Molecular and Cellular Endocrinology 221 (2004) 47–55 Bisphenol-A and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells David W. Singleton a,1 , Yuxin Feng a,1 , Yangde Chen b , Steve J. Busch b , Adrian V. Lee c , Alvaro Puga d , Sohaib A. Khan a,∗ a Department of Cell Biology, Neurobiology and Anatomy, Vontz Center for Molecular Studies, University of Cincinnati College of Medicine, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA b Aventis Pharmaceuticals, Bridgewater, NJ, USA c Breast Center, Baylor College of Medicine, Houston, TX, USA d Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0521, USA Received 29 December 2003; received in revised form 30 March 2004; accepted 12 April 2004 Abstract Xenoestrogens such as bisphenol-A (BPA) can mimic endogenous 17-estradiol (E2) in vitro and in vivo through binding the estrogen receptor (ER), and modulating target gene expression. In the present study, we compared global gene regulation by BPA and E2 in estrogen responsive (ER-HA) human breast cancer cells derived from the MCF-7 cell line. The ER-HA cells (stably over-expressing ER) were exposed to E2 (10 -8 M) or BPA (10 -6 M), for 3 h followed by analysis of global gene expression. More than 40 transcripts were significantly changed in ER-HA cells, with many being unique to BPA. At least 15 genes were modulated by BPA in the ER-null C4–12 cell line, indicating ER independent activity. Utilizing quantitative reverse transcription-polymerase chain reaction (RT-PCR), we confirmed BPA and E2 mediated regulation of four selected genes. A consensus Alu-type estrogen responsive element (ERE) was found in the Wiskott–Aldrich syndrome protein (WASP) gene, which conferred responsiveness to BPA and E2 in a reporter gene assay. Significant stimulation was seen only in ER expressing cells, thus indicating a functional ERE. Taken together these data illustrate novel gene regulation by BPA and E2, which has implications for in vivo actions and previous reports of additive and synergistic effects on breast cancer cell growth. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Bisphenol-A; Estrogen receptor; Wiskott–Aldrich syndrome protein; Estrogen responsive element; Endocrine disruptor; Xenoestrogen; Microarray 1. Introduction Endocrine disrupting chemicals (EDCs) are a large group of synthetic and naturally occurring agents that may perturb normal hormonal signaling in vivo. Among the most preva- lent EDCs are xenoestrogens, which mimic or disrupt pro- cesses mediated by the endogenous estrogen: 17-estradiol (E2). The xenoestrogen bisphenol-A (BPA) is an industrial monomer used in production of polycarbonates and epoxy resins. Traces of BPA are reported to leach from the lining Abbreviations: BPA, bisphenol-A; E2, 17-estradiol; ER, estrogen receptor; ERE, estrogen responsive element; HA, hemagglutinin epitope; RT-PCR, reverse transcription-polymerase chain reaction ∗ Corresponding author. Tel.: +1-513-558-7224; fax: +1-513-558-7223. E-mail address: sohaib.khan@uc.edu (S.A. Khan). 1 These authors contributed equally to this study. of some food cans and plastic ware, and from some den- tal sealants (Brotons et al., 1995; Olea et al., 1996). Evi- dence for possible endocrine disruptive effects by BPA has been reported in several studies showing activation of estro- gen receptors (ER) alpha and beta (Matthews et al., 2001; Routledge et al., 2000), stimulation of MCF-7 breast can- cer cell growth (Krishnan et al., 1993), and in vivo effects on rodent reproductive tract (Gupta, 2000; Morrissey et al., 1987; Ramos et al., 2001; Suzuki et al., 2002; Takao et al., 1999). BPA and other environmental contaminants are gen- erally much less potent than E2 in activating ER. Nonethe- less, because of the complexity of estrogen mechanisms of action, the potential exists for effects on human health by these agents. ER is a member of the steroid hormone nuclear recep- tor superfamily, whose members become active transcription factors when bound by cognate ligands. Liganded ER binds 0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.04.010