Ovarian Metabolism of Xenobiotics.

Ovarian xenobiotic exposures can destroy oocyte-containing follicles leading to infertility and premature entry into menopause, thus predisposing females to health complications associated with this physiological state. Of equal concern is the potential for chemical exposures to induce oocyte DNA damage, potentially leading to birth defects in the next generation. The occupational chemical 4-vinylcyclohexene (VCH) specifically depletes ovarian primordial and small primary follicles. Another ovotoxicant, 7,12-dimethylbenz[a]anthracene (DMBA), a polycyclic aromatic hydrocarbon, destroys follicles at all stages of development. The consequence of exposure to these ovotoxicants is largely determined by their metabolism through the action of ovarian expressed enzymes. VCH is bioactivated to a diepoxide ovotoxic metabolite 4-vinylcyclohexene diepoxide (VCD) by cytochrome P450 isoform 2E1 (CYP2E1) but can be biotransformed by microsomal epoxide hydrolase (EPHX1) to a less ovotoxic tetrol metabolite. DMBA undergoes a number of metabolism steps to yield the ovotoxic DMBA-3,4-diol, 1,2-epoxide catalyzed by CYP1B1 and EPHX1. Thus, the action of EPHX1 can lead to greater or lesser ovotoxic outcomes dependent on the chemical substrate. Another ovarian expressed enzyme, glutathione S-transferase pi (GSTP) negatively regulates the pro-apoptotic protein c-jun N-terminal kinase (MAPK8) and expression of Gstp mRNA and protein are up-regulated in response to both VCD and DMBA indicating a role in their metabolism. Additionally, in the presence of VCD and DMBA, the amount of MAPK8 bound to GSTP is increased and activity of MAPK8 is repressed, supporting a protective anti-apoptotic role for ovarian GSTP. The phosphatidylinositol-3 kinase (PIK3) pathway is involved in regulating primordial oocyte activation and viability. Inhibition of PIK3 signaling results in altered ovotoxicity-induced by VCD and DMBA. VCD-induced primordial follicle loss was prevented, while that induced by DMBA was accelerated, potentially indicating altered chemical metabolism gene expression. Postnatal day 4 (PND4) rat ovaries were cultured in control media or media containing the PIK3 specific inhibitor LY204002 (20 μM). Total RNA was isolated and cDNA amplified using qRT-PCR to measure mRNA expression of Ephx1 and Gstp. Protein levels of EPHX1 and GSTP were also quantified by Western blotting. mRNA and protein expression levels for Ephx1 and Gstp were increased (P < 0.05) by PIK3 inhibition, supporting increased detoxification of VCD and increased bioactivation of DMBA by EPHX1, and potentially indicating increased MAPK8 inhibition by GSTP. Another experiment was performed to activate PIK3 signaling using Kit Ligand (KITL) to confirm a role for PIK3 in chemical metabolism gene expression regulation. PND4 rat ovaries were cultured in control media or media containing KITL (400 nM). Ephx1 and Gstp mRNA levels were both decreased (P < 0.05) by PIK3 activation. Thus, these data support PIK3 signaling involvement in regulation of ovarian chemical metabolism and the extent of ovotoxicity observed is impacted by alterations to this pathway. In summary, ovarian chemical metabolism is important for protection of the primordial follicle pool and the action of these enzymes is critical to minimize the ovarian effects of ovotoxicant exposures. Supported by ES016818 from the National Institute of Environmental Health Sciences.