Quantification of Hemoglobin and White Blood Cell DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl Formed in Humans by Nanoflow Liquid Chromatography/Ion Trap Multistage Mass Spectrometry

Aromatic amines covalently bound to hemoglobin (Hb) as sulfinamide adducts at the cysteine 93 residue of the Hb beta chain have set-tea as biomarkers to assess exposure to this class of human carcinogens for the past 30 years. In this study, we report that 2-amino-9H-pyrido[2,3-b]indole (A alpha C), an abundant carcinogenic heterocyclic, aromatic amine formed in tobacco smoke,,and charred cooked meats, also reacts with Hb to form a sulfinamide adduct. A novel nanoflow liquid chromatography/ion trap multistage mass spectrometry (nanoLC-IT/MS3) method was established to assess exposure to A alpha C and the tobacco-associated bladder carcinogen 4-aminobiphenyl (4-ABP) through their Hb sulfinamide adducts. Following mild acid hydrolysis of Hb in vitro, the liberated A alpha C and 4-ABP were derivatized with acetic anhydride to form the N-acetylated amines, which were measured by nanoLC-IT/MS3. The limits of quantification (LOQ) for A alpha C- and 4-ABP-Hb sulfinamide adducts were <= 7.1 pg/g Hb. In a pilot study, the mean level of Hb sulfinamide adducts of A alpha C and 4-ABP were, respectively; 3.4-fold and 4.8-fold higher in smokers (>20,cigarettes/day) than nonsmokers. In contrast, the major DNA adducts of 4-ABP, N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl, and A alpha C, N-(2'-deoxyguanpsin-8-yl)-2-amino-9H-pyrido[2,3-b]indole, were below the LOQ (3 adducts per 109 bases) in white blood cell (WBC) DNA of smokers and nonsmokers. These findings reaffirm that tobacco smoke is a major source of exposure to A alpha C. Hb sulfinamide adducts are suitable biomarkers to biomonitor 4-ABP and A alpha C; however, neither carcinogen binds to DNA in WBC, even in heavy smokers; at levels sufficient for biomonitoring.