Abstract
2-Amino-9H-pyrido[2,3-b]indole (AαC) is a carcinogenic heterocyclic aromatic amine formed during the combustion of tobacco. AαC undergoes bioactivation to form electrophilic N-oxidized 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, 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), 2-nitroso-9H-pyrido[2,3-b]indole (NO-AαC), N-acetyloxy-2-amino-9H-pyrido[2,3-b]indole (N-acetoxy-AαC), and their [(13)C6]AαC-labeled 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(34), Tyr(140), and Tyr(150) residues when albumin was reacted with HONH-AαC or NO-AαC. Sulfenamide, sulfinamide, and sulfonamide adduct formation occurred at Cys(34) (AαC-Cys(34)). N-Acetoxy-AαC also formed an adduct at Tyr(332). 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(34) was the sole amino acid of albumin to form adducts with AαC. Albumin also served as an antioxidant and scavenged reactive oxygen species generated by metabolites of AαC in hepatocytes; there was a strong decrease in reduced Cys(34), whereas the levels of Cys(34) sulfinic acid (Cys-SO2H), Cys(34)-sulfonic acid (Cys-SO3H), and Met(329) sulfoxide were greatly increased. Cys(34) adduction products and Cys-SO2H, Cys-SO3H, and Met(329) sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with AαC and other arylamine toxicants present in tobacco smoke.
Highlights
The reactivity of A␣C, a tobacco smoke carcinogen, was investigated with DNA and albumin of human hepatocytes
Albumin served as an antioxidant and scavenged reactive oxygen species generated by metabolites of A␣C in hepatocytes; there was a strong decrease in reduced Cys34, whereas the levels of Cys34 sulfinic acid (Cys-SO2H), Cys34-sulfonic acid (Cys-SO3H), and Met329 sulfoxide were greatly increased
Primary human hepatocytes are an ideal ex vivo model system for studying metabolism, bioactivation, and mechanisms of toxicity of carcinogens, because cofactors are present at physiological concentrations, and biotransformation pathways may closely simulate those that occur in vivo [53]
Summary
The reactivity of A␣C, a tobacco smoke carcinogen, was investigated with DNA and albumin of human hepatocytes. Results: Hepatocytes bioactivate A␣C to metabolites, which adduct to DNA and albumin. Conclusion: Cys and Met329 of serum albumin are targets for A␣C electrophiles. Many genotoxicants and toxic electrophiles react with human serum albumin (albumin); the chemistry of reactivity of A␣C with proteins has not been studied. Albumin served as an antioxidant and scavenged reactive oxygen species generated by metabolites of A␣C in hepatocytes; there was a strong decrease in reduced Cys, whereas the levels of Cys sulfinic acid (Cys-SO2H), Cys34-sulfonic acid (Cys-SO3H), and Met329 sulfoxide were greatly increased. Cys adduction products and Cys-SO2H, Cys-SO3H, and Met329 sulfoxide may be potential biomarkers to assess exposure and oxidative stress associated with A␣C and other arylamine toxicants present in tobacco smoke
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