32P-postlabeling analysis of benzo[a]pyrene-DNA adducts formed in vitro and in vivo

Abstract

Benzo[a]pyrene (BP) was bound to DNA by horseradish peroxidase, rat liver microsomes, and rat liver nuclei in vitro and in mouse skin in vivo. The BP-DNA adducts formed were analyzed by the 32P-postlabeling technique. Activation by microsomes and nuclei resulted in the detection of five adducts, including a major adduct (55%) which cochromatographed with the adduct (+/-)-10 beta-deoxyguanosin-N2-yl-7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydro-BP (BPDE-N2dG) formed by reaction of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) with DNA or by microsomal activation of BP 7,8-dihydrodiol. Activation by horseradish peroxidase, which catalyzes one-electron oxidation, produced seven adducts, including a major one (30%) that coeluted with an adduct observed with microsomal (2%) and nuclear (14%) activation. The pattern of adducts formed in mouse skin treated with BP in vivo for 4 or 24 h contained four of the same adducts observed with nuclei or microsomes in vitro, and the predominant adduct detected (86%) was BPDE-N2dG. The adduct common to horseradish peroxidase, microsomes, and nuclei was also detected in mouse skin DNA (2%). These results demonstrate that multiple BP-DNA adducts are formed in these in vitro and in vivo systems and suggest that at least one adduct is formed in common in all of the systems. Thus, it appears that stable BP adducts can be formed in mouse skin DNA by both monooxygenation and one-electron oxidation.