Screening for DNA adducts in ovarian follicles exposed to benzo[a]pyrene and cigarette smoke condensate using liquid chromatography-tandem mass spectrometry.

Abstract A rapid mass spectrometric method was applied to non-targeted screening of DNA adducts in follicular cells (granulosa cells and theca cells) from isolated ovarian follicles that were exposed in-vitro to benzo[ a ]pyrene (B[ a ]P) and cigarette smoke condensate (CSC) for 13 days of culture. The method employed a constant neutral loss (CNL) scan to identify chromatographic peaks associated to a neutral loss of deoxyribose moiety of DNA nucleosides. These peaks were subsequently analyzed by a product ion scan in tandem mass spectrometry to elucidate structures of DNA adducts. The identification was further confirmed through synthesis of proposed DNA adducts where possible. Three DNA adducts, benzo[ a ]pyrene-7,8-dihydrodiol-9,10-epoxide-dG (BPDE-dG), phenanthrene 1,2-quinone-dG (PheQ-dG) and B[ a ]P-7,8-quinone-dG (BPQ-dG) were identified in the follicular cells from isolated ovarian follicles exposed to B[ a ]P. Along with these three, an additional DNA adduct, 4-aminobiphenyl-dG, was identified in the follicular cells from isolated ovarian follicles exposed to CSC. The amounts of the identified DNA adducts in follicular cells increased in a dose-dependent manner for both B[ a ]P (0, 1.5, 5, 15 and 45 ng/mL) and CSC (0, 30, 60, 90 and 130 μg/mL). The results revealed that B[ a ]P-related DNA adducts were the major adducts in the ovarian follicular cells exposed to CSC. The results also revealed that two oxidative biomarkers, 8-hydroxy-2-deoxy guanosine (8-OH-dG) and 8-isoprostane (8-IsoP), in both B[ a ]P-exposed and CSC-exposed ovarian follicles had strong correlations with the three DNA adducts, BPDE-dG, BPQ-dG and PheQ-dG. A pathway to describe formation of DNA adducts was proposed based on the DNA adducts observed.