DNA adduct formation of 4-aminobiphenyl and heterocyclic aromatic amines in human hepatocytes

Abstract

DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 10(7) DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation.

Figure 1

Chemical structures of 4-ABP and HAAs and their DNA adducts. The proposed structure of dG-N²-N⁴-4-ABP is tentative.

Figure 2

The kinetics of 4-ABP and HAA biotransformation in human hepatocytes (F and G identity codes). Data are the mean ± SD of three independent measurements.

Figure 3

Chemical structures of MeIQx and PhIP metabolites.

Figure 4

The kinetics of MeIQx (upper panels) and PhIP metabolite (lower panels) formation in human hepatocytes (F and G identity codes). Data are the mean ± SD of three independent measurements.

Figure 5

The LC-ESI/MS/MS³ traces of 4-ABP- and HAA-DNA adducts from (A) untreated hepatocytes and (B) hepatocytes treated with carcinogens for 8 h. The dG-N²-MeIQx adduct elutes at t_R 14.2 min and dG-N²-N⁴-ABP elutes at t_R 17.0 min.

Figure 6

The kinetics of 4-ABP- and HAA-DNA adduct formation in hepatocytes from 4 donors, including donors F and G, which were used for kinetics of MeIQx and PhIP metabolite formation (Figure 2). Data are the average and SD of two independent measurements, except for donor E, where a single measurement was done. Time points are from left to right: T 3, 8, and 24 h. The ordinate is set at the maximum adduct value for each data set.

Figure 7

The kinetics of 4-ABP- and HAA-DNA adduct formation in 3 different rat hepatocyte preparations. Data are the average and SD of two independent measurements. Time points are from left to right: T 3, 8, and 24 h. The ordinate is set at the maximum adduct value for each data set.