DETECTION OF THE MAJOR DNA ADDUCTS OF BENZO[J]FLUORANTHENE IN MOUSE SKIN - NONCLASSICAL DIHYDRODIOL EPOXIDES

Studies have demonstrated that the metabolic activation of benzo[j]fluoranthene (B[j]F) to a genotoxic agent proceeds through the formation of either the trans-4,5-dihydrodiol (B[j]F-4,5-diol) or the trans-9,10-dihydrodiol (B[j]F-9,10-diol) metabolite of B[j]F. Using P-32-postlabeling analysis, the profiles of DNA adducts formed in vivo in mouse skin from B[j]F-4,5-diol and B[j]F-9,10-diol were obtained to establish the contribution of each of these dihydrodiols to the formation of B[j]F-DNA adducts in vivo. B[j]F,B[j]F-4,5-diol and B[j]F-9,10-diol were applied to the shaved backs of mice (100 mug/mouse), and DNA adducts were isolated and separated using multidimensional TLC and reverse-phase HPLC. The greatest level of adducts was observed with B[j]F-4,5-diol, which resulted in the formation of 383 pmol of DNA adducts/mg of DNA. This level of DNA modification was more than 2 orders of magnitude greater than that observed with B[j]F or B[j]F-9,10-diol. The major adducts detected from the application of B[j]F-4,5-diol to mouse skin in vivo had chromatographic properties similar to those of the major adducts detected with B[j]F. In contrast, the major DNA adducts detected with B[j]F-9,10-diol had chromatographic properties distinctly different than the adducts formed from either B[j]F or B[j]F-4,5-diol. DNA adducts formed from the syn and anti isomers of the 4,5-dihydrodiol 6,6a-epoxide and the 9,10-dihydrodiol 11,12-epoxide of B[j]F were also evaluated. Each dihydrodiol epoxide derivative was reacted with calf thymus DNA in vitro and applied to mouse skin in vivo. The DNA adducts formed in vitro were qualitatively similar to the adducts formed in vivo in mouse skin. The DNA adducts of each dihydrodiol epoxide derivative were also chromatographically similar to the major adducts isolated from mouse skin after application of the corresponding precursor dihydrodiol metabolite. The anti isomer formed considerably more DNA adducts than the syn isomer for each of the dihydrodiol epoxide derivatives evaluated. Differences in the adduct levels observed for anti isomers of B[j]F-4,5-diol and B[j]F-9,10-diol, however, were not significant. In addition, the level of adducts formed from the syn isomers of each dihydrodiol was also similar. These results, along with the observation that B[j]F-4,5-diols forms exceptionally higher levels of DNA adducts than that observed with B[j]F-9,10-diol, indicate that B[j]F-4,5-diol has an increased preference for further metabolism to the dihydrodiol epoxide metabolite. These data indicate that B[j]F-4,5-diol is the major proximate tumorigenic metabolite of B[j]F on mouse skin. Studies performed with deoxyadenosine 3'-phosphate and deoxyguanosine 3'-phosphate indicate that the principal adducts formed with the anti-4,5-dihydrodiol 6,6a-epoxide of B[j]F are the result of the interaction with deoxyguanosine. These DNA adducts correspond to the major DNA adducts formed in vivo in mouse skin with B[j]F.