Synthesis and structure determination of the adducts formed by electrochemical oxidation of the potent carcinogen dibenzo[a,I]pyrene in the presence of nucleosides.

Because dibenzo[a,l]pyrene (DBP) is the most potent known carcinogenic aromatic hydrocarbon, reference adducts formed by reaction of deoxyribonucleosides with electrophilic intermediates of DBP are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of DBP in the presence of nucleosides leads to adducts from DBP.+. When 6.8 equiv of charge are consumed, three adducts are formed with dG: 7-(DBP-10-yl)Gua (89%), 8-(DBP-10-yl)dG (2%), and 8-(DBP-10-yl)Gua (2%). With 10 equiv of charge, however, only two adducts are formed: 7-(DBP-10-yl)Gua (89%) and 8-(DBP-10-yl)Gua (4%). Anodic oxidation of 8-(DBP-10-yl)dG yields 8-(DBP-10-yl)Gua. Anodic oxidation of DBP in the presence of G produces 7-(DBP-10-yl)Gua (27%) and 8-(DBP-10-yl)G (9%). Anodic oxidation of DBP in the presence of dA affords two adducts, N6-(DBP-10-yl)dA (28%) and 7-(DBP-10-yl)Ade (12%), whereas anodic oxidation in the presence of A produces only N6-(DBP-10-yl)A (24%). The structures of the adducts were elucidated by using UV, NMR, and MS. Formation of these adducts demonstrates that DBP.+ reacts at C-10 with nucleophiles. The most reactive nucleophilic groups for the Gua moiety are the N-7 and C-8, whereas for the Ade moiety they are N-7 and the 6-amino group.

Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin.

Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Model adducts of benzo[a]pyrene and nucleosides formed from its radical cation and diol epoxide.

Reference adducts formed by reaction of deoxyribonucleosides with the ultimate carcinogenic forms of benzo[a]pyrene (BP), BP radical cation and BP diol epoxide, are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of BP in the presence of dG or dA produces adducts from BP radical cation. When 8 equiv of charge are consumed, four adducts are formed with dG: 7-(BP-6-yl)Gua, 8-(BP-6-yl)Gua, N2-(BP-6-yl)dG and 3-(BP-6-yl)dG. With 2 equiv of charge, however, only 7-(BP-6-yl)Gua and 8-(BP-6-yl)dG (BP-6-C8dG) are formed. Anodic oxidation of BP-6-C8dG affords 8-(BP-6-yl)Gua. Anodic oxidation of BP in the presence of dA produces 7-(BP-6-yl)Ade. Reaction of BP diol epoxide with dG yields 10-(guanin-7-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP, whereas reaction with dA affords three adducts, 10-(adenin-7-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP and two isomers of 10-(deoxyadenosin-N6-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydroBP . On the basis of comparative kinetic studies among adducts of aromatic hydrocarbons and dG or G, only BP-6-C8dG easily loses the sugar moiety, providing a basis for a mechanism of hydrolysis of the glycosidic bond.

Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin.

The DNA adducts of benzo[a]pyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound at C-10 to the 2-amino of dG (BPDE-10-N2dG). Identification of the depurination adducts was achieved by HPLC and fluorescence line narrowing spectroscopy. The depurination adducts, BP-6-C8Gua (34%), BP-6-N7Gua (10%), and BP-6-N7Ade (30%), constituted 74% of the adducts found in mouse skin 4 h after treatment with BP. The stable adduct BPDE-10-N2dG accounted for 22% of the adducts. Treatment of the skin with BP-7,8-dihydrodiol or BP diolepoxide yielded almost exclusively the stable adduct BPDE-10-N2dG. When BP or BP-7,8-dihydrodiol was bound to RNA or denatured DNA in reactions catalyzed by rat liver microsomes, no depurination adducts were detected. The profiles of stable adducts were similar both qualitatively and quantitatively with native or denatured DNA. With activation of BP by horseradish peroxidase, the profiles of stable adducts differed with native and denatured DNA. The total amount of adducts with denatured DNA was only 25% of the amount detected with native DNA. No depurination adducts were detected with denatured DNA or RNA in the peroxidase system.(ABSTRACT TRUNCATED AT 250 WORDS)