Alternative Testing Methodologies
Environmental Health Perspectives, Volume 106, Supplement 2, April 1998

[Citation in PubMed] [Related Articles]

Identification and Assessment of Endocrine Disruptors: Limitations of in Vivo and in Vitro Assays

Tim Zacharewski

Department of Pharmacology and Toxicology, University of Western Ontario, London, Ontario, Canada

Abstract

It has been suggested that chemicals and complex mixtures capable of modulating the endocrine system may contribute to adverse health, reproduction, and developmental effects in humans and wildlife. These effects include increased incidence of hormone-dependent cancers, compromised reproductive fitness, and abnormal reproductive system development. In response to public concern, regulatory agencies in North America and Europe are formulating potential strategies to systematically test chemicals and complex mixtures for their endocrine-disrupting activities. Because of the complexity of the endocrine system and the number of potential endocrine disruptor targets, a tiered approach involving a complementary battery of short- and long-term in vivo and in vitro assays that assesses both receptor and nonreceptor-mediated mechanisms of action is being considered. However, the available established assays use a limited number of end points, and significant information gaps exist for other potential targets in the endocrine system. In addition to discussing the merits and limitations of the assays that may be adopted, this paper also highlights potential problems associated with the use of a tiered testing strategy. -- Environ Health Perspect 106(Suppl 2):577-582 (1998). http://ehpnet1.niehs.nih.gov/docs/1998/Suppl-2/577-582zacharewski/abstract.html

Key Words: endocrine disruptor, estrogen, androgen, in vivo and in vitro testing

Introduction

Epidemiologic studies have found significant increases in the incidence of hormone-dependent diseases including cancers of the breast, prostate, and testis (1-8). For example, the U.S. National Cancer Institute's Surveillance Epidemiology and End Results (SEER) Program reports that newly diagnosed cases of breast cancer increased at an annual rate of 1% between 1950 and 1979. This diagnosis rate tripled to 3% per year from 1980 to 1984 (9). Glass and Hoover have shown that the largest increases in incidence have occurred in women 60 years of age and older (74%), and in those 45 to 59 years of age (36%) (10). In women between 20 to 44 years of age, the rate has remained essentially unchanged. Less than one-third of the overall 15.3% increase in the age-adjusted rate for invasive breast cancers seen between 1972 and 1985 could be attributed to the increased use of screening mammography (5).

The occurrence of breast cancer has been associated with affluent societies, and studies have shown that the rates of occurrence can vary by as much as 5- to 10-fold between countries. Moreover, migrants who move from low- to high-risk countries adopt the rates of their new country. There have also been reports of significant increases in the incidence of male reproductive tract disorders (e.g., maldescent [cryptorchidism], urethal abnormalities [hypospadias]), decreases in semen volume and sperm counts, and compromised reproductive fitness in humans and wildlife (7,11-14). These results suggest that environmental factors may contribute to the increased incidence of these adverse effects (15,16).

This hypothesis is supported by several paradigms that have shown that the development of cancers and the occurrence of reproductive tract disorders can be influenced by exposure to estrogens or estrogenic drugs. These include

• experimental studies demonstrating the ability of sex steroids to promote tumor development (17-20);

• epidemiologic studies reporting the protective effect of ovariectomization, the increased risk of breast cancer in young women exposed to diethylstilbestrol, and the association between maternal estrogen concentrations and the frequency of testicular cancer and cryptorchidism (19,21-23);

• the prevalence of infertility and malformations of the genitalia in male rodents exposed prenatally to diethylstilbestrol (24,25); and

• the efficacy of hormone antagonists in treating cancers (26,27).

Consequently, it has been suggested that xenobiotics capable of mimicking or blocking the activities of sex steroids may play a role in the etiology of hormone-dependent cancers and disorders of the male reproductive tract in humans and wildlife (12,13,15,28-35).

Exogenous substances that can elicit sex steroidlike activities are commonly referred to as endocrine disruptors and have been defined as any exogenous agent, either synthetic or natural, that interferes with the production, release, transport, metabolism, binding, biologic action, or elimination of natural ligands in the body that are responsible for the maintenance of homeostasis and the regulation of developmental processes. In many cases, these endocrine disruptors share no apparent structural similarities to traditional steroids. Endocrine disruptors include natural products (phytoestrogens, e.g., genistein) (36-38), pharmaceuticals (i.e., diethylstilbestrol, ethynyl estradiol) (39), environmental pollutants (i.e., DDT, polychlorinated biphenyls, dioxins, polyaromatic hydrocarbons) (40-44), and industrially relevant chemicals (i.e., alkylphenols, bisphenol A) (45-47). The potential exposure and economic significance of several of these substances have made endocrine-disrupting chemicals a contentious health concern and environmental issue.

In contrast, several studies suggest that endocrine disruptors may not significantly contribute to the development of hormone-dependent disease or compromise reproductive fitness in humans. For example, Wilcox et al. (48) report that men who were prenatally exposed to diethylstilbestrol experienced no impairment of fertility based on the following parameters: incidence of impregnation, age and birth of first child, average number of children, diagnosis of fertility problems, and length of time to conception. In addition, no impairment of sexual function as indicated by the frequency of intercourse or reported episodes of decreased libido were reported (48). Consistent with the results of this study, several recent reports provide evidence refuting the findings that sperm counts and semen quality have been decreasing (49-51). Furthermore, women on vegetarian diets who consume large quantities of natural products with weak estrogenic activities (e.g., phytoestrogens) have a lower incidence of breast cancer (52,53). This evidence has led some researchers to suggest that weak estrogen agonists may function as antiestrogens in the presence of potent agonists, and therefore, do not contribute to the development of hormone-dependent diseases and developmental abnormalities and may even serve a protective role in some situations (54-56).

In Vivo Assessment of Endocrine Disruptors

As suggested by the endocrine disruptor definition, there are a number of potential mechanisms of action that may be susceptible to the adverse effects of endocrine disruptors. This diversity of potential targets and the complexity of feedback mechanisms significantly complicates evaluating the consequences of exposure to endocrine disruptors. Fortunately, many of the functions and mechanisms of the endocrine system are conserved among species, thereby providing scientists methodologies, initially developed for clinical medicine and drug discovery or evaluation, to investigate the activities of endocrine disruptors. A compendium listing the assays currently used to test for the estrogenic activities of a substance or complex mixture has recently been compiled (57). Several of these tests (e.g., enzyme induction, cell differentiation, effects on organ weights) take advantage of the receptor-mediated mechanism of action of sex steroids. The advantages and disadvantages of some of these in vitro and in vivo assays as well as emerging methodologies have recently been reviewed (58,59). Endocrine disruptors also elicit effects through receptor-independent mechanisms that may involve steroid transport (i.e., hormone-binding globulins) (60,61), steroid synthesis (i.e., inhibition of aromatase activity) (62), or interactions with target cell membranes (63,64). Therefore, a comprehensive evaluation of an endocrine disruptor requires a battery of complementary in vitro and in vivo assays that are based on receptor- and nonreceptor-mediated mechanisms.

There are a limited number of short-term, established in vivo assays that could be used to assess endocrine disruptors. However, it is questionable whether these assays alone can accurately identify and assess chemicals, natural products, environmental pollutants, and complex mixtures alleged to possess endocrine-disrupting activities. For example, two classical assays, namely the uterotrophic and vaginal cell cornification assays, are the most widely used in vivo assays for assessing estrogenic substances (58,65-70). Increases in uterine wet weight is an established measure of the estrogenicity of a compound and is the hallmark for the definition of an estrogen or the identification of an estrogenic substance (66,71). Previous studies examining the effects of estrogenic substances on uterine wet weight have used a number of different protocols and species; therefore, uterotrophic assays require standardized operating procedures that specify species, strain, age, and route of test compound administration in addition to other potential interventions (i.e., ovariectomy). For example, it has been reported that there are marked species differences in responsiveness and that the mouse uterus is much more sensitive to estrogens than the rat (66). In addition, although studies suggest that the uterotrophic assay exhibits greater sensitivity in immature, ovariectomized animals (69,72), the uterus also responds to progesterone, testosterone, and other agents that are not characteristically estrogenic, which can lead to confounding results (73-77).

In contrast, vaginal epithelial cell cornification in ovariectomized rodents can be induced only by compounds considered to be estrogenic. It is believed to be a definitive in vivo test for identifying estrogenic substances or complex mixtures (78). Although the assay has the advantage of being relatively simple and can use the same animals repeatedly provided the test compound does not bioaccumulate, it has been criticized as being largely qualitative as scoring is dependent on the evaluation of cellular contents of a vaginal lavage. In addition, the assay requires large numbers of animals to ensure accurate results (66,68). The qualitative nature of the assay has been somewhat addressed by introducing a grading system that involves scoring the degree of cornification using the disappearance of leukocytes and the appearance of cornified squamous cells (79).

There has also been some concern that short-term rodent assays may not possess sufficient sensitivity to identify substances and complex mixtures with weak or specific endocrine-disrupting activities. It is conceivable that endocrine disruptors may elicit responses at the gene expression level that may not be translated into immediate responses at the organ or tissue level but could subsequently predispose an individual or subpopulation to adverse effects at later stages of development. Assessment of endocrine disruptors is further complicated by the fact that many substances elicit species-, tissue-, cell-, and response-specific effects. For example, some estrogens are more effective for imbibition of uterine fluid, whereas others are more active in the promotion of uterine growth. Moreover, superior efficacy for one response does not indicate that the same rank order of potency will be exhibited for a different response (66). Another example is tamoxifen, which exhibits antiestrogenic activity in the breast and agonist activities in the uterus. These examples demonstrate the necessity of measuring a number of different end points in order to comprehensively evaluate the potential endocrine-disrupting activities of a substance or complex mixture.

The appropriateness of using rodents as models to assess the risk that endocrine disruptors pose to human and wildlife health has also been questioned as rodents do not express sex hormone-binding globulin (SHBG) following parturition. SHBG is a 17ß-estradiol-inducible circulating serum protein that exhibits significant changes in expression levels during development in all vertebrate species and is a major determinant of the metabolic clearance and the bioavailability of sex steroids (60,80,81). In addition, specific receptors for SHBG and ligand-bound SHBG have been identified in prostatic, placental, endometrial, and breast cells (82-91) that may be involved in a cAMP-dependent signaling pathway that induces cell growth (92-95). Intracellular SHBG has also been identified in these tissues, suggesting a physiologic function for this protein in cellular sex steroid uptake (88,90). Although it is unclear if endocrine disruptor interaction with SHBG plays a role in eliciting adverse effects, it is known that humans and some wildlife species express SHBG after parturition. Therefore, SHBG may be a potential target or protective measure against endocrine disruption that to date has not been adequately considered.

In Vitro Assays for Endocrine Disruptors

A number of in vitro assays are also based on known mechanisms of action of sex steroids. These include

• measuring the activity of enzymes involved in steroid synthesis (62,96,97);

• competitive ligand binding assays using binding globulins (61,98,99) and receptors (43,59,100);

• cell proliferation assays (101-104); and

• gene expression assays in mammalian cells and yeast (59,105-110).

However, many of these assays lack standardized operating procedures with suggested performance guidelines based on appropriate controls and proficiency samples. This is critical as in vitro assay performance can be fickle because of differences in media formulations, serum source, and cell line strains (59,111).

In addition, the ability to predict responses in vivo is questionable as it is not possible to accurately reproduce the in vivo pharmacokinetic and pharmacodynamic interactions in in vitro assays. For example, in vitro assays do not possess the same metabolic capabilities present in vivo and therefore may generate false positive results due to the inability to metabolically inactivate an estrogenic substance. This has been observed with selected phthalate esters that were found to induce weak estrogen receptor-mediated effects in vitro (112,113) but did not elicit a response in vivo, as evidenced by uterotrophic and vaginal cornification assays (113). Potentially more problematic are false negative results that are due to the inability of in vitro systems to bioactivate a proestrogen to its estrogenic metabolite. However, several in vitro systems possess some metabolic capabilities and, to date, there have been no reported examples of in vitro assays generating false negative results even with endocrine disruptors that are known to require bioactivation (i.e., methoxychlor, polychlorinated biphenyls) (41).

Summary

To comprehensively assess the potential endocrine disrupting activities of a substance or complex mixture, it is essential that a complementary battery of in vitro and in vivo assays be used. This battery could involve a tiered strategy consisting of computational models such as quantitative structure-activity relationship (QSAR), paradigms, in vitro assays and short-term in vivo assays in tier I, longer term in vivo assays in tier II, and if necessary, mulitigenerational studies in tier III. In this scheme, subsequent tier testing would be triggered following a review of the results obtained in the preceding tier. Uncertainties in this strategy arise when determining what constitutes sufficient data to warrant further testing. For example, there is no doubt that endocrine disruption in short-term in vivo studies in tier I would provide sufficient evidence to warrant further testing in tier II. However, the course of action may be less clear when there is a lack of an effect in vivo, but a response is observed using in vitro assays as well as positive predictions of endocrine-disrupting activities from QSAR models. Therefore, it may be prudent to establish guidelines that outline criteria which essentially exculpate a chemical or complex mixture that is suspected of eliciting endocrine-disrupting activities, in order to avoid a futile testing loop.

It is also clear that further research is required for the development of new in vitro and in vivo assays. Currently, there are inadequate in vitro and in vivo testing methodologies for several known potential targets such as the thyroid and androgen receptor systems. Moreover, there is a complete lack of knowledge regarding the impact of endocrine disruptors on other potential endocrine targets and mechanisms of action, including crosstalk between membrane-bound and nuclear receptors (114-117), the roles of new (118,119) and orphan receptors (120-122), and the effect on growth factor-mediated signal transduction. Needless to say, prior to the incorporation of any of these assays into a screening protocol, they should be subjected to a rigorous evaluation to determine their advantages and limitations, as well as to define how this information will be used in risk assessment and regulatory arenas.

References and Notes

1. Adami H, Bergstrom R, Mohner M, Zatonski W, Storm H, Ekbom A, Tretli S, Teppo L, Ziegler H, Rahu M. Testicular cancer in nine northern European countries. Int J Cancer 59:33-38 (1994).

2. Ahlborg UG, Lipworth L, Titus-Ernstoff L, Hsieh C-C, Hanberg A, Baron J, Trichopoulos D, Adami H-O. Organochlorine compounds in relation to breast cancer, endometrial cancer and endometriosis: an assessment of the biological and epidemiological evidence. Crit Rev Toxicol 25:463-531 (1995).

3. Forman D, Moller H. Testicular cancer. Cancer Surv 19/20:323-341 (1994).

4. Giwercman A, Skakkebaeck NE. The human testis--an organ at risk. Int J Androl 15:373-375 (1992).

5. Houghton DL, Ritter L. Organochlorine residues and risk of breast cancer. J Am Coll Toxicol 14:71-89 (1995).

6. Spitz MR, Sieder JG, Pollack ES, Lynch HK, Newell GR. Incidence and descriptive features of testicular cancer among United States whites, blacks and Hispanics. Cancer 58:1785-1790 (1986).

7. Osterlind A. Diverging trends in incidence and mortality of testicular cancer in Denmark, 1943-1982. Br J Cancer 53:501-505 (1986).

8. Wilkinson TJ, Colls BM, Schhluter PJ. Increased incidence of germ cell testicular cancer in New Zealand Maoris. Br J Cancer 65:769-771 (1992).

9. Newman PA. Breast cancer incidence is on the rise--but why? (News Report). J Natl Cancer Inst 82:998-1000 (1990).

10. Glass AG, Hoover RN. Rising incidence of breast cancer: relationship to stage and receptor status. J Natl Cancer Inst 82:693-696 (1990).

11. Carlsen E, Giwercman A, Keiding N, Skakkebaek NE. Evidence for decreasing quality of semen during past 50 years. Br Med J 305:609-613 (1992).

12. Colborn T, vom Saal FS, Soto AM. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ Health Perspect 101:378-384 (1993).

13. Giesy JP, Ludwig JP, Tillitt DE. Deformities in birds of the Great Lakes Region. Assigning causality. Environ Sci Technol 28:128A-135A (1994).

14. Jackson MB. The epidemiology of cryptorchidism. Hormone Res 30:153-156 (1988).

15. Sharpe RM. Another DDT connection. Nature 375:538-539 (1995).

16. Willet W. The search for the causes of breast and colon cancer. Nature 338:389-394 (1989).

17. Arai Y, Mori T, Suzuki Y, Bern HA. Long-term effects of perinatal exposure to sex steroids and diethylstilbestrol on the reproductive system of male mammals. Int Rev Cytol 84:235-268 (1983).

18. Herbst AL, Ulfelder H, Poskanzer DC. Adenocarcinoma of the vagina: association of maternal stilbestrol therapy with tumor appearance in young women. N Engl J Med 284:878-881 (1971).

19. Prener A, Hsieh C-C, Engholm G, Trichopoulos D, Jensen OM. Birth order and risk of testicular cancer. Cancer, Causes, Control 3:265-272 (1992).

20. Welsch CW. Hormones and murine mammary tumorgenesis: an historical view. In: Hormonal Regulation of Mammary Tumors (Leung BS, ed). Montreal:Eden Press, 1982;1-29.

21. Depue RH. Maternal and gestational factors affecting the risk of cryptorchidism and inguinal hernia. Int J Epidemiol 13:311-318 (1984).

22. Henderson BE, Ross R, Bernstein L. Estrogens as a cause of human cancer. Cancer Res 48:246-253 (1988).

23. Vessey MP. The involvement of estrogen in the development and progression of breast disease: epidemiological evidence. Proc R Soc Edinb 95B:35-48 (1989).

24. McLachlan JA, Newbold RR, Bullock B. Reproductive tract lesions in male mice prenatally exposed to diethylstilbestrol. Science 190:991-992 (1975).

25. McLachlan JA. Prenatal exposure to diethylstilbestrol in mice; toxicological studies. J Toxicol Environ Health 2:527-577 (1977).

26. Miller WR. Endocrine treatment for breast cancers: biological rationale and current progress. J Steroid Biochem Molec Biol 37:467-480 (1990).

27. Santen RJ, Manni A, Harvey H, Redmond C. Endocrine treatment of breast cancer in women. Endocr Rev 11:221-265 (1990).

28. Carter HB, Coffey DS. The prostate: an increasing medical problem. Prostate 16:39-48 (1990).

29. Chiarodo A. National Cancer Institute roundtable on prostate cancer: future research directions. Cancer Res 51:2498-2505 (1991).

30. Davis DL, Bradlow HL, Wolff M, Woodruff T, Hoel DG, Anton-Culver H. Medical hypothesis: xenoestrogens as preventable causes of breast cancer. Environ Health Perspect 101:372-377 (1993).

31. Davis DL, Bradlow HL. Can environmental estrogens cause breast cancer? Sci Am (October):167-172 (1995).

32. Falck F, Ricci A, Wolff MS, Godbold J, Deckers P. Pesticides and polychlorinated biphenyl residues in human breast lipids and their relation to breast cancer. Arch Environ Health 47:143-146 (1992).

33. Hileman B. Environmental estrogens linked to reproductive abnormalities, cancer. Chem Eng News 31:19-23 (1994).

34. Sharpe RM, Skakkebaek NE. Are estrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet 341:1392-1395 (1993).

35. Wolff MS, Toniolo PG, Lee EW, Rivera M, Dubin N. Blood levels of organochlorine residues and risk of breast cancer. J Natl Cancer Inst 85:648-652 (1993).

36. Martin PM, Horwitz KB, Ryan DS, McGuire WL. Phytoestrogen interaction with estrogen receptors in human breast cancer cells. Endocrinology 103:1860-1867 (1978).

37. Miksicek RJ. Commonly occurring plant flavonoids have estrogenic activity. Mol Pharmacol 44:37-43 (1993).

38. Verdeal K, Ryan D. Naturally-occurring estrogens in plant foodstuffs: a review. J Food Prot 42:577-583 (1979).

39. McLachlan JA, Korach KS, Newbold RR, Degen GH. Diethylstilbestrol and other estrogens in the environment. Fundam Appl Toxicol 4:686-691 (1984).

40. Clemons JH, Marvin CR, Wu ZF, McCarry BE, Allan L, Bryant DW, Zacharewski TR. Identification of estrogen- and dioxin-like activities in urban air particulate matter using recombinant receptor/reporter gene bioassays. In: 17th Annual Meeting of the Society of Environmental Toxicology and Chemistry, 17-21 November 1996, Washington, DC. Abstract 490. Pensacola, FL:SETAC Press, 1996;211.

41. Fielden M, Wu ZF, Chen I, Chittim B, Safe S, Zacharewski T. Examination of the estrogenicity of 2,2´,4,6,6´-pentachlorobiphenyl (PCB104) and its hydroxylated and chlorinated derivatives, 2,2´,4,6,6-pentachloro-4-biphenylol and 2,2´,4,4´,6,6´-hexachlorobiphenyl (PCB155). Toxicol Appl Pharmacol (Suppl) 36:159 (810) (1997).

42. Jansen HT, Cooke PS, Porcelli J, Liu T-C, Hansen LG. Estrogenic and antiestrogenic actions of PCBs in the female rat: in vitro and in vivo studies. Reprod Toxicol 7:237-248 (1993).

43. Kelce WR, Stone CR, Laws SC, Gray LE, Kemppalnen JA, Wilson EM. Persistent DDT metabolite p,p´-DDE is a potent androgen receptor antagonist. Nature 375:581-585 (1995).

44. Safe S. Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Pharmacol Ther 67:247-281 (1995).

45. Korach K. Editorial: Surprising places of estrogenic activity. Endocrinology 132:2277-2278 (1993).

46. Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132:2279-2286 (1993).

47. Nimrod AC, Benson WH. Environmental estrogenic effects of alkylphenol ethoxylates. Crit Rev Toxicol 26:335-364 (1996).

48. Wilcox AJ, Baird DD, Weinberg CR, Hornsby PP, Herbst AL. Fertility in men exposed prenatally to diethylstilbestrol. N Engl J Med 332:1411-1416 (1995).

49. Fisch H, Goluboff ET, Olson JH, Feldshuh J, Broder SJ, Barad DH. Semen analyses in 1,283 men from the United States over a 25-year period: no decline in quality. Fertil Steril 65:1009-1014 (1996).

50. Fisch H, Goluboff ET. Geographic variation in sperm counts: a potential cause of bias in studies of semen quality. Fertil Steril 65:1044-1046 (1996).

51. Paulsen CA, Berman NG, Wang C. Data from men in greater Seattle area reveals no downward trend in semen quality: further evidence that deterioration of semen quality is not geographically uniform. Fertil Steril 65:1015-1020 (1996).

52. Adlercreutz H, Fotsis T, Heikkinen R, Dwyer JT, Woods M, Goldin BR, Gorbach SL. Excretion of the lignans enterolactone and enterodiol and of equol in omniverous and vegetarian women and women with breast cancer. Lancet 11:1295-1299 (1982).

53. Messina MJ, Persky V, Setchell KDR, Barnes S. Soy intake and cancer risk: a review of in vitro and in vivo data. Nutr Cancer 21:113-131 (1994).

54. Barnes S, Grubbs C, Setchell KDR, Carlson J. Soybeans inhibit mammary tumors in models of breast cancer. In: Mutagens and Carcinogens in the Diet (Pariza M, ed). New York:Wiley-Liss, 1990;239-253.

55. Hirano T, Fukuoka K, Oka K, Naito T, Hosaka K, Mitsuhashi H, Matsumoto Y. Antiproliferative activity of mammalian lignan derivatives against the human breast cell line, ZR-75-1. Cancer Invest 8:595-601 (1990).

56. Safe SH. Environmental and dietary estrogens and human health: is there a problem? Environ Health Perspect 103:346-351 (1995).

57. ECETOC. ECETOC Document 33 "Environmental Oestrogens". Compendium of Test Methods 33. Brussels:European Centre for Ecotoxicology and Toxicology of Chemicals, 1997.

58. Reel JR, Lamb JC, Neal BH. Survey and assessment of mammalian estrogen biological assays for hazard characterization. Fundam Appl Toxicol 34:288-305 (1996).

59. Zacharewski T. In vitro assays used to assess estrogenic substances. Environ Sci Tech 31:613-623 (1997).

60. Hammond GL. Molecular properties of corticoid binding globulin and the sex-steroid binding proteins. Endocr Rev 11:65-79 (1990).

61. Lans MC, Klasson-Wehler E, Willemsen M, Meussen E, Safe S, Brouwer A. Structure-dependent, competitive interaction of hydroxy-polychlorobiphenyls, -dibenzo-p-dioxins and -dibenzofurans with human transthyretin. Chem-Biol Interact 88:7-21 (1993).

62. Khalil MW, Morley P, Glasier MA, Armstrong DT, Lang T. Formation of 4-oestrone-3,17-dione (19-norandrostenedione) by porcine granulosa cells in vitro is inhibited by the aromatase inhibitor 4-hydroxyandrostenedione and the cytochrome P-450 inhibitors aminoglutethimide phosphate and ketoconazole. J Endocrinol 120:251-260 (1989).

63. Pappas TC, Gametchu B, Watson CS. Membrane estrogen receptors identified by multiple antibody labelling and impeded-ligand binding. FASEB J 9:404-410 (1995).

64. Watson CS, Pappas TC, Gametchu B. The other estrogen recil Steril 24:284-291 (1973).

68. Lauson HD, Heller CG, Golden JB, Sevringhaus EL. The immature rat uterus in the assay of estrogenic substances and a comparison of estradiol, estrone and estriol. Endocrinology 24:35-44 (1939).

69. Martin L, Claringbold PJ. The mitogenic action of oestrogens in the vaginal epithelium of the ovariectomized mouse. J Endocrinol 20:173-186 (1960).

70. Rubin BL, Dorfman AS, Black L, Dorfman RI. Bioassay of estrogens using the mouse uterine response. Endocrinology 49:429-439 (1951).

71. Astwood EB. Estrogens and progestins. In: The Pharmacological Basis of Therapeutics. 4th Ed (Goodman LS, Gilamn A, eds). Toronto:The MacMillan Company, 1970;1538-1565.

72. Gellert RJ. Kepone, mirex, dieldrin and aldrin: estrogenic activity and the induction of persistent vaginal estrus and anovulation in rats following neonatal treatment. Environ Res 16:131-138 (1978).

73. Clark JH, Watson C, Upchurch S, McCormack S, Padykula H, Markaverich B, Hardin JW. Estrogen action in normal and abnormal cell growth. In: Estrogens in the Environment (McLachlan JA, ed). New York:Elsevier, 1980;53-67.

74. Drill VA. Endocrine properties and long-term safety of oral contraceptives. Metabolism 14:295-301 (1965).

75. Emmens CW, Martin L. Estrogens. In: Methods in Hormone Research, Vol III (Dorfman RI, ed). New York:Academic Press, 1964;1-80.

76. Korach KS, McLachlan JA. Techniques for detection of estrogenicity. Environ Health Perspect 103 (Suppl 7):5-8 (1995).

77. Nelson JA, Struck RF, James R. Estrogenic activities of chlorinated hydrocarbons. J Toxicol Environ Health 4:325-339 (1978).

78. Drill VA. Biological properties. In: Oral Contraceptives. New York:Mcgraw-Hill, 1966;16-43.

79. Terenius L. The Allen-Doisy test for estrogens reinvestigated. Steroids 17:653-661 (1971).

80. Rosner W. The functions of corticoid-binding globulin and sex hormone-binding globulin: recent advances. Endocr Rev 11:80-91 (1990).

81. Strel'chyonok OA, Avvakumov GV. Specific steroid-binding glycoproteins of human blood plasma: novel data on their structure and function. J Steroid Biochem 35:519-534 (1990).

82. Fortunati N, Fissore F, Fazzari A, Berta L, Giudici M, Frairia R. Sex steroid-binding protein interacts with a specific receptor on human premenopausal endometrium: modulating effect of estradiol. Steroids 56:341-346 (1991).

83. Fortunati N, Fissore F, Fazzari A, Berta L, Varvello L, Frairia R. The receptor for sex steroid-binding protein (SBP) of endometrial membranes: solubilization, partial characterization, and role of estradiol in SBP-soluble receptor interaction. Steroids 57:464-470 (1992).

84. Fortunati N, Frairia R, Fissore F, Berta L, Fazzari A, Gaidano G. The receptor for human sex steroid binding protein (SBP) is expressed on membranes of neoplastic endometrium. J Steroid Biochem Mol Biol 42:185-191 (1992).

85. Fortunati N, Fissore F, Fazzari A, Berta L, Benedusi-Pagliano E, Frairia R. Biological relevance of the interaction between sex steroid binding protein and its specific receptor on MCF-7 cells: effect on the estradiol-induced cell proliferation. J Steroid Biochem Mol Biol 45:435-444 (1993).

86. Hryb DJ, Kahn MS, Rosner W. Testosterone-estradiol-binding globulin binds to human prostatic cell membranes. Biochem Biophys Res Commun 128:432-466 (1985).

87. Hryb DJ, Kahn MS, Romas NA, Rosner W. Solubilization and partial characterization of the sex steroid binding globulin receptor from human prostate. J Biol Chem 264:5378-5383 (1989).

88. Hryb DJ, Kahn MS, Romas NA, Rosner W. The control of the interaction of sex hormone-binding globulin with its receptor by steroid hormones. J Biol Chem 265:6048-6054 (1990).

89. Krupenko NT, Avvakumov GV, Strel'chyonok OA. Binding of human sex hormone-binding globulin-androgen complexes to the placental synctiotrophoblast membrane. Biochem Biophys Res Commun 71:1279-1283 (1990).

90. Porto CS, Gunsalus GL, Bardin CW, Phillips DM, Musto NA. Receptor-mediated endocytosis of an extracellular steroid-binding protein (TeBG) in MCF-7 human breast cancer cells. Endocrinology 129:436-445 (1991).

91. Strel'chyonok OA, Avvakumov GV, Survilo LI. A recognition system for sex-hormone-binding protein-estradiol complex in human decidua endometrial membranes. Biochim Biophys Acta 802:459-466 (1984).

92. Lewin DL. From outside or in, sex hormones tweak prostate cells. J NIH Res 8:29-30 (1996).

93. Nakhla AM, Kahn MS, Rosner W. Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3´,5´-monophosphate. J Clin Endocrinol Metab 71:398-404 (1990).

94. Nakhla AM, Khan MS, Romas NP, Rosner W. Estradiol causes the rapid accumulation of cAMP in human prostate. Proc Natl Acad Sci 91:5402-5405 (1994).

95. Nakhla AM, Rosner W. Stimulation of prostate cancer growth by androgens and estrogens through the intermediacy of sex hormone-binding globulin. Endocrinology 137:4126-4129 (1996).

96. Laskey J, Berman E. Steroidogenic assessment using ovary culture in cycling rats: effects of bis(2-diethylhexyl) phthalate on ovarian steroid production. Reprod Toxicol 7:25-33 (1993).

97. Laskey JW, Klinefelter GR, Kelce WR, Ewing LL. Effects of ethane dimethanesulfonate on adult and immature rabbit Leydig cells: comparison with EDS treated rat Leydig cells. Biol Reprod 50:1151-1160 (1994).

98. Eil C, Nisula BC. The binding properties of pyrethroids to human skin fibroblast androgen receptors and to sex hormone binding globulin. J Steroid Biochem 35:409-414 (1990).

99. Hammond GL, Lahteenmaki PLA. A versatile method for the determination of serum cortisol globulin and sex hormone binding globulin binding capacities. Clin Chim Acta 132:101-110 (1983).

100. Wilson EM, French FS. Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis and prostate. J Biol Chem 251:5620-5629 (1976).

101. Damassa DA, Lin T-M, Sonnenschein C, Soto AM. Biological effects of sex hormone-binding globulin on androgen-induced proliferation and androgen metabolism in LNCaP prostate cells. Endocrinology 129:75-84 (1991).

102. Gierthy JF, Lincoln DW, Roth KE, Bowser SS, Bennett JA, Bradley L, Dickerman HW. Estrogen-stimulation of postconfluent cell accumulation and foci formation of human MCF-7 breast cancer cells. J Cell Biochem 45:177-187 (1991).

103. Soto AM, Lin T-M, Justicia H, Silvia RM, Sonnenschein C. An "in culture" bioassay to assess the estrogenicity of xenobiotics (E-Screen). Adv Mod Environ Toxicol 21:295-309 (1992).

104. Soto AM, Sonnenschein C, Chung LK, Fernandez MF, Olea N, Olea-Serrano MF. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ Health Perspect 103(Suppl 7):113-122 (1995).

105. Arnold SF, Robinson MK, Notides AC, Guillette LJ Jr., McLachlan JA. A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens. Environ Health Perspect 104:544-548 (1996).

106. Balaguer P, Joyeux A, Denison M, Vincent R, Gillesby B, Zacharewski T. Assessing the estrogenic and dioxin-like activities of chemicals and complex mixtures using in vitro recombinant receptor/reporter gene assays. Can J Physiol Pharmacol 74:216-222 (1996).

107. Connor K, Howell J, Chen I, Liu H, Berhane K, Sciarretta C, Safe S, Zacharewski T. Failure of chloro-S-triazine-derived compounds to induce estrogen receptor-mediated responses in vivo and in vitro. Fundam Appl Toxicol 30:93-101 (1995).

108. Moore M, Mustain M, Daniel K, Chen I, Safe S, Zacharewski T, Gillesby B, Joyeux A, Balaguer P. Antiestrogenic activity of hydroxylated polychlorinated biphenyl congeners identified in human serum. Toxicol Appl Pharmacol 142:160-168 (1997).

109. White R, Jobling S, Hoare SA, Sumpter JP, Parker MG. Environmentally persistant alkylphenolic compounds are estrogenic. Endocrinology 135:175-182 (1994).

110. Zacharewski T, Berhane K, Gillesby B, Burnison K. Detection of estrogen- and dioxin-like activity in pulp and paper mill black liquor effluent using in vitro recombinant receptor/reporter gene assays. Environ Sci Tech 29:2140-2146 (1995).

111. Wiese TE, Kral LG, Dennis KE, Butler WB, Brooks SC. Optimization of estrogen growth response in MCF-7 cells. In Vitro Cell Dev Biol 28A:595-602 (1992).

112. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally persistent chemicals, including some plasticizers, are weakly estrogenic. Environ Health Perspect 103:582-587 (1995).

113. Meek MD, Clemons J, Wu ZF, Zacharewski T. Assessment of the alleged estrogen-receptor mediated activity of eight commercial phthalate esters. Fundam Appl Toxicol 36(Suppl):295 (Abstract 1500) (1997).

114. Ignar-Trowbridge DM, Nelson KG, Bidwell MC, Curtis SW, Washburn TF, McLachlan JA, Korach KS. Coupling of dual signaling pathways: epidermal growth factor action involves estrogen receptor. Proc Natl Acad Sci USA 89:4658-4662 (1992).

115. Nazareth LV, Weigel NL. Activation of the human androgen receptor through a protein kinase A signaling pathway. J Biol Chem 271:19900-19907 (1996).

116. Mani SK, Allen JM, Clark JH, Blaustein JD, O'Malley BW. Convergent pathways for steroid hormone- and neurotransmitter-induced rat sexual behavior Science 265:1246-1249 (1994). [Published erratum appears in Science (1995) 268:1833].

117. Shalev A, Siegrist-Kaiser CA, Yen PM, Wahli W, Burger AG, Chin WW, Meier CA. The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin. Endocrinology 137:4499-4502 (1996).

118. Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature 383:728-731 (1996).

119. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A. Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925-5930 (1996).

120. Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW, Abbud R, Nilson JH, Parker KL. The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:2302-12 (1994).

121. Luo X, Ikeda Y, Schlosser DA, Parker KL. Steroidogenic factor 1 is the essential transcript of the mouse Ftz-F1 gene. Mol Endocrinol 9:1233-9 (1995).

122. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM. The nuclear receptor superfamily: the second decade. Cell 83:835-839 (1995).

Last Update: April 28, 1998