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 B6C3F1 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 (1μM; 6 h -15 days); and 3) DMBA (1μM) 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.

Physiological Aspects of Female Fertility: Role of the Environment, Modern Lifestyle, and Genetics

“…Both TCE and its metabolites led to a reduction in fertilization of mouse oocyte (68). Exposure to 7,12-dimethylbenz[a]anthracene (DMBA), found in cigarette smoke and car exhaust fumes, leads to a loss of mouse or rat follicles at all stages of development (161), and exposure to DMBA led to altered expression of genes regulating follicular development (322). A common by-product of pesticide, rubber, plastic, and flame retardant manufacture is 4-vinylcyclohexene (VCH).…”

All Your Eggs in One Basket: Mechanisms of Xenobiotic Induced Female Reproductive Senescence

“…This intermediate is then further modified by either Cyp1A1 or Cyp1B1 to form the ultimate ovotoxicant DMBA-3,4-diol-1,2-epoxide (Shimada and Fujii Kuriyama, 2004;Shimada et al, 2001). These three enzymes required for DMBA's biotransformation are all expressed and induced by DMBA exposure in the murine ovary (Igawa et al, 2009;Rajapaksa et al, 2007b;Shimada et al, 2003). In further support of localised DMBA bioactivation, inhibition of MeH in cultured rat ovaries inhibited DMBA induced ovotoxicity, while ovarian culture in the presence of DMBA-3,4-diol induced significantly more primordial follicle depletion than DMBA alone (Igawa et al, 2009;Rajapaksa et al, 2007b).…”

“…Microarray analysis confirmed via qPCR also showed VCD and MXC upregulated PI3K/Akt and mTOR signalling, two synergistic pathways intimately associated with primordial follicle activation (Reddy et al, 2010). Further evidence for PI3K/Akt signalling in VCD induced primordial follicle activation comes from a study conducted by Hoyer et al (2009), in which LY294002, an inhibitor of PI3K, prevented primordial follicle depletion in cultured rat ovaries (Vlahos et al, 1994).…”

“…Cellular toxicity occurs when these adducts disrupt the normal structure and/or function of these macromolecules, resulting in apoptosis, necrosis or carcinogenesis. The main site of xenobiotic biotransformation within the body is the liver, although the ovary is capable of both phase I and phase II metabolism (Igawa et al, 2009;Rajapaksa et al, 2007aRajapaksa et al, , 2007bShimada et al, 2003). Therefore, there is potential for the vulnerable primordial follicle to come into contact with bioactivated ovotoxic metabolites via several routes of exposure.…”

“…These three enzymes required for DMBA's biotransformation are all expressed and induced by DMBA exposure in the murine ovary (Igawa et al, 2009;Rajapaksa et al, 2007b;Shimada et al, 2003). In further support of localised DMBA bioactivation, inhibition of MeH in cultured rat ovaries inhibited DMBA induced ovotoxicity, while ovarian culture in the presence of DMBA-3,4-diol induced significantly more primordial follicle depletion than DMBA alone (Igawa et al, 2009;Rajapaksa et al, 2007b). Another example of localised bioactivation is the conversion of the industrial chemical 4-Vinylcyclohexene (VCH) into VCH diepoxide (VCD) (Fig.…”