Characterization of AαC-serum albumin adducts 2-Amino-9H-pyrido[2,3-b]indole (AαC) Adducts and Thiol Oxidation of Serum Albumin as Potential Biomarkers of Tobacco Smoke

2-Amino-9H-pyrido[2-3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine (HAA) formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic Noxidized metabolites that react with DNA to form adducts, which can lead to mutations. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); however, the chemistry of reactivity of AαC with proteins has not been studied. The genotoxic metabolites, 2hydroxyamino-9H-pyrido[2-3-b]indole (HONHAαC), 2-nitroso-9H-pyrido[2-3-b]indole (NOAαC), a N-acetoxy-9H-pyrido[2-3-b]indole (Nacetoxy-AαC), and their [C6]-AαC labelled homologues were reacted with albumin. Sites of adduction of AαC to albumin were identified, by data dependent scanning and targeted bottom-up proteomics approaches employing ion trap and Orbitrap MS. AαC–albumin adducts were formed at Cys, Tyr and Tyr residues, when albumin was reacted with HONH-AαC or NOAαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys (AαC-Cys). N-Acetoxy-AαC also formed an adduct at Tyr. Albumin-AαC adducts were characterized in human plasma treated with N-oxidized metabolites of AαC and human hepatocytes exposed to AαC. High levels of N(deoxyguanosin-8-yl)-AαC (dG-C8-AαC) DNA adducts were formed in hepatocytes. The Cys was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species (ROS) generated by metabolites of AαC in hepatocytes: there was a strong decrease in reduced Cys while the levels of Cyssulfinic acid (Cys-SO2H), Cys-sulfonic acid (Cys-SO3H) and Met sulfoxide were greatly increased. Cys-adduction products and Cys-SO2H, CysSO3H and Met sulfoxide may be potential biomarkers to assess exposure and oxidative