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

Abstract

7,12-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 4 days followed by single exposures of vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and maintained in culture for 4 or 8 days. Alternately, PND4 F344 rat ovaries were exposed to 1 μM DMBA at the start of culture for 2 days. 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 (12.5 nM and 1 μM) 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 8 days of DMBA exposure. Total ATM increased in a concentration-dependent temporal pattern (75 nM d4; 12.5 nM d8), while pATM was localized in large primary and secondary follicles and increased after 8 days of 75 nM DMBA exposure compared to both control and 12.5 nM 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.

Keywords: 7,12-Dimethylbenz[a]anthracene; DNA damage; DNA double strand break repair; Ovotoxicity.

Fig. 1

Effect of DMBA exposure on caspase-3 protein expression. Following 8 days of culture, total protein was isolated from PND4 rat ovaries exposed to control (CT), 12.5 or 75 nM DMBA. Caspase-3 protein was measured by Western blotting. (A) Densitometry data was normalized to Ponceau S and expressed as mean raw data ± SE; n = 3 (10 ovaries per pool). Statistical significance was defined as * = P < 0.05. (B) Western blot of caspase-3.

Fig. 2

Localization and quantification of γH2AX protein. Paraffin embedded ovarian sections from PND4 rat ovaries exposed to (A) control (CT), (B) 12.5 or (C) 75 nM DMBA were used to perform immunohistochemistry to determine localization of γH2AX protein after 4 days. (D) Quantification of γH2AX loci in large primary and secondary follicles; data is expressed as number of follicles positive for γH2AX ± SE; n = 3; Statistical significance was defined as * = P < 0.05. (E) Western blot (D2) to detect γH2AX in vehicle control (C) or 1 µM DMBA (D) treated ovaries and expressed as mean raw data ± SE; n = 3 (10 ovaries per pool). Statistical significance was defined as * = P < 0.05.

Fig. 3

Effect of DMBA exposure on DNA repair gene mRNA expression. Following (A) 4 or (B) 8 days of culture, RNA was isolated from PND4 rat ovaries exposed to control (CT), 12.5 or 75 nM DMBA and qPCR performed. Additionally, RNA was isolated from ovaries exposed to (C) control (CT) or 1 µM DMBA for qPCR. Values are expressed as mean fold change ± SE; n = 3 (10 ovaries per pool). Statistical significance was defined as * = P < 0.05.

Fig. 4

Effect of DMBA exposure on total ATM protein level. Following 4 or 8 days of culture, total protein was isolated from PND4 rat ovaries exposed to control (CT), 12.5 or 75 nM DMBA. ATM protein was measured by Western blotting and a representative blot fromd8 of exposure is presented. (A) Results were normalized to Ponceau S and expressed as mean raw data ± SE; n = 3 (10 ovaries per pool). Statistical significance was defined as * = P < 0.05. (B) Representative Western blot for ATM.

Fig. 5

Localization and quantification of pATM protein. Following 8 days of culture, paraffin embedded ovarian sections from PND4 rat ovaries exposed to (A) control (CT), (B) 12.5 or (C) 75 nM DMBA were used to perform immunohistochemistry to determine localization and (D) quantification of pATM protein. Data is expressed as number of follicles positive for pATM ± SE; n = 3; statistical significance was defined as * = P < 0.05.

Fig. 6

Effect of DMBA exposure on PARP1 protein level. Following 4 or 8 days of culture, total protein was isolated from PND4 rat ovaries exposed to control (CT), 12.5 or 75 nM DMBA. PARP1 protein was measured by Western blotting and a representative blot from d8 of exposure is presented. (A) Results were normalized to Ponceau S and expressed as mean raw data ± SE; n = 3 (10 ovaries per pool). Statistical significance was defined as * = P < 0.05. (B) Representative Western blot for PARP1.