Evaluation of ovotoxicity induced by 7, 12-dimethylbenz[a]anthracene and its 3,4-diol metabolite utilizing a rat in vitro ovarian culture system

Abstract

The polycyclic aromatic hydrocarbon 7, 12-dimethylbenz[a]anthracene, (DMBA), targets and destroys all follicle types in rat and mouse ovaries. DMBA requires bioactivation to DMBA-3,4-diol-1,2-epoxide for ovotoxicity via formation of the intermediate, DMBA-3,4-diol (catalyzed by microsomal epoxide hydrolase; mEH). mEH was shown to be involved in DMBA bioactivation for ovotoxicity induction in B6C3F 1 mouse ovaries. The current study compared DMBA and DMBA-3,4-diol mediated ovotoxicity, and investigated mEH involvement in DMBA-3,4-diol bioactivation in Fischer 344 (F344) rat ovary. F344 postnatal day (PND) 4 rat ovaries were cultured in vehicle control or media containing 1) DMBA or DMBA-3,4-diol (12.5 nM – 1 μM; 15 days); 2) DMBA (1 μM; 6 h – 15 days); and 3) DMBA (1 μM) or DMBA-3,4-diol (75 nM) ± the mEH activity inhibitor cyclohexene oxide (CHO; 2 mM; 4 days). Ovaries were histologically evaluated and mEH mRNA and protein were measured by reverse transcriptase PCR or Western blotting, respectively. Ovotoxicity following 15 days of culture occurred ( P < 0.05) at lower concentrations of DMBA-3,4-diol (12.5 nM — primordial; 75 nM — primary) than DMBA (75 nM — primordial; 375 nM — primary). The temporal pattern of mEH expression following DMBA exposure showed mRNA up-regulation ( P < 0.05) on day 2, with increased protein ( P < 0.05) on day 4, the earliest time of observed follicle loss ( P < 0.05). mEH inhibition prevented DMBA-induced, but not DMBA-3,4-diol-induced ovotoxicity. These results demonstrate a conserved response in mice and rats for ovarian mEH involvement in DMBA bioactivation to its ovotoxic, 3,4-diol-1,2-epoxide form.