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DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

ABSTRACT: Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (A?C) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. A?C and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of A?C and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with A?C and 4-ABP, whereas adduct formation was ?5-fold lower for MeIQx. dG-C8-A?C and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 ?M). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of A?C present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that A?C can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

SUBMITTER: Nauwelaers G

PROVIDER: S-EPMC3904354 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

Nauwelaërs Gwendoline G Bellamri Medjda M Fessard Valérie V Turesky Robert J RJ Langouët Sophie S

Chemical research in toxicology 20130816 9

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carc ...[more]

PMID: 23898916

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Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) is the most abundant carcinogenic heterocyclic aromatic amine (HAA) formed in mainstream tobacco smoke. A?C is a liver carcinogen in rodents, but its carcinogenic potential in humans is not known. To obtain a better understanding of the genotoxicity of A?C in humans, we have investigated its metabolism and its ability to form DNA adducts in human hepatocytes. Primary human hepatocytes were treated with A?C at doses ranging from 0.1-50 ?M, and the metabolites were characterized by ultra-performance LC/ion trap multistage mass spectrometry (UPLC/MSn). Six major metabolites were identified: a ring-oxidized doubly conjugated metabolite, N2-acetyl-2-amino-9H-pyrido[2,3-b]indole-6-yl-oxo-(?-d-glucuronic acid) (N2-acetyl-A?C-6-O-Gluc); two ring-oxidized glucuronide (Gluc) conjugates: 2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(?-d-glucuronic acid) (A?C-3-O-Gluc) and 2-amino-9H-pyrido[2,3-b]indol-6-yl-oxo-(?-d-glucuronic acid) (A?C-6-O-Gluc); two sulfate conjugates, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (A?C-3-O-SO3H) and 2-amino-9H-pyrido[2,3-b]indol-6-yl sulfate (A?C-6-O-SO3H); and the Gluc conjugate, N2-(?-d-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (A?C-N2-Gluc). In addition, four minor metabolites were identified: N2-acetyl-9H-pyrido[2,3-b]indol-3-yl sulfate (N2-acetyl-A?C-3-O-SO3H), N2-acetyl-9H-pyrido[2,3-b]indol-6-yl sulfate (N2-acetyl-A?C-6-O-SO3H), N2-acetyl-2-amino-9H-pyrido[2,3-b]indol-3-yl-oxo-(?-d-glucuronic acid) (N2-acetyl-A?C-3-O-Gluc), and O-(?-d-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN2-O-Gluc). The latter metabolite, A?C-HN2-O-Gluc is a reactive intermediate that binds to DNA to form the covalent adduct N-(2'-deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-A?C). Preincubation of hepatocytes with furafylline, a selective mechanism-based inhibitor of P450 1A2, resulted in a strong decrease in the formation of A?C-HN2-O-Gluc and a concomitant decrease in DNA adduct formation. Our findings describe the major pathways of metabolism of A?C in primary human hepatocytes and reveal the importance of N-acetylation and glucuronidation in metabolism of A?C. P450 1A2 is a major isoform involved in the bioactivation of A?C to form the reactive A?C-HN2-O-Gluc conjugate and A?C-DNA adducts.

Project description:2-Amino-9H-pyrido[2,3-b]indole (A?C) is a carcinogenic heterocyclic aromatic amine (HAA) that arises in tobacco smoke. UDP-glucuronosyltransferases (UGTs) are important enzymes that detoxicate many procarcinogens, including HAAs. UGTs compete with P450 enzymes, which bioactivate HAAs by N-hydroxylation of the exocyclic amine group; the resultant N-hydroxy-HAA metabolites form covalent adducts with DNA. We have characterized the UGT-catalyzed metabolic products of A?C and the genotoxic metabolite 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-A?C) formed with human liver microsomes, recombinant human UGT isoforms, and human hepatocytes. The structures of the metabolites were elucidated by (1)H NMR and mass spectrometry. A?C and HONH-A?C underwent glucuronidation by UGTs to form, respectively, N(2)-(?-D-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (A?C-N(2)-Gl) and N(2)-(?-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HON(2)-Gl). HONH-A?C also underwent glucuronidation to form a novel O-linked glucuronide conjugate, O-(?-D-glucosidurony1)-2-hydroxyamino-9H-pyrido[2,3-b]indole (A?C-HN(2)-O-Gl). A?C-HN(2)-O-Gl is a biologically reactive metabolite and binds to calf thymus DNA (pH 5.0 or 7.0) to form the N-(deoxyguanosin-8-yl)-A?C adduct at 20-50-fold higher levels than the adduct levels formed with HONH-A?C. Major UGT isoforms were examined for their capacity to metabolize A?C and HONH-A?C. UGT1A4 was the most catalytically efficient enzyme (V(max)/K(m)) at forming A?C-N(2)-Gl (0.67 ?l·min(-1)·mg of protein(-1)), and UGT1A9 was most catalytically efficient at forming A?C-HN-O-Gl (77.1 ?l·min(-1)·mg of protein(-1)), whereas UGT1A1 was most efficient at forming A?C-HON(2)-Gl (5.0 ?l·min(-1)·mg of protein(-1)). Human hepatocytes produced A?C-N(2)-Gl and A?C-HN(2)-O-Gl in abundant quantities, but A?C-HON(2)-Gl was a minor product. Thus, UGTs, usually important enzymes in the detoxication of many procarcinogens, serve as a mechanism of bioactivation of HONH-A?C.

Dataset Information

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

Publications

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

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