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

Abstract

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)-beta, which was down-regulated, whereas ERalpha 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 microg/kgxd or 100 mg/kg.d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERalpha and ERbeta genes in postnatal d 50-60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERbeta promoter regions (P < 0.05), whereas the ERalpha 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.

Endocrine-disrupting chemicals in ovarian function: effects on steroidogenesis, metabolism and nuclear receptor signaling

“…Studies using transfected cell lines have demonstrated that the active metabolites of MXC are agonists of ESR1, but antagonists for both ESR2 and AR (Gaido et al 2000). Other studies have shown that fetal and neonatal exposure to MXC (20 mg/kg and 100 mg/kg) alters methylation on the promoter region of ESR2 and therefore suppresses the expression of ESR2, causing ovarian dysfunction in the rat (Armenti et al 2008, Zama & Uzumcu 2009). Interestingly, antral follicles overexpressing ESR1 have been shown to be more sensitive to toxicity by MXC and its metabolites in both in vivo and in vitro experiments (Tomic et al 2006.…”

“…Early studies with BPA showed that it has estrogenic properties and its binding affinity to ESRs is lower than E 2 or DES (Zama & Uzumcu 2009). In vitro, BPA acts as an agonist for both ESR1 and ESR2 on a tissue-specific basis (Kurosawa et al 2002), although in vivo findings suggest that it can also antagonize ESR2 function (Susiarjo et al 2007).…”

The importance of ERβ signalling in the ovary

“…Methoxychlor (MXC), an organochloride pesticide with oestrogenic activity mediated primarily via ERb (Zama & Uzumcu 2009), caused ovarian dysfunction in the adult rodent following exposure to rats during the foetal or neonatal period. Follicle composition was altered with more preantral and early antral follicles present and fewer CL (Gray et al 1989).…”

Specific transgenerational imprinting effects of the endocrine disruptor methoxychlor on male gametes

“…by modulating gene expression without modifying the underlying DNA sequence. The perinatal exposure to MXC has, indeed, been shown to cause an hypermethylation of the ESR2 promoter and of ten other genes in the ovary (Zama & Uzumcu 2009).…”