Metabolism of the Tobacco Carcinogen 2-Amino-9H-pyrido[2,3-b]indole (AαC) in Primary Human Hepatocytes

Abstract

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

Quantification of Hemoglobin and White Blood Cell DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl Formed in Humans by Nanoflow Liquid Chromatography/Ion Trap Multistage Mass Spectrometry

“…19,20 These results signify that both AαC and 4-ABP are efficiently N -hydroxylated in human liver. AαC undergoes extensive metabolism by primary human hepatocytes to produce multiple phase I and phase II metabolites, 66 whereas the pathways of metabolism of 4-ABP in human hepatocytes are not well characterized. 20 On the basis of our understanding of 4-ABP metabolism in rodent and canine models, 32,67–69 we surmise that the amount of HONH-4-ABP circulating in blood and delivered to the erythrocyte is far greater than that of HONH-AαC, resulting in proportionately higher levels of 4-ABP-Hb adducts.…”

“…37 We previously reported that DNA adducts of AαC are formed at higher levels than the adducts formed with 4-ABP, PhIP and MeIQx in human T lymphocytes in vitro ; however, the DNA adducts are only detected in lymphocytes pre-activated with PMA/Ionomycin or CD3/CD28, which induce the expression of functional CYP1 activity for bioactivation. 76 Therefore, the capacity of cytochrome P450 enzymes expressed in peripheral WBC may be insufficient to N -hydroxylate 4-ABP and AαC. Alternatively, the phase II enzymes such as N -acetyltransferases (NAT) and sulfotransferases (SULT), which bioactivate HONH-4-ABP and HONH-AαC to the penultimate species that form DNA adducts, 48,77 may be expressed at insufficient levels in WBC to catalyze DNA binding of the N -hydroxylated metabolites.…”

Constructing Xenobiotic Maps of Metabolism to Predict the Role of Enzymes in DNA Adduct Formation

“…The ratio for the other HAAs ranges from 38,8% to 55,8%. MeIQx and PhIP have a relatively high ratio of metabolites reactive to DNA (45,2% and 44,5%) while AαC has a lower ratio (38,8%) in spite of its known higher reactivity towards DNA compared with MeIQx or PhIP [14]. This observation might be related to its smallest map of metabolism (average and median of numbers of metabolites and reactions in the map).…”

“…The size of these maps could be explained by the chemical structure of both HAAs that have few sub-structures on which SyGMa transformation rules can be applied. The maps predicted for two of the three well characterized HAAs in primary human hepatocytes [11,12,14], MeIQx -155 metabolites -and PhIP -128 metabolites, have a medium size. On the contrary, AαC, the third one, is associated with one of the smallest metabolic map.…”

“…The main cornerstone to use toxicity prediction tools on HAA is that compounds which may bind to DNA result from one or several metabolic transformations, which are unstable and cannot be experimentally characterized. As a consequence, the bioactivation metabolism and DNA adduct formation are fully characterized for only three HAAs i.e., AαC, MeIQx and PhIP in human liver [11,12,13,14]. This advocates for the use of in silico methods to predict HAA derivatives and potential DNA adducts derived from HAAs bioactivation in order to drive research about toxicity of HAAs family.…”