Polycyclic aromatic hydrocarbons (PAH) are activated by cytochrome P-450 via two main pathways monooxygenation and one-electron oxidation, with formation of the reactive intermediate diol epoxides and radical cations, respectively. When benzo[a]pyrene (BP) is bound to DNA by cytochrome P-450 in vitro or in vivo, the major adducts, formed by one- electron oxidation, contain BP bound at C-6 to N-7 of Gua (BP-6-N7Gua), C-8 of Gua (BP-6-C8Gua) and N-7 of Ade (BP-6-N7Ade), all lost from DNA by depurination. About 90% of all BP-DNA adducts formed are lost from the DNA by depurination. Rodents treated with BP excrete these adducts. These findings enable us to focus on PAH-DNA adducts formed in vivo and begin to study their biological significance. We propose to measure BP- 6-N7Gua and BP- 6-N7Ade as markers for exposure to BP by using monoclonal antibodies (Mabs). BALB/c mice will be immunized with BP-6- N7Gua (or BP-6-N7Ade) linked through succinimidyl-4-(N- maleimidoethyl)cyclohexane-1-carboxylate (SMCC) to keyhole limpet hemocyanin (KLH). Hybridomas will be developed and assayed for Mabs specific for the adducts. Selected Mabs will be used in ELISA to determine the presence of BP-6-N7Gua and/or BP-6-N7Ade in urine from rats and mice treated with BP and in human urine samples from cigarette smokers and non-smokers.
In Aims #1 and 2, we expect to develop an immunoassay for human exposure to the carcinogen BP in cigarette smoke and air pollution.
In Aim #4, we propose to examine the labile DNA adducts formed by BP and 7, 12-dimethylbenz[a]anthracene (DMBA) by analyzing alkali-labile lesions in a 229 base-pair fragment of pBR322 DNA treated with BP or DMBA in chemical and enzymatic reactions. With these experiments, we will identify the sequence specificity of apurinic sites arising by depurination of the labile adducts that constitute 90% of all BP-DNA. adducts. Recent results suggest that 7-hydroxyBP (7-OHBP) specifically inhibits metabolism of BP 7,8-dihydrodiol to BP diol epoxide, but does not interfere with formation of BP radical cation. If so, 7-OHBP may provide a method to identify mechanisms of carcinogenic activation of PAH. To explore this, we propose (Aim #3) to determine the inhibitory effects of 7-OHBP on 1) formation of BP-and DMBA-DNA adducts by one-electron oxidation and monooxygenation, and 2) initiation of tumors by BP, DMBA and 5-methylchrysene in mouse skin. We expect 7-OHBP to inhibit tumor initiation by BP 7,8-dihydrodiol, DMBA 3,4-dihydrodiol and 5-methylchrysene, but not by BP and DMBA, that we think are mainly activated by one-electron oxidation. If so, this will open a new era of research in elucidating mechanisms of carcinogenesis of PAH.
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