EXPANDED ANALYSIS OF BENZO[A]PYRENE-DNA ADDUCTS FORMED IN-VITRO AND IN MOUSE SKIN - THEIR SIGNIFICANCE IN TUMOR INITIATION

This paper reports expanded analyses of benzo[a]pyrene (BP)-DNA adduct s formed in vitro by activation with horseradish peroxidase (HRP) or 3 -methylcholanthrene-induced rat liver microsomes and in vivo in mouse skin. The adducts formed by BP are compared to those formed by BP-7,8- dihydrodiol and anti-BP diol epoxide (BPDE). First, activation of BP b y HRP produced 61% depurinating adducts: 7-(benzo[a]pyrene-6-yl)guanin e (BP-6-N7Gua), BP-6-C8Gua, BP-6-N7Ade, and the newly identified BP-6- N3Ade. As a standard, the last adduct was synthesized along with BP-6- N1Ade by electrochemical oxidation of BP in the presence of adenine. S econd, identification and quantitation of BP-DNA adducts formed by mic rosomal activation of BP showed 68% depurinating adducts: BP-6-N7Ade, BP-6-N7Gua, BP-6-C8Gua, BPDE-10-N7Ade, and the newly detected BPDE-10- N7Gua. The stable adducts were mostly BPDE-10-N(2)dG (26%), with 6% un identified. BPDE-10-N7Ade and BPDE-10-N7Gua were the depurinating addu cts identified after microsomal activation of BP-7,8-dihydrodiol or di rect reaction of anti-BPDE with DNA. In both cases, the predominant ad duct was BPDE-10-N(2)dG (90% and 96%, respectively). Third, when mouse skin was treated with BP for 4 h, 71% of the total adducts were the d epurinating adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and small amou nts of BPDE-10-N7Ade and BPDE-10-N7Gua. These newly detected depurinat ing diol epoxide adducts were found in larger amounts when mouse skin was treated with BP-7,8-dihydrodiol or anti-BPDE. The stable adduct BP DE-10-N(2)dG was predominant, especially with anti-BPDE. Comparison of the profiles of DNA adducts formed by BP, BP-7,8-dihydrodiol and anti -BPDE with their carcinogenic potency indicates that tumor initiation correlates with the levels of depurinating adducts, but not with stabl e adducts. Furthermore, the levels of depurinating adducts of BP corre late with mutations in the Harvey-ras oncogene in DNA isolated from mo use skin papillomas initiated by this compound [Chakravarti et al. (19 95) Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426]. The depurinating a dducts formed by BP in mouse skin appear to be the key adducts leading to tumor initiation.