimental setting (e.g., T, pH, ionic strength) of a given a solute/sorbant combination requires recalibration of some of the parameters in order to be able to fit the data to one of the isotherms. SUMMARY The main potential artifacts in sorption studies with trace elements have been described in this chapter. The major sources of artifacts are often associated to the separation procedures utilized. Filtration and centrifugation have been and are still the most popular methods used to separate "solid" and "dissolved" phases. This separation is purely an operational one as natural suspensions and sediments are known to be made of a "continuum" of particle sizes. Colloidal particles (diameter < 1 |im) are frequently accounted for as part of the "dissolved" fraction because they tend to pass through the pores of conventional filters. The amount of trace elements associated with colloidal matter may be quite significant. An overestimation of the "dissolved" fraction is a major source of error in sorption studies with environmental samples and one of the likely causes of the different distribution coefficients obtained at varying concentration of sorbent particles in suspensions. The entrapping of some solute in the interstices of the solid phase separated by centrifugation is also a source of errors. Field-flow fractionation has been used quite successfully in separation studies. The main drawback of this technique is the cost of the equipment required. Other sources of error include insufficient equilibration time in experimental conditions. Adsorption onto filters and containers cannot be avoided in the pH range (i.e., circumneutral and alkaline) typical of many environmental samples, and needs to be corrected by setting up a protocol that includes routine determinations on control samples. The main disadvantages of the use of trace amounts of radiolabels include the possibility that only interactions at high affinity sites may be studied and, if experiments are carried out on live cells, the reduction of biological activity due to emission of radiation. Considerable trace metal losses to the cell assembly are known to affect sorption experiments with voltammetric techniques, especially if environmental samples are used. A Scatchard plot can be a reliable method for measuring the number of surface sites and binding constants only if it has been established by other binding measurements that there is only one type of surface site, and when the concentration of metal ions or ligands used in the experiment is sufficiently high to saturate all the available surface sites. In experiments with biological material, data obtained at equilibrium can be interpreted using a Scatchard plot only when the free ligand (text continued on page 485)

Table 2 Summary of potential artifacts in sorption studies Procedure

Any stage of a sorption experiment

Cause of problem

Artifacts

Detection/correction

Contamination

High blanks Low accuracy and precision Inconsistent results

Routine blank determinations Interlaboratory calibrations

Sorption onto filtration apparatus Colloidal particles passing through filter pores Filter clogginghreakage

Overestimation of sorption

Blank determinations

Overestimation of dissolved fraction Lack of reproducibility Altered sorptioddesorption

Filtration tests and SEM/TEM studies Reproducibility tests Tests to be carried out a different flow rates Reduction in filtration flow rate Filtration of smaller volumes of suspension

SorbenUsorbateseparation Filtratiodultrafiltration

Dialysis

Centrifugation

Breakage of livingldead cells

Contamination

Binding to dialysis membrane

Overestimation of sorption

Long equilibration time

Underestimation of sorption

Particle agglomeration, compaction, alteration

Overestimation of dissolved fraction Reduction of centrifugation rate due to reduced surface site and potentially higher dissolution of trace elements from particles

Blank determinations at different equilibriation times Experiments needed at different equilibration times Dialysis method to be avoided if dissociation rates are high

I

0

Column Chromatography

Sample dilution at elution step Dissociation o f sorbate

Contamination Underestimation o f sorption

Binding of trace element to resin Incorrect phase separation and bed potential cross-contamination Dissolutiodalteration of resin Underestimation o f sorption bed

Sequential leaching

Potential leaching of wrong solid phases

Incorrect desorption measurements

Blank determinations Reduction of separation time depending on equilibration binding constants Choice of appropriate pH, ionic strength, and eluant composition Reduction of amount of resin used in the experiment Choice of appropriate pH and eluant composition Parallel elemental analysis o f untreated and leached solid phases

Potential readsorption of desorbed elements

Voltammetric techniques

Adsorption onto the cell assembly components

Underestimation of dissolved fraction

Long equilibration times at low Overestimation of dissolved fraction trace element concentration, high pH, and low ionic strength Underestimation of sorption Decreased sorption with increasing concentration of supporting electrolytes and complexing agents

Radioactive tracers

(table continued on next page)

Sorptive losses to filters and container walls

Overestimation of binding

0

E u)

I

s %= P)

$ -. The problem may be reduced by using cells made of polystyrene or glass, by measuring blanks at the pH of the ligand solution, or by determining adsorptioddesorption corrections The experiment must last until equilibration is reached

3

v)

4 z. s 2P

I sz 5

Choice of appropriate electrolyte Blank determinations Experiment with model solutions, with and without complexing agents Measurement of binding in the absence of particulate material

2 0 n, n,

2

2

Table 2 (Continued) Procedure

Incorrect data manipulation or interpretation

Cause of problem

Artifacts

Unbound radioligands trapped in the interstices of the separated solid fraction

Overestimation of binding

Low equilibrium time allowed in lab experiments

Underestimation of binding

Separation procedure too slow for ligand dissociation rates

Underestimation of binding

Death of cell material due to high tracer radioactivity Extrapolation of experimental data obtained at a specific sorbate/sorbent ratio to different environmental conditions Incorrect use of Scatchard plots

Unrealistic binding results

Linearization of the data for the purpose of analysis The fitting of experimental data to a specific equation (e.g., Lanemuir, Freundlich)

Wrong estimate of adsorption

Detectionlcorrection

Measurement of binding in additional sets of samples containing a substance known to have high affinity for the binding sites Equilibration time must be increased in line with kinetic data and with residence time of suspended particles Separation time must be reduced to take into account the dissociation rate constant of the radioligand at the temperature used in the experiment Use of control cultures

Meaningless trends Incorrect partition (distribution) coeffecients

Experiments must be carried out at different sorbate/sorbant ratios, and should mimic closely the environmental conditions of interest

Incorrect determination of maximum number of binding sites and adsorption constant

Scatchard plots may be used if only one type of binding site is known to be present The solute concentration used in the experiment must be sufficiently high to saturate all surface sites Preference should be given to nonlinear weighted regression analysis Other information must be available to assume an adsorption mechanism

Adsorption processes are assumed to be responsible for the observed trends

Causes of Artifacts in Sorption Studies with Trace Elements

485

(text continued from page 481)

concentration [D] is in the range O.lK^j-lO K^ [106]. The fitting of experimental data to a Langmuir or a Freundlich equation (or others available) is a necessary but not sufficient condition to conclude that adsorption is the mechanism involved. The potential problems arising in sorption studies are summarized in Table 2. It includes a description of the sources of artifacts in connection with the techniques used, a list of the resulting artifacts, and suggestions for their detection and correction. The nature of the artifacts encountered tends to become more complicated as the experimental parameters imitate closely those found in natural settings. In particular, the use of natural sorbents and sorbates shows that precise duplication of field conditions is quite often impossible. Once the artifacts affecting the experiments have been corrected for, the data obtained can be extrapolated to environmental conditions only with extreme caution. REFERENCES 1. Bennet, J. P. Jr. "Methods in binding studies," in Neurotransmitter Receptor Binding. H. I. Yamamura, S. J. Enna, and M. J. Kuhar (Eds.), New York: Raven Press, 1978, pp. 57-90. 2. Tipping, E., Hetherington, N. B., Hilton, J., Thompson, D. W., Bowles, E., and Hamilton-Taylor, J. "Artifacts in the use of selective chemical extraction to determine distributions of metals between oxides of manganese and iron:'Analytical Chemistry, Vol. 57, 1985, pp. 1,944-1,946. 3. Davison, W., De Mora, S. J., Harrison, R. M., and Wilson, S. "pH and ionic strength dependence of the ASV response of cadmium, lead and zinc in solutions which simulate natural waters." Science of the Total Environment, Vol. 60, 1987, pp. 35-44. 4. Giusti, L., Hamilton-Taylor, J., Davison, W., and Hewitt, C. N. "Artifacts in sorption experiments with trace metals." Science of the Total Environment, Vol. 152, 1994, pp. 227-238. 5. Lead, J. R., Hamilton-Taylor, J., and Kelly, M. "Artifacts in the determination of the binding of americium and europiun to an aquatic fulvic acid." The Science of the Total Environment (In press, 1995). 6. Weber, W. J. Jr., McGinley, P. M., and Katz, L. E. "Sorption phenomena in subsurface systems: concepts, models and effects on contaminant fate and transport." Water Research, Vol. 25, No. 5, 1991, pp. 499-528. 7. da Silva, J. J. R. F., and Williams, R. J. P. The Biological Chemistry of the Elements: The Inorganic Chemistry of Life, Oxford and New York: Oxford University Press, 1991. 8. Siegel, A. "Metal-organic interactions in the marine environment," in: Organic Compounds in Aquatic Environments, S. J. Faust and J. V. Hunter (Eds.), New York: Marcel Dekker, 1971, pp. 265-295.

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estuaries in England." Estuarine, Coastal and Shelf Science, Vol. 22, 1986, pp. 479-^86. 96. Diaz-Cruz, J. M., Esteban, M., van den Hoop, M. A. G. T., and van Leeuwen, H. P. "Stripping voltammetry of metal complexes: interferences from adsorption onto cell components." Analytical Chemistry, Vol. 64, 1992, pp. 1,769-1,776. 97. Edward, C. A. Anodic Stripping Voltammetry^A Review, Technical Report TR 22, Medmenhamn Bucks.: Water Research Centre, 1972. 98. Whitfield, M., and Jagner, D. Marine Electrochemistry. Chichester: Wiley, 1981. 99. Aston, S .R., and Duursma, E. K. "Concentration effects on ^^^Cs, ^^Zn, ^^Co and ^^^Ru sorption by marine sediments, with geochemical implications." Netherland Journal of Sea Research, Vol. 6, 1973, pp. 225-240. 100. Jackson, T. A., Kipphut, G., Hesslein, R. H., and Schindler, D. W. "Experimental study of trace metal chemistry in soft-water lakes at different pH levels." Canadian Journal of Fisheries and Aquatic Science, Vol. 37, 1980, pp. 387-402. 101. Li, Y.-H., Burkhardt, L., Buckholtz, M., O'Hara, P., and Santschi, P. H. "Partition of radiotracers between suspended particles and seawater." Geochimica et Cosmochimica Acta, Vol. 48, 1984, pp. 2,011-2,019. 102. Balls, P. W. "The partitioning of trace metals between dissolved and particular phases in European coastal waters: a compilation of field data and comparison with laboratory studies." Netherland Journal of Sea Research, Vol. 23, 1989, pp. 7-14. 103. Forster, T. H., Allan, D. J., Gobe, G. C , Harmon, B. V., Walsh, T. P., and Kerr, J. F. R. "Beta-radiation from tracer doses of phosphorus-32 induces massive apoptosis in a Burkitt's lymphoma cell line." International Journal of Radiation Biology, Vol. 61, No. 3, 1992, pp. 365-367. 104. Stumm, W. Chemistry of the Solid-Water Interface. New York: John Wiley and Sons, 1992, pp. 90-93. 105. Scatchard, G. "The attractions of proteins for small molecules and ions." Annals of the New York Academy of Sciences, Vol. 51, 1949, pp. 660-672. 106. Boon, N. A., Oh, V. M. S., Taylor, E. A., Johansen, T., Aronson, J. K., and Grahamr-Smith, D. G. "Measurement of specific [^H]-ouabain binding to different types of human leucocites." British Journal of Clinical Pharmacology, Vol. 18, 1984, pp. 153-161. 107. Klotz, I. M. "Number of receptor sites from Scatchard graphs: facts and fantasies." Science, Vol. 217, 1982, pp. 1,247-1,249.

CHAPTER 23 BIOACCUMULATION OF SURFACTANTS J. Tolls and D. T. H. M. Sijm Environmental Chemistry Group Research Institute of Toxicology NL-3508 TB Utrecht, The Netherlands CONTENTS INTRODUCTION, 493 CR-Values as a Quantitative Measure of Surfactant Bioconcentration, 495 UPTAKE OF SURFACTANTS, 496 SURFACTANT ELIMINATION, 496 ENVIRONMENTAL FACTORS INFLUENCING SURFACTANT BIOCONCENTRATION, 497 SURFACTANT STRUCTURE AND BIOCONCENTRATION, 497 REFERENCES, 498 INTRODUCTION In a recent article [1] we reviewed the existing literature on surfactant accumulation in aquatic organisms. Here we want to summarize our findings to give an overview of the current knowledge of this topic. For more detailed information, the reader is referred to our review [1] and the references cited therein. We conclude that the present surfactant bioconcentration data cannot be used to quantitatively describe surfactant bioconcentration. The database is largely restricted to bioconcentration of anionic surfactants in fish. Gastrointestinal uptake of surfactants and bioconcentration in other species have been neglected. However, the data on fish allow to state the following: 1. surfactant uptake occurs primarily across the gills. 2. surfactants are biotransformed by fish to a considerable extent. 3. surfactant bioconcentration appears to be hydrophobicity dependent. 4. a number of environmental factors appears to influence surfactant bioconcentration. Synthetic surfactants are compounds of widespread use in different fields of application. Their annual production rate is approximately 7 Mt [2]. About one-half of this amount is used in domestic cleaning agents for textiles, dishwashing, cosmetics, etc. Surfactants combine hydrophobic and hydropholic properties in one molecule. The hydrophobic moiety of surfactants consists most frequently of a long-chain aliphatic hydrocarbon. While there is little variation in the hydrophobic tail of the surfactants, the structural diversity of surfactants is largely accounted for 493

494

Health and Toxicology

by different types of polar head groups. Hence, the general classification of surfactants occurs according to the charge of the headgroup, which can either be noncharged, amphoteric, cationic, or anionic. Surfactants are of environmental interest because, after consumer use, the major portion of them is disposed of into wastewater. Even though wastewater treatment can largely reduce the concentrations of surfactants in wastewater treatment effluents [3], a certain amount of surfactants will reach natural waters [4]. Therefore, aquatic biota are exposed to surfactants. An important question in the assessment of the environmental risk posed by surfactants is whether and to what extent these compounds are accumulated by aquatic organisms. For aquatic organisms there exist two pathways to exposure to xenobiotics and thus to bioaccumulation. On the one hand, xenobiotics can be taken up via ingestion. On the other hand, uptake via the respiratory organs is often an important route because aquatic organisms have to pass large volumes of water across their gills in order to fulfill their oxygen demands. The large surface area of the gills is not only very efficient for O2 uptake, but also a port of entry for xenobiotics. Two terms, bioconcentration and biomagnification, have been defined to indicate whether the exposure occurred via the water (across the gills) or the food (ingestion), respectively. Most information concerning surfactant bioaccumulation stems from bioconcentration experiments. Only two studies investigated uptake in the gastrointestinal tract (G.I. tract) [5, 6]. Therefore, we will also deal predominantly with surfactant bioconcentration. All of the data were obtained for aquatic organisms, most of them fish but also for invertebrates and algae. Bioconcentration is the result of simultaneous uptake from and elimination into the water. This has been conceptualized in a simple model in which uptake as well as elimination are assumed to follow first-order kinetics and each fish is regarded to be one well-mixed compartment. The time course of a chemical's concentration in fish is then given by Equation 1 [7]. dCf/dt = + ki»Cw-k2*Cf

(1)

For steady-state, i.e., dCf/dt = 0, a time-dependent measure of bioconcentration can be derived (Equation 2). This measure is called the bioconcentration factor (BCF) as defined by the OECD [8]. The symbols used here are explained in Table 1. BCF = Cf/Q = ki/k2.

(2)

From Equation 2, it can be seen that the BCF can be determined as the quotient of the steady-state concentrations in fish and water or as the quotient of the uptake and elimination rate constants. Here, the term concentration ratio (CR) will be employed to refer to bioconcentration data because the surfactant bioconcentration data do not fulfill the definition of the BCF. The criteria used for assessing in how far the reported CRs are quantitative measures of surfactant bioconcentration have

Bioaccumulation of Surfactants

495

Table 1 Symbols used for the bioconcentration model

BCF kj k2 Cf C^

bioconcentration factor (L • kg"^ uptake rate constant (L • kg~^ • d~^) elimination rate constant (d~^) concentration in the organism (mol • kg"^) concentration in the water (mol • L~^)

been outlined in Reference 1. CR-values were selected if evidence for attainment of steady state was presented or if the CR was determined according to the kinetic method. Comparison of CR-values obtained using the kinetic and the steady-state approach indicate that both methods yield similar CR-values [9, 10]. Hence, the kinetic approach based on the one-compartment first-order model appears suitable for the investigation of the bioconcentration of surfactants. An overview of the available literature on bioconcentration of surfactants is provided in Table 2. Besides the references reporting the whole body CRs, nine further references contained tissue-specific CRs or information concerning the biotransformation of surfactants in fish. No information exists for amphoteric surfactants, while more than two-thirds of the data are reported for anionic surfactants and almost half of the data have been obtained for linear alkylbenzenesulfonates (LAS). CR-VALUES AS A QUANTITATIVE MEASURE OF SURFACTANT BIOCONCENTRATION In almost all experiments, liquid scintillation counting (LSC) was used for measuring surfactant concentrations. Using chromotographic techniques prior to LSC, a number of studies demonstrated qualitatively that the tested surfactants are biotransformed [1]. Because the CRs were determined without separation of parent Table 2 CRs grouped into two categories

All

Number of reported CRs Distributed among: Number of compounds tested Number of organisms tested Number of sources

Whole body CRs Selected

100

54

22 16 22

16 9 12

All data (all) and data selected to be consistent overestimates of bioconcentrations (selected). Also given is the distribution of the CRs among compounds, organisms and references.

496

Health and Toxicology

compounds from metabolites, the measurements include the biotransformation products as well as the parent compound. Therefore, the measured CRs overestimate the extent of accumulation of intact surfactant molecules. Furthermore, in a number of investigations, in which the CR was determined using the steady state approach, either steady state was not attained as evidenced by the experimental data or no evidence of the attainment of steady state is provided. Many of the bioconcentration data were obtained by quantitating mixtures of isomers (e.g., all data for LAS) or homologs by LSC. These data are not specific for an individual compound and do not fulfill the definition of the BCF, which pertains to individual compounds. Considering the points mentioned above, it can be stated that the available data are not suitable for the quantification of bioconcentration of surfactants. UPTAKE OF SURFACTANTS Tissue-specific analyses and whole-body autoradiograms of fish exposed to surfactants demonstrated that many surfactants are readily taken up and distributed within the fish [1]. The rapid increase of radioactivity in the gills as evidenced by whole-body autoradiograms in combination with the rapid increase of radioactivity in the blood indicates that the gills are the site of surfactant uptake. Exceptions with regard to rapid uptake are hexadecylpyridinium bromide [6, 11] and didecyldimethylammonium chloride [12]. Tissue-specific data for these two surfactants indicate that they are primarily enriched in the gills with slow overall surfactant uptake. The values of the uptake rate constant ki span a range of more than three orders of magnitude and range from 0.4 L • kg~^ • d~^ for octyltrimethylammonium chloride to 959 L • kg~^ • d~^ for tetradecylheptaoxethylene [1]. From data on homologous series, it appears that uptake rate constants increase with increasing hydrocarbon chain length. SURFACTANT ELIMINATION While there is a rather large spread in the values of ki, the values of the elimination rate constant k2 vary by a factor of less than 20, with a minimum of 0.12 d~^ and a maximum of 2.2 d~^ [1]. In this context, it has to be stressed that the data refer to measurements of total radiolabel, i.e., parent surfactant and biotransformation products. Therefore, the elimination data underestimate the elimination rates of the parent surfactant compound. The length of either the hydrocarbon chain (cationics) [13] or the oxyethylene chain (nonionics) [14] did not appear to significantly influence the elimination rate constant. Hence, there does not seem to exist a dependency of the elimination rate constant on hydrophobicity. It has been shown qualitatively that anionic surfactants (LAS, alkylsulfates, alkylethersulfates, and diethylhexylsulfosuccinate) and nonionic surfactants polyethylene glycol monoalkyl ethers are biotransformed by fish, while no evidence for

Bioaccumulation of Surfactants

497

biotransformation of cationic surfactants in the tested organisms has been presented. No information is available for the kinetics of surfactant biotransformation in fish. Hitherto, no biotransformation of LAS could be detected in Daphnia [15, 16]. 4-butyrolactone was identified as a biotransformation product of dodecylsulfate in goldfish [17]. This indicates that degradation by ca-oxidation of the alkyl terminus followed by (3-oxidation is a likely metabolic pathway for surfactants in fish because all surfactants possess an alkyl chain. The metabolites are predominantly excreted into the gall bladder. ENVIRONMENTAL FACTORS INFLUENCING SURFACTANT BIOCONCENTRATION Some evidence for an increase of bioconcentration with increasing water hardness has been provided for LAS [18] and dodecylsulfate [19]. In the presence of methylene blue active substances [20] and/or dissolved organic carbon [13] bioconcentration of cationic surfactants is reduced, likely due to reduced bioavailability [21]. A dependence of surfactant bioconcentration on the concentration of the surfactant solution has not been consistently demonstrated [9, 22]. Suspended matter, DOC, methylene blue active substances and the presence of micelles appear to reduce CR-values, probably by reducing bioavailability. Because field data on the bioaccumulation of surfactants are not available, the existing surfactant bioconcentration data cannot be compared to actual environmental conditions.

SURFACTANT STRUCTURE AND BIOCONCENTRATION In order to learn more about the relationship between surfactant bioconcentration and their structure, we defined a criterium to select a subset of CR-values that can be compared with each other [1]. Our criterium makes sure that the selected data are consistent overestimates of the real bioconcentration factors. The extent of overestimation is unknown, but is substantial in some cases [14-16]. Looking at comparable CRs from single experiments it appears that CRs increase with increasing length of the alkyl chain [13, 15]. Of the selected wet-weight based CR-values, the lowest was 0.8 L • kg~^ for octyltrimethylammonium chloride in the presence of dissolved organic carbon, and the highest was 1,960 L • kg~^ for a mixture of hexadecyl- and octadecylammonium chloride. For comparison, the measured BCF-values for poly chlorinated biphenyls range between 10^ and 10^ L • kg~^ [23]. In a detailed analysis of the relationships between surfactant bioconcentration and hydrophobicity we employed the critical micelle concentration (CMC) as measure of surfactant hydrophobicity. We found a significant positive relationship between the CR-values and the inverse of the CMC, indicating that surfactant bioconcentration increases with increasing hydrophobicity. There is also a significant positive relationship between the uptake rate constants kj and 1/CMC. The variation of elimination rate constant was small and not significantly correlated to 1/CMC.

498

Health and Toxicology

This bioconcentration behavior is, according to the diffusive mass transfer concept of bioconcentration, indicative of hpid-diffusion Hmited uptake of surfactants. Surfactant bioaccumulation therefore differs from the bioaccumulation of chlorinated organics in several aspects. Firstly, biotransformation appears to occur at a relatively high rate for surfactants. Secondly, it is the uptake rate constant that appears to be hydrophobicity-dependent, while it is the elimination rate constant in the case of polychlorinated benzenes (PCBzs) and biphenyls (PCBs). As a result, bioconcentration increases with increasing hydrophobicity for surfactants as well as for polychlorinated benzenes and biphenyls, indicating that surfactant bioconcentration is a hydrophobicity-driven process. A detailed analysis of the relationship between surfactant bioconcentration and hydrophobicity has been published elsewhere [24]. One of the major reasons for the limitations of the present database is the lack of analytical capabilities to extract, separate, and measure parent surfactant compounds. Due to the progress made in HPLC-MS, it seems likely that further maturation of this technique will eliminate the analytical obstacles that impeded the research of surfactant bioconcentration in the past. In the future, the use of analytical techniques that allow for separation and detection of surfactant parent compounds and biotransformation products will probably make it easier to quantitate surfactant bioconcentration. Compound-specific bioconcentration data for surfactants will contribute to deepening the understanding of the bioconcentration of polar and ionic organic substances. Derivation of quantitative relationships between surfactant bioconcentration and physical-chemical and/or structural properties will render prediction of the bioconcentration behavior possible. ACKNOWLEDGEMENTS The authors gratefully acknowledge a grant from N.V. Procter & Gamble S.A., European Technical Center, Brussels, Belgium. REFERENCES 1. J. Tolls, P. Kloepper-Sams, and D. T. H. M. Sijm, Chemosphere 693-717(1994).

29,

2. H. Stache, and K. Kosswig, Tensid Taschenbuch 3, Ausgabe, Hanser, Munchen, 1990. 3. D. C. McAvoy, W. S. Eckhoff, and R. A. Rapaport, Environ. Toxicol Chem. 12, 997-987 (1993). 4. D. J. Versteeg, R. Shimp, E. Meiers, B. Hamm, and T. Keough, Presentation at the 14th annual meeting of the Society of Environmental Toxicology and Chemistry, 14-18 November 1993, Houston.

Bioaccumulation of Surfactants

499

5. A. Neufarth, H. G. Eckert, H. Kellner, and K. Loetzsch, Report presented at the CED congress, March 1978, Madrid, pp. 205-216. 6. J. P. Knezovich, and L. S. Inoue, Ecotoxicol. Environ. Safety 26, l-bl^-lM (1993). 7. D. R. Branson, G. E. Blau, H. C. Alexander, and W. B. Neely, Trans. Am. Fish Soc. 104, 785-792 (1975). 8. Organization for Economic Cooperation and Development (1988) OECD, Guidelines for testing chemicals. Draft guideline 305, Bioconcentration: flow-through fish test. Paris, France. 9. W. E. Bishop, and A. W. Maki, "A critical comparison of bioconcentration test methods." In: Aquatic Toxicology, Eaton, J. G., P. R. Parrish, and A. C. Hendricks (Eds), American Society for Testing and Materials, ASTM STP 707, Washington, pp. 116-129 (1980). 10. W. Coenen, (1988) "Einfluss des linearen Alklybenzolsulfonates (LAS) auf die Kinetik von Lindan, 4-Nitrophenol und DDT beim Zebrabarbling." Diploma thesis. University of Mainz. 11. J. P. Knezovich, M. P. Lawton, and L. S. Inoue, Bull. Environ. Contam. Toxicol. 42, 87-93 (1989). 12. K. Yoshimura, "Fish bioconcentration of quartemary ammonium salt, didecyl dimethyl ammonium chloride," In: Umweltbundesamt, EEC-Workshop "Systematic Assessments on Surfactants," Working Document 1.5, Berlin, pp. 1-2 (1989). 13. D. J. Versteeg, and S. J. Shorter, Environ. Toxicol. Chem. 11, 571-580 (1992). 14. M. Wakabayashi, M. Kikuchi, A. Sato, and T. Yoshida, Ecotoxicol. Environ. Safety 13, 148-163 (1987). 15. R. A. Kimerle, R. D. Swisher, and R. M. Schroeder-Comotto, "Surfactant structure and aquatic toxicity." Proc. In: International Joint Commission, Great Lakes Research Advisory Board, IJC Symposium of Structure Activity Correlations in Studies on Toxicity and Bioconcentration with Aquatic Organisms, Burlington, Ontario, Canada, pp. 22-35 (1975). 16. R. M Comotto, R. A. Kimerle, and R. D. Swisher, "Bioconcentration and metabolism of linear alkylbenzene sulfonate by daphnids and fathead minnows." In: Aquatic Toxicology, Marking, L. L., and R. A. Kimerle (Eds.), American Society for Testing and Materials, ASTM STP 667, pp. 232-250 (1979). 17. P. W. A. Tovell, D. Howes, and C. S. Newsome, Toxicology 4, 17-29 (1975). 18. M. Wakabayashi, M. Kikuchi, H. Kojima, and T. Yoshida, Chemosphere 11, 917-924(1978). 19. P. W. A. Tovell, C. S. Newsome, and D. Howes, Water Res. 8, 291-296 (1974). 20. M. A. Lewis, and V. T. Wee, Environ. Toxicol. Chem. 2, 105-118 (1983). 21. L. H. Huber, J. Am. Oil. Chem. Soc. 61, 377-381 (1984).

500

Health and Toxicology

22. M. Wakabayashi, M. Kikuchi, A. Sato, and T. Yoshida, Bull. Jap. Soc. Sci. Fisheries 47, 1,383-1,387(1981). 23. D. Mackay, W. Y. Shiu, and K. C. Ma, Illustrated Handbook of physical-chemical properties and environmental fate for organic chemicals. Vol. 1, Monoaromatic hydrocarbons, chlorobenzenes and PCBs, Lewis, Boca Raton (1992). 24. J. Tolls, and D. T. H. M. Sijm, Environ. Toxicol. Chem. 14, 1,675-1,685 (1995).

INDEX application of liquid paints 275 applications of paint 283 aquatic environments 461 aromatic amines 26, 27 aromatic amino acids 226 aromatic hydrocarbons 272 arsenic 396 artificial sweeteners 383 asbestos 27, 111, 129 asbestos dust 93 asbestos exposure 94,118,127 asbestos fibers 24 asbestos fiber type 130 asbestos in urban populations 129 asbestos minerals 128 asbestos regulations 102 asbestos utilization 93 asbestos-associated diseases 108 asbestos-containing construction materials 94 asbestos-containing products 128 asbestos-related lung cancer 132 asbestosis 108, 131 aspergillus flavus 6 Aspergillus species 337 asthma 332,341 asthma-like symptoms 69 asthmatics 51 atmospheric acidity 356 atopy 79

absorption 208, 462 acetate derivatives 274 acid aerosols 356 acid precipitation 355,361 acid rainfalls 358 Acid Summer Haze Effect 336 acoustic conditions 75 acrylics 280 active site 226 acute poisoning 446 acute toxicity 260, 273 acute toxicosis 78 additives 271 adenocarcinoma 171, 175, 178 adsorption 464 adsorption energy 480 adsorption isotherms 475 adsorption of metals 464 adsorption patterns 475 aerosol 357 airborne asbestos 98 airborne dust 310 airborne infectious disease 76 airborne ions 75 airless pulverization 278 airmix pulverization 278 air pollutants 47, 49, 330, 331 air pollution 17, 336 air quality 344 alachlor 440 alcohols 272 aliphatic hydrocarbons 272 allergens 78,321,345 allergic bronchial asthma 308 allergic contact dermatitis 308 allergic diseases 78 aluminum 358, 405 American Cancer Society 219 ammonium vanadate 402 amphiboles 94 animal breeding 21 animal models 397 animal studies 260, 261 animal toxicity tests 437 anions 464 anti-corrosion 287 anti-corroSion undercoating 286

B bacteria 77 BaP suspentions 231 BaP-induced carcinomas 223 barometric pressure 333 base coat 286 benzene 207,208,209,213,286 benzene leukemogenesis 216 benzene oxide 212 benzene rings 220 benzene secondary metabolism 210 benzene toxicity 210 benzene toxicocinetics 207, 212 benzene toxicology 213 binders 269 binding site 226 bioaccumulation of surfactants 493 bioaerosols 76 501

502

Health and Toxicology

bioassays 417 bioassay system 420 biochemical constituents 365 biochemistry of organisms 367 bioconcentration 497 bioconcentration behavior 498 bioconcentration experiments 494 bioconcentration of surfactants 495 bioeffluents 78 biogeochemical 9 biogeochemical cycles 462 biological contaminants 76 biological dissipative structures 33 Biological Exposure Indices (BEI) 255 biological material 61 biological monitoring 453 biological sampling 255 biological systems 219 biology 33 bioluminescence 12 biomarker-based biological monitoring 62 biomarkers 54, 55, 56 biotransformation 259 bladder cancer 377, 388, 389 bladder cancer epidemiology 382 bladder cancer risk factors 382 blood cells 10 boiling point 264 breast cancer 6 bromodichloromethane 379 bromoform 379 bronchial bifurcations 191, 197 bronchial provocation test 50 bronchoconstriction 333 bronchospasm 333 bronchus 111 butiminous coal 144 cabinetmakers 181 cabinet making 173 cadmium 359, 397 calcium 358 cancer 1,8,34,56,78 cancer cell 37 cancer clasters 16 cancer incidence 24 cancer of urinary bladder 377 cancer risk 15 cancers 207, 261

cancers of the trachea 111 can coating 283 carbon dioxide 333 carbonless copy paper 82 carbon monoxide 48 carbon tetrachloride 274 carboxyhemoglobin level 263 carcinogen 192,221,222 carcinogenecity 223 carcinogenesis 41, 55, 59 carcinogenic effects of wood dust 185 carcinogens 30, 219 carcinomas 194 case-control studies 179 cathaphoresis 281 causal mechanisms 70 cell cultures 240 central nervous system 273 centriacinar (centrilobular) emphysema 140 centrifugation 470 chelating agent 402 chemical carcinogenesis 234 chemical curing 268 chemical plants 58 chemical reaction 266 chemical stripping 256 chemical stripping agents 256 chemiluminescence 15, 16 chest X-ray photographs 120 chloramination 380 chlorinated hydrocarbons 272 chlorinated water 378 chlorination 379 chlorination of drinking water 377 chlorination of water supplies 380 chlorophenols 381 chromate lung cancer 192 chromatographic techniques 472 chromium 403 chromium (VI) 25 chromium accumulation 191 chronic respiratory disease 329, 330, 331,343 chronic toxicity 261, 274 chronic toxicology studies 439 chronic weakness 440 chrysolite 94 cigarettes 27 cigarette smoke 219 clear coat 286

Index climate 345 climate change 343 climatic conditions 441 coal 150, 165 coal-emphysema 161 coalescence 268 coalworkers 137, 144 coal workers' pneumoconiosis 143 cobalt 307,310,403 cobalt exposure 323 co-carcinogens 30 coffee 26 coffee drinking 383 collagenous tissue 138 colloidal pumping 467 computer terminals 82 conductivity of paint 279 connective tissue network 138 construction industry 94, 98, 172 construction materials 252 construction workers 93 contamination 466 conventional dipping 280 copper 359 corrosion-resistant superalloys 307 Cr accumulations 197 Cr bronchial distribution 194 Cr compounds 202 Cr concentration 196, 203 Cracow region 11 creatinine 456 critical levels 357 curtain machines 282 cutaneous pathways 272 cyclohexane 274 cytochrome 225 cytochrome P450 224,225 cytology tests 121 cytotoxic 212

D DDT 444 death certificates 385 destruction in emphysema 139 dialysis 470 dialysis membranes 468 diamond abrasive production 312,313 dichlorodiphenyl tricloroethane (DDT) 434 dichloromethane 259, 262, 263 dichloromethane-based stripping

503

agents 265 diffusing capacity of the lung 48 difractometer 456 digestive tract 272 dinamic quenching studies 231 diol-epoxide 223 direct impact of climate 332 diseases 1 disinfection 381 disinfection process 388 disinfection processes 378 dispersion 338 distribution 208 DNA 58 DNA moieties 425 DNAs 221,222 domestic activities 252 drinking water 3 drinking water disinfection methods 388 Drosophila melanogaster All dry-process photocopy 83 dust conditions 184 dust content 164 dysplasia 36

E ecologic studies 384 ecology 33 ecotoxicology 1, 3, 7 effective exposure 208 effects of solvents 273 effects of tannery effluent 366 electrochemical potential 12 electrochemical techniques 473 electrodeposited alloys 307 electrodeposition 280 electromagnetic radiation 75 electrostatic pulverization 279 elimination 209 emotional stress 29 emphysema 137, 138, 144, 145, 162, 163, 165 emphysematous lungs 161 endocrinological activity 13 endotoxin 77 entropy 42 environmental acidification 361 environmental conditions 38 environmental contaminants 70 environmental contamination 435 environmental control 346

504

Health and Toxicology

environmental control systems 67, 80, 81 environmental factors 493 environmental pollutants 20, 219 environmental pollution 357 environmental risk factor 24 environmental risk factors 26, 39 environmental surveillance 315 environmental tobacco smoke 73 enzyme acetylclolinesterase 440 epidemics 342 epidemiological studies 285, 292 epidemiologic evidence 389 epidemiologic studies 174, 185 epidemiology 130 epithelial cells 19 epoxides 280 esters 272 ethanol 150 etiologic fraction of asbestos exposure 131 exporting wastes 30 exposure sources 359 exposure to acid aerosols 356 extenders 271 external stressors 80 extra-fetal mechanisms 397 ferrous metals 284 Field-Flow Fractionation (FFF) 472 filters 468 filtration 466,467,468 finished coat 286 finishing 287 floor coverings 83 flow-coating 282 fluorescence 229 fluorescence observations 229 focal emphysema 141 Food and Drug Administration (FDA) 422 forestry workers 181 formaldehyde 72 free-living amoebae 78 free radicals 15 fuel consumption 67 fungal spores 337 fungi 77,337 fungicides 422,433 furniture industry 184 furniture workers 181

gallium 405 General Agreement on Tariffs and Trade (GATT) 444 genetic activity 417 genetic evaluation 415 genotoxic carcinogenicity 416 genotoxicity of pesticides 416 genotoxity testing 427 geochemical environment 3 glycogen 369 glycol 272 glycol ethers 274 grinding 312,313 grinding processes 318 grinding-sharpening of hard metal pieces 317

H hair dye 384 haloacetonitriles 381 hard metal alloy filling 313 hard metal production 315 health control 120 health disorders 121 health effects 56,445 health examination 120 health problem 186 health status 3 heavier elements 8 herbicides 433 hexachlorocyclohexane (HCH) 434 high-exposure environments 404 high-strength steels 307 house dust mite 338 human bioeffluents 71 human studies 261 humidity of indoor air 347 hydrophilic substrates 227 hyperplasia 19 hypoplasia 207

I incidence of asthma 344 individual sensitivity 79 indoor air pollution 70 indoor surface pollution 73 industrial exposure to solvents 292 industrial machines 312 industrial metals 402

Index industrialized countries 437 infections 345 infectious diseases 78 insecticides 433 insulation materials 101 international trade 443 International Agency for Research on Cancer (lARC) 174 International Air Transport Association (lATA) 265 intoxication 20 intracellular dissipative structures 36 invertebrates 368 ion exchange 462 ionizing radiation 24 ionizing radiation 27 irritants 344 isoforms 241 isopropyl alcohol 274 job satisfaction

80

K ketones 272 kidney cells 235 kinetics 255 Langmuir equation 477 Langmuir isotherm 476 latent viruses 341 leaching of elements 461 lead 359,398 leukemia 27 leukemic cattle 9 leukemic cells 5 leukemic subjects 10 limit values for occupational exposure 262 liposomal system 232 liquid paints 267, 281 lithium 406 liver microsomes 225 logging workers 181 long-chain aliphatic hydrocarbon 493 long-term chronic health effects 447 low pressure pulverization (HVLP) 278 lung 111 lung cancer 127,129,191 lung cancer risk 130 lung disease 308

505

lung fibrosis 110 lung function 48 lung function measurements 153 lung function test 121 lung mineral content 137 lungs 5 lung silica content 163 lung tissues of construction workers 105 lymphatic cells 10 lymph vessels 221

M Macromia cingulata 369 macroscopic measurements 164 magnesium 10, 360 magnetic alloys 307 malignant mesothelioma 111, 113, 127 manganese 403 marine 275 massive fibrosis 162 medical elementology 1 medical surveillance 186 membrane-bound cytochrome P450 227 membrane-bound P450s 226 membrane topology 225 mercury 18,360,399 mercury in blood 361 metabolic disturbances 55 metabolism 221,259 metabolism of solvents 275 metabolites 212,221 metabolites of BaP 228 metal adsorptive losses 469 metal alloy filling 311 metal binding 464 metal exposure in farmers 360 metallic cobalt 307 metallic finishers 286 metals 395 metal turning 313 metal-organic interactions 472 methamidophos 446 methanol/water solution 230 method of curing 267 methylmercury 399 methyl-mercury aggravates 20 methyl mercury chloride 19 Mg-rich mineral water 22 microenvironmental variations 229 micronucleated erythrocites 424 microwaves 28

506

Health and Toxicology

Millipore apparatus 469 minerals 22 mining 165 mining regions 163 moisture control 347 mold growth 341 moldy dwellings 341 molecular cytogenetic techniques 427 monitoring of residues 442 morphology 139 mucous membrane irritation 69 mucous lining 357 multi-stage process 241 mycotoxins 6, 26

occupational safety and heals measures in the construction industry 100 odor complaints 69 office buildings 67 office machinery 82 oncoproteins in saliva 58 organic compounds 378 organic solvents 251,252 organogenesis 402 osmolality 454 outdoor air 73 outdoor air ventilation rates 81 oxygen-binding 226 ozone 330

N nasal carcinoma 172 nasal impairments 186 neoplasia 202 neoplasms 2, 3, 33, 38, 39 neoplastic disease 60 neoplastic disease risk 61 neoplastic tissue 37 neurotoxic 18 neurotoxic symptoms 69 neutron irradiation 197 nitrogen dioxide 330 nitrosoamines 26 non-ferrous metals 284 non-genotoxic mechanisms 41 non-industrial environments 68 non-ionic surfactants 496 non-ionizing radiation 24 non-metallic finishers 286 non-occupational exposure 185 non-smokers 161 non-specific reactions 273 nutritional prevention 32 nutritional prevention of diseases 23

o occupational cobalt exposure 314 occupational exposure 25,251 occupational exposure to organic solvents 252 occupational exposure to paint solvent 285 occupational heals 98 occupational health education 121 occupational limit values 255 occupational safety and heals 98

P. judaica 338 PAH carcinogenesis 223, 241 PAH in living cells 234 PAH in microsomes 232 PAHmetaboUsm 230 PAHs 222 PAHs in liposomes 230 paint baths 280 paint film 252 paint formulations 288 painting 251 paints 265,269 paint stripping 251, 256 paint-stripping techniques 264 panacinar (panlobular) emphysema 142 particle size 192 partition coefficients 467 pathological cells 8,12 pathology 55 penicillium 6 pesticide regulation 437 pesticide research 415 pesticide residues 443, 445 pesticide residues in food 433 pesticides 20,415,421,422 pesticide use 445 petroleum refining 25 phagocytic activity 16 photocopying 82 photon emission 15 physical curing 268 physico-chemical curing 268 physiological equilibrium 35 physiological functions 22 pig kidney 240

Index

pigments 271 pipefitters 110 placenta 19 plastic (PVC) 284 plastic material 266 pleural plaques 108,110,132 pneumatic pulverization 276 pneumoconiosis 47, 101, 163 pneumoconiosis measurement 145 pollen 338 polluted air 47 polycyclic aromatic hydrocarbons 26, 58 polycyclic aromatic hydrocarbons (PAHs) 219 polynuclear aromatic compounds 25 population exposure burden 207 porous polymers 253 post-mortem examination 165 presintering 317 pressing 317 pressurized spraying of paint 276 prevention of asbestos exposure 118 prevention of sinonasal cancer 186 prevention of tumors 31 primary prevention 1,41 production of asbestos 129 propylene glycol ethers 272 prostate glands 5 proteins 369 psychological factors 79 public health 1,23 pulmonary Cr burden 192,193 pulmonary effects of air pollutants 51 pulmonary emphysema 138 pulmonary function parameters 48 pulp and paper workers 181

R radioactive tracers 220, 474 radiochemical methods 474 radon-222 24 rate model 235 red cells 9 regulation of asbestos 102 relative humidity 74 residue levels 444 resin 472 respiratory diseases 47, 330 respiratory disorders 105,118 respiratory function changes 47 respiratory sensitivity 336

507

respiratory symptoms 341 respiratory tract 272 risk factor for bladder cancer 378 risk factors 1,17,20,28,47 risk of lung cancer 131, 132 risk of sinonasal cancer 174 RNAs 221 rodenticides 433 S-phenylmercapturic acid 210 salts 22 sampling 253 saturated amines 280 sawmill workers 181 scanning microscopy 9 Scatchard plot 478,479,481 seafood 18 seasonality 334 seed germination 367 selenium 10 selenium compounds 32 SEM 8 separation techniques 466 sequential leaching 473 settling particles 464 sharpening hard metal tools 314 short-term toxicity studies 441 sick building syndrome 67 sideroblastic anemia 9 silica 24, 144, 150, 162 sinonasal cancer 171,172,175 sinonasal cancer in woodworkers 179 sintering 319 skin cancer 27 skin irritation 69 smoking 17,111,153,157,378 sorbent material 466 sorption processes 462 sorption studies 461, 462, 466 source waters 378 specific gravity 455 spot urine 453 stages of carcinogenesis 36 standards 441 statistical methods 156 styrene 274 substitution 118 sugars 369 sulfur dioxide 330 sulphate 464

508

Health and Toxicology

suppressor genes 56 surfactants 493 surfactants bioconcentration 494, 495, 497,498 surfactants elimination 496 surfactant structure 497 suspended particulate matter 72 synergistic effects 342 synergistic relationships 347 tannery effluent 365 testing of pesticides 417 therapeutic metals 404 thermal comfort 74 tissue materials 196 tissue preparation 145 tobacco smoking 25 toluene 274 tool production 313 topology 228 toxic aluminum compounds 358 toxic chemicals 53 toxicity 20 toxicity in mammals 395 toxicity of dichloromethane 259 toxicity of environmental toxic elements 396 toxicity of gallium 405 toxicity of solvents found in paint 272 toxic metals 355 toxic pollutants 461 toxicological testing of pesticides 438 trace elements 2,3,461,462,475 trace metal losses 481 trans, trans-muconic acid 212 transport 221 trichloroethylene 426 trihalomethanes 381 tumor 37 tumors 38,421 types of wood dusts 176

u ultrafilters 468 ultrafiltration 466 ultraviolet radiation 24 under coat 286 uptake of surfactants 496

uranium 400 urban populations 127 urinary bladder 26 urinary bladder cancer 27 urine 61,454 use of asbestos 128

V vanadium 400 vapor pressure 264 ventilation 347 vibration syndrome 101 visual display units 83 vitamins 32 volatile organic compounds 71, 78 volatilization 436 voltammetric methods 473 volume parameters 48

W waterbome pathogens 380 water chemistry 378 water disinfection 377, 384 water pollution 365 water sources 389 weather phenomena 330 wet-process photocopy 83 wind 336 wood 284 wood dust 173, 174 wood dust exposure 171, 172 wooden furniture manufacture 173 wood panels 281 wood product makers 181 woodworkers 176 woodworking 175 work control 119 working conditions 171 working environment 185 working environmental control 119 X-ray analysis 105 X-ray microanalysis 8,21 xylenes 274

Z zinc 404