Dataset Information

Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking.

ABSTRACT: 2-Amino-9H-pyrido[2,3-b]indole (A?C) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and during the high-temperature cooking of meats. Human enzymes biotransform A?C and PhIP into reactive metabolites, which can bind to DNA and lead to mutations. We sought to understand the relative contribution of smoking and diet to the exposure of A?C and PhIP, by determining levels of A?C, its ring-oxidized conjugate 2-amino-9H-pyrido[2,3-b]indole-3-yl sulfate (A?C-3-OSO3H), and PhIP in urine of smokers on a free-choice diet before and after a six week tobacco smoking cessation study. A?C and A?C-3-OSO3H were detected in more than 90% of the urine samples of all subjects during the smoking phase. The geometric mean levels of urinary A?C during the smoking and cessation phases were 24.3 pg/mg creatinine and 3.2 pg/mg creatinine, and the geometric mean levels of A?C-3-OSO3H were 47.3 pg/mg creatinine and 3.7 pg/mg creatinine. These decreases in the mean levels of A?C and A?C-3-OSO3H were, respectively, 87% and 92%, after the cessation of tobacco (P < 0.0007). However, PhIP was detected in <10% of the urine samples, and the exposure to PhIP was not correlated to smoking. Epidemiological studies have reported that smoking is a risk factor for cancer of the liver and gastrointestinal tract. It is noteworthy that A?C is a hepatocellular carcinogen and induces aberrant crypt foci, early biomarkers of colon cancer, in rodents. Our urinary biomarker data demonstrate that tobacco smoking is a significant source of A?C exposure. Further studies are warranted to examine the potential role of A?C as a risk factor for hepatocellular and gastrointestinal cancer in smokers.

SUBMITTER: Konorev D

PROVIDER: S-EPMC4699441 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking.

Konorev Dmitri D Koopmeiners Joseph S JS Tang Yijin Y Franck Thompson Elizabeth A EA Jensen Joni A JA Hatsukami Dorothy K DK Turesky Robert J RJ

Chemical research in toxicology 20151203 12

2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are carcinogenic heterocyclic aromatic amines (HAAs) formed during the combustion of tobacco and during the high-temperature cooking of meats. Human enzymes biotransform AαC and PhIP into reactive metabolites, which can bind to DNA and lead to mutations. We sought to understand the relative contribution of smoking and diet to the exposure of AαC and PhIP, by determining levels of AαC, its ring-oxidize ...[more]

PMID: 26574651

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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: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.

Dataset Information

Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking.

Publications

Measurement of the Heterocyclic Amines 2-Amino-9H-pyrido[2,3-b]indole and 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Urine: Effects of Cigarette Smoking.

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