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 amon g 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 mic rosomes, and DB[a,l]P was also bound to DNA by horseradish peroxidase. The ''stable'' (remaining intact in DN4 under normal conditions of pu rification) 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 P-32-p ostlabeling technique. Depurinating adducts were identified by compari son of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by m eans of fluorescence line-narrowing spectroscopy. When DB[a,l] was act ivated 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 microsome s, 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 (+/-)-sy n-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 th e 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 depu rinating adducts arising from DB[a,l]PDE were found, but they constitu ted only 19% of the adducts because the amount of stable adducts was m uch higher than with DB[a,l]P. Analysis of stable DNA adducts by the P -32-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, result ing in apurinic sites in the DNA.