The Role of Ovarian Metabolism in 4-Vinylcyclohexene Metabolites and 7,12-Dimethylbenz[a]anthracene Induced Ovotoxicity in Mice

Abstract

Ovarian toxicants 4-vinylcychlohexene (VCH) and 7,12-dimethylbenz[a]anthracene (DMBA) requires bioactivation to induce follicle loss. VCH is bioactivated to monoepoxides (1,2-VCM and 7,8-VCM), and subsequently to an ovotoxic diepoxide (VCD) by hepatic CYP 2A and CYP 2B. DMBA is sequentially bioactivated to the ovotoxicant DMBA-3,4-diol-1,2-epoxide by hepatic CYP 1B1, microsomal epoxide hydrolase (mEH), and CYP 1A1/1B1 enzymes. Even though the liver is the primary organ metabolizing VCH and DMBA to reactive intermediates, several studies suggest that the ovary can also metabolize these two compounds. Studies were designed to investigate the role of ovarian metabolism in the resulting ovotoxicity of these two compounds using a novel mouse ovarian culture system. The hypothesis was that the ovary can participate in bioactivation and detoxification of VCH/VCM and DMBA and thereby influence the resulting ovotoxicity.Postnatal day 4 CYP 2E1 wild-type, null and B6C3F1 mouse ovaries were incubated with 1,2-VCM, VCD or DMBA for various lengths of time. 28 day old female CYP 2E1 wild-type and null mice were dosed (15d, i.p) with VCH, 1,2-VCM, VCD, or sesame oil (control). Following incubations and dosing, ovaries were prepared for histological evaluation of follicle numbers, mEH mRNA level, or mEH protein level. Medium from cultures were analyzed by LC/MS for VCD-GSH adducts.DMBA was found to be a potent ovotoxicant compared to VCH/VCM/VCD. In the ovarian culture system, VCM-induced toxicity required the CYP 2E1 enzyme. However, in vivo dosing studies indicated that in the presence of hepatic metabolism the ovary plays a minimal role in VCH/VCM-induced toxicity. Studies utilizing LC/MS showed that once bioactivated to VCD, this ovotoxic metabolite can be detoxified by glutathione conjugation in the ovary. Follicle loss induced by the ovotoxicant DMBA was found to involve mEH enzyme in culture.Collectively, these studies show that the ovary has the capacity to bioactivate and detoxify ovotoxicants. In the presence of hepatic metabolism ovarian effects might play only a minimal role in the resulting toxicity. The role of ovarian metabolism in the whole animal needs to be further investigated, especially for potent toxicants such as DMBA that can induce ovotoxicity at nanomolar concentrations.