Synthesis of Depurinating DNA Adducts Formed by One-Electron Oxidation of 7H-Dibenzo[c,g]carbazole and Identification of These Adducts after Activation with Rat Liver Microsomes Liang Chen, † Prabu D. Devanesan, † Jaeman Byun, ‡ Jonathan K. Gooden, ‡ Michael L. Gross, ‡ Eleanor G. Rogan, † and Ercole L. Cavalieri* ,† Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805, and Department of Chemistry, Washington University, One Brookings Drive, St. Louis, Missouri 63130 Received August 19, 1996 X It is hypothesized that 7H-dibenzo[c,g]carbazole (DBC) is metabolically activated by one- electron oxidation in accordance with its propensity to be easily oxidized to its radical cation. Iodine oxidation of DBC produces a radical cation that subsequently binds to nucleophilic groups of dG or Ade. Oxidation of DBC in the presence of dG produces three adducts: DBC-5-N7Gua, DBC-6-N7Gua, and DBC-6-C8Gua, whereas in the presence of Ade, four adducts are obtained: DBC-5-N7Ade, DBC-5-N3Ade, DBC-5-N1Ade, and DBC-6-N3Ade. Formation of these adducts demonstrates that the DBC radical cation reacts at C-5 or C-6 with the reactive nucleophiles N-7 and C-8 of dG and N-7, N-3, and N-1 of Ade. Formation of DNA adducts by DBC was studied by using horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes for activation. Identification of the biologically-formed depurinating adducts was achieved by comparison of their retention times on HPLC in two different solvent systems and by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Quantitation of the adducts formed by rat liver microsomes shows that 96% are depurinating adducts, DBC- 5-N7Gua (11%), DBC-6-N7Gua (32%), and DBC-5-N7Ade (53%), and 4% are unidentified stable adducts. Activation of DBC by horseradish peroxidase affords 32% stable unidentified adducts and 68% depurinating adducts: 19% DBC-5-N7Gua, 13% DBC-6-N7Gua, 27% DBC-5-N7Ade, and 9% DBC-5-N3Ade. Thus, activation of DBC by cytochrome P450 predominantly forms depurinating adducts by one-electron oxidation. Introduction Elucidation of the mechanisms of metabolic activation of polycyclic aromatic hydrocarbons (PAH) 1 is essential for understanding their mechanisms of tumor initiation. Two major pathways of activation are involved in the formation of the ultimate carcinogenic metabolites of PAH: one-electron oxidation to form intermediate radical cations and monooxygenation to produce bay-region diol epoxides (1-4). The predominant mechanism of activa- tion for some potent carcinogenic PAH appears to be one- electron oxidation (5-11). Activation of PAH by formation of radical cations can be predicted on the basis of two major factors: a low oxidation potential that allows easy removal of one electron by cytochrome P450 or peroxidases (1, 2, 12) and sufficient charge localization that allows efficient and specific reaction of the radical cations with the nucleo- philic groups of DNA (1, 2). These rules have been applied for predicting the occurrence of this mechanism for alternate PAH (condensed aromatic rings containing an even number of carbon atoms), but the same rules are not applicable to nonalternate PAH (condensed aromatic rings containing one or more rings with an odd number of carbon atoms, e.g., cyclopenta[c,d]pyrene) or heterocyclic PAH. 7H-Dibenzo[c,g]carbazole (DBC), an N-heterocyclic PAH, is a potent environmental carcinogen that has been shown to induce tumors in dogs, rats, hamsters, and mice (13, 14). Both local and systemic induction of tumors has been observed in mouse skin, liver, and lung (14-16). DBC has a relatively low oxidation potential (anodic peak potential ) 1.07 eV), that is similar to that of 3-methylcholanthrene (3-MC, 1.08 eV) (12) and lower than that of benzo[a]pyrene (BP, 1.12 eV) (12). Thus, DBC can be easily oxidized by both cytochrome P450 and peroxidases. The low value of the oxidation potential is necessary, although not sufficient, for metabolic activa- tion by one-electron oxidation. To assess the importance of this mechanism for DBC, this compound was oxidized by iodine in the presence of dG or Ade. Investigation of the adducts formed by one-electron oxidation of DBC serves several purposes: (1) to determine the specific reactivity of one or more positions in the DBC radical cation, (2) to determine the nucleophilic groups in the nucleic acid bases that participate in adduct formation, (3) to provide evidence for the mechanism of adduct * To whom correspondence should be addressed. † Eppley Institute for Research in Cancer. ‡ Washington University. X Abstract published in Advance ACS Abstracts, January 15, 1997. 1 Abbreviations: BCC, 4-(benzyloxy)-R-cyanocinnamic acid; BP, benzo[a]pyrene; CA, collisional activation; CAD, collisional activation decomposition(s); COSY, two-dimensional chemical shift correlation spectroscopy; DBA, dibenz[a,j]acridine; DB[a,l]P, dibenzo[a,l]pyrene; DBC, 7H-dibenzo[c,g]carbazole; DMF, dimethylformamide; FAB MS/ MS, fast atom bombardment tandem mass spectrometry; FWHM, full width at half-maximum; Gly/TFA, glycerol/1% trifluoroacetic acid; HRP, horseradish peroxidase; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; 3-MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon(s); PDA, photodiode array; PSD, post source decay(s). 225 Chem. Res. Toxicol. 1997, 10, 225-233 S0893-228x(96)00149-X CCC: $14.00 © 1997 American Chemical Society