[OR33-4] Bisphenol-A (BPA) Exposure In Utero Leads to Epigenetic Changes and Altered Developmental Programming.

JG Bromer, YP Zhou, M Taylor, HS Taylor, Obstetrics, Gynecology, and Reproductive Scis, Yale Univ Sch of Med, New Haven, CT

Introduction: Bisphenol-A (BPA) is a ubiquitous endocrine disrupting chemical (EDC) that has recently been linked to adverse health outcomes. We have shown previously that in utero exposure to BPA induces permanently increased expression of HOXA10 in the adult. HOXA10 controls uterine organogenesis during embryonic development, and is necessary for implantation in the adult. There is increasing evidence that epigenetic changes in gene expression may occur through aberrant DNA methylation. We hypothesized that BPA-induced changes in HOXA10 were due to alterations in DNA methylation.
Methods: 6 pregnant CD-1 mice were treated with intraperitoneal injections of BPA (5 mg/kg) or vehicle on days 9-16 of gestation. 2 weeks after birth, the uteri of female offspring were isolated and DNA and mRNA extracted. Genomic DNA was bisulfite-modified and a CpG-rich region of the Hoxa10 promoter was analyzed via bisulfite sequencing. DNA methytransferase (DNMT) mRNA expression was quantified using real time RT-PCR, and normalized to -actin. Oligonucleotides specific to known Estrogen Respone Element (ERE) and SP1 binding sites of the HOXA10 promoter were treated with CpG methyltransferase. Electrophoretic Mobility Shift Assay (EMSA) was used to assess whether increased DNA methylation led to altered Estrogen Receptor (ER) or SP1 binding. All assays were repeated in triplicate. Differences in methylation between groups and in DNMT expression were compared using the Student's T-test.
Results: In the offspring of BPA-treated animals (n=8), the percentage of methylated sites in the Hoxa10 promoter was significantly lower than in controls (n=7), (2.9% vs. 57.0%, p=0.002). Relative fold change mRNA expression in the 3 DNMT's was not permanently altered with BPA exposure to a significant extent (DNMT1: 1.17 fold, p=0.459, DNMT3a: 1.13 fold, p=0.751, and DNMT3b: 1.34 fold, p=0.165). EMSA demonstrated that decreased DNA methylation resulted in increased ER and SP1 binding to the HOXA10 promoter.
Conclusions: In utero exposure to BPA produces persistently decreased DNA methylation in the Hoxa10 gene, despite normalization of DNMT expression. This decreased methylation is associated with increased ER and SP1 binding to the HOXA10 promotor. Decreased methylation may therefore lead to increased estrogen responsiveness and increased expression of HOXA10 in the adult. Epigenetic modification is a novel mechanism of endocrine disruptor action leading to altered developmental programming.

Date: Saturday, June 13, 2009
Session Info: ORAL SESSION: BASIC/TRANSLATIONAL - The Widespread Disruption of Endocrine Signaling (1:15 PM-2:30 PM)
Presentation Time: 1:15 PM
Room: 144A