In vitro models in endocrine disruptor screening.

The public and scientific concern that chemicals present in the human diet and the environment and their ability to disrupt the normal hormonal milieu in humans and wildlife have become a high-profile international issue. In 1998, the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) convened by the Environmental Protection Agency (EPA) recommended a tiered testing approach for the evaluation of estrogen, androgen, and thyroid-related effects of some 87,000 commercial chemicals and environmental contaminants. The function of this committee concluded with its final report, and the further implementation of the recommended testing strategy has now been carried forward with the assistance of the Endocrine Disruptor Methods Validation Subcommittee. The function of this body is to provide advice to the EPA on scientific and technical issues related specifically to the conduct of studies required for the validation of assays proposed by the EDSTAC as part of the tiered screening program. The EDSTAC recommended and alternative screening batteries encompass four in vitro mammalian assays. The current methodologies and validation status of the proposed in vitro EDSTAC assays are discussed and consist of estrogen/androgen receptor binding, estrogen/androgen gene transactivation, and minced testis, and one alternate (placental aromatase) in vitro screening assay.

[1]  B. Ozenberger,et al.  Selective uptake of estrogenic compounds by Saccharomyces cerevisiae: a mechanism for antiestrogen resistance in yeast expressing the mammalian estrogen receptor. , 1995, Endocrinology.

[2]  L. Gray,et al.  Persistent DDT metabolite p,p'–DDE is a potent androgen receptor antagonist , 1995, Nature.

[3]  E. Berman,et al.  The use of cultured ovarian fragments to assess toxicant alterations in steroidogenesis in the Sprague-Dawley rat. , 1995, Reproductive toxicology.

[4]  Martin van den Berg,et al.  A comparison of human H295R and rat R2C cell lines as in vitro screening tools for effects on aromatase. , 2004, Toxicology letters.

[5]  Y. Nishi,et al.  Printed in U.S.A. Copyright © 2001 by The Endocrine Society Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor , 2000 .

[6]  G. Cooke.  Effect of organotins on human aromatase activity in vitro. , 2002, Toxicology letters.

[7]  M. van den Berg,et al.  Effects of some persistent halogenated environmental contaminants on aromatase (CYP19) activity in the human choriocarcinoma cell line JEG-3. , 1998, Toxicology and applied pharmacology.

[8]  L. Gray,et al.  A novel cell line, MDA-kb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists. , 2002, Toxicological sciences : an official journal of the Society of Toxicology.

[9]  J. Cook,et al.  Ex vivo and in vitro testis and ovary explants: utility for identifying steroid biosynthesis inhibitors and comparison to a Tier I screening battery. , 1998, Toxicological sciences : an official journal of the Society of Toxicology.

[10]  L. Gray,et al.  Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. , 2002, Toxicological sciences : an official journal of the Society of Toxicology.

[11]  F. Orio,et al.  A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects , 2000, Molecular and Cellular Endocrinology.

[12]  B. Katzenellenbogen,et al.  Synergistic activation of estrogen receptor-mediated transcription by estradiol and protein kinase activators. , 1993, Molecular endocrinology.

[13]  B. O’Malley,et al.  Ligand-dependent and -independent function of the transactivation regions of the human estrogen receptor in yeast. , 1992, Molecular endocrinology.

[14]  J. Tesarik,et al.  Nongenomic actions of steroid hormones in reproductive tissues. , 1998, Endocrine reviews.

[15]  S. Serizawa,et al.  Estrogenic and thyroid hormone activity of a series of hydroxy-polychlorinated biphenyls. , 2003, Chemosphere.

[16]  J. Ashby,et al.  Assessment of the effects of metabolism on the estrogenic activity of xenoestrogens: a two-stage approach coupling human liver microsomes and a yeast estrogenicity assay. , 2001, The Journal of pharmacology and experimental therapeutics.

[17]  R. Evans,et al.  The steroid and thyroid hormone receptor superfamily. , 1988, Science.

[18]  R. Chapin,et al.  Six high-priority organochlorine pesticides, either singly or in combination, are nonestrogenic in transfected HeLa cells. , 2000, Reproductive toxicology.

[19]  Martin van den Berg,et al.  Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. , 2002, Toxicology and applied pharmacology.

[20]  M Sue Marty,et al.  Evaluation of Tier I Screening Approaches for Detecting Endocrine-Active Compounds (EACs) , 2002, Critical reviews in toxicology.

[21]  Lin Yang,et al.  Activation of Phosphatidylinositol 3-Kinase/Akt Pathway by Androgen through Interaction of p85α, Androgen Receptor, and Src* , 2003, Journal of Biological Chemistry.

[22]  I. Nakatsuka,et al.  Lack of significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor alpha-mediated mechanisms. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[23]  W. Schoonen,et al.  Effects of two classes of progestagens, pregnane and 19-nortestosterone derivatives, on cell growth of human breast tumor cells: I. MCF-7 cell lines , 1995, The Journal of Steroid Biochemistry and Molecular Biology.

[24]  J. Fishman,et al.  Mechanism of estrogen biosynthesis. Stereochemistry of C-1 hydrogen elimination in the aromatization of 2 beta-hydroxy-19-oxoandrostenedione. , 1981, The Journal of biological chemistry.

[25]  S. Safe,et al.  Differential Interaction of the Methoxychlor Metabolite 2,2-Bis-(p-Hydroxyphenyl)-1,1,1-Trichloroethane with Estrogen Receptors α and β1. , 1999, Endocrinology.

[26]  R. McPherson,et al.  Linkage of Rapid Estrogen Action to MAPK Activation by ER-Shc Association and Shc Pathway Activation , 2001 .

[27]  M E Hurtt,et al.  Effect of the peroxisome proliferator, ammonium perfluorooctanoate (C8), on hepatic aromatase activity in adult male Crl:CD BR (CD) rats. , 1996, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[28]  C. Corbin,et al.  Structural and functional differences among purified recombinant mammalian aromatases: glycosylation, N-terminal sequence and kinetic analysis of human, bovine and the porcine placental and gonadal isozymes , 2003, Molecular and Cellular Endocrinology.

[29]  B. Bhavnani,et al.  Applicability of the product isolation and the radiometric aromatase assays for the measurement of low levels of aromatase: lack of aromatase activity in the human endometrium. , 1990, The Journal of endocrinology.

[30]  J. Sumpter,et al.  Effects of flavonoids on aromatase activity, an in vitro study , 1996, The Journal of Steroid Biochemistry and Molecular Biology.

[31]  R. Muccitelli,et al.  Interactions of methoxychlor, methoxychlor base-soluble contaminant, and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane with rat uterine estrogen receptor. , 1978, Journal of toxicology and environmental health.

[32]  B. Bhaskar Gollapudi,et al.  Incorporation of S-9 activation into an ER-α transactivation assay☆ , 2000 .

[33]  M D Shelby,et al.  Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. , 1996, Environmental health perspectives.

[34]  Jaap Keijer,et al.  Development of a rapid yeast estrogen bioassay, based on the expression of green fluorescent protein. , 2004, Gene.

[35]  D. W. Bryant,et al.  Evidence of Estrogen- and TCDD-Like Activities in Crude and Fractionated Extracts of PM10 Air Particulate Material Using in Vitro Gene Expression Assays , 1998 .

[36]  K. Kostial,et al.  Assessment of steroid disruption using cultures of whole ovary and/or placenta in rat and in human placental tissue , 2002, International archives of occupational and environmental health.

[37]  E. Simpson,et al.  Assay of aromatase activity. , 1991, Methods in enzymology.

[38]  T. Zacharewski,et al.  Activity of benzo[a]pyrene and its hydroxylated metabolites in an estrogen receptor-alpha reporter gene assay. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[39]  R. Pieters,et al.  AHTN and HHCB show weak estrogenic--but no uterotrophic activity. , 1999, Toxicology Letters.

[40]  A M Vinggaard,et al.  Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. , 2000, Toxicology in vitro : an international journal published in association with BIBRA.

[41]  Anne Marie Vinggaard,et al.  Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. , 2002, Toxicology and applied pharmacology.

[42]  S. Bulun,et al.  Aromatase gene expression in adipose tissue: Relationship to breast cancer , 1994, The Journal of Steroid Biochemistry and Molecular Biology.

[43]  T. Santa,et al.  Study on interactions of endocrine disruptors with estrogen receptor using fluorescence polarization. , 2003, The Analyst.

[44]  W. Wahli,et al.  Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements. , 1995, Molecular endocrinology.

[45]  T. Sugawara,et al.  Development of a simple screening system for endocrine disruptors. , 2002, Medical science monitor : international medical journal of experimental and clinical research.

[46]  Zbigniew Dauter,et al.  Molecular basis of agonism and antagonism in the oestrogen receptor , 1997, Nature.

[47]  Y. Osawa,et al.  Aromatization of 16α-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[48]  J. Gustafsson,et al.  Cloning of a novel receptor expressed in rat prostate and ovary. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[49]  S. Imazato,et al.  Estrogenicity of Fissure Sealants and Adhesive Resins Determined by Reporter Gene Assay , 2000, Journal of dental research.

[50]  R. Pieters,et al.  Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. , 1999, Toxicology and applied pharmacology.

[51]  Alexius Freyberger,et al.  Development and standardization of a simple binding assay for the detection of compounds with affinity for the androgen receptor. , 2004, Toxicology.

[52]  M E Hurtt,et al.  Effects of ammonium perfluorooctanoate on Leydig cell function: in vitro, in vivo, and ex vivo studies. , 1995, Toxicology and applied pharmacology.

[53]  T. Wiese,et al.  Rapid screening of environmental chemicals for estrogen receptor binding capacity. , 1998, Environmental health perspectives.

[54]  R. Turner,et al.  Raloxifene inhibits bone turnover and prevents further cancellous bone loss in adult ovariectomized rats with established osteopenia. , 1996, Endocrinology.

[55]  K. Sumida,et al.  An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes. , 2001, Biochemical and biophysical research communications.

[56]  J. Corton,et al.  Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor β. , 1998, Endocrinology.

[57]  K. Sumida,et al.  Limited species differences in estrogen receptor alpha-medicated reporter gene transactivation by xenoestrogens , 2003, The Journal of Steroid Biochemistry and Molecular Biology.

[58]  S. Ohta,et al.  Metabolic activation of bisphenol A by rat liver S9 fraction. , 2001, Toxicological sciences : an official journal of the Society of Toxicology.

[59]  S. O. Mueller,et al.  Activation of estrogen receptor α and ERβ by 4-methylbenzylidene-camphor in human and rat cells: comparison with phyto- and xenoestrogens , 2003 .

[60]  S. Sikka,et al.  In vitro inhibition of testosterone biosynthesis by ketoconazole. , 1985, Endocrinology.

[61]  J. Caillaud,et al.  Pig Leydig cell culture: a useful in vitro test for evaluating the testicular toxicity of compounds. , 1991, Toxicology and applied pharmacology.

[62]  C. Gennings,et al.  Optimization of an estrogen receptor‐α transcriptional activation assay for testing a diverse spectrum of chemicals , 2000, Journal of applied toxicology : JAT.

[63]  P. Argos,et al.  Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A , 1986, Nature.

[64]  James C. Lamb,et al.  Regular ArticleSurvey and Assessment of Mammalian Estrogen Biological Assays for Hazard Characterization , 1996 .

[65]  L. G. Davis,et al.  Evaluation of a Tier I screening battery for detecting endocrine-active compounds (EACs) using the positive controls testosterone, coumestrol, progesterone, and RU486. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[66]  J. Gustafsson,et al.  Update on estrogen signaling , 2003, FEBS letters.

[67]  M. Denison,et al.  Recombinant cell bioassays for endocrine disruptors: development of a stably transfected human ovarian cell line for the detection of estrogenic and anti-estrogenic chemicals. , 2000, In vitro & molecular toxicology.

[68]  S. D. Turner,et al.  A high-throughput screen to identify inhibitors of aromatase (CYP19). , 2000, Analytical biochemistry.

[69]  J R Reel,et al.  Survey and assessment of mammalian estrogen biological assays for hazard characterization. , 1996, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[70]  F. S. French,et al.  The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate. , 1988, Molecular endocrinology.

[71]  J. Laskey,et al.  In vitro/in vivo effects of ethane dimethanesulfonate on Leydig cells of adult rats. , 1991, Toxicology and applied pharmacology.

[72]  N. Itoh,et al.  Trialkyltin compounds enhance human CG secretion and aromatase activity in human placental choriocarcinoma cells. , 2002, The Journal of clinical endocrinology and metabolism.

[73]  H. Sauerwein,et al.  Development of an immuno-immobilized androgen receptor assay (IRA) and its application for the characterization of the receptor binding affinity of different pesticides. , 2002, Chemosphere.

[74]  Y. Osawa,et al.  Catalytic differences between porcine blastocyst and placental aromatase isozymes. , 2000, European journal of biochemistry.

[75]  H Fang,et al.  The estrogen receptor relative binding affinities of 188 natural and xenochemicals: structural diversity of ligands. , 2000, Toxicological sciences : an official journal of the Society of Toxicology.

[76]  A D Vethaak,et al.  Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. , 1999, Toxicological sciences : an official journal of the Society of Toxicology.

[77]  T. Zacharewski,et al.  Ability of structurally diverse natural products and synthetic chemicals to induce gene expression mediated by estrogen receptors from various species , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[78]  L. Gray,et al.  Environmental hormone disruptors: evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. , 1994, Toxicology and applied pharmacology.

[79]  S. Hill,et al.  Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays. , 1996, Environmental health perspectives.

[80]  K. Gaido,et al.  Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. , 1998, Toxicology and applied pharmacology.

[81]  G. Greene,et al.  Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha and ERbeta expressed in Chinese hamster ovary cells. , 1999, Molecular endocrinology.

[82]  L. Gray,et al.  Hamster Leydig cells are less sensitive to ethane dimethanesulfonate when compared to rat Leydig cells both in vivo and in vitro. , 1995, Toxicology and applied pharmacology.

[83]  T. Zacharewski,et al.  Hydroxylated benzo[a]pyrene metabolites are responsible for in vitro estrogen receptor-mediated gene expression induced by benzo[a]pyrene, but do not elicit uterotrophic effects in vivo. , 2001, Toxicological sciences : an official journal of the Society of Toxicology.

[84]  C. Portier,et al.  Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. , 1997, Toxicology and applied pharmacology.

[85]  J. Larsen,et al.  Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic effects of environmental chemicals. , 1999, Toxicology and applied pharmacology.

[86]  J. Sumpter,et al.  Structural Features of Alkylphenolic Chemicals Associated with Estrogenic Activity* , 1997, The Journal of Biological Chemistry.

[87]  J. Toppari,et al.  Endocrine, paracrine and autocrine regulation of testicular steroidogenesis. , 1995, Advances in experimental medicine and biology.

[88]  Seiji Amano,et al.  Bone anabolic effects of S-40503, a novel nonsteroidal selective androgen receptor modulator (SARM), in rat models of osteoporosis. , 2003, Biological & pharmaceutical bulletin.

[89]  E. Wilson,et al.  Androgen Receptor Antagonist versus Agonist Activities of the Fungicide Vinclozolin Relative to Hydroxyflutamide (*) , 1995, The Journal of Biological Chemistry.

[90]  T. Zacharewski.  In Vitro Bioassays for Assessing Estrogenic Substances , 1997 .

[91]  S. Safe,et al.  Differential activation of wild-type and variant forms of estrogen receptor α by synthetic and natural estrogenic compounds using a promoter containing three estrogen-responsive elements , 2001, The Journal of Steroid Biochemistry and Molecular Biology.

[92]  C. A. Harris,et al.  Issues arising when interpreting results from an in vitro assay for estrogenic activity. , 2000, Toxicology and applied pharmacology.

[93]  J. Ashby.  Validation of In Vitro and In Vivo Methods for Assessing Endocrine Disrupting Chemicals , 2000, Toxicologic pathology.