Effect of Cytochrome P450 Reductase Deficiency on 2-Amino-9H-pyrido[2,3-b]indole Metabolism and DNA Adduct Formation in Liver and Extrahepatic Tissues of Mice

Abstract

2-Amino-9H-pyrido[2,3-b]indole (AαC), a carcinogen formed during the combustion of tobacco and cooking of meat, undergoes cytochrome P450 (P450) metabolism to form the DNA adduct N-(deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole (dG-C8-AαC). We evaluated the roles of P450 expressed in the liver and intestine to bioactivate AαC by employing male B6 wild-type (WT) mice, liver-specific P450 reductase (Cpr)-null (LCN) mice, and intestinal epithelium-specific Cpr-null (IECN) mice. Pharmacokinetic parameters were determined for AαC, 2-amino-9H-pyrido[2,3-b]indol-3-yl sulfate (AαC-3-OSO3H), and N2-(β-1-glucosidurony1)-2-amino-9H-pyrido[2,3-b]indole (AαC-N2-Glu) with animals dosed by gavage with AαC (13.6 mg/kg). The uptake of AαC was rapid with no difference in the plasma half-lives (t1/2) of AαC, AαC-3-OSO3H and AαC-N2-Glu among mouse models. The maximal plasma concentrations (Cmax) and the areas under concentration-time curve (AUC0-24h) of AαC and AαC-N2-Glu were 4-24 fold higher in LCN than in WT mice, but were not different between WT and IECN mice. These findings are consistent with the ablation of hepatic P450 activity in LCN mice. However, the Cmax and AUC0-24h of AαC-3-OSO3H in plasma were not substantially different among the mouse models. Similar pharmacokinetic parameters were obtained with WT and LCN mice treated with a lower AαC dose (1.36 mg/kg). dG-C8-AαC was detected at similar levels in the livers of all three mouse models at the high AαC dose; levels of dG-C8-AαC in colon, bladder, and lung were greater in LCN than in WT mice and were the same in colon of IECN and WT mice. At the low AαC dose, dG-C8-AαC occurred at ~40% lower levels in liver of LCN mouse than in WT mouse liver, but adduct levels remained higher in extrahepatic tissues of LCN mice. Therefore, hepatic P450 plays an important role in detoxication of AαC, but other hepatic enzyme(s) contributes to the bioactivation of AαC. P450s expressed in the intestine do not appreciably contribute to bioactivation of AαC in mice.

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

“…41 The level of DNA adduct formation of AαC in liver was also unaffected in the liver specific P-450 reductase null mice treated with high doses of AαC. 43 Our data show that the contribution of non-P450 oxidases to the metabolism of AαC and bioactivation in human hepatocytes is minor.…”

“…The UV spectra of each hydrolysis products was compared to those spectra of AαC-6-OH and AαC-3-OH, which were produced using human liver microsome as previously reported. 34,41 …”

“…41 However, knowledge about the major metabolic pathways of AαC and the key enzymes involved in bioactivation of this carcinogen in liver and extrahepatic tissues of humans are limited. There is only one study reported the metabolism of AαC in human hepatocarcinoma cell line HepG2.…”

High-throughput and sensitive analysis of urinary heterocyclic aromatic amines using isotope-dilution liquid chromatography–tandem mass spectrometry and robotic sample preparation system

“…Urinary concentrations of PhIP and the two beta-carboline HCAAs evaluated, harman and norharman, were similar in smokers and non-smokers. As there are reports that the half-lives of HCAA are relatively short (< 24 hours), the urinary HCAA levels probably reflect the short-term exposure to HCAA [27, 28]. Measurement of HCAA in hair could be an alternative analysis to determine the long-term exposure to HCAA.…”

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

“…11–14 Moreover, high levels of DNA adducts are detected in the liver, colon and other extrahepatic tissues of mice upon AαC treatment. 13,15,16 The carcinogenic potential of AαC in humans is unknown, however, AαC is genotoxic to human lymphoblastoid cells (MLC-5) and peripheral blood lymphocytes cells. 17,18 We previously reported that primary human hepatocytes also efficiently bioactivate AαC to its N -hydroxylated metabolite, 2-hydroxamino-9 H -pyrido[2,3-b]indole (HONH-AαC), 19,20 which binds to DNA and can lead to mutations.…”