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ISSUES IN ENVIRONMENTAL SCIENCE AND TECHNOLOGY

EDITORS: R. E. HESTER AND R. M. HARRISON

ROYAL SOCIETY OF CHEMISTRY ISBN 0-85404-255-5 ISSN 1350-7583

A catalogue record for this book is available from the British Library

@ The Royal Society of Chemistry 1999

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Ronald E. Hester, BSc, DSc(London), PhD(Cornell), FRSC, CChem Ronald E. Rester is Professor of Chemistry in the University of York. He was for short periods a research fellow in Cam bridge and an assistant professor at Cornell before being appointed to a lectureship in chemistry in Y orkin 1965. Hehas been a full professor in York since 1983. His more than 300 publications are mainly in the area of vibrational spectroscopy, latterly focusing on time-resolved studies of photoreaction intermediates and on biomolecular systems in solution. He is active in environmental chemistry and is a founder member and former chairman of the Environment Group of the Royal Society ofChemistry and editor of'lndustry and the Environment in Perspective' (RSC, 1983) and 'Understanding Our Environment' (RSC, 1986). As a member of the Council of the UK Science and Engineering Research Council and several of its sub-committees, panels and boards, he has been heavily involved in national science policy and administration. He was, from 1991-93, a member of the UK Department of the Environment Advisory Committee on Hazardous Substances and is currently a member of the Publications and Information Board of the Royal Society of Chemistry.

Roy M. Harrison, BSc, PhD, DSc (Birmingham), FRSC, CChem, FRMetS, FRSH Roy M. Harrison is Queen Elizabeth II Birmingham Centenary Professor of Environmental Health in the University of Birmingham. He was previously Lecturer in Environmental Sciencesat the University ofLancaster and Reader and Director of the Institute of Aerosol Science at the University Qf Essex. His more than 250 publications aremainlyin the field of environmental chemistry, although his current work includes studies of human health impacts of atmospheric pollutants as well as research into the chemistry of pollution phenomena. He is a past Chairman of th~ Environment Group of the Royal Society ofChemistryfor whom he has edited 'Pollution: Causes, Effects and Control' (RSC, 1983; Third Edition, 1996) and 'Understanding our Environment: An Introduction to Environmental Chemistry and Pollution' (RSC, Third Edition, 1999). He has a close interest in scientific and policy aspects of air pollution, having been Chairman of the Department of Envi- ronment Quality of Urban Air Review Group as well as currently being a member of the DETR Expert Panel on Air Quality Standards and Photochemical Oxidants Review Group, the Department ofHealth Committee on the Medical Effects of Air Pollutants and Chair of the DETR Atmospheric Particles Expert Group.

XI Contributors

z. Billinghurst, Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PIA BAA, UK C. Botham, MRC Institute for Environment and Health, University of Leicester, 94 Regent Road, Leicester LEI 7DD, UK M. H. Depledge, Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PIA BAA, UK T. S. Galloway, Plymouth Environmental Research Centre, University of Plymouth, Drake Circus, Plymouth PIA BAA, UK P. Harrison, MRC Institute for Environment and Health, University of Leicester, 94 Regent Road, Leicester LEI 7DD, UK P. Holmes, MRC Institute for Environment and Health, University of Leicester, 94 Regent Road, Leicester LEI 7DD, UK D. E. Kime, Department of Animal and Plant Sciences, University of Sheffield, Sheffield SIO 2TN, UK A. F. Maciorowski, Prevention, Pesticides and Toxic Substances, Office of Science Coordination and Policy, US Environmental Protection Agency, 401 M Street SW, Washington, DC 20460, USA B. Phillips, MRC Institute for Environment and Health, University of Leicester, 94 Regent Road, Leicester LE1 7DD, UK G. E. Timm, Prevention, Pesticides and Toxic Substances, Office of Science Coordination and Policy, US Environmental Protection Agency, 401 M Street SW, Washington, DC 20460, USA K. J. Turner, MRC Reproductive Biology Unit, Centrefor Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, UK

XII] Contents

Overview of the Endocrine Disrupters Issue 1 Barry Phillips and Paul Harrison 1 The Emergence of Endocrine Disruption as a Toxicological Problem

2 The Expanding Definition of Endocrine Disruption 4

3 Human Health and Endocrine Disrupters 5

4 Endocrine Disruption and Wildlife 8

5 Proposed Mechanisms of Endocrine Disruption 11

6 Suspected EDs and Sources of Exposure 13

7 Methods of Identifying EDs 16

8 Estimation of Risk from EDs 19 9 Positions and Activities of Governments and International Organizations 21 10 Conclusions and Unanswered Questions 25

Environmentally Induced Endocrine Abnormalities in Fish 27 David E. Kime

1 Background 27 2 The Nature of Aquatic Pollution 28 3 The Endocrine System of Fish 30 4 Hypothalamic and Pituitary Abnormalities 34 5 Male Reproductive Problems in Fish 34 6 Female Reproductive Problems in Fish 37 7 Abnormalities in Growth, Metabolism and the Stress Response 42 8 Abnormal Liver Function 43 9 The Implications of Endocrine Dysfunction for Fish 44 10 Fish, Wildlife and Humans-A Warning 46 11 Conclusion 47

Issues in Environmental Science and Technology No.12 Endocrine Disrupting Chemicals @ The Royal Society of Chemistry, 1999

VU Contents

Effects of Endocrine Disrupting Chemicals in Invertebrates 49 Michael H. Depledge, Tamara S. Galloway and Zoe Billinghurst I Introduction 49 2 Endocrine Disrupting Chemicals 50 3 Endocrine Disrupters in Invertebrates 51 4 Invertebrate Endocrine Function 54 5 Evidence of Endocrine Disruption in Invertebrates 55 6 Detection and Assessment of Endocrine Disrupting Chemicals 57 7 Biomarkers of Endocrine Disruption 58 8 A Strategy for the Detection of Endocrine Disruption 59 9 Summary and Conclusions 60

Endocrine Disruption in Mammals, Birds, Reptiles and Amphibians 61 Catherine Botham, Philip Holrnes and Paul Harrison 1 Introduction 61 2 Evidence for Endocrine Disruption in Wildlife 62 3 Biological and Physicochcmical Modifiers of Endocrine Disrupter Exposure 75 4 Geographical Considerations and Implications for Recovery Rates 79 5 Conclusions 81 6 Acknowledgements 82

Oestrogens, Environrnentai Oestrogens and Male Reproduction 83 Katie J. Twner Introduction 83 Is Male Reproductive Health Deteriorating? 83 Is There a Common Aetiology? 87 Determinants of Fertility in Adulthood 88 Exposure to Oestrogen Is Associated with Impaired Male Reproductive Health 93 6 Effects of Oestrogcn on the Development and Function of the Male Reproductive System 95 7 Environmental Oestrogens 101 8 Are Humans at Risk? 105 9 Endocrine Disruption-Concluding Remarks 107 10 Acknowledgements 108

Human Health EHects of Phytoestrogens 109 Philip Holmes and Barry Phillips 1 Introduction 109 2 Potency of the Phytoestrogens 114

... Vlll Contents

3 Potential Beneficial Effects 114 4 Possible Causes for Concern 129 5 Conclusions 132 6 Acknowledgements 133

Endocrine Disrupter Research and Regulation in the United States 135 Anthony F. Maciorowski and Gary E. Timm

1 Introduction 135 2 The Need for Research and Science Policy 136 3 Endocrine Disrupter Screening, Testing and Regulatory Implementation 139 4 Implementation of the Endocrine Disrupter Screening Program 145 5 Conclusions 146

Subject Index 147

IX Overview of the Endocrine Disrupters Issue

BARRY PHILLIPS AND PAUL HARRISON

1 The emergence of Endocrine Disruption as a Toxicological Problem For a number of years, concern has been growing over changes in the health and fecundity of both humans and wildlife which may be associated with the disruption of hormonal systems by environmental chemicals.— The issue of environmental endocrine disrupters has become a focus of considerable media attention throughout the world and is now on the agenda of many expert groups, panels and steering committees of governmental organizations, industry and academia in Europe, the USA and Japan. The major findings driving this interest are derived from experimental and epidemiological studies on humans and wildlife, particularly those pertaining to effects on reproductive health which may result from exposure to endocrine disrupters early in life. It is pertinent to ask why endocrine disruption has become such an active and controversial issue in the last decade, and whether toxicology has neglected effects on the endocrine system in the past. It might reasonably be assumed that the effects of chemicals on the endocrine system, a vital and integral part of the biology of higher organisms, would be detected by long-established tests for

T. Colborn and C. Clement, Advances in Modern Environmental Toxicology: Volume XXI. Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, Princeton Scientific, New Jersey, 1992. J. Toppari, J. C. Larsen, P. Christiansen, A. Giwercman, P. Grandjean, L. J. Guillette Jr., B. Jegou, T. K. Jensen, P. Jouannet, N. Keiding, H. Leffers, J. A. McLachlan, O. Meyer, J. Muller, E. Rajpert-De Meyts, T. Scheike, R. Sharpe, J. Sumpter and N. E. Skakkebaek, Environ. Health Perspect., 1996, 104, 741. Institute for Environment and Health; Assessment A1, Environmental Oestrogens: Consequences to Human Health and Wildlife, IEH, Leicester, 1995. R. J. Kavlock, G. P. Daston, C. DeRosa, P. Fenner-Crisp, L. E. Gray, S. Kaatari, G. Lucier, M. Luster, M. J. Mac, C. Maczka, R. Miller, J. Moore, R. Rolland, G. Scott, D. M. Sheehan, T. Sinks and H. A. Tilson, Environ. Health Perspect., 1995, 104 (suppl. 4), 715. US Environmental Protection Agency, Special Report on Environmental Endocrine Disruption: An Effects Assessment and Analysis, EPA, Washington, 1997, EPA Report No. EPA/630/R-96/012.

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

1 B. Phillips and P. Harrison toxicity in experimental animals. For example, one might expect standard regulatory tests for reproductive toxicity in rodents to detect the consequences of disruption of sex hormone action. If a chemical had a biologically significant effect on reproductive capacity, then such tests would be expected to detect it, regardless of the mechanism involved. Indeed, many compounds have been tested for adverse effects on the reproductive system and in some cases these effects can be ascribed to, or at least include, disruption of part of the endocrine system. Ethanol, for example, could be said to be an endocrine disrupter in that it causes a variety of hormonal disturbances in experimental animals and humans. In female mice, rats, rabbits and monkeys it causes disturbances of the oestrus cycle, ovulatory function and fertility. In male rats, testicular atrophy and a decrease in the plasma levels of testosterone and luteinizing hormone has been observed. A lowering of plasma testosterone levels, leading sometimes to testicular atrophy and impotence, was also found in male alcoholics. When these effects were discovered, they were regarded as interesting and important but were not sufficient to trigger the rapid growth of a distinct new area of toxicology dedicated to endocrine disruption. With regard specifically to oestrogenic chemicals, the range of toxicological effects that they can produce, and their detectability by rodent toxicity tests, is well illustrated by work on the synthetic oestrogen diethylstilboestrol (DES). Used pharmaceutically from the late 1940s to the early 1970s to prevent abortions and pregnancy complications in women, DES was eventually found to increase abortions, neonatal deaths and premature births and to increase, post-pubertally, the incidence of clear-cell adenocarcinoma of the vagina of girls exposed in utero. A study of men exposed in utero showed that 31.5% had abnormalities of the reproductive tract compared with 7.8% of controls. The abnormalities included cryptorchidism and hypospadias. Sperm concentration and quality were also lower, although reduced fertility has not been observed in these men. Exposure of mice in utero induced very similar effects to those seen in humans. In 1979, the International Agency for Research on Cancer (IARC) concluded from the evidence then available that DES was causally associated with the occurrence of cancer in humans. At the same time, there was ‘sufficient evidence’ for its carcinogenicity in experimental animals; studies as early as the 1940s showed an increase in mammary tumours in mice. It is not certain that all the effects of DES can be ascribed to its oestrogenic activity (that is to say, directly related to its ability to bind to the oestrogen receptor), but it would appear from experience with this compound that rodent assays are able to detect the relevant toxicological effects. What then was the

T. J. Cicero, E. R. Meyer and R. D. Bell, J. Pharmacol. Exp. Ther., 1979, 208, 210. J. S. Gavaler and D. H. Van Thiel, Mutat. Res., 1987, 186, 267. A. L. Herbst, H. Ulfelder and D. C. Poskanzer, N. Engl. J. Med., 1971, 284, 878. W. B. Gill, G. F. B. Schumacher, M. Bibbo, F. H. I. Straus and H. W. Schoenberg, J. Urol., 1979, 122,36. A. J. Wilcox, D. D. Baird, C. R. Weinberg, P. P. Hornsby and A. L. Herbst, N. Engl. J. Med., 1995, 332, 1411. J. A. McLachlan, in Developmental Eﬀects of Diethylstilboestrol (DES) in Pregnancy, ed. A. L. Herbst and H. A. Bern, Thieme-Stratton, New York, 1981, p. 148. IARC, Evaluation of the Carcinogenic Risk of Chemicals to Humans; Volume 21: Sex Hormones (II), IARC, Lyon, 1979.

2 Overview of the Endocrine Disrupters Issue stimulus to the rapid growth of the endocrine disruption issue in the 1990s? As is usually the case, the convergence of several lines of enquiry was crucial. A number of worrying trends had been reported relating to male human reproductive health: declining sperm counts and increases in the incidence of testicular cancer, hypospadias and cryptorchidism. One suggested explanation for these trends was increasing exposure to certain environmental chemicals. By this time, a variety of adverse trends in the reproductive health of wildlife had also been noted and ascribed to pollution. In some cases, specific chemicals were implicated and endocrine disruption already suspected as a common mechanism. At the same time, evidence was emerging from a variety of experimental studies that many extensively used chemicals, often widely distributed in the environment, had the ability to bind to, and activate, oestrogen receptors. In general, their affinity for the receptor was very weak compared with the natural ligand or with synthetic oestrogens such as DES. However, their activity was seen as sufficient to support a working hypothesis that environmental chemicals might be damaging the reproductive health of human and wildlife populations by disrupting sex hormone action. A crucial factor in fuelling concern was the suspicion that chemicals acting through the medium of hormone receptors might, like the natural hormones, have profound effects at very low concentrations. The perceived conjunction of a threat to the survival of both human and wildlife populations led to a rapid and vigorous response from governments, international organizations, non-governmental environmental organizations and from the chemical industry. The nature of the response differed between organizations but encompassed the needs both for further research and practical measures to obviate the possible threat. In general, the following requirements were identified:

E Further research was needed to confirm the existence and severity of the reported adverse trends in the reproductive health of both humans and wildlife. E In cases where an adverse effect was confirmed, a definite, causative link with exposure to an environmental chemical or chemicals needed to be established. E Reliable methods were required for the detection of chemicals with the potential to cause the adverse effects identified. Existing methods might be sufficient but modifications or entirely new methods might be necessary. E Known and suspected endocrine disrupting chemicals needed to be ranked in order of priority for possible regulatory action. E Where appropriate, action should be taken to limit release of certain chemicals into the environment.

The priority given to each of these requirements is of course the most contentious issue. There is considerable disagreement about the standard of scientific proof needed to trigger regulation of a suspected endocrine disrupting chemical, reflecting the various interpretations of the ‘Precautionary Principle’ (broadly speaking, the concept of taking prudent action in advance of scientific certainty). Some action has already been taken to replace or reduce the use

T. O’Riordan and J. Cameron, Interpreting the Precautionary Principle, Earthscan, London, 1994.

3 B. Phillips and P. Harrison and/or release of particular chemicals where evidence of adverse effects due to endocrine disruption is clear, even in the absence of specific legislation or an agreed testing strategy. This has happened where field studies have suggested effects of particular chemicals on wildlife species, for example the effects of breakdown products of alkylphenol polyethoxylates (used in industrial detergents) on fish in some UK rivers. It should be emphasized that no such action has been based on effects on human health, since there is, at this time, no evidence directly to link such effects with exposure to endocrine disrupting chemicals.

2 The Expanding Definition of Endocrine Disruption Originally, the concern over endocrine disruption was based almost entirely on perceived effects on the reproductive system and it was usual to refer to the chemicals concerned as oestrogen mimics or oestrogenic chemicals. Later, chemicals were found that could block oestrogenic responses (anti-oestrogens) or androgenic responses (anti-androgens) and it was soon recognized that chemicals could affect other elements of the endocrine system via interaction with hormone receptors other than those of the sex steroids. The term endocrine disrupter is now preferred because it allows inclusion of health effects thought to result from interference with any part of the endocrine system, including thyroid, thymic and pituitary hormones. In order to establish consensus on the scope of the endocrine disrupter issue, to facilitate the identification of active chemicals and, ultimately, to underpin any future regulatory control, it is essential to agree a precise definition of an endocrine disrupter (ED). Such a definition was proposed at a major European Workshop on EDs. ‘An endocrine disrupter is an exogenous substance that causes adverse health effects in an intact organism, or its progeny, subsequent to changes in endocrine function.’ It was agreed at the workshop that endocrine disrupting activity could only be adequately defined in terms of effects in intact animals, be they juvenile or adult, or in the offspring of exposed parents. For many chemicals, evidence of endocrine disrupting activity has been obtained only by the use of in vitro models, such as hormone binding assays. It was accepted, therefore, that chemicals active in such models should be considered only as ‘potential’ EDs and should be distinguished from those established as active in vivo. For such chemicals, an alternative definition was recommended: ‘A potential endocrine disrupter is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism.’ However, several different definitions are also in current use:

E The US Environmental Protection Agency (EPA) Risk Assessment Forum: ‘An endocrine disrupter is an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural P. T. C. Harrison, P. Holmes and C. D. N. Humfrey, Sci. Total Environ., 1997, 205, 97. European Commission DGXII, Report EUR 17549; European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife, Brussels, 1997.

4 Overview of the Endocrine Disrupters Issue

hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development and/or behaviour.’ E The International Programme on Chemical Safety (IPCS): ‘An endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.’ and ‘A potential endocrine disrupter is an exogenous substance or mixture that possesses properties that might be expected to lead to endocrine disruption in an intact organism , or its progeny, or (sub)populations.’ E The working definition used in the final report of the US EPA’s Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC): ‘An endocrine disruptor is an exogenous chemical substance or mixture that alters the structure or function(s) of the endocrine system and causes adverse effects at the level of the organism, its progeny, populations, or subpopulations of organisms, based on scientific principles, data, weight-of-evidence, and the precautionary principle.’

A major difficulty which has been encountered with these definitions (identified as a particular problem by EDSTAC) is the definition of the term ‘adverse’. For a chemical to be judged an ED, it is important to show that the response seen has an adverse effect on the health or reproductive capacity of affected organisms or populations and is not just a change which falls within the normal range of physiological variation. A second problem concerns delimiting the mechanisms of action which should be included in the definition, to exclude effects which are a secondary consequence of overt toxicity in other body systems. For example, disruption to the endocrine system caused by general metabolic disturbance, such as in severe liver damage, should not be grounds for calling a chemical an ED.

3 Human Health and Endocrine Disrupters Eﬀects in humans for which links with exposure to endocrine disrupters have been suggested include the following.

Temporal Reductions in Sperm Counts and Quality A study of sperm counts conducted worldwide suggested that an annual fall of 0.8% had occurred between 1938 and 1990. Since then, falling sperm count and quality have been reported in a number of countries— and a recent study of International Programme on Chemical Safety, Report of IPCS/OECD Scoping Meeting on Endocrine Disrupters (EDCs), 16–18 March 1998, Washington, 1998. Endocrine Disruptor Screening and Testing Advisory Committee, Final Report,EPA,Washington, 1998. E. Carlsen, A. Giwercman, N. Keiding and N. E. Skakkebaek, Br. Med. J., 1992, 305, 609. J. Auger, J. M. Kunstmann, F. Czyglik and P. Jouannet, N. Engl. J. Med., 1995, 332, 281. S. Irvine, E. Cawood, D. Richardson, E. MacDonald and J. Aitken, Br. Med. J., 1996, 312, 467. K. Van Waeleghem, N. De Clercq, L. Vermeulen, F. Schoonijans and F. Comhaire, Hum. Reprod., 1996, 11, 325.

5 B. Phillips and P. Harrison testicular morphology in Finland suggested a reduction in spermatogenesis between 1981 and 1991. In contrast, no evidence for a decline in sperm counts or quality has been found at a number of locations within the USA, although considerable geographical variations in sperm counts were noted.— Despite uncertainties, there is general consensus that, in some countries at least, semen quality (sperm count, sperm morphology and/or sperm physiology) has declined. It is now apparent that determining trends in sperm counts and quality is extremely difficult, owing to geographical and cyclic variation and the influence of bias in the selection of subjects for study. There are also considerable concerns and uncertainties about the consistency and reliability of baseline data and measurement methodologies. While considerable attention is given to the question of male fertility, the incidence of infertility or sub-fertility in the population is difficult to measure and it is not known to what extent measures of semen quality, for example, are indicators of male fertility as such.

Increased Incidence of Testicular and Prostate Cancer The incidence of testicular cancer has increased quite dramatically in many countries with cancer registries, including Scandinavia, the countries around the Baltic Sea, Germany, the UK, the USA and New Zealand.— It is interesting that although the incidences of testicular cancer in Denmark and Finland are rising, rates in Finland are several times lower than in Denmark; further study of this apparent geographical gradient may give important clues on aetiology. The incidence of prostate cancer also appears to have risen in many countries.

Increased Incidence of Cryptorchidism and Hypospadias The incidence of congenital malformations such as cryptorchidism (undescended testes) and hypospadias (malformation of the penis) may have increased, but J. De Mouzon, A. Spira, P. Thonneau and L. Multigner, Br. Med. J., 1996, 313, 43. J. Pajarinen, P. Laippala, A. Penttila and P. J. Karhunen, Br. Med. J., 1997, 314, 13. H. Fisch, E. T. Goluboff, J. H. Olson, J. Feldshuh, S. J. Broder and D. H. Barod, Fertil. Steril., 1996, 64, 1009. H. Fisch and E. T. Goluboff, Fertil. Steril., 1996, 65, 1044. H. Fisch, E. F. Ikeguchi and E. T. Goluboff, Urology, 1996, 48, 909. C. A. Paulsen, N. G. Berman and C. Wang, Fertil. Steril., 1996, 65, 1015. P. Bromwich, J. Cohen, I. Stewart and A. Walker, Br. Med. J., 1994, 309, 19. L. Lerchl and E. Nieschlag, Exp. Clin. Endocrinol. Diabetes, 1996, 104, 301. L. M. Brown, L. M. Pottern, R. N. Hoover, S. S. Devesa, P. Aselton and J. T. Flannery, Int. J. Epidemiol., 1986, 15, 164. T. Hakulinen, A. A. Andersen, B. Malker, E. Rikkala, G. Shou and H. Tulinius, Acta Pathol. Microbiol. Immunol. Scand., 1986, 94A, (suppl. 288), 1. The Annual Report of the Chief Medical Officer of the Department of Health, On the State of The Public Health, HMSO, London, 1992. T. J. Wilkinson, B. M. Colls and P. J. Schluter, Br. J. Cancer, 1992, 65, 769. H.-O. Adami, R. Bergstro¨m,M.Mo¨ hner, W. Zatonski, H. Storm, A. Ekbom, S. Tretli, L. Teppo, H. Zeigler, M. Rahu, R. Gurevicius and A. Stengrevics, Int. J. Cancer, 1994, 59, 33. P. Boyle, P. Maisonneuve and P. Napalkov, J. Urol., 1995, 46, 47. R. M. Merrill and O. W. Brawley, Epidemiology, 1997, 8, 126.

6 Overview of the Endocrine Disrupters Issue these trends are diﬃcult to evaluate because of problems with recruiting individuals for analysis and registration of abnormalities at birth.

Altered Sex Ratios There have been suggestions of alterations in sex ratios following accidental environmental exposure to dioxin in Seveso, Italy, in 1976. Between 1977 and 1984, 74 births occurred in the most heavily contaminated zone which showed an excess of females (26 males and 48 females born). Preliminary evidence suggests that the excess was associated with high dioxin exposure in both parents. Over a later period, between 1985 and 1994, the ratio declined (60 males and 64 females) and was no longer statistically signiﬁcant.

Increased Incidence of Female Breast Cancer In women, the incidence of breast cancer has increased steadily over the past few decades in a number of countries including Finland, Denmark, USA and the UK. In Finland, for example, the incidence rose from 25 per 100 000 in 1953 to more than 40 per 100 000 in 1980. Although improved detection may be partly responsible, the underlying upward trend is estimated as about 1% per year since 1940. A number of factors that increase breast cancer risk have been identiﬁed, including diet, calorie intake and alcohol consumption, but lifetime exposure to oestrogens (age at menarche and menopause, use of contraceptive pill, etc.) is of major importance and environmental oestrogens might contribute to overall exposure and thereby to the rising incidence of the disease.

Neurological Effects It is known that the brain is one of the most sensitive sites of action of steroids in utero, and recently there have been suggestions that EDs may affect normal brain development and behaviour. For example, it has been alleged that in utero exposure to polychlorinated biphenyl compounds (PCBs) resulted in adverse effects on neurologic and intellectual function (memory and attention) in young children born to women who had eaten PCB contaminated fish in the USA. It has also been speculated that exposure to environmental pollutants with steroidal activity may be influencing human sexual development and sexually controlled behaviour.

Other Possible Eﬀects It has also been suggested that endocrine disruption may play a part in an World Health Organization, Congenital Malformations Worldwide: A Report from the International Clearinghouse for Birth Defects Monitoring Systems, Elsevier, Oxford, 1991, p. 113. P. Mocarelli, P. Brambilla, P. M. Gerthoux, D. G. Patterson and L. L. Needham, Lancet, 1996, 348, 409. M. S. Wolﬀ, P. G. Toniolo, E. W. Lee, M. Rivera and N. Dublin, J. Natl. Cancer Inst., 1993, 85, 648. M. Quinn, and E. Allen, Br. Med. J., 1995, 311, 1391. J. L. Jacobson and S. W. Jacobson, N. Engl. J. Med., 1996, 335, 783. P. L. Whitten, Adv. Mod. Environ. Toxicol.,1992, 21, 311.

7 B. Phillips and P. Harrison increased incidence of polycystic ovaries and endometriosis in women, cardiovascular disease, thyroid disorders and deﬁciencies in the immune system.

General Considerations It is not yet clearly established that any of the conditions listed above is evoked by, or associated with, endocrine disruption. However, they all have a component of sensitivity towards endocrine-active compounds. For example, tumours of the testis, prostate and female breast are generally sensitive and responsive to sex hormones. In fact, substances that inhibit the action of sex hormones are routinely used in the treatment of these cancers. Moreover, the production of sperm is under the control of sex hormones and may, therefore, be influenced by sex hormone-mimicking compounds. In addition, cryptorchidism and hypospadias are indices of disturbances in gonadal development which may be the result of alterations in sex hormonal function and/or metabolism in utero. Indeed, these congenital abnormalities may be biologically associated with testicular cancer and decreased sperm quality. Testicular cancer is more common in patients with cryptorchidism, and so an increase in the incidence of cryptorchidism parallel to the well-documented increase in testicular cancer would not be unexpected. This association has given rise to the so-called ‘unifying hypothesis’ of sex hormone disruption. On the other hand, there may be other reasons for the observed trends. As the changes have occurred over one or two generations, it is possible that the changing environment (including changes in lifestyle) may be at least partly responsible. A number of possible factors that might affect sperm production have been suggested, including dietary deficiency of selenium (a vital component of selenoenzymes which have a number of roles including the maintenance of normal sperm motility, testicular morphology and testosterone metabolism). Intakes of selenium are reported to be falling in Britain and Europe. Different types of underwear have also been reported to affect the production of sperm brought about by, for example, temperature changes caused by an increased tightness of fit. The epidemiological observations, in particular the apparent upward trends in testis, prostate and breast cancer incidences, are of concern and should be followed closely whether or not chemical endocrine disrupters are involved.

4 Endocrine Disruption and Wildlife Endocrine disruption has been postulated as the cause of a large number of adverse aﬀects on the health of various species of animals in the wild. The majority of cases involve reproductive abnormalities that might be linked to population declines. Data supporting causative associations between the biological

A. Giwercman, J. Grindsted, B. Hansen, O. M. Jensen and N. E. Skakkebaek, J. Urol., 1987, 138,1214. A. Prener, G. Engholm and O. M. Jensen, Epidemiology, 1996, 7, 14. R. M. Sharpe and N. E. Skakkebaek, Lancet, 1993, 341, 1392. M. P. Rayman, Br. Med. J., 1997, 314, 387. C. H. J. Tiemessen, J. L. H. Evers and R. S. G. M. Bots, Lancet, 1996, 347, 1844.

8 Overview of the Endocrine Disrupters Issue effects and exposure to a specific chemical agent are limited to a small number of cases, where exposure of animals to the suspect chemical under controlled conditions, sometimes in the laboratory, has reproduced the effects observed in the wild. In still fewer cases has a chemical effect in individual animals been demonstrated to have had a significant effect on the wild population as a whole. The impact of chemical pollution on the reproductive success and population sizes of wildlife species is often difficult to assess. In many cases, environmental factors such as habitat restriction, stress due to human intrusion and changes in natural food supplies owing to hunting, fishing and restocking policies may have a significant, even predominant, effect on population size. This makes it difficult to determine to what extent, if any, environmental endocrine disrupters may be contributing to observed effects on reproduction or population size in wildlife species. Effects on wildlife are important in their own right, but are also of significance to human health concerns because of the information that may be conveyed regarding possible parallel changes in humans. The adverse effects that have been identified and the compounds that have been implicated, with at least some degree of certainty, are summarized below.

Mammals Disturbed fertility of male Florida panthers, Baltic grey seals and Baltic ringed seals, common seals, and Beluga whales has been attributed to pollution by PCBs and other organochlorine chemicals. Disturbance of the immunological system of seals by a mechanism involving thymic hormone disruption by PCBs has been suggested as a causative factor in deaths from virus infection. Also, associations between reduced population growth in the European otter and increased tissue levels of PCBs have been demonstrated in the UK.

Birds Populations of several species of birds of prey, notably the peregrine falcon, declined worldwide during the 1950s and 1960s. It was found that eggshell thinning, with consequent reproductive failure, was being caused by exposure to organochlorine pesticides such as DDT. Reproductive failure, including eggshell thinning and embryo mortality in fish-eating birds in the Great Lakes area, has also been attributed to organochlorine contamination. A multigeneration study on captive American kestrels, in which females were treated with o,p-dicofol (closely related to DDT), showed effects in first and second generation C. F. Facemire, T. S. Gross and L. J. Guillette, Environ. Health Perspect., 1995, 103, (suppl. 4), 79. A. Bergman and M. Olsson, Finn. Game Res., 1985, 44, 47. P. J. H. Reijnders, Nature, 1986, 324, 456. A. Brouwer, P. J. H. Reijnders and J. H. Koeman, Aquatic Toxicol., 1989, 15, 99. S. De Guise, D. Martineau, P. Beland and M. Fournier, Environ. Health Perspect., 1995, 103, 73. P.Ross,R.deSwart,R.Addison,J.VanLoveren,J.VosandA.Osterhaus,Toxicology, 1996, 112,157. C. F. Mason, Cah. Etol., 1995, 15, 307. C. H. Walker, S. P. Hopkin, R. M. Sibly and D. B. Peakall, Principles of Ecotoxicology, Taylor & Francis, London, 1996.

9 B. Phillips and P. Harrison oﬀspring which included eggshell thinning, embryo mortality and feminization of the gonads of male embryos. Altered reproductive behaviour, following feminization of male embryos by DDT, was suggested as the cause of abnormal nesting behaviour in gulls, as demonstrated by abnormal clutch size and female-female pairing.

Reptiles One of the most extensively studied cases of possible endocrine disruption in wildlife was that of the alligators in Lake Apopka in Florida, USA, whose population declined in the 1980s following a major spillage of chemicals, including the organochlorine pesticide dicofol. Subsequent studies found that, compared with alligators from a less polluted site (Lake Woodruff), both male and female animals from Apopka had abnormally developed reproductive organs and altered blood hormonal patterns. Fertility in males was adversely affected and egg viability in the contaminated lake was significantly reduced. Eggs of the snapping turtle from sites in the Great Lakes were found to have high organochlorine levels and a significant increase in embryo deformity and mortality compared with those from less polluted sites. PCBs have also been shown to modify the sex determination mechanisms of red-eared slider turtles.

Fish Substances in sewage and industrial effluent have been shown to cause a number of changes in fish, including intersex, production in adult males and juvenile forms of the female yolk precursor lipoprotein vitellogenin, and abnormal testicular development. A combination of laboratory and field studies, mainly on rainbow trout and roach, have suggested that the effects, in the UK, are caused by oestrogenic hormones from human waste and also by alkylphenols.— Most of the work to date on oestrogenic effects of sewage on fish has been conducted on freshwater species, although laboratory and field experiments have shown vitellogenin induction in the males of a number of estuarine and marine

K. N. M. MacLellan, D. M. Bird, D. M. Fry and J. L. Cowles, Arch. Environ. Contam. Toxicol., 1996, 30, 364. D. M. Fry, Environ. Health Perspect., 1995, 103 (suppl. 7), 165. G. A. Fox, in Advances in Modern Environmental Toxicology: Volume XXI. Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, ed. T. Colborn and C. Clement, Princeton Scientific, New Jersey, 1992. L. J. Guillette Jr. and D. A. Crain, Comment Toxicol., 1996, 5, 381. D. Crews, J. M. Bergeron and J. A. McLachlan, Environ. Health Perspect., 1995, 103, 73. C.E. Purdom, P. A. Hardiman, V. J. Bye, N. C. Eno, C. R. Tyler and J. P. Sumpter, Chem. Ecol., 1994, 8, 275. C. Desbrow, E. Routledge, D. Sheehan, M. Waldock and J. Sumpter, in The Identification and Assessment of Oestrogen Substances in Sewage Treatment Works Effluents, Environment Agency, Bristol, 1996. J. E. Harries, D. A. Sheahan, S. Jobling, P. Matthiessen, P. Neall, E. J. Routledge, R. Rycroft, J. P. Sumpter and T. Tylor, Environ. Toxicol. Chem., 1996, 15, 1993. S. Jobling, D. Sheahan, J. A. Osborne, P. Matthiessen and J. P. Sumpter, Environ. Toxicol. Chem., 1996, 15, 194.

10 Overview of the Endocrine Disrupters Issue species. In the US, delayed sexual development, altered blood sex hormone profiles, impaired reproductive performance and masculinization have been found in fish exposed to Kraft pulp mill effluents. The active chemical is unknown, although b-sitosterol, a major constituent, has been shown to be oestrogenically active. The significance of these effects on fish at the population level is as yet unknown.

Marine Molluscs Changes in the reproductive organs of mature female neogastropods, particularly the dogwhelk (Nucella lapillus), have been shown to be caused by exposure to tributlytin (TBT), used in anti-fouling paints on boats and harbour equipment. In marinas and harbours polluted with TBT, female molluscs were found with a condition commonly called ‘imposex’ where the reproductive organs of females partly resemble those of males, preventing reproduction. This observation is currently the only example of chemical-mediated endocrine disruption which has resulted in a demonstrable effect at the population level. Other effects reported in molluscs attributable to TBT, for which mechanisms have yet to be elucidated, include altered larval behaviour and development in the juvenile stages of the bivalve Scrobicularia. Few studies have been conducted on other invertebrate taxa but pentachlorophenol exposure has been shown to affect daphnid reproductive capacity and sex steroid hormone metabolism and a high incidence of intersex has been observed in harpacticoid copepods near an Edinburgh sewage outfall.

5 Proposed Mechanisms of Endocrine Disruption There are many possible mechanisms by which chemicals may interact with the endocrine system, some of which are discussed below.

Interaction with Hormone Receptors In the initial phase of development of endocrine disruption research, attention Y. Allen, J. E. Thain, P. Matthiessen and S. Haworth, in Seventh Annual Meeting of SETAC-Europe, Amsterdam, Netherlands, Society of Environmental Toxicology and Chemistry, Brussels, 1997, p. 45. K. Hylland, B. Braaten, F. R. Knudsen and C. Haux, in Seventh Annual Meeting of SETAC-Europe, Amsterdam, Netherlands, Society of Environmental Toxicology and Chemistry, Brussels, 1997, p. 45. K. R. Munkittrick, C. B. Portt, G. J. van der Kraak, I. R. Smith and D. A. Rokosh, Can. J. Fish Aquat. Sci., 1991, 48, 1371. W. P. Davis and S. A. Bortone, Advances in Modern Environmental Toxicology: Volume XXI. Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, ed. T. Colborn and C. Clement, Princeton Scientiﬁc, New Jersey, 1992, p. 113. D. L. MacLatchy, Z. Yao, L. Tremblay and G. J. Van Der Kraak, in Proceedings of the 5th International Symposium on Reproductive Physiology of Fish, University of Texas, Austin, 1997, p. 189. P. E. Gibbs and G. W. Bryan, in Biomonitoring of Coastal Waters and Estuaries, ed. K. J. M. Kramer, CRC Press, Boca Raton, FL, 1994, p. 205. W. J. Langston, Toxicol. Ecotoxicol. News/Rev., 1996, 3, 179. J. M. Ruiz, G. W. Bryan, G. D. Wigham and P. E. Gibbs, Mar. Environ. Res., 1995, 40, 363. L. G. Parks and G. A LeBlanc, Aquatic Toxicol., 1996, 34, 291. C. G. Moore and J. M. Stevenson, J. Nat. History, 1994, 28, 1213

11 B. Phillips and P. Harrison

Table 1 Hormonal Type of activity! cross-reactivity of three progestogens (adapted Anti- Andro- Gluco- from Edgren) Compound Progestational Oestrogenic oestrogenic genic corticoid Progesterone ; 9;9; Norethisterone ; ;;;9 Medroxy- ; 9;;; progesterone

!; Active, 9 inactive.

was focused almost exclusively on so-called oestrogen mimics: chemicals which induced biological responses normally associated with the action of natural oestrogens. Since oestrogens act by binding to specific receptors in target tissues, the ability of a chemical to bind to the same receptors was taken as sufficient evidence to define it as an oestrogen mimic. Of course, the situation is not that simple. Chemicals may bind to the oestrogen receptor with vastly different affinities and the responses induced are not necessarily related directly to binding affinity. Thus, some chemicals which bind poorly can nevertheless induce a response while others may bind avidly but, inducing a very weak response, serve only to block the binding of endogenous oestrogen. The situation has recently become more complex with the discovery of two distinct classes of oestrogen receptor (alpha and beta) with different tissue distributions and binding characteristics. With the expansion of the definition of endocrine disruption, EDs now include chemicals which can interact with any hormone receptor, most notably androgen, thyroid and progestogen hormone receptors. Some compounds can bind to more than one receptor and a complex array of interactions is possible. As shown in Table 1, progesterone, norethisterone and medroxyproges- terone are all recognized progestagens but also have other types of hormonal activity. More subtle modes of action are also possible since the response to hormone receptor binding is complex and could be affected by chemical interference with receptor-related proteins, DNA methylation or histone acetylation. Dioxin (TCDD), for example, reduces the ability of the oestrogen—receptor complex to bind to the oestrogen response element of DNA, reducing gene transcription.

Eﬀects on Hormone Metabolism The levels of circulating natural hormones can be altered by chemical interference with the synthesis or breakdown of the hormone by mechanisms not necessarily mediated through hormone receptors. For example, the phytoestrogen b-sitosterol is able to reduce gonadal steroid biosynthesis by either aﬀecting cholesterol availability or by altering the activity of P450-dependent enzymes. Tributyltin inhibits the conversion of androgens to oestrogens in neogastropods by inhibiting aromatase or by inhibiting testosterone metabolism and excretion. I. Kharat and F. Saatcioglu, J. Biol. Chem., 1996, 271, 10 533. G. Majdic, R. M. Sharpe, P. J. Oshaughnessy and P. T. K. Saunders, Endocrinology, 1996, 137, 1063. C. Bettin, J. Oehlmann and E. Stroben, Helgol. Meersunters., 1996, 50, 299.

12 Overview of the Endocrine Disrupters Issue

PCBs and dioxins are well known for their ability to induce certain iso-enzymes of P450 in the mammalian liver. Some of these iso-enzymes are involved in the metabolism of steroids, and it is possible that changes in rates of metabolism might disturb hormone levels.

Eﬀects on Hormone Receptor Numbers The responsiveness of a tissue to a hormone depends on the density of receptors within its component cells. The number of receptors is determined by their rate of synthesis and catabolism, which is itself controlled by complex feedback mechanisms involving hormone action. Some chemicals are known to interfere with this regulation. For example, TCDD can act to increase or decrease the expression of the oestrogen receptor.

Effects on Synthesis, Storage, Release, Transport and Clearance of Hormones There are many ways in which endocrine disrupters might affect the levels of hormones circulating in the bloodstream. For example, lipid soluble hormones, including sex steroids, thyroid hormones and glucocorticoids, are transported bound to carrier proteins and their effects are to some extent influenced by the levels of these proteins in the blood. In mammals, oestrogens increase the concentration of sex hormone binding globulin (SHBG) in the plasma, whereas androgens decrease it. It is possible, therefore, that EDs might have similar effects.

6 Suspected EDs and Sources of Exposure Very many substances are suspected of being EDs. These include both naturally occurring and anthropogenic chemicals and some of the most important are listed in Table 2. The main sources of human exposure to EDs are as follows.

Food Food is the most important source of human exposure to EDs, with the possible exception of pharmaceutical hormone administration, and the majority is as naturally occurring phytoestrogens. Phytoestrogens, principally lignans (e.g. enterodiol and enterolactone which are formed in the intestine) and isoﬂavones (e.g. daidzein and genistein), are constitutents of many foodstuﬀs including beans, sprouts, cabbage, spinach, soybean, grains and hops. These naturally occurring chemicals have many structural similarities to 17b-oestradiol and are more

M. J. J. Ronis and A. Z. Mason, Mar. Environ. Res., 1996, 42, 161. J. A. Goldstein and S. Safe, in Halogenated Biphenyls, Naphthalenes, Bibenzodioxins and Related Compounds, ed. R. D. Kimborough and A. A. Jensen, Elsevier, Amsterdam, 1989, p. 239. M. Romkes, J. Piskorskapliszczynska and S. Safe, Toxicol. Appl. Pharmacol., 1987, 87, 306. S. H. Ingbar and K. A. Woeber, in Textbook of Endocrinology, ed. R. H. Williams, Saunders, Philadelphia, 1974, p. 95. S. H. Safe, Environ. Health Perspect., 1995, 103, 346.

13 B. Phillips and P. Harrison

Table 2 Some known or Compounds Occurrence suspected endocrine disrupting chemicals Natural and synthetic Natural hormones, augmented by hormonal drugs hormones such those used as oral contraceptives, are excreted by humans and animals and occur in sewage Phytoestrogens Natural constitutents of many foodstuffs including beans, sprouts, cabbage, spinach, soybean, grains and hops. The major classes are lignans and isoflavones (e.g. daidzein and genistein) Mycotoxins Produced by fungi which can contaminate crops. Some, such as zearalenone, are oestrogenic Organochlorine pesticides DDT, lindane and beta-HCH are common, persistent environmental pollutants Polychlorinated biphenyls Widespread, persistent environmental pollutants (PCBs) Alkylphenol Non-ionic surfactants used in detergents, paints, polyethoxylates (APEs) herbicides, pesticides and plastics. Breakdown products, such as nonylphenol and octylphenol, are found in sewage and industrial effluents Dioxins Products of combustion of many materials Phthalate esters Widely used as plasticisers for PVC. Common environmental pollutants Bisphenol A A component of polycarbonate plastics and epoxy resins used to line food cans

potent oestrogens in vitro than many of the man-made chemicals tested to date. There is much current interest and debate regarding the significance of human exposure to phytoestrogens in the diet, particularly in respect of infant diets. As mentioned above, there is evidence that hormonal disturbance may have its greatest effect during the early stages of life, including the first few months after birth. The inclusion in infant milk formulae of soya products, containing isoflavones, has therefore caused some concern. However, phytoestrogens do not appear to bioaccumulate and the relative significance of exposure to natural and synthetic EDs remains to be clarified. It has been speculated that animals may have become adapted to dietary phytoestrogens through co-evolution with plants, while exposure to man-made chemicals is too recent for this to have occurred. Meat, dairy products and eggs contain low levels of natural hormones, including oestrogens, progesterone and testosterone, possibly enhanced by the use of hormones for veterinary purposes or as growth enhancers. Many materials used for food packaging contain chemicals which can migrate into the food in small quantities. These include plasticizers such as phthalate esters, used in wrapping materials, and bisphenol A, used in the resin linings of

R. E. Chapin, J. T. Stevens, C. L. Hughes, W. R. Kelce, R. A. Hess and G. P. Daston, Fundam. Appl. Toxicol., 1996, 29,1. S. Hartmann, M. Lacorn and H. Steinhart, Food Chem., 1998, 62,7. UK Ministry of Agriculture Fisheries and Food, Food Surveillance Information Sheet Number 82: Phthalates in Food, MAFF Publications, London, 1996.

14 Overview of the Endocrine Disrupters Issue food cans. Organochlorine pesticides such as DDT, chlorinated cyclohexanes and vinclozolin, many of which are no longer in general use, are very persistent and can be found in food many years after they were last used in the area of origin. Food may sometimes be contaminated also by oestrogenic mycotoxins.

Pharmaceuticals Drugs with endocrinological functions or side-eﬀects are common, but oestrogenic hormones in oral contraceptives are particularly widely used. Pharmaceuticals and their metabolites eventually ﬁnd their way into the environment, predominantly via excretion into sewage.

Occupational Exposure Occupational exposure to EDs presents a potential risk which needs to be further evaluated. There is good experimental and epidemiological evidence to suggest that occupational exposure to chemicals such as inorganic lead, manganese and mercury, and to organic chemicals such as dibromochloropropane (DBCP), ethylene glycol and carbon disulﬁde, can produce adverse eﬀects on the male reproductive system. For many other agents, including most EDs, associations are either only suggested or suspected.

Air A number of airborne chemical contaminants are EDs, particularly products of combustion such as dioxins and polycyclic aromatic hydrocarbons.

Drinking Water Groundwater is vulnerable to pollution by chemicals carried by rainwater, leaching from waste sites or from waste water carrying industrial or agricultural eﬄuent. Treatment of drinking water may remove some, but not all, of these contaminants. Some polycarbonate or metal water pipes that are lined with epoxy resin lacquers may release bisphenol A. Exposure of wildlife to EDs also occurs via their food and in most ecosystems there is a tendency for persistent chemicals to bioaccumulate and biomagnify; organisms higher up the food chain accumulate more of the chemical than those

J. A. Brotons, M. F. Olea-Serrano, M. Villalobos, V. Pedraza and N. Olea, Environ. Health. Perspect., 1995, 103, 608. UK Ministry of Agriculture Fisheries and Food and Health and Safety Executive, Annual Report of the Working Party on Pesticide Residues in Food (1997), MAFF Publications, London, 1997. IARC, Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans; Volume 56: Some Naturally Occurring Substances: Food Items and Constituents, Heterocyclic Aromatic Amines and Mycotoxins, WHO, Geneva, 1979, p. 397. L. D. Arcand-Hoy, A. C. Nimrod and W. H. Benson, Int. J. Toxicol., 1998, 17, 139. S. Tas, R. Lauwerys and D. Lison, Crit. Rev. Toxicol., 1996, 26, 261. J. H. Clemons, L. M. Allan, C. H. Marvin, Z. Wu, B. E. McCarry, D. W. Bryant and T. R. Zacharewski, Environ. Sci. Technol., 1998, 32, 1853.

15 B. Phillips and P. Harrison lower in the chain. Aquatic wildlife is especially sensitive to water-borne EDs to which they are constantly exposed. Surface waters, rivers and lakes, and the sea are very vulnerable to contamination by waste and airborne pollutants. Of all the uncertainties surrounding the hypothesis that environmental chemicals with endocrine disrupting properties are responsible for the observed eﬀects in humans and wildlife, one of the major unknowns relates to exposure. Humans and wildlife can be, and sometimes are, exposed to these substances in the environment but our knowledge of the levels, routes and timing of exposure is poor. The types of chemical likely to be of most importance include:

E Persistent and semi-persistent lipophilic (fat soluble) substances known to accumulate in fatty tissue. E Persistent or semi-persistent metabolites of compounds with lipophilic properties. E Substances, including metabolites, that bind to transport proteins and/or receptors in the body. E Hydrophilic substances and/or compounds with a relatively high reactivity, present at a steady-state concentration in biota due to high external exposures.

Human exposure to environmental contaminants has been investigated through the analysis of adipose tissue, breast milk, blood and the monitoring of faecal and urinary excretion levels. However, while levels of persistent contaminants in human milk, for example, are extensively monitored, very little is known about foetal exposure to xenobiotics because the concentrations of persistent compounds in blood and trans-placental transmission are less well studied. Also, more information is needed in general about the behaviour of endocrine disruptive compounds (and their metabolites) in vivo, for example the way they bind to blood plasma proteins. It is also important to develop an understanding of the movement of chemicals through the environment by investigating their fate and behaviour. Based on a chemical’s inherent physicochemical properties, it is possible to predict with some degree of certainty which environmental compartment it is likely to reside in and to what extent it is likely to be bioavailable and accumulate through the food chain.

7 Methods of Identifying EDs Many diﬀerent test systems have been used to investigate the ability of chemicals to interact with components of the endocrine system. The usefulness and applicability of the available methods has been the subject of much debate. Four key texts are particularly helpful in reviewing and giving guidance on currently available test methods and strategies for testing EDs.—

SETAC-Europe/OECD/EC, Expert Workshop on Endocrine Modulators and Wildlife: Assessment and Testing, SETAC-Europe, Brussels, 1997. ECETOC, Document No. 33; Environmental Oestrogens: A Compendium of Test Methods, ECETOC, Brussels, 1996.

16 Overview of the Endocrine Disrupters Issue

The methods range from cell-free in vitro systems for examining specific hormone receptor binding capacity to multigeneration reproductive studies in experimental animals. The in vitro methods have advantages in terms of rapidity, low cost and saving on the use of animals. They can also be very helpful in elucidating mechanisms of action. Many chemicals which are currently suspected of being EDs have been implicated principally on the basis of in vitro test results, notably the stimulation of cell proliferation in the oestrogen-responsive human breast cancer cell line MCF-7, or induction of marker enzyme synthesis in genetically engineered yeast cells containing the human oestrogen receptor. However, in vitro tests cannot take account of all the complex interactions possible in a whole organism, including metabolism and excretion of the test chemical and interactions between the various elements of the endocrine system. They cannot accurately predict the outcome of exposure of an organism to a chemical at a particular concentration. The in vitro tests are therefore useful for identifying potential EDs and investigating mechanisms of action. For the definitive demonstration of ED disrupting activity it is necessary to perform animal tests with specific, validated endpoints of relevance to the health of animals and humans. For the detection of oestrogenic activity, for example, the mouse uterotrophic assay, measuring the ability of a chemical to induce cell proliferation in the uterus, is a widely used short-term test. However, a comprehensive assessment of all possible reproductive effects of a chemical requires a multi-generation study, including examination of many aspects of fertility and reproductive health in both males, females and their offspring. There is a broad international consensus on the general strategy for detecting EDs, although there are at present no internationally agreed standardized test methods. The overall approach can be summed up as follows:

E Testing to identify hazards should be tiered, starting with short-term screening, followed by subchronic and chronic tests. E Tests should be standardized. E In vivo tests will be required in all three tiers, including the short-term screen. E Several vertebrate groups will be needed for testing. E Much basic research is needed on tests using invertebrates. E Research is needed on developing the triggers to move from one hazard tier to the next and on risk assessment methods.

The main conclusion of an expert workshop on endocrine modulators and wildlife in 1997 was that some existing test methods, as defined in guidelines published by the Organization for Economic Cooperation and Development (OECD), could be adapted to incorporate specific endocrine disrupting endpoints, but that there might also be the need to develop new tests, e.g. for fish. On behalf of the UK Government, the MRC Institute for Environment and Health (IEH)

Organization for Economic Co-operation and Development, Draft Detailed Review Paper: Appraisal of Test Methods for Sex-Hormone Disrupting Chemicals. OECD Environmental Health and Safety Publications, Environment Directorate, OECD, Paris, 1997. G. T. Ankley, R. D. Johnson, G. Toth, L. C. Folmar, N. E. Detenbeck and S. P. Bradbury, Rev. Toxicol., 1998, 1, 71.

17 B. Phillips and P. Harrison produced a Detailed Review Paper for the OECD on test methods for sex hormone disrupting chemicals, covering oestrogen and androgen agonists and antagonists. This critically assessed existing OECD test methods for their ability to identify sex hormone disrupting activity and suggested modifications or additions to these tests, where appropriate. In addition, a number of in vitro and in vivo assays were identified as having potential for use in a testing strategy. In the USA, the EDSTAC has recently published a strategy for testing chemicals for endocrine disrupting activity. This involves a number of steps: initial sorting of chemicals (based on existing data), priority setting (based on knowledge of exposure, effects and statutory criteria), tier 1 screening and tier 2 testing. Tier 1 screening is designed to detect direct interaction with the endocrine system and includes the following tests:

In vitro Assays E Oestrogen receptor binding/Reporter gene assay. E Androgen receptor binding/Reporter gene assay. E Steroidogenesis assay with minced testis.

In vivo Assays E Rodent 3-day uterotrophic assay: increase in uterine weight in ovariectomised rat. E Rodent 20-day pubertal female with thyroid: age of rats at time of vaginal opening. E Rodent 5—7-day Hershberger assay: change in weight of prostate and seminal vesicles in castrated rats. E Frog metamorphosis assay: rate of tail resorption in Xenopus laevis. E Fish gonadal recrudescence assay: eﬀects on light and temperature sensitive sexual maturation.

Tier 2 is intended to determine and characterize the eﬀects of the chemical on the endocrine system and includes:

E Two-generation mammalian reproductive toxicity study or a less comprehensive test. E Avian reproduction test. E Fish life-cycle test. E Mysid (shrimp) life-cycle test. E Amphibian development and reproduction test.

The state of development of the EDSTAC strategy, and progress to date, is described in Section 9, below.

18 Overview of the Endocrine Disrupters Issue

8 Estimation of Risk from EDs

Risk of What? EDs pose particularly difficult problems for risk assessment. Ideally, estimates of risk should be based on observations of relevant biological responses in a suitable organism, using a range of concentrations wide enough to define the dose—response relationship. For the vast majority of EDs, the necessary data are not available and a major stumbling-block to obtaining the data is disagreement, or genuine uncertainty, about what constitutes a ‘relevant’ biological response. The wide variety of adverse health effects that are of concern, in humans and many other species, cannot as yet be modelled by one test or even a reasonable battery of tests. Before valid risk assessments can be made, it will be necessary to define precisely which risks are considered important and to conduct validated tests to estimate quantitatively the ability of a chemical to cause the relevant changes.

Exposure-based Risk Assessment One approach to assessing the likely impact of environmental EDs on the health of humans and wildlife is to compare the endocrine modulating activity of anthropogenic EDs with that of naturally occurring hormones and related substances and then to relate this ‘relative potency’ to exposure to the two types of chemical. Such estimates have been made for exposure to chemicals with oestrogenic (and anti-oestrogenic) properties. For example, Safe carried out a mass/potency balance analysis to estimate daily human exposure to environmental and dietary oestrogens in terms of oestrogen equivalents (EQs) and anti-oestrogen equivalents (2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD] anti-oestrogen equivalents; TEQs), based on oestrogenic or anti-oestrogenic activity in vitro. It was concluded that, with the exception of pharmaceutical hormone administration, the major human intake of oestrogenic chemicals was from naturally occurring oestrogens in foods. Against this background, the contribution to oestrogenic activity of exposure to organochlorine compounds appears negligible. Estimates of relative potency must be treated with caution. The potency of two substances can only be compared if their dose—response curves are well defined and parallel. In addition, a clear distinction should be made between affinity and efficacy when considering interactions with receptors. While affinity describes the ability of a substance to bind to a receptor, efficacy describes its ability to produce an effect. Thus, a full agonist has both high affinity and high efficacy, a partial agonist may have either high affinity and low efficacy or vice versa, and an antagonist may have varying degrees of affinity but low, or no, efficacy (i.e. binds but does not produce an effect). In different assays, and under different conditions, an agonist may be either partial of full. For example, using a recombinant yeast screen, Harris et al. showed that butyl benzyl phthalate could be considered as either a partial oestrogen agonist (following a 4-day

C. A. Harris, P. Henttu, M. G. Parker and J. P. Sumpter, Environ. Health Perspect., 1996, 105, 802.

19 B. Phillips and P. Harrison

Table 3 An estimate of Estimated Potency daily human exposure to Source exposure/day in vitro! EQ (kg/day)" oestrogenic and anti- oestrogenic chemicals Oestrogens (adapted from Safe) Morning after pill 333.5 mg 1 333 500 Birth control pill 16.675 mg 1 16 675 Post-menopausal therapy 3.35 mg 1 3350 Flavonoids in food 1020 mg 0.0001 102 Organochlorine environmental oestrogens 2.5 kg 0.000001 0.0000025 Anti-oestrogens TEQ (kg/day) TCDD and organochlorines# 80—120 pg 1 0.000080—0.00012 Polycyclic aromatic hydrocarbons in food 1.2—5.0 kg 0.001 0.0012—0.005 Indolo[3,2-b]carbazole in 100 g brussels sprouts 0.25—1.28 kg 0.001 0.000250—0.00128

!Potency of compound relative to 17b-estradiol (oestrogenicity) or TCDD (anti-oestrogenicity) determined from cell culture assays. "Daily exposure in dose equivalents, using 17b-estradiol activity for oestrogenicity (EQ) and TCDD for anti-oestrogenic activity (TEQ). #TCDD : 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

incubation period) or as a full oestrogen agonist (following a 13-day incubation period). Edgren carried out a number of studies in which bioassays were used to compare the widely differing potencies of a number of analogues of 17b-oestradiol that were modified in the 18-position. Commenting on the use of potency ratios, Edgren concluded that they were only valid for specific substances and test systems and ‘useless for product safety testing’. These problems could have important consequences for any attempt to establish the potency of specific environmental EDs or environmentally relevant mixtures.

Interactions between EDs Risk assessment for EDs is further complicated by the fact that any hormonal activity of an exogenous chemical will occur against a background of the activity of endogenous hormones, the levels of which may themselves be variable. In addition, EDs may be either agonistic or antagonistic in relation to normal hormone activity and combinations of different EDs may not have an additive effect. The issue of interactions between EDs in mixtures has received much attention. A paper purporting to show synergistic effects of binary combinations of some environmentally relevant chemicals (dieldrin, chlordane, endosulfan and toxaphene) in an in vitro yeast-based oestrogen assay was later withdrawn.

J. A. Edgren, in Pharmacology of the Contraceptive Steroids, ed. J. W. Goldzieher, Raven Press, New York, 1994, p. 81. S. F. Arnold, D. M. Klotz, B. M. Collins, P. M. Vonier, L. J. Guillette Jr. and J. A. McLachlan, Science, 1996, 272, 1489.

20 Overview of the Endocrine Disrupters Issue

Many groups throughout the world tried to repeat the experiments using similar in vitro assays and other test systems, but without success. Current experimental data suggest additive interactions do occur between EDs, but the issue of interactive eﬀects, and synergism in particular, will undoubtedly remain a topic of intense debate for some time to come.

Problems of Extrapolation In addition to exposure, a number of other parameters need to be considered in an overall assessment of the likely effects of EDs. Rates of absorption, metabolism and excretion, bioaccumulation and protein binding (e.g. to sex hormone binding globulin) may significantly affect the outcome of a given exposure. One important point that has already been mentioned is that critical periods of sensitivity to EDs may exist during the life cycle of animals and humans. For example, exposure to EDs could be particularly important during foetal development, around the time of birth and perhaps at puberty when unique changes in morphology and physiology are taking place. For example, in salmonid fish, exposure to exogenous oestrogens can cause feminization, but only if this occurs during a narrow period of about 10 days either side of egg hatching. Timing of exposure will also determine whether or not effects are likely to be reversible; effects on maturation can be reversible while effects on sexual differentiation are usually irreversible. A fundamental question which has caused considerable controversy concerns the validity of assuming that the effects of EDs are related to exposure in a linear fashion. It is well known in endocrinology that whilst low concentrations of hormones may induce a tissue response which increases with concentration, hormone concentrations above a certain level often result in an increasingly suppressive effect. Experime’ntal data have indicated just such an inverted-U-shaped dose—response relationship for DES. In this study, prostate weight in 8-month-old male offspring of pregnant mice fed between 0.02 and 200 kg DES/kg bodyweight/day on gestation days 11—18 increased relative to controls at low doses but decreased at the highest dose. It remains to be seen whether this kind of dose—response relationship is common for other substances with endocrine disrupting activity.

9 Positions and Activities of Governments and International Organizations The European Commission (EC) Within the EC, two Directorates General have a major involvement with the

K. Ramamoorthy, F. Wang, I.-C. Chen, S. Safe, J. D. Norris, D. P. McDonnell, K. W. Gaido, W. P. Bocchinfuso and K. S. Korach, Science, 1997, 275, 405. J. Ashby, P. A. Lefevre, J. Odum, C. A. Harris, E. J. Routledge and J. P. Sumpter, Nature, 1997, 385, 494. F. Piferrer and E. M. Donaldson, Aquaculture, 1989, 77, 251. F. S. vom Saal, B. G. Timms, M. M. Montano, P. Palanza, K. A. Thayer, S. C. Nagel, M. D. Dhar, V. K. Ganjam, S. Parmigiani and W. V. Welshons, Proc. Natl. Acad. Sci. USA, 1997, 94, 2056.

21 B. Phillips and P. Harrison issue of EDs: DG XII (Science, Research and Development) and DG XXIV (Consumer Policy and Consumer Health Protection). DG XII co-sponsored a meeting, held in Weybridge in the UK in December 1996, entitled ‘European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife’. This Workshop led to the publication of a report, known as the ‘Weybridge’ report. A number of key research and monitoring recommendations were made, covering human epidemiology, wildlife studies, mechanisms and models, exposure assessment and methodology. The Directorate also co-sponsored (together with SETAC-Europe and OECD) a Workshop held in the Netherlands in April 1997 entitled ‘Expert Workshop on Endocrine Modulators and Wildlife: Assessment and Testing’. This concentrated on hazard identification for terrestrial and aquatic wildlife, from all chemicals and contaminated environmental media. Having identified the need for research in this area, DGXII initiated a number of projects as part of its Environment and Climate Programme. EC research projects are incorporated in multiannual Framework Programmes. The Fourth Framework Programme (1994—1998) did not specifically include research on EDs but, in the Fifth Framework Programme (1998—2002), EDs will appear in two programmes: ‘Quality of Life and Management of Living Resources’ and ‘Preserving the Ecosystem’. DG XXIV relies on high quality scientific advice for the drafting and amendment of Community rules regarding consumer protection. The system of scientific advice was reformed recently and a Scientific Steering Committee and eight new Scientific Committees were established. One of these, the Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE), has the mandate to consider scientific and technical questions relating to examination of the toxicity and ecotoxicity of chemical, biochemical and biological compounds whose use may have harmful consequences for human health and the environment. This includes the issue of EDs and recently the CSTEE has reviewed phthalates in soft PVC toys and child-care articles. Existing legislative instruments, such as Directives relating to classification, packaging and labelling, are being examined with a view to possible modification for the purpose of regulating EDs.

The UK and Germany Many European governments are actively involved in research into endocrine disruption. In the UK, the Department of the Environment, Transport and the Regions (DETR) takes the lead, in active collaboration with the Department of Health, the Environment Agency, the Ministry of Agriculture, Fisheries and Food, the Health and Safety Executive and other government departments and agencies. An interdepartmental group consisting of these government departments and agencies, together with representatives from the research councils, was set up in 1995 to coordinate research activities and exchange views and information. The Interdepartmental Endocrine Disrupter Research Group is concerned with assessment of the risks currently posed to human health and wildlife by EDs in the environment and whether current controls are adequate. In 1994, the MRC Institute for Environment and Health (IEH) was commissioned to prepare an

22 Overview of the Endocrine Disrupters Issue assessment of the then current state of knowledge, which resulted in the publication in 1995 of ‘Environmental Oestrogens: Consequences to Human Health and Wildlife’. In 1996, the DETR co-sponsored the European Workshop on the Impact of Endocrine Disrupters on Human Health and Wildlife, referred to above. Since this meeting, a number of other government-funded meetings and workshops have taken place, including a Health and Safety Executive Workshop on Male Fertility and Reproductive Health organized and held at the IEH in November 1996, which resulted in a joint HSE/DETR/DH call for research proposals and, in May 1997, an IEH workshop on behalf of the DETR on the Ecological Signiﬁcance of Endocrine Disruption: Eﬀects on Reproductive Function and Consequences for Natural Populations. Other activities include the development by the IEH, on behalf of the DETR, of a prioritization database of potential endocrine disrupters. In July 1997, the German Federal Environmental Agency (FEA) issued a press release containing information related to the German inventory of current research projects on endocrine disruption. The statement indicated that over 130 research projects are underway in 15 European countries, with substances investigated including phthalates, alkylphenol polyethoxylates (APEs), polychlorinated biphenyls (PCBs) and organotin compounds. The FEA also indicated that the previous year it had commissioned research projects at a cost of over DM1 million with a further DM3.5 million to be made available for future projects. Overall, Germany announced plans during 1997 to increase research into endocrine disruption by four times the previous year’s level to DM5.2 million.

The United States In the USA, the Environmental Protection Agency (EPA) has taken the major role in research and regulatory action on EDs. The EPA’s Office of Research and Development held workshops in 1994 and 1995 to solicit opinions on research requirements. In 1996, the Food Quality Protection Act (FQPA) and Amendments to the Safe Drinking Water Act (SDWA) required the EPA to ‘Develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring oestrogen, or other such endocrine effect as the Administrator may designate’. The EPA formed the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) to provide advice on how to design a screening and testing program (EDSTP) for EDs. The EDSTP was designed to address both human and ecological (wildlife) effects, to examine effects on oestrogen, androgen and thyroid hormone-related processes and to evaluate endocrine disrupting properties of both chemical substances and common mixtures. The findings of EDSTAC were published in August 1998 (see above; Detection of EDs). The EPA has begun to implement the recommendations of EDSTAC by setting priorities for testing, starting with a list of approximately 87 000 chemicals. The availability of relevant data on these compounds is extremely variable. To assist with prioritization, a programme of testing has been started, based on the use of the tier 1 in vitro assays (oestrogen and androgen binding and steroidogenesis) in

23 B. Phillips and P. Harrison an automated, high-throughput screening mode. The EPA plan to test 15 000 high-tonnage chemicals in 1999. A demonstration programme, using 150 chemicals, is due to be completed in March 1999. Another section of the EPA, the Oﬃce of Prevention, Pesticides, and Toxic Substances (OPPT), has recently updated and harmonized its testing guidelines for evaluating the developmental and reproductive eﬀects of pesticides and industrial chemicals to include an assessment of endocrine disrupting properties. These guidelines will be used in future testing of pesticides under both the Toxic Substances Control Act (TSCA) and the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA).

Japan The Japanese Environment Agency (JEA) set up an Exogenous Endocrine Disrupting Chemical Task Force in March 1997 and, in May 1998, they announced their strategic programmes on environmental endocrine disrupters (SPEED’98/JEA). These included research on the present status of ED pollution, including surveys of possible eﬀects in wildlife and human health, improvement of the research base, promotion of environmental risk assessment and strengthening of international cooperation. A budget of more than 10 billion yen was allocated to endocrine disrupter research. At least eight other ministries and agencies have an interest in the issue and coordination of their activities will be an important part of the work of the JEA.

Organization for Economic Cooperation and Development (OECD) The OECD publishes a large number of test guidelines which are widely accepted as basic guides for the testing of chemicals. Endocrine disruption is not a speciﬁc OECD programme but an issue dealt with in the context of many ongoing activities. At a meeting of the National Coordinators of the OECD Test Guidelines Programme in September 1996, it was proposed that new and/or existing test guidelines should be developed and/or revised for the testing and hazard characterization of EDs. In November 1996, these recommendations were endorsed by the OECD Chemicals and Management Groups and the OECD Pesticide Forum and the OECD Endocrine Disrupters Project was launched. One major part of these activities has been the development of a Detailed Review Paper (see above; Detection of EDs) which included:

E A description of the scientific progress in the area and new techniques available. E An inventory of existing test methods, together with an appreciation of the scientific validity, sensitivity, specificity and reproducibility of these methods. E Identification of gaps in existing OECD Test Guidelines for these endpoints. E Proposal(s) for the development of new Guidelines or the revision of existing Guidelines.

The OECD has been very active in the development of a concensus on tests for endocrine disruption in ﬁsh, including selection of enhancements to guidelines

24 Overview of the Endocrine Disrupters Issue such as numbers 416 and 407, and in the selection of a group of reference chemicals. There have also been discussions as to what mammalian toxicity tests might need enhancement for ED detection, but there is currently no consensus on what enhancements would be most appropriate. Before test methods can be adopted by the OECD, it is essential that they are carefully validated to establish reliability, reproducibility and relevance. This process will require the testing of reference chemicals in a large number of laboratories. The OECD has identiﬁed the uterotrophic and Hershberger assays and additions to the 28-day rodent toxicity study (guideline 407) as suitable for immediate validation.

The International Programme on Chemical Safety (IPCS) The IPCS is administered by the World Health Organization (WHO). In collaboration with other international agencies including the OECD, the IPCS has been charged with creating a global inventory of all research on EDs and providing an international assessment of the state of the science on these chemicals. The ﬁrst meeting on this topic was held in March 1998 in Washington, USA. The IPCS has taken the lead role in coordinating the establishment of the database and soliciting contributions of information. A report, ‘Global State of the Science Assessment’, is being compiled under the direction of the IPCS by an international group of scientiﬁc experts and is expected to be completed within two years.

The European Chemical Industry Council (CEFIC) CEFIC has undertaken a wide-ranging programme of research in cooperation with similar organizations throughout the world. The major topics covered by this three-year programme, costing $7m in Europe and $20m worldwide, are male reproductive health, wildlife exposure and testing methods.

10 Conclusions and Unanswered Questions There is increasing evidence for adverse trends in certain measures of human reproductive health, most notably testicular cancer and female breast cancer. In some cases, such as decreasing sperm counts, the existence of an adverse trend is still a matter of debate. Even for those health trends for which there is good evidence, a causal link with exposure to environmental chemicals has not been established, and other explanations involving diet and lifestyle are plausible. However, environmental chemicals have been implicated on the grounds that the involvement of hormonal changes in the effects in question can be readily envisaged and that various widely distributed environmental chemicals can be shown, in the laboratory, to possess oestrogenic or other hormonal activities. In wildlife, there is more convincing evidence for a link between exposure to environmental chemicals with endocrine disrupting activity and certain adverse reproductive and developmental effects, such as imposex in neogastropods and feminization in fish. This adds weight to the contention that endocrine disruption by environmental chemicals may pose a threat to both wildlife and humans.

25 B. Phillips and P. Harrison

A great deal of research and monitoring will be required to address certain key questions regarding endocrine disruption. There is a need to refine and clarify the measurement of trends in human reproductive health, especially with respect to semen quality and fertility. Where the existence of a trend is established, identification of the cause or causes will be a major challenge and concentration on endocrine disruption should not distract attention from other possible explanations. Further work is also needed to evaluate the significance for wildlife populations, communities and ecosystems of the observed effects of endocrine disrupters on individuals. The identification of environmental chemicals with significant endocrine disrupting activity will require a continued refinement and validation of test methods that can accurately predict the effects of chemicals on the health of human and wildlife populations. Risk assessment will need to address the problem of interactions between chemicals because, in practice, exposure is more likely to be to mixtures than to single EDs. The final, but most pressing, question is how should chemicals that are suspected of endocrine disrupting activity be regulated? In cases where a chemical can be demonstrated to have caused a detrimental effect on human health or on wildlife, there is little need to establish its mechanism of action before taking regulatory action. However, if chemicals are to be regulated on the basis of a property such as the ability to bind to a hormone receptor, it is necessary to establish clearly how that property is related to putative adverse effects, preferably quantitatively as well as qualitatively. Controversy will continue to surround the need for regulation of potential EDs until this relationship is thoroughly established.

26 Environmentally Induced Endocrine Abnormalities in Fish

DAVID E. KIME

1 Background Publication of ‘Silent Spring’ by Rachel Carson in 1962 provided a warning of the dangers of the mass spraying of agricultural land with pesticides. The high incidence of dead and dying birds and fish was immediately apparent in areas subjected to this practice, and humans in affected areas were also experiencing symptoms attributable to use of the pesticides. With the cessation of such eradication programmes, which in many cases had not brought about the benefits that had been claimed, it was assumed that the problem had disappeared. Indeed, the birds returned and sang again and fish were once more found in the rivers. For two decades the world became complacent and concentrated on increasing the living standards of a minority of its population in the northern hemisphere. Motor traffic increased in cities, the network of motorways expanded and there was a massive growth in air traffic as world trade and both business and leisure travel expanded. Use of timber pulp for packaging and printing increased greatly, while the innovation of plastics has revolutionised our way of life. Together with the production and disposal of many thousands of different consumer goods, this has resulted, in less than half a century, in the production and release of several hundreds of thousands of synthetic chemicals and the release of abnormally high amounts of many natural chemicals in timber and mining wastes. Whether such release was into the atmosphere, onto land or into the rivers, it all eventually comes to rest in the aquatic ecosystem. Fish, as inhabitants of the rivers, lakes and oceans, inevitably receive the greatest exposures. In areas such as the Great Lakes, the North and Baltic Seas, situated close to areas of high industrial activity, with a relatively low water flow or where sedimentation can occur, fish can be subjected to extremely high exposures. The organochlorine pesticides, which were of greatest concern to Carson, were

R. Carson, Silent Spring, Houghton Miﬄin, Boston, 1962 (1965 paperback edition, Penguin, Harmondsworth, UK).

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

27 D. E. Kime banned in Western Europe and North America in the 1970s, as were the polychlorinated biphenyls (PCBs) widely used as electrical insulators. Both of these classes of chemical are extremely persistent, resisting biodegradation and in many areas their concentrations in fish tissues remain unchanged several decades after production ceased. To these have been added a vast array of other chemicals which may reside in lake, river and ocean sediments for varying periods and that can be metabolised into a wide range of products of unknown toxicity. Except in cases of localised acute pollution, dead fish are now seen much more rarely, but there is increasing evidence that their health is being compromised by long-term low-level pollution. At one level this may lead to premature mortality, resulting in a decreased number of breeding seasons and thence fewer offspring. Evidence is now accumulating, however, that even lower levels of pollutants can disrupt the functioning of the endocrine system of fish, leading to decreases in immune and stress responses, energy metabolism, osmoregulatory ability and reproductive function. Because of their exposure in the aquatic ecosystem, as the major repository of environmental pollutants, fish can provide an early warning of effects that may later become apparent in other wildlife and ultimately in humans themselves. Indeed, there is increasing evidence that some of the problems found in fish, including decreased fertility, genital abnormalities, altered behaviour patterns and response to stress and disease, are now appearing in human populations. This review will summarise the main evidence for such changes in fish exposed both in the laboratory and in polluted habitats to a wide range of the pollutants that now contaminate our environment. References have been kept to the minimum for simplicity and the reader is referred elsewhere for fuller references to much of the data cited.

2 The Nature of Aquatic Pollution The aquatic environment is subject to an ever increasing range of man-made (anthropogenic or xenobiotic) pollutants, reflecting the ever more rapid innovations of our technology to manufacture goods to satisfy a perceived increase in consumer demand on which our economy is based. Some of the pollutants that are now present in the tissues of fish, wildlife and humans also reflect past usage of chemicals, such as the organochlorine insecticides and PCBs, which have been banned or restricted in use for several decades. Measurements of tissue concentrations are, however, overwhelmingly limited to a range of pollutants such as pesticides, polyaromatic hydrocarbons (PAHs) and PCBs that are known to be present in the aquatic environment and for which measurement methods do exist. Alkylphenolics and phthalates have only been measured in tissues during the last few years after in vitro studies had shown that they could mimic the action of the natural estrogen estradiol, i.e. they were estrogenic. It cannot be over-emphasised that there are many other xenobiotic chemicals present in the aquatic environment that have never been measured in fish tissue and whose identity and potential endocrine disrupting activity is wholly unknown.

D. E. Kime, Endocrine Disruption in Fish, Kluwer, Boston, 1998. T. Colborn, D. Dumanoski and J. P. Myers, Our Stolen Future, Little Brown, London, 1996.

28 Environmentally Induced Endocrine Abnormalities in Fish

Demonstration of endocrine dysfunction in fish taken from a polluted habitat, together with high tissue concentration of a chemical such as a PCB, does not demonstrate that the PCB is the cause of endocrine dysfunction. The high PCB level shows only that the fish is inhabiting a polluted habitat and reflects a potential tissue burden of other pollutants released by the same urban or industrial complex as the PCB. Only by exposing fish under controlled laboratory conditions to a single chemical can cause and effect be clearly established, and even then this may be the action of just one chemical in a vastly complex cocktail. The very high concentrations of heavy metals, PCBs, PAHs and pesticides in fish taken from the North Sea, the Baltic and the Great Lakes is simply a reflection of proximity to industrial, agricultural and urban outlets. Such fish have clearly defined endocrine disorders that may or may not be caused by the chemicals measured, but nevertheless they provide evidence that our present economic and industrial system is unsustainable if we wish to live in a healthy environment. For convenience, anthropogenic pollutants can be divided into three classes: heavy metals, pesticides and industrial chemicals. Cadmium, lead and mercury, which are released from mining activity or metallurgical processing, play no natural role in any living organism and, as known neurotoxins, are especially damaging to the neuroendocrine system. Metals accumulate in the endocrine system and even 0.03 kgl\ mercury in a river can accumulate to 0.3 mg kg\ in the body of fish, a level that is well within the range that can cause endocrine disruption, while even higher tissue levels have been found in fish from some polluted areas. Although zinc and copper are essential for endocrine function, they may cause dysfunction at abnormally high exposures. The organochlorine pesticides, such as DDT, have been progressively banned in much of North America and Europe owing to their long-term persistence and high toxicity. Exposure to such pesticides comes now both from accumulation in the sediments, where they were deposited from past usage, and from aerial dispersal from other areas of the world in which they are still in use. Increasingly, the organochlorines are being replaced by organophosphorus, carbamate and other less persistent pesticides. Although such pesticides may be more rapidly degraded, they are still potential endocrine disruptors and even exposure for a short period can have serious consequences for fish if it coincides with key phases of the reproductive cycle. Relatively few of these newer pesticides have been tested for endocrine disruption. Popular concern has focused on the estrogenic hazards of sewage effluents and the alkylphenolic detergents, but a very wide range of other chemicals released into the aquatic ecosystem from industrial and domestic wastes also have potential endocrine disrupting activity. Polychlorinated biphenyls (PCBs), like the organochlorine pesticides, are both persistent in the aquatic environment and have some estrogenic activity. Polyaromatic hydrocarbons (PAHs) are released from both motor vehicles and power stations during combustion of fossil fuels, while downstream of pulp and textile mills the effluent can cause major endocrine

K. Matsunaga, Nature, 1975, 257, 49.

29 D. E. Kime malfunction. Tests for endocrine disrupting activity have so far centred on persistent chemicals such as PAHs and PCBs, and the few chemicals like alkylphenolics and phthalates that have been discovered to have endocrine disrupting activity only by chance. High concentrations of these chemicals are found in fish in polluted areas such as the North and Baltic Seas and the Great Lakes. Endocrine dysfunction in fish from such areas has been demonstrated, but cannot simply be causally related to the high levels of these particular chemicals since they are inevitably associated with many of the hundreds of thousands of other chemicals that are released from other industrial processes in the same waterways. Commercial secrecy is a major impediment to identifying and restricting release of endocrine disrupting chemicals. The scale of the problem is exemplified by pesticide formulations in which the active ingredient is mixed with an ‘inert dispersant’. One such widely used ‘inert’ dispersant was the alkylphenolic detergent, which is now known to degrade to persistent and estrogenic products. Measurement of contaminants in fish has concentrated on muscle tissue since the aim has generally been to protect the health of the consumer rather than that of the fish. Endocrine tissue such as the gonads has been much more rarely examined, while data for adrenal, thyroid and pituitary levels are virtually non-existent. More data are available for the liver, as a lipid rich tissue and the major site of xenobiotic catabolism, but the concentrations have rarely been related to its capacity to produce vitellogenin or metabolise endogenous hormones. Tissue concentrations of a wide range of chemicals, are at a level which suggests that, either alone or in combination, they will cause significant endocrine disruption in fish in many polluted habitats.

3 The Endocrine System of Fish To fully understand how these pollutants can affect fish at very low levels, it is necessary to outline very briefly how the endocrine system works. It is a control system of the body which responds to internal and external signals to maintain the body in a chemical equilibrium, to regulate sexual development and the seasonal reproductive cycles, and to evoke a stress response to external threats. At its core are the hypothalamus and pituitary, which respond to neural signals from the brain and convert them into hormone messengers which act on the individual glands such as the gonads, the thyroid and the adrenal (Figure 1). The endocrine systems of all vertebrates have essentially the same components, which originated during the early evolution of fish. It is not clear how many of the differences between mammals and fish are due to evolutionary divergence and how much is attributable to the very different habitats in which they have developed. Clearly the regulation of water and mineral balance in the body fluids (osmoregulation) requires very different control in mammals to that of fish inhabiting fresh- or seawater environments, while regulation of reproduction in the predominantly egg-laying (oviparous) fish is very different from that which is required in placental mammals. In many cases it is not the hormones but the uses to which they are put which differs, although some of the steroid hormones in fish do show marked differences to those of mammals. Once they have elicited the appropriate action in their target tissue, hormones

30 Environmentally Induced Endocrine Abnormalities in Fish

Figure 1 A schematic diagram of the endocrine system of ﬁsh. TRH : thyrotrophin releasing hormone; GnRH : gonadotrophin releasing hormone; CRH : corticotrophin releasing hormone; TSH : thyroid stimulating hormone; GtH : gonadotrophins I and II; ACTH : adrenocortico- trophic hormone; : T thyroxine; : T triiodothyronine; : E estradiol; T : testosterone; are converted by the liver to metabolites which are more easily excreted. There is 17,20bP : 17,20b- dihydroxy-4-pregnen-3-one; a negative feedback of the circulating hormones to the pituitary and hypothalamus KT : 11-ketotestosterone; which maintains the normal hormonal equilibrium. VTG : vitellogenin.

The Control of Reproduction in Fish Fish possess the same essential components of the reproductive endocrine system as mammals in that external cues, such as seasonal changes in temperature or daylength, behaviour patterns of a potential mate, etc., are translated by the brain and hypothalamus into the release of gonadotrophin releasing hormone (GnRH). This in turn causes the pituitary gland, situated at the base of the brain, to release gonadotrophin which stimulates steroid synthesis in the gonads. At least some fish possess two gonadotrophins (GtH-I and GtH-II) analogous to the follicle stimulating and luteinising hormones (FSH and LH) which regulate the female cycle in mammals. GtH-I stimulates the ovary to produce estradiol which induces production of a yolk protein (vitellogenin) by the liver, while GtH-II predominates just before spawning when it stimulates ovarian synthesis of a progestogen (17,20b-dihydroxy-4-pregnen-3-one, usually abbreviated to 17,20bP), which induces maturation of the oocytes prior to ovulation. This progestogen may also play a role in sperm maturation but progesterone, which is an essential hormone in the female mammal, has no known role in fish. Male fish also differ from mammals in that the major product of the testis is 11-ketotestosterone rather than testosterone. In fish, unlike mammals, the gonads of both sexes synthesise testosterone, which may play an important role in feedback to the pituitary. The gonads of fish also have some of the properties associated with the liver in mammals in that they can convert steroid hormones into metabolites. In some species of fish these metabolites may act as sexual signals (pheromones) to members of the opposite sex.

31 D. E. Kime

Growth, Metabolism and the Stress Response

The hormones secreted by the thyroid [thyroxine (T) and triiodothyronine (T)] and adrenal (cortisol and adrenaline) tissues are identical in both fish and mammals. In fish, however, these tissues do not form discrete glands; the thyroid is scattered around the ventral aorta, while adrenal tissue is dispersed within the kidney. This makes it very difficult to measure the changes induced in their structure by chemical pollutants. The hormones of the thyroid regulate general metabolic rate, growth and possibly embryonic development, while those of the adrenal are involved in the stress response, osmoregulation and carbohydrate metabolism. Growth in fish is continuous and does not cease at puberty as in mammals. Fish size is therefore not only dependent on secretion of growth hormone by the pituitary gland as in mammals, but on age and the rate of metabolic activity and energy utilisation as determined by both the thyroid and interrenal glands. Thyroid and adrenal activities are also involved in the osmoregulatory adaptations required by some species, such as salmon, which migrate between salt and fresh water.

Endocrine Disruption

Definitions. The Weybridge Workshop defined ‘An endocrine disruptor is an exogenous substance that causes adverse health effects in an intact organism, or its progeny, consequent to changes in endocrine function’ while ‘A potential endocrine disruptor is a substance that possesses properties that might be expected to lead to endocrine disruption in an intact organism’. It is important to note that the primary site of action must be the endocrine system. This therefore does not include pollutants if their main action is as an irritant which leads to alteration in endocrine function as a result of stress. In such cases, tissue which is in direct contact with the pollutant, often the gills or skin in fish, will be irritated, causing discomfort to the fish and evoke a stress response involving increased secretion of adrenaline and cortisol by the adrenal tissue. This is analogous to a respiratory or skin irritation in mammals, which induces the stress response of adrenal secretion of cortisol which in turn initiates an anti-inflammatory reaction. This is a natural response by the endocrine system to restore equilibrium and is not endocrine disruption. The key point in the definition is that specific components of the endocrine system are affected by a pollutant at concentrations that have no effect on the functioning of non-endocrine tissue in the animal. There are, however, many substances which can affect reproductive capacity by altering the viability of sperm or eggs even though they do not affect gonadal hormone secretion. Endocrinology, however, now encompasses a very wide range of effects and includes the many mechanisms that regulate inter- and intracellular communication, many of which are not yet fully understood. Some of the effects on both the gametes and the immune system may well therefore be European Commission, European Workshop on the Impact of Endocrine Disruptors on Human Health and Wildlife, Weybridge, 2–4 Dec. 1996, Environment and Climate Research Programme, European Commission, DG XII, Brussels, Report EUR 17549.

32 Environmentally Induced Endocrine Abnormalities in Fish included in the deﬁnition, but clearly the boundaries of endocrine disruption, like those of endocrinology, cannot be too clearly delineated.

Mechanisms of Endocrine Disruption. Classical toxicology deals with the effects of chemicals which act by causing damage to the function of the animal cell, and as such they have relatively little tissue specificity. Xenobiotics which disrupt the endocrine system, however, can act via a very wide variety of mechanisms. They can mimic the effect of a natural hormone (agonist), or they can block its action (antagonist). This may cause changes in the response to the hormone at its target site by affecting its ability to bind to receptors, affect its feedback action to the higher sites of endocrine control such as the hypothalamus and pituitary, or alter the hormone-dependent behaviour of the exposed animal. They can also affect the action of key enzymes of biosynthesis and metabolism that will alter circulating hormone levels, or cause structural changes which affect the function of an endocrine gland. The complexity of the issue is clearly seen in Figure 1, where disruption of any single component can cause dysfunction of a whole range of inter-related mechanisms.

Fish as Monitors of Endocrine Disruption. Since fish are, perhaps, the class of vertebrate most at risk of endocrine disruption in that their habitat receives the greatest imput of anthropogenic pollution, they might be considered the obvious animal for experimental investigation. Their commercial and dietary importance certainly warrants an investigation of any factors which might lead to a decrease in their populations. In addition, there are some endocrine functions that are far more easily investigated in fish than in mammals. Recent reports in the popular press concentrating on the threat to male fertility posed by environmental estrogens have largely obscured both the fact that female fertility may be equally well compromised and that such effects can be caused by many compounds that are not estrogenic. Although such bias may be attributable to the need to make a good story, or to a male dominated society, they also reflect the fact that males continually produce sperm in extremely large amounts which are physically accessible for research. In contrast, the female mammal releases only a very small number of eggs at infrequent intervals and they are not accessible without invasive surgery. Fish, which can spawn thousands or millions of eggs to the external environment, therefore provide an excellent model with which to examine the effects of endocrine disruptors on female fertility. The high fecundity also makes it much more feasible to examine the effects of such disruptors on embryonic development and on the effects of parental or embryonic exposure on the endocrine function of the offspring. Fish also have a major advantage since it is far easier to give a regular exposure of known concentration via the holding water or diet, and to examine the effects of exposure during defined periods of development or during different phases of the reproductive cycle. The ready availability of small fish such as zebrafish, fathead minnows and medaka with a very short life cycle also makes it much easier to examine the effects of exposure at specific periods on endocrine function over several generations.

33 D. E. Kime

4 Hypothalamic and Pituitary Abnormalities The close neural relationship of the hypothalamus and the pituitary gland with the brain makes them particularly vulnerable to neurotoxins such as the organophosphate pesticides and the heavy metals lead and mercury. Although the numbers of studies on these tissues are few, reflecting their very small size and inaccessibility, there is clear evidence that both these heavy metals, and the organochlorine and carbamate pesticides, can damage the neurones of the hypothalamus which are responsible for GnRH release, leading to failure of ovaries and testes to produce yolky eggs and viable sperm. Organochlorine and organophosphate pesticides, cyanide, PAHs, PCBs, cadmium and mercury can all cause degeneration of the secretory cells of the pituitary gland and decrease its release of hormones. Industrial pollutants, such as paper mill effluents, can also affect the responsiveness of the pituitary to GnRH released by the hypothalamus. The feedback signal to the pituitary, which regulates plasma steroid hormone balance, can be disrupted by any xenobiotic that has hormone mimicking properties. Organochlorine pesticides, such as the lindane impurity b-HCH, mimic natural estrogen and induce a negative feedback response in the pituitary. This could account for many of the reports of reproductive failure of fish in waters such as the Great Lakes that are heavily polluted with organochlorines.

5 Male Reproductive Problems in Fish Much of the impetus for the recent upsurge in activity on endocrine disruption was the finding of decreased sperm counts and an increased incidence of genital abnormalities in humans. While some of these findings have been disputed, the much more extensive evidence from studies with fish adds considerable weight to the argument that such changes are occurring and that they are caused by exposure to anthropogenic pollutants rather than to changes in human lifestyle during the last few decades. Although the abnormalities in human populations were attributed to ‘environmental estrogens’, and this has been the subject of much popular concern, it is important to realise that there are very many chemicals that affect male reproductive function in fish which have no estrogenic activity. Such chemicals, even if they are not hormone mimics, can either disrupt the activity of the tissues that secrete or metabolise hormones or the receptors for these hormones. A wide variety of chemicals which cause direct changes in endocrine function of isolated tissues can be considered as potential endocrine disruptors, but too frequently there has been a failure to demonstrate that in the intact animal such chemicals act on the endocrine system at concentrations which do not cause either a stress response or are toxic to the animal. Many organochlorine pesticides, for example, are estrogenic, but they also have a very high toxicity and estrogenic action is of little consequence to a dead or dying fish. Even nonylphenol, which has been of such recent concern for its estrogenic

G. J. Van der Kraak, K. R. Munkittrick, M. E. McMaster, C. B. Portt and J. P. Chang, Toxicol. Appl. Pharmacol., 1992, 115, 224. P. W. Wester, J. H. Canton and A. Bisschop, Aquat. Toxicol., 1985, 6, 271. R. M. Sharpe and N. E. Skakkebaek, Lancet, 1993, B41, 1392.

34 Environmentally Induced Endocrine Abnormalities in Fish

\ activity, has a 96 h LC of only 0.12 mg l in salmon. It remains to be shown how many potential endocrine disruptors are in fact endocrine disruptors in intact fish. Abnormal function of the reproductive endocrine system in male and female fish can be caused by disruption of hypothalamic, pituitary or gonadal function, and by changes in the liver that affect the enzymes which deactivate steroid hormones. Both gonads and liver are fatty tissues and rapidly bioaccumulate both non-biodegradable organic pollutants and heavy metals such as mercury and cadmium that can be present at levels several orders of magnitude greater than in either the surrounding water or in muscle tissue. Endocrine dysfunction in the testis may be apparent by changes in its structure, its secretion of hormones, the activity of the enzymes that are necessary for steroid synthesis, the quality and quantity of the sperm produced and in the hormone dependent behaviour patterns of the male fish.

Testicular Structure and Hormones Endocrine disruption can cause changes in cellular structure or organisation that are specific to the endocrine tissues rather than due to non-specific cytotoxic action. Clear evidence for endocrine disruption of the testis is apparent when its steroid producing cells show increased or decreased activity, the proportions of sperm at different stages of development differ from those of unexposed fish or there is a complete arrest in sperm production. Such changes may be a result of either primary action on the testis or secondary action consequent upon action at the hypothalamic—pituitary complex. Endocrine dysfunction may also result from specific toxic action on the testis owing to selective bioaccumulation so that the endocrine tissues are exposed to higher concentrations than other cells in the organism. There is evidence of such disruption of the male reproductive system following exposure to heavy metals such as arsenic, cadmium, copper, lead and mercury, the organochlorine pesticides DDT, chlordecone and endosulfan, some carbamate and organophosphate pesticides, and industrial chemicals including pulp mill effluents, PCBs and alkylphenolics. Contrary to popular belief, there is no evidence that exposure of adult male fish to any environmental estrogen can cause it to change sex. Inhibition of gonadal development certainly occurs and the effects of the weakly estrogenic alkylphenolics closely follow those obtained with the synthetic estrogen ethynylestradiol at very much lower concentrations. Such inhibition is not, however, necessarily indicative of exposure to estrogens since many non-estrogenic chemicals have similar effects. Xenobiotics as diverse as cadmium, pulp mill effluents, crude oil, PCBs and alkylphenolics all decreased concentrations of the major testicular steroid 11-ketotestosterone in fish plasma, but it is often not clear whether such changes are the cause or result of retarded development of the testis. Although they can generally be assumed to be a good indicator that testicular function has been compromised, they can also be a result of general stress. Although it may appear obvious to compare fish sampled at clean and polluted S. Jobling, D. Sheahan, J. A. Osborne, P. Matthiessen and J. P. Sumpter, Environ. Toxicol. Chem., 1996, 15, 194.

35 D. E. Kime sites, this can be misleading since they may constitute two distinct populations that spawn at different times even if they are not exposed to a pollutant. In rivers, large lakes and oceans there can be significant migrations so that the fish captured in a clean habitat may have been feeding in polluted areas or vice versa. Considerations of spawning migrations are particularly important since early life is one of the most sensitive periods for inducing endocrine disruption and exposure at a polluted spawning ground could lead to abnormalities that only become apparent when the fish is caught later as an adult in clean water perhaps some hundreds of kilometres away. Conversely, fish captured in polluted water may be apparently unaffected because they were spawned in distant clean water. It is also clear that it is difficult to relate cause and effect to any specific chemical since, with the exception of point source effluents, many waterways contain a multitude of chemicals, of which the active endocrine disruptor may not be that which has been measured in the water or tissue. For such reasons, many studies have used in vitro experiments in which isolated tissue, either from a control animal or one captured in a polluted water system, is exposed to a single pollutant in the laboratory. Such experiments have shown significant disruption to testicular activity by a wide range of xenobiotics, including cadmium, lindane, DDT, cythion, hexadrin and PCBs.

Gamete Viability Although all male vertebrates produce vastly more sperm than eggs, evidence from both fish and mammals suggests that even a small decrease in sperm quality or quantity can decrease the male’s fertilising ability. Viability of sperm in fish, as in other vertebrates, is dependent on the correct hormonal and nutritional environment during their development within the testis. This may in turn be affected by the internal hormonal environment during the early life stages in which the testis is differentiated. Endocrine disruption can therefore lead to abnormal development of the sperm and decrease its viability. This may take the form of either abnormal sperm structure or a decrease in its energy supply, both of which can alter its swimming ability and therefore its capacity to reach and fertilise the egg. Computer technology can now make a quantitative assessment of the effects of xenobiotics on sperm quality and predict fertilisation success.— Recent studies suggest that mercury can cause an instant decrease in the sperm viability of fish at concentrations comparable to those which are permitted in drinking water (1 kgl\). The bioconcentration of the metal to levels in the testis considerably higher than this from water containing only 1/30 of permitted levels suggests that current legal limits are much too high.

Other Eﬀects on the Male Courtship rituals in ﬁsh can involve a complex communication between males D. E. Kime, M. Ebrahimi, K. Nysten, I. Roelants, E. Rurangwa, H. D. M. Moore and F. Ollevier, Aquat. Toxicol., 1996, 36, 223. G. P. Toth, S. A. Christ, H. W. McCarthy, J. A. Torsella and M. K. Smith, J. Fish. Biol., 1995, 47, 986. E. Rurangwa, I. Roelants, G. Huyskens, M. Ebrahimi, D. E. Kime and F. Ollevier, J. Fish Biol., 1998, 53, 402.

36 Environmentally Induced Endocrine Abnormalities in Fish and females via chemicals released into the surrounding water. These pheromones are, in many cases, the hormones secreted by the gonads and are probably a selective adaptation enabling males to detect females that are ready to spawn. Successful courtship is therefore dependent on the ability of the female to secrete the pheromone, the male to detect it, and for his neural and endocrine system to elicit a response that both triggers his production of sperm and his appropriate sexual behaviour. Carbofuran and the organophosphate diazinon can both disrupt the ability of male fish to detect such pheromones. Xenobiotics might also cause changes to other aspects of androgen-dependent breeding behaviour, particularly in species in which the male builds or guards a nest or spawning territory. This may be particularly important in species such as the stickleback which produces an androgen stimulated glue from the kidney for nest-building. Development of male secondary sexual characteristics such as the red belly of sticklebacks or the hooked jaw in salmonids is dependent upon testicular secretion of 11-ketotestosterone, and any decrease in production of this steroid or effects on its receptors could inhibit his ability to attract a mate. Such changes can make a useful biomonitor for reduction in testicular function. In mammals, sex-dependent behavioural patterns are imprinted during a very brief period of testicular activity at birth, but such patterns are only expressed at puberty. Nothing is known about whether such patterns are also imprinted in fish, and if so, whether, as in mammals, the sensitive window for such exposure differs from that in which the genitalia become structurally differentiated. In mammals a hormonal imbalance, either natural or xenobiotic induced, during this ‘imprinting’ period can result in behaviour patterns characteristic of the opposite sex. Similarly, the elaborate courtship ritual which is essential to induce ovulation in many fish species could be disrupted by alterations in endocrine function during key early life stages. There have, however, been few studies of the effects of xenobiotics on endocrine mediated behaviour patterns.

6 Female Reproductive Problems in Fish Xenobiotic induced disruption of female fertility follows essentially the same pattern as that of the male and can be caused by changes in pituitary—hypothalamic function, primary disruption of ovarian structure or hormone secretion, or changes in the rate of hormone deactivation. In addition, there may be changes in the synthesis of estrogen induced production of the yolk protein by the liver (vitellogenesis), which in turn can lead to failure to lay down suﬃcient yolk in the developing oocytes. Vitellogenesis provides a valuable biomarker for endocrine dysfunction in both sexes, but is more properly considered as part of the liver function.

A. Moore and C. P. Waring, J. Fish Biol., 1996, 48, 758. C. P. Waring and A. Moore, Fish Physiol. Biochem., 1997, 17, 203. F. S. Vom Saal, M. M. Montano and M. H. Wang, in Chemically Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, ed. T. Colborn and C. Clement, Princeton Scientiﬁc, Princeton, 1992, p. 17. D. E. Kime, J. P. Nash and A. P. Scott, Aquaculture, 1999, 117, 345.

37 D. E. Kime

Ovarian Structure and Hormones The main factor causing the increase in ovarian weight during the reproductive development of fish is the deposition of yolk into the developing eggs. Abnormal development of the ovary, like the testis, can be caused by lack of stimulation by pituitary hormones, failure of steroid synthesis or direct cellular damage. The most commonly observed effect in the ovary is a decrease in the numbers of large yolky eggs together with increased numbers of immature oocytes, suggesting a primary effect on pituitary function. Such changes have been observed with heavy metals (arsenic, cadmium, lead, mercury), organochlorine (DDT, endosulfan, aldrin, chlordecone, c-BHC, methoxychlor), organophosphate (monocrotophos, fenthion, cythion, fenitrothion, phenthoate, malathion, chlorfenvinphos, tetrachlorvinphos, mevinphos) and carbamate (carbaryl, carbofuran) pesticides. Among industrial chemicals, effects have been shown predominantly for PCBs and PAHs, and contaminated aquatic ecosystems such as those off the west coast of the USA which contain high residues of these chemicals. A variety of other industrial sources, such as textile and pulp mills containing high concentrations of natural and synthetic phenolic compounds, also inhibit ovarian development. Some sediments from the Rhine, however, had the opposite effect in that they actually advanced ovarian development. Synthesis of the steroid hormones testosterone and 17,20bP is identical in both ovary and testis, and dependent upon the functional integrity of both the steroid producing enzymes and the receptors for pituitary gonadotrophins. Xenobiotics that affect the components of the testis will therefore also have an effect on the ovary. Inhibition of ovarian steroid synthesis has been demonstrated for the heavy metals cadmium and lead, and the pesticides DDT, c-BHC, malathion, metacid-50 and carbaryl. Hormone levels were also depressed in plasma of fish exposed to the effluent of pulp mills or taken from the Great Lakes and other areas rich in PCBs and PAHs. Male and female gonads differ in that only the ovary has an active aromatase enzyme that converts testosterone to estradiol. Inhibition of this ovarian aromatase activity in female fish will decrease estrogen synthesis, and have consequential effects on the ability of its liver to synthesise yolk proteins, leading to retarded growth of the oocytes. Such aromatase inhibiting activity has been demonstrated for imidazole fungicides, PAHs, pulp mill effluents and tributyltin (TBT) which was widely used in antifouling paints on boats. These compounds may also affect steroid feedback to the pituitary in both sexes since this is dependent upon aromatase activity. Since all-male populations of fish can be produced in aquaculture by treatment of the larvae with a synthetic aromatase inhibitor during a 2 h critical window, any of

P. A. H. Janssen, J. G. D. Lambert, A. D. Vethaak and H. J. Th. Goos, Aquat. Toxicol., 1997, 39, 195. G. Monod, A. de Mones and A. Fostier, Mar. Environ. Res., 1993, 35, 147. L. O. B. Afonso, P. M. Campbell, G. K. Iwama, R. H. Devlin and E. M. Donaldson, Gen. Comp. Endocrinol., 1997, 106, 169. M. E. McMaster, C. J. Van der Kraak and K. R. Munkittrick, Comp. Biochem. Physiol., 1995, 112C, 169. K. Fent, Crit. Rev. Toxicol., 1996, 26,1. F. Piferrer, S. Zanuy, M. Carrillo, I. I. Solar, R. H. Devlin and E. M. Donaldson, J. Exp. Zool., 1994, 270, 255.

38 Environmentally Induced Endocrine Abnormalities in Fish these xenobiotic inhibitors could have a similar eﬀect and alter the sex ratio of exposed populations.

Ovulation and Spawning The majority of the literature on endocrine disruption in fish is concerned with ovarian development, rather than ovulation and spawning. This is undoubtedly due to the difficulty in assessing changes that can occur extremely rapidly during the 1—2 days which precede spawning. Ovulation is stimulated by a range of external stimuli such as the presence of a male and his courtship behaviour, spawning substrate, as well as temperature or water flow. Absence of the correct stimuli is a frequent cause of ovulatory failure in captive fish. Failure to ovulate in the presence of xenobiotics may not therefore be due to true endocrine disruption of the female but to indirect factors which affect the male or to environmental factors such as loss of spawning vegetation. The final maturation of the eggs just prior to ovulation and spawning is dependent on a very rapid change in the pattern of secretion of pituitary and ovarian hormones. Any disruption to this pattern can have major consequences for the ability of the fish to spawn. While long-term exposure to such chemicals would lead to delayed ovarian development, their release into rivers for only a short period that coincided with the spawning period, as occurs for example with organophosphorus sheep dips, could have serious consequences for the future fish population. Such disruption has been observed for the pesticides malathion, phosdrin, gardona and endosulfan. In many waters polluted by PCBs, pulp mill effluents or acid, however, spawning can proceed without difficulty, but is delayed by days or even weeks. Such late spawning is usually a result of decreased steroid production causing slower ovarian development than normal, but even if the full complement of eggs is eventually spawned this delay may have serious consequences for the numbers of offspring which survive to form a breeding population. Fish have evolved so that seasonal cues, mediated by the reproductive endocrine system, ensure that the spawning date is such that eggs will hatch at a time that coincides with a plentiful food supply and/or an absence of predators. Any delay in spawning will therefore decrease the competitive advantage of the young of the species that will both be too small to feed on their natural prey and become the target of other predators. In temperate species it will also lead to lower energy reserves with which to survive the first winter.

Egg Numbers and Viability Decreased production of yolk protein resulting from inhibition of either ovarian or liver function presents the ﬁsh with a similar choice to that resulting from decreased food availability. It can produce the same number of smaller eggs, or a smaller number of eggs of normal size, but the mechanism by which that choice is made is unclear. Since the decreased nutrient content of small eggs will result in smaller larvae with a decreased survival rate, the net result in both cases may be a

C. R. Tyler and J. P. Sumpter, Rev. Fish Biol. Fisheries, 1996, 6, 287.

39 D. E. Kime decreased survival of the progeny. Furthermore, there is evidence that many xenobiotics can be passed on from the mother to the offspring by incorporation into the developing eggs. Although this off-loading of the pollutant burden will decrease its toxicity to the mother, it will inevitably increase the exposure of the developing offspring at critical stages of embryonic development. Since eggs rapidly become impermeable in water after spawning, it is probable that in most cases the embryo receives the majority of the toxicant from its mother rather than directly from the surrounding water. While fertilisation and hatch rates have long formed the basis of many standard toxicity tests owing to the rapidity with which they can be carried out with small fish such as danios and minnows, they also provide valuable information on endocrine disruption. In many cases, abnormal development may be caused by the chemicals interfering with key developmental processes involving cellular signalling mechanisms, rather than by simple toxicity or chromosomal damage. Although the exact mechanism by which embryonic and larval development is controlled is not yet fully understood, it involves both the steroid and thyroid hormones and a range of intra- and intercellular messengers that are now covered by the broader definitions of endocrinology. As a result of the widespread use of early life stages for toxicity testing there is a vast literature on the types of developmental abnormalities which can occur, but so far very little on how much of this is really endocrine disruption and how much simply random toxicity or genetic damage which leads to deformity or mortality. Hatching failure can be due to a multiplicity of causes, but some syndromes of abnormality such as blue-sac disease and the similar PCB-induced M74 syndrome affecting Baltic salmon appear to be surprisingly common, which suggests disruption of key stages of embryonic or larval development. Until the site of action involved can be clarified, and whether the xenobiotic acts via hormonal or cellular signalling can be determined, it must remain open as to whether this can truly be called endocrine disruption. Sexual differentiation is more easily understood since in fish, as in mammals, it is dependent on the internal hormonal environment at critical early life stages. In mammals, both structural differentiation of the gonads and sexual behaviour patterns in adults can be affected by abnormalities in the internal hormonal environment during very early life. Such effects may be more pronounced in fish in which sex determination is more labile. The ‘hermaphrodite’ fish found after exposure to water containing estrogenic alkylphenolics that have been the subject of recent concern are much more likely to have been the result of pollutants altering the hormonal balance at key stages in early life than to exposure of adult males. Indeed, as long ago as 1986, b-hexachlorocyclohexane (an impurity in the insecticide lindane) was shown to cause an ova-testis in medaka hatched from exposed eggs. Since both alkylphenolics and b-HCH have estrogenic activity, it is probable that such compounds could affect sexual differentiation by mimicking the action of estradiol that might normally be produced by female larvae. Similar effects could also be expected in young fish

S. Jobling, M. Nolan, G. Brighty, C. R. Tyler and J. P. Sumpter, Environ. Sci. Technol., 1998, 32,1498. M. A. Gray and C. D. Metcalf, Environ. Toxicol. Chem., 1997, 16, 1082. P. W. Wester and J. H. Canton, Aquat. Toxicol., 1986, 9, 21.

40 Environmentally Induced Endocrine Abnormalities in Fish feeding on the contaminated egg yolk inherited from a mother exposed to pollutants on the spawning grounds. The effects of estrogens are not always so clear, since in some cases they can induce cellular damage in embryonic fish and there is an arrest in embryonic development of eggs from danios exposed to ethynylestradiol. In neither case is it clear whether this is due to simple toxicity or to exposure of the embryo to a hormone at an inappropriate stage or dose. By contrast, chemicals that affect the natural production of estrogen by the young could masculinise genetic females. Anthropogenic aromatase inhibitors, such as TBT, pulp mill effluents, imidazole fungicides and PAHs, could, like the synthetic inhibitors, induce masculinisation if such exposure coincided with a critical window of sensitivity during development. Female sexual development and behaviour in mammals occurs by default and requires no ovarian secretion, and it is only in genetic males that the testis can secrete hormones which destroy this female pattern and superimpose that of the male. Sexual differentiation is not so well defined in fish, and larval exposure to both synthetic estrogens and androgens is widely used in aquaculture to produce monosex cultures. Endocrine disruption of sexual differentiation in fish may therefore reflect both the complexity and diversity of such processes between different species. Some care is required in use of the terms ‘hermaphrodite’ and ‘sex-reversal’ since a true hermaphrodite has both functional testes and ovaries and a sex-reversed fish is fully functional as its final sex—both produce the appropriate viable gametes. Such functional sex-reversal is not possible in mammals, but in some species of fish it is the normal developmental pattern. In most of the cases of hermaphroditism or sex-reversal reported in the non-scientific press, there is evidence only for a few ovarian follicles within a functional testis. This may be considered as feminisation or a form of intersex, and is very clearly endocrine disruption, but it is certainly neither sex-reversal nor hermaphroditism. In some cases the terms have even been used to infer induction of a single female characteristic such as production of yolk-protein by males.

Other Effects on the Female Xenobiotics can also affect secondary sexual characteristics in species in which they are present. The most documented such case is in mosquitofish and least killifish in which the anal fin of the male is modified to form a gonopodium that is used to transfer sperm to the female during internal fertilisation. Such development has also been observed in female fish captured below pulp mills in Florida and has been taken to indicate the presence of ‘androgenic’ chemicals in the effluent. Since pulp mill effluent contains aromatase inhibitors, it is possible that this masculinisation could have been due to the absence of estrogen during sexual differentiation rather than to the presence of a true androgen. As in so many cases, the absence of a hormone can be just as disrupting as the presence of an inappropriate one. N. N. Huffman, R. W. Jones and A. A. Katzberg, Cancer, 1957, 10, 707. D. E. Kime and J. P. Nash, Sci. Total Environ., 1999, in press. S. A. Bortone, W. P. Davis and C. M. Bundrick, Bull. Environ. Contam. Toxicol., 1989, 43, 370.

41 D. E. Kime

7 Abnormalities in Growth, Metabolism and the Stress Response Abnormal Thyroid Activity The hormones of the thyroid, in concert with those of the adrenal gland and growth hormone from the pituitary, regulate energy utilisation, metabolic rate and growth. Since thyroid hormones are incorporated into the developing egg from maternal sources, they may also be involved in larval development, although their role in this is still far from clear. The main thyroid hormones, thyroxine (T) and triiodothyronine (T), are tetra- and triiodinated derivatives of 4-hydroxydiphenyl ether and as such have close structural similarities to other planar halogenated aromatic compounds such as PCBs, dioxins and DDT. It is therefore not surprising that such chemicals are those most suspected of inducing thyroid dysfunction by competing for receptors. As in mammals, thyroid function can be disrupted by action of pollutants at several sites, including production of thyrotrophin releasing hormone (TRH) from the hypothalamus, release of thyroid stimulating hormone (TSH) from the pituitary, synthesis of T by the thyroid or its conversion in the thyroid and liver into T (Figure 1). The study of thyroid activity has undoubtedly been inhibited by the difficulty of isolating the tissue which, unlike mammals, does not form a distinct gland but is scattered as isolated cells around the ventral aorta. The most extensive studies of thyroid dysfunction have been of salmon in the Great Lakes where massive goitres, with thyroid enlargements of up to a million million fold, were observed. Goitre is usually attributed to iodine deficiency and loss of negative feedback to the pituitary resulting from failure of the thyroid to synthesise T and T. This was not the case in the Great Lakes, nor was there a direct correlation between tissue PCB levels and the presence of goitres. The Great Lakes, however, contain such a cocktail of chemicals that it is impossible to analyse for all xenobiotics. Rodents fed Great Lakes salmon showed similar excessive thyroid growth which was related to the PCB content of the fish diet, but this does not demonstrate a cause and effect unless all xenobiotics in the fish could be identified. The high incidence of human goitres in the State of Michigan reinforces the suggestion that environmental pollution is the cause of the goitres in fish, and emphasises the value of fish as monitors of human health hazards. Other studies have shown that thyroid activity can be affected by DDT, fenitrothion, malathion, endosulfan, c-BHC and carbamate pesticides, organic and inorganic mercury, and lead. In addition to its function in metabolic control, the thyroid plays an important role in fish migration and especially in the ability of young salmon to adapt to saltwater during their seaward migration. There is evidence that pre-migratory patterns and sea-water adaptation can be affected by PCBs, fuel oil, arsenic, copper and zinc. Such disruption could have a major impact on the stocks of such migratory fish.

J. F. Leatherland, in Chemically-induced Alterations in Sexual and Functional Development: The Wildlife-Human Connection, ed. T. Colborn and C. Clement, Princeton Scientiﬁc, Princeton, 1992, p. 129. W. H. Beierwaltes, Washington City Med. Soc. Bull., 1987, Sept 1987, 3.

42 Environmentally Induced Endocrine Abnormalities in Fish

Abnormal Stress Responses Many pollutants increase the activity of the adrenal gland of fish as a result of their action as irritants or of their inhibition of the respiratory function of the gills. It is important to recognise that these changes are simply a normal stress response and differ little from that which occurs in a fish subject to capture or attack by a predator. Similarly, xenobiotic induced changes in the gills may lead to an imbalance in mineral or water balance and the observed hormonal changes are simply a natural effort to restore equilibrium, while failure to do so may cause a stress response. These effects cannot be considered as endocrine disruption since the primary site of action is not disruption of the endocrine system. Measurement of basal levels of stress hormones are in fact very difficult since capture of the fish almost inevitably involves stress. This, however, provides a good method for determining whether there is endocrine disruption of the adrenal gland since impairment of adrenal activity as the primary effect of the xenobiotic will also lead to an impaired response to stress. Perch taken from three sites contaminated with heavy metals, pulp mill effluents or PCBs, PAHs and heavy metals all showed an inhibited stress response. Since the steroidogenic pathways of the interrenal are very similar to those of the gonads, many of the chemicals that inhibit gonadal production of steroids could similarly inhibit the stress response. The adrenal hormones, adrenaline and cortisol, also suppress the immune response, while the immune system itself comprises a range of intracellular messengers that can be affected by pollutants. Endocrine disruption of adrenal activity can therefore also affect both the ability of the fish to cope with natural stress and that induced by pollutants, and its susceptibility to disease.

8 Abnormal Liver Function The liver plays an important role in the endocrine system. The concentrations of hormones in plasma, and the activity of the glands which secrete them, are determined by the rate at which they are deactivated by the liver. The liver also has a major function in female reproduction since it is the target tissue of ovarian estrogen, to which it responds by producing the yolk protein vitellogenin. Xenobiotics that aﬀect either of these functions can therefore be considered to be potential endocrine disruptors.

Steroid Deactivation The liver contains cytochrome P-450 dependent mono-oxygenases, and reducing and conjugating enzymes that convert hormones into water soluble products which can be more easily excreted. Mono-oxygenase enzymes of the liver, which are closely related to enzymes in the adrenal gland and the gonads, are affected by copper, mercury and organotin, PCBs, PAHs, pulp mill effluent and municipal wastewater, but are unaffected by most organochlorine pesticides. Such changes A. Hontela, Rev. Toxicol., 1997, 1, 159. G. Iwama and T. Nakanishi (eds), The Fish Immune System, Academic Press, San Diego, 1996. C. R. Tyler, J. P. Sumpter, H. Kawauchi and P. Swanson, Gen. Comp. Endocrinol., 1991, 84, 291.

43 D. E. Kime in enzyme activity will alter the rate at which the hormones are deactivated and inevitably aﬀect the overall endocrine balance.

Vitellogenesis The fish liver possesses receptors which specifically respond to estrogen by synthesising the yolk protein vitellogenin. Male, female and juvenile fish all possess such receptors in the liver, but under normal conditions only the female produces sufficient estrogen to induce vitellogenin synthesis. This is usually a seasonal phenomenon and coincides with the need of the developing egg to incorporate yolk protein. Measurement of vitellogenin in plasma therefore provides both a useful indicator of the reproductive status of the female and a method of distinguishing the two sexes. Since vitellogenin is not normally detectable in significant amounts in male, juvenile or sexually regressed female fish, its presence is a clear indication of abnormal stimulation by an environmental estrogen. The extremely high levels of vitellogenin in the plasma of estrogen stimulated fish (up to 10%), the ease with which it can be measured and the specificity of the response has made this one of the most widely used tests for endocrine disruption. Unfortunately, it has also increased the general misconception that endocrine disruptors and environmental estrogens are synonymous. It is very important to realise that environmental estrogens are just one of many different endocrine disruptors and that chemicals or waterways that do not induce vitellogenesis in fish, and therefore do not contain estrogenic pollutants, can still affect many other components of the endocrine system. Induction of vitellogenesis must be seen as just one more test for a specific type of endocrine disruptor. With this proviso, it has produced valuable evidence of endocrine disruption caused by both natural estrogens in sewage effluents and by alkylphenolic detergents in effluents from textile mills. By contrast, the presence of non-estrogenic endocrine disruptors of pituitary, ovarian or liver function in an effluent can be apparent by a decrease in the normal vitellogenin production of females, leading to smaller or fewer eggs. Plasma vitellogenin measurement therefore provides valuable information about exposure to a wide range of endocrine disruptors, and not only to environmental estrogens. While production of vitellogenin by males and juveniles can be a useful bio-indicator of estrogen exposure, it is important to show whether this is actually harmful to the male. In some cases, induction of vitellogenin occurs at levels at which there is also clear evidence of inhibition of testicular activity and steroid secretion by the estrogen exposure, while in other cases the high level of the protein can cause a range of non-endocrine related problems such as kidney failure.

9 The Implications of Endocrine Dysfunction for Fish There is now very clear evidence that a wide range of anthropogenic chemicals can be considered as potential endocrine disruptors in ﬁsh (Table 1), but it is rather less clear whether they cause actual endocrine disruption in intact animals J. E. Harries, D. A. Sheahan, S. Jobling, P. Matthiessen, P. Neall, J. P. Sumpter, T. Tylor and N. Zaman. Environ. Toxicol. Chem., 1997, 16, 534.

44 Environmentally Induced Endocrine Abnormalities in Fish

Table 1 Chemicals which Heavy metals can be considered as Arsenic, cadmium, chromium, copper, iron, lead, mercury, tin, zinc potential endocrine disruptors in fish Organochlorine pesticides Aldrin, chlordecone (Kepone), 2,4-D, DDT and metabolites, dieldrin, endosulfan, endrin, b- and c-HCH (lindane), linuron, methoxychlor, mirex Organophosphate pesticides Chlorfenvinphos, cythion, diazinon, elsan, fenitrothion, fenthion, malathion, methyl parathion, mevinphos, monocrotophos, parathion, quinalphos, temephos, TEPA, tetrachlorvinphos Other pesticides Atrazine, carbofuran, carbaryl, esfenvalerate, imidazole fungicides Industrial chemicals Alkylphenols, ammonia, asbestos, chlorinated paraffins, 4-chloroaniline, cyanide, detergents, di-n-butyl phthalate, polyaromatic hydrocarbons (PAHs; e.g. anthracene, benzopyrene, methylcholanthrene, b-naphthoflavone), nitrate, nitrite, petroleum oil, phenol, pentachlorophenol, 4-nitrophenol, dinitro-o-cresol, polychlorinated biphenyls (PCBs; especially coplanar), polychlorinated dioxins, polybrominated naphthalenes, b-sitosterol, sulfide, thiourea, urea, acid water, coal dust Effluents of oil refineries, textile mills, power stations, pulp mills, sewage treatment works, vegetable oil factories Rhine sediment, St. Lawrence and Tennessee Rivers, Great Lakes, Puget Sound, Rhode Island coast, North Sea, Baltic Sea

since much of the data originated from experiments on isolated tissue. It is often not clear whether the concentrations used in such experiments would be toxic in an intact animal, and in many cases little attempt has been made to determine the lowest concentration that had an adverse effect. Nevertheless, it is clear that even of the known pollutants that are present in our aquatic ecosystems, a very high proportion can be considered as potential endocrine disruptors, and these are always present as a very complex cocktail. Although each of these chemicals may be present at levels well below the present legal limits, the cumulative effect of large numbers of such chemicals can nevertheless be significant. Legal limits have, however, generally been determined from simple toxicity testing based on lethal dose or on carcinogenicity and not on the very much lower levels that can induce endocrine disruption. Endocrine disrupting effects so far observed on the reproduction of fish include delay in gonadal development and maturation, decreased numbers and quality of eggs and sperm, inhibited response to pheromones, altered courtship patterns, and decreased fertilisation rates Many of the incidences of production of abnormal larvae may be a result of exposure during very short critical periods during early life, including exposure to contaminated yolk passed on from mothers who have accumulated high pollutant burdens during their whole life. Most studies so far have examined only survival rates and developmental abnormalities in such fish, but it is highly probable that some of the effects of early pollutant exposure will only become apparent when these fish, or even their

45 D. E. Kime offspring, attain sexual maturity. Such transgenerational consequences of pollutant exposure is potentially one of the most insidious effects of endocrine disruptors. The probability of such transgenerational transmission of xenobiotic induced abnormalities occurring in fish and other vertebrates has been significantly increased by recent understanding of the possible mechanisms of epigenetic inheritance by which mammalian parents can transmit the effects of drug and hormone treatment or starvation to their offspring and even to their offsprings offspring. In addition to reproductive effects, fish exposed to endocrine disruptors may have a decreased response to stress or decreased growth and metabolism which can affect their ability to survive, or to defend themselves against predators. All of these factors can affect the ability of the species to survive and to reproduce itself in sufficient numbers to maintain the stocks on which our commercial and sport fisheries are based. Not all fish species will be equally susceptible to the effects of endocrine disruptors. Selective sensitivity to such effects, especially those affecting reproduction, may well lead to major changes in the flora and fauna of some of our major aquatic ecosystems as the balance between fish, mammals, invertebrates and plants, and between predators and prey, is destabilised Fish are an important source of protein to much of the world’s population. Current concern has focused on the effects of overfishing and its effects on depletion of the world’s fish stocks. If commercial species, which are overwhelmingly top predators with the greatest bioaccumulation of xenobiotics, are affected by endocrine disruptors such that they have decreased fertility, they will be less able to compensate for even moderate catch rates. The combination of compromised reproductive function induced by exposure to xenobiotics with increasingly sophisticated fisheries equipment is indeed a recipe for a catastrophic decline in fish stocks. It also emphasises the transnational dimension of endocrine disruption, since rivers can transport their chemical burden across many international frontiers, and marine fish can feed, spawn and be captured in waters receiving effluent from countries with very different levels of emission and standards of pollution control. Volatile endocrine disrupting organochlorine pesticides such as DDT, which have been banned for decades in North America and Europe, are still being deposited there by aerial transmission from the Third World where they are still in use, while the pesticides, PCBs and mercury used in North America and Europe can be found even in remote Arctic regions as a result of global distillation. Protection of fish stocks from the effects of endocrine disruptors can therefore only be brought about by international agreement to limit release into the environment.

10 Fish, Wildlife and Humans—A Warning Fish have many advantages as experimental models in the study of endocrine disruption, and although they do have some signiﬁcant diﬀerences in their endocrine system to that of mammals, the underlying basis is very similar. Chemicals which are shown to be either actual or potential endocrine disruptors

G. Vines, New Sci., 28 Nov. 1998, 26.

46 Environmentally Induced Endocrine Abnormalities in Fish in fish can therefore certainly be considered as potential endocrine disruptors in mammals, including humans. Fish thus provide an excellent biomonitor of potential hazards of endocrine disruption to humans of exposure to similar chemicals. Although many xenobiotics bioconcentrate in the gonads, liver and other viscera, the edible muscle tissue still comprises the bulk of the fish and a high intake of dietary fish may lead to human exposure of high levels of potential endocrine disruptors. Such effects are most likely to be seen in wildlife and human communities with a diet rich in fish from contaminated waters. Evidence of endocrine disruption in fish-eating birds, including reproductive failure, developmental abnormalities and diminished parental care attributable to dietary exposure to PCBs, dioxins, organochlorine pesticides and mercury, has been well documented. Similar effects have been found in fish-eating mammals such as mink, seals and whales. The recent episodes of high mortality of marine mammals has been attributed to immunosuppression resulting from adrenal overactivity, but the seals also suffered from reproductive dysfunction and had high tissue PCB concentrations. Most urban communities consume a mixed diet in which fish play a minor part, but in fish-eating rural societies, around the Great Lakes and in the Arctic, fish can be the main protein source. In both areas the diet is similar to that consumed by fish-eating birds and marine mammals and contains very high levels of at least PCBs. Such communities will be the first in which signs of endocrine dysfunction become apparent. Indeed, there is already evidence that they have similar behavioural disturbances to those found in fish-eating mammals and birds. For urban societies, the risk is less obvious, but the diversity of chemicals is much greater and now includes exposure to high levels of PAHs from fossil fuel combustion, pesticide residues, food additives and colourants, wood preservatives, plasticizers etc., few of which have had even basic testing for endocrine disruption.

11 Conclusion There is now very clear evidence that fish captured in polluted habitats suffer from a variety of endocrine disorders, including reduced fertility, abnormal sexual differentiation, developmental abnormalities and decreased response to stress. Such effects have been replicated in laboratory exposures and in in vitro experiments. Similar problems have been found in fish-eating birds and mammals, which have diets similar to those of fish-eating human communities. There are increasing numbers of reports of decreased sperm counts and an increased incidence of genital abnormalities in human males. While endocrine disruption in the human population will first become manifest at levels which do not attain the levels of clinical concern, even the slight decreases in fertility, response to stress and behavioural changes now apparent in fish and other wildlife could threaten the cohesion of human society. There is also some evidence that such effects can be transmitted across generations so that both past and present exposures could affect even our children’s children. There are many G. A. Fox, J. Great Lakes Res., 1993, 19, 722. D. M. Fry, Environ. Health Perspect. 1995, 103 (Suppl. 7), 165. J. L. Jacobson and S. O. Jacobson N. Engl. J. Med., 1996, 335, 783.

47 D. E. Kime parallels between the problems of climate change and endocrine disruption since both result from the unnecessarily high levels of consumption which form the basis of our dominant but unsustainable socio-economic system. Even though there is strong evidence that the dangers exist, commercial and political vested interests are correct in their view that there is as yet no absolutely conclusive proof that either is a threat to human health. If, however, we wait until there is such deﬁnitive evidence it will be too late to take remedial action and the inevitable consequences could cause catastrophic political and social upheaval. The condition of the ﬁsh in our rivers and oceans, like the recent rise in global temperatures, is a warning to us all that the present high living standard of a minority of the world’s human population has a price which is paid by all of humanity and by the other species with whom we share this planet.

48 Eﬀects of Endocrine Disrupting Chemicals in Invertebrates

MICHAEL H. DEPLEDGE, TAMARA S. GALLOWAY AND ZOE BILLINGHURST

1 Introduction Evidence is accumulating that many common environmental toxicants are capable of disrupting developmental processes by interfering with the actions of endogenous hormones. Public attention has focused on apparent increases in hormone dependent cancers of both male and female reproductive organs, which cannot be accounted for simply by improvements in detection methods. In wildlife species, an array of abnormal physiological and morphological responses likely to compromise reproductive fitness has been noted. Hormonal disturbances in wildlife include sex changes in riverine fish and marine snails, reproductive failure in birds and abnormalities in the reproductive organs of alligators and polar bears. The term endocrine disruption has been used to describe a range of such effects that may be acting through entirely different mechanisms. For the purpose of this review, the term endocrine disruptor is defined as any exogenous agent that causes adverse health effects in an intact organism, or its progeny, consequent to changes in endocrine function. Endocrine disruptors may act to (1) mimic the effects of hormones, (2) antagonise the effects of hormones, (3) alter the pattern of synthesis and metabolism of hormones and (4) modify hormone receptor levels. The most widely studied of these effects to date is the action of diverse substances which mimic the action of endogenous estrogen, either through interaction with the estrogen receptor, or through other mechanisms. These are referred to as estrogenic xenobiotics, exoestrogens or xenoestrogens. In this article, the effects of endocrine disrupting chemicals in a range of

R. M. Sharpe and N. E. Skakkebaek, Lancet, 1993, 341, 1392—5. T. Colborn, J. Peterson Myers and D. Dumanoski, Our Stolen Future, Little, Brown, Boston, 1996. P. Grandjean et al., presented at the European Workshop on the Impact of Endocrine Disruptors on Human Health and Wildlife, Weybridge, UK, 1996, Environment and Climate Research Programme, DGXII, European Commission Publication EUR 17549, p. 5. A. M. Soto, C. Sonnenschien, K. L. Chung, M. F. Fernandez, N. Olea and F. O. Serrano, Environ. Health Perspect., 1995, 103 (suppl.), 113.

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

49 M. H. Depledge, T. S. Galloway and Z. Billinghurst invertebrate species are considered and the potential ecological signiﬁcance discussed.

2 Endocrine Disrupting Chemicals Many compounds introduced into the environment by human activity have been implicated in endocrine disruption. Because of the diversity of structure and mechanism of action of these compounds, and as yet uncertain synergistic effects of mixtures, the development of management strategies (e.g. by environmental agencies and legislators) has been difficult. Compounds implicated are often ubiquitous and persistent, and may bioaccumulate at chronically polluted sites. For instance, the bioaccumulation concentration factor (BCF) for DDT in aquatic animals ranges from 25 000 to 100 000 whilst that of nonylphenol approaches 300. The duration and timing of exposure are also important. Short term exposure of the echinoderm Asterias rubens to 200 kgl\ of cadmium caused a reduction in ovary growth. Long term exposure to 25 kgl\ caused a delay in ovarian growth which was obvious after 5 months exposure but by the end of the reproductive cycle the difference had become smaller. Some of the many compounds implicated in endocrine disruption are shown in Table 1. The difficulty of predicting the endocrine disruptive potential of any chemical is illustrated by the diverse variety of structures that possess estrogenic activity. Naturally occurring estrogens, such as 17b-estradiol, are 18-carbon steroids with a phenolic  ring and a b-hydroxy group or ketone at position 17 of the d ring (Figure 1). The conformation of the estrogen receptor contains a binding pocket into which the phenolic ring fits. The structure of the remainder of the molecule then determines the affinity of binding and the mode of action as an agonist or antagonist, depending on its ability to allow the correct conformational changes by the receptor. Yet while most xenoestrogens showing high receptor affinity possess a para-substituted phenolic ring, some have more than one (methoxychlor metabolites and diphenolic isoflavonoids) and others have none (chlordecone and o,p-DDT). Structural rigidity imparted by conformational restriction may explain the ability of compounds such as para-substituted hydroxylated metabolites of PCBs to bind the estrogen receptor, and explain the fact that the o,p-isomer of DDT is estrogenic while the p,p-isomer is only weakly estrogenic. The QSAR (quantitative structure—activity relationship) approach has been considered for the identification of toxicants that bind to steroid and aryl

A. C. Nimrod and W. H. Benson, Crit. Rev. Toxicol., 1996, 26, 335. R. Ekelund, A. Bergman, A. Granno and M. Berggren, Environ. Pollut., 1990, 64, 107. P. J. Den Besten, J. R. Maas, D. R. Livingstone, D. I. Zandee and P. A. Voot, Comp. Biochem. Physiol. C, 1991, 100, 165. L. E. Gray Jr., E. Monosson and W. R. Kelce in Interconnections between Human and Ecosystem Health, ed. E. Monosson and R. T. Di Giulio, Chapman and Hall, London, pp. 45—82. W. L. Daux and J. F. Griﬃn, J. Steroid Biochem., 1987, 27, 271. V. C. Jordan, S. Mittal, B. Gosden, R. Koch and M. E. Lieberman, Environ. Health Perspect., 1985, 61, 97. J. D. McKinney and C. L. Waller, Environ. Health Perspect., 1994, 102, 290. D. Kupfer and D. H. Bulger, Estrogenic Properties of DDT and Its Analogues, in Estrogens in the Environment, ed. J.A. McLachlan, Elsevier, New York, 1980, p. 239.

50 Eﬀects of Endocrine Disrupting Chemicals in Invertebrates

Table 1 Examples of Environmental estrogens: estrogen receptor mediated chemicals suspected of Chlordecone causing endocrine Polychlorinated biphenyls (PCBs) disruption listed according o,p-DDT to suspected mechanism of action Environmental antiestrogens Dioxin p,p-DDT/DDE Endosulfan Environmental antiandrogens Vinclozolin Procymidone Kraft mill eﬄuent Toxicants that alter circulating steroid hormone levels Dioxin Endosulfan Aroclor 1254 Toxicants that act via the CNS Dithiocarbamate pesticides Carbon disulﬁde Manganese Other mechanisms Dibutyl phthalate Benzidine-based dyes Vinylcyclohexene Antithyroid endocrine disruptors PCBs Herbicides, e.g. nitrofen Phthalic acid esters Adrenal endocrine disruptors Aniline dyes Ketoconazole fungicides PCBs

Adapted from Gray et al.

hydrocarbon (Ah) receptors. This procedure is limited by the extent of information available to form a reliable database. The promiscuous nature of estrogen and other steroid binding sites within the steroid receptor superfamily, and the level of interspecies variability, also complicate the issue.

3 Endocrine Disruptors in Invertebrates Whilst the impact of xenoestrogens has been the most widely studied eﬀect in vertebrates, it is unclear whether endocrine disruption in invertebrates proceeds

C. Waller and J. McKinney, Toxicologist, 1995, 15, 271.

51 M. H. Depledge, T. S. Galloway and Z. Billinghurst

Figure 1 Structures of some estrogenic chemicals

predominantly through similar mechanisms to those outlined above, or via other routes. A list of chemicals which have been shown to act as endocrine disruptors in invertebrates is shown in Table 2. An appreciation of the extent to which invertebrate species may be exposed to such chemicals comes from considering the eﬀects of complex mixtures. In the North Atlantic ecosystem alone, hundreds of pollutant chemicals have been identiﬁed. These include metals, synthetic and chlorinated organics and polycyclic aromatic hydrocarbons. Over 300 aromatic hydrocarbons have been detected in some regions of the Chesapeake Bay, and high concentrations of PCBs have been

52 Eﬀects of Endocrine Disrupting Chemicals in Invertebrates

Table 2 Chemicals Herbicides PCBs/alkylphenols suspected of causing Diquat bromide Clophen A50 endocrine disruption in Atrazine Aroclor 1242 invertebrate species Simazine Nonylphenol Diuron Pentylphenol Metals Insecticides Cadmium Pyriproxyfen Selenium DDT Zinc Endrin Mercury Methoprene Lead Diflubenzuron Tributyltin (TBT) Kelthane Vertebrate steroids Complex mixtures Diethylstilbestrol (DES) Tannery and Kraft mill effluent Testosterone Sediment extract Estradiol Sewage effluent

found in sediments of New Bedford Harbour. The potential for them to interact is therefore great. Much controversy has arisen regarding the ability of mixtures of weakly estrogenic compounds to act synergistically, notably the synergistic potential of mixtures of PCBs or of the insecticides dieldrin and toxaphene. Recent studies suggest that the action of mixtures is at least additive. The determination of routes of uptake, patterns of bioactivation, biotransformation and excretion of endocrine disruptors has yet to be carried out. The estrogenic action of some compounds is known to be enhanced during metabolism, such as the metabolic hydroxylation of PCBs to yield the more potent estrogenic polychlorinated hydroxybiphenyls. The issue is further complicated by the range of naturally occurring estrogenic substances to which diﬀerent species may be exposed through their diet. For example, Farnsworth et al. list over 400 species of plants that contain potentially estrogenic isoﬂavonoids or coumestans. Some plant extracts also have antispermatic activity, either acting directly on the testis, or altering hypothalamic—pituitary function. In assay sytems, many of these phytoestrogens are able to bind more avidly to the estrogen receptor than estrogen itself. Why plant products should possess such avid estrogenic compounds is unclear, but it has been suggested that consumption of phytoestrogens by insects may result in alterations in the sex

S. F. Arnold, B. M. Collins, M. K. Robinson, L. J. Guillette and J. A. McLachlan, Steroids, 1996, 61, 642. D. M. Klotz, B. S. Beckman, S. M. Hill, J. A. McClachlan, M. R. Walters and S. F. Arnold, Environ. Health Perspect., 1996, 104, 1084. T. E. Wiese, C. R. Lambright and W. R. Kelce, Fundam. Appl. Toxicol., 1997, 36, 294. K. S. Korach, P. Sarver, K. Chae, J. A. McClachlan and J. D. McKinney, Mol. Pharmacol., 1987, 33, 120. N. M. Farnsworth, A. S. Bingel, G. A. Cordell, F. A. Crane and H. H. S. Fong, J. Pharm. Sci., 1975, 64, 717. D. K. Salunkhe, R. N. Adsule and K. I. Bhonsle in Toxicants of Plant Origin, ed. P. Cheeke, CRC Press, Boca Raton, 1989, vol. 4, pp. 53—81.

53 M. H. Depledge, T. S. Galloway and Z. Billinghurst ratio which ultimately reduce population size, thereby sparing the plant. Some phytosteroids, the brassinosteroids, are structurally similar to invertebrate ecdysteroids in that they contain the entire C cholesterol skeleton complete with its C side chain. This section is removed in vertebrates through the action of cytochrome P450## side chain cleavage enzyme. As a consequence, the brassinosteroids are very soluble in water. More than 100 of these so-called phytoecdysteroids have been identified, and many have been shown to possess hormonal activity in insects. For example, moulting is arrested or delayed in some aquatic insects following exposure to paper mill or tannery effluents, which contain high concentrations of phytoestrogens, presumably through interference with ecdysone metabolism. Exposure to estrogenic compounds through diet will differ for herbivores and carnivores, the latter being most likely to encounter endogenous steroids in their prey. Efficient uptake of steroids in mammals is illustrated by the use of the contraceptive pill, but routes of absorption in invertebrates remain to be determined. The relationship between endocrine disruption and metabolic toxicity, with reduced reproductive viability a secondary consequence of metabolic disturbance, also merits further study in invertebrate species.

4 Invertebrate Endocrine Function The invertebrate phyla are often neglected in ecotoxicological testing protocols. A token invertebrate species such as the copepod Daphnia may be used to evaluate the effects on extremely diverse phyla. This neglects the diversity of biochemical and physiological functions that may render different phyla vulnerable to different classes of compound at different stages of their life cycles. The use of hormones to control and co-ordinate physiological and behavioural processes is common to all the major invertebrate taxa. Neuropeptide signalling mechanisms involving the peptide products of specialised neurosecretory cells feature predominantly, for instance crustacean hyperglycaemic hormone (CHH) which is synthesised and secreted from the neuroendocrine eyestalk gland. Vertebrate-like steroids have been identified in the tissues of many species, although their precise functional role remains unclear. In situ synthesis of testosterone and other steroids appears to occur in echinoderms, molluscs and crustaceans and is linked to reproduction and developmental processes. It is not always possible, however, to predict the effects of vertebrate steroids in other species. Injection of concentrations of androgenic steroids capable of causing virilisation in vertebrates failed to elicit a definable response in the crustaceans Orchestria gammarellus or Pachygraspus crassipes. Insects, crustaceans, platehelminthes, nematodes and annelids use homoses- quiterpenoid epoxides (juvenile hormones) and ecdysteroids (ecdysone, 20-

B. Luu and W. Werner, Presic. Sci., 1995, 46, 49. J. H. Adler and R. J. Grebenok, Lipids, 1995, 30, 257. M. A. Subramanian and G. Varadarag, Odonatlogica, 1993, 22, 229. L. Swevers, J. Lanbert and A. De Loof, Comp. Biochem. Physiol. B, 1991, 99, 35. H. Charniaux-Cotton, Gen. Comp. Endocrinol., 1962, suppl. 1L, 241. D. S. King, Gen. Comp. Endocrinol., 1964, 4, 533.

54 Effects of Endocrine Disrupting Chemicals in Invertebrates hydroxyecdysone) in the control of reproduction, moulting, feeding and behaviour. Crustaceans also use methyl farnesoate, an unepoxidised precursor of juvenile hormone, in the control of ecdysis. The structure of these steroids differs from vertebrate steroids in that they are lipophilic and larger in size, with the C cholesterol skeleton intact, and are altered in shape, having an ab cis configuration, which leaves the a ring perpendicular to the rest of the molecule (Figure 1). These hormones have their own binding proteins and intracellular receptor systems. The juvenile hormone receptor shows some homology to vertebrate steroid hormone receptors but is not classed as a member of the steroid receptor superfamily. None of these specialised steroid hormones is present in vertebrates.

5 Evidence of Endocrine Disruption in Invertebrates A wealth of laboratory-based evidence is available to illustrate endocrine disruption in marine invertebrates. Fingerman et al. has reviewed the effects of trace metals in crustaceans, in which disturbances of the hormonal control of moulting, limb regeneration, blood glucose level, colour changes and reproduction were seen. Cadmium and selenium are both strongly implicated in a range of effects in, for example, crustaceans and echinoderms. Both cadmium and selenium affect moulting in Daphnia magna, with proecdysis delayed and the intermoult period lengthened. The size and viability of progeny is also reduced. When the sea urchin Strongylocentrotus intermedius is exposed short term to 100 mg l\ cadmium, oogenesis is adversely affected. Short-term exposure of the sea star Asterias rubens to cadmium or zinc led to altered steroid metabolism in the gonads and pyloric caeca, and caused a consequent reduction in progesterone and testosterone levels. A similar mechanism may be responsible for the abnormal reproductive development seen in Asterias following exposure to PCBs. The effects of cadmium may be modulated by environmental factors, including salinity and the presence of other compounds. The synergistic inhibition of limb regeneration in the hermit crab Uca pugilator caused by combinations of cadmium and methylmercury is only evident in water of high salinity. Many pesticides cause endocrine disruption in vertebrate and invertebrate species at concentrations that are not overtly metabolically toxic. The insect growth inhibitor diflubenzuron can affect the reproduction, development and behaviour of estuarine crustaceans at concentrations of just 10 mg l\ (reviewed

D. S. King in Endocrinology of the Insects, ed. R. Downer and H. Laufer, Liss, New York, 1983, pp. 57—64. J. Koolman and K. D. Spindler in Endocrinology of the Insects, ed. R. Downer and H. Laufer, Liss, New York, pp. 179—201. M. Fingerman, M. Devi, P. S. Reddy and R. Katyayani, Zool. Stud., 1996, 35,1. C. Bodar, P. Voogt and D. Zandee, Comp. Biochem. Physiol. C, 1990, 90, 341. T. W. Schultz, S. R. Freeman and J. N. Dumont, Arch. Environ. Contam. Taxicol., 1980, 9, 23. N. K. Khristoforova, S. M. Gnezdilova and G. A. Vlasova, Mar. Ecol. Press Ser., 1984, 17,9. P. J. Den Besten, H. J. Herwig, D. I. Zandee and P. A. Voogt, Ecotoxicol. Environ. Safety, 1989, 18,173. P. J. Den Besten, J. M. Elenbaas, J. R. Maas, D. R. Livingstone, D. I. Zandee and P. A. Voot, Aquat. Toxicol., 1991, 20, 95.

55 M. H. Depledge, T. S. Galloway and Z. Billinghurst by Cunningham). Concentrations of diflubenzuron as low as 0.075 mg l\ led to reduced fecundity in the estuarine crustacean Mysidopsis bahia. The organochlorine pesticide endrin delays the onset of spawning and embryo viability of the shrimp Palaeomenetes pugio at concentrations of 30 mg l\. Endrin can also alter the reproduction and development of the malacostracan Gammarus fasciatus. The toxicity of the non-systemic acaricide kelthane was investigated by Rao et al. in the prawn Metapenaeus monoceros, who concluded that the toxicity of the pesticide was modulated by ecdysteroid products of the neuroendocrine eyestalk gland. Many insecticides designed to inhibit the growth and moulting of insects can affect crustaceans. Methoprene is a juvenile hormone mimic, which completely inhibits larval development in the estuarine shrimp Palaemonetes pugio. The first and last larval stages were particularly sensitive. A similar study in the mud crab Rhithropanopeus harrisii showed that this effect was isomer-specific. Few clear examples of endocrine disruption in invertebrates in situ exist. One of the best documented and understood examples is that of ‘imposex’ and ‘intersex’ in gastropod molluscs exposed to anti-fouling paints containing tributyltin (TBT). The term ‘imposex’ was first coined in the early 1980s to describe the superimposition of male characteristics onto females which was being observed with increasing frequency in molluscs exposed to TBT in the vicinity of marinas. Neogastropods are gonochoristic (sexes are separate). Females exposed to TBT develop a penis-like structure, vas deferens and convoluted gonoduct. In some species, malformations in the oviduct, which prevent copulation and capsule formation, are seen, and this condition has been termed ‘intersex’. The frequency and degree of malformation is related to the degree of exposure and the species in question. The mechanism by which TBT causes these effects has been extensively studied. The androgenic effects of TBT appear to be caused by interference with steroid biosynthesis rather than by mimicking the action of testosterone at the androgen receptor. Exposure of female molluscs to TBT leads to an elevation in testosterone in the haemolymph. Much of the experimental evidence

P. A. Cunningham, Environ. Pollut., 1986, 40, 63. D. R. Nimmo, T. L. Hamaker, J. C. Moore and C. A. Sommers, Bull. Environ. Contam. Toxicol., 1979, 22, 767. D. B. Tyler-Schroeder, Use of Grass Shrimp Paleomonotes pugio in a Life Cycle Toxicity Text, American Society for Testing and Materials, Philadelphia, 1979. K. J. Macek, K. S. Buxton, S. S. Sauter, S. Gnilka and J. W. Dean, Chronic Toxicity of Atrazine to Selected Aquatic Invertebrates and Fishes, Environmental Research Laboratory, US Environmental Protection Agency, Deluth, MN, 1976, EPA 600/3-76-047. K. V. Rao, P. Surendranath and Ramanaiah, Bull. Environ. Contam. Toxicol., 1992, 49, 582. C. L. McKenney and E. Matthews, Environ. Pollut., 1990, 64, 169. D. M. Celestial and C. L. McKenney, Environ. Pollut., 1994, 85, 169. B. S. Smith, Proc. Malacol. Soc. London, 1971, 39, 377. B. Bauer, P. Fioroni, I. Ide, S. Liebe, J. Oehlmann, E. Stroben and B. Waterman, Hydrobiologica, 1995, 309, 15. G. W. Bryan, P. E. Gibbs, L. G. Hummerstone and G. R. Burt, J. Mar. Biol. Soc. UK, 1986, 66, 611. P. Matthiessen and P. E. Gibbs, Environ. Toxicol. Chem., 1998, 17, 37. R. F. Lee, Mar. Environ. Res., 1991, 32, 29. N. Spooner, P. E. Gibbs, G. W. Bryan and L. J. Goad, Mar. Environ. Res., 1991, 32, 37.

56 Effects of Endocrine Disrupting Chemicals in Invertebrates available points to a competitive inhibition of cytochrome P450 aromatase, which converts testosterone to 17b-estradiol, leading to a build up of testosterone. Additional possibilities include inhibition of testosterone sulfur conjugate formation, which would hinder excretion. It should be noted, however, that TBT does not appear to induce imposex in many gastropods for reasons that are unclear, and that agents other than TBT do cause imposex in species in which TBT is ineffective. Feral and LeGall have suggested that TBT may affect the neuroendocrine system, interfering with the release of a retrogressive factor from the cerebropleural ganglia. The effects of TBT on neuroendocrine function merit further study. Another possible example of endocrine disruption in situ relates to the findings of Moore and Stevenson of altered sex ratios and intersex harpacticoid copepods in the vicinity of Edinburgh’s long sea sewage outfall. Intersexuality is common in some crustaceans but is extremely rare in harpacticoid copepods. There was no relationship between the frequency of intersex and distance from the most contaminated sites and nor was there evidence that other benthic species had been adversely affected. Additional work is required to prove conclusively that endocrine disruption is involved in this case.

6 Detection and Assessment of Endocrine Disrupting Chemicals Several recent expert reviews and workshops have discussed the effects of endocrine disruption on wildlife and especially invertebrate species. These include the EU workshop on the impact of endocrine disruptors on human health and wildlife (Weybridge, 1996), the IEH workshop (Leicester, May 1997), the Environment Agency Consultative report (January 1998) and the Tyndall Forum at the Royal Institution (February 1998). They have concluded that endocrine disruption may have far-reaching adverse consequences for biodiversity and the sustainability of natural ecosystems. More comprehensive bioassay systems are required to identify and assess chemicals alleged to produce endocrine modulating effects. A number of in vitro estrogenicity assays are currently in use including competitive ligand binding, cell proliferation, post-confluent cell accumulation, endogenous protein expression, recombinant receptor/reporter gene assays, quantitative reverse transcriptase—polymerase chain reaction and yeast-based assays such as the YES assay. These assays provide an efficient and cost-effective means of screening large numbers of chemicals at a mechanistic

S. Evans, personal communication. C. Feral and S. LeGall, in Molluscan Neuroendocrinology, ed. J. Lever and H. H. Boer, North Holland, Amsterdam, 1983, pp. 173—175. C. G. Moore and J. M. Stevenson, J. Nat. Hist., 1994, 28, 1213. T. S. Ruh, B. S. Katzenellenbogen, J. A. Ketzenellenbogen and J. Gorski, Endocrinology, 1973, 92,125. J. F. Gierthy, D. W. Lincoln, K. E. Roth, S. S. Bowser, J. A. Bennett, L. Bradley and H. W. Dickerman, J. Cell. Biochem., 1991, 45, 177. S. A. Heppel, N. D. Denslow, L. C. Folmar and C. V. Sullivan, Environ. Health Perspect., 1995, 103,9. P. Balaguer, A. Joyeux, M. S. Denison, R. Vincent, B. E. Gillesby and T. Zacharewski, Can. J. Physiol. Pharmacol., 1996, 74, 216. T. R. Zacharewski, K. L. Bondy, P. McDonell and Z. F. Wu, Cancer Res., 1994, 54, 2707. B. E. Gillesby and T. R. Zacharewski, Fundam. Appl. Toxicol., 1997, 36, 130.

57 M. H. Depledge, T. S. Galloway and Z. Billinghurst level, but provide no information concerning the importance of estrogenicity at the level of whole organisms or populations. Routine androgenicity bioassays are not yet available, nor are bioassays for the eﬀects of endocrine disrupting chemicals in invertebrates.

7 Biomarkers of Endocrine Disruption Biomarkers are defined as biological responses that can be measured in tissue samples, body fluids or at the level of the whole organism, which signal exposure to or adverse effects of anthropogenic chemicals and radiations. The biomarker approach may be an extremely useful means of detecting endocrine disruption in situ, providing early indications of ecologically relevant effects. Ecotoxicology is concerned with ensuring that environmental contamination has a minimal impact on population dynamics, community structure and ecosystem processes. The induction or inhibition of a particular enzyme is of little use as a biomarker if it has no relevance to the overall well-being of the organism. Thus, biomarker responses should ideally be linked to measures of growth rate, reproductive output and viability of offspring. Such considerations have been discussed in detail by Depledge. Biomarkers of endocrine disruption have been described in a variety of species. In lower vertebrates (fish and amphibians), production of the egg yolk protein vitellogenin is altered by exposure to xenoestrogens and assays for vitellin (a precursor protein) and vitellogenin have been developed as biomarkers of exposure. For instance, Palmer et al. developed an ELISA for vitellin in the African clawed toad and used it to demonstrate induction of vitellin following exposure to the potent estrogenic compound diethylstilbestrol (DES). Zona radiata protein, which is involved in egg shell production, shows an even stronger response than does vitellin, and may be a potentially useful biomarker. Whilst these assays have proved useful in the detection of chemicals with estrogenic activity, it is still unclear what physiological significance this increase in protein production has, if any, in relation to the growth and development of the organism. The identification of phenotypic alterations that occur in response to endocrine disrupting chemicals may be extremely useful, particularly in field studies. The nonionic surfactant 4-n-nonylphenol has been shown to cause a range of effects depending on the life stage during which exposure occurs. Prenatal exposure of Daphnia led to morphological abnormalities in a proportion of juveniles, whilst exposure of adult females led to reduced fecundity. Nonylphenol can accumulate on particulate matter in interstitial environments, posing a potential threat to organisms which live there including the polychaete worm Dinophylus gyrociliatus.WhenD. gyrociliatus isexposedtoenvironmentally M. H. Depledge, in Toxic Impacts of Wastes on the Aquatic Environment, ed. J. F. Tapp, S. M. Hunt and J. R. Wharfe, Royal Society of Chemistry, Cambridge, 1996, pp. 104—115. M. H. Depledge, Environ. Health Perspect., 1994, 102, 101. J. P. Sumpter, Toxicol. Lett., 1995, 82/83, 737. B. D. Palmer, L. K. Huth, D. L. Pito and K. W. Selcer, Environ. Toxicol. Chem., 1998, 17, 30. A. Aruwke, F. R. Knudsen and A. Goksyor, Environ. Health Perspect., 1997, 105, 418. M. H. Comber, T. D. Williams and K. M. Stewart, Water Res., 1993, 27, 273. J. B. Shurin and S. I. Dodson, Environ. Toxicol. Chem., 1997, 16, 1269.

58 Effects of Endocrine Disrupting Chemicals in Invertebrates realistic concentrations of nonylphenol, an increase in egg production coupled to a reduction in egg viability was seen. Growth rate and sex ratio were unaffected. D. gyrociliatus has a short life cycle and is easy to culture, making it a promising candidate for the study of reproductive and transgenerational effects. The benthic amphipod Corophium volutator has also been examined for potential morphological changes. Exposure to environmentally realistic sub-lethal concentrations of nonylphenol for 100 days led to a decrease in the average length, density and longevity of mature organisms. A range of striking phenotypic alterations was seen, including a significant increase in the length of antennae, which might prove a useful morphological biomarker. The LC for nonylphenol in C. volutator was 1.67 mg l\, indicating that these organisms are relatively insensitive to its toxic effects. In contrast, the 48 h LC value for nonylphenol in rainbow trout is reported to be just 230 mg l\. This is an important point, as it remains possible that nonylphenol exposure could lead to lethality prior to the occurrence of estrogenic responses. The threshold concentration of nonylphenol reported to induce vitellogenesis in rainbow trout is 20—50 mg l\. Cyprid major protein is a larval storage protein necessary for successful metamorphosis. Production of cyprid major protein was increased in the barnacle Balanus amphitrite following exposure to both nonylphenol and estradiol, suggesting that it may be a potential biomarker of estrogen exposure in invertebrates such as barnacles. Another potential histopathological biomarker of endocrine disruption is the development of ovotestes in the lobster Homarus americanus.

8 A Strategy for the Detection of Endocrine Disruption A proper assessment of the ability of chemicals, or mixtures of chemicals, to act as endocrine disruptors requires a multi-tiered approach which is able to identify mechanisms, give early warning of adverse eﬀects and identify which of these eﬀects is likely to be ecologically relevant. A strategy has been proposed by Depledge and Billinghurst and is outlined below.

1. Culture a range of invertebrate species from the major phyla, preferably species with short life cycles. The effects of potential endocrine disrupting chemicals on growth rate, reproductive output, viability of offspring and sex ratio, and the vulnerability of different stages of the life cycle, can then be determined. 2. Develop biomarkers of endocrine disruption. Further evidence of endocrine disruption is required in addition to population parameters. Biochemical and histopathological biomarkers are required to identify which hormone systems are affected.

M. H. Depledge, Mutat. Res., 1998, 399, 109. R. J. Brown, M. Conradi and M. H. Depledge, Sci. Total Environ., 1999, in press. K. Shimizu, W. Saikawa and N. Fusetani, Comp. Biochem. Physiol. B, 1996, 115, 111. Z. Billinghurst, A. S. Clare, Matsumara and M. H. Depledge, Aquat. Toxicol., 1999, in press. G. Sangalang and G. Jones, Can. Tech. Rep. Fish Aquat. Sci., 1997, 2163, 46. M. H. Depledge and Z. Billinghurst, Mar. Pollut. Bull., 1999, in press.

59 M. H. Depledge, T. S. Galloway and Z. Billinghurst

3. Mechanistic studies to identify how endocrine disrupting chemicals interact with hormone systems are required. Although population effects coupled with biomarkers of exposure are strongly suggestive of endocrine disruption, the effect could be secondary to metabolic toxicity. Establishing mechanisms may avoid the need to make decisions on a weight of evidence approach alone. 4. Conduct field studies based on the results of population studies and biomarker responses. The extent to which endocrine disruption occurs in situ is important in considering the scale of remedial action required.

The success of the above approach depends strongly on the identification of an adequate range of sentinel species. Sentinel species should be common and widespread, reproduce sexually and be sexually dimorphic, be relatively insensitive to pollutants other than endocrine disruptors and have well described hormonal systems. Selection of species with rapid generation times will allow the identification of transgenerational effects. These criteria were recently discussed at an Institute of Health workshop on endocrine disruption (Leicester, 1997). Suites of species designed to cover as wide a range of habitat and feeding habit as possible will increase the power of this approach.

9 Summary and Conclusions Endocrine disrupting chemicals are a structurally diverse group of compounds that may adversely affect endocrine function in both human and wildlife populations. They may act through a variety of mechanisms, the most widely studied being through interaction with the estrogen receptor. This diversity of structure and mechanism makes prediction particularly difficult and it is not yet clear to what extent the effects noted in laboratory experiments are of ecological significance. The best documented example of endocrine disruption affecting invertebrates in the environment is the description of imposex and intersex in marine gastropods exposed to TBT. Evidence also exists of vitellogenin induction in crabs, other decapods and molluscs, the development of intersex in marine copepods and freshwater Daphnids, morphological changes in the antennae of the marine amphipod C. volutator and alterations in larval settlement rate and development in barnacles. These observations highlight some of the varied endpoints which may occur in invertebrate species in response to endocrine disrupting chemicals. It is not yet clear to what extent the route of exposure may affect the end result, nor is the vulnerability of different phyla at different stages of the lifecycle defined. In view of the ecological and commercial importance of invertebrate species, it is crucial that a strategy for the identification of endocrine disruption in invertebrates is developed and a comprehensive approach combining both in vivo and in vitro measures is advocated. Finally, it is worth noting that endocrine disruption in invertebrates may encompass other systems in addition to those controlling sexual development and reproduction. For example, interference with the ecdysone-mediated process of moulting and growth rate may have profound implications for the survival of the organism.

60 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans

CATHERINE BOTHAM, PHILIP HOLMES AND PAUL HARRISON

1 Introduction Throughout the latter half of this century, there has been growing concern about the potential dangers from anthropogenic chemicals in the environment. Recently, a growing body of evidence has suggested that certain groups of these chemicals can disrupt normal endocrine functions, and that this could constitute a threat to humans and wildlife. Concern initially focused on the ability of some chemicals to mimic the activity of endogenous oestrogens, but the scope has now broadened to include not only the gonads and reproductive system, but also other endocrine glands (e.g. the pituitary, thyroid, thymus and adrenal) and a number of other endocrine-modulated physiological systems, including the immune and neurological systems. Evidence of reproductive abnormalities has been noted in individuals of a number of wildlife populations, although this has usually been restricted to the vicinity of polluted locations, e.g. sites of accidental chemical spillage or close to point sources of effluent discharge. It is also recognised that endocrine disruption could result from exposure to naturally occurring or plant-derived substances, for example compounds in Kraft paper mill effluent. The term endocrine disrupter (ED) has tended to be used for those chemicals which act specifically at the level of the hormone receptor present in the target cells of various organs. Such chemicals may either mimic the action of the natural hormone (agonistic activity) or are sufficiently similar in molecular shape to the naturally produced hormone to interfere with the interaction between the hormone and receptor, thus blocking or impeding the activation of the receptor (antagonsitic activity). Such effects may occur at very low concentrations (as with the endogenous hormone), compared with the concentrations normally required to elicit the more ‘traditional’ toxic effects attributed to chemicals. Recently,

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

61 C. Botham, P. Holmes and P. Harrison wider definitions of endocrine disruption have been proposed, which incorporate mechanisms such as the disruption of receptor imprinting during neonatal stages and the modification of hormonal biotransformation pathways (for example, by induction of members of the cytochrome P450 super family of enzymes), thus affecting the synthesis or degradation of hormones or indirectly suppressing associated hormones via feedback mechanisms. Particularly sensitive exposure windows for endocrine disruption are now thought to exist at the organisational level in the developing embryo or during metamorphosis, and at the activational level in juvenile or adult animals.

2 Evidence For Endocrine Disruption Endocrine disruption in wildlife was first reported in the 1950s, though the observations were not immediately associated with disturbance of the endocrine system. The deleterious effects of organochlorine pesticides, in particular DDT and its metabolites, became apparent when breeding failure in raptors in the USA was observed, leading to dramatic falls in populations in exposed areas. The subsequent restrictions in use of such pesticides in Western countries has been associated with a gradual recovery in most affected areas. Nonetheless, owing to the marked environmental stability of these compounds, residues and metabolites are still detected globally. Other chemicals suspected of causing disruption have been identified, including the polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), dioxins (PCDD) and furans (PCDFs), non-ionic surfactants (used in detergents) and phthalate esters (used as plastisers), together with a number of agrochemicals (such as lindane and endosulfan). In addition, natural hormones and synthetic steroids used as birth control agents, re-activated or released during sewage effluent treatment, have been implicated at certain sites. Concerns have also been expressed regarding the potential effects of phytoestrogens present in certain plants and plant products. This section reviews the basic mechanisms of endocrine function and the evidence for EDs causing adverse effects on the reproductive, thyroid and immune systems of mammals, birds, reptiles and amphibians.

The Neuro-Endocrine-Immune System Among vertebrate species, the neuro-endocrine-immunesystem is responsible for many complex, inter-related physiological processes including neuronal, homeostatic, reproductive and immune functions. There are four main types of hormone: polypeptides, eicosanoids, steroids and thyroid hormones. Reﬂecting the inter-dependency of the neuro-endocrine and immune systems, hormones, neuropeptides and other neurotransmitters are known to be produced by some immune cells and play a role in the regulation of the immune system, while endocrine and nervous tissues express receptors for many substances produced by the immune system. The major focus of interest in endocrine disruption has

The Institute for Environment and Health, The Ecological Signiﬁcance of Endocrine Disruption: Eﬀects on Reproductive Function and Consequences for Natural Populations, IEH, Leicester, 1999. B.Marchetti,M.C.Morale,F.Gallo,N.Baiticone,Z.FarinellaandM.Cioni,Endocrine, 1995, 3,845.

62 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans been reproduction and sexual differentiation and development, with most attention being given to effects associated with steroid hormones and, to a lesser extent, the thyroid hormones. The steroid hormones are metabolically derived from cholesterol, and are biologically stable, lipophilic chemicals which are active at low concentrations. Many steroid hormones exist, and are generally named after their principal organ of origin or their main biological function. Because of the complexity and evolutionary history of the endocrine system, these hormones may serve several functions within different tissues of an organism. For example, the sex hormones are primarily produced by the gonads, and comprise the androgens (male hormones) and oestrogens (female hormones). These chemicals have vital roles in the control of reproductive functions such as sperm or ova development, and the development and maintenance of secondary sexual characteristics. During development,the balance of these hormones also plays a key role in the formation and functioning of sexually dimorphic parts of the brain (with consequent implications for sexual behaviour) and in the development of a sexually dimorphic immune system. The glucocorticoid hormones, such as cortisol, are produced by the adrenal glands. In addition to their principal effects on metabolism, they influence the immune and reproductive systems. Less well known is thymulin, a polypeptide hormone found mainly in the cortex and medulla of the thymus and thought to be involved in the education and maturation of the immune system’s T-cells. Interference with the production of this hormone seriously compromises the thymus’s ability to produce mature T-cells. Trace amounts of thymulin have also been detected in the gonads, and this hormone appears to play a role in gonadogenesis and the embryonic development of oocytes. There are thought to be feedback mechanisms linking thymulin to testosterone, oestrogen, cortisol and thyroid hormone metabolism.

Potential Effects of Endocrine Disrupting Chemicals on Reproductive Function In order to fully appreciate the potential implications of endocrine disruption, it is important to consider the normal role of hormones in controlling the development and functioning of the reproductive systems of the different vertebrate classes. In the majority of vertebrates, sex is determined by genetic composition, but this is by no means universal. In most species, the genes of an individual direct all of the later ontogenetic processes involved in male—female differentiation of the genitalia and associated structures. The two major types of genotypic sex determination, the mammalian and the avian types, can be referred to as XX/XY or ZZ/ZW, respectively. In animals expressing the XX/XY system, the male is the heterogametic sex (XY) while the female is homogametic (XX). The avian equivalent to the Y-chromosome of mammals is designated by the letter W, and the X-chromosome denoted by the C. J. Grossman, G. A. Roselle and C. L. Mendenhall, J. Steroid Biochem. Mol. Biol., 1991, 40, 649. M. S. Vacchio, J. D. Ashwell and L. B. King, Ann. N.Y. Acad. Sci., 1998, 840, 317. J. A. Marsh and C. G. Scanes, Poult. Sci., 1994, 73, 1049.

63 C. Botham, P. Holmes and P. Harrison letter Z. However, in this case it is the homozygous (ZZ) condition which produces males while the heterozygous (ZW) produces females. In some species the situation is more complex. Recent research suggests that certain autosomal genes may play a role in sex determination. The sex or ‘SRY’ genes influence sexual development via hormones, leading to dimorphically different sex, brain and immune development. There is evidence that exogenous endocrine disrupting chemicals can affect this control. For example, in neonates the imprinting of hormone—receptor systems can be disrupted by the presence of exogenous chemicals, resulting in the non-recognition of a designated receptor by the appropriate hormone. The sex of some poikilothermic reptiles and amphibians is determined by environmental temperature during development of the fertilised egg, whilst in others, sex appears to be an interaction between environmental temperature and the genetic system.

Mammals. During early development, the mammalian embryo displays both male (the Wolffian ducts) and female (the Mu¨ llerian ducts) characteristics. Development is governed genotypically, although differentiation is modulated by the steroid and thyroid hormones. Female sexual differentiation appears to be the default pathway, with female characteristics developing unless dictated otherwise by genetically directed production of male hormones. The presence of a Y-chromosome is responsible for induction of the Leydig cells to produce testosterone, under which influence the embryo develops a male phenotype. During critical periods of fetal development, exposure to sex steroids can exert irreversible changes on sexual development and subsequent adult behaviour. Thus, the mammalian system is potentially at risk from endocrine disruption during early differentiation; the period of sexual maturation is clearly also a particularly susceptible phase. In contrast, exposure to hormonally active chemicals following maturation may temporarily interfere with reproductive functions but is unlikely to elicit permanent effects. Associations between various environmental pollutants and disruption of the endocrine system have been suggested as possible explanations for population declines and abnormalities in reproductive function of several mammalian species, including the Florida panther (Felis concolor coryi), Baltic grey seal (Halichoerus grypus), Baltic ringed seal (Phoca hispida botnica), common seal (Phoca vitulina) and Beluga whale (Delphinapterus leucas). S. S. Wachtel, G. Wachtel and D. Nakamura, in Vertebrate Endocrinology. Fundamentals and Biomedical Implications. Part B. Reproduction, ed. P. K. T. Pang and M. P. Schreibman, Academic Press, London, 1991, p. 149. G. Csaba and A. Inczefi-Gonda, Hum. Exp. Toxicol., 1998, 17, 88. C. Pieau, M. Girondot, G. Desvages, M. Dorizzi, N. Richerd-Mercier and P. Zaborski, in The Difference Between the Sexes, ed. R. V. Short and E. Balahan, Cambridge University Press, Cambridge, 1994, p. 433. R. J. Nelson, in An Introduction to Behavioural Endocrinology, ed. R. J. Nelson, Sinauer Associates, Sunderland, MA, 1995, p. 81. C. F. Facemire, T. S. Gross, and L. J. Guillette, Jr., Environ. Health Perspect., 1995, 103, 79. S. Jensen, B. Jansson and B. Olsson, Ann. N.Y. Acad. Sci., 1979, 320, 436. A. Bergman and M. Olsson, Finn. Game Res., 1985, 44, 44. P. J. H. Reijnders, Nature, 1986, 324, 456. S. de Guise, D. Martineau, P. Beland and M. Fournier, Environ. Health Perspect., 1995, 103, 73.

64 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans

By 1995, the Florida panther population numbered only 30—46 individuals, with many of the surviving animals suffering from various physiological abnormalities of the reproductive, endocrine and immune systems. Reproductive defects included sperm abnormalities, low sperm density and cryptorchidism. The level of serum 17b-oestradiol was noted to be similar for males (whether cryptorchidic or normal) and females, suggestive of demasculinisation and/or feminisation. The oestradiol: testosterone ratio was significantly higher in females and not significantly different between normal and cryptorchidic males. Because of the rarity of the species, comparisons could not be made with reference populations. Inbreeding has been suggested as a possible cause of such abnormalities. However, on the basis of the high residue levels of mercury, selenium and several organochlorine compounds found in tissue samples taken from a dead individual, and work in south Florida which showed that racoons (a major prey item of the panther) have bioaccumulated organochlorine pesticides, exposure to potential endocrine disrupters has also been suggested as a possible cause of the effects seen in these panthers. A causal link between the reproductive abnormalities and exposure to endocrine disrupting chemicals has however not been established. Declines in seal populations occurred in the Baltic Sea and the western-most part of the Wadden Sea (north of the Netherlands) between 1950 and 1975; numbers fell from more than 3000 to less than 500. This was accompanied by a sharp decline in pup production. Tissue levels of organochlorine compounds in female grey and ringed seals in the Baltic have been associated with reproductive problems, with higher levels of PCB and DDT metabolites being detected in the tissues of non-reproductive females than in the tissue of pregnant females. Associated damage to the endocrine, genital and urinary systems, adrenocortical hyperplasia, uterine stenoses and occlusions were also observed. Again it is not known whether or not these observations are causally linked. Organochlorine accumulation in the European otter (Lutra l. lutra) has been investigated throughout Europe. A relationship has been identified between tissue levels of organochlorine chemicals and population declines in the UK and Ireland. Surveys of river catchments in Ireland have shown high PCB levels in individual animals. In the upper and lower reaches of three river catchments in Wales and the west Midlands, the otter populations in the lower river catchment areas were found not to be in equilibrium and levels of organochlorines in spraints (otter faeces) in these regions were sufficiently high to raise concern in over 50% of samples analysed. In the upper catchments, populations were stable and PCB spraint concentrations were low. It has been suggested that organochlorine contamination within the food chain in the lower catchment areas might be inhibiting the growth of otter populations at these sites. Similar associations have been found in otter populations in Sweden and Norway.

C. F. Mason, Cah. Etol., 1995, 15, 307. C. F. Mason and W. M. O’Sullivan, Biol. Environ.: Proc. R. Ir. Acad., 1993, 93B, 187. C. F. Mason, S. M. Macdonald, H. C. Bland and J. Ratford, Water Air Soil Pollut., 1992, 64, 617. M. D. Smit, P. E. G. Leonards, A. J. Murk, A. W. J. J. de Jongh and B. van Hattum, in Development of Otter Based Quality Objectives for PCBs, Institute for Environmental Studies, De Boelelaan, Netherlands, 1996.

65 C. Botham, P. Holmes and P. Harrison

The declining pod (a small school or herd of marine mammals) of beluga whales in the St. Lawrence river estuary has been the subject of much research. Possible mechanisms for this decline have been recently reviewed. Reproductive abnormalities and immunosuppression have been noted in these animals compared with a pod of Arctic belugas. Histological examination of ovary sections from the St. Lawrence belugas have shown little ongoing ovarian activity. Additionally, an adult beluga from the St. Lawrence has been found to have hermaphroditic characteristics (two ovaries, two testes and the genital tracts of both sexes except for a cervix, vagina and vulva). A complex mixture of contaminants, including organochlorines, is present at high levels in the tissues of the St. Lawrence whales, although the signiﬁcance of this to the reproductive abnormalities observed is unclear. A number of other chemicals suspected of having endocrine disrupting potential also occur at high levels in the tissues of marine mammals. For example, tributyltin compounds are present in the tissues of the Steller sea lion (Eumetopias jubatus) from Hokaido, Japan, and in stranded bottlenose dolphins (Tursiops truncatus) found along the US Atlantic and Gulf coasts.— Additional chemicals detected include PAHs, toxaphene and chlordane.

Birds. Embryonic development in birds begins with the migration of primordial germ cells to the seminiferous tubules in the male; this constitutes the default developmental pattern. In the absence of oestrogen, regression of the developing oviducts occurs in males under the influence of a glycoprotein, Mu¨ llerian regression factor (MRF). Development of the ovarian architecture only occurs when oestradiol is synthesised by the gonad, resulting in localisation of the primordial germ cells in the cortex of the left ovary. Further, release of oestradiol also causes regression of the right gonad and suppression of the synthesis of MRF. Thus, oestradiol is necessary for the retention of the left Mu¨ llerian duct and its subsequent differentiation into the functional left oviduct and shell gland. Exposure to exogenous oestrogen during embryonic development may cause altered differentiation of the testes and the accessory ducts of both males and females. Androgens appear to play an important role in the development of secondary sexual characteristics of most birds, influencing plumage and bill colouration. Oestrogens can actively suppress the development of these attributes in males, and are important in territorial display behaviour. Courtship rituals seem to involve a negative feedback of testosterone on follicle stimulating hormone (FSH) production, while copulatory behaviour is related to decreases in circulating testosterone level. Thus, steroid hormones play a major role in both structural organisation and the activation of reproductive behaviour in birds.

G. B. Kim, S. Tanabe, R. Tatsukawa, T. R. Loughlin and K. Shimasaki, Environ. Toxicol. Chem., 1996, 15, 2043. G. B. Kim, S. L. Lees, S. Tanabe, H. Iwata, R. Tatsukawa and K. Shimasaki, Mar. Pollut. Bull., 1996, 32, 558. K. Kannan, K. Senthilkumar, B. G. Loganathan, S. Takahashi, D. K. Odell and S. Tanabe, Environ. Sci. Technol., 1997, 31, 296. R. J. Law and J. A. Whinnett. Mar. Pollut. Bull., 1992, 24, 550. O. Andersson and A. Watanian, AMBIO, 1992, 21, 550. D. M. Fry, Environ. Health Perspect., 1995, 103 (suppl. 7), 165.

66 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans

Many environmental factors (e.g. daylight length) also affect the breeding cycle of birds. However, these tend to be mediated through the hypothalamus—pituitary axis, resulting in gonadotrophin release of FSH and leutenising hormone (LH), which in turn control testosterone and oestrogen production in males and females. The detrimental effect of organochlorine pesticides on reproductive success in birds of prey is well established following the crash of some populations during the 1950s and 1960s. Links have been established with the DDT metabolite, DDE, the cyclodiene pesticides and PCBs. Although many raptor species have been affected globally, particular attention has focused on the peregrine falcon (Falco peregrinus). Levels of DDE in the eggs correlated with reduced eggshell thickness in failed nests in North America. Although the exact mechanism has not been established, it is now generally accepted that the transport of calcium across the eggshell gland mucosa is affected by DDE and that decreased activity of Ca-ATPase and effects on prostaglandins are key mechanisms. A dose-dependent inhibition of progesterone binding to its cytoplasmic receptor in the shell gland mucosa with increasing levels of DDE, possibly contributing to the toxic effects on reproduction, has been described in vitro. There appear to be significant interspecies variations in sensitivity to the effect of DDE on eggshell thickness. Bird-eating raptors and some fish-eating birds, such as cormorants and pelicans, appear sensitive irrespective of body burden. In the 1970s, reproductive abnormalities were observed in terns and cormorants, including behavioural changes in adults, death of embryos and abnormal embryonic development, such as crossed beaks in chicks. Experimental egg transfer from ‘clean’ to contaminated colonies, and vice versa, has suggested that both embryo development and parental behaviour could be affected by organochlorine contamination. Although eggshell thinning attributable to DDE exposure has occurred in birds in the UK, the lethal and sublethal effects of the cyclodiene pesticides aldrin, dieldrin and heptachlor are also believed to have contributed to the population effects, particularly in the case of the sparrowhawk and peregrine falcon. Following the withdrawal of DDT and the cyclodienes from use in the UK, Europe and North America, bird of prey populations that were severely affected have shown partial or complete recovery. In several species of gull, female—female pairing, supernormal clutches, a high incidence of embryo developmental abnormalities, chick mortality and abnormal parental behaviour have been recorded. Observations made in western gull (Larus occidatalis) colonies in the Channel Islands off the California coast showed skewed sex ratios in favour of females, thought to be the result of embryo feminisation/demasculinisation by chemically mediated endocrine disruption

J. B. Hutchison, in Advances in the Study of Behavior, ed. J. Rosenblatt, Academic Press, New York, 1992, p. 159. D. B. Peakall and J. L. Lincer, Environ. Pollut., 1996, 91, 127. C. H. Walker, S. P. Hopkin, R. M. Sibly and D. P. Peakall, Principles of Ecotoxicology, Taylor & Francis, London, 1996. C. E. Lundholm, Comp. Biochem. Physiol., 1988, 89C, 361. G. A. Fox, in Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection, ed. T. Colborn and C. Clement, Princeton Scientiﬁc, Princeton, 1992, p. 147.

67 C. Botham, P. Holmes and P. Harrison causing the absence of males from the breeding colony. Most reports of female—female pairing have been in colonies of western gulls and herring gulls (L. argentatus) in the UK and in the Great Lakes (particularly Lakes Ontario and Michigan), USA. Injection of western gull eggs with DDT/DDE at environmentally relevant concentrations resulted in embryo feminisation, indicated by the localisation of primordial germ cells in a thickened ovary-like cortex in the left testis. At high chemical concentrations, there was a marked reduction in the number of seminiferous tubules and the development of ovary-like cortical and medullary tissue. However, the cortex was histologically less well organised than in genetic females.

Reptiles. Considerable interspecies differences exist in the control systems for sex determination and development and reproductive processes in reptiles. During embryo development in many reptiles, feminisation requires the presence of oestrogen; in its absence, testes develop. Certain members of the crocodilian and turtle families exhibit temperature-dependent sexual development, whereas other species of snake, turtle and lizard show the mammalian/avian type of genotypic sex determination. In alligators, the incubation temperature can determine the embryo’s sex. Incubation at extremes of the temperature range induce an all-or-nothing response, so that an embryo will be either male or female; few intersex forms are produced. It has, however, been shown that the influence of environmental temperature can be modified by treating eggs with oestrogens or oestrogen analogues. Perhaps the best example of chemically mediated endocrine disruption in a wild population of reptiles is the alligators of Lake Apopka, Florida, USA. This lake is highly polluted as a result of agricultural run-off, sewage effluent and a major chemical spill in 1980 of the organochlorine dicofol (contaminated with DDT, DDD, DDE, chloro-DDT and sulfuric acid). Prior to this time the alligator population in Lake Apopka was highly successful but by 1984 the juvenile alligator population had declined by 90% (presumably due to the acute toxic effects of the spill). A decrease in clutch viability was also observed in 1984. Elevated levels of p,p-DDE, p,p-DDD, dieldrin and cis-chlordane were found in eggs collected from Lake Apopka when compared with levels in eggs collected from Lake Woodruff (a relatively unpolluted ‘reference’ lake). Males from Lake Apopka eggs had histologically poorly organised testes with aberrant structures, while six-month-old females were found to have abnormal ovaries with prominent polyovular follicles and an unusually large number of multinucleated oocytes compared with those from Lake Woodruff. In older alligators, differences in plasma steroid levels were apparent. Female alligators aged 2—5 years from Lake Apopka had higher plasma testosterone levels than those from Lake Woodruff. Plasma 17b-oestradiol was elevated in males from Lake Apopka compared with males from Lakes Woodruff and another lake, Lake Orange, and plasma testosterone was significantly lower in Lake Apopka males than those from Lake Woodruff but not those from Lake Orange. From this, it was concluded that other lakes, such as Lake Orange, may be affected to some degree by chemical pollution. Altered

D. M. Fry and C. K. Toone, Science, 1981, 213, 922.

68 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans steroidogenesis was apparent in alligators from Lake Apopka, and it has been suggested that exposure to certain chemicals may alter the circulating sex steroid and thyroid hormone levels, resulting in suppression of testosterone levels in males. Phallus length was reduced in Lake Apopka males compared with those from Lake Woodruff, and there is a suggestion that the reduction was most marked in the region of Lake Apopka where the chemical spill occurred. A potential link between reductions in phallus length and an anti-androgenic effect is supported by research showing that the development and growth of crocodilian phalli is androgen dose-dependent. It is uncertain which chemicals are responsible for the observed effects, as many suspected endocrine disrupters have been detected in alligator eggs from Lake Apopka. Contamination may occur through maternal transfer of pollutants to the eggs, although in ovo environmental exposure in the nest is also a possibility. Experimentally, external contamination of turtle eggs has been shown to affect developing embryos.

Amphibians. Amphibians show either XX/XY, ZZ/ZW or temperature-dependent sex determination. The male gonaducts are derived from paired Wolffian ducts of the pronephric and mesonephric kidneys, and function as urinary and sperm ducts. Development is promoted by testosterone or dihydrotestosterone, but is antagonised by oestrogen. In the female, Mu¨ llerian ducts develop into oviducts, and normal differentiation is controlled by oestrogen. In certain species, males and females retain both Mu¨ llerian and Wolffian ducts, which can lead to sexual reversal in mature animals. Studies on two species of salamander, Pleurodeles waltl and P. poireti, have shown the influence of temperature alongside a genetically based sex determination. Both species display ZZ/ZW genotypic sex determination, and at an ambient temperature of 20 < 2 °C a sex ratio of 1: 1 occurs. High temperatures (30 °C) produce opposite effects in the two species; in P. waltl the sex ratio becomes biased towards males whereas in P. poireti more females develop. Because a fairly large temperature change during a critical developmental period is necessary for such effects, it seems unlikely that this will be a factor in the natural environment. Amphibians posses a similar repertoire of hormones to other vertebrates, and these are involved in the development and maintenance of reproductive functions. The pituitary synthesises the gonadotrophins FSH and LH, while the gonads produce steroids such as progesterone, 17b-oestradiol, testosterone and 5a-dihydrotestosterone. As well as having developmental roles, hormones control sexual behaviour in amphibians. Testicular hormones have been linked with calling, courtship and clasping behaviour (although, in certain species, high brain oestrogen concentrations in males are also important for courtship). Thyroid hormones in amphibians have been shown to influence a wide range of processes including reproduction, metabolism, moulting and metamorphosis. In the larval form, triiodothyronine (T3) levels are restricted. At the onset of metamorphosis, the formation of T3 from thyroxine (T4) is enhanced, resulting in the transformation from larval to adult form.

D. A. Crain, L. J. Guillette, A. A. Rooney and D. B. Pickford, Environ. Health Perspect., 1997, 105,528. F. L. Moore, in Fundamentals of Comparative Vertebrate Endocrinology, ed. I. Chester-Jones, P. M. Ingleton and J. G. Phillips, Plenum Press, London, 1987, p. 207.

69 C. Botham, P. Holmes and P. Harrison

There is some evidence for chemically mediated endocrine disruption in amphibians. The egg yolk protein, vitellogenin, is inducible in amphibians by exposure to DDT. Males of the short clawed toad Xenopus laevis given 250 kg/g or 1 kg/g o,p-DDT for seven days have been shown to produce vitellogenin, although the induction was less than that achieved by treatment with 1 kg/g of either 17b-oestradiol or diethylstilboestrol. Research has also shown that endocrine disrupting chemicals can alter sex ratios in wild populations of certain species; PCB congeners and organochlorine compounds have been linked with male domination of sex ratios in polluted compared to unpolluted sites.

Potential Effects of Endocrine Disrupting Chemicals on Thyroid Function The thyroid gland is present in all vertebrates. Its activity is principally controlled by the thyroid stimulating hormone (TSH) released by the pituitary. The hormones produced by the thyroid gland are iodinated tyrosine compounds, and are found in the body in two forms: thyroxine (T4) and, at lower levels, the more active form in mammals, triiodothyronine (T3). In addition to their role in modulating the general metabolic activity of an organism, these hormones, operating via permissive action, have been found to influence embryo differentiation and development, the functioning of the reproductive and immune systems, the development of olfactory senses, social behaviour and intelligence. Disruption of thyroid functions in vertebrates has been suggested to constitute a potential threat to many vital functions. For example, there is a possibility that disruption to the thyroid hormone levels during embryogenesis could result in disturbed behaviour patterns in the adult form, possibly interfering with migration in certain species and sonar functions in cetaceans. In anurans, thyroid hormones are essential for initiating metamorphosis.

Mammals. Exposure to persistent organic compounds has been associated with eﬀects on thyroid hormone levels in mammals. Even at low levels, PCBs have been alleged to suppress thyroid hormone levels in grey seal pups in the Baltic sea. Functional and morphological changes in the thyroid gland have been associated with high body burdens in St. Lawrence beluga whales, and with phocine distemper infection in harbour seals and harbour porpoises, compared to healthy controls. In otters environmentally exposed to PCBs, there was a strong negative correlation between vitamin A and PCB concentrations, with a high incidence of infectious disease apparent in contaminated animals.

D. O. Norris, in Vertebrate Endocrinology, ed. D. O. Norris, Academic Press, San Diego, 1996, chap. 8, p. 286. B. D. Palmer and S. K. Palmer, Environ. Health Perspect., 1995, 103 (suppl. 4), 19. A. L. Reeder, G. L. Foley, D. K. Nichols, L. G. Hansen, B. Wikoﬀ, S. Faeh, J. Eisold, M. B. Wheeler, R. Warner, J. E. Murphy and V. R. Beasley, Environ. Health Perspect., 1998, 106, 261. D. O. Norris, in Vertebrate Endocrinology, ed. D. O. Norris, Academic Press, San Diego, 1996, chap. 7, p. 243. B. M. Jenssen and J. U. Skaare, Chemosphere, 1996, 32, 2115. U. Schaumacher, S. Zahler, H. P. Horny, G. Heidemann, K. Skirnisson, and U. Welsh, J. Wildl. Dis., 1993, 29, 103.

70 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans

Birds. The activity of the thyroid hormones in birds is similar to that in mammals. Thyroxine has been functionally associated with embryonic development and gonadal development and function, and may be involved in migratory behaviour. During and after hatching there are signiﬁcant changes in thyroid metabolism. In precocial birds (i.e. species in which the young show only a limited degree of dependency on their parents), the conversion of thyroxine to triiodothyronine is high, and there is an accompanying low rate of degradation of the latter. The opposite is found in altricial species (requiring high parental care) during the period of dependency. Several studies have identiﬁed a relationship between body burden of EDs and disruption of thyroid hormone concentrations in avians. Investigations in the Netherlands, in two cormorant colonies (Phalacrocorax carbo), found that chicks environmentally exposed in ovo to PCBs and dioxins demonstrated reduced thyroid hormone levels when compared to the ‘control’ colony. Another Dutch study found almost identical results in exposed cormorant chicks, with circulating thyroid hormone and hepatic vitamin A levels depressed by approximately 50%, whereas a 19-year study of herring gull colonies around the Great Lakes in North America showed consistent depressed levels of hepatic vitamin A in relation to PCB and DDE body burden. A link has been found with polyhalogenated hydrocarbon (PHH) levels in the yolk sacs of common terns (Stena spp.), which constitute the main food source of adult gulls prior to mating. A relationship has been proposed between observed PHH concentrations in yolk sacs and late egg laying, prolonged incubation periods, smaller eggs and chicks, and lowered thyroid hormone levels present in the yolk sac. Experimentally exposed mallards showed a dose-dependent relation between triiodothyronine and vitamin A.

Reptiles. In many reptiles, thyroid function changes have been associated with seasonal changes in spermatogenesis, ovulation and mating. However, there is little evidence of a similar relationship in viviparous reptiles, although in one case thyroidectomy of a viviparous female has been shown to cause abortion of the majority of the eggs, with retained eggs failing to hatch. Evidence for chemically mediated disruption of thyroid function in wild reptile populations includes the ﬁnding of elevated thyroxine levels in male alligators from Lake Apopka, although a causal relationship with speciﬁc chemicals has not been established.

A. J. Murk, P. E. G. Leonards, B. van Hattum, R. Luit, M. E. J. van der Weiden and M. Smit, Environ. Toxicol. Pharmacol., 1998, 6, 91. M. Vandenburg, B. L. K. J. Craane, T. Sinnige, S. van Mourik, S. Dirken, T. Boudewijn, M. Vandergugg, I. J. Spenkelink and A. Brouwer, Environ. Toxicol. Chem., 1994, 13, 803. M. Vandenburg, B. L. K. J. Craane, T. Sinnige, S. van Mourik, A. Brouwer, 1995, Ardea, 83, 299. G. A. Fox, S. Trudeau, H. Won, K. A. Grassman, Environ. Monit. Assess., 1998, 53, 147. A. J. Murk, T. J. Boudewijn, P. L. Meininger, A. T. C. Bosveld, G. Rossaert, T. Ysebaert, P. Meire and S. Dirks, Arch. Environ. Contam. Toxicol., 1996, 31, 128. J. R. Fowles, A. Fairbrother, K. A. Trust and N. I. Kerlevliet, Environ. Res., 1997, 75, 119. D. O. Norris, in Vertebrate Endocrinology, ed. D. O. Norris, Academic Press, San Diego, 1996, chap. 8, p. 292.

71 C. Botham, P. Holmes and P. Harrison

Amphibians. Amphibians are highly susceptible to endocrine disruption during development of the larval form and during metamorphosis. The action of metamorphosis is triggered and controlled by the thyroid gland via an increase in triiodothyronine and a decrease in thyroxine, and differs greatly between oviparous and viviparous species. Experimentally, it has been shown that disruption during this sensitive period can lead to malformations and adverse impacts on immune and reproductive functions. A review of possible correlations between environmental toxicants and declines in global amphibian populations found several potential mechanisms other than lethal toxicity, including increased susceptibility to disease, impairment of predator avoidance, retardation of growth and development, and reproductive impairment. However, the authors concluded that the few studies available did not identify the causes of the population declines. Thus, the amphibian population declines, in particular among anuran species, might conceivably be related to many factors, including exposure to increasing levels of UV light, disappearance of natural habitat, parasitism, disease and endocrine disruption. Both parasitism by trematode (flatworm) and exposure to chemicals have been linked to the appearance of gross malformations in the United States and Canada. The majority of these malformations are fatal prior to sexual maturity and are therefore only apparent in tadpoles and juveniles. In Japan, research has linked these malformations with high levels of PCBs and organochlorine pesticides, and high concentrations of PCBs have been associated with the population decline of green frogs in southern Ontario. American research has singled out a group of insecticides which contain S-methoprene (a juvenile hormone mimic used in the control of mosquitoes) as the major active ingredient. This compound is known to undergo degradation by UV light to form isomers of retinol, a well-documented teratogen. Laboratory studies exposing developing frog embryos to water from ponds where high incidences of malformations have been noted was found to result in a high rate of malformation compared to control groups using water from ‘clean’ ponds. However, where only single chemicals have been tested in the laboratory, malformations were only witnessed at concentrations well above those found in the environment, possibly suggesting an additive or synergistic effect between a number of different chemicals. Exposure of the anurans Amybstoma maculatum and Xenopus laevis to UV radiation and PAHs has also been shown to cause malformations and mortality. Current evidence points towards chemical disruption of the thyroid hormones and retinoid receptors, as these are instrumental in regulating development, differentiation and metamorphosis. Considering the evidence so far, it is credible that disruption of thyroid

C. Carey and J. C. Bryant, Environ. Health Perspect., 1995, 103, 13. M. Ouellet, J. Bonin, J. Rodrigue, J. L. DesGranges and S. Lair, J. Wildl. Dis., 1997, 33, 95. K. Kadokami and M. Takeishi, NIEHS Statement, http://www.npwrc.usgs.gov [15/12/98], 1998. R. W. Russell, K. A. Gillan and G. D. Haﬀner, Environ. Toxicol. Chem., 1997, 16, 2258. J. J. La Clair, J. A. Bantle and J. Dumont, Environ. Sci. Technol., 1998, 32, 1453. J. Manuel, Environ. Health Perspect., 1997, 105, 1046. G. S. Schuytema and A. V. Nebeker, Arch. Environ. Contam. Toxicol., 1998, 34, 370. A. C. Hatch and G. A. Burton, Environ. Tox. Chem., 1998, 17, 1777.

72 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans hormones (and the changes consequent to such disruption) could be a chemically mediated endocrine receptor based eﬀect.

Potential Eﬀects of Endocrine Disrupting Chemicals on Immune Function The mammalian and avian immune systems function similarly; both incorporate humoral and cell-mediated cytotoxic mechanisms, and are thought to share a 160m year old relationship with the reptilian immune system. The immune system of mammals shows sexual dimorphism; a greater immune response is normally observed in females, which has been attributed to diﬀerences in steroid hormone concentration. In the toad Bufo regularis, sexual dimorphism of the immune system is also apparent.

Mammals. Many studies have shown that synthetic and natural oestrogens suppress the immune system, and that, during pregnancy, the female immune system is naturally suppressed, accompanied by a decrease in thymulin levels and an increase in circulating oestrogen levels. Oestrogen receptors in the thymus have been located in thymulin-producing cells, indicating a possible mechanism by which the T-cell mediated immune response is co-ordinated by these two hormones and manifested as a lowered maternal immunity to infections. Increased concentration of testosterone has a positive, enhancing effect on the immune system, whereas increase or decrease in thyroid hormones results in a negative effect. High body levels of several EDs, in particular PCBs and organochlorine compounds, have been associated with suppression of the immune system. Impaired immune function has been associated with mass mortality among sea mammals following infection with a strain of the phocine distemper virus, morbillivirus. Observations have been made in harbour seals, grey seals and harbour porpoises in north eastern Europe, in striped dolphins in the Mediterranean, and in baikal seals in Siberia. The same virus has been identified in polar bears (A. maritimus) in North America and Siberia, though the mortality noted in marine mammals has not occurred. Bottle-nosed dolphin (Tursiops truncatus) mortalities have occurred along the eastern seaboard and the Gulf of Mexico, USA. Pathological examination showed opportunistic infections associated with immunosuppression,and effects were found to correlate with high PCB and other organochlorine levels in adipose tissue. T-cell response was also suppressed in

J. M. Sharma, Vet. Immunol. Immunopathol., 1991, 30, 199. C. R. Pope, Vet. Immunol. Immunopathol., 1991, 28, 173. A. H. Saad and B. Plytycz, Folia Biol.(Krakow), 1994, 42, 63. A. H. Saad, Zool. Sci., 1992, 9, 1081. B. Milholjcic, L. Radic and Z. Aleckovic, Acta Vet.(Beograd), 1990, 7, 269. A. G. Rijhsinghani, K. Thompson, S. K. Bahtia and T. J. Waldschmidt, Am. J. Reprod. Immunol., 1996, 36, 269. M. D. Kendall, B. Saﬁeh, J. Harwood and P. P. Pomroy, Sci. Total Environ., 1992, 115, 133. A. D. M. E. Osterhaus, R. E. de Swart, H. W. Voss, P. S. Ross, M. J. H. Kenter and T. Barrett, Vet. Microbiol., 1995, 44, 219. E. H. Follman, G. W. Garner, J. F. Everman and A. J. McKeirnan, Vet. Rec., 1996, 138, 615.

73 C. Botham, P. Holmes and P. Harrison live pelagic dolphins from the same geographical area. A possible mechanism for the reduced T-cell activity is suppression of the circulating thymulin level. Chemical-mediated immune suppression has been identified from the experimental study of several wildlife species. Harbour seals fed either chemically contaminated fish from the Wadden Sea or uncontaminated fish were found to have differing immune responses, with the exposed group showing lowered immune response to microbial infections and certain types of cancer. Mink fed fish taken from below a discharge point for bleached Kraft pulp mill effluent have also shown impaired immune function, showing that the non-accumulative chemicals in this effluent can actively disrupt endocrine associated functions.

Birds. In avian populations, organochlorine-associated suppression of T-cell mediated immune response has been found in herring gull and caspian tern colonies adjacent to the Great Lakes in Northern America. In the most severe cases, immune response was suppressed by up to 50%. Similarly, seabirds exposed to petrochemicals from oil spills have been found to have suppressed immune function.

Amphibians. Immunosuppression has been shown to occur during metamorphosis in several amphibians, and appears to be hormonally regulated. The disappearance of 11 populations of toad from Colorado was associated with immune suppression, and frog mortalities have been associated with infection by a commonly occurring bacteria, Aeromonas hydrophila; the cause of the immunosuppression in these animals has not been elucidated.

Effects at the Population and Community Level Establishing causal relationships between a particular pollutant and significant changes in community and ecosystem structure and function is difficult, owing to the complexity of the inter-relationships present, which involve both biotic and abiotic interactions. Until population level effects are better understood, attempts to investigate endocrine disruption at the community or ecosystem level are unlikely to be profitable. Organisms with a short life cycle plus a quick reproductive cycle (e.g. daphnia or rats) are probably least affected long-term by EDs as they are able to evolve more quickly to changes within their environment and to acquire some form of resistance. Organisms which have a long life cycle and are slow to reproduce (e.g. raptors and cetaceans) will be the most affected long-term, with impaired reproductive function restricting the ability to adapt to a changing environment. Recovery in the latter organisms would be slow and could be seriously affected by a restrictive gene pool caused by loss of individuals.

G. P. Lahvis, R. S. Wells, D. W. Kvehl, J. L. Stewart, H. L. Rhinehart and C. S. Via, Environ. Health Perspect., 1995, 103 (suppl. 4), 67. R. de Swart, P. S. Ross, L. J. Vedder, H. H. Timmerman and S. Heistercamp, AMBIO, 1994, 23, 155. J. E. G. Smits, B. R. Blakely and G. A. Wobeser, J. Wildl. Dis., 1996, 32, 199. K. A. Grasman, G. A. Fox, P. F. Scanlon and J. P. Ludwig, Environ. Health Perspect., 1996, 104, 829. K. T. Briggs, S. H. Yoshida and M. E. Gershwin, Reg. Toxicol. Pharmacol., 1996, 23, 145. C. Carey, Conserv. Biol., 1993, 7, 355.

74 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans

Therefore, at a population level, endocrine disruption could culminate in the elimination of long-lived organisms over a short period of time. This would result in an unfilled ecological niche, potentially leading to an overall drop in biodiversity at the community level, as populations of short-lived organisms expand to fill the niches left by the longer-lived species. Since longer-lived species tend to be top predators, there would be disturbance within the different trophic levels. At the population level, evolution of a single species is likely to be restricted by the reduction of the gene pool.

3 Biological and Physicochemical Modifiers of Endocrine Disrupter Exposure From observations in the field and laboratory experiments, it is apparent that, for an endocrine disrupting chemical, exposure level is not in itself an adequate predictor of the type or degree of effect that any given species may display. A number of aspects relating to the organism may have a profound influence on the outcome. These include ecological niche, gender, stage of the life cycle and reproductive status, as well as species-specific aspects such as sensitivity, metabolic status, physiology and endocrinology. For example, aquatic organisms appear at particular risk because they are exposed to chemicals though ingestion, respiratory tissues and dermal contact with the surrounding medium. Compromised immune and reproductive function have been observed in several aquatic species of mammal, amphibian, reptile and bird. Aquatic top predators, in particular territorial species, appear to be most susceptible. In addition to these biologically based factors, a number of physicochemical and environmental aspects are known to influence or modify the potential exposure of an organism to an ED.

Biological Modifiers of Endocrine Disrupter Exposure The life stage of an organism is known to influence the type and extent of any effects of a given exposure to an ED. Depending on the stage of development, effects are likely to be either reversible or irreversible. Organisms exposed during embryonic development are believed to be particularly vulnerable as, to a lesser extent, are animals at the time of sexual maturation. The life cycle of amphibians suggests that they may be particularly susceptible since they undergo metamorphosis, with specific species exhibiting experimentally induced sex reversal during adulthood. Laboratory studies have also shown that alligators and several species of turtle that show temperature-dependent sex determination can undergo anomalous sexual differentiation if the developing embryos are exposed to an oestrogenic compound during specific periods, suggesting a mechanism by

G. A. Fox, Environ. Health Perspect., 1995, 104 (suppl. 4), 93. R. W. Luebke, P. V. Hodson, M. Faisal, P. S. Ross, K. A. Grasman and J. Zelikoﬀ, Fund. Appl. Toxicol., 1997, 37,1. C. R. Tyler, S. Jobling and J. P. Sumpter, Crit. Rev. Toxicol., 1998, 28, 319. D. O. Norris, in Vertebrate Endocrinology, ed. D. O. Norris, Academic Press, San Diego, 1996, chap. 12, p. 442.

75 C. Botham, P. Holmes and P. Harrison which EDs might be able to influence sex ratios in exposed wildlife populations. The sex of an organisms may also affect the length of time to which it is exposed to an ED and its bioaccumulation rate, as well as influencing the effects observed. For example, in Atlantic minke whales, significant differences have been reported in the body burden of & PCB and & DDT in males compared to female or juveniles. Many studies also have provided evidence of relationships between age or sex and organochlorine body burden in pinnipeds. Male seals accumulate organochlorine compounds throughout their life cycle. In contrast, females only accumulate these compounds until reproductive maturity since, during subsequent seasons of gestation and lactation, part of the mother’s body burden is transferred to their offspring as a result of mobilisation from fat tissue in which the accumulated chlorinated organic compounds tend to be stored. However, the transfer of chemicals as a result of lactation may be highly selective. For example, in two species of Arctic seal there were lower concentrations of the higher chlorinated PCBs in the pups than the mother; a similar pattern has also been noted in the polar bear, particularly during times of maternal fasting and lactation. Interestingly, increasing concentrations of PCB’s were found in the milk as fasting continued. Body fat content is also a factor affecting the bioaccumulation of chemicals. Organisms with a high body fat content will bioaccumulate larger amounts than those with a lower body fat content. Compartmentalisation of compounds within an organism depends on the body fat content and distribution, with higher concentrations apparent in lipid-storing organs.

Physicochemical Modifiers Many anthropogenic pollutants find their way into watercourses, either through leaching, spillage, run-off or through licensed effluent discharge. A number of volatile or gaseous chemicals, such as dioxins, may contaminate terrestrial as well as aquatic environments at a local or global level. PCBs of low molecular weight are more prone to volatilisation and are thus more likely to spread through the atmosphere, causing terrestrial pollution via deposition onto soil or biota. For example, high concentrations of PCBs have been detected in the Arctic polar regions, as a result of dispersal by aerial transport. A chemical must have certain physicochemical properties to elicit an endocrine disrupting effect. For example, the ability to enter the body and to cross the cell membrane into the cellular medium requires a degree of lipophilicity. Lipophilic potentials may be compared by reference to the chemical’s octanol—water coefficient (usually expressed as log K). This property, together with molecular size and chemical structure, has an important influence on the bioaccumulation D. Crews, J. M. Bergeron and J. A. McLachlan, Environ. Health Perspect., 1995, 103 (suppl. 7), 73. L. Kleivane and J. U. Skaare, Environ. Pollut., 1998, 101, 231. J. D. Hutchinson and M. P. Simmonds, Rev. Environ. Contam. Toxicol., 1994, 15, 1979. O. Espeland, L. Kleivane, S. Haugen and J. V. Skaare, Mar. Environ. Res., 1997, 44, 315. A. Bernhoft, O. Wiig and J. U. Skaare, Environ. Pollut., 1997, 95, 151. S. C. Polishuk, R. J. Letcher, R. J. Nostrom and R. A. Ramsey, Sci. Total Environ., 1995, 161, 465. B. T. Elkin and R. W. Bethke, Sci. Total Environ., 1995, 161, 307. D. J. Thomas, B. Tracey, H. Marshall and R. J. Nostrom, Sci. Total Environ., 1992, 122, 135.

76 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans potential of a chemical and determines the maximum effective exposure concentration. Reduced planarity of a compound lowers uptake and bioaccumulation potential. Prey—predator interactions can result in biomagnification within the food chain, resulting in a constant internal dose; effects may then manifest themselves accordingly. However, not all EDs with a high log K possess or require the ability to bioaccumulate in order to be biologically active. For example, phthalate plasticisers, chlorophenols from Kraft mill effluents and natural or synthetic hormones can influence an organism’s hormone profile and affect reproductive function and immune response without exhibiting bioaccumulation. It might be assumed that a prerequisite for an endocrine disrupting chemical to operate through a receptor-mediated process is a molecular structure similar to that of the natural hormone for which it shows agonistic or antagonistic activity. However, it has been demonstrated that, at least in the case of the oestrogen receptor, a chemical may show activity without having obvious structural similarity. This has resulted in the oestrogen receptor being labelled ‘promiscuous’ by some researchers. Nonetheless, it appears that an active, charged chemical group at the para position (usually phenolic and, in the case of oestrogen mimics, mirrored at the opposite position) may be a key structural requirement for attachment to the cellular receptor. In addition to their endocrine disrupting properties, it must be appreciated that many of the chemicals in question possess more general toxic properties, which may be potentiated by metabolism by the organism. Several PAHs, PCBs and PCDDs are carcinogenic, while certain phthalate esters can enhance the excretion of zinc, potentially leading to zinc deficiency. Zinc, an essential element, plays a vital role in spermatogenesis and mature T-cell production. Deficiency may result in abnormalities of the male reproductive system, depletion of spermatogenesis and suppression of the immune system.

Bioconcentration, Bioaccumulation and Biomagnification. These aspects are determined by the physicochemical properties of a chemical, an organism’s ability to excrete the chemical, the organism’s lipid content and its trophic level. Bioconcentration relates to the difference between the environmental concentration and that of the body tissues. A high bioconcentration factor (BCF) predisposes to bioaccumulation. The upper limit of bioaccumulation is determined by lipid levels in the organism’s tissues. Whether the resultant body burden causes biomagnification in the food chain depends upon the metabolic capabilities of the exposed organism. Different species within a given environment will be exposed to varying concentrations and mixtures of compounds, and will exhibit different accumulation patterns. Congener patterns (in the case of PCBs, for example) may also vary in the same species at different locations, reflecting the varying concentrations in their major food source.

J. E. Harries, D. A. Sheaham, S. Jobling, P. Matthiessen, M. Neall, J. P. Sumpter, T. Taylor and N. Zaman, Environ. Toxicol. Chem., 1997, 16, 534. A. E. Karels, M. Soimasuo, J. Lappivarara, H. Leppanen, T. Aaltonen, P. Mellanen and A. O. J. Oikari, Ecotoxicology, 1998, 7, 123.

77 C. Botham, P. Holmes and P. Harrison

Biotransformation and Excretion. Selective biotransformation is known to influence the biological effects of chemicals at both the species and individual level. Specific cytochrome P450 subfamilies (CY P450) of the mixed function oxidase system have evolved throughout the vertebrate classes as a means of chemically modifying steroid hormones and exogenous lipophilic compounds into forms suitable for conjugation reactions to form more hydrophilic and hence more excretable compounds. An explosion of genetic diversity in these enzymes has occurred over the last 400 million years, especially in the case of the CYP2B superfamily. The CYP1A superfamily, responsible for metabolising planar compounds, is present in all marine vertebrates and is highly inducible in fish and birds. In contrast, CYP2B, responsible for metabolising non-planar compounds, appears undeveloped in most groups but is well developed in terrestrial mammals and some birds. The development of plant chemical defence mechanisms seems to have played a major role in the evolution of the mixed function oxidase systems of animals via selective evolutionary pressure. Emergence of the CYP2B superfamily coincides with the development of terrestrial animals and exposure to a more varied diet, and has been found to be absent in animals with a monophagus diet. Interestingly, pinnipeds (having readopted a marine environment and become largely monophagus) are restricted in their ability to metabolise organochlorine compounds compared to terrestrial mammals, although some species, specific differences are apparent. Aquatic organisms, such as fish and invertebrates, can excrete compounds via passive diffusion across membranes into the surrounding medium and so have a much reduced need for specialised pathways for steroid excretion. It may be that this lack of selective pressure, together with prey—predator co-evolution, has resulted in restricted biotransformation ability within these animals and their associated predators. The resultant limitations in metabolic and excretory competence makes it more likely that they will bioaccumulate EDs, and hence they may be at greater risk of adverse effects following exposure to such chemicals. In general, terrestrial vertebrates must biotransform endogenous steroids and many xenobiotic compounds to more soluble forms before being able to excrete them via the urine and/or bile. In addition, terrestrial animals are more likely than their aquatic counterparts to be exposed to a varied diet containing exogenous lipophilic compounds. This appears to have resulted in evolutionary pressure to develop more elaborate metabolic and excretory systems. Research on the mixed function oxidase systems of birds has shown differences associated with both species and diet. For example, cormorants, sparrowhawks and peregrine falcons possess only low aldrin epoxidase activity (the enzyme responsible for biotransforming co-planar chlorinated compounds in birds), compared to omnivorous species such as herring gulls. A larger proportion of the higher-chlorinated PCBs (relative to total body burden) have been reported for the European otter (Lutra l. lutra), Arctic fox, and polar bear. Similar PCB

J. P. Boon, E. van Arnhem, S. Jansen, N. Kannan, G. Petrick, D. Schultz, J. C. Duinker, P. J. H. Reijnder and A. Goksoyr, in Persistent Pollutants in Marine Ecosystems, ed. C. H. Walker and R. Livingstone, Pergamon Press, Oxford, 1992, p. 83. M. C. Fossi, A. Massi, L. Lari, C. Leonzio, S. Focardi, I. Marsili and A. Renzoni, Sci. Total Environ., 1995, 171, 221.

78 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans patterns have been found in polecats feeding on either terrestrial (small rodents) or aquatic (amphibian) prey despite different exposures, leading to the suggestion that the similarities were attributable to metabolic rather than dietary factors. A recent study on selective dietary accumulation in the European otter showed that although the lower chlorinated PCBs are more prevalent at the lower trophic levels of the foodweb, biomagnification through the foodchain resulted in the higher chlorinated PCBs making up approximately 80% of the total PCB body burden compared to a level of 30—50% in their diet. This has been attributed to metabolic differences between species, with otters being able to excrete the lower chlorinated PCBs and thus gaining a higher body burden of co-planar compounds. In many of the organisms so far studied, EDs are also active inducers of the lower P450 subfamilies (i.e. CYP1A1, CYP2B, CYP3B), which are involved with steroid metabolism. Each subfamily appears to have a particular affinity for specific isomers of the steroid hormones. Compounds induce different subfamilies and this can lead to preferential metabolism of a particular isomeric form altering the hormonal profile of the organism. Alternatively, this may occur through EDs acting as antagonists of particular CYP 450s, which could potentially result in the build-up of particular hormones within the organism.

4 Geographical Considerations and Implications for Recovery Rates As with all pollutants, effects of EDs are likely to be greatest when dispersion does not occur and there is consequently an increase in local environmental levels. Endocrine disrupting chemicals that are lipophilic will preferentially bioconcentrate in sediments and biota, thus dispersing only slowly into the wider environment. As previously noted, the aquatic environment is the likely fate of many of these chemicals and certain geographical areas are more susceptible to accumulation, such as areas which accumulate sediment, or locations where dispersion to the wider environment is compromised. Prime examples are estuaries, slow moving sediment-rich rivers, benthic coastal regions and lakes. For example, a nine-year study found that beluga whales in the St. Lawrence estuary have greater body burdens of potential endocrine disrupting chemicals than belugas inhabiting the Arctic Ocean. Other particularly susceptible environments are H. Kruuk and J. W. H. Conroy, Environ. Pollut., 1996, 92, 165. G. Wangendersen, J. U. Skaare, P. Prestrud and E. Steinnes, Environ. Pollut., 1993, 82, 269. G. Norheim, J. U. Skaare and O. Wiig, Environ. Pollut., 1992, 77, 51. P. E. G. Leonards, B. van Hattum, W. P. Cofino and U. A. T. Brinkman, Environ. Toxicol. Chem., 1994, 13, 129. P. E. G. Leonards, Y. Zierikzee, U. A. T. Brinkman, W. P. Cofino, N. M. van Straalan and B. van Hattum, Environ. Toxicol. Chem., 1997, 16, 1807. H. J. Drenth, M. van den Berg and C. Bouwman, Reproductive effects of PCBs, the role of cytochrome P450 induction and steroid hormone production, Research Institute of Toxicology, Utrecht University, Utrecht, Netherlands, 1994. G. H. Panter, R. S. Thompson and J. P. Sumpter, Aquat. Toxicol., 1998, 42, 243. C. F. Mason and S. M. Macdonald, Sci. Total Environ., 1993, 138, 127. P. Beland, S. de Swart, C. Girard, A. Lagace, D. Martineau, R. Michaud, D. C. G. Muir, R. J. Nostrom, E. Pelletier, S. Ray and L. R. Schugart, J. Great Lakes Res., 1993, 19, 766.

79 C. Botham, P. Holmes and P. Harrison the polar regions. The poles are subject to anthropogenic pollution as a result of global aerial and aquatic migration of pollutants. Since the 1970s, bioaccumulation of organochlorine compounds and PAHs has occurred in the polar regions. This has been demonstrated in the fur seal (Callorhinus ursinus), ringed seal (Phoca hispida), hooded seal (Cystophora cristata), bearded seal (Erignathus barbatus), walrus (Obdobenus rosmarus divergens), Beluga whale (Delphinapterus leucas), porpoise (Phocoena phocoena) and polar bear. Worryingly, there have been recent reports of pseudohermaphrodism in female polar bears in Svalbard, Norway. Even in the Arctic there are regional differences in concentration, dictated by emission source and abiotic factors. PCB congeners analysed in adipose tissue from polar bears at Wrangel Island in Russia, North America, eastern Greenland and Svalbard demonstrated significantly higher levels in Svalbard, eastern Greenland and the Canadian Arctic Ocean. Concentrations of DDE and dieldrin are most concentrated in bears from the Hudson Bay area and are generally higher on the North American continent. In areas severely affected by chemical pollution, recovery of the affected wildlife has been influenced by the degree of chemical dispersion from the site. In the UK, recovery in several species has been seen in terrestrial and inland river environments where chemical dispersion has occurred. For example, population recovery has been noted for the European otter, sparrowhawk and peregrine falcon, alongside the withdrawal of organochlorine chemicals from agricultural use and the gradual dispersal of these chemicals into the wider environment. Pollution remains a problem, however, in estuaries and marine areas associated with large human populations and industry. Recovery of populations of Baltic grey seal and ringed seal have been noted in the northern Baltic sea and northern Gulf of Bothnia (between Sweden and Finland), respectively. Population increases of about 10% for the Baltic grey seal and about 6% for the ringed seal have been recorded, and these recoveries have been associated with declines in the incidence of uterine occlusions and decreases in the level of PCBs in the ecosystem and in seal tissues. In the USA, recovery has been slower, reflecting the larger environmental loads of chlorinated organic chemicals from agricultural and industrial use. Bald eagle populations close to the Great Lakes and associated fish spawning grounds have shown lower recovery rates than populations associated with rivers flowing into barriered dams which prevent migration of contaminated fish into the area. Recovery has also been noted in cormorants, herring gulls and Caspian terns. Observed recovery in raptors has been slow owing to their lifecycle; this is also an indication of the environmental movement and dispersal of endocrine disrupting chemicals from areas of intense agriculture to the aquatic environmental

R. J. Nostrom and D. C. G. Muir, Sci. Total Environ., 1994, 154, 107. O. Wiig, A. E. Derocher, M. M. Cronin, J. U. Skaare, J. Wildl. Dis., 1998, 34, 792. R. J. Nostrom, S. E. Belikov, E. W. Born, G. W. Garner, B. Malone, S. Olpinski, M. A. Ramsey, S. Schleibe, I. Stirling, M. S. Sitshov, M. K. Taylor and O. Wiig, Arch. Environ. Contam. Toxicol., 1998, 35, 354. M. Mattson and E. Helle, Eleventh Biennial Conference on the Biology of Marine Mammals, The Society for Marine Mammalogy, Lawrence, Kansas, USA, Abstract p7A. W.W.Bowerman,J.P.Giesy,D.A.BestandV.C.Kramer,Environ. Health Perspect., 1995, 103,51. K. R. Solomon, Int. J. Toxicol., 1998, 17, 159.

80 Endocrine Disruption in Mammals, Birds, Reptiles and Amphibiıans sinks. The pattern of dispersal and effect now appears to be changing, leading to the focusing of attention on environmental sinks within the environment, such as river corridors and marine and polar regions (rather than riparian agricultural land), and on the top marine/estuarine and terrestrial mammalian predators (rather than the riparian top predators). However, it must be appreciated that an estimated 65% of global PCBs are currently resident in landfills with 31% mobile and 4% destroyed. Hence, there remains a long-term potential for continued environmental exposure to potential EDs which could have local implications for biodiversity as well as for the aquatic and polar environments. In addition, with the continued detection of abnormalities suggestive of endocrine disruption in amphibians and fish, more generalised environmental concerns appear justified.

5 Conclusions There is clear evidence of abnormalities suggestive of endocrine disruption in individuals of various wildlife species. Examples include the changes seen in several aquatic mammalian and avian species, which have to varying degrees been associated with exposure to chlorinated organic compounds. Evidence of endocrine disruption has also been noted in alligators and turtles, although the causative agents have yet to be identified, and there are increasing reports of developmental abnormalities in many populations of wild amphibians. Recent findings imply a possible chemical origin to these effects. The evidence suggests that all vertebrates could potentially be at risk, especially those exposed through their surrounding media and by ingestion of contaminated food, i.e. aquatic species. The possibility and biological plausibility that endocrine disruptive chemicals could affect other physiological systems, such as the immune system, raises concerns that non-reproductive adverse effects might also occur. The apparent correlation between high levels of chlorinated organic compounds and pseudohermaphrodism in polar bears suggests that, globally, a wide range of organisms could potentially be at risk. Research has identified multiple forms of CY P450 in the polar bear capable of biotransforming diverse forms of substrate, for example co-planar chlorinated compounds, suggesting that even in species possessing a well-developed excretory system, exposure can result in intersex. Concentrations of chlorinated organic compounds in milk have been found to be similar for polar bears and the local Inuit population, raising concerns for particular human populations. In response to these concerns, the Faroe Island authorities have recommended restrictions in consumption of whale products. They have recommended that women and girls should not consume whale blubber until child-bearing is over, should not eat liver and kidneys at all, and should not eat whale meat three months prior to conception, because of recognised toxicity to reproductive organs pre- and post-puberty and

D. L. Swankhamer, in Chlorinated Organic Micropollutants, ed. R. E. Hester and R. M. Harrison, The Royal Society of Chemistry, Cambridge, 1996, p. 137. S. M. Bandiera, S. M. Torok, R. J. Letcher and R. J. Norstrom, Chemosphere, 1995, 34, 1469. R. J. Letcher, R. J. Nostrom, S. Lin, M. A. Ramsey and S. M. Bandiera, Toxicol. Appl. Pharmacol., 1996, 137, 127. M. Oehme, A. Biseth, M. Schlabach and O. Wiig, Environ. Pollut., 1995, 90, 401.

81 C. Botham, P. Holmes and P. Harrison fetal development of the central nervous system and reproductive organs. In conclusion, given the current body of evidence relating to the potential role of chemically mediated endocrine disruption in causing the adverse eﬀects seen in various vertebrate organisms, there is clearly some cause for concern regarding the potential risk to human as well as to wildlife populations. However, our present state of knowledge is inadequate for a robust and reliable assessment of the magnitude of the potential risk to be made on either count.

6 Acknowledgements The authors would like to thank Dr Mark Taylor, Dr Raquel Duarte-Davidson and the other participants who contributed to our understanding during a workshop on the ecological eﬀects of sex hormone disrupters held at the Institute in January 1998. We also acknowledge the ﬁnancial support provided by the UK Department of the Environment, Transport and the Regions for the work at IEH on endocrine disruption. However, the opinions expressed in this paper are those of the authors and do not necessarily represent those of any government department or agency.

Press Release, Faroe Islands Authorities, 1998.

82 Oestrogens, Environmental Oestrogens and Male Reproduction

KATIE J. TURNER

1 Introduction Over the past five years, concerns have been raised by the scientific community and the media about the potential of endocrine disrupters to harm the health of both humans and wildlife. There are now many reports describing the increasing incidence of disorders of the male reproductive tract, from poor semen quality to testicular cancer. So far the emphasis has been on implicating chemicals in the environment with oestrogenic activity as causal factors, since the weight of epidemiological and biological evidence suggests that inappropriate exposure to oestrogen during early development can induce the types of disorders reported. Currently, there is no definitive evidence that environmental oestrogens are responsible. This article will review the data which has linked endocrine disrupters, specifically environmental oestrogens, with effects on male reproduction. The aim is to provide an overview of the current understanding about endocrine disruption in males as well as highlighting the many difficulties faced in order to establish whether the presence of endocrine disrupters in our environment is a health risk.

2 Is Male Reproductive Health Deteriorating? Semen quality The issue of whether sperm counts are declining has been the focus of most media attention. A debate still continues as to whether the reported changes in sperm quality are genuine. In 1992, an article described the meta analysis of available data on semen quality in normal men, taken from reports published between the period 1938—1990. This analysis showed a 50% decline in sperm concentration

D. S. Irvine, Balliere’s Clin. Obstet. Gynaecol., 1997, 11, 655. E. Carlsen, A. Giwercman, N. Keiding and N. E. Skakkebaek, Br. Med. J., 1992, 305, 609.

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

83 K. J. Turner from 113 million/ml in 1940 to 66 million/ml in 1990. A recent re-analysis of this data also concluded that there was evidence of a decline in sperm concentrations in the USA and in Europe but not in non-Western countries. There are data which suggests that semen quality has deteriorated in some regions of France, Scotland and Belgium, although other studies conducted in the USA, Finland and France have not confirmed this decrease in sperm quality with time. One explanation of these differences is that the decline in sperm counts may be a regional phenomenon. There is no conclusive message available from the current literature on this issue. This stems from the fact that all the data available on semen quality suffer from a variety of limitations including subject selection bias, such that many of the studies are based on men who are unlikely to be representative of the general population, a lack of standardisation of the methodology used to analyse semen quality and all of the data are retrospective. There is some doubt as to whether it will ever be possible to gain definitive evidence of a decline in semen quality. In some of the studies on semen quality there is additional data which suggest that the change observed is strongly associated with year of birth. A study based in Paris has shown that in a large group of fertile men, sperm counts had declined by an average of 2.6% for each later year of birth during a 20 year period. A Scottish study analysed sperm counts from unselected men donating semen for research who were born between 1951 and 1973. A significant decline in sperm counts was observed over this period such that median sperm concentration fell from 98 million/ml amongst donors born before 1959 to 78 million/ml amongst donors born after 1970. A much larger French study analysed sperm counts in men whose partners were attending IVF clinics because of tubal damage. Whilst unable to show a decrease in semen quality with time, the data did show a decrease in sperm counts in relation to the men’s year of birth. One implication of the birth cohort effect is that it suggests that the perinatal period is an important time for determining sperm counts in adulthood (see section on testicular cancer below). Controversy will continue to persist over allegations that sperm counts are declining. Prospective studies on semen quality in fertile men involving centres in

S. Swan, E. Elkin and L. Fenster, Environ. Health Perspect., 1997, 105, 1228. J. Auger, J. M. Kunstmann, F. Czyglik and P. Jouannet, N. Engl. J. Med., 1995, 332, 281. S. Irvine, E. Cawood, D. Richardson, E. MacDonald and J. Aitken, Br. Med. J., 1996, 312, 467. J. De Mouzon, P. Thonneau, A. Spira and I. Multigner, Br. Med. J., 1996, 313, 43. K. Van Waeleghem, N. De Clercq, L. Vermuelen, F. Schoonjans and F. Comhaire, Human Reprod., 1996, 11, 325. H. Fisch, E. Goluboff, J. Olson, J. Feldshuh, S. Broder and D. Barad, Fertil. Steril., 1996, 65, 1009. C. Paulsen and N. Berman, C. Wang, Fertil. Steril., 1996, 65, 1015. M. Vierula, M. Niemi, A. Keiski, M. Saatanen, S. Saarikoski and J. Suominen, Int. J. Androl., 1996, 19, 11. L. Bujan, A. Mansat, F. Pontonnier and R. Mieusset, Br. Med. J., 1996, 312, 471. H. Fisch and E. T. Goluboff, Fertil. Steril., 1996, 65, 1044. J. Toppari, J. C. Larsen, P. Christiansen, A. Giwercman, P. Grandjean, L. J. Guillette Jr., B. Je´gou, T. K. Jensen, P. Jouannet, N. Keiding, H. Leffers, J. A. McLachlan, O. Meyer, J. Mu¨ ller, E. Rajpert-De Meyts, T. Scheike, R. M. Sharpe, J. P. Sumpter and N. E. Skakkebaek, Environ. Health Perspect., 1996, 104, 741. John Radcliffe Hospital Cryptorchidism Study Group, Arch. Dis. Child., 1992, 67, 892.

84 Oestrogens, Environmental Oestrogens and Male Reproduction

Europe, the USA and Japan are under way. In these studies, semen analysis methodology and subject selection criteria have been standardised to address whether there are region-specific differences in semen quality. These data can then be used as a reference point for any studies on future decline in sperm counts. More importantly, it still remains to be demonstrated whether the reported decrease in semen quality is sufficient to compromise fertility.

Malformations of the Male Genital Tract The incidence of male genital tract abnormalities such as cryptorchidism (undescended testes) and hypospadias (abnormal site of urethral opening due to incomplete closure of the urethral folds during penis development) are also thought to be increasing in Western countries. A prospective study performed in Oxford between 1984 and 1988 reported that the incidence of cryptorchidism had increased by approximately 35% at birth and by 93% at three months in comparison to a similar study performed in the 1950s. The authors of this study used the same criteria for diagnosis of cryptorchidism in the 1980s as were applied in the 1950s, thus minimising the possibility that their results stemmed from altered diagnostic criteria. In contrast, a prospective study performed in the USA between 1984 and 1990 reported that the incidence of cryptorchidism had remained unchanged when compared to those reported several decades earlier. However, this study has been criticised as it is heterogeneous with respect to the ethnicity of the subjects; it is well established that American blacks have a much lower incidence of cryptorchidism than do American whites. Unfortunately, it is is diﬃcult to gauge whether cryptorchidism is becoming more prevalent as there is a lack of studies in which the incidence of cryptorchidism has been examined with time, within the same population or geographical area using standardised criteria. Epidemiological trends in incidence of hypospadia are suggestive of an increase in several European countries, Australia and New Zealand, especially as there is thought to be substantial under-reporting of the mild cases. A multicentre study of seven malformation surveillance systems around the world showed that geographical diﬀerences existed in the prevalence of hypospadia at birth and that not all countries showed an increase in incidence. However, more convincing evidence of a change in prevalence comes from a recent American study based on data from two established surveillance systems, which has shown that the total number of cases of hypospadias has nearly doubled over the period 1968—1993. Both studies showed an increase in incidence of 2—3% per year; furthermore, the number of severe cases of hypospadia (the minority, but not under-reported) showed a similar or higher

G. Berkowitz, R. Lapinski, S. Dolgin, J. Gazella, C. Bodian and I. Holzman, Pediatrics, 1993, 92, 44. B. Ka¨ lle´n, R. Bertollini, E. Castilla, A. Czeizel, L. Knudsen, M. Martinez-Frias, P. Mastroiacovo and O. Mutchinick, Acta Paediatr. Scand., 1986, suppl. 324,1. L. Paulozzi, J. Erickson and R. Jackson, Pediatrics, 1997, 100, 831. H.-O. Adami, R. Bergstro¨m,M.Mo¨ hner, W. Zatonski, H. Storm, A. Ekbom, S. Tretli, L. Teppo, H. Ziegler, M. Rahu, R. Gurevicius and A. Stengrevics, Int. J. Cancer, 1994, 59, 33. R. Bergstro¨ m, H.-O. Adami, M. Mo¨ hner, W. Latonski, H. Storm, S. Tretli, L. Teppo, O. Akre and T. Hakulinen, J. Natl. Cancer Inst., 1996, 88, 727.

85 K. J. Turner rate of increase, conﬁrming that the results were not due to an improvement in the accuracy of reporting.

Testicular Cancer The best evidence of a growing problem with male reproductive health is the fact that the incidence of testicular cancer, which predominantly affects young men aged 15—45 years, is known to be on the increase in most countries. Testis cancer is caused by the abnormal development of germ cells during fetal life (see Section 4 on determinants of fertility in adulthood below). The diagnosis and reporting of testicular cancer has always been accurate, which means that these data do not suffer some of the limitations which affect the data collected on incidence of hypospadia and cryptorchidism. Cancer incidence has increased by 2—4% per year since the 1960s in Great Britain, the Nordic and Baltic countries, Australia, New Zealand and North America. — It should be noted that there is substantial geographical variation in the incidence of testicular cancer and in the observed rate of increase. This is one of the reasons why environmental factors have been implicated as having a causal role in the increased incidence. Currently, Denmark has the highest incidence of testicular cancer, which has increased threefold from the period 1943—1945 to the period 1985—1989, and the lifetime risk of a male developing testis cancer is 0.8%. As an example of geographical variation, the incidence of testicular cancer is four times higher in Denmark than in Finland. Interestingly, this higher rate of cancer incidence appears to be correlated with the opposite difference in semen quality, since the available studies suggest that men in Denmark have lower sperm counts than those observed in the Finnish population. However, some of the countries with the lowest incidence of testicular cancer are showing some of the highest rates of increase; for example, the annual change in incidence in Finland is 3.4% and 4.8% in Poland. Interestingly, the observed increases in testicular cancer can also be correlated to year of birth and this appears to be independent of whether the country has a high or low incidence of testicular cancer. A major study has analysed over 30 000 cases of testis cancer from 1945 to 1989 in men aged 20—84 from Denmark, Norway, Sweden, East Germany, Finland and Poland. In all six countries it was found that there was a much stronger correlation of testis cancer risk with year of birth than with calendar time. For example, men born in 1965 have a risk of cancer that is between four times higher (Sweden) to 11 times higher (East Germany) in comparison with men born in 1905. A similar study conducted in the USA also concluded that the increase in incidence of testis cancer seen in men born after 1910 was a birth cohort effect. It should be noted that although there is marked geographical variation in incidence of testicular cancer, there is an increase with time in most populations. Reports suggest that caucasians are three times more susceptible to developing testicular cancer than American blacks.

S. Devesa, W. Blot, B. Stone, B. Miller, R. Tarone and J. J. Fraumeni, J. Natl. Cancer Inst., 1995, 87,175. T. Zheng, T. Holford, Z. Ma, B. Ward, J. Flannery and P. Boyle, Int. J. Cancer, 1996, 65, 723. T. Jensen, A. Giwercman, E. Carlsen, T. Scheike and N. Skakkebaek, Lancet, 1996, 347, 1844. A. Ekbom and O. Akre, Acta Pathol. Microbiol. Immunol. Scand., 1998, 106, 225.

86 Oestrogens, Environmental Oestrogens and Male Reproduction

However, incidence of testicular cancer is increasing in American blacks and Japansese, both of whom have a low incidence of testis cancer. Extrapolation of the epidemiological trends suggests that the incidence rate of testis cancer will continue to double every 15—25 years.

3 Is there a Common Aetiology? The fact that there might be common aetiology for the increase in incidence of testicular cancer, congenital malformations and the decline in semen quality was highlighted in 1993. A hypothesis was published in the Lancet which proposed that exposure of the fetus and/or neonate to excessive oestrogens could impair development of the male genitalia, descent of the testes and sperm production, as well as increase the likelihood of testicular cancer in later life. Support for an adverse effect during fetal/perinatal life involving a common mechanism comes from two sources. Firstly, epidemiological data suggest that year of birth, birthweight and the presence of genital tract malformations are all risk factors for testicular cancer (see below). Secondly, that perturbation of male differentiation and development by hormones during fetal/perinatal life can result in the types of disorders described above (see Section 5 on exposure to oestrogen below). It is well established that a history of cryptorchidism is a strong risk factor for developing testicular cancer later in life, there being a three- to five-fold increase in risk. Current data suggest that about 5% of boys diagnosed with cryptorchidism subsequently develop testicular cancer. However, there is no decrease in risk associated with early correction of maldescent, which suggests that the association between cryptorchidism and testis cancer is not due to testicular damage induced in the inguinal canal, but rather due to a common causal factor. Some studies also indicate that congenital malformations of the reproductive tract including inguinal hernia, hypospadias and hydrocele are other risk factors for developing testicular cancer, but this is less well established than for cryptorchidism. Testicular maldescent has also been linked to hypospadias. It has been noted that in boys operated on for cryptorchidism there is often an inguinal hernia, which may impede testicular descent. There is substantial epidemiological data to support the idea of a common cause for testicular cancer and cryptorchidism, but further studies are needed to establish whether a causal link can be extended to include other genital tract malformations. It is widely accepted that men with testicular cancer have a higher incidence of abnormalities associated with impaired spermatogenesis, both in the cancerous testis but also in the contralateral testis. Men diagnosed as having testicular cancer often have very poor semen quality, with sperm concentrations of less than 10 million/ml compared to healthy men with 9 50 million/ml. It is thought that gonadal function is abnormal even before testicular cancer develops,

W. R. Miller and R. M. Sharpe, Endocrine-Relat. Cancer, 1998, 5, 69. R. M. Sharpe and N. E. Skakkebaek, Lancet, 1993, 341, 125. H. Moller, A. Prener and N. Skakkebaek, Cancer Causes Control, 1996, 7, 264. A. Prener, G. Engholm and O. Jensen, Epidemiology, 1996, 7, 14. P. Petersen, N. Skakkebaek and A. Giwercman, Acta Pathol Microbiol. Immunol. Scand., 1998, 106,24.

87 K. J. Turner especially in view of the fact that testicular cancer is more common in individuals with conditions associated with abnormal sexual differentiation during fetal life. Unpublished data by Auger and colleagues also supports a relationship between increasing incidence in testicular cancer and a decline in sperm counts. They have found that in men with testicular cancer there is a 6.2% decrease in sperm counts with each later year of birth (J. Auger and P. Jouannet, personal communication). In addition, cryptorchidism is associated with poor semen quality and higher risk of infertility. There appears to be a wide spectrum in the severity of affects on male reproduction, with men with testicular cancer showing a profound decrease in sperm numbers compared with normal men who show a more modest decrease in sperm numbers. Currently, there is no explanation as to why these trends are occurring. It is well established that low birth weight is a risk factor for cryptorchidism, hypospadias and testicular cancer.— Interestingly, a recent publication suggests that oligozoospermia is also associated with low birthweight. In addition, there are increasing numbers of reports in the scientific literature that indicate that the intrauterine environment is a key determinant of other aspects of health in later life. Studies on populations in Europe and the USA suggest that low birthweight is linked with an increased incidence of hypertensive disease, non-insulin-dependent diabetes and cardiovascular disease in adulthood. It has been hypothesised that increased production of the hormones involved in the hypothalamic—pituitary—adrenalaxis during fetal life might be one mechanism of inducing insulin resistance in adulthood. All of these epidemiological associations strongly suggest that there are critical periods of development during early life, and that alteration of hormone levels or other factors can adversely affect development such that the effects will be permanent and impair health in adulthood.

4 Determinants of Fertility in Adulthood In order to discuss the biological mechanisms involved in the aetiology of genital tract malformations, testicular cancer and lowered sperm counts, some knowledge of the processes involved in the diﬀerentiation and development of the male reproductive tract and determination of normal testicular function is required. An early embryo has the potential to develop either a male or a female

J. Mu¨ ller, J. Clin. Endocrinol. Metab., 1984, 59, 785. M. Savage and D. Lowe, Clin. Endocrinol., 1990, 32, 519. J. Hutson, S. Hasthorpe and C. Heyns, Endocrine Rev., 1997, 18, 259. C.Chilvers,N.E.Dudley,M.H.Gough,M.B.JacksonandM.C.Pike,J. Pediatr. Surg., 1986, 21,691. H. Moller and N. Skakkebaek, Cancer Causes Control, 1997, 8, 904. L. Brown, L. Pottern and R. Hoover, Cancer Res., 1986, 46, 4812. O. Akre, A. Ekbom, C.-C. Hsieh, D. Trichopoulos and H.-O. Adami, J. Natl. Cancer Inst., 1996, 88,883. I. Francois, F. de Zegher, C. Spiessens, T. D’Hooghe and D. Vanderschueren, Pediatr. Res., 1997, 42, 899. D. Barker and P. Clark, Rev. Reprod., 1997, 2, 105. D. Phillips, D. Barker, C. Fall, J. Seckl, C. Whorwood, P. Wood and B. Walker, J. Clin. Endocrinol. Metab., 1998, 83, 757. J. Seckl, Steroids, 1997, 62, 89.

88 Oestrogens, Environmental Oestrogens and Male Reproduction reproductive system. They possess two sets of ducts, called Mu¨ llerian and Wolffian ducts, which will go on to develop a female or male reproductive tract, respectively. Development of the male phenotype requires activation of the testis-determining gene (Sry) on the Y chromosome, which initiates a cascade of events leading to the formation of the testis. In the absence of Sry, and thus hormonal signals, the embryo will develop as a female, and is therefore considered to be the default pathway. Differentiation of the testis occurs once the primordial germ cells have migrated into the gonadal ridge, which is completed by week 5 of fetal life in humans. By week 7, the Sertoli cells have become organised into cord-like structures, called seminiferous cords. These contain the primordial germ cells, renamed gonocytes once they become embedded into the Sertoli cells. Within the surrounding interstitium the fetal Leydig cells differentiate from mesenchymal cells and steroidogenesis starts almost immediately. Differentiation of the male reproductive tract begins once the testes have formed, and involves the secretion of two hormones by the testis, anti-Mu¨ llerian hormone (AMH; also called Mu¨ llerian inhibiting substance) and testosterone. AMH produced by the fetal Sertoli cells induces regression of the Mu¨ llerian ducts which would otherwise differentiate to form the oviducts, uterus and upper third of the vagina of the female reproductive system. The fetal Leydig cells produce testosterone which acts on the Wolffian ducts to induce differentiation of the epididymis, vas deferens and seminal vesicles. Testosterone synthesis is also needed for masculinisation of the urogenital sinus, external genitalia and the brain; however, it is first converted to the more potent androgen 5a- dihydrotestosterone. Masculinisation occurs around weeks 7—12 of human gestation. Hypospadia is the result of incomplete masculinisation due to a lack of androgen action; it is common in men with androgen receptor defects. Testicular descent begins around this time and involves other factors in addition to testosterone (see below). The Sertoli cells are thought to co-ordinate the processes relating to testicular development and masculinisation. In addition to producing AMH, it is likely that they regulate the differentiation and multiplication of the early germ cells, as well as the fetal Leydig cells and their production of testosterone through the secretion of paracrine factors. The Sertoli cells multiply in number during late fetal life and for the first year of childhood with a probable further increase during puberty. In the rat, replication commences around 19—20 days gestation and ceases at around 15 days of postnatal life. Sertoli cell multiplication is controlled in part

F. W. George and J. D. Wilson, in The Physiology of Reproduction, 2nd edn., ed. E. Knobil and J. D. Neill, Raven Press, New York, 1994, pp. 3—28. J. Marshall-Graves, BioEssays, 1998, 20, 264. A. G. Byskov and P. Hoyer, in The Physiology of Reproduction, 2nd edn., ed. E. Knobil and J. D. Neill, Raven Press, New York, 1994, pp. 487—540. N. Skakkebaek, E. Rajpert-De Meyts, N. Jorgensen, E. Carlsen, P. Petersen, A. Giwercman, A. Andersen, T. Jensen, A.-M. Andersson and J. Mu¨ ller, Acta Pathol. Microbiol. Immunol. Scand., 1998, 106,3. C. Quigley, A. De Bellis, K. B. Marschke, M. El-Awady, E. M. Wilson and F. S. French, Endocrine Rev., 1995, 16, 271. D. Cortes, J. Mu¨ ller and N. E. Skakkebaek, Int. J. Androl, 1987, 10, 589.

89 K. J. Turner by the follicle stimulating hormone (FSH) from the pituitary gland, which increases the rate at which they divide. The number of Sertoli cells is of critical importance as it determines the number of sperm that can be made in adulthood and therefore testicular size, as each Sertoli cell can only support the development of a fixed number of germ cells. It has been demonstrated in animal models that manipulation of Sertoli cell number alters testis size and daily sperm production. Furthermore, Sertoli cells only divide for a fixed period, and once they have matured they stop proliferating and cannot be stimulated to divide again. Sertoli cells are also thought to regulate the proliferation of gonocytes and their differentiation into spermatogonia during the second and third trimesters of pregnancy. In normal gonadal development, differentiation should be complete by the end of the first year of post-natal life such that all gonocytes should be absent. Spermatogonia undergo several mitotic divisions during perinatal life, then enter a quiescent phase until they enter meiosis during puberty, thus initiating spermatogenesis. The regulation of spermatogenesis is poorly understood but it is generally thought that the Sertoli cells provide a unique environment which ensures the normal development of the germ cells into spermatozoa. It is obvious from the above information that testosterone and AMH are important for male differentiation and development, although it should be noted that other hormones produced by the pituitary and thyroid glands are also involved. The pituitary secretes gonadotrophins, luteinising hormone (LH), which is needed to maintain testosterone production by the fetal Leydig cells, and FSH, which is involved in regulating Sertoli cell replication together with thyroid hormone. Suppression of gonadotrophin secretion results in small testes due to inhibition of Sertoli cell proliferation. A negative feedback system operates in which the steroid hormones produced by the testis, testosterone and oestrogen act on the brain to regulate gonadotrophin secretion by the pituitary (Figure 1). Oestrogens have the potential to act at various points within this feedback loop to alter hormone levels and thus testicular function.

Testicular Descent and Cryptorchidism Testicular descent is a complex process regulated by hormones. It can be divided into two stages, transabdominal descent and inguinal descent. Two structures called the gubernaculum and cranial suspensory ligament are important for guiding the testis during descent to its correct position. The ﬁrst stage occurs between weeks 8—15 of fetal life and involves descent of the testis through the abdomen to lie in the groin area. This phase may be regulated in part by AMH, which is thought to induce swelling of the gubernaculum and regression of the cranial suspensory ligament. The second stage occurs between weeks 28—35 of fetal life and involves the migration of the testis into the scrotum. This phase is

J. M. Orth, Anat. Rec., 1982, 203, 485. J. M. Orth, Endocrinology, 1984, 115, 1248. L. Johnson, R. S. Zane, C. S. Petty and W. B. Neaves, Biol. Reprod., 1984, 31, 785. J. M. Orth, G. M. Gunsalus and A. A. Lamperti, Endocrinology, 1988, 122, 787. R. M. Sharpe, in The Physiology of Reproduction, 2nd edn., ed. E. Knobil and J. D. Neill, Raven Press, New York, 1994, pp. 1363—1434.

90 Oestrogens, Environmental Oestrogens and Male Reproduction

Figure 1 The major hormones involved in growth and function of the fetal/neonatal testis illustrating how exogenous, environmental oestrogens could disrupt the normal balance of these mechanisms.

modulated by testosterone, which acts on the gubernaculum and on the genitofemoral nerve which innervates it. The cause of cryptorchidism is likely to be multifactorial and is not really understood. The most common cause of cryptorchidism is thought to be due to a lack of androgen action which impairs the second phase of descent. Spermatogenesis is abnormal in cryptorchid testes but this is generally thought to be related to the higher temperature of the testes, 37 °C, instead of 33 °C when the testes reside in the scrotum. Steroidogenesis is also abnormal and is most likely a reﬂection of a reduction in Leydig cell number. Interestingly, it has also been found that cryptorchid testes possess increased numbers of gonocytes during early postnatal life, suggesting that their ability to diﬀerentiate into spermatogonia is impaired; a similar situation is thought to be involved in the generation of testicular cancer (see below). This would result in fewer germ cells available to enter spermatogenesis, and it is known that rats made cryptorchid at birth have decreased semen quality but are still fertile (see Hutson et al. for references).

Abnormal Germ Cell Development and Testicular Cancer Testicular cancer is thought to be caused by the abnormal development of early germ cells, giving rise to pre-malignant cells during fetal life. By studying the early stage of testicular cancer known as carcinoma in situ (CIS), it has been demonstrated that CIS is a precursor of most types of germ cell cancer and that a CIS cell is actually a malignant gonocyte. This is based on the histological D. Huﬀ, F. Hadziselimovic, H. M. Snyder, B. Blyth and J. Duckett, J. Urol., 1991, 146, 624. N. Skakkebaek, J. Berthelsen, A. Giwercman and J. Mu¨ller, Int. J. Androl., 1987, 10, 19.

91 K. J. Turner

Figure 2 Key events in fetal/neonatal life which predetermine reproductive function of the adult human male. Note that the Sertoli cells are involved in the orchestration and co-ordination of all of the key events. The relationship between the key events indicated and disorders of male reproductive development and function are indicated in boxes.

similarities between CIS cells and gonocytes, and the expression of common antigens such as c-kit, a growth factor receptor. Testes with CIS possess increased numbers of gonocyte-like cells which would normally have differentiated into spermatogonia. It has been hypothesised that external factors somehow block this differentiation process, presumably a reflection of impaired function of the Sertoli cells and fetal Leydig cells. It has also been hypothesised that increased exposure to hormones induces the malignant gonocytes to become invasive. This seems plausible, as the time at which gonocytes become invasive coincides with periods during postnatal life and puberty when Sertoli cells are producing high amounts of hormones. The malignant gonocytes overexpress c-kit so it is possible that they might proliferate in response to stimulation by stem cell factor secreted by the Sertoli cells. In summary, several processes are involved in determining adult reproductive function and the Sertoli cells appear to play a key role in their co-ordination (Figure 2). Firstly, the Sertoli cells secrete AMH which ensures regression of the Mu¨ llerian ducts. Secondly, they regulate the production of testosterone by Leydig cells which ensures masculinisation of the male reproductive tract, external genitalia and the brain. Thirdly, they have to multiply for a fixed length of time in order to establish sufficient numbers to support spermatogenesis at a level which ensures fertility. Finally, Sertoli cells are responsible for supporting germ cell development from gonocytes onwards. It is important to note that all these processes occur during fetal/perinatal life and that various hormones play key roles in regulating these processes. Thus, exposure to a factor(s) during this critical period of development which is capable of altering one or more of these

A. Holstein, B. Schu¨tte, H. Becker and M. Hartmann, Int. J. Androl., 1987, 10,1. N. Jorgensen, E. Rajpert-De Meyts, N. Graem, J. Mu¨ller, A. Giwercman and N. Skakkebaek, Lab. Invest., 1995, 72, 223. E. Rajpert-De Meyts, N. Jorgensen, K. Brondum-Nielsen, J. Mu¨ller and N. Skakkebaek, Acta Pathol. Microbiol. Immunol. Scand., 1998, 106, 198.

92 Oestrogens, Environmental Oestrogens and Male Reproduction key processes will potentially cause adverse eﬀects on male reproductive function in adult life (Figure 2).

5 Exposure to Oestrogen is Associated with Impaired Male Reproductive Health A variety of epidemiological data suggest that the hormonal environment of the fetus may be involved in the development of testicular cancer. Testicular cancer is most common in individuals with maldeveloped gonads, notably in patients with intersex conditions in the presence of a Y chromosome such as gonadal dysgenesis and androgen insensitivity syndrome (AIS) and also in cryptorchidism. The common factor in these disorders is that Sertoli cell function and/or differentiation is severely impaired. In AIS, LH and testosterone levels are elevated owing to insensitivity to normal androgen feedback mechanisms and, as a consequence, oestrogen levels are also raised. Although neither Sertoli cells nor gonocytes express androgen receptors during fetal life, there is now evidence that they express oestrogen receptor beta (ERb) (see section on sites of oestrogen action, below). This provides a route by which oestrogens might act directly to impair Sertoli cell and gonocyte development. Oestrogens have been implicated as a risk factor for testicular cancer since the early 1980s. Studies have shown that oestrogen levels are higher during a first pregnancy and it is known that there is an increased risk of testis cancer associated with the first born boy in comparison with subsequent male births. Mothers of cryptorchid sons were found to have a higher level of unbound oestradiol during the first trimester of pregnancy in comparison with mothers of boys with normally descended testes. Use of exogenous hormones during pregnancy has also been associated with increased risk of testicular cancer. The main evidence which suggests that prenatal exposure to oestrogens has a major influence on the development of the male reproductive tract comes from the treatment of pregnant women with the synthetic oestrogen diethylstilbestrol (DES). Between the 1940s and the 1970s, approximately 2—3 million women in the USA and some countries in Europe were treated in early pregnancy with DES. Subsequently it was shown that the offspring of the DES-treated mothers had an increased range of abnormalities of the reproductive tract. The male progeny were reported to show an increased incidence of smaller testes, cryptorchidism, hypospadias and epididymal cysts together with poor semen quality. The prevalence of genital malformations was double in those men exposed during the first 10 weeks of pregnancy in comparison to men exposed during the later stages of pregnancy. Based on these findings it was suspected that these men would have fertility problems. However, a recent study based on a cohort of DES-exposed males from the Chicago area was unable to find any

P. T. K. Saunders, J. S. Fisher, R. M. Sharpe and M. R. Millar, J. Endocrinol., 1998, 156, R13. B. Henderson, B. Benton, J. Jing, M. Yu and M. Pike, Int. J. Cancer, 1979, 23, 598. R. Depue, M. Pike and B. Henderson, J. Natl. Cancer Inst., 1983, 71, 1151. W. J. Dieckmann, M. E. Davis and L. M. Rynkiewicz, Am. J. Obstet. Gynecol., 1953, 66, 1062. R. J. Stillman, Am. J. Obstet. Gynecol., 1982, 142, 905. A. Wilcox, D. Baird, C. Weinberg, P. Hornsby and A. Herbst, N. Engl. J. Med., 1995, 332, 1411.

93 K. J. Turner evidence of impaired fertility or increased incidence of testicular cancer. It is well established that the male offspring of DES-treated pregnant mice also show the same pattern of abnormalities observed in humans, such as undescended and small testes, infertility, epididymal cysts, sperm abnormalities, hypospadias and incomplete regression of the Mu¨ llerian ducts. Prenatal exposure to a high dose of DES (100 kg/kg/day) results in sterility in 60% of offspring, the majority of which are cryptorchid and have morphologically abnormal sperm and non-motile sperm. When mice exposed in utero to DES are then examined once they have reached 10—18 months of age, a spectrum of degenerative changes in the testes are observed ranging from mild to severe: normal spermatogenesis within smaller testes, impaired spermatogenesis, atrophied seminiferous tubules and, at worst, necrotic testes. The incomplete regression of the Mu¨ llerian ducts results in remnants which become enlarged and cystic and are thought to give rise to epididymal cysts. A recent study has shown that administration of DES to pregnant mice using the same treatment regimen results in a delay in the formation of the Mu¨ llerian ducts by approximately 2 days; although the ducts begin to regress remnants still remain at the distal (urogenital sinus) end. The authors hypothesise that incomplete regression of the Mu¨ llerian ducts is due to the delay in their formation so that they miss the window of regression. In some animals, proliferation and hyperplasia of the rete testis is observed and occasionally this becomes neoplastic, resulting in adenocarcinoma. Similarly, treatment of pregnant mice with the synthetic oestrogen ethinyloestradiol increases the incidence of cryptorchidism and testicular cancer. Exposure to ethinyloestradiol also affects testicular differentiation, resulting in increased numbers of gonocytes, decreased numbers of Sertoli cells and smaller fetal Leydig cells and is associated with gonadal dysgenesis. Furthermore, perinatal exposure of male mice to oestrogen causes cryptorchidism and lesions of the reproductive tract, notably of the efferent ducts and epididymis. It is stressed that all of these studies have used high doses of DES or ethinyloestradiol to induce the effects reported. In 1992 a mechanism was hypothesised to explain how exposure of the male fetus or neonate to exogenous oestrogens might impair development, resulting in lowered sperm counts, cryptorchidism or even testicular cancer. It was suggested that the effects on sperm counts could be a consequence of suppression of FSH levels by oestrogen negative feedback, thus inhibiting Sertoli cell proliferation. The reduction in Sertoli cell numbers would result in reduced capacity of the testis to make sperm. In addition, the secretion of AMH and other factors by the Sertoli cells, involved in germ cell and Leydig cell development as well as steroid biosynthesis, might be inhibited. In support of this hypothesis it

J. A. McLachlan and R. R. Newbold, Science, 1975, 190, 991. J. Visser, A. McLuskey, M. Verhoef-Post, P. Kramer, J. Grootegoed and A. Themmen, Endocrinology, 1998, 139, 4244. R. R. Newbold, B. C. Bullock and J. A. McLachlan, Am. J. Pathol., 1986, 125, 625. Y. Yasuda, T. Kihara and T. Tanimura, Teratology, 1985, 32, 113. A. Walker, L. Bernstein, D. Warren, N. Warner, X. Zheng and B. Henderson, Br. J. Cancer, 1990, 62, 599. Y. Yasuda, T. Kihara, T. Tanimura and H. Nishimura, Teratology, 1985, 32, 219. Y. Arai, T. Mori, Y. Suzuki and H. Bern, Int. Rev. Cytol., 1983, 84, 235.

94 Oestrogens, Environmental Oestrogens and Male Reproduction was found that exposure of pregnant rats to low doses of DES or the environmental oestrogens (octylphenol and butyl benzyl phthalate), throughout gestation and lactation, encompassing fetal development and the period of Sertoli cell proliferation, resulted in a modest but significant reduction in testis weight and daily sperm production in the adult offspring. Testicular histology was normal in these animals; there was no indication of damage to spermatogenesis, which suggested that the reduction in sperm numbers was due to a reduced number of Sertoli cells. Furthermore, another study has demonstrated that the exposure of pregnant sheep to DES or octylphenol is able to suppress blood FSH levels within the fetus. The accumulating evidence suggests that inappropriate exposure to exogenous oestrogens during fetal/perinatal life can permanently impair testicular descent, masculinisation of the reproductive tract and sperm production in adulthood. Perhaps the most reassuring conclusion from these data is that these effects appear to be induced by exposure to high levels of oestrogens or very potent oestrogenic chemicals such as DES. It seems unlikely that exposure to environmental oestrogens which are only weakly oestrogenic would be able to induce such severe effects by acting through the mechanism described above. However, in the next section we will describe some more recent studies which show that this is not the only route by which oestrogens can adversely affect differentiation and development of the male reproductive tract.

6 Effects of Oestrogen on the Development and Function of the Male Reproductive System The question of whether environmental oestrogens are a threat to human health has served to highlight our ignorance of the role that oestrogens play in normal development and function in males. A concerted effort has begun to address this deficiency in our knowledge with the aim that a better understanding of oestrogen action will make it easier to establish whether environmental oestrogens pose a risk. So far I have given the impression that oestrogens act indirectly on the male reproductive tract by the modulation of other hormones such as FSH and AMH to cause adverse changes. However, there is increasing evidence that oestrogen may have a necessary and beneficial role in male development and function. Oestrogen can also act directly on various cellular targets within the male reproductive system via its receptors. In addition, it is well established that, within the testis, immature Sertoli cells, Leydig cells and germ cells possess the enzyme aromatase which is responsible for the synthesis of oestradiol from testosterone (see below). The next section will describe the most likely targets of oestrogen action within the male reproductive tract based on the localisation of ERs and aromatase activity. This will be followed by a brief

R. M. Sharpe, J. S. Fisher, M. R. Millar, S. Jobling and J. P. Sumpter, Environ. Health Perspect., 1995, 103, 1136. T. Sweeney and A. N. Brooks, J. Reprod. Fertil., 1996, Abstr. Ser. 17, Abstr. 46, p. 18. R. M. Sharpe, Trends Endocrinol. Metab., 1998, 9, 371. P. T. K. Saunders, Rev. Reprod., 1998, 3, 164.

95 K. J. Turner overview of some of the potential functions that oestrogen might have within the testis and excurrent duct system.

Sites of Oestrogen Action and Production in the Male Reproductive Tract Oestrogen action is mediated through specific receptors localised within the nucleus of target cells (see Carson-Jurnica et al. and Tsai and O’Malley for more detailed information on steroid action). ERs belong to a large family of hormone activated transcription factors. Once oestrogen has bound to its receptor, the receptor can dimerise and thus acquires the ability to interact with genes possessing a specific region called an oestrogen response element. This activates gene transcription and induces an oestrogenic response within the cell. At least two ERs have been identified, named ERa and ERb. The first ER was cloned in 1986 from a human breast cancer cell line. A decade later a second ER was cloned from a rat prostate cDNA library, which subsequently became named ERb. This has led to a complete re-evaluation of the cellular targets of oestrogen action in both males and females. Furthermore, this has added another level of complexity to our understanding of oestrogen action since there is now the possibility of interaction between the two receptors if they are expressed within the same cell. Immunocytochemistry has demonstrated that within the testis of the rat and marmoset, ERa is expressed in the fetal and the adult generations of Leydig cells. However, the most abundant expression of ERa is found in the efferent ducts, with expression detectable from neonatal life through to adulthood. The efferent ducts extend from the rete region of the testis to the initial segment of the epididymis and their main role is thought to be the resorption of fluid surrounding the sperm. It has been shown that : 90% of the fluid entering the efferent ducts is resorbed before reaching the epididymis. This increases sperm concentration, which is thought to be important for sperm maturation and their acquisition of fertilising ability in the epididymis. In fetal life, expression of ERa can be detected in the mesonephric tissue surrounding the mesonephric tubules from which the efferent ducts derive. The efferent ducts and Leydig cells appear to be the most important sites of oestrogen action via ERa, although there is some expression of ERa in the rete testis of the rat (see Table 1).

M. A. Carson-Jurnica, W. T. Schrader and B. W. O’Malley, Endocrine Rev., 1990, 11, 209. M.-J. Tsai and B. O’Malley, Annu. Rev. Biochem., 1994, 63, 451. S. Green, P. Walter, V. Kumar, A. Krust, J.-M. Bornert, P. Argos and P. Chambon, Nature, 1986, 320, 134. G. G. J. M. Kuiper, E. Enmark, M. Pelto-Hukko, S. Nilsson and J.-A. Gustafsson, Proc. Natl. Acad. Sci. USA, 1996, 93, 925. J. S. Fisher, M. R. Millar, G. Majdic, P. T. K. Saunders, H. M. Fraser and R. M. Sharpe, J. Endocrinol., 1997, 153, 485. R. A. Hess, D. H. Gist, D. Bunick, D. B. Lubahn, A. Farrell, J. Bahr, P. S. Cooke and G. L. Greene, J. Androl., 1997, 18, 602. T. T. Turner, J Reprod. Fertil., 1984, 72, 509. R. A. Hess, D. Bunick, K.-H. Lee, J. M. Bahr, J. A. Taylor, K. S. Korach and D. B. Lubahn, Nature, 1997, 390, 509.

96 Oestrogens, Environmental Oestrogens and Male Reproduction

Table 1 Sites of androgen Location AR ERa ERb Aromatase and oestrogen receptors and aromatase activity in Testis the testis and associated Sertoli cells 99;; ducts during fetal/perinatal Gonocytes 99;; life in the rat! Peritubular cells ;; 9 ; Fetal Leydig cells 9;;;(?) Associated ducts Mesenchymal cells ; (;) ; surrounding Wolﬃan ducts Mesonephric tissue 9;; Mesonephric ducts 99; Eﬀerent ducts (derived 9 ;;; ; (?) from mesonephric ducts)

!The pattern of expression of the receptors is based on immunolocalisation studies conducted by colleagues where ; represents the intensity of immunostaining and 9 where expression could not be detected. The presence of aromatase activity in Sertoli cells has been reported in the literature. Aromatase may also be expressed in fetal Leydig cells and eﬀerent ducts (K. J. Turner, unpublished observations).

The localisation of ERb expression within the body is less well established owing to a lack of good antibodies, so most of the available data is based on mRNA expression determined using RT-PCR or in situ hybridisation. Interestingly, ERb expression has been immunolocalised to many more cell types within the male reproductive tract in comparison to ERa (Table 1). The most important difference is within the testis, as ERb is expressed in the nuclei of the Leydig cells, the Sertoli cells and some of the germ cells (spermatogonia and pachytene spermatocytes) of the adult rat, and in marmoset and humans as well (P. T. K. Saunders, personal communication). In the fetal rat testis, ERb is present in peritubular cells, fetal Leydig cells, Sertoli cells and gonocytes. It is also expressed in seminal vesicles and the prostate. ERb mRNA expression has been detected in the efferent ducts (although at much lower levels than ERa), epididymis, vas deferens and the prostate of the adult rat. Like ERa,ERb is expressed in the mesonephric tissue but in addition is expressed within the ducts themselves (J. S. Fisher and P. T. K. Saunders, unpublished data). In contrast to the androgen receptor (AR), ERs are expressed more widely in the male. In the fetal testis, AR is only expressed in the peritubular cells, interstitial cells and the mesenchymal cells surrounding the Wolffian ducts, whereas in the adult testis, AR are expressed in the Sertoli cells, Leydig cells and peritubular cells. The presence of aromatase in the testis and in sperm further emphasises the likelihood that the local production of oestradiol within the male reproductive

E. Enmark, M. Pelto-Huikko, K. Grandien, S. Lagercrantz, J. Lagercrantz, G. Fried, M. Nordenskold and J.-A. Gustafsson, J. Clin. Endocrinol. Metab., 1997, 82, 4258. P. T. K. Saunders, S. M. Maguire and J. Gaughan, M. R. Millar, J. Endocrinol., 1997, 154, R13. G. Majdic, M. R. Millar and P. T. K. Saunders, J. Endocrinol., 1995, 147, 285. W. J. Bremner, M. R. Millar, R. M. Sharpe and P. T. K. Saunders, Endocrinology, 1994, 135, 1227.

97 K. J. Turner tract may have important consequences. There are some data to suggest that the fetal testis has low levels of aromatase activity (Table 1). It is well established that adult Leydig cells express aromatase, as do immature Sertoli cells. More recently, it has been discovered that spermatocytes, spermatids and spermatozoa possess aromatase activity, leading to the suggestion that germ cells may modulate their own environment via oestrogen production, both in the testis and as they pass through the excurrent ducts.

Possible Functions of Oestrogen in Male Reproduction Sertoli Cells. The fact that oestradiol is produced whilst Sertoli cells are proliferating has led to speculation that oestrogen might be involved in regulating Sertoli cell number. Treatment of neonatal rats with a high dose of DES (10 kg on alternate days between day 2 and day 12) during the period of peak Sertoli cell replication results in a 40% decrease in Sertoli cell numbers at day 18, and this is reflected by changes in testis weight which is reduced by 58% in adulthood. As expected, a similar reduction in daily sperm production was observed. Another study investigating the effect of in utero exposure to 2.5 ppm 17b-oestradiol on male rats failed to demonstrate any alteration in Sertoli cell number even though levels of oestrogen were significantly increased and testosterone was reduced in these rats in comparison to control values. One possible explanation for this discrepancy is that alteration of Sertoli cell number requires exposure to a high dose of oestrogen. Oestrogen may also be involved in the maturation of Sertoli cells and their support of germ cell development. Treatment of neonatal rats with either DES or oestradiol benzoate, 500 kg on day one, resulted in a delay of Sertoli maturation as judged by immunoexpression of marker proteins days 18—25 or morphological appearance at 45 days of age. An indirect measurement of Sertoli cell functionality is their ability to support germ cell development. This can be represented by the measurement of germ cell volume/number expressed relative to Sertoli cell volume/number. Neonatal DES treatment alters this ratio, indicating that the capacity of the Sertoli cells to support developing germ cells is impaired and this appears to be a permanent change. However, in utero

T. L. Greco and A. H. Payne, Endocrinology, 1994, 135, 262. J.-P. Weniger, J. Steroid Biochem. Mol. Biol., 1993, 44, 459. L. E. Valladares and A. H. Payne, Endocrinology, 1979, 105, 431. S. Inkster, W. Yue and A. Brodie, J. Clin. Endocrinol. Metab., 1995, 80, 1941. J. H. Dorrington and S. A. Khan, in The Sertoli Cell, ed. L. Russell and M. Griswold, Cache River Press, Clearwater, 1993, pp. 538—549. H. Nitta, D. Bunick, R. A. Hess, L. Janulis, S. C. Newton, C. F. Millettte, Y. Osawa, Y. Shizuta, K. Toda and J. M. Bahr, Endocrinology, 1993, 132, 1396. L. Janulis, R. A. Hess, D. Bunick, H. Nitta, S. Janssen, Y. Osawa and J. M. Bahr, J. Androl., 1996, 17, 111. R. A. Hess, D. Bunick and J. M. Bahr, Environ. Health Perspect., 1995, 103 (suppl. 7), 59. R. M. Sharpe, N. Atanassova, C. McKinnell, P. Parte, K. J. Turner, J. S. Fisher, J. B. Kerr, N. P. Groome, S. Macpherson, M. R. Millar and P. T. K. Saunders, Biol. Reprod., 1998, 59, 1084. J. Cook, L. Johnson, J. O’Connor, L. Biegel, C. Krams, S. Frame and M. Hurtt, Toxiocol. Sci., 1998, 44, 155. F. Gaytan, L. Pinilla, R. Aguilar, M. Lucena and R. Paniagua, J. Androl., 1986, 7, 112.

98 Oestrogens, Environmental Oestrogens and Male Reproduction exposure to 2.5 ppm 17b-oestradiol had no eﬀect on the ratio of spermatids to Sertoli cells. Examination of testicular morphology in neonatally oestrogen-treated rats shows that spermatogenesis is impaired. Widespread degeneration and exfoliation of germ cells occurs, although there is considerable variation in the severity of damage between animals. Treatment can result in Sertoli cell-only seminiferous tubules or even maldescended and necrotic testes, implying that the Sertoli cells are incapable of supporting normal germ cell development. It can also be argued that oestrogen might have a direct role in the regulation of spermatogenesis, based on the fact that germ cells have aromatase activity and express ERs. It is well established that testosterone is needed to maintain spermatogenesis; however, there is obviously the potential for germ cells to convert testosterone to oestradiol which can then act in an autocrine or paracrine fashion via its receptors. In addition, DES treatment causes distension of the seminiferous tubule lumens and the rete testis which is indicative of ﬂuid accumulation (see below for further discussion). This gives further support to the idea that oestrogens can act at other sites in addition to the Sertoli cells.

Gonocytes. The oestradiol synthesised by immature Sertoli cells might stimulate proliferation of gonocytes. In rats, it is known that gonocytes proliferate on days 3 and 4 of neonatal life, after which they migrate from the centre of the seminiferous cords to the base where they will later diﬀerentiate into spermatogonia. A recent study has shown that gonocytes isolated from rat pups at days 3 will proliferate in response to oestradiol and that this can be blocked using an ER antagonist. Gonocytes isolated at an earlier age failed to proliferate in response to oestradiol, supporting the idea that, like Sertoli cells, gonocytes have a ﬁxed window in which they can undergo mitosis. It is not known whether in vivo treatment with oestrogen on day 3 of postnatal life can increase gonocyte numbers. It is interesting to note that gonocytes can respond to oestrogen in vitro since exposure to oestrogen has been implicated in the development of testis cancer.

Leydig Cells. It has been speculated for a long time that oestrogens may play a role in the development of Leydig cells, in the determination of their numbers and the regulation of testosterone biosynthesis. Fetal Leydig cells and their precursors, as well as the adult generation of Leydig cells, are all potential targets of oestrogen action as they possess ERs and are capable of aromatising testosterone to oestradiol. However, evidence of this has been more diﬃcult to demonstrate. There are some data from animal models to suggest that oestrogens can inhibit adult Leydig cell development and extensive data showing inhibitory eﬀects of oestrogen on adult Leydig cell steroidogenesis. More recently, it has been shown that administration of high doses of DES (500 kg/kg) or an environmental

J. Blanco-Rodriguez and C. Martinez-Garcia, Tiss. Cell, 1996, 28, 387. J. S. Fisher, K. J. Turner, H. M. Fraser, P. T. K. Saunders, D. Brown and R. M. Sharpe, Endocrinology, 1998, 139, 3935. H. Li, V. Papadopoulos, B. Vidic, M. Dym and M. Culty, Endocrinology, 1997, 138, 1289. T. O. Abney and R. B. Myers, J. Androl., 1991, 12, 295. S. B. Cigorraga, S. Sorrell, J. Bator, K. J. Catt and M. L. Dufau, J. Clin. Invest., 1980, 65, 699.

99 K. J. Turner oestrogen, octylphenol (600 mg/kg), to pregnant rats during the period of sexual differentiation can cause inhibition of one of the enzymes essential for testosterone biosynthesis. In this study, both immunoexpression of 17a-hydroxylase, C17,20-lyase by the fetal Leydig cells and enzyme activity were decreased in fetal life. This decrease was the result of inhibition of enzyme expression as Leydig cell numbers remained unchanged. These effects would imply that testosterone production was impaired, although actual suppression of testosterone levels was not determined. Similarly, in utero exposure to DES and octylphenol was shown to reduce the expression of a transcription factor, steroidogenic factor-1 (SF-1) which is essential for gonadal differentiation, within fetal testes. It is therefore biologically plausible that exposure to oestrogens may impair testosterone production by Leydig cells and thus affect male differentiation and masculinisation as a consequence. At present there is no evidence to indicate that exposure to weakly oestrogenic chemicals will have severe effects on testicular steroid production.

Rete Testis/Efferent Ducts. The first clear indication of a physiological function for oestrogen came from studies of transgenic mice (ERKO) in which the gene for ERa was disrupted. These have shown that oestrogen plays a role in regulating fluid resorption within the efferent ducts and that the disruption of fluid resorption, which occurs in ERKO mice, is the most likely explanation of the progressive damage to spermatogenesis and sperm fertilising ability seen in these mice. Neonatal oestrogen treatment also causes fluid problems, as mentioned previously. Administration of DES neonatally to rats was accompanied by pronounced distension of the rete testis and efferent ducts and this was sustained through puberty and into adulthood. Fluid accumulation within the rete testis can also be induced by neonatal treatment with 500 kg oestradiol. Examination of the morphology of the efferent ducts in neonatally DES-treated rats revealed that the epithelium was reduced in height, thus giving the cells a cuboidal rather than a columnar appearance due to the loss of the brush border and apical cytoplasm. The efferent ducts in ERKO mice also have this morphological appearance. Furthermore, immunoexpression of a water channel protein, aquaporin-1 which is thought to be involved in water resorption, within the efferent duct epithelium was reduced at postnatal days 10, 18 and 25 following neonatal treatment with DES up to day 12. Corresponding with the times when severe distension of the efferent ducts was observed. However, treatment with lower doses of DES, bisphenol-A or octylphenol reduced epithelial cell height in the efferent ducts but without inducing any gross morphological distension of the efferent ducts or rete testis.? There is good evidence to suggest that oestrogens are involved in fluid regulation within the efferent ducts, especially in view of the fact that this is the site of highest ER expression. Curiously, it seems that under-exposure to

G. Majdic, R. M. Sharpe, P. J. O’Shaughnessy and P. T. K. Saunders, Endocrinology, 1996, 137,1063. G. Majdic, R. M. Sharpe and P. T. K. Saunders, Mol. Cell. Endocrinol., 1997, 127, 91. E. M. Eddy, T. F. Washburn, D. O. Bunch, E. H. Goulding, B. C. Gladen, D. B. Lubahn and K. S. Korach, Endocrinology, 1996, 137, 4796. J. Aceitero, M. Llanero, R. Parrado, E. Pena and A. Lopez-Beltran, Anat. Rec., 1998, 252, 17. ? J. S. Fisher, K. J. Turner, D. Brown and R. M. Sharpe, Environ. Health Perspect., 1999, 107, 397.

100 Oestrogens, Environmental Oestrogens and Male Reproduction oestrogen, as seen in the ERKO mice, and over-exposure to oestrogen both result in fluid accumulation and permanent morphological changes to the rete and efferent ducts, although there are some differences in that the DES-treated rats show overgrowth of the rete, and distension is evident much earlier in development (before puberty) and does not get progressively worse as seen in the ERKO mice. There are potentially more direct pathways by which oestrogens could impair sperm production and fertility rather than indirectly through the suppression of FSH. It seems likely that endogenous oestrogen production will be involved in ensuring normal development and function of the reproductive tract. This means that under- or over-exposure to oestrogen during critical periods of development of the male reproductive tract may lead to permanent alteration of structure and function. However, the results from animal studies would suggest that only exposure to extremely high doses of oestrogen during fetal or neonatal life results in permanent effects. It remains to be demonstrated whether fertility is compromised by oestrogen treatment during fetal/neonatal life, and this is difficult to resolve in rodent studies as neonatally DES-treated animals do not mate (R. M. Sharpe, unpublished observations).

7 Environmental Oestrogens This term is used to describe man-made chemicals and phytoestrogens which are present in the environment and have oestrogenic activity in vitro and/or in vivo.A large number of compounds within several major groups of chemicals have been identiﬁed as being weakly oestrogenic by in vitro screening methods; however, very few have been adequately tested in vivo (see Table 2 below). Many of these compounds are widespread and persistent in the environment. They are likely to be present in the food chain, drinking water, plastics, household products, cosmetics, toiletries and food packaging, though which is the most important route of human exposure is unclear. However, exposure to the weak oestrogenic activity of man-made chemicals might not be the real cause for concern. It can be argued that exposure to industrial chemicals or agricultural products with oestrogenic activity is likely to be minimal in comparison to our dietary intake of naturally occurring plant-derived oestrogens, the phytoestrogens. The topic of hormone-disrupting chemicals has been expanding rapidly over the past few years, and for more information the reader is referred to the reviews. —

Man-made Chemicals and Eﬀects on Wildlife Those man-made chemicals which have been identiﬁed as being oestrogenic belong to several major groups of chemicals including organochlorine pesticides, polychlorinated biphenyls, alkylphenolic compounds,

S. Safe, Environ. Health Perspect., 1995, 103, 346. K. J. Turner and R. M. Sharpe, Rev. Reprod., 1997, 2, 69. W. Kelce and E. Wilson, J. Mol. Med., 1997, 75, 198. C. Tyler, S. Jobling and J. Sumpter, Crit. Rev. Toxicol., 1998, 28, 319. W. Kelce, C. Stone, S. Laws, L. Gray, J. Kemppainen and E. Wilson, Nature, 1995, 375, 581. A. Soto, K. Chung and C. Sonnenschein, Environ. Health Perspect., 1994, 102, 380.

101 K. J. Turner phthalate esters and bisphenol-A. Only some of the chemicals within each group have been shown to be oestrogenic and only a small fraction of man-made chemicals present in our environment have been tested (Table 2). It is reasonably certain that other chemicals with oestrogenic activity will be identified over the next few years. Increasingly, it appears that phenolic compounds are likely to be oestrogenic and these comprise 9 50% of all man-made chemicals. It should be noted that all the chemicals identified as having oestrogenic activity are approximately 1000- to 100 000-fold less potent than oestradiol, and recent data would suggest that they have a similar binding affinity for ERa and ERb. Furthermore, new data are beginning to emerge which suggests that chemicals which act as weak oestrogen receptor agonists in vitro may also be weak androgen receptor antagonists in vitro (Table 2, and see concluding remarks below), thus, making it even more difficult to predict the in vivo bioactivity of an individual chemical. However, these in vitro measures of potency take no account of bioaccumulation or whether the chemical binds to steroid-binding proteins in plasma. Nevertheless, it is presumed that biological effects of individual compounds in animals or man will only occur when exposure is reasonably high. The strongest evidence which implicates exposure to man-made chemicals as a causal factor in abnormalities of the reproductive tract comes from effects on wildlife. There are a number of reports of decreased reproductive success and developmental defects in a few species associated with areas of industrial pollution. The best example of a known endocrine disrupting chemical causing severe reproductive abnormalities is the effect of tributyltin (TBT) on invertebrates. The phenomenon of pseudohermaphroditism or ‘imposex’ in marine gastropods has been described world-wide. TBT is used as an anti-fouling agent which is painted onto the hulls of ships. It inhibits aromatase activity, resulting in elevated androgen levels and thus masculinisation of female gastropods as it induces growth of a penis, thus acting as an anti-oestrogen. In Florida, male reproductive abnormalities have been K. Connor, K. Ramamoorthy, M. Moore, M. Mustain, I. Chen, S. Safe, T. Zacharewski, B. Gillesby, A. Joyeux and P. Balaguer, Toxicol. Appl. Pharmacol., 1997, 145, 111. E. Routledge and J. Sumpter, J. Biol. Chem., 1997, 272, 3280. S. Jobling, D. Sheahan, J. A. Osborne, P. Matthiessen and J. P. Sumpter, Environ. Toxicol. Chem., 1996, 15, 194. S. Jobling, T. Reynolds, R. White, M. G. Parker and J. P. Sumpter, Environ. Health Perspect., 1995, 103, 582. C. Harris, P. Henttu, M. Parker and J. Sumpter, Environ. Health Perspect., 1997, 105, 802. N. Ben-Jonathan and R. Steinmetz, Trends Endocrinol. Metab., 1998, 9, 24. J. Gould, L. Leonard, S. Maness, B. Wagner, K. Conner, T. Zacherewski, S. Safe, D. McDonnell and K. Gaido, Mol. Cell. Endocrinol., 1998, 142, 203. G. Kuiper, J. Lemmen, B. Carlsson, J. Corton, S. Safe, P. Van der Saag, B. Van der Burg and J.-A. Gustafsson, Endocrinology, 1998, 139, 4252. P. Sohoni and J. Sumpter, J. Endocrinol., 1998, 158, 327. S. Nagel, F. Vom Saal, K. Thayer, M. Dhar, M. Boechler and W. Welshons, Environ. Health Perspect., 1997, 105, 70. P. Gibbs, P. Pascoe and G. Bryan, Comp. Biochem. Physiol., 1991, 100C, 231. J. Oehlmann, U. Schulte-Oehlmann, E. Stroben, B. Bauer, C. Bettin and P. Fiorni, in Endocrinologically Active Chemicals in the Environment, Umweltbundesamt, Berlin, 1996, pp. 111—118.

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Table 2 Some Receptor In vitro environmental chemicals Chemical binding action In vivo action with the ability to mimic Organochlorine pesticides hormones in vitro and in DDT metabolites vivo o,p-DDT ERa,ERb Agonist Uterotrophic—ER agonist p,p-DDE AR Antagonist Effects on androgen regulated endpoints—AR antagonist Methoxychlor ERa,ERb Agonist ER agonist Fungicides Vinclozolin AR Antagonist Effects on androgen regulated endpoints—AR antagonist Industrial chemicals (a) Polychlorinated biphenyls Over 200 congeners ERa,ERb Agonist/ Agonist and antagonist antagonist effects on rodent uteri 3,4,3,4- Tetrachlorobiphenyl ERa Agonist Uterotrophic—agonist (b) Alkylphenolic compounds Nonylphenol ERa, Agonist Induces vitellogenin ERb , expression in fish—ER AR agonist Octylphenol ERa, ERb Agonist As for nonylphenol (c) Alkyl hydroxy benzoate Preservatives (parabens) ERa Agonist Uterotrophic—ER agonist (d) Phthalate esters Butyl benzyl phthalate ERa Agonist AR Antagonist Di-n-butyl phthalate ERa Agonist Effects on androgen AR Antagonist regulated endpoints—AR antagonist? (e) Bisphenol-A ERa, Partial Uterotrophic—ER ERb agonist agonist AR Antagonist Partial ER agonist Food additives Butylated hydroxyanisoleERa Agonist Phytoestrogens Isoflavones: genistein ERb,ERa Agonist Induces vitellogenin expression, Uterotrophic—ER agonist Coumestans: coumesterolERb,ERa Agonist Induces vitellogenin expression, Uterotrophic—ER agonist

103 K. J. Turner described in alligators, panthers and black bears. The incidence of cryptorchidism has increased in both panthers and black bears. Furthermore, many Florida panthers are sterile as their semen quality is so poor. It has been suggested that they may also be feminised as their serum oestradiol levels are comparable to those seen in female panthers. Environmental contaminants such as the organochloride compounds, p,p-DDE and methoxychlor, as well as mercury, have been implicated as possible factors involved in the reproductive impairment. Reproductive abnormalities have also been described in alligators at Lake Apopka which became heavily polluted with dicofol and sulfuric acid in the 1980s. Juvenile male alligators were found to have lower testosterone levels and smaller genitalia in comparison to alligators in less contaminated lakes, and as a consequence there has been a population decline. Initially, p,p-DDE was implicated in reproductive abnormalities observed in Lake Apopka alligators, but it now seems more likely that a variety of reproductive toxicants are involved. In the UK, it has been well documented that exposure of male fish to industrial effluent or sewage effluent increases the incidence of hermaphroditism and induces a massive elevation in plasma vitellogenin levels, an oestrogen-inducible protein normally only expressed in female fish. The presence of oestrogenic chemicals in the aquatic environment is widespread, as demonstrated by surveys on the effects of placing caged male trout in various sewage effluent outfalls as well as on wildfish in rivers throughout the UK. Originally, it was thought that exposure to alkylphenols in effluent was responsible for inducing oestrogenic effects, but it now appears most likely that the main source of oestrogenic activity is due to the presence of natural oestrogens (17b-oestradiol, estrone, estriol) and synthetic oestrogen (ethinyloestradiol) excreted by humans which accumulate in sewage. Field studies have described reproductive abnormalities in other species, but for the most part they all involve environments which are known to be heavily contaminated with a variety of chemicals. So far, there is only limited evidence which establishes a link between an individual chemical and disruption of reproductive function in wildlife.

L. J. Guillette Jr., D. Pickford, D. Crain, A. Rooney and H. Percival, Gen. Comp. Endocrinol., 1996, 101, 32. C. Facemire, T. Gross and L. J. Guillette Jr., Environ. Health Perspect., 1995, 103 (suppl. 4), 79. M. Dunbar, M. Cunningham, J. Wooding and R. P. Roth, J. Wildl. Dis., 1996, 32, 661. L. J. Guillette Jr., T. Gross, G. Masson, J. Matter, H. Percival and A. Woodward, Environ. Health Perspect., 1994, 102, 680. J. Semenza, P. Tolbert, C. Rubin, L. J. Guillette, Jr. and R. Jackson, Environ. Health Perspect., 1997, 105, 1030. J. Harries, D. Sheahan, S. Jobling, P. Matthiessen, P. Neall, E. Routledge, R. Rycroft, J. Sumpter and T. Tylor, Environ. Toxicol. Chem., 1996, 15, 1993. S. Jobling, M. Nolan, C. Tyler, G. Brighty and J. Sumpter, Environ. Sci. Technol., 1998, 32, 2498. C. Desbrow, E. Routledge, G. Brighty, J. Sumpter and M. Waldock, Environ. Sci. Technol., 1998, 32, 1549. E. Routledge, D. Sheahan, C. Desbrow, G. Brighty, M. Waldock and J. Sumpter, Environ. Sci. Technol., 1998, 32, 1559.

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Phytoestrogens Phytoestrogens are present in plants (soya, beans, grains, vegetables and fruit) and are consumed by both man and animals. They are structurally and functionally similar to oestradiol and there are three main types: isoflavones, coumestans/lignans and mycoestrogens. Unlike many of the chemicals, phytoestrogens do not bioaccumulate in body fat and are readily metabolised. However, they are considerably more potent oestrogenically. Interestingly, there are data which suggest that some phytoestrogens, for instance genistein, may have a stronger binding affinity for ERb than ERa. Since soy-derived protein is being used increasingly in processed foods in the West and approximately 60% of such foods are thought to contain soy derivatives, human intake could be substantial especially for Orientals and vegetarians whose diet will contain higher quantities of phytoestrogens. There is some evidence from a number of animal species that phytoestrogen consumption can interfere with reproductive development and function. At present there are only meagre data on the effects of exposure to phytoestrogens during gestation or neonatal life on the male reproductive tract in animal models. Only a high dose of genistein (500 mg/kg bodyweight) was found to have a permanent oestrogenic effect on the mouse urethroprostatic complex when administered during the neonatal period. Neonatal treatment of rats with 4 mg/kg/day genistein (this dose is equivalent to a baby’s daily intake from soy-formula milk; see below) had no effect on testis weight in adulthood (R. M. Sharpe, unpublished data). It is possible that phytoestrogens may be able to act as anti-oestrogens in some situations, as a study in mice found that a soy-rich diet could reduce the severity of the effects induced on the prostate by neonatal DES treatment. In humans, it has been shown that a high soy-containing diet can mimic oestrogen by prolonging the follicular phase of the menstrual cycle in women by suppressing FSH secretion. A recent study found that babies who were fed a 100% soy-formula milk diet had blood concentrations of isoflavenoids approximately 1000 times higher than Asian infants who were breast-fed by mothers on a soy-rich diet. This level of phytoestrogens is 10-fold higher than needed to suppress FSH in adult women. However, it is not known if FSH levels in these infants were suppressed and if there was any effect on Sertoli cell number. The future health implications for babies fed on soy-formula milk diets remains to be clarified.

8 Are Humans at Risk? It is biologically plausible that exposure to a sufficient level of an oestrogenic chemical could cause harmful biological effects. However, it is far more difficult to establish that environmental oestrogens are responsible for adverse effects on

R. Kaldas and C. J. Hughes, Reprod. Toxicol., 1989, 3, 81. R. Santti, S. Ma¨kela¨, L. Strauss, J. Korkman and M.-L. Kostian, Toxicol. Ind. Health, 1998, 14, 223. L. Strauss, S. Ma¨ kela¨ , S. Joshi, I. Huhtaniemi and R. Santti, Mol. Cell. Endocrinol., 1998, 144, 83. A. Cassidy, S. Bingham and K. Setchell, Am. J. Nutr., 1994, 60, 333. K. Setchell, L. Zimmer-Nechemias, J. Cai and J. Heubi, Lancet , 1997, 350, 23.

105 K. J. Turner male reproduction. At present, there is no direct evidence to demonstrate such a cause and effect relationship in humans. In order to allow an unequivocal asssessment of whether humans are at risk from endocrine disrupters, data are needed to establish the routes and types of exposures, the level of exposure throughout life and in vivo bioactivity of individual and complex mixtures of chemicals. Obtaining this information will be extremely difficult. The general presumption for wildlife is that exposure to environmental oestrogens is via food and water. Whether the same is true for humans is less certain. Food as well as containers for foodstuff and drinks may be important sources of some environmental oestrogens such as phytoestrogens, phthalate esters and bisphenol-A. There may be other more important routes of human exposure to such chemicals from substances such as the many lotions, cosmetics, detergents and shampoos that we now apply to our skins daily. Recent data suggest that that these products are likely to be oestrogenic, as they contain alkyl hydroxybenzoate preservatives. Humans are exposed to a variety of endocrine disrupting chemicals throughout their lifetime; establishing the level of exposure accurately presents a difficult task. Furthermore, there is the possibility of additive effects of chemicals in vivo, since this is clearly evident in vitro. Further information on oestrogenic chemicals and routes of exposure has been published. Most environmental oestrogenic chemicals have been identified using in vitro screening systems (such as human breast cancer cells, fish hepatocytes or transfected yeast cells). There is much less data on their bioactivity in vivo and whether they are able to exert effects at environmentally relevant concentrations. It is argued that humans are unlikely to be exposed to sufficient levels of oestrogenic chemicals to cause harm. Some recent work suggests that in vitro potency of a single chemical may bear no resemblance to actual potency in vivo. Bisphenol-A and octylphenol have been shown to be weak oestrogens by a variety of screening methods. However, there is some doubt as to whether these methods provide an acurate prediction of in vivo bioactivity. Serum contains steroid binding proteins which alter the availability of free steroids to bind to receptors. There is little information on whether these proteins have the same capacity to bind environmental oestrogens as they would endogenous oestradiol. The inclusion of serum in an in vitro oestrogenicity assay suggested that bisphenol-A would be more oestrogenic and octylphenol less oestrogenic than previously predicted. This was confirmed in vivo; administration of 2 or 20 kg/kg of bisphenol-A to pregnant mice resulted in enlarged prostates in the adult male offspring. A previous study had demonstrated that a small increase in fetal serum oestradiol levels could induce a permanent 25% increase in prostate

R. Kavlock, G. Daston, C. DeRosa, P. Fenner-Crisp, L. Gray, S. Kaattari, G. Lucier, M. Luster, M. Mae, C. Maczka, R. Miller, J. Moore, R. Rolland, G. Scott, D. Sheehan, T. Sinks and H. Tilson, Environ. Health Perspect., 1996, 104 (suppl. 4), 715. J. Ashby, E. Houthoﬀ, S. Kennedy, J. Stevens, R. Bars, F. Jekat, P. Campbell, J. Van Miller, F. Carpanini and G. Randall, Environ. Health Perspect., 1997, 105, 164. E. J. Routledge, J. Parker, J.Odum, J. Ashby and J. P. Sumpter, Toxicol. Appl. Pharmacol., 1998, 153, 12.

106 Oestrogens, Environmental Oestrogens and Male Reproduction size in adult life. The extreme sensitivity of the fetus to its hormonal environment is further illustrated by studies in mice demonstrating that intrauterine fetal position can influence male sexual behaviour and androgen responsiveness. In addition, exposure of pregnant rats during pregnancy and lactation to 1 mg/l of an oestrogenic chemical (octylphenol, butyl benzyl phthalate) via the drinking water is sufficient to induce a small but significant decrease in testis weight in the adult offspring. However, as a note of caution, there is growing awareness that it may be difficult to reproduce endocrine disruption data using weakly active compounds between different laboratories or even between different studies. Without good data on effects in vivo of the suspect chemicals in animals and parallel information on the extent of human exposure, it will be impossible evaluate risk to man. Other obstacles to risk assessment are the difficulties associated with investigating the additive effect of several compounds in vivo and the absence of definitive biological endpoints of oestrogen exposure in males. More basic knowledge is required about oestrogen action and its effects on the body. It is already becoming quite clear that different tissues will have different sensitivities to oestrogen exposure.

9 Endocrine Disruption—Concluding Remarks It has been known for a few years that DDT has both oestrogenic (o,p-DDT) and anti-androgenic (p,p-DDE) isomers. New data demonstrate that a number of chemicals identified as being oestrogenic (some of the phthalates and alkylphenols) may also be anti-androgenic in vitro. Furthermore, there is in vivo evidence that exposure of rats to high doses of di-n-butyl phthalate (DBP; 250—750 mg/kg/day) throughout gestation and lactation results in abnormalities of the male reproductive tract, most likely through an anti-androgenic rather than oestrogenic mechanism. DBP exposure affected various processes known to be regulated by testosterone, resulting in reduction of anogenital distance, hypospadias, underdeveloped or absent epididymides, testicular maldescent and atrophy as well as under development of the seminal vesicles and prostate, but with little effect on the female offspring. These observations are strongly reminiscent of the effects of prenatal exposure to the anti-androgen flutamide (see Mylchreest et al. for references) and the fungicide vinclozolin, which also acts as an androgen receptor antagonist. Data have been presented at scientific meetings which indicate that several phthalates possess anti-androgenic activity and can induce effects in vivo at levels which begin to approach levels of human intake (R. M. Sharpe, personal communication). Furthermore, both oestradiol and DES have F. Vom Saal, B. Timms, M. Montano, P. Palanza, K. Thayer, S. Nagel, M. Dhar, V. Ganjam, S. Parmigiani and W. Welshons, Proc. Natl. Acad. Sci. USA, 1997, 94, 2056. D. Nonneman, V. Ganjam, W. Welshons and F. Vom Saal, Biol. Reprod., 1992, 47, 723. R. M. Sharpe, K. J. Turner and J. P. Sumpter, Environ. Health Perspect., 1998, 106, A220. J. Ashby and B. Elliott, Regul. Toxicol. Pharmacol., 1997, 26, 94. E. Mylchreest, R. Cattley and P. Foster, Toxicol. Sci., 1998, 43, 47. L. Gray, J. Ostby and W. Kelce, Toxicol. Appl. Pharmacol., 1994, 129, 46. W. Kelce, E. Monosson, M. Gamcsik, S. Laws and L. E. Gray Jr., Toxicol. Appl. Pharmacol., 1994, 126, 276.

107 K. J. Turner some affinity for the androgen receptor. It now appears that the emphasis on chemicals acting via an oestrogenic mechanism as a risk to human health may have been misleading. Many of the effects on the male reproductive system that have been described could also be caused by compounds acting as antiandrogens. This illustates the complex nature of endocrine disruption and how easy it is to draw premature conclusions from in vitro data. It will be of extreme importance to establish the range of potential agonist and antagonist activities a compound may have when binding to steroid receptors both in vitro and in vivo. It is well established that processes involved in differentiation of the male reproductive tract and masculinisation of genitalia during fetal and neonatal life are controlled by hormones and constitute a vulnerable period of development. It is obvious that any disruption of these processes could have serious implications for future fertility. A variety of data is accumulating which suggests that male reproductive health is declining, some of which is widely accepted (the testis cancer data) and some of which remains controversial (semen quality data). At present the only sensible (and most cautious) option is to treat the situation with concern and to perform prospective studies to establish whether men really are at risk. However, this will not give an answer for another 10—20 years, by which time male reproduction may be severely compromised. In the meantime, studies must continue to address whether endocrine disrupters present in the environment are capable of adversely affecting the health of humans and wildlife. It remains a plausible hypothesis that inappropriate exposure to oestrogen during development could have permanent consequences which might impair fertility. However, this is only one mechanism by which the endocrine system may be perturbed and investigation of other mechanisms should not be neglected, as any compound which can mimic hormone action either as an agonist or antagonist, or interfere with the biosynthesis or metabolism of both androgen and oestrogen and their receptors, has the ability to be an endocrine disruptor.

10 Acknowledgements I am especially grateful to Richard Sharpe for his constructive comments on this article. I would also like to thank Philippa Saunders, John Sumpter, Jane Fisher and Stewart Irvine for their helpful discussions and for allowing me to include some of their unpublished data. Thanks to Ted Pinner for help with the ﬁgures. This work was supported by a Zeneca Strategic Research Fund Award.

K. Gaido, L. Leonard, S. Lovell, J. Gould, D. Babai, C. Portier and D. McDonnell, Toxicol. Appl. Pharmacol., 1997, 143, 205. R. Gellert, W. Heinrichs and R. Swerdloﬀ, Endocrinology, 1972, 91, 1095. W. Kelce, C. Lambright, L. E. Gray Jr. and K. Roberts, Toxicol. Appl. Pharmacol., 1997, 142, 192. L. E. Gray Jr., J. Ostby, J. Ferrell, G. Rehnberg, R. Linder, R. Cooper, J. Goldman, V. Slott and J. Laskey, Fund. Appl. Toxicol., 1989, 12, 92. K. Nesaretnam, D. Corcoran, R. Dils and P. Darbre, Mol. Endocrinol., 1996, 10, 923. R. White, S. Jobling, S. Hoare, J. Sumpter and M. Parker, Endocrinology, 1994, 135, 175. J. Ashby and H. Tinwell, Environ. Health Perspect., 1998, 106, 719. R. Santell, Y. Chang, M. Nair and W. Helferich, J. Nutr., 1997, 127, 263. K. Medlock, W. Branham and D. Sheehan, Proc. Soc. Exp. Med., 1995, 208, 313.

108 Human Health Eﬀects of Phytoestrogens

PHILIP HOLMES AND BARRY PHILLIPS

1 Introduction Phytoestrogens are naturally occurring plant compounds that may be defined on the basis of their structural and functional similarity to 17b-oestradiol or their ability to elicit oestrogenic and/or anti-oestrogenic effects in animals. The phytoestrogens fall into three main chemical classes, the isoflavones, coumestans and lignans, although it must be stressed that while a large number of chemicals from these classes have been identified in plants, only relatively few have been demonstrated to possess oestrogenic activity. Phytoestrogens occur in many plants, including those used in human foodstuffs. Isoflavones occur predominantly in legumes and beans, coumestans in germinating beans and fodder crops, while lignans occur widely in cereals, fruit and vegetables. Other oestrogenically active compounds, such as the resorcylic acid lactones, may be introduced into foodstuffs through contamination by moulds or fungi. However, these so-called mycoestrogens are outside the scope of this review. Scientific interest in the hormonal effects of phytoestrogens arose in the 1940s from breeding problems in sheep in Western Australia. The syndrome, termed Clover Disease, was characterised by cystic ovaries, irreversible endometriosis and a failure to conceive, and was attributed to ingestion of subterranean clover (Trifolium subterraneum) which was found to contain high levels of isoflavone. The phytoestrogens may perform various functions in plants including acting as anti-fungal agents, pigments or as precursors for lignification. They may also act as defensive agents against herbivores through their hormonally mediated effects on the reproductive functions of animals.

D. C. Knight and J. A. Eden, Maturitas, 1995, 22, 167. H. W. Bennetts, E. J. Underwood and F. L. Shier, Aust. Vet. J., 1946, 22,2. R. B. Bradbury and D. E. White, Vitam. Horm., 1954, 12, 207. H. Naim, B. Gestetrer, S. Zilkah, Y. Bisk and A. Bondi, J. Agric. Food Chem., 1974, 22, 806. S. Clevenger, Sci. Am., 1964, 210, 84. C. M. Francis and I. D. Hume, Aust. J. Biol. Sci., 1971, 24,1. C. L. Hughes Jr., Environ. Health Perspect., 1988, 78, 171.

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

109 P. 110 omsadB. and Holmes Phillips

Figure 1 Comparison of structure of the principal phytoestrogens and 17b-oestradiol Human Health Eﬀects of Phytoestrogens

The structures of important phytoestrogens are shown in Figure 1. The principal isoflavone phytoestrogens are daidzein and genistein, their glucosides daidzin and genistin, and their methyl ether precursors formononetin and biochanin A. These precursors are converted to daidzein and genistein by intestinal glucosidases while daidzein may itself be further metabolised to equol and O-desmethylangolensin. The coumestans are structurally similar and biosynthetically related to the isoflavones; coumestrol and 4-methoxycoumestrol are of greatest interest. Lignans contain a 2,3-dibenzylbutane structure and exist as minor plant constituents in many species, where they form precursors to lignin in the cell walls. Lignans were first identified in humans after observations in monkeys of cyclical patterns in urinary excretion of compounds having mass spectral similarities to urinary steroid hormone metabolites. The lignans of particular significance to humans are enterolactone and enterodiol, which are synthesised by the gut microflora from their plant precursors matairesinol and secoisolariciresinol. The pharmacokinetics of phytoestrogens are complex. Absorbed phytoestrogens or their metabolites undergo enterohepatic circulation (excretion into the bile, followed by deconjugation by gut microflora, reabsorption and reconjugation in the liver) and may ultimately be excreted in the urine. The amount of phytoestrogens to which an individual is exposed will depend upon the types and amounts of food they consume, and the composition of the foodstuffs. The level of the various phytoestrogens in foodstuffs varies widely (e.g. soy products tend to be high in isoflavones, alfalfa contains high levels of coumestrol, and celery, linseed oil and onions contain lignans and flavonoids). The levels present depend not only on the genetic constitution of the plants used, but also on external factors during the growing of the plants (e.g. agricultural practice and environmental conditions) and post-harvest storage. In addition, subsequent food processing practices may have a profound influence on the levels occurring in the final foodstuff. Overall, daily intakes of phytoestrogens can vary markedly. For example, in Japan, intakes appear at least 30 times greater than in the UK.— Increasingly, the suggested health benefits of phytoestrogens are giving rise to the marketing of various ‘health’ supplements and drinks, including the proposed sale of tablets of isoflavone extracts as a ‘natural’ hormone replacement therapy. Such products would be expected to increase exposures to the phytoestrogens, at least among certain population subgroups. There is compelling evidence that both short- and long-term differences in diet result in alterations in the levels of phytoestrogens present in humans. Analysis of

K. R. Price and G. R. Fenwick, Food Addit. Contam., 1985, 2, 73. A. L. Murkies, G. Wilcox and S. R. Davis, J. Clin. Endocrinol. Metab., 1998, 83, 297. K. D. R. Setchell and H. Aldercreutz, in Role of the Gut Flora in Toxicity and Cancer, ed. I. R. Rowland, Academic Press, San Diego, 1988, pp. 315—345. J. T. Dwyer, B. R. Goldin, N. Saul, L. Gualtieri and H. Adlercreutz, J. Am. Diet. Assoc., 1994, 94, 739. K. Reinli and G. Block, Nutr. Cancer, 1996, 26, 123. Statement by the Committee of Toxicity of Chemicals in Food, Consumer Products and the Environment on phytoestrogens, Food Surveillance Paper No. 57, MAFF, London, 1996, pp. 59—81. A. Cassidy, S. Bingham and K. D. R. Setchell, Am. J. Clin. Nutr., 1994, 60, 333. M. Messina, Am. J. Clin. Nutr., 1995, 62, 645. B. J. Wilcox, K. Fuchigami and D. C. Wilcox, Am. J. Clin. Nutr., 1995, 61, S901. S. A. Bingham, C. Atkinson, J. Liggins, L. Bluck and A. Coward, B. J. Nutr., 1998, 79, 393.

111 P. Holmes and B. Phillips plasma, urine or faecal samples from individuals who normally consume different diets (e.g. omnivores versus vegetarians), or from participants in short-term dietary modification studies, has demonstrated effects on plasma concentrations and the urinary and faecal excretory rates of both phytoestrogens and their metabolites.— The differences in diet may also be reflected in variations in the composition of the gut microflora which could, in turn, influence the bioavailability and metabolism of important and potentially active dietary constituents, or may influence other aspects of the host’s physiological status (e.g. by altering bile flow). However, the situation is complex since considerable inter-individual variation has been found in terms of phytoestrogen levels in various physiological matrices, gut microfloral composition and ability to metabolise the phytoestrogens.— The biological effects of phytoestrogens on reproductive functions have been investigated in many animal species including cattle, cheetahs, mice, quail, rabbits and sheep. The overall effect in non-human animals appears relatively consistent and is that of depressed fertility; the effects appear more distinct in females than males. Potential sites of action include the genital tract, ovaries, pituitary and the central nervous system, with adverse effects on fertility mainly occurring in females. The presence of such effects in animals may give cause for concern over the consumption of phytoestrogens by humans. However, there is considerable epidemiological evidence to suggest that consumption of foodstuffs containing phytoestrogens may exert beneficial rather than harmful effects in humans, at least in adults. This relates particularly to protecting against several of the common hormone-dependent diseases, such as cancer of the breast (in women), endometrium and prostate, and cancers of the colon, rectum, stomach and lung, as well as some non-neoplastic conditions, for example osteoporosis, post-menopausal symptoms and cardiovascular disease. In addition, experimental work has suggested that some of these effects could be mediated through changes

X. Xu, H. J. Wang, P. A. Murphy, L. Cook and S. Hendrich J. Nutr., 1994, 124, 825. H. Adlercreutz, H. Markkanen and S. Watanabe, Lancet, 1993, 342, 1209. H. Adlercreutz, T. Fotsis, J. Lampe, K. Wa¨ha¨la¨ , T. Ma¨kela¨, G. Brunow and T. Hase, Scand. J. Clin. Lab. Invest., 1993, 53,5. S. M. Morton, G. Wilcox, M. L. Wahlqvist and K. Griﬃths, Endocrinology, 1994, 142, 251. M. Axelson, J. Sjovall, B. E. Gustafsson and K. D. R. Setchell, Endocrinology, 1984, 102, 46. A. M. Hutchins, J. L. Slavin and J. W. Lampe, J. Am. Diet. Assoc., 1995, 95, 545. A. M. Hutchins, J. W. Lamp. M. C. Martini, D. R. Campbell and J. L. Slavin, J. Am. Diet. Assoc., 1995, 95, 769. M. S. Kurzer, J. W. Lampe, M. C. Martini and H. Adlercreutz, Cancer Epidemiol. Biomark. Prev., 1995, 4, 353. M. S. Finegold, R. H. Attebery and L. V. Sutter, Am. J. Clin. Nutr., 1974, 27, 1456. W. E. C. Moore, E. P. Cato, I. J. Good and L. V. Holdeman, in Banbury Report Number 7: Gastrointestinal Cancer: Endogenous Factors, eds. W. R. Bruce, P. Correa, M. Lipkin, S. R. Tannenbaum and T. D. Wilkins, Cold Spring Harbor Laboratory, New York, 1981, pp. 11—-24. W. E. C. Moore and L. H. Moore, Appl. Environ. Microbiol., 1995, 61, 3203. K. D. R. Setchell, S. P. Borriello, P. Hulme, D. N. Kirk and M. Axelson, Am. J. Clin. Nutr., 1984, 287, 569. G. E. Kelly, G. E. Joannou, A. Y. Reeder, C. Nelson and M. A. Waring, Proc. Soc. Exp. Biol. Med., 1995, 208, 40. R. E. Chapin, J. T. Stevens, C. L. Hughes, W. R. Kelce, R. A. Hess and G. P. Daston, Fundam. Appl. Toxicol., 1996, 29,1.

112 Human Health Effects of Phytoestrogens in an individual’s hormonal profile associated with the oestrogenic activity of the phytoestrogens. This review briefly summarises the available evidence on the relative potency of the phytoestrogens, and discusses the evidence that beneficial effects on human diseases may potentially arise from consumption of foodstuffs containing these compounds, with particular regard to those effects that have been suggested as possibly being related to the hormone-receptor mediated activities of the phytoestrogens. Certain causes for concern regarding these compounds are also addressed. Before proceeding further, however, it must be appreciated that hormone-receptor mediated effects may not be the only, or the principal, explanation for the apparent influence of these chemicals (or of the foodstuffs rich in them) on incidences of certain disease conditions, because in addition to direct oestrogenic activity, phytoestrogens are known to have other important activities. For example, the major source of oestrogen in postmenopausal women is the aromatization (by the enzyme aromatase) of androstenedione to oestrone in peripheral tissues (e.g. adipose tissue), and inhibition of aromatase by some * phytoestrogens has been noted: enterolactone is a moderate inhibitor (I k 9 k value of 74 M), enterodiol a weak inhibitor (I 100 M), while coumestrol k has an I value of 17 M. Isoflavones have also been shown to have anticarcinogenic activity in vitro (through effects on inhibition of tyrosine kinases), to inhibit the DNA repair enzyme topoisomerase, to inhibit angiogenesis and cell cycle progression, to stimulate sex-hormone binding globulin (SHBG) synthesis (with consequent implications for hormone pharmacokinetics) and to show antioxidant and digitalis-like properties. It is also difficult to separate the effects of the phytoestrogens from those of the many other biologically active compounds present in plants. For example, linolenic acids, such a a-linolenic acid, occur in linseed and have been suggested to be responsible for its beneficial effects on reducing blood lipids and platelet aggregation. For soya, active constituents include protease inhibitors such as Bowman-Birk protease inhibitor (BBI) and Kunitz trypsin inhibitor: BBI can inhibit or prevent development of experimentally induced colon, oral, lung, liver and oesophageal cancers. Other active constituents of soya include phytosterols, saponins and inositol hexaphosphate (phytic acid). Phytosterols such as b-sitosterol appear to inhibit cholesterol absorption, and may also have anti-carcinogenic properties. A negative correlation has also been established between levels of inositol hexaphosphate (present in cereals, fruits and vegetables) and the incidence of colon cancer. This compound can chelate metals (e.g. calcium, zinc and iron) and this, particularly with regard to iron, may explain its action in reducing oxidant activity and inhibiting lipid peroxidation and experimentally induced colon cancer. Hexaphosphate may also be involved with regulating cell differentiation following dephosphorylation to inositol trisphosphate, an important intracellular second messenger.

* I: inhibitor concentration reducing V ! by 50%. C. Wang, T. Ma¨ kela¨, T. Hase, H. Adlercreutz and M. S. Kurzer, J. Steroid Biochem. Mol. Biol., 1994, 50, 205. M. Messina and S. Barnes, J. Natl. Cancer Inst., 1991, 83, 541.

113 P. Holmes and B. Phillips

2 Potency of the Phytoestrogens Oestrogenic activity has been demonstrated for phytoestrogens in animal models or in vitro test systems. However, data on relative potency are quite variable, and very dependent on the test model used and the specific end-point considered. Available evidence suggests that in vitro assays are capable of detecting biological activity at lower concentrations than the in vivo models, probably reflecting the ameliorating influence, in vivo, of factors such as absorption, distribution, protein binding, metabolism and degredation/ elimination. Ultimately, the physiological significance of exposure to a phytoestrogen will depend on the activity shown by the substance in the in vivo situation. Data on the relevant potencies are summarised in Table 1. Provisionally, the most potent appear to be the coumestans, particularly coumestrol. Isoflavones appear less active, with the highest activity in this class being shown by genistein and daidzein. There is a noticeable absence of data on potency for lignans. Interestingly, a recent study has shown that the oestrogen receptor binding activities of several phytoestrogens differ, depending on the oestrogen receptor subtype considered; the highest affinities appear to be shown for the b-subtype. This possibility needs to be investigated further since, given the varying distribution of receptor subtypes in the organs and tissues of the body, differential activity could have important implications for the potential susceptibility of the potential target tissues and processes suspected of being modulated by the phytoestrogens. A further factor of importance to the biological actions of phytoestrogens is their ability to bind to SHBG. This protein, present in the blood of mammals, is responsible for the sequestration of the physiologically present steroid hormones (i.e. the bodies natural hormones), and thus acts to limit their bioavailability and thereby modulates their actions. Thus, the low relative binding affinity of the phytoestrogens to SHBG suggests that they might be relatively more bioavailable than the endogenous hormones, which could enhance their relative impact on the intact organism.

3 Potential Beneficial Effects The evidence for potential beneficial effects on various human conditions and diseases arising from the consumption of dietary phytoestrogens has been extensively reviewed. The effects of these substances in various biological systems and during different phases of life have also been reviewed; for example, neonatal injection of rodent pups with genistein has been shown to reduce the incidence and multiplicity of mammary tumours arising from post-weaning treatment with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), suggesting a possible protective effect against chemical-induced neoplasia. Although lifestyle and changing socio-economic status can be expected to play a role, it appears that dietary practice may significantly influence the occurrence

G. G. J. M. Kuiper, J. G. Lemmen, B. Carlsson, J. C. Corton, S. H. Safe, P. T. van der Saag, B. van der Burg and J.-A. Gustafsson, Endocrinology, 1998, 139, 4252. A. Cassidy, Breast, 1996, 5, 389. D. C. Knight and J. A. Eden, Obstet. Gynecol., 1996, 87, 897.

114 Table 1 Summary of relative potencies of phytoestrogens (as % of 17b-oestradiol value)!

RBA to oestrogen receptor ROP in in vitro cell systems

Rat RBA ROP to Substance MCF-7 SheepRat ERA" ERB" ERA# ERB# Recept$ MCF-7 MCF-7 LeC- Ish in vivo SHBG 9 Natural/synthetic steroids Diethylstilboestrol 117.0 100 Oestrone 7.7 6.9 Coumestans Coumestrol 10 5 5 20 140 34 100 0.03 0.001 0.06 0.202 0.035 14 Isoﬂavones Daidzein 0.1 0.09 0.1 0.5 0.2 1 0.002 0.003 0.013 0.00075 Genistein 2 1/3 4 87 0.7 13 3.0 0.01 0.04 0.084 0.001 Daidzin 0.0012 Genistin 0.0048 27 Formononectin : 01 : 0.01 : 0.01 : 0.01 ND ND 0.001 0.00025 0.0006 0.00026 24 Biochanin A 0.07 : 0.01 : 0.01 ND ND 0.0012 0.00053 : 0.006 Equol 0.4 0.061 Angolensin 0.03 O-desmethylangolensin 0.05 Lignans Enterolactone Enterodiol

!RBA, relative binding affinity; ROP, relative oestrogenic potency; Ish, Ishikawa cell line; SHBG, sex hormone binding globulin; ERA, oestrogen receptor subtype a; ERB, oestrogen receptor subtype b;ND, none detected. "By solid phase competition assay. #By solubilized competition assay. $cross-reactivity assay. 115 P. Holmes and B. Phillips and progression of certain diseases within human populations. In particular, consumption of diets rich in phytoestrogens has been suggested as exerting a protective effect against a number of the common disease conditions affecting adult women and men. It is, however, important to note that the known inter-individual variations may result in subpopulations who are either sensitive or resistant to the effects of phytoestrogens, and that the effects may differ depending upon the stage of life of an individual at the time of exposure (i.e. whether during early developmental, pre-pubertal, reproductive or post- reproductive periods). For example, if phytoestrogens act as oestrogen mimics in postmenopausal women, they might be expected to exert effects similar to hormone replacement therapy, such as reducing the bone loss associated with osteoporosis and reducing the risk of cardiovascular disease, but not in earlier life. Historically, the incidence of various human cancers and other disease conditions has shown marked geographical variations. For example, rates of breast and prostate cancer in Asian countries are significantly lower than in the West. Although such reports must be treated with caution because of national differences in health services and diagnostic and recording practice, it appears that it may be lifestyle and environment, not genetics, that play the most significant roles in many of the common diseases. In particular, associations have been found between diet and cancers of the gastrointestinal tract and hormonally responsive tissues. Both breast cancer incidence and mortality also associate with diet, even after adjustment for differences in height, weight and age at menarche. Studies on differences between rural and urban populations in Japan (e.g. for gastric cancer) and changes in disease patterns in migrant populations (e.g. female breast cancer incidences in Asians moving to the USA) also provide strong supporting evidence for the influence of diet.

Eﬀects in Women Breast Cancer. Many studies have observed low incidences of hormone-dependent cancers, particularly breast cancer, in Asian countries compared with Western countries and it is becoming increasingly accepted that dietary factors play an important role. Although breast cancer can occur in either males or females, only about 1% of all cases occur in men, and male breast cancer is a rare disease in all parts of the world. Although there appear to be some similar risk factors for breast cancer in males and females, there is no indication in the literature that diet is either a risk or a protective factor for male breast cancer. The development of breast cancer is known to be highly dependent on the hormones associated with female reproductive functions, while established genetic factors have been

B. Armstrong and R. Doll, Int. J. Cancer, 1975, 15, 617. G. E. Gray, M. C. Pike and B. E. Henderson, Br. J. Cancer, 1979, 39, 1. Y. Tsubono, M. Kobayashi and S. Tsugane, Nutr. Cancer, 1997, 27, 60. R. G. Ziegler, R. N. Hoover, M. C. Pike, A. Hildesheim, A. M. Y. Nomura, D. W. West, A. H. Wu-Williams, L. N. Kolonel, P. L. Horn-Ross, J. F. Rosenthal and M. B. Hyer, J. Natl. Cancer Inst., 1993, 85, 1819. A. J. Sasco, A. B. Lowenfels, D. E. Pasker and P. Jong, Int. J. Cancer, 1993, 53, 538.

116 Human Health Effects of Phytoestrogens estimated only to account for approximately 4% of these cancers in Western populations. Events occurring premenopausally, and perhaps during adolescence, are known to influence the occurrence of this disease in postmenopausal women. Established risk factors include early menarche, late age at menopause, late age of first pregnancy and, in postmenopausal women, elevated plasma levels of free oestradiol. Several epidemiological studies have suggested that women either diagnosed as having breast cancer, or living in geographical areas associated with high risk of breast cancer, may have low intakes of lignans and isoflavones (as assessed by analysis of levels of these substances in their urine) when compared with either matched controls unaffected by breast cancer or with women living in areas with low risk of hormone-dependent cancers. More recently, a case-control study of women newly diagnosed with early breast cancer, and randomly selected controls, reported that high urinary phytoestrogen excretion was associated with substantial reductions in breast cancer risk. However, the ability of these types of study (in which women having diagnosed cancers are compared to apparently healthy women) to establish a causal relationship has been questioned because of the difficulty of ascribing biological relevance to phytoestrogen levels in subjects after the development of a disease condition when the important factor would be the levels that existed during the initial induction or subsequent progression of that condition. Given that the half-life of phytoestrogens in humans is of the order of hours, measurements of urinary levels after diagnosis cannot be assumed to reflect dietary practice over the many years required for the development of this disease. Studies specifically focused on the influence of soya consumption on cancer of the breast and other sites, published between 1957 and 1992, have been reviewed by Messina et al. The findings were quite variable for fermented and unfermented soya and soya-containing foods. For studies reporting specifically unfermented soya products, one showed a decreased breast cancer risk while two showed no association or a non-significant effect. In two studies reporting the effects of fermented soya products, one found a decreased risk and the other found either no association or a non-significant effect. In a study not included in that review, a negative association was found between intake of soya protein and total soya products and breast cancer risk in premenopausal but not postmenopausal women in Singapore. However, in a recent study of two Chinese populations in whom the intake of soya protein was reported to be similar to the population in the Singapore study, breast cancer risk was not related to intake of soya protein for either pre- or postmenopausal women. While there is limited evidence to suggest that consumption of diets high in fruit and vegetables is associated with a reduction in risk of breast cancer, the H. Adlercreutz, E. Ha¨ma¨la¨ inen, I. S. Gorbach, R. B. Goldin, N. M. Woods and T. J. Dwyer, Am. J. Clin. Nutr., 1989, 49, 433. D. Ingram, K. Sanders, M. Kolybaba, and D. Lopez, Lancet, 1997, 350, 990. C. D. N. Humfrey, Natural Toxins, 1998, 6, 51. P. Mangtani and S. I. dos Santos, Lancet, 1998, 351, 137. M. J. Messina, V. Persky, K. D. R Setchell and S. Barnes, Nutr. Cancer, 1994, 21, 113. H. P. Lee, L. Gourley, S. W. Duffy, J. Esteve, J. Lee and N. E. Day, Lancet, 1991, 337, 1197. J. M. Yuan, Q. S. Wang, R. K. Ross, B. E. Henderson and M. C. Yu, Br. J. Cancer, 1995, 71, 1353.

117 P. Holmes and B. Phillips overall epidemiological evidence that the phytoestrogens in the diet are protective in premenopausal women is inconclusive and there is no evidence for a protective effect in postmenopausal women. The original intention of most of the studies performed to date has been to identify possible associations between breast cancer risk and general dietary factors, rather than to investigate the specific effects on risk of the phytoestrogens present in the foodstuffs. As a result, levels of intake in these studies have been assessed in terms of various food items rather than as intake of specific phytoestrogens. Although some estimation of intake can be made from the particular food items consumed, it is perhaps not surprising that these have not been consistent, since even similar food types may differ quite markedly in phytoestrogen content. Other limitations, associated with the studies conducted on soya consumption, include the relatively small differences in soya intake, the inability to separate soya from other dietary variables and the inherent difficulty in relating estimated dietary intakes with the development of cancer several decades later. Epidemiological evidence links the consumption of foodstuffs containing phytoestrogens with physiological changes that would be expected to decrease breast cancer risk. For example, fibre intake has been correlated with delayed menarche in girls. More direct information comes from controlled dietary studies that have measured physiological change in response to modifications of the diet. In premenopausal women, dietary supplementation with linseed has been associated with lengthening of the luteal phase and altered progesterone—oestradiol ratios, although follicular phase and overall cycle lengths appeared unaffected. Soya has been shown to increase the length of the menstrual cycle and/or to delay menstruation, and to reduce the levels of LH, FSH and progesterone at various stages of the cycle. Reports of the effect of soya on blood 17b-oestradiol level are inconsistent: one study reported reduced levels throughout the cycle, another reported increases during the follicular phase only and a third detected no change in levels. Similarly, the evidence for changes in levels of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) is conflicting. Low levels of DHEAS have been reported in women with breast cancer and in populations at high risk of breast cancer (particularly in the case of premenopausal women), and positive correlations have been found between soya intake and DHEAS level in adolescent girls. In contrast, in a controlled dietary study, a negative correlation with DHEAS was shown with soya-milk consumption. Further data must be awaited to clarify the significance of the available evidence. It is, however, of note that plasma levels of DHEAS are known to vary with energy intake; for example, a recent study in premenopausal women found that for each additional 1 MJ (239 kcal) consumed, levels of

V. Persky and L. Van Horn, J. Nutr., 1995, 125, 709S. H. Adlercreutz, Environ. Health Perspect., 1995, 103 (suppl. 7), 103. W. R. Phipps, M. C. Martini, J. W. Lampe, J. L. Slavin and M. S. Kurzer, J. Clin. Endocrinol. Metab., 1993, 77, 1215. A. Cassidy, S. Bingham and K. D. R Setchell, Br. J. Nutr., 1995, 74, 587. L. J. W. Lu, K. E. Anderson, J. J. Grady and M. Nagamani, Cancer Epidemiol. Biomark. Prev., 1996, 5, 63. D. D. Baird, D. M. Umbach, L. Lansdell, C. L. Hughes, K. D. R Setchell, C. R. Weinberg, A. F. Haney, A. J. Wilcox and J. A. McLachlan, Clin. Endocrinol. Metab., 1995, 80, 1685.

118 Human Health Eﬀects of Phytoestrogens

DHEAS decreased by 5.1%. To ensure that this does not confound intervention studies, it would be sensible for future studies to utilise diets that are isocalorific. Both lignans and isoflavones appear to increase plasma SHBG levels. As noted earlier, this could reduce the availability of biologically active sex hormones and thus influence cancer risk. Again, however, the evidence is conflicting: lower levels of SHBG have been found in postmenopausal women with breast cancer than in vegetarians or omnivores. In contrast, another study found no difference between postmenopausal breast cancer patients and their controls. In controlled trials, in which lignans (as flax seed or linseed) or soya (as textured vegetable protein and miso) were added to the diets of premenopausal women, no significant changes were seen in SHBG. Overall, the available evidence suggests that phytoestrogens are biologically active in women and able to influence sex hormone levels, thereby potentially contributing to a reduced breast cancer risk. The effects seen have not always been consistent between studies, although this may reflect experimental design differences and the generally small numbers studied. In order to further elucidate the potential beneficial effects of phytoestrogens in breast cancer risk reduction, controlled studies in larger populations of premenopausal women are warranted.

Post-menopausal Symptoms. Reports of the incidence of hot flushes (one of the most common symptoms of the menopause) vary markedly between countries; levels are high in Europe (from 70—80% of postmenopausal women), intermediate in Malaysia (57%), and low in China (18%) and Singapore (14%). Several studies have investigated the possible role of soya product consumption in modulating these symptoms. In addition, many plants of the family Leguminosae (which contain high levels of isoflavones and their glucosides) have a long history of use by Chinese herbal doctors in the treatment of menopausal symptoms. The physiological basis for the apparent effects on post-menopausal symptoms is, however, unclear. A study in which postmenopausal women consumed a diet supplemented in turn for two-week intervals with soya flour, red clover sprouts or linseed did not appear to affect leuteinizing hormone (LH) or follicle stimulating hormone (FSH) levels at the end of each treatment phase, but showed a marginal cumulative effect on FSH levels over the entire study period. Also, evidence of oestrogenic activity was established by cytological examination of the vaginal epithelium. However, a control group was not included, and the findings have not been confirmed by other studies. A study of postmenopausal women reporting more than 14 hot flushes per week who consumed diet supplemented with soya flour or wheat flour for 12 weeks showed no effect on vaginal cell

J. F. Dorgan, M. E. Reichman, J. T. Judd, C. Brown, C. Longcope, A. Schatzkin, M. Forman, W. S. Campbell, C. Franz, L. Kahle and P. R. Taylor, Am. J. Clin. Nutr., 1996, 64, 25. H. Adlercreutz, Y. Mousavi, J. Clark, K. Ho¨ ckerstedt, E. Ha¨ma¨ la¨inen, K. Wa¨ha¨la¨ , T. Ma¨ kela¨ and T. Hase, J. Steroid Biochem. Mol. Biol., 1992, 41, 331. P. F. Bruning, J. M. G Bonfrerand and A. A. M. Hart, Br. J. Cancer, 1985, 51, 479. A. Cassidy, M. Faughnan, R. Hughes, C. Fraser, A. Cathcart, N. Taylor, K. D. R. Setchell and S. Bingham, Am. J. Clin. Nutr., 1998, (suppl.), 15315. L. L. Lien and E. J. Lien, J. Clin. Pharmacol. Therap., 1996, 21, 101. G. Wilcox, M. L. Wahlqvist, H. G. Burger and G. Medley, Br. Med. J., 1990, 301, 905.

119 P. Holmes and B. Phillips maturation, although the incidence of hot flushes was significantly reduced. Subjective assessment of menopausal symptoms also showed significant reductions with either treatment, despite the fact that urinary levels of daidzein, equol and enterolactone were significantly raised at the end of the study only in the soya flour group (although the wheat flour also contained phytoestrogens). However, a placebo effect cannot be discounted. In a further study, in which postmenopausal women consuming normal diets were compared with women on a diet supplemented with soya foods for four weeks, no significant differences in vaginal maturation index or levels of FSH, LH or SHBG were noted either between the groups or when comparing levels within a group before and after the intervention. This was despite an average 105-fold increase in urinary excretion of isoflavone phytoestrogens in the soya diet group and an average two-fold increase in the control group (not statistically significant). Serum oestradiol levels were slightly but not statistically significantly decreased in both groups over the course of the study. The possibility that the different sampling technique employed in this study might have falsely lowered the estimate of vaginal maturation has been suggested. In addition, the lack of information on achieved dosages makes it difficult to determine whether the apparent lack of effect was real or due to the achieved dosages actually being too low to elicit a detectable biological response. Certainly, inclusion of measurement of a biological marker of effect, such as a reduction in level of blood cholesterol, would have enabled any effect of the administered dose to be monitored more clearly. The current evidence suggests that there might be some beneficial effects on postmenopausal symptoms, but it is not possible to be more categorical at present given the conflicting observations in the limited number of studies undertaken and the possibility that placebo effect may explain the changes noted in the incidence of subjective symptoms.

Osteoporosis. Osteoporosis may be defined as the abnormal rarefaction of bone. As a result of an imbalance between formation and resorbtion of its mineral structure, the bone becomes porous and thus prone to structural failure and fracture. This potentially debilitating condition occurs most commonly in the elderly, particularly women. Depending on the extent of bone demineralisation, the condition may be accompanied by pain, deformity and pathological fractures. Interestingly, its incidence increases with duration of the menopause, and rates vary between different geographical areas. For example, the incidence in Asian women is lower than those in Western countries, and the risk of hip fracture is lower in Japanese women than in Caucasian women. Diet is one factor postulated to be involved in these differences. However, epidemiological data directly supporting a link between osteoporosis and dietary phytoestrogen intake is scant. Evidence for the possible role of phytoestrogens comes from a small number of animal and in vitro studies on phytoestrogens, and from studies on

A. L. Murkies, C. Lombard, B. J. G. Strauss, G. Wilcox, H. G. Burger and M. S. Morton, Maturitas, 1995, 21, 189. WHO, Report of a WHO Study Group Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (Technical Report Series 843), World Health Organization, Geneva, 1994.

120 Human Health Effects of Phytoestrogens ipriflavone (a synthetic isoflavone derivative, 7-isopropoxyisoflavone) on prevention of osteoporosis in postmenopausal women. Overall, data on humans indicate that ipriflavone is beneficial in maintaining bone density in postmenopausal women. This compound is believed to function by inhibiting bone resorbtion, thus improving bone density, rather than acting directly through oestrogen receptor-mediated mechanisms.— Ipriflavone has also been used successfully to prevent the rapid bone loss that occurs following induction of hypogonadism in premenopausal women treated with gonadotrophin hormone-releasing hormone agonists, further underlining its ability to operate via a non-hormonal mechanism. One of the main metabolites of ipriflavone in humans is daidzein, which constitutes approximately 10% of all its metabolites. While these findings cannot be directly extrapolated to the natural isoflavones, or indeed other phytoestrogens, they suggest a basis for further investigation. Experimental evidence that naturally occurring phytoestrogens could exert a moderately beneficial effect on osteoporosis comes from studies showing these substances to be biologically active in some relevant experimental models. Oestrogen receptors have been demonstrated in osteoblast cells, the cells responsible for laying down new bone. Additionally, oestrogens have a negative effect on the activity of osteoclast cells which are responsible for bone resorbtion and remodelling, and genistein has been shown to suppress osteoclastic activity both in vitro and in rats in vivo. The mechanism(s) of action is not clearly understood. Possible modes of action include interactions with the classical oestrogen receptor process, inhibition of receptor-mediated tyrosine kinase activation, or antioxidant effects. However, the recent discovery of a second human oestrogen receptor (ERb) with apparently different binding and activation characteristics may prove to have important implications in the interpretation of the relative importance of the various proposed mechanisms.

Effects in Men Prostate Cancer. Evidence that phytoestrogens can influence the incidence of male-specific diseases is restricted to prostate cancer, and is largely of an observational nature. In the UK, prostate cancer is the most common hormone-related cancer in men. However, like breast cancer in women, it is comparatively rare as a clinically evident disease in men living in Asian countries.

D. Agnusdei, S. Adami, R. Cervetti, G. Crepaldi, O. Di Munno, L. Fantasia, G. C. Isaia, G. Letizia, S. Ortolani, M. Passeri, U. Serni, L. Vecchiet and C. Gennari, Bone Miner., 1992, 19, S43. A. B. Kovacs, Agents Actions, 1994, 41, 86. G. B. Melis, A. M. Paoleti, A. Cagnacci, L. Bufalino, A. Spinetti, M. Gambacciani and P. Fioretti, Endocrinol. Invest., 1992, 15, 755. M. Petilli, G. Fiorelli, S. Benvenuti, U. Frediani, F. Gori and M. L. Brandi, Calcif. Tissue Int., 1995, 56, 160. M. Gambacciani, A. Spinetti, L. Piaggesi, B. Cappagli, F. Taponeco, P. Manetti, C. Weiss, G. C. Teti, P. Commare and V. Facchini, Bone Miner., 1994, 26, 19. M. L. Brandi, Bone Miner., 1992, 19 (suppl.), S3. J. J. B. Anderson and S. C. Garner, Nutr. Res., 1997, 17, 1617. H. C. Blair, S. E. Jordan, T. G. Peterson and S. Barnes, J. Cell. Biochem., 1996, 61, 629. G. G. J. M. Kuiper and J. A. Gustafsson, FEBS Lett., 1997, 410, 87.

121 P. Holmes and B. Phillips

Migrant populations moving from areas with low prostatic cancer incidences to areas of high incidence have been shown to suffer increased rates, approaching those of the ‘host’ country, suggesting that environmental factors may be involved aetiologically. For example, age-standardised rates of prostate cancer in different Japanese regions between 1983 and 1987 were between 6.6 and 10.0 per 100 000, while the rate among migrant Japanese in Hawaii was approximately four-fold higher at 34 per 100 000. In turn this is only about half the rate among white Hawaiians (63 per 100 000). It has been argued, however, that the incidence of this condition in Japan is actually much higher than it appears owing to regional differences in diagnostic practice. After adjustment for differences between USA and Japan in the proportion of latent prostate carcinoma and of localised tumours among all carcinomas of the prostate, the incidence rate was estimated as between 25 and 33 per 100 000 population. This would suggest that clinically significant prostate cancer may be over-diagnosed in the USA, and migration may not play as significant a role as previously reported. Notwithstanding this possibility, Japanese men have low mortality rates from prostatic cancer with small latent carcinomas only infrequently developing into clinically apparent disease. The high levels of dietary isoflavones and lignans consumed throughout the lifetime of Japanese men have been suggested as being protective against disease progression. Thus, it is suggested that high levels of isoflavones may influence the growth of cancer cells, slowing the development of the latent carcinomas. In Finland, despite a high fat intake, the incidence of prostate cancer is much lower than in the USA (although higher than in Japan), and this has been suggested to be due to the high production of lignans in their gut resulting from the relatively high intake of whole-grain products (particularly rye bread). Several studies have investigated the role of dietary factors in prostate cancer risk, but results appear inconsistent. Significant effects have not been detected for dietary soya products; certain vegetables, beans, fruit, rice and seaweed appear to be protective in some studies, while another has shown no protective effect from seaweed or vegetable consumption. In addition, a number of other risk factors have been shown to be associated with an increased risk of this cancer, including meat and dairy products and carotenoids. Evidence for effects of phytoestrogens on the hormonal profile of men is limited. Although a study of young men consuming dietary supplements of linseed (13.5 g/day) for six weeks showed marked increases in urinary levels of enterodiol and enterolactone, no significant effects were observed in plasma levels of total and free testosterone or SHBG. In a further experiment, daily dietary supplements of 60 g textured vegetable protein (i.e. soya, providing 45 mg/day

R. K. Severson, A. M. Y. Nomura, J. S. Grove and G. N. Stemmermann, Cancer Res., 1989, 49, 1857. IARC, Cancer incidence in Five Continents, Volume VI (IARC Scientiﬁc Publication No 120), International Agency for Research on Cancer, Lyon, 1992. H. Shimizu, R. K. Ross and L. Bernstein, Jpn. J. Cancer Res., 1991, 82, 483. H. Adlercreutz, Scand. J. Clin. Lab. Invest., 1990, 50, 3. T. Hirayama, Natl. Cancer Inst. Monogr., 1979, 53, 149. K. Oishi, K. Okada, O. Yoshida, H. Yamabe, Y. Ohno, R. B. Hayes and F. H. Schroeder, Prostate, 1988, 12, 179. T. D. Schultz, W. R. Bonorden and W. R. Seaman, Nutr. Res., 1991, 11, 1089.

122 Human Health Effects of Phytoestrogens isoflavones) for four weeks showed no significant changes in levels of leuteinising (LH) or follicle stimulating (FSH) hormones. However, middle aged men given a larger supplement of linseed (40 g/day providing 27 mg/day secoisolariciresinol) for four weeks showed significant reductions in FSH and LH levels. Mean levels of serum testosterone and dihydrotestosterone (DHT) did not change significantly and neither the DHT—testosterone ratio nor total urinary androgens were affected. With the limited human data currently available, a direct protective role for phytoestrogens on prostate cancer risk cannot be confirmed. However, it has been proposed that there is sufficient evidence to suggest that genistein and related compounds may be suitable for clinical intervention trials on the prevention of prostate cancer.

Other Possible Beneﬁts Phytoestrogens have been suggested as potentially protective against a number of other disease conditions which can aﬀect both men and women.

Cardiovascular Disease and Atherosclerosis. Coronary heart disease and other cardiovascular diseases are multifactorial conditions: risk factors include raised serum cholesterol, high blood pressure and smoking. The rates of coronary heart disease vary depending on geographical location, sex, age and dietary practice. Thus, the incidence of cardiovascular disease in general is lower in Asian than Western countries. For example, in 1986, age-standardised mortality rates for coronary heart disease for men and women (40—69 years old) in the USA were approximately 300 and 100 per 100 000 respectively and, in Japan, 50 and 15 per 100 000 respectively. Rates are also known to be lower in vegetarians than omnivores. These diseases contribute significantly to mortality. In the UK, for example, coronary heart disease accounts for 30% and 23% of mortality in men and women, respectively. The finding that the incidence of cardiovascular disease is lower for premenopausal women than men of similar age, but rises in women postmenopausally to a level approaching that in males, is suggestive of a hormonal influence on risk of this condition. This is further supported by the finding that oestrogen replacement therapy reduces the risk of cardiovascular disease. This is thought to occur as a result of lowering of low density lipoprotein (LDL) cholesterol levels and raising levels of high density lipoprotein (HDL) cholesterol, and through effects on the blood vessels. Indeed, a high level of LDL cholesterol is believed to be a causal factor in the development of coronary artery disease, and plasma LDL may be the primary source of the lipid that accumulates in arterial walls as part of the atherosclerotic process. It has been estimated that a 1% lowering of plasma cholesterol would result in a 2—3% reduction in population rates of coronary heart disease. Lignans inhibit the activity of cholesterol-7a-hydroxylase, the rate limiting enzyme in the formation

J. E. Karp, A. Chiarodo, O. Brawley and G. J. Kelloﬀ, Cancer Res., 1996, 56, 5547. R. Beaglehole, Epidemiol. Rev., 1990, 12,,1. J. N. Wilcox and B. F. Blumenthal, J. Nutr., 1995, 125 (suppl.), S631. Department of Health, Nutritional Aspects of Cardiovascular Disease, Report on Health and Social Subjects, no. 46, HMSO, London, 1994.

123 P. Holmes and B. Phillips of primary bile acids from cholesterol, and would thus be expected to decrease cholesterol catabolism, while oestrogen treatment has been shown to be one of the few effective methods of reducing lipoprotein (a), an independent risk factor for coronary disease. Such observations suggest that phytoestrogens might be cardioprotective. However, the potential influence of other dietary components must be recognised, such as the cardioprotective action of flavonoids from tea consumption, shown in Dutch men. Postulated mechanisms by which flavonoids might act include their action on platelet function (anti-aggregatory and inhibitory of cyclo-oxygenase activity), through free radical scavenging, or by effects on the vasculature. A number of in vitro studies have suggested other potential mechanisms by which phytoestrogens might be protective against oxidative events. Indeed, the isoflavones, being structurally similar to the flavonoids, are capable of acting as phenolic antioxidants, their activity depending on the number of hydroxyl groups and their positions and arrangement in the chemical structure. Dietary soya protein is generally considered anti-atherogenic when compared with animal proteins, although the role of phytoestrogens in this protective activity is unclear, with some researchers, such as Setchell, supporting the role of phytoestrogens and others suggesting that the amino acid composition is at least partly responsible. Dietary substitution of soya protein for animal protein, or addition of soya protein to the diet, is known to lower total and LDL cholesterol.— The effects appear variable but are generally greater in hypercholesterolemic subjects (changes ranging from 9 34% to ; 6%) than in those who are normocholesterolemic (changes ranging from 9 12% to ; 1%). Most of the hypocholesterolemic effect of the soya protein has been attributed to phytoestrogens, although this has recently been questioned. Soya protein diets have also been shown to reduce triglyceride levels, particularly in hypertriglyceridemic subjects, but appear to have little action on HDL cholesterol levels. Other recent studies of the effect on blood lipids of soya, miso, Arcon F (an isoflavone-free soyabean product) and linseed are summarised in Table 2. It is also of note that beneficial effects of soya on blood lipid profiles and atherosclerosis development have been reported in dietary manipulation studies using non-human primates. In vitro studies also suggest that genistein may interfere with aspects of the coagulation system thought to promote vascular lesion development. However, a diet modification study in men using soya D. M. Tham, C. D. Gardner and W. L. Haskell, J. Clin. Endocrinol. Metab., 1998, 83, 2223. M. G. L. Hertog, P. C. H. Hollman, M. B. Katan and D. Kromhout, Nutr. Cancer, 1993, 20, 21. M. B. Ruiz-Larrea, A. R. Mohan, G. Paganga, N. J. Miller, G. P. Bolwell and C. A. Rice-Evans, Free Radical Res., 1997, 26, 63. T. B. Clarkson, M. S. Anthony and M. S. Hughes Jr., Trends Endocrinol. Metab., 1995, 6, 11. K. D. R. Setchell, in Estrogens in the Environment, ed. J. A. McLachlan, Elsevier, New York, 1985, pp. 69—85. W. J. Erdman and J. E. Fordyce, Am. J. Clin. Nutr., 1989, 49, 725. K. K. Carroll, J. Am. Diet. Assoc., 1991, 91, 820. K. K. Carroll and E. M. Kurowska, J. Nutr., 1995, 125 (suppl. 3), 594S. W. J. Anderson, M. B. Johnstone and E. M. Cook-Newell, N. Engl. J. Med., 1995, 333, 276. S. M. Potter, Curr. Opin. Lipids, 1996, 7, 260. C. R. Sirtori, E. Gianazza, C. Manzoni, M. R. Lovati and P. A. Murphy, Am. J. Clin. Nutr., 1997, 65, 166.

124 Human Health Eﬀects of Phytoestrogens

Table 2 Summary of Subjects and dietary information! Effects Ref. investigations into the effect of soya or linseed on 15 healthy non-vegetarian women 52 blood lipid levels in men One control cycle (normal diet) then Mean cholesterol and women one cycle on 60 g TVP/day (45 mg/day levels significantly conjugated isoflavones, n : 6) decreased (by 9%) One control cycle (normal diet) then No significant changes one cycle on 28 g TVP/day (23 mg/day conjugated isoflavones, n : 6) One control cycle (normal diet) then No significant changes one cycle on 50 g miso/day (25 mg/day unconjugated isoflavones, n : 3) One control cycle (control diet) then Significant increases in one cycle on diet supplemented with LDL, total cholesterol 60 g Arcon F (n : 5) and LDL: HDL ratio" Pre- (n : 14) and post- (n : 10) No significant effect 121 menopausal caucasian women (aged on levels of plasma 29—58) cholesterol, HDL Normal ad libitum diet supplemented cholesterol or with 38 g soya protein/day (38 mg/day triglyceride genistein) for 6 months Postmenopausal women (n : 6) No significant effect 58 Diet supplemented with 60 g TVP/day on levels of total (45 mg/day isoflavones) for 4 weeks cholesterol, HDL or LDL Middle-aged men (n : 6) No significant effect 58 Diet supplemented with 60 g TVP/day on levels of total (45 mg/day isoflavones) for 4 weeks cholesterol, HDL or LDL Healthy young adults (5 male, 5 female, Plasma LDL 139 aged 25 < 3 years) cholesterol reduced by Diet supplemented with 50 g linseed/day up to 8% for 4 weeks Postmenopausal women (n : 7) Levels of total 139 Diet supplemented with 40 g linseed/day cholesterol, LDL and (27 mg/day secoisolariciresinol) for 6 HDL significantly weeks reduced Middle-aged men (n : 6) Levels of LDL 58 Diet supplemented with 40 g linseed/day significantly reduced (27 mg/day secoisolariciresinol) for 4 weeks !TVP, textured vegetable protein. "Possibly attributable to fatty acids in the margerine used to make biscuits containing Arcon F.

protein powder or casein supplement has detected no signiﬁcant eﬀects on platelet aggregation. Linseed oil has been shown to impact platelet composition and function in humans, but this has been attributed to the presence of high concentrations of a-linolenic acid rather than to the lignan content. M. J. Gooderham, H. Adlercreutz, S. T. Ojala, K. Wa¨ha¨la¨ and B. J. Holub, J. Nutr., 1996, 126, 2000. M. A. Allman, M. M. Pena and D. Pang, Eur. J. Clin. Nutr., 1995, 49, 169.

125 P. Holmes and B. Phillips

Thus, it is apparent that soya, some soya products and linseed oil influence blood lipid levels, particularly cholesterol and LDL cholesterol. While the extent of the reduction appears to largely depend on an individual’s initial serum cholesterol level, the maximum reductions observed are of the order of 10—15%. For hyperlipidemic individuals this may not be a marked reduction, but such an effect on the general population may well have a beneficial effect on the overall incidence of cardiovascular disease and atherosclerosis. The possibility that non-phytoestrogenic dietary components may contribute to the hypocholesterolemic properties cannot, however, be discounted. Indeed, certain types of dietary fibre have been shown to have a hypolipidemic effect via their ability to increase faecal excretion rates.

Colorectal Cancer. Colorectal cancer occurs frequently in the UK population but is historically rare in Asia. Rates in Japan have, however, increased rapidly in recent years. Interestingly, there appears to be an association between oestrogen exposure and colon cancer: risk has been shown to increase in women with increasing age of first live birth, and to decrease with increasing parity (number of children). In addition, many colon tumours express sex hormone receptors, and this is thought to play a part in development of the tumours. As noted above, geographical and temporal differences in the incidence of colon cancer are well established (see also Table 3). This has led to the suggestion that diet may play a key role, with low colon cancer incidences being linked with vegetarian or semi-vegetarian diets. For rectal cancer, the effects are less clear; incidence rates between 1981 and 1987 were similar for native Japanese women (6—9 per 100 000) and those in Hawaii (7 per 100 000), although rates were slightly higher in the Hawaiian-resident Japanese men (20 per 100 000 compared with 11—16 per 100 000 in Japan). The changes in incidence rates in male Japanese migrants have, however, been suggested to reflect the consequences of a move towards a more Western-type diet containing lower levels of soya products and phytoestrogens. It is also of note that the incidence of and mortality from colorectal cancer in Japan has increased such that the age-adjusted incidence now approaches that of the white population of the USA. This dramatic increase has been attributed to the adoption by many Japanese of a more Western-type diet and, in particular, to the increased amounts of fat and decreased amounts of fibre consumed. It is important to note that diet is a complex mixture that contain compounds with varying activity. Chemical stimulators of colon cancer growth include bile acids, 1,2-diglycerides and prostaglandins which stem from consumption of fat. In contrast, fruits and vegetables contain substances such as carotenoids, flavonoids and fibre, which may inhibit cancer cell growth, and the risk of colon cancer appears to be mirrored by the ratio of plant sterols to cholesterol in the

K. Tamura, S. Ishiguru, A. Munakata, Y. Yoshida, S. Nakaji and K. Sugawara, Cancer, 1996, 78, 1187. K. Furukawa, I. Yamamoto, N. Tanida, T. Tsujiai, M. Nishikawa, T. Narisawa and T. Shimoyama, Cancer, 1995, 75 (suppl.), 1508. B. Marian, Onkologie, 1996, 19, 132. P. P. Nair, N. Turjman, G. Kessie, B. Calkins, T. G. Goodman, H. Davidovitz and G. Nimmagadda, Am. J. Clin. Nutr., 1984, 40, 927.

126 Human Health Eﬀects of Phytoestrogens

Table 3 Incidence rates of White Hawaiians Native Japanese Japanese in Hawaii colon cancer (per 100 000) Period Men Women Men Women Men Women 1962—65 19 1978—81 8.1 6.7 34 19 1981—87 32 24 14—21 10—14 37 22

diet. Dietary fibre appears to be protective against colon cancer, possibly as a result of its high lignan levels. Indeed, excretion of urinary lignans (assumed to be a reflection of intake) is higher in subjects living in areas of low colon cancer risk. However, there have also been suggestions that the historically low risk of colon cancer in the Japanese might not be due to high dietary fibre intake; preparation of composite diets based on food consumption data for 1959, 1970 and 1979 suggest that average intakes of non-starch polysaccharides did not exceed 13 g per day, and were similar to intakes of the Scandinavians and the British, who have a high colon cancer risk. It thus appears that if any source of dietary fibre is protective against cancer, it is probably vegetables, although other factors may also play a role. Studies of the relationship between soya product consumption and colon cancer risk suggest that soya-based foods may exert a significant protective effect, with a relative risk of less than one being reported for three studies (although these failed to attain statistical significance); findings for the risk of rectal cancer were inconsistent. In a case-control study of subjects with colorectal cancer, a significant interaction was established between total fibre and total vegetables, with a protective effect only being shown for the high fibre and high vegetable combination. An independent protective effect of cruciferous vegetables (broccoli, brussel sprouts, cabbage, cauliflower, swede, turnip and kale) was suggested as possibly due to indoles present in these vegetables. However, the intake of cruciferous and leafy green vegetables was measured with the least reliability of all the variables studied and hence these findings should be interpreted with caution. The effects of fruit and vegetable consumption on colon cancer risk have also been investigated in a prospective cohort study of postmenopausal women in Iowa, USA, from 1986 to 1991; most associations between colon cancer risk and fruit/vegetable intake were weak and not statistically significant. Thus, although there is evidence that consumption of fruit and vegetables may be protective against colon and rectal cancer in humans, the role of phytoestrogens has yet to be fully defined. Some animal studies do suggest a protective role for some phytoestrogens. For example, linseed decreases some early markers of colon carcinogenesis (aberrant crypts and foci), owing, in part, to the presence of

M. Kuratsune, T. Honda, H. N. Englyst and J. H. Cummings, Jpn. J. Cancer Res.(Gann), 1986, 77, 736. L. R. Jacobs, Proc. Soc. Exper. Biol. Med., 1986, 183, 299. S. Kune, G. A. Kune and L. F. Watson, Nutr. Cancer, 1987, 9,5. S. Kune, G. A. Kune and L. F. Watson, Nutr. Cancer, 1987, 9, 21. K. A. Steinmetz, L. H. Kushi, R. M. Bostick, A. R. Folsom and J. D. Potter, Am. J. Epidemiol., 1994, 139,1.

127 P. Holmes and B. Phillips lignan precursors such as secoisolariciresinol diglycoside, while in vitro experiments have shown that genistein, but not daidzein, inhibits inducible nitric oxide synthase activity in human intestinal cell cultures (the expression and activity of this enzyme is up-regulated during inﬂammation and carcinogenesis in humans).

Other Diseases. Although lacking a definitive basis, it has been suggested that dietary practice, and hence potentially phytoestrogen intake, may play a modifying role in the incidence of a number of other disease conditions. Although mortality from stomach cancer has declined in recent years in Japan, the incidence in this country is still one of the highest. Age standardised rates in the USA between 1983 and 1987 (6—17 per 100 000 in males; : 3—8 per 100 000 in females) were markedly lower than in Japan (74—93 per 100 000 in males; 32—43 per 100 000 in females). A study of risk factors for gastric cancer among Japanese living in Hawaii has identified significant protective effects only for vegetables (mostly cabbage, lettuce and tomatoes) and fruits (mostly papayas, oranges, apples, mangoes and guavas). Increased risk was attributed to prior infection with Helicobacter pylori, cigarette smoking and low serum ferritin levels. A review by Messina et al. concluded that the evidence linking soya intake to altered risk of stomach cancer was inconsistent. Increased risks were noted more often with fermented soya products such as miso soup, while protective effects were observed more frequently with intake of unfermented products including beans, bean curd (tofu) and soya-milk. Thus, it is uncertain to what extent the apparent protective effects of fruit and vegetable consumption on risk of stomach cancer can be attributable to their phytoestrogen content. This appears not to have been studied directly, and other constituents such as ascorbic acid (vitamin C), a-tocopherol (vitamin E) and b-carotene may be potentially protective. In areas with cancer registries, the rates of endometrial cancer vary significantly, being highest in part of the USA (25 per 100 000) and lowest in Singapore and Japan (3 per 100 000). Reflecting the situation with breast cancer, there is an apparent association between the consumption of high quantities of soya products and a low incidence of endometrial cancer. However, no studies appear to have directly investigated associations between phytoestrogen intake and the risk of endometrial cancer. An inverse relationship between endometrial cancer risk and intake of green vegetables and fruit has been reported, although only a limited number of the potential modifying factors were considered in this study. Nonetheless, many case-control studies have shown that combined oral contraceptives protect against endometrial cancer and, in menopausal women,

M. Serraino and L. U. Thompson, Cancer Lett., 1992, 63, 159. M. Jenab and L. U. Thompson, Carcinogenesis, 1996, 17, 1343. A. L. Salzman, A. G. Denenberg, I. Ueta, M. O’Connor, S. C. Linn and C. Szabo, Am. J. Physiol. Gastroenterol. Liver Physiol., 1996, 270, G565. S. Tominaga, Cancer Chemother., 1995, 22,1. A. M. Y. Nomura, G. N. Stemmermann and P.-H. Chyou, Jpn. J. Cancer Res., 1995, 86, 916. IARC, Cancer Incidence in Five continents, Volume V, International Agency for Research on Cancer, Lyon, 1987. C. La Vecchia, A. Decarli, M. Fasoli and A. Gentile, Cancer, 1986, 57, 1248.

128 Human Health Effects of Phytoestrogens unopposed oestrogen replacement therapy can cause endometrial cancer, suggesting that hormonally active substances can indeed exert a modifying role in this disease. Similarly, several case-control studies have suggested that dietary factors may modify the risk of ovarian cancer, high consumption of animal fat and/or meat being associated with increased risk and increased vegetable consumption associated with decreased risk.— There is limited epidemiological evidence— that high intakes of fruit, vegetables and soya products might also be protective against lung cancer. However, the compounds involved are unknown. Similarly, some studies have investigated the association between soya or soya product consumption and cancers of the oesophagus, liver, bile duct and pancreas, but the very limited data have generally shown no significant effects.

4 Possible Causes for Concern Although, as previously noted, interest in phytoestrogens arose from the detection of adverse effects in a number of domestic and other species, there is a lack of evidence of obvious adverse effects in human populations that have traditionally consumed diets high in fruits or vegetables (e.g. the Asian countries). There are only isolated reports of adverse oestrogen-related effects, for example menstrual disorders in Dutch women who apparently consumed large quantities of tulip bulbs during the Second World War. However, even here the effects have not been established as being causally related to phytoestrogens. The human experimental and epidemiological evidence is generally supportive of the beneficial nature of a diet rich in foodstuffs containing phytoestrogens. Thus, it might at first sight be surprising that concerns have been expressed over the potential for phytoestrogens to cause adverse effects. In adults, a few areas may require further study. For example, there is a report of soya consumption causing an increased incidence of hyperplastic epithelial cells in the nipple aspirate fluid of pre- and postmenopausal women. This could constitute a risk factor for breast cancer. Also, the use in herbal medicine of particular plants emphasises that these species have the potential to cause physiological changes. Consequently, the increasing public interest in the use of herbal medicines could lead to unintended (adverse) effects, particularly as most

M. P. Vessey, J. Roy. Soc. Med., 1984, 77, 542. C. La Vecchia, A. Decarli, E. Negri, F. Parazzini, A. Gentile, G. Cecchetti, M. Fasoli and S. Franceschi, J. Natl. Cancer Inst., 1987, 79, 663. A. Engle, J. E. Muscat and R. E. Harris, Nutr. Cancer, 1991, 15, 239. M. Fukushima, M. Mori, M. Hara and R. Kudo, Tumor Res., 1993, 28, 1. H. A. Risch, M. Jain, L. D. Marrett and G. R. Howe, J. Natl. Cancer Inst., 1994, 86, 1409. R. Sankaranarayanan, C. Varghese, S. W. Duﬀy, G. Padmakumary, N. E. Day and M. K. Nair, Int. J. Cancer, 1994, 58, 644. Y. X. Lei, W. C. Cai, Y. Z. Chen and Y. X. Du, Lung Cancer, 1996, 14 (suppl. 1), S121. Y. X. Du, Q. Cha, X. W. Chen, Y. Z. Chen, L. F. Huang, Z. Z. Feng, X. F. Wu and J. M. Wu, Lung Cancer, 1996, 14 (suppl. 1), S9. J. B. Labov, Comp. Biochem. Physiol., 1977, 57,3. N. L. Petrakis, S. Barnes, E. B. King, J. Lowenstein, J. Wiencke, M. M. Lee, R. Miike, M. Kirk and L. Coward, Cancer Epidemiol. Biomark. Prev., 1996, 5, 785.

129 P. Holmes and B. Phillips herbal medicines have received little toxicological assessment. However, these potential problems should be balanced against the extensive experimental evidence suggesting a preventive (beneficial) role for phytoestrogens. It has been suggested that the timing of exposure to oestrogens is an important determinant of their effect. Thus, for example, prepubertal exposure potentially initiating precocious maturation of the breast terminal buds would afford subsequent protection against neoplastic development later in life, whereas increased postpubertal exposure to oestrogens in the absence of the early breast maturation (as a result of either significant phytoestrogen ingestion during childhood or full-term pregnancy) might increase the risk of breast cancer developing. There currently are some concerns regarding the potential effect that phytoestrogen exposure could have on the developing fetus and the neonate. The publication in 1993 of the ‘Sharpe—Skakkebaek’ hypothesis drew considerable public and media attention to the potential adverse effects that might be associated with exposure to hormonally active substances during early life. This hypothesis proposed that early exposure to oestrogens might, at least in part, explain the apparent trends noted for several hormone-dependent conditions such as an apparent decline in semen quality in some geographical areas, increases in male congenital abnormalities and increased testicular cancer rates. Other effects that have been suggested to be linked potentially to oestrogen exposure include increases in prostatic and breast cancer, polycystic ovaries and altered mental and physical development of children. Such considerations have given rise to concerns that harmful effects might result from exposure to high levels of phytoestrogens during the fetal period or neonatally from the milk in the case of mothers who themselves consume high levels of phytoestrogens, or from the feeding of infants with soya-based infant formula. It is of interest to note the apparent international differences in usage of soya-based formulae, with soya-formula constituting approximately 25% of the US infant formula market while only 2% of UK infants are so exposed. Adverse effects do not appear to have been detected in the children of Asian populations consuming diets high in phytoestrogens, despite evidence that children of women consuming traditional Japanese diets are exposed to high levels of phytoestrogens during pregnancy. For example, a recent study has found high levels of phytoestrogen metabolites in maternal cord blood and amniotic fluid of women consuming traditional Japanese diets (see Table 4), thus establishing that the high levels of phytoestrogens to which the mother is exposed when consuming a traditional Asian diet are normally transferred to the fetus. Isoflavones have also been found in mothers’ milk following consumption of roasted soyabeans or if eating a traditional Chinese diet. The possibility that feeding of soy-based infant milk could be harmful has been noted by Turner and D. M. Sheehan, Proc. Soc. Exp. Biol. Med., 1998, 217, 379. R. M. Sharpe and N. E. Skakkebaek, Lancet, 1993, 341, 1392. P. T. C. Harrison, P. Holmes and C. D. N. Humfrey, Sci. Total Environ., 1997, 205, 97. K. O. Klein, Nutr. Rev., 1998, 56, 193. Plant Oestrogens in Soya-based Infant Formulae, Food Surveillance Paper No. 167, MAFF, London, 1998. H. Adlercreutz, T. Yamada, K. Wahala and S. Watanabe, Steroids, 1997, 62, 733. A. A. Franke and L. J. Custer, Aust. J. Biol. Sci., 1996, 24,1.

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Table 4 Levels of Concentration (nmol/l) phytoestrogen found in the blood and amniotic fluid Phytoestrogen Maternal plasma Cord plasma Amniotic fluid of women consuming traditional Japanese diets Total isoflavinoids 232 (19—744) 299 (58—831) 233 (52—779) at time of giving birth Total lignans 14.6 (1—85) 3.5 (0.1—16) 15.1 (0.1—61)

Sharpe, who also quoted Setchell (at the 2nd International Symposium of Soy Foods and the Prevention of Chronic Disease, Brussels, 1996) as indicating that babies fed 100% soy-formula milk had blood concentrations of isoflavonoids approximately 1000-times that found in breast fed infants. Bingham et al. have also expressed concern regarding the issue of neonatal exposure, and have drawn attention to experimental evidence of developmental effects. For example, a study was noted in which rat pups were postnatally exposed for 10 or 21 days via the milk of dams fed 100 kg/g coumestrol. In those female pups exposed to coumestrol for 21 days, persistent oestrus was subsequently noted, accompanied by a failure of the expected LH surge (indicating defeminisation). Testing of the mating behaviour of male pups exposed for 10 days also revealed increases in the latency to mount and ejaculate, and reductions in mounting and ejaculation rates, despite gonadal weight and plasma testosterone level being unaffected. These findings clearly demonstrate neuroendocrine developmental effects in a rodent model at relatively low levels of coumestrol exposure. Suggestions have also been made regarding the possible risk of autoimmune thyroid disease in infants consuming soy-formula milk. The present concern about the safety of soya-derived infant milk is not without critics, and the potential benefits of soya-based formulae for infants with a family history of allergy or signs of milk intolerance, or as an aid to recovery from diarrhoea, kwashiorkor or colic have been noted. The use, without evidence of adverse effects, of soya-based infant formulae in the USA for 50—60 years has been stressed. Postnatal development of the gut and its microflora will also influence absorption patterns. While acknowledging the higher levels of isoflavones in infants fed soya-based formula compared with either human or cows milk based formula (Table 5), the lack of any biological or clinical changes in these infants argues that since these substances are much less potent than the endogenous hormones, their relative potencies will only be comparable to those of the endogenous oestrogens present in all infants. In addition, it has been argued that infants are able to extensively metabolise soya isoflavones into glucuronide and sulfate metabolites of low or negligible activity which would rapidly be excreted. In a 1996 review, the Committee of Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) estimated the intake of isoflavones from soya-milk to be approximately 4 mg/kg/day over the first 4 months of life. This is greater than that associated with hormonal effects in premenopausal women, but the COT nonetheless supported the existing Department of Health’s advice that

K. J. Turner and R. M. Sharpe, Rev. Reprod., 1997, 2, 69. P. L. Whitten, C. Lewis, E. Russell and N. Frederick, J. Nutr., 1995, 125, 771S. A. C. Huggett, S. Pridmore, A. Malnoe, F. Haschke and E. A. Oﬀord, Lancet, 1998, 350, 815.

131 P. Holmes and B. Phillips

Table 5 Isoﬂavone levels Isoﬂavone level (ng/ml) in infants fed various forms of milk or milk Matrix Age substitute Infant’s food analysed (months) Genistein Daidzein Equol Soya-based formula plasma 4 684 < 443 295 < 59.9 2.0 urine 2 26 451 < 8559 25 399 < 8081 6.4 < 6.4 urine 4 8758 < 3808 17 577 < 5452 1.5 < 1.5 Cow’s milk based plasma 4 3.2 < 0.7 2.1 < 0.3 4.1 < 0.5 formula urine 2 205 < 52 155 < 64 42.6 < 2.3 urine 4 536 < 393 706 < 555 40.5 < 3.3 Human milk plasma 4 2.8 < 0.7 1.5 < 0.1 0.5 urine 2 1284 < 1089 697 < 653 2.9 < 2.9 urine 4 161 < 108 179 < 80 59 < 42

the small number of non-breast fed babies that are unable to tolerate cow’s milk should continue to receive soya-based infant formulae. The COT expressed concern over the potential for adverse effects, while acknowledging the lack of reported adverse effects for populations habitually consuming large quantities of soya. A recent survey of soya-based infant milk formulae in the UK found total isoflavone concentrations of between 18 and 41 mg/l of made-up formulae, giving estimated average intakes of 5 mg/kg/day for 1—2 month old infants and 4.5 mg/kg/day at 4—6 months of age. These intakes are comparable to the earlier estimate used by the COT and enabled the COT to reaffirm its earlier advice. Given the current lack of definitive data on the potential health effects of early developmental exposure to phytoestrogens (in utero and neonatally), there is a need for further research in this field.

5 Conclusions The phytoestrogens are a diverse range of naturally occurring plant compounds that can interact with cellular oestrogen receptors. Although their potencies are low compared with the endogenous hormones, levels of exposure may be high. There is also evidence that they can bind to SHBG found in mammalian blood, which may further modulate hormonal profiles. In addition to their endocrinological properties, these compounds have other biological activities and it must also be remembered that plants contain many other biologically active compounds. Therefore, effects attributed to the consumption of foodstuffs rich in phytoestrogens cannot automatically be attributed to the endocrine modulating properties of their phytoestrogen constituents. While the evidence from animal studies is that foodstuffs rich in phytoestrogens cause adverse health effects, particularly with regard to fertility, the data for humans suggests that overall they are beneficial. There is compelling evidence from human epidemiological studies that, at least for adults, consumption of a diet rich in foodstuffs containing phytoestrogens is linked with a reduced risk of a wide range of common human diseases. Such conditions include cancer of the breast (in women), endometrium, prostate, colon, rectum, stomach and lung, and

132 Human Health Effects of Phytoestrogens non-neoplastic conditions such as osteoporosis, post-menopausal symptoms and cardiovascular disease. It is of note that although these diseases are known to be multifactorial in origin, all have in common a degree of hormonal dependency. Available epidemiological evidence is uncertain regarding the protective effect of diets rich in phytoestrogens on female breast cancer in premenopausal women, and there is a lack of information regarding a protective effect in postmenopausal women. However, particularly in premenopoausal women, phytoestrogens and phytoestrogen-containing foods can elicite physiological changes that would be expected to reduce breast cancer risk. Similarly, high vegetable intakes are associated with low ovarian cancer incidence and high soya consumption is linked to reduced endometrial cancer rates. It is plausable that the hormonal activity of phytoestrogens may be associated with these effects. Epidemiological data supporting a beneficial effect on osteoporosis is scant, although some support comes from animal and in vitro studies on phytoestrogens and the synthetic isoflavone derivative ipriflavone. It is also possible that there are some beneficial effects on the incidence of postmenopausal symptoms. Although requiring further investigation, there is some epidemiological and experimental evidence suggesting that the progression, if not the initiation, of prostate neoplasia can be favourably modified by diets rich in phytoestrogens. For cancers of the colon, stomach and lung, the evidence supports a protective role for high dietary intakes of plant material, although a causal relationship with phytoestrogens has yet to be established. The influence of diets high in plant material on rectal cancer is unclear. There is also evidence that soya foods and linseed oil can influence blood lipid profiles in a manner that would be expected to protect against cardiovascular disease and atherosclerosis. Although the extent of the effect on blood lipids appears relatively small, this could be sufficient to result in beneficial effects at the population level. There are isolated reports of adverse change following the consumption of phytoestrogens, but in adults the evidence overwhelmingly supports the beneficial nature of a diet rich in foodstuffs containing phytoestrogens. Based on some laboratory findings of adverse developmental effects in neonatal animals exposed to high levels of phytoestrogen, concerns have been raised over the possible adverse consequence of exposure of children to high levels of phytoestrogens (for example, in soy-based milk formula). These concerns must, however, be weighed against the absence of reports of adverse effects in such children. In summary, in adults, a diet rich in plant material appears to be protective against many of the common chronic diseases of the developed countries, and there appears no convincing evidence of adverse effects. Evidence to link the benefical effects specifically to phytoestrogens is, however, insufficient, although some experimental data are suggestive of such a link. The potential for adverse effects arising from fetal or neonatal exposure to phytoestrogens has been raised, although there is as yet insufficient evidence to establish the potential risks and benefits of such exposure. There remain, therefore, a number of outstanding questions that will require further studies to resolve.

133 P. Holmes and B. Phillips

6 Acknowledgements The authors would like to thank Dr Paul Harrison for his helpful comments during the preparation of early drafts of this manuscript, part of which is based on a review of this topic undertaken by the MRC Institute for Environment and Health (IEH). The contributions made by Dr Charles Humfrey and by participants at a peer-review workshop held at the Institute in March 1997 are also gratefully acknowledged. The authors acknowledge the ﬁnancial support of the UK Ministry of Agriculture, Fisheries and Food for the work on phytoestrogens at IEH. The opinions expressed in this paper are those of the authors and do not necessarily represent those of any government department or agency.

K. Verdeal and D. S. Ryan, J. Food Prot., 1979, 42, 577. A. J. Oosterkamp, B. Hock, M. Seifert and H. Irth, Trends Anal. Chem., 1997, 16, 544. U. Mayr, A. Butsch and S. Schneider, Toxicology, 1992, 74, 135. A. M. Soto, T.-M. Lin, H. Justicia, R. M. Silvia and C. Sonnenschein, J. Clean Technol. Environ. Toxicol. Occup. Med., 1998, 7, 331. L. Markiewicz, J. Garey, H. Aldercreutz and E. Gurpide, J. Steroid Biochem. Mol. Biol., 1993, 45,399. E. M. Bickoﬀ, A. L. Livingston, A. P. Hendrickson and A. N. Booth, Agric. Food Chem., 1962, 10,410. P. M. Martin, K. B. Horwitz, D. S. Ryan and W. L. McGuire, Endocrinology, 1978, 103, 1860. C. S. Cunnane, M. J. Hamadeh, A. C. Liede, L. U. Thompson, T. M. S. Wolever and D. J. A. Jenkins, Am. J. Clin. Nutr., 1995, 61, 62.

134 Endocrine Disruptor Research and Regulation in the United States

ANTHONY F. MACIOROWSKI AND GARY E. TIMM

1 Introduction Scientific evidence has accumulated that humans, domestic animals, and fish and wildlife species have been impacted by exposure to chemicals that interact with the endocrine system. To date, the best documented episodes involve exposure to high concentrations of organochlorine compounds (e.g., DDT and its metabolite DDE, PCBs, and dioxins), and to a few naturally occurring plant estrogens. There is general consensus that free-ranging fish and wildlife populations exhibit endocrine related anomalies attributable to contaminants. However, the role of environmental concentrations of contaminants to human disease trends remains unclear. Conflicting reports have appeared regarding declines in the quality and quantity of sperm production in humans. Increases in cancers of endocrine sensitive tissues (e.g., breast, prostate, testicle) have been reported, but direct evidence linking disease trends with exposure to low levels of environmental contaminants remains scant. Nevertheless, there is little doubt that small disturbances in endocrine function, particularly during certain highly sensitive stages of the life cycle (e.g., in utero or in ovo, exposure, early development, sexual differentiation, etc.) can lead to profound irreversible adverse effects. Taken collectively, the existing body of scientific evidence in human epidemiology, animal laboratory studies, and fish, bird, reptile, and invertebrate population

EC, European Workshop on the Impact of Endocrine Disruptors on Human Health and Wildlife, European Commission, Brussels, 1996, EUR 17549. L. Tattersﬁeld, P. Matthiessen, P. Campbell, N. Grandy and R. La¨nge, Expert Workshop on Endocrine Modulators and Wildlife: Assessment and Testing, SETAC-Europe, Brussels, 1997. R. Kavlock, G. Daston, C. DeRosa, P. Fenner-Crisp, E. Gray, S. Kaattari, G. Lucier, M. Luster, M. Mac, C. Maczka, R. Miller, J. Moore, R. Rolland, G. Scott, D. Sheehan, T. Sinks and H. Tilson, Environ. Health Perspect., 1996, 104, 715. EPA, Special Report on Environmental Endocrine Disruption: An Eﬀects Assessment and Analysis. Environmental Protection Agency, Washington, DC, 1997, EPA/630/R-96/012.

Issues in Environmental Science and Technology No. 12 Endocrine Disrupting Chemicals © The Royal Society of Chemistry, 1999

135 A. F. Maciorowski and G. E. Timm studies provide a plausible scientific hypothesis that environmental contaminants can disrupt the endocrine system leading to adverse health and ecological consequences. However, difficult unanswered questions contribute to a sense of scientific and regulatory uncertainty:

E Are observed effects the result of isolated, high exposure incidents or a broad environmental problem? E What specific chemicals act as endocrine disruptors? E Have endocrine effects been missed by conventional toxicological testing and hazard assessment practices? E What endocrine disruptors are humans and wildlife exposed to either singly or in combination? E Are breast and testicular cancer and other human diseases linked to endocrine disruptors? E Are the low environmental concentrations of most contaminants sufficiently high to exert adverse effects on populations?

The scientific resolution of such questions is ultimately dependent upon continued multidiscplinary research involving epidemiology, ecology, mammalian toxicology, ecological toxicology, and environmental chemistry. Nevertheless, the endocrine disruptor debate is not restricted to the scientific community, but has already passed into the broader realm of public policy. Within both the scientific and public policy arenas, opinions regarding endocrine disruption range from ‘an obvious and apparent environmental problem’ to ‘just another example of junk science’. Regardless of one’s point of view regarding the endocrine disruption issue, two laws passed in 1996 by the United States Congress (the Food Quality Protection Act and the Safe Drinking Water Act) require the EPA to develop and implement an endocrine disruptor screening and testing program. As such, the Agency was faced with initiating a screening and testing program in an atmosphere of complex and rapidly evolving science, coupled with a similarly complex public policy debate. Under the circumstances, the Agency developed a dual strategy for implementation. Firstly, a research program was initiated to increase understanding of the basic scientific issues concerning endocrine disruption. Secondly, the Agency convened a multi-stakeholder panel, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), to provide advice and recommendations regarding the development of a scientifically sound strategy for practical regulatory implementation. The present review briefly summarizes major endocrine disruptor research and regulatory initiatives within the U.S. EPA.

2 The Need for Research and Science Policy In 1994, the U.S. EPA held an Agency-wide colloquium to examine the then emerging endocrine disruptor issue. This original colloquium was a galvanizing event for Agency endocrine disruptor initiatives and called for a science assessment of the overall issue, a focused research agenda, collaboration with other government agencies, and the development of science and science policies.

136 Endocrine Disruptor Research and Regulation in the United States

Research Strategies Research strategies were realized in 1995, when two workshops were convened to assist the U.S. government in identifying research needs for endocrine disruptors. One workshop focused on mammalian toxicology and human health, whereas the second workshop concerned ecological toxicology and environmental eﬀects. The workshops identiﬁed salient questions to be addressed by research:

E What types of adverse effects are caused by endocrine disruptors? E What chemicals are responsible for causing endocrine disruption? E What exposure levels are required to cause adverse effects? E Are effects on fish and wildlife due to isolated high exposure incidents or the result of a broader environmental contamination problem? E How much exposure to endocrine disrupting chemicals occurs for humans, fish, and wildlife? E What is the shape of the dose—response curve? E Are breast and testicular cancers due to endocrine disruptors? E Are existing test protocols adequate to detect the effects of endocrine disruptors?

Over the past two years, the research strategy has been implemented through intramural and extramural research grant programs. In addition, the Agency has developed a United States Federal Research Inventory to facilitate understanding of the kinds of research being conducted or funded by U.S. Federal Agencies.

U.S. Federal Research Inventory Under the auspices of the U.S. Office of Science and Technology Policy, Committee on the Environment and Natural Resources (CENR), 14 U.S. federal agencies came together to develop a comprehensive endocrine disruptor research inventory. The research inventory is intended to coordinate research among U.S. agencies, eliminate redundancies, and identify knowledge gaps that require research by the U.S. federal agencies (www.epa.gov/endocrine). The inventory lists 396 research projects, of which nearly 70% are directed toward human health, with the remaining projects evenly split between ecological effects and exposure. One limitation of ongoing research that became evident from the inventory was a focus on very few endocrine active chemicals (e.g., PCBs, dioxins, and persistent pesticides). Further, the majority of projects focused attention on reproductive effects followed by cancer, neurotoxicity, and immunotoxicity, in decreasing order. Most projects in the inventory were basic research oriented, followed by hazard characterization, the development of risk models, investigations of biomarkers, and the development of exposure measurement methods. The CENR workgroup on endocrine disruptors identified research needs in three major areas: methods development; model development; and laboratory and

G. Ankley, R. Johnson, G. Toth , L. Folmar, N. Detenbeck and S. Bradbury, Rev. Toxicol., 1997, 1, 71. NSTC, A Framework for Planning, National Science and Technology Council, Committee on Environment and Natural Resources, Washington, DC, 1996.

137 A. F. Maciorowski and G. E. Timm

ﬁeld data measurements. The highest priority research topics identiﬁed for each research need area follow.

Methods development research topics E Establish the eﬀects of endocrine disruptors during stages of development. E Develop non-invasive biomarkers for wildlife. E Develop techniques for assessing exposure during critical life stages. E Develop biomarkers of exposure for use in screening techniques.

Models development research topics E Develop methods for using mechanistic and biomarker data in risk models. E Characterize dose-response relationships under environmentally relevant conditions. E Develop risk models for ecological risk assessments. E Develop fate and transport models for soil, air and water.

Laboratory and field measurements research topics E Establish relationships between exposure and adverse endocrine effects. E Develop exposure and effects data for amphibians, reptiles, and invertebrates. E Quantify endocrine disruptor body burdens in humans and wildlife.

The U.S. Federal research inventory has also served as a prototype for international expansion under the auspices of the World Health Organization, International Program on Chemical Safety. Under this program, the research inventory has been expanded to include endocrine disruptor research being conducted by Canada, Japan, and the European Union countries.

EPA Science Policy Council Interim Position In 1997, the EPA released a special report on environmental endocrine disruption. The report was intended as an interim assessment to inform Agency risk assessors of the major ﬁndings and uncertainties and to provide a basis for the following Science Policy Council interim position statement:

E EPA is aware of and concerned about information indicating the possibility of adverse impacts on human health and the environment associated with exposure to endocrine disruptors. E At the present time, there is little knowledge of or agreement on the extent of the problem. E Based on the current state-of-the-science, the Agency does not consider endocrine disruption to be an adverse endpoint per se, but rather a mode or mechanism of action potentially leading to other outcomes. Examples include carcinogenic, reproductive, or developmental eﬀects routinely used in reaching regulatory decisions. E Evidence of endocrine disruption alone can inﬂuence priority setting for

138 Endocrine Disruptor Research and Regulation in the United States

further testing. E EPA continues to stay abreast of scientiﬁc developments and will take regulatory action whenever sound scientiﬁc information and prudent public policy dictate. E We are currently committed to pursuing domestic and international opportunities for exposure/risk assessment related to endocrine disruptors.

Although the EPA Science Policy Council interim position represents the Agency’s ﬁrst formal statement concerning endocrine disruptor policy, science policy is subject to change as new research data and information become available. Additionally, science policy should not be confused with other types of public policy, such as legislation, or regulatory policy stemming from the implementaion of public policy. Indeed, while the above science policy statement was under review, legislative action occurred that mandated the EPA to implement endocrine disruptor screening as part of its regulatory programs. Although science remains important in regulation, it should be emphasized that legal, socio-economic, and political considerations are of equal importance in ﬁnal regulatory policy development and implementation.

3 Endocrine Disruptor Screening, Testing and Regulatory Implementation Endocrine disruptor research remains an important and ongoing initiative within the EPA. However, the passage of laws that mandate screening of chemicals for estrogenic activity instantaneously required additional Agency emphasis on regulatory implementation. The Food Quality Protection Act of 1996 (P.L. 104—170), 21 U.S.C. § 346a(p) requires the EPA to:

‘develop a screening program, using appropriate validated test systems and other scientifically relevant information, to determine whether certain substances may have an effect in humans that is similar to an effect produced by a naturally occurring estrogen, or such other endocrine effect as the Administrator may designate.’

In 21 U.S.C. § 346a(p)(3), the FQPA also states that in carrying out its screening program, the EPA:

‘(A) shall provide for the testing of all pesticide chemicals and (B) may provide for the testing of any other substance that may have an eﬀect that is cumulative to an eﬀect of a pesticide chemical if the Administrator determines that a substantial population may be exposed to such a substance.’

Additionally, Congress amended the Safe Drinking Water Act (42 U.S.C. § 300j-17) authorizing the EPA to screen ‘contaminants in drinking water to which substantial numbers of people would be exposed.’ The congressional requirement to develop a screening program in a controversial and rapidly emerging area of science led the EPA to seek a consensus strategy

139 A. F. Maciorowski and G. E. Timm regarding how to best meet its mandate. In October 1996, the Agency established The Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC). EDSTAC consisted of 39 members representing a broad range of constituencies including pesticide and commodity chemical manufacturers, state and Federal government agencies, worker protection organizations, and environmental and public health advocacy organizations. EDSTAC was charged with advising the Agency on the development of a practical, scientifically defensible endocrine disruptor screening strategy. At its first meeting, EDSTAC decided to include the estrogen, androgen, and thyroid systems in its deliberations. The Committee cited numerous examples of estrogen, anti-estrogen, anti-androgen, and anti-thyroid agent impacts on reproduction, growth, and development as the rationale for their inclusion. EDSTAC recognized that estrogen, androgen, and thyroid were not the only possibilities, but reasoned that these three hormone systems were well studied and represented a logical starting point. They also believed that additional hormone systems could be considered in the future as the underlying science and related methods became available. Ecological effects were also deemed important for screening and testing in that ecological data presently provides the strongest evidence for endocrine disruption effects in natural populations. Finally, EDSTAC concluded that chemicals other than pesticides and drinking water contaminants should be considered as candidates for screening. Over a two-year period, EDSTAC formulated a total of 71 detailed consensus recommendations that it offered to the Agency. The consensus aspect of the recommendations is significant given: the diverse constituency of the committee; the complex and controversial nature of the science surrounding endocrine disruptors; and the often difficult arena of pesticide and commodity chemical evaluation and regulatory decision making. Following issuance of its final report to the EPA, EDSTAC completed its work and the committee was disbanded. The responsibility then fell to the EPA to act on the EDSTAC recommendations by proposing an Endocrine Disruptor Screening Program responsive to the FQPA and SDWA, and to implement the program.

EPA’s Proposed Endocrine Disruptor Screening Program The Endocrine Disruptor Screening Program (EDSP) developed by the EPA closely follows the EDSTAC consensus recommendation and is only summarized here. EPA’s proposed EDSP consists of four discrete stages: (1) initial sorting of chemicals; (2) establishment of screening priorities; (3) Tier 1 screening; and (4) Tier 2 testing.

Initial Sorting. The universe of chemicals to be sorted included approximately 900 pesticide active ingredients; 2500 pesticide formulation inert ingredients; 75 500 industrial chemicals; and 8000 cosmetics, food additives, and nutritional

EPA, Endocrine Disruptor Screening and Testing Advisory Committee: Final Report, Environmental Protection Agency, Washington, DC, 1998; available at www.epa.gov/opptintr/opptendo. Federal Register, 1998, 63, 4285. Federal Register, 1998, 63, 71452.

140 Endocrine Disruptor Research and Regulation in the United States supplements. It should be emphasized that the EPA has no regulatory purview over the latter three chemical groups, and will focus its efforts on pesticides, industrial chemicals, and environmental contaminants. However, in keeping with the EDSTAC recommendations, the EPA will continue to collaborate with other appropriate federal agencies (e.g., Food and Drug Administration, National Institute of Environmental Health Sciences) to facilitate examination of the remaining groups of chemicals. The initial sorting stage is intended to separate the universe of chemicals into four discrete categories, based upon a review of all existing relevant scientific information. Category 1 includes those chemicals that are unlikely to exhibit endocrine activity and should not be screened (e.g., strong mineral acids and bases, amino acids, sugars, certain polymers, etc.). Category 2 consists of chemicals with insufficient data to determine their potential for endocrine activity. Category 3 includes those chemicals that have sufficient data to bypass screening, but need testing. Finally, Category 4 consists of substances with adequate data which will be referred to the appropriate agency for hazard assessment. Category 1 chemicals deserve some comment. As examples, polymers with a numerical average molecular weight (NAMW) greater than 1000 daltons and other kinds of chemicals (e.g., certain biologically inactive ingredients or highly reactive substances such as strong mineral acids and bases that will react at the portal of entry) are unlikely to display endocrine activity. Polymers with the specified NAMW will be set aside pending a review of their monomers, which will be scrutinized separately. Such polymers were thought to pose little risk because they are generally too large to pass through biological membranes and interact with the endocrine system. Although initial sorting is a fairly straightforward exercise, relatively few chemicals (hundreds) will have sufficient screening and testing data to be placed into Categories 3 and 4. Presumably the vast majority of chemicals will fall into Category 2.

Priority Setting. The Category 2 chemicals (those with insufficient information and data to determine if they are endocrine active) constitute the largest category, and are therefore of the greatest interest to the EPA for priority setting. EDSTAC recommended several approaches for setting priorities among these chemicals. One approach consisted of ranking chemicals with known exposure and effects as the highest priority for screening. However, some EDSTAC members were concerned that this would inappropriately focus screening priorities on the most studied chemicals, and jeopardize priority setting for potentially problematic but little studied chemicals. In addition, EDSTAC understood that priority setting was not generally an objective, data- driven process. Equitable and uniform data are simply not available for commodity chemicals, pesticide active ingredients, pesticide formulation ingredients, environmental contaminants, and other groups of chemicals. As an example, ranking a chemical that may have extensive monitoring data in wildlife, against one with known human exposure but entirely lacking any effects data, becomes an exceptionally difficult task. A priority setting approach using like information and data for comparative purposes in priority setting was viewed as an ideal. However, a uniform approach was viewed as not

141 A. F. Maciorowski and G. E. Timm practically achievable given the uneven nature of existing product chemistry, fate and exposure, use information, effects, and human and environmental monitoring data for different chemicals. EDSTAC also grappled with the fact that priority setting often necessitated a range of subjective value judgements that were difficult to quantify. Nevertheless, they believed that such value judgements must be clearly defined and transparent in the priority setting process to engender public confidence. The priority setting approach that ultimately emerged from EDSTAC was a conceptual plan that requires additional development by the EPA prior to implementation. The overall concept consists of first developing a comprehensive relational database to consolidate existing databases and information for chemicals. The database could then be queried for the existence of empirical data such as product chemistry, fate and transport, presence in environmental samples, presence in human tissues and fluids, chronic toxicity information, epidemiology, etc. The queries could take the form of specific information types (e.g., effects only, exposure only, or various combinations of exposure and effects). Where only limited information occurred (e.g., product chemistry data), it was though that models could be used to estimate certain useful parameters (e.g., bioaccumulation potential from octanol—water partition coefficients, quantitative structure—activity relationships for the estrogen and androgen receptor, probable environmental partitioning, etc.). The database could then be used to estimate the number of chemicals that might occur under different exposure, effect, or exposure and effects scenarios. EDSTAC developed a prototype Endocrine Disruptor Priority Setting Database that proved the concept was feasible, but had insufficient time and resources to adequately refine the model and verify the integrity and accuracy of data in the prototype. Accordingly, continued development of the database as a tool to establish ‘compartments’ or ‘sets’ of chemicals was recommended. Ranking within sets would then be used for overall priority setting. This approach was called a ‘compartment-based’ priority setting strategy. In the compartment-based strategy, a number of compartments (or sets) of chemicals are defined and the individual chemicals within each set are prioritized. One might think of mathematics and sets of numbers as an analogy. In mathematics, sets might be real numbers, integers, irrational numbers, even numbers, etc. Numbers within each set can then be ordered in some fashion. Some numbers may belong to more than one set, and the same will presumably hold true for chemicals. Specific compartments or sets that might be defined include high production volume chemicals, chemicals measured in biota, chemicals in consumer products, chemicals detected in the workplace, etc. Given the number of chemicals to be prioritized for screening, implementation will undoubtedly occur in phased batches. Once the compartments of chemicals are defined and prioritized, a batch of chemicals will be selected for the initial phase of the screening program. The contribution of each set of chemicals to this batch is the key subjective judgment that must be made in the priority setting process. The size of the first batch and spacing of subsequent batches of chemicals depends largely on the available laboratory capacity of the system, the ability of industry to pay for testing, and the resources of the EPA to review submitted data. The

142 Endocrine Disruptor Research and Regulation in the United States

EPA is presently in the process of advancing the prototype Endocrine Disruptor Priority Setting Database, as well as the compartment-based priority setting approach. Although the compartment-based priority setting approach holds promise, EDSTAC recognized an additional problem for priority setting. Very few chemicals actually have any relevant existing effects data germane to endocrine disruption. Chemical effects data are often scant, and limited to short-term mutagenicity or acute toxicity tests. Even chemicals that have been evaluated for reproductive and developmental effects were most likely tested using conventional toxicological protocols that were not designed to detect endocrine effects. Among the most innovative EDSTAC recommendations was the suggestion to use automated ‘high throughput screening’ technology to provide estrogen and androgen receptor binding data on high production volume chemicals and pesticides. The receptor binding and reporter gene in vitro assays in the Tier 1 screening battery (see below) are amenable to automated processing in a high throughput mode. These assays are specific to receptor binding mechanisms of action. Moreover, all of the 15 000 high production volume chemicals could be assayed in a 3—6 month window at a relatively modest cost. Called high throughput pre-screening (HTPS), such data were viewed as important adjunct effects data for priority setting. The HTPS data could be used with existing production and exposure information and data to assist priority setting for further in vivo screening. A positive result in the HTPS, coupled with existing production, use, and exposure information, would raise the priority for additional screening. Concomitantly, negative HTPS results would not raise or lower priority, because mechanisms other than receptor binding might be involved.

Tier 1 Screening. The Tier 1 screening battery recommended by EDSTAC and proposed by the EPA was the culmination of work begun by other scientific workgroups regarding candidate screening assays for regulatory application. Prior to EDSTAC, the groups reviewed numerous existing screening assays and concluded that no single assay could detect estrogen, androgen, and thyroid agonists and antagonists. Rather, a battery of in vitro and in vivo assays was thought necessary to evaluate endocrine disruption potential. The former are advantageous in that they are inexpensive and specific for a particular mode of action. Conversely, they lack the metabolic and response complexity of intact animals. After reviewing approximately 80 existing assays, EDSTAC recommended a Tier 1 screening battery consisting of three in vitro and five in vivo assays for Tier 1 screening. The in vitro assays include an estrogen receptor binding or reporter

G. Ankley, E. Mihaich, R. Stahl, D. Tillit, T. Colborn, S. McMaster, J. Bantle, P. Campbell, N. Denslow, R. Dickerson, L. Folmar, M. Fry, J. Giesy, E. Gray, P. Guiney, T. Hutchinson, S. Kennedy, V. Kramer, G. LeBlanc, M. Mayes, A. Nimrod, R. Patino, R. Peterson, R. Purdy, R. Ringer, P. Thomas, L. Touart, G. Van Der Kraak and T. Zacharewski, Environ. Toxicol. Chem.. 1998, 17, 68. E. Gray, W. Kelce, T. Weis, R. Tyl, K. Gaido, K. Cook, G. Klinefelter, D. Desaulnier, E. Wilson, T. Zacharewski, C. Waller, P. Foster, J. Laskey, J. Giesey, S. Laws, J. Mclachlan, W. Breslin, R. Cooper, R. Di Giulio, R. Johnson, R. Purdy, E. Mihaich, S. Safe, T. Colborn T. et al., Reprod. Toxicol., 1997, 11, 719.

143 A. F. Maciorowski and G. E. Timm gene assay, an androgen receptor binding or reporter gene assay, and a steroidogenesis assay using minced testes. Functional reporter gene assays were viewed as preferable to receptor binding assays because the former are more sensitive, and capable of differentiating agonist from antagonist activity. Permanently transfected reporter gene assays use cell lines permanently modified by the introduction of DNA and produce a particular marker protein. Luciferase, the enzyme involved in the production of firefly light, is the specific marker selected by the EPA for the estrogen (ER) and androgen (AR) receptor assays currently under development. A positive response in the assay produces light that can be measured photometrically. The reporter gene assays are being developed with high throughput technology. Once developed and validated, the EPA will use the assay results to assist priority setting for the remainder of the Tier 1 battery. The in vivo assays selected for the Tier 1 battery include a rodent 3-day uterotrophic assay, a rodent 20-day pubertal female assay with thyroid endpoints, a rodent 5—7-day Hershberger assay, a frog metamorphosis assay, and a fish gonadal recrudescence assay. Endpoint complementarity was deliberately incorporated into the entire screening battery to provide a weight-of-the-evidence determination. Under this scenario, the ER reporter gene, uterotrophic, and pubertal female assays screen for estrogenicity and anti-estrogenicity. The AR reporter gene and Hershberger assays screen for androgenicity and anti- androgenicity. The frog assay and pubertal female assays screen for thyroid activity. Finally, the fish assay represents the vertebrate class most removed from mammals with respect to metabolism and hormone systems. The presence of diverse taxa in the Tier 1 screening battery may also provide some indication of consistent or variable response patterns among organisms. In keeping with the weight-of-the-evidence approach, in vivo results will generally outweigh in vitro results when interpreting the battery. Additionally, chemicals that are negative in Tier 1 screening would be considered to have a low potential for estrogen, androgen, or thyroid activity. Conversely, chemicals with positive Tier 1 results move to Tier 2 testing for a more comprehensive evaluation.

Tier 2 Testing. The Tier 2 tests are intended to identify adverse effects due to endocrine disruption and establish dose—response relationships. The Tier 2 tests are all designed to include the most sensitive developmental life stages (in utero or in ovo fetal development) and multi-generational effects for the five major taxonomic groups represented (mammals, birds, fish, amphibians, and invertebrates). The Tier 2 test battery can also be specifically tailored to coincide with salient exposure and effects information. For instance, if it can be demonstrated that human worker exposure is the only concern with the particular use pattern of a chemical, it may only be necessary to conduct the two-generation test with rodents. Similarly, if a particular use pattern suggests that only aquatic systems are of concern, it may only be necessary to conduct tests with fish and a representative aquatic invertebrate.

144 Endocrine Disruptor Research and Regulation in the United States

4 Implementation of the Endocrine Disruptor Screening Program The EPA must implement its Endocrine Disruptor Screening Program (EDSP) under strict deadlines mandated by the FQPA. Specifically, the FQPA required the Agency to develop a plan by August 1998, implement the plan by August 1999, and report progress to Congress by August 2000. The EPA has met its initial deadline through publication of a notice in the Federal Register outlining its plan, followed by a proposed statement of policy that provides additional detail. Nevertheless, EPA’s proposed EDSP, as well as its individual elements, remain subject to considerable development, scientific validation, appropriate scientific peer review, and public notice and comment prior to full regulatory implementation. Recognizing the amount of work facing it, the Agency has already initiated a number of key steps necessary for implementation. The EPA has published its proposed EDSP to obtain public comments on the program as well as its implementation plans. Following receipt of public comments, the proposed program will also be subjected to external peer review by a joint Panel of the FIFRA Scientific Advisory Panel and the EPA Science Advisory Board. Such steps will assist the EPA in improving its proposal and ensuring scientific integrity early in the program implementation phase. In addition to the overall program review, a number of key activities regarding EDSP elements have also been initiated. Initial sorting and priority setting are key steps in the EDSP that are dependent upon completion of the Endocrine Disruptor Priority Setting Database, definition of the actual compartments in the ‘compartment-based’ priority setting approach, and a feasibility demonstration of using the reporter gene assays on a wide range of chemicals. EPA has established workgroups to complete development of the Endocrine Disruptor Priority Setting Database and to define the compartments. The products of the workgroups will be reviewed in ad hoc multistakeholder workshops, and published for public comment. The foregoing tasks should be completed by the end of 1999. A pilot demonstration of the reporter gene assays has also been initiated to determine the suitability of using such automated assays for commercial chemicals and pesticides. If the pilot demonstration proves successful, it will be followed by an additional study of approximately 300—500 chemicals. Assuming successful completion and appropriate scientific peer review, automated screening of up to 15 000 high production volume chemicals and pesticides could begin in 1999. Completion of the priority setting database, compartments, and automated screening of up to 15 000 chemicals would allow the completion of priority setting for additional screens in the year 2000. Although the target completion dates for the above tasks are ambitious, they are considered achievable. The most difficult task facing the Agency remains standardization and validation of the individual screens and tests in the EDSP. All of the proposed screens and tests in the EDSP require some degree of standardization and validation to ensure that they give reliable and repeatable results prior to being used in regulatory decision making. Toward this end, the EPA has established an Endocrine Disruptor Screening and Testing Validation Task Force. The task force is charged with developing the technical work

145 A. F. Maciorowski and G. E. Timm necessary to standardize protocol design, conduct any pre-validation studies necessary to optimize the protocol, ensure it can be run in multiple laboratories, and design and conduct any multiple laboratory trials that may be necessary to ensure reliability and reproducibility. The task force will coordinate the standardization and validation effort with other U.S. government agencies, industry, and several public interest groups. The task force also has close liaison and membership overlap with the Organization for Economic Cooperation and Development (OECD) Endocrine Disruptor Testing and Assessment Task Force. The OECD task force will coordinate standardization, validation, and international harmonization efforts for those screens and tests of international interest. The domestic and international standardization efforts are decidedly ambitious. At least two to three years will be required to standardize and validate the in vitro and in vivo mammalian screens and tests assays which are the most highly developed. The ecotoxicological screens and tests will require three to five years.

5 Conclusions The scientific and policy debates surrounding endocrine disruptors remain complex and are frequently controversial. Some observers have suggested that legislation and environmental policies are ahead of the rapidly emerging science and its ability to resolve uncertainty. Others maintain that the scientific evidence is clear and convincing, and that more stringent policies are necessary to protect human health and the environment. Given the current scientific, regulatory, and advocacy issues at play in the endocrine disruptor milieu, debate and controversy should come as no surprise. After all, complexity and controversy have long been part and parcel of science, policy, and regulation. Further, the endocrine disruptor debate is somewhat unique in that the underlying science and emerging environmental policies are evolving simultaneously. With time, scientific issues and uncertainties will be resolved through research. Similarly, screening and testing issues will undergo refinement and standardization through the validation processes. Certainly, as data become available, they will inform and shape aspects of environmental and regulatory policy, as well as our ability to determine the relative risk of endocrine disruptors to human health and ecological integrity. Just as certainly, broader endocrine disruptor policy questions concerning socio-economic, legal, and aesthetic values may well be acted upon before the resolution of all scientific questions. In any event, endocrine disruptor regulatory policy in the United States will begin with the implementation of the EPA’s Endocrine Disruptor Screening Program, and continue using established Agency procedures under the Food Quality Protection Act, The Safe Drinking Water Act, the Toxic Substances Control Act, The Federal Insecticide, Fungicide, and Rodenticide Act, and other environmental legislation as appropriate.

146 Subject Index

Abnormal development, 40 of prey, 9 Abnormalities, 81 Bisphenol A, 14, 102, 103 Acid, 39 Blood lipid levels, 125 Adrenal glands, 30, 32 Body fat, 76 Adrenal endocrine disruptors, 51 Bowman-Birk protease inhibitor, Alkyl hydroxy benzoate, 103 113 Alkylphenolic compounds, 28, 30, 35, Brassinosteroids, 54 40, 10), 103 Breast cancer, 7,103,116, 117,'118,119, Alkylphenolic detergents, 29, 44 129, 103, 133 Alligators, 10, 68 Amphibians, 69, 72, 74 Cadmium, 29, 34, 35, 36, 38, 45, 50 Androgen, 66, 97 Cancer incidence, 86 Anthropogenic chemicals, 61 Carbamate, 29, 34, 38 Anthropogenic pollutants, 76 Cardiovascular disease, 112, 123, 126, Anticarcinogenic properties, 113 132 Antiest,ogens, 51 Cell proliferation, '57 Antispermatic activity, 53 Chemical dispersion, 72, 80 Antithyroid endocrine disruptors, 51 Children, 130, 133 Aquatic organisms, 78 Chlordecone, 52 Aquatic top predators, 75 Cholesterol, 123, 124, 126 Aromatase, 38, 41 Clover disease, 109 Arsenic, 35, 38, 45 Colon, 133 Atherosclerosis, 123, 126 Colorectal cancer, 126, 127 Competitive ligand binding, 57 Baltic Sea, 29, 30, 45 Congenital malformations, 87 Beluga whales, 66 Copper,29 Beneficial effects, 114 Cormorants,71 Bioaccumulation, 77 Coumestans, 53, 109, 111 concentration factor (BCF), 50 Coumestrol, 52 Bioconcentration, 77 Courtship, 36, 37 Biodiversity, 75 Crustacean hyperglycaemic hormone Biomagnification, 77, 79 (CHH), 54 Biomarkers, 58 Cryptorchidism, 6, 85, 90, 93 Birds, 47,66,71,74 Cyprid major protein, 59

147 Subject Index

Cytochrome P-450, 43, 78 Endometriosis, 8 P450 aromatase, 57 Endrin, 56 EP A Science Policy Council, 138 Daily intakes, 111 Epididymal cysts, 93 DCB, 4H2'5'-, 52 17p-Estradiol, 50,52 DDE, 67 Ethynylestradiol, 35 DDT, 15,29, 36,42, 50, 52 European Chemical Industry Council Defeminisation, 131 {CEFIC), 25 Definition of endocrine disruption, 4, European Commission {EC), 21 32 Exposure, 13 Dehydroepiandrosterone sulfate, 118 Developmental effects, 131 Falcons, 67 pattern, 66 Fate of endocrine disrupting chemicals, DHEAS,119 79 Dieldrin, 53 Female Teproduction, 37 Diet, 116, 122, 127 Fertilisation, 36, 40 Diethylstilbestrol (DES), 2, 52, 58, 93 Fertility, 33, 88 Differentiation, 64 Fetal development, 82 Diflubenzuron, 55, 56 Fibre intake, 118 Dioxins, 42 Fish, 10 Dogwhelk, 11 Florida panther, 9, 65 Dose-response relationship, 21 Follicle stimulating hormone, 90 Drinking water, 15 Food additives, 103 Quality Protection Act, 139 Earlylife stages, 36 Foodstuffs, III Ecdysteroids (ecdysone, Fruit, 127 20-hydroxyecdysone), 55 Fungicides, 103 Ecosystem structure, 74 Ecotoxicology, 58 Genital tract, 85 EDSTAC, 136, 140 Germ cell development, 91 Efferent ducts, 100 German Federal Environmental Eggs, 40 Agency (FEA), 23 numbers, 39 Glucocorticoid hormones, 63 Eggshell thickness, 67 Gonadotrophins, 31 ELISA,58 Gonads, 30 Embryo feminisation, 68 Gonocytes, 99 Embryogenesis, 70 Great Lakes, 29, 30, 38 Embryonic development, 71 Gross malformations, 72 Endocrine disruptors, 61 Growth, 32, 42, 46 disruption, 49, 81 rate, 59 disruptor research, 135 Gulls, 10 Disruptor Screening and Testing Advisory, Committee (EDST AC), Half-life, 117 -23,140 Hatch rates, 40 function, 62 Herbal medicine, 129 Endogenous protein, 57 Hermaphrodites, 40,41 Endometrial cancer, 128, 133 Hombsesquiterpenoid epoxides

148 Subject Index

(juvenile hormones), 54 Menstruation, 118 Hormonal profile, 79 Mercury, 29, 34, 35, 36, 38, 43, 45 Hormone metabolism, 12 Metabolism, 32 Hormone receptors, 11 Metamorphosis, 69, 72 Human health, 5 Methoprene, 56 Human populations, 81 Migration, 42, 122 Hydrocarbon (Ah) receptors, 51 Molluscs, II Hypospadia, 6, 85, 89, 93 Morphological abnormalities, 58 Hypothalamus, 30, 33, 34 Mycotoxins, 15

Immune systems, 73 Neuroendocrine developmental effects, Impaired immune function, 73 131 Imposex, 11, 56 Neuroendocrine function, 57 In vitra assays, 18 Neuroendocrine~immune system, 62 estrogenicity, 57 Neurological effects, 7 In viva assays, 18 Nonylphenol, 34 Infants, 131, 132 North Sea, 29, 30 Interdepartmental Endocrine Disrupter Research Group, 22 Oestrogen, 66, 95, 97, 98 International Program on Chemical receptor, 3, 12; 77 Safety, 25, 138 receptor subtype, 114 Intersex, 10, 56 replacement therapy, 123 Isoflavonoids, 53, 109, 111 Oligozoospermia, 88 Organization for Economic Japanese Environment Agency (JEA), Cooperation and Development 24 (OECD), 24, 146 Organochlorine compounds, 29, 34, 35, 11-Ketotestosterone, 31, 35, 37 38, 45, 80 Kunitz trypsin inhibitor, 113 pesticides, 15, 101, 103 Organophosphates, 34,35,37,38,45 Lake Apopka, 68 Organophosphorus pesticides, 29, 39 Larvae, 40, 45 Osmoregulation, 30 Lead, 29, 35, 38 Osteoporosis, 112, 120, 121, 132 Otters, 9, 65 , Leydig cells, 99 Life cycle, 75 Ovarian cancer, 129 Lifestyle, 116 Ovaries, 8, 38 Lignans, 109, III Ovotestes, 59 Linseed, 125 Ovulation, 31, 39 Liver, 35, 43 Lung cancer, 129 PARs, 29,34, 38,41,43,45 Paper mill effluents, 34 Male fertility, 6,47 Parental care, 47 reproduction, 98 PCBs, 7,29,34,35,36,38,39,40,42,43, reproductive health, 34, 83 45,51,101,103 Malformations, 85 Peregrine falcons, 67 Mammals, 46, 47, 64, 70, 73 Pesticides, 15,29,39,45, 101, 103 Masculinisation, 41 Phenolic compounds, 102

149 Subject Index

Phenotypic alterations, 59 Science Policy, 136 Pheromones,37 Scientific Committee on Toxicity, Phthalate esters, 14, 28, 102 Ecotoxicity and the Environment Physicochemical properties, 76 (CSTEE), 22 Phytoecdysteroids, 54 Screening, 17, 139, 142 Phytoestrogens, 13, 103, 105 Seals, Pituitary gland, 30, 33, 34, 35, 90 fertility, 9 Plasma steroid levels, 68 populations, 65 Polar regions, 80 Selective biotransformation, 78 Polychlorinated biphenyls, see PCBs Selenium, 55 Polycyclic aromatic hydrocarbons, Semen quality, 83 52 Sentinel species, 59 ovaries, 8 Sertoli cells, 89, 92, 93, 95, 99 Post-confluent cell accumulation, 57 Sewage effluents, 29, 44 Post-menopausal symptoms, 112,119, treatment, 45 120, 121, 132 Sex, 76 Potency,114 determination, 69 Precautionary Principle, 3 hormone binding globulin (SHBG), Progestogen, 31 13, 113 Prostate cancer, 6, 121, 122 hormones, 63 Pulp mills, 38, 45 ratios, 7, 76 effluents, 35, 39,41, 43 Sexual behaviour, 69 development, 64 Quantitative reverse differentiation, 40, 41 transcriptase-polymerase chain dimorphism, 73 reaction, 57 Sharpe-Skakkebaek hypothesis, 130 Recombinant receptor/reporter gene SHBG, 13,113, 132 assays, 57 level, 119 Recovery of affected wildlife, 80 Soya, 117,118,120, 124,125,127,128, Rectal cancer, 126, 127 129 Reduced T -cell activity, 74 Soya-based infant formula, 130, 131, Regulation, 135 132 Reproduction, 30 Spawning, 36, 39 Reproductive abnormalities, 65 Species-specific effects, 75 Reproductive health, 34, 83, 93 Sperm, 36 Reproductive output, 59 counts, 5, 84 Reproductive systems, 63 Steroids, 50, 62 Reproductive tract, 89 Stomach cancer, 128, Reptiles, 10, 68, 71 Stress, 32, 42, 43, 46 Research inventory, 137 Suppression of testosterone, 69 Research strategies, 137 Synergistic effects, 20 Rete testis, 100 Risk, 105 TBT,41 assessment, 19 Terrestrial vertebrates, 78 Testing, 144 Safe Drinking Water Act, 139 Test systems, 114

150 Subject Index

Testicular cancer, 6, 83, 86, 91, 93 US Environmental Protection Agency Testicular descent, 90 (USEPA), 23, 136 Testis, 35 Uterotrophic assay, 18 Testosterone, 56 suppression, 69 Vegetables, 127,128 synthesis, 89 Viability of offspring and sex ratio, Thyroid gland, 30, 32, 40, 70 59 Thyroid hormones, 63, 69, 71 Vitellin, 58 Tin, 45 Vitellogenesis, 37 Top predators, 75, 81 Vitellogenin, 10, 43, 44, 58, 70 Toxaphene,53 Transgenerational, 46 Whales, 9 Tributlytin (TBT}, 11,38,56,102 Wildlife, 8, 61, 101 Triiodothyronine, 32 World Health Organization, 138 Turtles, 10

YES assay, 57 UK Department of the Environment, Transport and the Regions Zinc, 29,45, 55 (DETR), 22 Zona radiata protein, 58

151