The role of ovarian metabolism in 4-vinylcyclohexene-induced ovotoxicity in B6C3F(1) mice

Abstract

4-Vinylcyclohexene (VCH), an industrial chemical, causes destruction of small pre-antral follicles (F1) in mice. Previous studies suggested that VCH is hepatically bioactivated to its ovotoxic metabolite, vinylcyclohexene diepoxide (VCD), by the cytochrome P450 enzymes, likely Cyp 2E1, Cyp 2A, and Cyp 2B. Additionally, microsomal epoxide hydrolase (mEH) likely participates in detoxifying the epoxide metabolites. The role of ovarian metabolism (bioactivation/detoxification) of VCH and its metabolites is not known. The hypothesis of this dissertation research was that ovarian metabolism contributes to VCH-induced ovotoxicity. These studies investigated whether the mouse ovary expresses (a) mRNA, (b) total protein, and (c) functional protein for several metabolic enzymes (Cyp 2E1, Cyp 2A, Cyp 2B, mEH), as well as, (d) the effects of VCH/VCD dosing on these parameters. Female B6C3F1 mice were dosed (i.p.) daily (15 d) with VCH (7.4 mmol/kg) or VCD (0.57 mmol/kg). Ovaries were removed and enzymatically digested and sorted into specific ovarian fractions (F1, small preantral; F2, large preantral; F3, antral; Int, interstitial cells) for mRNA analysis by realtime PCR, fixed for immunohistochemistry by confocal microscopy, or homogenized for functional assays. Basal expression was detected for mRNA encoding Cyp 2E1, Cyp 2A, Cyp 2B, and mEH in all ovarian fractions. In vivo dosing with VCH/VCD differentially altered expression, as expression increased for all enzymes in targeted F1 follicles. All enzymes were also distributed throughout the ovary, with high immunostaining intensity in the Int. In vivo dosing with VCH/VCD also affected protein distribution. Utilizing model substrates, catalytic activity was evaluated in ovarian fractions or whole ovaries. Basal activity was detected for Cyp 2E1, Cyp 2B, and mEH, while VCH dosing only induced activities for Cyp 2E1 and mEH. Taken together, the ovary has the metabolic capacity to be involved in metabolic reactions. Bioactivation is likely via Cyp 2E1 in the Int. cells. Although the relative contribution of ovarian metabolism in VCH-induced ovotoxicity is not known, the ovary likely plays a greater role in detoxification, due to greater levels of mEH activity. Thus, the ovary may provide a metabolic contribution to mediating the effects of ovotoxicants.