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 10⁷ 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.

INTRODUCTION

4-Aminobiphenyl (4-ABP) is a prototypical aromatic amine and a recognized human urinary bladder carcinogen (1–3). 4-ABP served as an antioxidant in the rubber industry prior to discovery of its carcinogenic activity (2,3). In the United States and many developed countries, strict federal regulations have drastically diminished the industrial usage of 4-ABP and other carcinogenic aromatic amines, but 4-ABP can still be found as a contaminant in color additives (4,5), paints (6), food colors (7), leather and textile dyes (8,9). 4-ABP and other aromatic amines also arise in diesel-exhaust particles (10–12) and cooking oil fumes (13). A major source of exposure to 4-ABP occurs through tobacco smoke: 4-ABP is present in mainstream tobacco smoke at levels ranging from 0.1 to 4.3 ng/cigarette (14,15) Another potential source of exposure to 4-ABP occurs through the usage of commercial hair dyes (16,17).

Heterocyclic aromatic amines (HAAs) are carcinogens in experimental laboratory animals and potential human carcinogens (18). HAAs are structurally related to aromatic amines: both classes of compounds undergo common pathways of bioactivation (19,20). The principal source of exposure to HAAs occurs through consumption of well-done cooked meats: the concentrations of HAAs can range from <1 part-per-billion up to the 500 ppb level (18,21,22). Several HAAs are also formed during the high-temperature burning of tobacco. 2-Amino-9H-pyrido[2,3-b]indole (AαC) was one of the first chemicals in a series of HAAs identified in protein pyrolysates (23,24). Thereafter, AαC was detected in mainstream tobacco smoke at levels ranging from 60 to 258 ng/cigarette (25,26). These levels are ~25 to 100-fold higher than the levels of 4-ABP, 2-naphthylamine, or benzo(a)pyrene (27) present in mainstream smoke and are comparable to the levels of tobacco specific nitrosamines, such as 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (28). The latter compounds are known human carcinogens. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) occurs in mainstream tobacco smoke at levels that range from 11 to 23 ng/cigarette (29). PhIP also forms during the combustion of organic material, and it is present in air-borne and diesel exhaust particles (12). 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) was reported at levels below 1 ng/cigarette (30), whereas 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was not detected. Thus, there is much concern about the health risk associated with the exposure to these structurally related class of chemicals found in tobacco smoke and cooked meats.

One approach that has been used to assess the carcinogenic potential of genotoxic chemicals is the carcinogen binding index (31). This approach has been used in rodent models, where the DNA adduct levels of carcinogens measured in liver at the given dose are normalized to the dose which resulted in a 50% tumor incidence under the conditions of a 2-year bioassay (TD₅₀ dose). The carcinogen binding index has been used as a preliminary assessment of the carcinogenic potential of a chemical (31); however, the development of cancer is a multifactorial process, that requires a number of steps, including mutations, genetic alterations, and cell proliferation (18). Moreover, the extrapolation of animal carcinogenicity data to assess human health risk has many uncertainties, which can lead to tenuous risk assessment estimates. Perhaps the most critical and variable parameters in interspecies extrapolation are the effects of dose, species differences in catalytic activities of xenobiotic metabolism enzymes, and the influence of genetic polymorphisms that encode enzymes that lead to interindividual differences in metabolism of carcinogens (32).

The liver is the most metabolically active organ in the biotransformation of 4-ABP and HAAs to genotoxic metabolites (33,34). We have previously shown that freshly isolated human hepatocytes in primary culture effectively metabolize a variety of chemical carcinogens (35), including aflatoxin B₁ (36), MeIQx (37), and PhIP (38) to reactive metabolites capable of damaging DNA. In this study, we have investigated the capacity of human and rat hepatocytes to bioactivate 4-ABP and several HAAs, to form DNA adducts, which are believed to be the initiating events of chemical carcinogenesis (39). Our data show that HAAs covalently bind to DNA in human hepatocytes at considerable levels. Moreover, the levels of AαC adducts formed are comparable to the levels of DNA adducts formed with 4-ABP, a known human carcinogen. The amounts of HAA-DNA adducts formed in human hepatocytes are much greater than the levels of adducts produced in hepatocytes of rats, a species that is used in long-term carcinogen bioassays and extrapolation of toxicity data for human risk assessment. Thus, the data on the carcinogen binding index of HAAs in rat hepatocytes greatly underestimates the genotoxic potential of HAAs in human hepatocytes.

Materials and Methods

Caution

AαC, 4-ABP, IQ, MeIQx, PhIP are carcinogens, and they should only be handled in a well-ventilated fume hood with the appropriate protective clothing.

Chemicals

MeIQx, 3-[²H₃C]-MeIQx, IQ, 3-[²H₃C]- IQ and PhIP, and 1-[²H₃C]-PhIP, were purchased from Toronto Research Chemicals (Toronto, ON, Canada). AαC and [¹³C₆]-AαC were kindly provided by Dr. Dan Doerge NCTR (Jefferson, AR). The isotopic purities of all compounds exceeded 99.5%, except for 3-[²H₃C]-MeIQx, which was 96.5% isotopically pure. 4-ABP was purchased from Aldrich (Milwaukee, WI). 2-Nitro-9H-pyrido[4,5-b]indole was a kind gift from Dr. D. Miller, NCTR (Jefferson, AR). [²H₉]-4-Nitrobiphenyl (99.3% isotopically pure) was purchased from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec, Canada). [²H₉]-4-ABP was prepared by reduction of [²H₉]-4-nitrobiphenyl with Zn in C₂H₅OH/HCl at 60 °C for 2 h as previously described (16). [¹³C₁₀]-dG (99.9% isotopically pure) was purchased from Cambridge Isotopes (Andover, MA). Alkaline phosphatase (from E. coli), and nuclease P1 (from Penicillium citrinum) were purchased from Sigma (St. Louis, MO). Phosphodiesterase I (from Crotalus adamanteus venom) was from GE Healthcare (Piscataway, NJ). All solvents used were high-purity B & J Brand from Honeywell Burdick and Jackson (Muskegon, MI). ACS reagent-grade formic acid (88%) was purchased from J.T. Baker (Phillipsburg, NJ). HyperSep^™ filter SpinTips C18 (20 mg) were from Thermo Scientific (Palm Beach, FL). Oragene-DNA saliva kits were from Genotek Inc. (Ontario, Canada).

Preparation of MeIQx and PhIP metabolites

The following metabolites and their trideuterated internal standards were prepared by chemical means (37) or biosynthetically as previously reported (40): 2-Amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH); 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH₂OH-IQx); (N²-(β-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) (MeIQx-N²-Gl); N²-(β-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HON-MeIQx-N²-Gl), N²-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl-sulfamic acid (MeIQx-N²-SO₃H); N²-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N²-Gl); N3-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N3-Gl); N²-(β-1-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-N²-Gl); and N3-(β-1-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-N3-Gl).

Preparation of the DNA adducts

N-(Deoxyguanosin-8-yl)-AαC (dG-C8-AαC) (41), N-(deoxyguanosin-8-yl)-IQ (dG-C8-IQ), N-(deoxyguanosin-8-yl)-IQ, 5-(deoxyguanosin-N²-yl)-2-amino-3-methylimidazo[4,5-flquinoline (dG-N²-IQ), N-(deoxyguanosin-8-yl)-MeIQx (dG-C8-MeIQx), and 5-(deoxyguanosin-N²-yl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (dG-N²-MeIQx) (42,43), and N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP) (44,45) were prepared by reaction of their N-acetoxy-HAA derivatives with dG or [¹³C₁₀]-dG (5 mg) in 100 mM potassium phosphate buffer (pH 8.0). N-(Deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP) was prepared by reaction of N-hydroxy-4-ABP with pyruvonitrile, followed by reaction with dG or [¹³C₁₀]-dG (46). For the case of dG-C8-MeIQx, the internal standard was prepared by reaction of the N-acetoxy derivative of 3-[²H₃C]-MeIQx with dG. The adducts were purified by solid phase extraction, followed by HPLC purification (42,43).

Cell Isolation, Culture, and Metabolism Studies

Human liver samples were obtained from eight patients undergoing liver resection for primary or secondary hepatomas through the Biological Resource Center (CHRU Pontchaillou, Rennes, France). The research protocol was conducted under French legal guidelines and fulfilled the requirements of the local institutional ethics committee. This study was approved by the Institutional Review Board at the Wadsworth Center. Hepatocytes were isolated by a two-step collagenase perfusion procedure. The liver parenchymal cells were seeded in Petri dishes at a density of 3 × 10⁶ viable cells/19.5 cm² dish, in 3 mL of Williams’ medium with the following supplements: bovine serum albumin (1g/L), glutamine (2 mM), bovine insulin (5μg/mL), penicillin (10 U/mL), streptomycin (10 μg/mL) and fetal calf serum (10% v/v). The cell culture medium was renewed daily. After 36 h cell seeding, the media was replenished with cell media lacking fetal calf serum and hydrocortisone hemisuccinate (54 μM) was added. Then, 4-ABP or HAAs (10 μM) in DMSO (0.1% v/v) was added to the medium, and the cells were incubated for 3, 8 or 24 h. The incubations were terminated by collecting the culture medium. Adult male Sprague Dawley rats (200–250 g) were purchased from Janvier (Genest-Saint-Isle, France). Liver parenchymal cells were prepared and seeded at the same density, and treated with carcinogens as described above. No toxicity was observed with any compounds on the basis of the methylthiazoltetrazolium test for either human or rat hepatocytes.

The cells were scraped from the P60 Petri dishes with a spatula and combined with the cell media in Eppendorf tubes, followed by centrifugation. The supernatant containing the cell culture media was retrieved from the cellular pellets containing DNA, and both fractions were stored at −80 °C, prior to analyses. The cell culture media (5 μL) containing the equivalent of 11.2 ng of PhIP and its metabolites (50 pmol of PhIP) or the equivalent of 10.7 ng of MeIQx and its metabolites (50 pmol of MeIQx) were diluted with water (45 μL), followed by the addition of 750 pg of isotopically labeled internal standard for each metabolite of PhIP or MeIQx and either 250 pg of 3-[²H₃C]-MeIQx or 1-[²H₃C]-PhIP. The cell culture media of hepatocyte samples of 4-ABP, AαC and IQ (5 μL, 50 pmol of carcinogen) were diluted with water (45 μL), and were spiked with 250 pg of [²H₉]-4-ABP, 3-[²H₃C]-IQ, or [¹³C₆]-AαC. The samples were added to CH₃OH (150 μL) and incubated on ice for 1 h, to promote precipitation of salts and protein. The samples were then centrifuged at 15000 g for 3 minutes at 4 °C, and the supernatants were transferred to capLC recovery vials and concentrated to dryness by vacuum centrifugation. The extracts were dissolved in water and assayed by LC-ESI/MS/MS (vide infra).

Ethoxyresorufin O-deethylation (EROD) associated with P450 1A1/2 activity was measured in cultured hepatocytes essentially as described by Burke and Mayer (47). Methoxyresorufin O-demethylation (MROD) was used as a substrate for specific P450 1A2 activity (48). The reaction rates were linear with time and proportional to protein concentration. Cellular protein content was estimated by the Bradford procedure (49). Values (pmol/min/mg protein) are the mean ± SD of quadruplicate measurements.

Isolation and Enzymatic Digestion of DNA, and Solid Phase Enrichment of DNA Adducts

DNA was isolated from the cellular pellets by a chloroform/phenol extraction method (50). The amount of DNA recovered from each carcinogen treatment was determined by UV spectroscopy, assuming a concentration of DNA (50 μg/mL) is equal to 1.0 absorbance unit at 260 nm. Isotopically labeled internal standards of each DNA adduct were added to the DNA recovered from the treated hepatocytes at a level of 1 adduct per 10⁶ DNA bases. The DNA from the respective time points of the individually treated 4-ABP and HAA hepatocyte samples were then pooled. The enzymatic digestion conditions used for the hydrolysis of DNA (~2 – 10 μg) in 5 mM Bis-Tris-HCl buffer (pH 7.1, 50 μL) employed DNAse I for 1.5 h, followed by incubation with nuclease P1 for 3 h, and then digested with alkaline phosphatase and phosphodiesterase for 18 h (45). These enzyme digestion conditions were shown to be highly efficient in the recovery of the dG-C8 adducts of PhIP, MeIQx, IQ, and 4-ABP from calf thymus DNA modified with these carcinogens (42–45,51,52). The DNA adducts were purified by solid phase extraction, using HyperSep^™ filter SpinTips, as previously described (52,53).

LC-ESI-MS/MS Measurements for Metabolites

The analyses of 4-ABP and HAAs, and the metabolites of MeIQx and PhIP were performed with a NanoAcquity^™ UPLC system (Waters Corporation, Milford, MA) equipped with a Michrom C18 AQ column (0.3 × 150 mm, 3 μm particle size, Michrom Bioresources Inc., Auburn, CA), as previously described (54). The analytes were separated by a gradient. The A solvent was 0.01% HCO₂H in H₂O, and the B solvent contained 0.01% HCO₂H and 5% H₂O in CH₃CN. The flow rate was set at 6 μL/min, starting at 100% A increased by a linear gradient to 65% B in 13 min, and then to 100% B at 14 min holding for 1 min. The gradient was reversed to the 100% A over 1 min and at the same time the flow rate was increased from 6 μL/min to 10 μL/min. A post-run time of 3 min was required for re-equilibration. The back pressure of the column was 3,700 psi at the initial solvent conditions. The sample injection volume was 0.4 μL. The manipulation of UPLC system was done by MassLynx software (Waters Corp., Milford, MA).

The mass-spectral data were acquired on a Finnigan^™ Quantum Ultra Triple Stage Quadrupole MS (Thermo Fisher, San Jose, CA) and processed with Xcalibur version 2.07 software. Analyses were conducted in the positive ionization mode and employed an Advance CaptiveSpray^™ source from Michrom Bioresources Inc. (Auburn, CA). The spray voltage was set at 1400 V; the in-source fragmentation was 5 V; and the capillary temperature was 200 °C. There was no sheath or auxiliary gas. The peak widths (in Q1 and Q3) were set at 0.7 Da. The measurement of analytes was done by selected reaction monitoring (SRM). The following transitions and collision energies were used for the quantification of MeIQx, PhIP, and their metabolites: MeIQx and [²H₃C]-MeIQx: 214.1 → 199.1 and 217.1 → 199.1, at 30 eV; 8-CH₂OH-IQx and [²H₃C]-8-CH₂OH-IQx: 230.1 → 197.1 and 233.1 → 197.1, at 35 eV; IQx-8-COOH and [²H₃C]-IQx-8-COOH: 244.1 → 183.1 and 247.1 → 183.1, at 38 eV; MeIQx-N²-SO₃H and [²H₃C]-MeIQx-N²-SO₃H: 294.1 → 214.1 and 297.1 → 217.1 @ 35 eV; MeIQx-N²-Gl and [²H₃C]-MeIQx-N²-Gl: 390.1 → 214.1, 212.1, 199.1 and 393.1 → 217.1, 215.1, 199.1 @ 55 eV; HON-MeIQx-N²-Gl and [²H₃C]-HON-MeIQx-N²-Gl: 406.1 → 214.1, 213.1, 212.1 and 409.1 → 217.1, 216.1, 215.1 @ 35 eV; PhIP and [²H₃C]-PhIP: 225.1 → 210.1 and 228.1 → 210.1 at 33 eV; PhIP-N²-Gl, PhIP-N3-Gl and [²H₃C]-PhIP-N²-Gl, [²H₃C]-PhIP-N3-Gl: 401.1 → 210.1 and 404.1 → 210.1 at 55 eV; HON-PhIP-N²-Gl, and [²H₃C]-HON-PhIP-N²-Gl: 417.1 → 225.1 and 224.1 and 420.1 → 228.1 and 227.1 @ 34 eV; HON-PhIP-N³-Gl and [²H₃C]-HON-PhIP-N³-Gl: 417.1 → 225.1 and 224.1 and 420.1 → 228.1 and 227.1 @ 34 eV. The transitions employed for IQ and [²H₃C]-IQ were 199.1 → 184.1 and 202.1 → 184.1 @ 32 eV; the transitions of AαC and [¹³C₆]-AαC were 184.1 → 167.1 and 190.1 → 173.1 @ 20 eV; and the transitions employed for 4-ABP and [²H₉]-4-ABP were 170.1 → 153.1 and 179.1 → 160.1 @ 30 eV. The dwell time for each transition was 10 ms. Argon was used as the collision gas and was set at 1.5 mTorr. Product ion spectra were acquired on the protonated molecules [M + H]⁺, scanning from m/z 50 to 250 or 500 at a scan speed of 500 amu/s using the same acquisition parameters as above.

LC-ESI/MS/MS³ Measurements for DNA Adducts

The DNA adduct analyses were conducted with an Agilent 1100 Series capillary LC system (Agilent Technologies, Palo Alto, CA) equipped with an Aquasil C18 column (0.32 × 250 mm) from Thermo Fisher (Bellafonte, PA). Samples (2 μL) were injected, and analytes were separated with a gradient. The solvent conditions were held at 100% A (solvent composition: 0.01% HCO₂H and 10% CH₃CN) for 2 min, followed by a linear gradient to 100% B (solvent composition: 95% CH₃CN containing 0.01% HCO₂H) over 30 min at a flow rate of 6 μL/min. The MS instrumentation was a linear quadrupole ion trap mass spectrometer (LTQ, Thermo Fisher, San Jose, CA), and Xcalibur version 2.07 software was used for data manipulations. Analyses were conducted in the positive ionization mode and employed an Advance CaptiveSpray^™ source from Michrom Bioresource Inc. (Auburn, CA). Representative optimized instrument tuning parameters were as follows: capillary temperature 220 °C; source spray voltage 1.5 kV; source current 0.3 μA; no sheath gas, sweep gas or auxiliary gas was employed; capillary voltage 32 V; tube lens voltage 110 V; and in-source fragmentation 10 V.

The LTQ MS was employed in the tandem MS/MS scan mode to monitor the loss of deoxyribose from the protonated molecules of the adducts ([M+H-116]⁺), followed by the consecutive reaction monitoring scan mode at the MS³ scan stage, to characterize the product ions of the aglycone adducts [BH₂]⁺ (45,53,55). The total ion counts produced at the MS³ scan stage were used for quantitative measurements for all of the adducts except for dG-C8-IQ and [¹³C₁₀]-dG-C8-IQ. The ions monitored in MS → MS² → scan modes were as follows: dG-C8-PhIP (m/z 490.1 → 374.1); [¹³C₁₀]-dG-C8-PhIP (m/z 500.1 → 379.1); dG-C8-MeIQx (m/z 479.1 → 363.1); dG-C8-[²H₃C]-C8-MeIQx (m/z 482.1 → 366.1); dG-AαC (m/z 449.1 → 333.1); [¹³C₁₀]-dG-AαC (m/z 459.1 → 338.1); dG-C8-4-ABP (m/z 435.1 → 319.1); [¹³C₁₀]-dG-C8-4-ABP (m/z 445.1 → 324.1), dG-C8-IQ (m/z 464.1 → 348.1); [¹³C₁₀]-dG-C8-IQ (m/z 474.1 → 353.1). Isobaric interferences precluded the use of total ion counts for measurement of dG-C8-IQ and ([¹³C₁₀]-dG-C8-IQ; the extracted ions at m/z 302.2 and 331.1, and 307.2 and 336.2, respectively, produced at the MS³ scan stage, were used for quantitative measurements. The [¹³C₁₀]-dG-C8-4-ABP, [¹³C₁₀]-dG-C8-IQ and [¹³C₁₀]-dG-C8-MeIQx were employed as internal standards to estimate the levels of dG-N²-N⁴-ABP, dG-N²-IQ and dG-N²-MeIQx.

The normalized collision energies were set at 32 and 40, and the isolation widths were set at 3.0 and 1.0 Da, respectively, for the MS² and MS³ scan modes, for all adducts. The activation Q was set at 0.35 and the activation time was 10 ms, for both scan modes. Helium was used as the collision damping gas in the ion trap and was set at a pressure of 1 mTorr. One μscan was used for data acquisition. The automatic gain control settings were full MS target 30,000 and MSⁿ target 10,000, and the maximum injection time was 10 ms.

Calibration curves

Estimates of MeIQx and PhIP metabolites were done with external calibration curves using 50 pg of isotopically labeled parent compounds and 150 pg of isotopically labeled metabolites. The unlabeled metabolite calibrant levels ranged from 0.5 to 30-fold the amount of the internal standards. The calibration curves were fitted to a straight line (area of response of the metabolite/internal standard versus the amount of metabolite/internal standard) using ordinary least-squares with equal weightings. The coefficient of determination (r²) values of the slopes exceeded 0.99.

Calibration curves were constructed with the stable, isotopically labeled DNA adducts set at 14.3 pg and a 6 point calibration curve with unlabeled DNA adducts was established with 0, 1.43 pg - 143 pg of each adduct added to the DNA digest matrix (10 μg), following solid phase extraction. Assuming an average recovery of 10 μg of DNA (30 nmol of deoxynucleoside) from each hepatocyte incubation, the level of internal standard represented 10 adducts per 10⁷ bases and the levels of unlabeled adduct ranged from 1 to 100 adducts per 10⁷ bases The calibration curves were done in triplicate at each level, and the data were fitted to a straight line (area of response of the adduct/internal standard versus the amount of adduct/internal standard) using ordinary least-squares with equal weightings. The coefficient of determination (r²) values of the slopes exceeded 0.98.

Results

4-ABP and HAA Metabolism in Human Hepatocytes

4-ABP, AαC, IQ, MeIQx, and PhIP undergo metabolic activation at the exocyclic amine group by cytochrome P450 enzymes to produce the genotoxic N-hydroxylated metabolites. These intermediates undergo esterification by phase II enzymes, including N-acetyltransferases (NATs) or sulfotransferases (SULTs), to produce electrophiles that react with DNA (19,20,56–58). The structures of these carcinogens and their DNA adducts are shown in Figure 1.

Figure 1.

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

The kinetics of metabolism and DNA adduct formation of the carcinogens were determined following 3, 8 or 24 h of incubation. These time points were selected on the basis of our previous studies on PhIP and MeIQx (10 μM) with human hepatocytes, which showed that up to 97% of the doses underwent metabolism after 24 h incubation. The extent of metabolism was strongly correlated to the level of P4501A2 enzyme activity (37,38). The rates of metabolism of HAAs and 4-ABP were measured in hepatocyte preparations from two donors. The metabolism of HAAs was more rapid than that of 4-ABP (Figure 2). At the 24 h time point, 71 – 98% of the doses of HAAs underwent metabolism, whereas 59 to 77% of the amount of 4-ABP underwent biotransformation. N-hydroxy-4-ABP was reported to undergo reduction to form 4-ABP at greater rates than the N-hydroxy-HAA substrates formed the parent HAAs, by an NADPH-dependent reductase, when employing rat or human microsomes, rat hepatocytes or human HepG2 cells (59). This reductase activity could influence the relative amounts of 4-ABP or HAAs remaining in primary culture.

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.

We previously characterized the major pathways of metabolism of MeIQx and PhIP in freshly cultured human and rat hepatocytes employing the ¹⁴C-labelled HAAs as substrates (37,38). In this current study, we measured the amounts of the principal metabolites of MeIQx and PhIP produced by human hepatocytes, employing LC-ESI/MS/MS with stable, isotopically labeled internal standards for quantitative measurements. The metabolic pathways of MeIQx and PhIP are depicted in Figure 3, and the kinetics of metabolite formation are shown in Figure 4. The LC-ESI/MS/MS traces of MeIQx and PhIP metabolites (formed at T 24 h) and their product ion spectra are shown in Supporting Information, Figures S-1 and S-2. The findings on metabolite formation are consistent with our earlier studies that employed radiolabelled substrates (37,38). Both MeIQx and PhIP undergo N-oxidation by P450 1A2, to produce HONH-MeIQx and HONH-PhIP as principal oxidation products. These N-hydroxylated HAAs undergo conjugation with glucuronic acid to form HON-MeIQx-N²-Gl and HON-PhIP-N²-Gl. The isomeric glucuronide conjugate HONH-N3-PhIP is formed, but it occurs at ~10-fold lower levels than HON-PhIP-N²-Gl. P450 1A2 also mediates oxidation of the 8-CH₃ group of MeIQx, to produce the alcohol IQx-8-CH₂OH and the carboxylic acid, IQx-8-COOH, as other prominent metabolites (60). The phase II conjugation products MeIQx-N²-SO₃H, MeIQx-N²-Gl and the isomeric N-glucuronide conjugates PhIP-N²-Gl and PhIP-N3-Gl were also formed, the latter PhIP-N-Gl metabolites were produced at considerably lower levels than the HON-PhIP-N-Gl metabolites.

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.

We have also examined the metabolism of AαC in human hepatocytes, by LC-ESI/MS/MS, in positive and negative ionization modes (Supporting Information, Figure S-3 A and Figure S-3 B). There is evidence for the formation of two glucuronide conjugates of AαC; four glucuronide conjugates of ring- or N-oxidized AαC; and two sulfate conjugates of AαC. The elucidation of the sites of AαC oxidation and phase II conjugation requires further study.

Monitoring 4-ABP and HAA DNA Adduct Formation in Human Hepatocytes

The LTQ MS was employed at the MS³ stage, to characterize and measure product ions of the aglycone adducts [BH₂]⁺. The LC-ESI/MS/MS³ traces of DNA adducts formed in hepatocytes treated with carcinogens for 8 h are shown in Figure 5. The dG-C8 DNA adducts of all carcinogens were readily identified. In addition, an isomeric dG-N² adduct of 4-ABP (tentatively assigned) and a minor 4-ABP adduct formed at the C8 atom with dA were detected (55,56). The dG-N² adducts of MeIQx and IQ were also identified. The LC-ESI/MS/MS³ product ion spectra of the DNA adducts and the respective internal standards are shown in Supporting Information, Figure S-4. The product ion spectra provide rich structural information about the structures of the adducts and corroborate their identities (45,52,53,58).

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.

The kinetics of DNA adduct formation were investigated in human hepatocytes of 4 donors (Figure 6), including donors F and G, which were examined for the metabolism of MeIQx and PhIP. At the earliest time point measured (3 h), the amount of the dG-C8-AαC formed was far greater than the levels of DNA adducts formed by any of the other carcinogens. The levels of dG-C8-HAA adduct formation peaked between 3 and 8 h of incubation, whereas the amount of dG-C8-4-ABP adduct formation continued to increase during the course of the 24 h incubation, perhaps reflective of a slower rate of uptake or metabolism of 4-ABP in comparison to the HAAs (Figure 2). The kinetics of DNA adduct formation were similar in the hepatocytes of the 4 donors. The levels of dG-C8-HAA and dG-C8-4-ABP adduct formation at 24 h for the 8 donors are reported in Table 1. The highest levels of adducts were consistently formed by AαC and 4-ABP, followed by PhIP, MeIQx and IQ.

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.

Table 1.

4-ABP- and HAA-DNA Adduct Formation in Human Hepatocytes Following 24 h Incubation

	Adducts per 107 Bases					pmol/min/mg protein
Hepatocyte	dG-C8-4-ABP	dG-C8-AαC	dG-C8-PhIP	dG-C8-MeIQx	dG-C8-IQ	EROD	MROD
HH A	N.A.	N.A.	13.4 ± 1.7	3.4 ± 0.3	9.8 ± 1.1	N.A.	N.A.
HH B	150	32.5 ± 1.8	22.0	12.4 ± 0.6	10.3 ± 0.6	N.A.	N.A.
HH C	38.8 ± 4.8	44.4 ± 5.6	7.5 ± 1.6	13.9 ± 0.2	18.3 ± 0.8	N.A.	N.A.
HH D	100	109 ± 6.4	7.7 ± 1.0	12.2 ± 3.5	26.2 ± 1.9	N.A.	N.A.
HH E	136	9.1	9.1	27.2	N.A.	N.A.	N.A.
HH F	49.0 ± 1.7	72.0 ± 5.9	14.0 ± 0.6	19.0 ± 1.9	N.A.	N.D.	0.43 ± 0.20
HH G	87.9 ± 8.3	82.0 ± 15.4	30.6 ± 2.4	16.1 ± 1.0	13.5 ± 1.5	0.78 ± 0.48	0.38 ± 0.13
HH H	70.0 ± 5.8	31.4 ± 3.3	6.0 ± 1.0	5.1 ± 0.1	7.0 ± 2.2	0.18 ± 0.09	0.90 ± 0.27

N.A. = Not assayed; N.D. = Not detected

DNA adduct data are either single or average of duplicate measurements and the SD

EROD and MROD data (Mean ± SD, N = 4)

4-ABP and HAA DNA Adduct Formation in Rat Hepatocytes

The kinetics of 4-ABP- and HAA-DNA adduct formation were also determined in hepatocytes isolated from 3 different rats (Figure 7). The EROD activities ranged from 0.4 to 2.7 pmol resorufin/min/mg protein, and MROD activities ranged from 0.8 to 1.6 pmol resorufin/min/mg protein. These P450 enzyme activities were comparable, if not superior to the P450 enzyme activities measured in the hepatocytes of 3 human donors (Table 1).

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.

The amounts of dG-C8-4-ABP ranged from 4.2 to 7.2 adducts per 10⁶ bases following 24 h of incubation; these levels of adduct formation are similar to the amounts of dG-C8-4-ABP formed in human hepatocytes (Figure 4). dG-C8-AαC was the second most abundant adduct formed: the levels ranged from 0.5 to 3.1 adducts per 10⁶ bases at 24 h of incubation, followed by dG-C8-MeQx (0.1 – 0.3 adducts per 10⁶ bases/24 h), and then by dG-C8-PhIP (0.01 – 0.02 adducts per 10⁶ bases/24 h).

Discussion

Human hepatocytes are highly efficient in the bioactivation of 4-ABP and HAA and in ensuing DNA adduct formation. The highest levels of adducts were formed with AαC, followed by 4-ABP, with lesser but appreciable amounts of adducts formed with PhIP, MeIQx, and IQ. 4-ABP induces cancer of the urinary bladder, liver, and mammary gland in rodents (61,62), and 4-ABP is a recognized human urinary bladder carcinogen (2). Cigarette smokers are at elevated risk for urinary bladder cancer, and the role of aromatic amines, including 4-ABP, as causal agents of this disease is well documented (2,63,64). It is noteworthy that the amounts of AαC present in cigarette smoke are far greater than the levels of 4-ABP: up to 258 ng AαC is produced in mainstream smoke per cigarette versus 0.1 to 4.3 ng 4-ABP/cigarette (14,15). AαC causes liver and blood vessel tumors in CDF₁ mice (18). AαC is a potent lacI transgene mutagen in the colon of C57BL/6 mice (Big Blue mouse) (65), and it induces aberrant crypt foci, early biomarkers of tumors, in the colon of this species (66); although carcinogen bioassays with AαC have not been conducted in this mouse strain. We note that even though AαC forms DNA adducts at high levels in rat hepatocytes (Figure 6), AαC is not a hepatocarcinogen in the rat model (18). HAAs and other genotoxicants form adducts in a number of tissues of experimental animals, where tumors do not develop during long-term feeding studies (67). The development of cancer is complex and requires a number of steps, including multiple mutations, genetic alterations, and cell proliferation (18,68). AαC present in tobacco smoke may act as an initiating agent in the development of human cancer during chronic smoking, in which many other mutagens-carcinogens, tumor promoters, and factors stimulating tumor progression exist (18).

The global DNA adduct formation and the genotoxicity of MeIQx, PhIP, AαC and 4-ABP in the hypoxanthine phosphoribosyl transferase (hprt) gene of Chinese hamster ovary cells (CHO) stably transfected with P450 1A1 or P450 1A2 and either NAT2*4 or NAT2*5B alleles were recently examined (53,69–71). On the basis of estimates of dG-C8-HAA and dG-C8-4-ABP adduct formation and assuming that these adducts are the principal genotoxic lesions, we estimate that one dG-C8-HAA or dG-C8-4-ABP adduct per 10⁷ bases in the CHO genome induced about 1.5 hprt mutants for MeIQx; 2.5 hprt mutants for AαC, 2.8 hprt mutants for PhIP, and ~0.7 hprt mutants for 4-ABP per one million cells, at the dose concentrations of 1.5 μM (MeIQx and AαC), 1.2 μM (PhIP) and 2 μM (4-ABP) (53,69–71). The potential of dG-C8-HAA and dG-4-ABP adducts to induce mutations at the hprt locus appear similar; the different levels of mutation induced by these carcinogens in CHO cells were driven by the differing levels of adduct formation, which are dependent upon the levels of N-oxidation and ensuing activation of the N-hydroxylated metabolites by phase II enzymes. In our current study, the levels of 4-ABP and HAA adducts formed in human hepatocytes well exceed the threshold adduct levels that induced hprt mutations in CHO cells.

HAA-DNA adduct formation for MeIQx and PhIP was up to 100-fold greater in human hepatocytes than in rat hepatocytes (Figures 6 and 7), whereas the levels of 4-ABP-DNA adducts were similar in hepatocytes of both species. Human and rat hepatocytes displayed comparable levels of MROD activity: a measure of P450 1A2 and the principal enzyme involved in N-oxidation of HAAs (72). The discrepancy in MeIQx- and PhIP-DNA adduct formation between species can be explained, in part, by the different catalytic activities of human and rat P4501A2 orthologues towards these procarcinogens. The catalytic efficiency of human P450 1A2 is 11- and 19-fold superior than rat P450 1A2 in the N-oxidation of MeIQx and PhIP, respectively, whereas the catalytic efficiency of MROD is quite comparable for human and rat P450 1A2 (72). MeIQx and PhIP are extensively metabolized in human hepatocyte preparations (>75% of the 10 μM dose); however, both carcinogens are poorly metabolized in rat hepatocytes, and a majority of the dose remains unmetabolized, even after 24 h (37,38). There are also interspecies differences in regioselectivity of the P450 1A2 oxidation of PhIP and MeIQx. Rat P4501A2 catalyzes oxidation at the heterocyclic rings of PhIP and MeIQx to produce detoxication products; N-oxidation products of both HAAs account for less than 2% of the dose in rat hepatocytes (37,38). In contrast to the rat P450 1A2 orthologue, the catalysis of ring oxidation products of PhIP and MeIQx, by human P450 1A2, is negligible (72,73), and a principal metabolic pathway for both HAAs in human hepatocytes occurs by P450 1A2-mediated N-oxidation (Figure 4) (37,38). In the case of 4-ABP, up to 23% of the dose (10 μM) was converted into N-oxidation products in rat hepatocytes (74). The DNA binding data show that both human and rat P450 1A2 orthologues efficiently carry out the N-oxidation of 4-ABP and comparable levels of DNA adducts are formed in hepatocytes of both species (Figures 6 and 7).

The major pathways of metabolism of MeIQx and PhIP have been extensively characterized in rat and human hepatocytes (37,38,75,76), and in vivo of both species (40,77), but knowledge about the metabolism of AαC is limited. The metabolism of AαC was examined in the human liver tumor HepG2 cell line (78); however, these cells do not express P450 1A2 (79) and are devoid of UGTs (78), and thus, the AαC metabolites formed may not accurately reflect the biotransformation pathways that occur for AαC in humans. Recombinant human P450 1A2 enzyme does catalyze N-oxidation of the exocyclic amine group of AαC, as well as ring oxidation at the C3 and C6 atoms of the AαC heteronucleus (80,81). Both human and rodent uridine diphosphate-glucuronosyltransferases are involved in the detoxication of MeIQx and PhIP (40,75,77,82). There is also evidence for the formation of N-glucuronide conjugates of AαC in rodents (83). Glutathione S-transfereases catalyze the detoxication of N-oxidized PhIP metabolites (84,85), but this family of enzymes has not been found to be involved in the detoxication of N-oxidized metabolites of MeIQx (84) or AαC (86). We have detected a number of metabolites of AαC in human hepatocytes, by LC-ESI/MS/MS (Supporting Information, Figure S-3 A and Figure S-3 B): AαC undergoes extensive oxidation, and acetyl, sulfate or glucuronide conjugates are formed. We plan to elucidate the pathways of AαC metabolism and the principal pathways involved in AαC-DNA adduct formation in hepatocytes, to establish human biomarkers of this HAA.

AαC undergoes N-oxidation by human P450 1A2, as well as P450s 1A1 and 2C9/2C10, which are highly expressed in the colon and respiratory tract (80). Moreover, human NAT1 and NAT2, and SULT1A1, which are expressed in liver and extrahepatic tissues, catalyze the binding of HONH-AαC to DNA (86,87). Thus, the potential for AαC to undergo metabolism and induce DNA damage in multiple organs that are in direct or indirect contact with tobacco smoke is great. Given the large amounts of AαC that arise in tobacco smoke (25,26) and the elevated levels of AαC found in urine of smokers in comparison to non-smokers (88), studies investigating the potential role of AαC in the carcinogenicity of liver and gastrointestinal cancers associated with tobacco smoking (89) are warranted.

ABBREVIATIONS

CHO

Chinese hamster ovary cells

EROD

ethoxyresorufin O-deethylation

HAA

heterocyclic aromatic amine

LC–ESI/MS/MS

liquid chromatography/electrospray ionization-tandem mass spectrometry

LC-ESI/MS/MSⁿ

liquid chromatography-electrospray ionization/multi-stage tandem mass spectrometry

LIT MS

linear quadrupole ion trap mass spectrometer

MROD

methoxyresorufin O-deethylation

NAT

N-acetyltransferase

SULT

sulfotransferase

4-ABP

4-aminobiphenyl

IQ

2-amino-3-methylimidazo[4,5-f]quinoline

AαC

2-amino-9H-pyrido[2,3-b]indole

HONH-AαC

2-hydroxyamino-9H-pyrido[2,3-b]indole

MeIQx

2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

HONH-MeIQx

2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline

IQx-8-COOH

2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid;

8-CH₂OH-IQx

2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline

MeIQx-N²-Gl

N²-(β-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

HON-MeIQx-N²-Gl.

N²-(β-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline

MeIQx-N²-SO₃H

N²-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl-sulfamic acid;2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

PhIP

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

HONH-PhIP,HON-PhIP-N²-Gl

N²-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine

HON-PhIP-N3-Gl

N3-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine

PhIP-N²-Gl

N²-(β-1-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

PhIP-N3-Gl

N3-(β-1-glucosiduronyl-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

dG-C8-4-ABP

N-(deoxyguanosin-8-yl)-4-ABP

dG-N²-N⁴-ABP

N-(deoxyguanosin-N²-yl)-4-aminobiphenyl

dG-C8-IQ

N-(deoxyguanosin-8-yl)-IQ

dG-N²-IQ

5-(deoxyguanosin-N²-yl)-IQ

dG-C8-AαC

N-(deoxyguanosin-8-yl)-AαC dG-C8-MeIQx, N-(deoxyguanosin-8-yl)-MeIQx

dG-N²-MeIQx

5-(deoxyguanosin-N²-yl)-MeIQx

dG-C8-PhIP

N-(deoxyguanosin-8-yl)-PhIP