Benzo[a]pyrene-7,8-quinone-3′-mononucleotide adduct standards for 32P postlabeling analyses: Detection of benzo[a]pyrene-7,8-quinone–calf thymus DNA adducts

Abstract Benzo[ a ]pyrene-7,8-quinone (BPQ) is one of the reactive metabolites of the widely distributed archetypal polycyclic aromatic hydrocarbon, benzo[ a ]pyrene (B[ a ]P). The formation of BPQ from B[ a ]P through trans -7,8-dihydroxy-7,8-dihydroB[ a ]P by the mediation of aldo-keto reductases and its role in the genotoxicity and carcinogenesis of B[ a ]P currently are under extensive investigation. Toxicity pathways related to BPQ are believed to include both stable and unstable (depurinating) DNA adduct formation as well as reactive oxygen species. We previously reported the complete characterization of four novel stable BPQ–deoxyguanosine (dG) and two BPQ–deoxyadenosine (dA) adducts (Balu et al., Chem. Res. Toxicol. 17 (2004) 827–838). However, the identification of BPQ–DNA adducts by 32 P postlabeling methods from in vitro and in vivo exposures required 3′-monophosphate derivatives of BPQ–dG, BPQ–dA, and BPQ–deoxycytidine (dC) as standards. Therefore, in the current study, BPQ adducts of dGMP(3′), dAMP(3′), and dCMP(3′) were prepared. The syntheses of the BPQ–3′-mononucleotide standards were carried out in a manner similar to that reported previously for the nucleoside analogs. Reaction products were characterized by UV, LC/MS analyses, and one- and two-dimensional NMR techniques. The spectral studies indicated that all adducts existed as diastereomeric mixtures. Furthermore, the structural identities of the novel BPQ–dGMP, BPQ–dAMP, and BPQ–dCMP adducts were confirmed by acid phosphatase dephosphorylation of the BPQ–nucleotide adducts to the corresponding known BPQ–nucleoside adduct standards. The BPQ–dGMP, BPQ–dAMP, and BPQ–dCMP adduct standards were used in 32 P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32 P postlabeling analysis revealed the formation of 8 major and at least 10 minor calf thymus DNA adducts. Of these BPQ–DNA adducts, the following were identified: 1 BPQ–dGMP adduct, 2 BPQ–dAMP adducts, and 3 BPQ–dCMP adducts. This study represents the first reported example of the characterization of stable BPQ–DNA adducts in isolated mammalian DNA and is expected to contribute significantly to the future BPQ–DNA adduct studies in vivo and thereby to the contribution of BPQ in B[ a ]P carcinogenesis.