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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 32P 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 32P postlabeling studies to identify BPQ adducts formed in vitro with calf thymus DNA and DNA homopolymers. 32P 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.

Benzo[b]fluoranthene (B[b]F) was administered (100 mg/kg by i.p. injection) to male Sprague--Dawley rats. Lungs, livers and peripheral blood lymphocytes (PBLs) were harvested 1, 3, 5, 7, 14, 28 and 56 days after treatment. Several DNA adducts were observed in each tissue, with maximal levels occurring at approximately 7 days after treatment. Lung DNA exhibited consistently higher adduct levels than liver or PBL DNA. At 56 days after B[b]F administration, the adducts in liver and PBL DNA were present at < 10 amol/microgram DNA, while in lung there were 100 amoles/microgram DNA. No significant differences were observed between tissues in the types of adducts produced. Co-chromatography with synthetic standards showed that only a minor adduct produced in vivo is derived from trans-9,10-dihydro-9,10-dihydroxybenzo[b]fluoranthene-11,12-oxide. Sister chromatid exchanges (SCEs) from whole blood cultures were significantly increased relative to concurrent controls between 1 and 14 days after B[b]F administration, with maximum levels at 14 days. By 28 days after treatment, SCEs had essentially returned to control levels. SCE induction did not correlate with the amount of B[b]F--DNA adducts remaining in the PBLs at harvest time.