7,12-Dimethylbenz[a]anthracene exposure induces the DNA repair response in neonatal rat ovaries

Abstract7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles at all stages of development. This study investigated DMBA-induced DNA double strand break (DSB) formation with subsequent activation of the ovarian DNA repair response in models of pre-antral or pre-ovulatory follicle loss. Postnatal day (PND) 4 Fisher 344 (F344) rat ovaries were cultured for 4days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5nM or 75nM) and maintained in culture for 4 or 8days. Alternately, PND4 F344 rat ovaries were exposed to 1μM DMBA at the start of culture for 2days. Total RNA or protein was isolated, followed by qPCR or Western blotting to quantify mRNA or protein level, respectively. γH2AX and phosphorylated ATM were localized and quantified using immunofluorescence staining. DMBA exposure increased caspase 3 and γH2AX protein. Additionally, DMBA (12.5nM and 1μM) increased levels of mRNA encoding Atm, Xrcc6, Brca1 and Rad51. In contrast, Parp1 mRNA was decreased on d4 and increased on d8 of DMBA exposure, while PARP1 protein increased after 8days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75nM d4; 12.5nM d8), while pATM was localized in large primary and secondary follicles and increased after 8days of 75nM DMBA exposure compared to both control and 12.5nM DMBA. These findings support that, despite some concentration effects, DMBA induces ovarian DNA damage and that DNA repair mechanisms are induced as a potential mechanism to prevent follicle loss