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Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901-8525

Address all correspondence and requests for reprints to: Mehmet Uzumcu, Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, 84 Lipman Drive, New Brunswick, New Jersey 08901-8525. E-mail: uzumcu{at}aesop.rutgers.edu.

Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ER was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 µg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ER and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ER promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.