Effect of 4-Vinylcyclohexene Diepoxide on Xenobiotic Metabolizing Enzyme Expression in the Rat Ovary.

Abstract

4-Vinylcyclohexene diepoxide (VCD) is an occupational chemical that selectively destroys small pre-antral follicles in ovaries of rats and mice. VCD can be detoxified in the ovary to an inactive tetrol metabolite, 4-[1,2-dihydroxy] ethyl-1,2-dihydroxycyclohexane by microsomal epoxide hydrolase (EPHX1) or by conjugation with glutathione by glutathione-S-transferase (GST). This selective destruction is via acceleration of the natural process of atresia (apoptosis) and requires repeated daily dosing (in vivo) or continuous exposure (in vitro). In mice, a previous study determined that increased expression of ovarian Ephx1, Gstp and Gstm mRNA and protein caused by VCD preceded onset of VCD-induced ovotoxicity. Therefore, the present study was designed to investigate whether this effect would also be seen in rats. A post-natal day 4 (PND4) rat whole ovary culture system (enriched in targeted small pre-antral follicles) was employed to examine a timeline for VCD-induced ovotoxicity. Previously it was determined in cultured neonatal rat ovaries that VCD (30μM) exposure for 8 days results in loss (P<0.05) of small pre-antral ovarian follicles. PND4 Fischer 344 (F344) rat ovaries were incubated in control medium or medium containing VCD (30μM) for 2, 4, 6, or 8d (n=3; 10 ovaries per pool). Expression of mRNA encoding the detoxification enzymes EPHX1, GSTP and GSTM were examined using Real-Time PCR at each timepoint. Additionally, western blotting was employed to evaluate changes in EPHX1, GSTP and GSTM protein levels after 4 and 8 days of VCD exposure. There was no difference in Ephx1, Gstp or Gstm mRNA levels between control and VCD-exposed ovaries on day 2. Relative to control, VCD exposure resulted in an increase (P<0.05) in Ephx1 mRNA by 0.61-fold on day 4 and in Gstp mRNA by 0.9-, 1.2- and 1-fold on days 4, 6 and 8, respectively. In contrast, VCD exposure did not have an effect (P>0.05) on Gstm mRNA expression at any timepoint. No changes in protein levels of the three enzymes occurred after 4 days of VCD exposure. However, on day 8, relative to controls, protein for EPHX1, GSTP and GSTM increased (P<0.05) by 17%, 27% and 10%, respectively, in response to VCD. These findings demonstrate that, as with mice, increased expression of ovarian detoxification enzymes in response to VCD precedes VCD-induced ovotoxicity in rats. This suggests a model in which up-regulation of these pathways by xenobiotic (VCD) exposure affords initial protection by detoxification of VCD but eventually the ovary becomes overwhelmed by repeated exposure, resulting in ovotoxicity. (ES09246 and Center grant 06694).