ABSTRACT A diverse range of endocrine disrupting chemicals (EDCs) was examined, using an in vitro test system, for critical events required for the onset of carcinogenesis in vivo. The initiation stage of carcinogenesis is a genotoxic process. 4-Octylphenol (alkylphenol), bisphenol A (plasticiser), coumestrol and genistein (phytoestrogens), 2,4-dichlorophe- noxyacetic acid and toxaphene (pesticides) and ethinylestradiol (synthetic hormone) were investigated for potential mutagencicity, DNA strand breakage, clastogenicity and DNA repair. Significant induction in the percentage of cells containing micronuclei was observed for all the EDCs. Toxaphene and coumestrol were mutagenic in the Ames assay. They also induced significant levels of unscheduled DNA synthesis and DNA strand breakage. Bisphenol A induced low level DNA strand breakage in HepG2 cells in the comet assay. The EDCs, with the exception of toxaphene, induced transcriptional activation in the yeast estrogen screen (YES) assay. They were potently oestrogenic in the mammalian based MVLN (transactivation) and E-SCREEN (proliferation) assays. This report on the transactivational, proliferative and genotoxic ability of the EDCs suggests that these chemicals may play a role in the etiology of male and female reproductive cancers.
Cite this paper
K. Quinn-Hosey, J. Roche, A. Fogarty and C. Brougham, "Screening for Genotoxicity and Oestrogenicity of Endocrine Disrupting Chemicals in Vitro," Journal of Environmental Protection, Vol. 3 No. 8, 2012, pp. 902-914. doi: 10.4236/jep.2012.328105.
 J. D. Yager and J. G. Liehr, “Molecular Mechanisms of Estrogen Carcinogenesis,” Annual Review of Pharmacology and Toxicology, Vol. 36, 1996, pp. 203-232. doi:10.1146/annurev.pa.36.040196.001223
 J. Russo and I. H. Russo, “The Role of Estrogen in the Initation of Breast Cancer,” The Journal of Steroid Biochemistry and Molecular Biology, Vol. 102, No. 1-5, 2006, pp. 89-96. doi:10.1016/j.jsbmb.2006.09.004
 J. R. Karavan and M. E. Pepling, “Effects of Estrogenic Compounds on Neonatal Oocyte Development,” Reproductive Toxicology, Vol. 34, No. 1, 2012, pp. 51-56. doi:10.1016/j.reprotox.2012.02.005
 J. Matthews, T. Celius, R. Halgren and T. Zacharewski, “Differential Estrogen Receptor Binding of Estrogenic Substances: A Species Comparison,” The Journal of Steroid Biochemistry and Molecular Biology, Vol. 74, No. 4, 2000, pp. 223-234. doi:10.1016/S0960-0760(00)00126-6
 K. Morito, T. Hirose, J. Kinjo, T. Hirakawa, M. Okawa, T. Nohara, S. Ogawa, S. Inoue, M. Muramatsu and Y. Masamune, “Interaction of Phytoestrogens with Estrogen Receptors α and β,” Biological and Pharmaceutical Bulletin, Vol. 24, No. 4, 2001, pp. 351-356. doi:10.1248/bpb.24.351
 J. C. Gould, L. S. Leonard, S. C. Maness, B. L. Wagner, K. Conner, T. Zacharewski, S. Safe, D. P. McDonnell and K. W. Gaido, “Bisphenol A Interacts with the Estrogen Receptor α in a Distinct Manner from Estradiol,” Molecular and Cellular Endocrinology, Vol. 142, No. 1-2, 1998, pp. 203-214. doi:10.1016/S0303-7207(98)00084-7
 C. Wohlfahrt-Veje, H. R. Andersen, I. M. Schmidt, L. Aks-glaede, K. Sorensen, A. Juul, T. K. Jensen, P. Grand- jean, N. E. Skakkebaekand and K. M. Main, “Early Breast Development in Girls after Prenatal Exposure to Non-Persistent Pesticides,” International Journal of Andrology, Vol. 35, No. 3, 2012, pp. 273-282. doi:10.1111/j.1365-2605.2011.01244.x
 C. R. Jefcoate, J. G. Liehr, R. J. Santen, T. R. Sutter, J. D. Yager, W. Yue, S. J. Santner, R. Tekmal, L. Demers, R. Pauley, F. Naftolin, G. Mor and L. Berstein, “Tissue-Specific Synthesis and Oxidative Metabolism of Estrogens,” Journal of National Cancer Institute Monographs, Vol. 27, 2000, pp. 95-112.
 A. M. Soto, C. Sonnenschein, K. L. Chung, M. F. Fernandez, N. Olea and F. Serrano, “The E-SCREEN Assay as a Tool to Identify Estrogens: An Update on Estrogenic Environmental Pollutants,” Environmental Health Perspect, Vol. 103, 1995, pp. 113-122. doi:10.1289/ehp.95103s7113
 M. Pons, D. Gagne, J. C. Nicolas and M. Mehtali, “A New Cellular Model of Response to Estrogens: A Bioluminescent Test to Characterise (anti) Estrogen Molecules,” Biotechniques, Vol. 9, 1990, pp. 450-459.
 E. J. Routledge and J. P. Sumpter, “Estrogenic Activity of Surfactants and Some of Their Degradation Products Assessed Using a Recombinant Yeast Screen,” Environmental Toxicology and Chemistry, Vol. 15, No. 3, 1996, pp. 241-248. doi:10.1002/etc.5620150303
 K. Mortelmansand E. Zeiger, “The Ames Salmo-Nella/ microsome Mutagenicity Assay,” Mutation Research, Vol. 455, No. 1-2, 2000, pp. 29-60. doi:10.1016/S0027-5107(00)00064-6
 M. Fenech, “The Cytokinesis-Block Micronucleus Technique: A Detailed Description of the Method and Its Application to Genotoxicity Studies in Human Populations,” Mutation Research, Vol. 285, No. 1, 1993, pp. 35-44. doi:10.1016/0027-5107(93)90049-L
 N. P. Singh, M. T. McCoy, R. R. Tice and E. L. Schneider, “A Simple Technique for Quantitation of Low Levels of DNA Damage in Individual Cells,” Experimental Cell Research, Vol. 175, No. 1, 1988, pp. 184-191. doi:10.1016/0014-4827(88)90265-0
 C. R. Kent, J. J. Eady, G. M. Ross and G. G. Steel, “The Comet Moment as a Measure of DNA Damage in the Comet Assay,” International Journal of Radiation Biology, Vol. 67, 1995, pp. 655-660.
 N. J. Duker, “Chromosome Breakage Syndromes and Cancer,” American Journal of Medical Genetics, Vol. 115, No. 3, 2002, pp. 125-129. doi:10.1002/ajmg.10688
 E. Pfeiffer, B. Rosenberg, S. Deuschel and M. Metzler, “Interference with Microtubules and Induction of Micronuclei in Vitro by Various Bisphenols,” Mutation Research, Vol. 390, No. 1, 1997, pp. 21-31.
 L. Lehmann and M. Metzler, “Bisphenol A and Its Methylated Congeners Inhibit Growth and Interfere with Microtubules in Human Fibroblasts in Vitro,” Chemico- Biological Interactions, Vol. 147, 2004, pp. 273-285.
 L. A. Haighton, J. J. Hlywka, J. Doull, R. Kroes, B. S. Lynch and I. C. Munro, “An Evaluation of the Possible Carcinogenicity of Bisphenol A to Humans,” Regulatory Toxicology and Pharmamcology, Vol. 35, No. 2, 2002, pp. 238-254. doi:10.1006/rtph.2001.1525
 S. E. Kulling, B. Rosenberg, E. Jacobs and M. Metzler, “The Phytoestrogens Coumestrol and Genistein Induce Structural Chromomsomal Aberrations in Cultured Human Peripheral Blood Lymphocytes,” Archives in Toxicology, Vol. 73, 1999, pp. 50-54.
 M. Berwick and P. Vineis, “Markers of DNA Repair and Susceptibility to Cancer in Humans: An Epidemiologic Review,” Journal National Cancer Institute, Vol. 92, No. 11, 2000, pp. 874-897. doi:10.1093/jnci/92.11.874
 H. J. De Geus, H. Besselink, A. Brouwer, J. Klungsoyr, B. McHugh, E. Nixon, G. G. Rimkus, P. G. Wester and J. de Boer, “Environmental Occurrence, Analysis, and Toxicology of Toxaphene Compounds,” Environmental Health Perspectives, Vol. 107, 1999, pp. 115-144.
 M. Yoshikane, W. R. Kay, Y. Shibata, M. Inoue, T. Yanai, R. Kamata, J. S. Edmonds and M. Morita, “Very High Concentrations of DDE and Toxaphene Residues in Crocodiles from the Ord River, Western Australia: An Investigation into Possible Endocrine Disruption,” Journal Environmental Monitoring, Vol. 8, No. 6, 2006, pp. 649-661. doi:10.1039/b518059g
 L. C. Terriere, U. Kiigemagi, A. R. Gerlach and R. L. Borovicka, “The Persistence of Toxaphene in Lake Water and Its Uptake by Aquatic Plants and Animals,” Journal Agricultural and Food Chemistry, Vol. 14, No. 1, 1966, pp. 66-69. doi:10.1021/jf60143a021
 W. Liao, M. A. McNutt and W. G. Zhu, “The Comet Assay: A Sensitive Method for Detecting DNA Damage in Individual Cells,” Methods, Vol. 48, No. 1, 2009, pp. 46-53. doi:10.1016/j.ymeth.2009.02.016
 A. M. Soto, T.-M. Lin, H. Justicia, R. M. Silvia and C. Sonnenschein, “An ‘in Culture’ Bioassay to Assess the Estrogenicity of Xenobiotics,” In: T. Colborn and C. Clement, Eds., Chemically Induced Alterations in Sexual Development: The Wildlife/Human Connection, Princeton Scientific Publishing, Princeton, 1992, pp. 295-309.
 G. E. Fahraeus-van Ree and J. F. Payne, “Effect of Toxaphene on Reproduction of Fish,” Chemosphere, Vol. 34, 1997, pp. 855-867. doi:10.1016/S0045-6535(97)00016-7
 C. C. Hedli, R. Snyder, F. K. Kinoshita and M. Steinberg, “Investigation of Hepatic Cytochrome P-450 Enzyme Induction and DNA Adduct Formation in Male CD/1 Mice Following Oral Administration of Toxaphen,” Journal of Applied Toxicology, Vol. 18, No. 3, 1998, pp. 173-178.
 M. D. Reuber, “Carcinogenicity of Toxaphene: A Review,” Toxicology and Environmental Health, Vol. 5, No. 4, 1979, pp. 729-748. doi:10.1080/15287397909529783
 R. White, S. Jobling, S. A. Hoare, J. P. Sumpter and M.G. Parker, “Environmentally Persistent Alkylphenolic Compounds Are Estrogenic,” Endocrinology, Vol. 135, No. 1, 1994, pp. 175-182. doi:10.1210/en.135.1.175
 A. Rivas, M. Lacroix, F. Olea-Serrano, I. Laios, G. Leclercq and N. Olea, “Estrogenic Effect of a Series of Bisphenol Analogues on Gene and Protein Expression in MCF-7 Breast Cancer Cells,” Journal of Steroid Biochemistry and Molecular Biology, Vol. 82, No. 1, 2002, pp. 45-53. doi:10.1016/S0960-0760(02)00146-2
 M. Shimizu, K. Ohta, Y. Matsumoto, M. Fukuoka, Y. Ohno and S. Ozawa, “Sulphation of Bisphenol A Abolished Its Estrogenicity Based on Proliferation and Gene Expression in Human Breast Cancer MCF-7 Cells,” Toxicology in Vitro, Vol. 16, 2002, pp. 549-556. doi:10.1016/S0887-2333(02)00055-3
 B. Gutendorf and J. Westendorf, “Comparison of an Array of in Vitro Assays for the Assessment of the Oestrogenic Potential of Natural and Synthetic Oestrogens, Phytoestrogens and Xenoestrogens,” Toxicology, Vol. 166, No. 1-2, 2001, pp. 79-89. doi:10.1016/S0300-483X(01)00437-1
 D. M. Harries, E. Besselink, S. M. Henning, V. L. W. Go and D. Heber, “Phytoestrogens Induce Differential Estrogen Receptor Alpha- or Beta-Mediated Responses in Transfected Breast Cancer Cells,” Experimental. Biology and Medicine, Vol. 230, 2005, pp. 558-568.
 K. Morito, T. Hirose, J. Kinjo, T. Hirakawa, M. Okawa, T. Nohara, S. Ogawa, S. Inoue, M. Muramatsu and Y. Masamune, Interaction of Phytoestrogens with Estrogen Receptors α and β,” Biological Pharmaceutical Bulletin, Vol. 24, No. 4, 2001, pp. 351-356. doi:10.1248/bpb.24.351
 H. H. Jury, T. R. Zacharewski and G. L. Hammond, “Interactions between Human Plasma Sex Hormone-Binding Globulin and Xenobiotic Ligands,” Journal Steroid Biochemistry and Molecular Biology, Vol. 75, 2000, pp. 167-176. doi:10.1016/S0960-0760(00)00168-0,
 H. Dechaud, C. Ravard, F. Claustrat, A. B. de la Pierriere and M. Pugeat, “Xenestrogen Interaction with Human Sex Hormone-Binding Globulin (hSHBG),” Steroids, Vol. 64, pp. 1999, 328-334.
 A. Reid and J. Roche, “Hormone Modulating Substances in the Irish Midlands Shannon Catchment: Extraction, Analysis and Quantification,” Proceedings ESAI ENVIRON 2005.
 A. M. Reid, C. A. Brougham, A. M. Fogarty and J. J. Roche, “Approaches to Enhance the Liquid Chromatographic Determination of Phthalates in Environmental Matrices,” Proceedings ESAI ENVIRON 2006.
 A. M. Reid, C. A. Brougham, A. M. Fogarty and J. J. Roche, “Isocratic LC Methods for the Trace Analysis of Phthalates and 4-Nonylphenol in Varying Types of Landfill and Adjacent Run-Offs,” Toxicological and Environmental Chemistry, Vol. 89, No. 3, 2007, pp. 399-410. doi:10.1080/02772240601116613
 A. M. Reid, C. A. Brougham, A. M. Fogarty and J. J. Roche, “Accelerated Solvent-Based Extraction and Enrichment of Selected Plasticisers and 4-Nonylphenol, and Extraction of Tin from Organotin Sources in Sediments, Sludges and Leachate Soils,” Analytica Chimica Acta, Vol. 634, No. 2, 2008, pp. 197-204.
 A. M. Reid, C. A. Brougham, A. M. Fogarty and J. J. Roche, “Analysis of Bio-Obtainable Endocrine Disrupting Metals in River Water and Sediment, Sewage Influent/Effluent, Sludge, Leachate, and Concentrated Lea- chate, in the Irish Midlands Shannon Catchment,” International Journal of Analytical Chemistry, Vol. 2009, 2009, Article ID: 325496, pp. 1-12. doi:10.1155/2009/325496
 H. Tarrant, N. Llewellyn, A. Lyons, N. Tattersall, S. Wylde, G. Mouzakitis, M. Maloney and C. McKenzie, “Endocrine Disruptors in the Irish Aquatic Environment. Environmental RTDI Programme 2000-2006,” Johnstown Castle, Co. Wexford, 2005.
 C. Desbrow, E. J. Routledge, G. C. Brighty, J. P. Sumpter and M. Waldock, “Identification of Estrogenic Chemicals in STW Effluent. 1. Chemical Fractionation and in Vitro Biological Screening,” Environmental Science and Technology, Vol. 32, No. 11, 1998, pp. 1549-1558. doi:10.1021/es9707973
 S. Jobling, R. Williams, A. Johnson, A. Taylor, M. Gross- Sorokin, M. Nolan, C. R. Tyler, R. van Aerle, E. Santos and G. Brighty, “Predicted Exposures to Steroid Estrogens in U.K. Rivers Correlate with Widespread Sexual Disruption in Wild Fish Populations,” Environmental Health Perspectives, Vol. 114, 2006, pp. 32-39. doi:10.1289/ehp.8050
 A. C. Belfroid, A. Van der Horst, A. D. Vethaak, A. J. Schafer, G. B. J. Rijs, J. Wegener and W. P. Cofino, “Analysis and Occurrence of Estrogenic Hormones and Their Glucuronides in Surface Water and Waste Water in The Netherlands,” The Science of the Total Environment, Vol. 225, No. 1-2, 1999, pp. 101-108.
 P. Spengler, W. Korner and J. W. Metzger, “Substances with Estrogenic Activity in Effluents of Sewage Treatment Plants in South-Western Germany. 1. Chemical Analysis,” Environmental Toxicology and Chemistry, Vol. 20, No. 10, 2001, pp. 2133-2141. doi:10.1002/etc.5620201001
 W. Korner, P. Spengler, U. Bolz, W. Schuller, V. Hanf and J. W. Metzger, “Substances with Estrogenic Activity in Effluents of Sewage Treatment Plants in South-Western Germany. 2. Biological Analysis,” Environmental Toxicology and Chemistry, Vol. 20, No. 10, 2001, pp. 2142-2151. doi:10.1002/etc.5620201002
 R. A. Rudel, S. J. Melly, P. W. Geno, G. Sun and J. G. Brody, “Identification of Alkylphenols and Other Phenolic Compounds in Wastewater, Septage, and Groundwater on Cape Cod, Massachusetts,” Environmental Science Technology, Vol. 32, No. 7, 1998, pp. 861-869. doi:10.1021/es970723r
 J. H. Shen, B. Gutendorf, H. H. Vahl, L. Shen and J. Westendorf, “Toxicological Profile of Pollutants in Surface Water from an Area in Taihu Lake, Yangtze Delta,” Toxicology, Vol. 166, No. 1-2, 2001, pp. 71-78.
 M. A. Kelly, A. M. Reid, K. M. Quinn-Hosey, A. M. Fogarty. J. J. Roche and C. A. Brougham, “Investigation of the Estrogenic Risk to Feral Male Brown Trout (Salmotrutta) in the Shannon International River Basin District of Ireland,” Ecotoxicology and Environmental Safety, Vol. 73, 2010, pp. 1658-1665.
 K. M. Quinn-Hosey, J. J. Roche, A. M. Fogarty and C. A. Brougham, “A Toxicological Assessment of Endocrine Disrupting Chemicals Found in the BMW (Border Midland and Western) Region of Ireland,” Journal of Environmental Protection, Vol. 3, No. 4, 2012, pp. 304-315. doi:10.4236/jep.2012.34039
 C. McGee, C. Brougham, J. Roche and A. Fogarty, “First Report of Intersex Roach Residing in Irish Rivers Downstream of Several Wastewater Treatment Plants,” Biology and Environment, Vol. 112, No. 1, 2012, pp. 1-9. doi:10.3318/BIOE.2011.23
 V. Pelekanou and G. Leclercg, “Recent Insights into the Effect of Natural and Environmental Estrogens on Mammary Development and Carcinogenesis,” International Journal Developmental Biology, Vol. 55, 2011, pp. 869- 878. doi:10.1387/ijdb.113369vp
 S. A. Bidgoli, R. Ahmadi and M. D. Zavarhei, “Role of Hormonal and Environmental Factors on Early Incidence of Breast Cancer in Iran,” Science Total Environment, Vol. 408, No. 19, 2010, pp. 4056-4061.
 A. Giwercman and Y. L. Giwercman, “Environmental Factors and Testicular Function,” Best Practice Research Clinical Endocrinology Metabolism, Vol. 25, No. 2, 2011, pp. 391-402. doi:10.1016/j.beem.2010.09.011
 K. M. Main, N. E. Skakkebaek, H. E. Virtanen and J. Toppari, “Genital Anomalies in Boys and the Environment,” Best Practice Research Clinical Endocrinology Metabolism, Vol. 24, No. 2, 2010, pp. 279-289. doi:10.1016/j.beem.2009.10.003
 D. Klingmuller and A. Allera, “Endocrine Disruptors: Hormone-Active Chemicals from the Environment: A Risk to Humans?” Dtsh Med Wochenschr, Vol. 136, 2011, pp. 967-972.
 L. F. Doherty, J. G. Bromer, Y. Zhou, T. S. Aldad and H. S. Taylor, “In Utero Exposure to Diethylstilbestrol (DES) or Bisphenol-A (BPA) Increases EZH2 Expression in the Mammary Gland: An Epigenetic Mechanism Linking Endocrine Disruptors to Breast Cancer,” Hormones Cancer, Vol. 1, No. 3, 2010, pp. 146-155. doi:10.1007/s12672-010-0015-9