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 (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 carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC 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 AαC 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 AαC and 4-ABP, whereas adduct formation was ~5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a ten thousand-fold concentration range (1 nM – 10 µM). 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 AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes, suggests that AαC can contribute to DNA damage and the risk of hepatocellular cancer in smokers.

2-Amino-9H-pyrido[2,3-b]indole (AαC) Adducts and Thiol Oxidation of Serum Albumin as Potential Biomarkers of Tobacco Smoke

“…The research protocol was conducted under French legal guidelines and approved by the local institutional ethics committee. Hepatocytes were isolated by a two-step collagenase perfusion, and the parenchymal cells were seeded at a density of ϳ3 ϫ 10 6 viable cells/19.5-cm 2 Petri dish in 3 ml of Williams' medium with supplements as reported (20), except fetal calf serum was replaced with human albumin (1 g/liter) pretreated with ␤-mercaptoethanol. After 2 days, the differentiated cells were incubated with A␣C (20,33).…”

“…An internal lock mass at m/z 371.1012 (polysiloxane) was employed while using the Orbitrap mass analyzer. The tune parameters were optimized with LQQCPFEHVK and LQQCPF (20). DNA (5 g) was spiked with the isotopically labeled internal standard ([ 13 C 10 ]dG-C8-A␣C) at 1 adduct per 10 6 bases, followed by enzymatic digestion of DNA (40).…”

“…1) (17), which reacts with DNA to form covalent adducts, leading to mutations (18). The genotoxic potential of A␣C has been shown in human peripheral blood lymphocytes (19), Chinese hamster ovary cells (18), and human hepatocytes (20), where high levels of A␣C-DNA adducts are formed. However, long term stable biomarkers of A␣C must be developed for implementation in molecular epidemiology studies that seek to address a role for this chemical in human cancer risk.…”

“…DNA adducts of A␣C can be measured by sensitive liquid chromatography/mass spectrometry (LC/MS)-based methods (20). However, DNA from biopsy specimens is often unavailable, which restricts the use of this biomarker.…”

Application of liquid chromatography-mass spectrometry and differential mobility spectrometry-mass spectrometry for detection and quantitation of biomarkers in complex biological matrices

“…The study differs from earlier work in that it focuses on the relatively long term 7-day response with urine sample collection at 7 d only, while earlier studies addressed response up to 72 h post-exposure. The whole-body doses administered from a 60 Co gamma source at 0.5 Gy/min (0 Gy, 2 Gy, 4 Gy, 6 Gy, 7 Gy, and 10 Gy) cover a range that is appropriate for the species [85][86][87][88], and 60 Co radiation approximates the spectrum of weapons detonation [87]. The LD50/60 level (50% mortality at 60 d) is at approximately 7 Gy, and the change from 10% to 90% mortality occurs over a range of about 2 Gy.…”