Dataset Information

DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in mouse liver and extrahepatic tissues during a subchronic feeding study.

ABSTRACT: Tobacco smoking is a risk factor for cancers of the liver and gastrointestinal (GI) tract, but the causal agents responsible for these cancers are uncertain. 2-Amino-9H-pyrido[2,3-b]indole (A?C) is an abundant heterocyclic aromatic amine present in tobacco smoke. A?C is a liver carcinogen and both a transgene mutagen and inducer of aberrant crypt foci in the colon of mice. We hypothesize that A?C may contribute to DNA damage and tumorigenesis in these organs of smokers. The potential of A?C to induce DNA adduct formation in liver, organs of the GI tract, lung, and urinary bladder, which are target organs of cancer in smokers, was examined using the C57BL/6 mouse as an animal model. A?C (400 or 800 ppm) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) (300 ppm), a liver and colon carcinogen in C57BL/6 mice, were given in the diet for up to 12 weeks. Liquid chromatography/mass spectrometry was employed to measure DNA adducts. The major DNA adducts of both carcinogens were identified as deoxyguanosine-C8 adducts. The levels of formation of A?C- and MeIQ-DNA adducts were similar in liver and extrahepatic tissues when adjusted for dose. The highest levels of adducts occurred in liver, followed by urinary bladder, and then in cecum and colon; lower DNA adduct levels were formed in the lung and pancreas following 12 weeks of feeding. The high levels of A?C adduct formed in liver, GI tract, and bladder of C57BL/6 mice reinforce the notion that A?C may contribute to DNA damage and cancer of these organs in smokers.

SUBMITTER: Tang Y

PROVIDER: S-EPMC3663563 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in mouse liver and extrahepatic tissues during a subchronic feeding study.

Tang Yijin Y Kassie Fekadu F Qian Xuemin X Ansha Buzayew B Turesky Robert J RJ

Toxicological sciences : an official journal of the Society of Toxicology 20130327 2

Tobacco smoking is a risk factor for cancers of the liver and gastrointestinal (GI) tract, but the causal agents responsible for these cancers are uncertain. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is an abundant heterocyclic aromatic amine present in tobacco smoke. AαC is a liver carcinogen and both a transgene mutagen and inducer of aberrant crypt foci in the colon of mice. We hypothesize that AαC may contribute to DNA damage and tumorigenesis in these organs of smokers. The potential of AαC to i ...[more]

PMID: 23535364

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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.

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-1,7-dimethylimidazo[4,5-g]quinoxaline (MeIgQx) is a recently discovered heterocyclic aromatic amine (HAA) that is formed during the cooking of meats. MeIgQx is an isomer of 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), a rodent carcinogen and possible human carcinogen that also occurs in cooked meats. MeIgQx is a bacterial mutagen, but knowledge about its metabolism and carcinogenic potential is lacking. Metabolism studies on MeIgQx and MeIQx were conducted with human and mouse liver microsomes, and recombinant human P450s. DNA binding studies were also investigated in mice to ascertain the genotoxic potential of MeIgQx in comparison to MeIQx. Both HAAs underwent comparable rates of N-oxidation to form genotoxic N-hydroxylated metabolites with mouse liver microsomes (0.2-0.3 nmol/min/mg protein). The rate of N-oxidation of MeIQx was 4-fold greater than the rate of N-oxidation of MeIgQx with human liver microsomes (1.7 vs 0.4 nmol/min/mg protein). The rate of N-oxidation, by recombinant human P450 1A2, was comparable for both substrates (6 pmol/min/pmol P450 1A2). MeIgQx also underwent N-oxidation by human P450s 1A1 and 1B1 at appreciable rates, whereas MeIQx was poorly metabolized by these P450s. The potential of MeIgQx and MeIQx to form DNA adducts was assessed in female C57BL/6 mice given [(14)C]-MeIgQx (10 μCi, 9.68 mg/kg body wt) or [(14)C]-MeIQx (10 μCi, 2.13 mg/kg body wt). DNA adduct formation in the liver, pancreas, and colorectum was measured by accelerator mass spectrometry at 4, 24, or 48 h post-treatment. Variable levels of adducts were detected in all organs. The adduct levels were similar for both HAAs, when adjusted for dose, and ranged from 1 to 600 adducts per 10(7) nucleotides per mg/kg dose. Thus, MeIgQx undergoes metabolic activation and binds to DNA at levels that are comparable to MeIQx. Given the high amounts of MeIgQx formed in cooked meats, further investigations are warranted to assess the carcinogenic potential of this HAA.

Dataset Information

DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in mouse liver and extrahepatic tissues during a subchronic feeding study.

Publications

DNA adduct formation of 2-amino-9H-pyrido[2,3-b]indole and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline in mouse liver and extrahepatic tissues during a subchronic feeding study.

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