Shanthi Ganesan

7,12-dimethylbenz[a]anthracene (DMBA), a polycyclic aromatic hydrocarbon, is liberated through organic matter combustion, thus cigarette smoke and car exhaust fumes are human exposure sources. DMBA destroys ovarian follicles in mice and rats, and in extra-ovarian tissues causes DNA damage through covalent binding. Ataxia telangiectasia mutated (ATM) initiates the cellular genotoxic response after double-stranded DNA break (DSB) occurrence. Also, H2AX phosphorylation (gammaH2AX) occurs rapidly at DNA DSB to recruit DNA repair molecules to damage sites until repair is complete. Acute single DMBA exposure to cultured postnatal day 4 (PND4) F344 rat ovaries caused loss of large primary and secondary follicles at 12.5 nM DMBA, while 75 nM DMBA only caused large primary follicle loss. Ovarian DMBA-induced DNA damage is not characterized, thus, this study investigated DMBA-induced DNA DSB and ovarian repair response activation in rat ovaries after acute DMBA exposure. PND4 F344 rat ovaries were cultured for 4 d followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and culture maintained for 4 or 8 d for quantification of mRNA or protein level, respectively. Total RNA was isolated (n = 3; 6-10 ovaries/pool) or ovaries (n = 3) were fixed in formalin for immunostaining. Quantitative RT-PCR was used to measure Atm mRNA expression. Immunofluorescence staining to measure gammaH2AX, total ATM and pATM protein level was performed. Both DMBA concentrations increased (P < 0.05) Atm mRNA expression relative to controls. Higher numbers (P < 0.05) of gammaH2AX foci were observed in large primary and secondary follicles at 75 nM DMBA compared to control and 12.5 nM DMBA. No change in total ATM across treatments was observed. pATM foci were increased (P < 0.05) in secondary follicles at 75 nM DMBA compared to control and 12.5 nM DMBA. These data support activation of the ovarian response to DNA damage at both DMBA concentrations at a transcriptional level, but at the translational level only at the 75 nM DMBA concentration. Thus, these results suggest that the ovarian protective DNA repair response is critical for survival of ovarian follicles exposed to DMBA. (Supported by ES016818 to AFK.)