Xenoestrogen Bisphenol A Inhibits Postembryonic Vertebrate Development by Antagonizing Gene Regulation by Thyroid Hormone

T H Y R O I D - T R H - T S H The Xenoestrogen Bisphenol A Inhibits Postembryonic Vertebrate Development by Antagonizing Gene Regulation by Thyroid Hormone Rachel A. Heimeier, Biswajit Das, Daniel R. Buchholz, and Yun-Bo Shi Section on Molecular Morphogenesis (R.A.H., B.D., Y.-B.S.), Program on Cell Regulation and Metabolism, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892; and Department of Biological Sciences (D.R.B.), University of Cincinnati, Cincinnati, Ohio 45221-0006 Bisphenol A (BPA), a chemical widely used to manufacture plastics, is estrogenic and capable of disrupting sex differentiation. However, recent in vitro studies have shown that BPA can also antagonize T3 activation of the T3 receptor. The difficulty in studying uterus-enclosed mammalian embryos has hampered the analysis on the direct effects of BPA during vertebrate development. This study proposed to identify critical T3 pathways that may be disrupted by BPA based on molecular analysis in vivo. Because amphibian metamorphosis requires T3 and encompasses the postembryonic period in mammals when T3 action is most critical, we used this unique model for studying the effect of BPA on T3-dependent vertebrate development at both the morphological and molecular levels. After 4 d of exposure, BPA inhibited T3-induced intestinal remodeling in premetamorphic Xenopus laevis tadpoles. Importantly, microarray analysis revealed that BPA antagonized the regulation of most T3-response genes, thereby explaining the inhibitory effect of BPA on metamorphosis. Surprisingly, most of the genes affected by BPA in the presence of T3 were T3-response genes, suggesting that BPA predominantly affected T3-signaling pathways during metamorphosis. Our finding that this endocrine disruptor, well known for its estrogenic activity in vitro, functions to inhibit T3 pathways to affect vertebrate development in vivo and thus not only provides a mechanism for the likely deleterious effects of BPA on human development but also demonstrates the importance of studying endocrine disruption in a developmental context in vivo. (Endocrinology 150: 2964 –2973, 2009) E ndocrine disruption by environmental contaminants poses a great concern for global ecology and human health. Endocrine disrupting compounds (EDCs) have been defined as exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations (1–3). Some EDCs act as antiestrogenic and antiandrogenic agents to affect reproductive function and sexual development (4), suggesting that EDCs are responsible for the increased appearance of reproductive health problems in both human and wildlife. In humans, the trend for increased breast and testicular cancers, reduced sperm counts, and early puberty has been attributed to increased exposure to EDCs (5–7). In wildlife, decreased species populations and increased animal malformations, including feminization and hermaphroditism, have been reported worldwide (8 –11). There is also increasing concern that EDCs may affect other endocrine systems, such as the T3 system. T3 plays a central role in vertebrate development, growth, and metabolism (12–18). The effects of EDCs on T3 signaling will undoubtedly pose a threat to human and wildlife health (19 –22). Keyed by the discovery of nuclear T3 receptors (TRs) that function as transcription factors, recent advances have been made in examining the mechanisms of T3 action at the molecular level (12, 13, 15, 23–31). Concurrently, studies have also revealed a broad array of EDCs that can bind to TR and affect T3-regulated gene expression in vitro (32). However, the lack of a suitable in vivo model to study EDCs’ effects on TR function in vertebrate development impedes our understanding on whether and how ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2009 by The Endocrine Society doi: 10.1210/en.2008-1503 Received October 28, 2008. Accepted February 11, 2009. First Published Online February 19, 2009 Abbreviations: BMP, Bone morphogenetic protein; BPA, bisphenol A; DMSO, dimethyl sulfoxide; EDC, endocrine disrupting compound; EF-1␣, elongation factor-1␣; ER, estrogen receptor; MMP, matrix metalloproteinase; rpl8, ribosomal protein L8; qRT-PCR, quantitative RT-PCR; RXR, retinoid X receptor; ST3, stromelysin-3; TH/bZIP, T3-responsive basic leucine zipper transcription factor; TIMP, tissue inhibitor of metalloproteinase; TR, T3 receptor. 2964 endo.endojournals.org Endocrinology, June 2009, 150(6):2964 –2973 Endocrinology, June 2009, 150(6):2964 –2973 persistent exposure to these bioaccumulative compounds affects human health. One such compound is bisphenol A (BPA), an established EDC of the reproductive system. BPA is used in the production of plastics and has widespread applicability, making its manufacturing and processing an important economical factor as well as a source of BPA release into the environment (33– 40). BPA studies have primarily focused on its estrogenic activity (4, 41). Recently based on extensive review of the existing data, the National Toxicology Program of the National Institutes of Health raised concerns for neural and behavioral effects of BPA in fetuses, infants, and children at the currently allowed human exposures (www.niehs.nih.gov/news/media/questions/sya-bpa. cfm#2). The concerns from this reviewing panel were primarily focused on the estrogenic effects of BPA, even though the role of estrogens on mammalian neural development is unclear. On the other hand, neural and behavioral development is dependent on T3, raising the possibility that the developmental effects of BPA in humans may be manifested through the T3 pathway. Given the possible cross talks between the T3 and estrogenic pathways (42, 43), BPA may indirectly affect T3 signaling by influencing estrogenic pathways. On the other hand, in vitro studies have shown that BPA can bind to and antagonize T3 activation of TR (44), and a study using cultured mouse oligodendrocyte precursor cells found that BPA inhibited T3-induced differentiation (45). In addition, a study with rats showed that BPA exposure during development produced an endocrine profile similar to that observed in patients with T3 resistance syndrome (46). The ability of BPA to bind to both estrogen and thyroid receptors to elicit disruption makes it very difficult to study the actions of BPA during mammalian development. Suitable alternative in vivo models are urgently needed to evaluate the effects of BPA on T3 function during development. Amphibian metamorphosis represents an attractive model due to its absolute dependence on T3 but not estrogens (14, 15), although sex steroids can alter larval development in amphibians (47– 49). Recent studies have shown that BPA blocks metamorphosis and affects T3-signaling in amphibians (50 –53). 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