In vivo exposure to benzo(a)pyrene induces significant DNA damage in mouse oocytes and cumulus cells

Abstract

Does in vivo exposure to benzo(a)pyrene (BaP) induce DNA damage in oocytes and cumulus cells (CCs) in mice? Significant increases in DNA strand breaks in oocytes and CCs and in BaP-induced DNA adducts in CCs were detected in exposed mice compared with controls. BaP has well-known mutagenic and carcinogenic effects on somatic cells, and is also registered as potential reproductive toxicant by several environmental protection agencies. It has been shown to cause a significant increase in DNA adducts in ovarian tissues; however, to our knowledge, the genotoxic effects of BaP on oocytes and CCs have not been studied to date. Female CD1 mice were exposed to BaP via the oral administration of a single dose of 13 mg/kg body weight (bw); matched controls were exposed to the vehicle only (soya oil). A total of 15 groups of 6 mice (exposed or controls) were sacrificed 2, 4, 6, 15 or 22 days after BaP exposure, and after collection of oviducts, the oocyte-CC complexes (COC) were released. The alkaline comet assay was used to quantify the DNA breaks in oocytes and CCs; DNA damage was expressed as the Olive Tail Moment (OTM). Immunofluorescent staining was used to quantify BaP-induced DNA adducts in CCs. Fluorescence was expressed as the average grey value (AGVA; arbitrary units). The differences between the exposed and control groups were assessed using analysis of variance (ANOVA) and the non-parametric Mann-Whitney test. Higher levels of DNA damage were observed in the oocytes and CCs of BaP-exposed mice than in those of vehicle controls. Significant increases in OTM (mean ± SE) were detected in (i) oocytes from females exposed for 4 (10.5 ± 0.9 versus 3.1 ± 0.4, P < 0.0001) or 6 days before collection (15.6 ± 2.0 versus 3.6 ± 0.9, P < 0.0001) and (ii) CCs from females exposed 2 (6.4 ± 0.6 versus 2.1 ± 0.2, P < 0.0001), 4 (7.8 ± 0.4 versus 2.4 ± 0.1, P < 0.0001) or 6 days before collection (7.3 ± 0.3 versus 3.2 ± 0.5, P < 0.0001) compared with controls. A significant increase in benzo(a)pyrene-7,8-9,10 diol epoxide (BPDE)-DNA adducts and higher AGVA (mean ± SE) scores were observed in CCs from females exposed 2 (6.1 ± 0.3 versus 3.6 ± 0.5, P < 0.0001), 4 (7.5 ± 0.1 versus 3.4 ± 0.1, P < 0.0001) or 6 days before collection (11.6 ± 0.4 versus 3.7 ± 0.1, P < 0.0001) compared with control mice. Mice were given one treatment via the oral route because this dose and mode of administration have been shown to induce detectable BPDE-DNA adduct levels in mouse organs and sperm cells. Additional data are needed to assess DNA damage in oocytes and CCs after chronic exposure to BaP in vivo. To our knowledge, this is the first study examining the in vivo genotoxicity of BaP in oocytes and CCs. We observed significant DNA damage in the oocytes and CCs of mice after acute BaP exposure. BPDE-DNA adducts result directly from BaP metabolism while DNA breaks could result mainly from BPDE-DNA adduct excision and repair and/or through direct genotoxicity from increased reactive oxygen species. These results add new and important insights regarding the recently suggested toxicity of chronic BaP exposure in the ovary. This work was supported by a grant (93-CPQ 2012-05) from the DIRRECTE, Provence Alpes Côte d'Azur, France. None of the authors have any conflict of interest to declare.