Identification and quantitation of dibenzo[a,l]pyrene-DNA adducts formed by rat liver microsomes in vitro: Preponderance of depurinating adducts

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB[a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The "stable" (remaining intact in DNA under normal conditions of purification) and "depurinating" (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the ³²P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N3Ade, 33%), 7-(DB[a,l]P-10-yl)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-yl)guanine (DB[a,l]P-10-N7Gua, 5%) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P-10-N3Ade (28%), DB[a,l]P-10-N7Ade (14%), DB[a,l]P-10-N7Gua (2%), and DB[a,l]P-10-C8Gua (6%), as well as the diol epoxide depurinating adducts (±)-syn-DB[a,l]P-diol epoxide (DE)-14-N7Ade (31%) and (±)-anti-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected. When DB [a,l]P-11,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB[a,l]PDE were found, but they constituted only 19% of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the ³²P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[a,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA.