Detection of the major DNA adducts of benzo[b]fluoranthene in mouse skin: role of phenolic dihydrodiols.

The primary mechanism of activation of benzo[b]fluoranthene (B[b]F) to a genotoxic agent does not involve the bay region dihydrodiol epoxide trans-9,10-dihydroxy-anti- 11,12-epoxy-9,10,11,12-tetrahydrobenzo[b]fluoranthene (B[b]F-9,10-DE). Previous results have suggested that the major DNA adduct(s) formed in vivo with B[b]F are more polar than those formed from B[b]F-9,10-DE. Employing newly-developed synthetic methods for the formation of B[b]F derivatives, trans-9,10-dihydro-5,9,10-trihydroxybenzo[b]fluoranthene (5-OH-B[b]F-9,10-diol) and trans-9,10-dihydro-6,9,10-trihydroxybenzo[b]fluoranthene (6-OH-B[b]F-9,10-diol) were synthesized and evaluated for tumor-initiating activity at initiation doses of 0.04, 0.12, and 0.4 mumol per mouse. In contrast to previous bioassay results, both of these phenolic dihydrodiols exhibited tumor-initiating activity. 5-OH-B[b]F-9,10-diol was the more potent tumor initiator, inducing a 90% incidence of tumor-bearing mice with an average of 7.50 tumors per mouse at a total initiator dose of 0.4 mumol. 6-OH-B[b]F-9,10-diol was significantly less active as a tumor initiator in this bioassay, inducing a 50% incidence of tumor-bearing mice with an average of 0.60 tumor per mouse at a total dose of 0.4 mumol. 32P-Postlabeling analysis was employed to evaluate the DNA adducts formed in vivo in mouse skin from both 5- and 6-OH-B[b]F-9,10-diol. HPLC analysis of B[b]F-DNA adducts formed in mouse skin demonstrated the presence of a single major adduct together with four additional minor adducts. The DNA adducts formed with 5-OH-B[b]F-9,10-diol had identical HPLC retention to the major and one of the minor adducts observed for B[b]F. These two DNA adducts account for 58% of the modified nucleotides detected in mouse skin following the topical application of B[b]F. Methods for the preparation of trans-9,10-dihydroxy-anti-11,12-epoxy-5-hydroxy-9,10,11,12- tetrahydrobenzo[b]fluoranthene (5-OH-B[b]F-9,10-DE) were developed. The DNA adducts formed with 5-OH-B[b]F-9,10-DE were similar to the adducts formed with 5-OH-B[b]F-9,10-diol. The results of the DNA binding studies along with the tumor-initiating data indicate that 5-OH-B[b]F-9,10-diol is the major proximate tumorigenic metabolite of B[b]F on mouse skin. Studies performed with deoxyguanosine 3'-phosphate suggest that the principal adducts formed with anti-5-OH-B[b]F-9,10-DE are the result of the interaction with deoxyguanosine.