3-Aminobenzanthrone, a Human Metabolite of the Environmental

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Chem. Res. Toxicol. 2004, 17, 1092-1101

3-Aminobenzanthrone, a Human Metabolite of the Environmental Pollutant 3-Nitrobenzanthrone, Forms DNA Adducts after Metabolic Activation by Human and Rat Liver Microsomes: Evidence for Activation by Cytochrome P450 1A1 and P450 1A2 Volker M. Arlt,*,† Alan Hewer,† Bernd L. Sorg,‡ Heinz H. Schmeiser,‡ David H. Phillips,† and Marie Stiborova§ Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom, Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany, and Department of Biochemistry, Charles University, Faculty of Science, Albertov 2030, 128 40 Prague 2, The Czech Republic Received March 19, 2004

3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the 32P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by R-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat hepatic microsomes, we showed that both human and rat microsomes lead to the same 3-ABA-derived DNA adducts. Pretreatment of rats with β-naphthoflavone or Sudan I, inducers of P450 1A1 and 1A2, and P450 1A1 alone, respectively, significantly stimulated the levels of 3-ABA-derived DNA adducts formed by rat liver microsomes. Utilizing purified rat recombinant P450 1A1, the participation of this enzyme in DNA adduct formation by 3-ABA was corroborated. In summary, our results strongly suggest a genotoxic potential of 3-ABA for humans. Moreover, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA.

Introduction Lung cancer is the most common malignant disease worldwide and is the major cause of death from cancer (1). Environmental factors and individual genetic susceptibility play an important role in many human cancers (2). Tobacco smoking is the overwhelming cause of lung * To whom correspondence should be addressed. Tel: +442087224405. Fax: +44-2087224052. E-mail: [email protected]. † Institute of Cancer Research. ‡ German Cancer Research Center. § Charles University.

cancer, but ambient air pollution is also implicated (3). Moreover, epidemiological data have shown that occupational exposure to diesel exhaust is associated with an increased risk of lung cancer (4, 5), and diesel exhaust is considered a probable human carcinogen by the IARC (6). Although traditional industrial emission levels are tending to decrease in Western countries, vehicular exhaust remains a continuing, even increasing, problem. The particulate phase of diesel exhaust is the predominant source of human exposure to nitro-PAHs (7), many of which are mutagens and rodent carcinogens (6, 8).

10.1021/tx049912v CCC: $27.50 © 2004 American Chemical Society Published on Web 06/25/2004

Activation of 3-Aminobenzanthrone by P450 Enzymes

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Figure 1. Proposed pathways of metabolic activation and DNA adduct formation of 3-NBA and 3-ABA. See text for details. R ) -COCH3 or -SO3H.

Their detection in the lungs of nonsmokers with lung cancer has led to considerable interest in assessing their potential cancer risk to humans (9). The aromatic nitroketone 3-NBA (3-nitro-7H-benz[de]anthracen-7-one)1 (Figure 1) is one of the most potent mutagens and a suspected human carcinogen (10, 11) that was discovered only a few years ago in diesel exhaust and bound to the surface of airborne particulate matter (10, 12, 13). It is most likely formed during incomplete combustion of diesel or by reaction of the parent aromatic hydrocarbon with nitrogen oxides in the atmosphere (10). More recently, 3-NBA was detected in surface soil and rainwater, suggesting that 3-NBA is an ubiquitous environmental contaminant (14-16). 3-NBA is genotoxic in various short-term tests (10, 17-19). The genotoxicity of this suspected carcinogen was further documented by the detection of specific DNA adducts in vitro, in cell culture, and in vivo in rats (20-29). Although human exposure to diesel exhaust occurs widely in the general environment, it may also occur at much higher levels occupationally, for example, in miners, professional drivers, bus garage workers, and mechanics. 3-ABA (Figure 1), a major metabolite of 3-NBA, was recently detected in the urine of smoking and nonsmoking salt mining workers (12). Several human biomonitoring studies using the detection of DNA adducts by the ultrasensitive 32P-postlabeling method have reported higher levels of bulky DNA adducts among subjects heavily exposed to diesel exhaust (30, 31). This correlates with the increased cancer risk (6). Determining the capability of humans to activate 3-NBA to form DNA adducts and understanding which human enzymes are involved in its metabolic activation are important in the assessment of susceptibility to this environmental contaminant. We recently showed that 3-NBA is activated by cytosolic nitroreductases and human hepatic microsomes forming DNA adduct patterns qualitatively similar to those found in vivo in rats treated with 3-NBA (21, 25, 27, 29). The correlation of P450-linked enzyme activities with the level of DNA 1 Abbreviations: 3-NBA, 3-nitrobenzanthrone; 3-ABA, 3-aminobenzanthrone; N-OH-ABA, N-hydroxy-3-aminobenzanthrone; P450, cytochrome P450; nitro-PAH, nitropolycyclic aromatic hydrocarbon; R-NF, R-naphthoflavone; β-NF, β-naphthoflavone; PB, phenobarbital; PCN, pregnenolone-16R-carbonitrile; CHAPS, 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfate; HEPES, N-[2-hydroxyethyl]piperazineN′-[2-ethanesulfonic acid]; NAT, N,O-acetyltransferase; SULT, sulfotransferase; TLC, thin-layer chromatography; RAL, relative adduct labeling.

binding indicated that most of the hepatic microsomal activation of 3-NBA was attributed to NADPH:P450 reductase (29). Moreover, we found recently that 3-ABA forms the same DNA adducts as 3-NBA in vivo in rats (27). Whereas 3-ABA can be used as a suitable biomarker of exposure to 3-NBA in humans, its participation in the metabolic activation of 3-NBA leading to the formation of DNA adducts is not known. The objective of this study was to investigate the capability of human and rat enzyme systems to activate 3-ABA and to identify those enzymes involved in DNA adduct formation by 3-ABA.

Materials and Methods Caution: 3-NBA is a potent mutagen and potential carcinogenic and should be handled with care. Exposure to 32P should be avoided, by working in a confined laboratory area, with protective clothing, plexiglass shielding, Geiger counters, and body dosimeters. Wastes must be discarded according to appropriate safety procedures. Chemicals. Chemicals were obtained from the following sources: R-NF, β-NF, PB, PCN, NADPH, CHAPS, dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, dilauroyl phosphatidylserine, HEPES, deoxyadenosine 3′-monophosphate, deoxyguanosine 3′-monophosphate, and calf thymus DNA were from Sigma Chemical Co. (St. Louis, MO); Sudan I was from BDH (Poole, United Kingdom); and furafylline was from New England Biolabs (Beverly, MA). All other chemicals were of analytical purity or better. Enzymes and chemicals for the 32Ppostlabeling assay were obtained commercially from sources described previously (26, 27). Synthesis of 3-NBA and 3-ABA. 3-NBA was synthesized as described previously (26). 3-ABA was synthesized as described (28). The authenticity of 3-NBA and 3-ABA was confirmed by UV, ES-MS, and high field proton NMR spectroscopy. Cell Culture of MCL-5 Cells and Treatment with 3-ABA. Human B-lymphoblastoid MCL-5 cells (32) were obtained under license from GENTEST (Woburn, MA). MCL-5 cells were cultivated as described previously (33). For treatment, aliquots (10 mL) of suspensions of MCL-5 cells (∼8.0 × 105 cells/mL) were incubated at 37 °C for 24 h with 0.1, 1, or 10 µM 3-ABA or 3-NBA (dissolved in 16.6 µL of DMSO). The controls were treated with DMSO only. The cell viability was determined by the trypan blue exclusion assay as described (26). The DNA from cells was isolated by the phenol extraction method as described previously (34). Preparation of Microsomes and Assays. Microsomes were isolated from the livers of 10 male Wistar rats, either untreated or pretreated with β-NF inducing P450 1A1 and 1A2 as described (35); those pretreated with Sudan I induced P450 1A1

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Table 1. P450-Dependent Catalytic Activities and DNA Adduct Formation by 3-ABA in Human Liver Microsomes RALd (mean ( SD/108 nucleotides)

P450-dependent catalytic activitiesc no. HK23a HK34a HG42a HG43a HG56a HG89a HG93a HG112a R1b R2b R3b R4b R5b

1A1 and 1A2 1A1

1A2 2A6

0.0088 0.0055 0.0067 0.0077 0.0268 0.0125 0.0182 0.0069 ND ND ND ND ND

0.96 1.00 0.70 0.59 2.30 0.75 0.89 0.32 ND ND ND ND ND

0.085 0.047 0.051 0.059 0.135 0.051 0.047 0.044 0.039 0.416 0.421 0.016 0.095

1.10 1.50 2.20 0.67 1.40 0.49 0.35 0.49 ND ND ND ND ND

2B6 0.024 0.039 0.150 0.027 0.045 0.030 0.027 0.150 ND ND ND ND ND

2C8 2C9 2C19 2D6 2E1 3A4 0.16 0.22 0.48 0.06 0.18 0.12 0.15 0.18 ND ND ND ND ND

2.10 1.90 1.60 1.70 3.10 1.80 1.70 4.00 ND ND ND ND ND

0.11 0.05 0.01 0.63 0.41 0.12 0.04 0.38 ND ND ND ND ND

0.140 0.100 0.095 0.022 0.100 0.010 0.047 0.032 ND ND ND ND ND

2.10 6.00 1.60 1.20 1.90 1.10 1.50 2.40 ND ND ND ND ND

6.80 5.20 15.0 4.80 4.00 6.00 1.70 25.0 ND ND ND ND ND

nmol P450 4A per mg protein 0.78 1.10 1.40 1.90 2.30 1.10 1.20 1.80 ND ND ND ND ND

0.38 0.50 0.67 0.26 0.48 0.40 0.32 0.56 0.62 1.32 1.56 1.55 2.74

nuclease P1

butanol

4.7 (3.4, 6.0) 4.8 (3.7, 5.9) 4.0 (4.1, 3.9) 4.0 (3.5, 4.4) 10.4 (10.2, 10.5) 3.9 (3.2, 4.7) 3.4 (3.7, 3.0) 2.4 (2.1, 2.7) 1.3 (1.4, 1.2) 21.2 (20.9, 21.4) 32.1 (39.4, 24.9) 2.4 (2.6, 2.3) 4.1 (3.6, 4.7)

8.7 (8.9, 8.5) 8.6 (8.7, 8.5) 9.0 (8.3, 9.4) 7.9 (6.3, 9.5) 14.5 (12.2, 16.7) 5.3 (5.0, 5.7) 5.5 (4.7, 6.2) 5.8 (5.4, 6.3) 3.0 (2.7, 3.3) 34.0 (25.2, 42.7) 29.2 (25.3, 31.1) 4.3 (5.5, 3.2) 4.8 (6.4, 3.2)

a HK23, HK34, HG42, HG43, HG56, HG89, HG93, and HG112, human hepatic microsomal samples. b R1-R5, rat hepatic microsomal samples; uninduced rat microsomes (R1), β-NF-induced rat microsomes (R2), Sudan I-induced rat microsomes (R3), PCN-induced rat microsomes (R4), and PB-induced rat microsomes (R5). c Each microsomal sample was evaluated for specific P450 activities by monitoring the following reactions: 7-ethoxyresorufin O-deethylation (P450 1A1 and 1A2), Sudan I oxidation (P450 1A1), phenacetin-O-deethylation (P450 1A2), coumarin 7-hydroxylation (P450 2A6), benzyloxyresorufin O-debenzylation (P450 2B6), paclitaxel 6R-hydroxylation (P450 2C8), diclofenac 4-hydroxylation (P450 2C9), (S)-mephenytoin 4-hydroxylation (P450 2C19), bufuralol 1′-hydroxylation (P450 2D6), chlorzoxazone 6-hydroxylation (P450 2E1), testosterone 6β-hydroxylation (P450 3A4), and lauric acid 12-hydroxylation (P450 4A). P450 activities in nmol/min/mg protein. ND ) not determined. d Mean RAL of four determinations (duplicate analyses of two independent in vitro incubations).

as described (36), those pretreated with PB induced P450 2B1 and 2B2 and NADPH:P450 reductase as reported (37), and those pretreated with PCN induced P450 3A1 and 3A2 and NADPH: P450 reductase as reported (38). Microsomes from livers of eight human donors were obtained from GENTEST (catalog nos. HK23, HK34, HG42, HG43, HG56, HG89, HG93, and HG112). Supersomes, microsomes isolated from insect cells transfected with baculovirus constructs containing cDNA of one of the following human P450s, P450 1A1, 1A2, 1B1, 2A6, 2B6, 2D6, 2C9, 2E1, or 3A4, and/or expressing NADPH:P450 reductase, were also obtained from GENTEST. Protein concentrations in the microsomal fractions were assessed using the bicinchoninic acid protein assay (Pierce, Rockford, IL) with serum albumin as a standard (39). The concentration of P450 was estimated as described previously (40). The content of P450 in each rat and human hepatic microsomes is shown in Table 1. Each human microsomal sample was evaluated for specific P450 activities by monitoring the following reactions: 7-ethoxyresorufin O-deethylation (P450 1A1 and 1A2), Sudan I oxidation (P450 1A1), phenacetin O-deethylation (P450 1A2), coumarin 7-hydroxylation (P450 2A6), benzyloxyresorufin O-debenzylation (P450 2B6), paclitaxel 6R-hydroxylation (P450 2C8), diclofenac 4-hydroxylation (P450 2C9), (S)-mephenytoin 4-hydroxylation (P450 2C19), bufuralol 1′-hydroxylation (P450 2D6), chlorzoxazone 6-hydroxylation (P450 2E1), testosterone 6β-hydroxylation (P450 3A4), and lauric acid 12-hydroxylation (P450 4A) (36, 41, and references therein). These activities are shown in Table 1. Rat microsomes were examined for Sudan I oxidation (P450 1A1) only (Table 1). Microsomal Incubations. Incubation mixtures, in a final volume of 750 µL, consisted of 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH, 0.1-1 mg of microsomal protein, 100 µM 3-ABA (dissolved in 12.5 µL of DMSO), and 0.5 mg of calf thymus DNA. The reaction was initiated by adding 3-ABA. Incubations with rat and human microsomes were carried out at 37 °C for 2 h. The control incubations were carried out either (i) without activating system (microsomes), (ii) with activating system and 3-ABA but without DNA, or (iii) with activating system and DNA but without 3-ABA. Incubation mixtures, in which microsomes containing human recombinant P450s and NADPH:P450 reductase (Supersomes) were used to activate 3-ABA, were the same composition except that 10, 25, or 50 pmol of P450s were added instead of hepatic microsomes. Supersomes containing human recombinant NADPH:P450 reductase alone were used for comparison (control). Supersomes incubations were carried out for 3 h at 37 °C. After the incubation, DNA

was isolated from the residual water phase by the phenol/ chloroform extraction method as described (42). Inhibition Studies. The following chemicals were used to inhibit the activation of 3-ABA in human hepatic microsomes: R-NF, which inhibits P450 1A1 and 1A2, and some reactions catalyzed by P450 3A, and furafylline, which inhibits P450 1A2 (43). The inhibitors were dissolved in 7.5 µL of methanol and were added to the incubation mixtures to yield final concentrations of 100 µM as used previously (43). The incubation mixtures containing the inhibitors were then incubated at 37 °C for 10 min with NADPH prior to adding 3-ABA and then for a further 60 min at 37 °C. An equal volume of methanol alone was added to the control incubations. Cell Culture of V79 Cells and Treatment with 3-ABA. The parental V79 Chinese hamster lung fibroblast subclone V79MZ (44) and the recombinant cells V79MZ-h1A2 expressing human P450 1A2 (45) were kindly provided by Prof. H. R. Glatt (German Institute of Human Nutrition, Potsdam, Germany). The recombinant cells V79MZ-h1A1 (46) and V79MZ-h3A4 (47) expressing human P450 1A1 or 3A4, respectively, were purchased from PharmBioDyn (Freiburg, Germany). All V79 cells were cultivated and treated with 3-ABA as described recently (26, 28). Cells treated with DMSO only were used as the control. As a positive control for oxidative activation by human P450 1A1, cells were treated with 1 µM benzo[a]pyrene. As a positive control for oxidative activation by human P450 3A4, cells were treated with 1 µM ellipticine. The cell viability (% of control) was determined by the trypan blue exclusion assay as described above. DNA from cells was isolated as described (34). Enzyme Preparations. Recombinant rat P450 1A1 was purified as described (48) from membranes of Escherichia coli transfected with a modified P450 1A1 cDNA. Rabbit liver NADPH:P450 reductase was purified as described (49). Recombinant rat P450 1A1 was reconstituted with rabbit NADPH: P450 reductase as follows: 0.5 µM P450s, 0.5 µM NADPH:P450 reductase, 0.5 µg/µL CHAPS, 0.1 µg/µL liposomes [dilauroyl phosphatidylcholine, dioleyl phosphatidylcholine, and dilauroyl phosphatidylserine (1:1:1)], 3 mM reduced glutathione, and 50 mM HEPES/KOH (pH 7.4). An aliquot containing appropriate amounts of reconstituted P450 (50-250 pmol) was used for activation of 3-ABA by its adding, instead of microsomes, to incubation mixtures of composition described above. In the control incubation, the P450 was omitted from the reconstitution mixture. After incubation (37 °C, 60 min), the incubation mixtures were extracted twice with ethyl acetate (2 × 2 mL). DNA was isolated from the residual water phase as described (42).



Figure 2. Autoradiographic profiles of 3-ABA-derived DNA adducts by using the nuclease P1 (upper panels) or butanol (lower panels) enrichment version of the 32P-postlabeling assay. Adduct profiles obtained from liver DNA of rats treated with 2 mg of 3-ABA per kg body weight [these profiles are representative of adduct profiles obtained with DNA from other rat tissues including lung, kidney, spleen, heart, and colon (27)] (A). Adduct profiles obtained in human MCL-5 cells treated with 1 µM 3-ABA (B). Adduct profiles obtained from calf thymus DNA treated with 3-ABA (100 µM) after activation by β-NF-induced hepatic microsomes from rat (C). Adduct profiles obtained from calf thymus DNA treated with 3-ABA (100 µM) after activation by human hepatic microsomes (HG56) (D). Adduct profiles in V79MZ-h1A1 cells expressing recombinant human P450 1A1 after exposure to 0.1 µM 3-ABA (E). Adduct profiles obtained from calf thymus DNA treated with 3-ABA (100 µM) after activation with 100 pmol of purified rat P450 1A1 (F). Adduct profiles from calf thymus DNA treated with 3-ABA (100 µM) after activation with Supersomes containing 50 pmol of human P450 1A1 (G). Preparation of Reference Adducts. Wistar rats were treated with a single dose of 3-NBA or 3-ABA (2 mg/kg body weight, ip), and DNA was isolated and analyzed as described recently (27). Deoxyadenosine and deoxyguanosine 3′-monophosphate (4 µmol/mL) (Sigma) were incubated with 3-NBA (0.3 mM) enzymatically activated by xanthine oxidase (1 U/mL) in 50 mM potassium phosphate buffer, pH 7.0, in the presence of 1 mM hypoxanthine as described (25). Aliquots of the incubation were used directly for the butanol extraction-mediated 32Ppostlabeling procedure. 32P-Postlabeling Analysis and HPLC Analysis of 32PLabeled 3′,5′-Deoxyribonucleoside Bisphosphate Adducts. 32P-postlabeling analysis using nuclease P1 and butanol enrichment and TLC and HPLC chromatography was performed as described recently (26, 27). DNA adduct spots were numbered as reported recently (27-29). Statistical Analysis. Statistical associations between P450linked catalytic activities in human and rat hepatic microsomal samples and levels of total 3-ABA-derived DNA adducts formed by the same microsomes were determined by linear regression using Statistical Analysis System software version 6.12. Correlation coefficients (r) were based on a sample size of eight for humans and five for rat microsomes, respectively. All P values are two-tailed and considered significant at the 0.05 level.

Results Comparison of DNA Adduct Formation of 3-ABA in Human B-Lymphoblastoid MCL-5 Cells and in Vivo in Rats. On the basis of previous studies that demonstrated specific sensitivity of arylamine- and nitroPAH-derived DNA adducts to nuclease P1 treatment, both butanol extraction and nuclease P1 digestion of the 32 P-postlabeling assay were employed to evaluate DNA adducts derived from 3-ABA (50, 51). The DNA adduct pattern detected by 32P-postlabeling induced by 3-ABA in vivo in rats consisted of a cluster of four adducts either with nuclease P1 (spots 1, 2, 3, and 6) (Figure 2Aa) or butanol enhancement (spots 1, 2, 3, and 4) (Figure 2Ab). Essentially, the same DNA adducts were observed in MCL-5 cells treated with 3-ABA (Figure 2Ba,b). No DNA adducts were found in DNA isolated from rat tissue or

Figure 3. Total DNA binding in human MCL-5 cells treated with 3-NBA and 3-ABA. The nuclease P1 and the butanol enrichment versions of the 32P-postlabeling assay were used. Values represent means ( SD of triplicate separate cell incubations; each DNA sample was determined by two postlabeled analyses. Cell viability (% of control) was always greater than 90%. DNA adduct levels in MCL-5 cells treated with 3-NBA have been published before (29). N.d. ) not detected.

cells treated with vehicle only (data not shown). In cells, DNA adduct formation was concentration-dependent (Figure 3). However, no DNA adducts were detected at the lowest 3-ABA concentration tested (0.1 µM). Total DNA binding in MCL-5 cells by 3-NBA was 4.4-7.5-fold (1 µM) and 8.1-21.0-fold (10 µM) higher as compared to 3-ABA (Figure 3). In contrast to the incubations of cells with 3-NBA, one additional adduct spot (assigned spot 7) was detected with 3-ABA (Figure 2B). Interestingly, adduct spot 7 was only detectable in MCL-5 cells treated with 1 µM 3-ABA but not at a 10-fold higher concentration (10 µM) (Table S1 in Supporting Information). A similar adduct spot was also observed previously in calf thymus DNA after activation of 3-NBA by reduction with zinc (21) or by human hepatic microsomes (29). However, low adduct levels prevented further characterization by HPLC cochromatographic analysis. Human and Rat Hepatic Microsomes Activate 3-ABA to Species that Bind to DNA. We determined the formation of DNA adducts by 3-ABA in calf thymus


Arlt et al.

Table 2. Effect of P450 1A1 and P450 1A2 Inhibitor, r-NF, and P450 1A2 Inhibitor, Furafylline, on DNA Adduct Formation by 3-ABA in Human Liver Microsomes RALa (mean ( SD/108 nucleotides) nuclease P1 without R-NF/furafylline in the presence of R-NF (100 µM) in the presence of furafylline (100 µM) a

butanol

HK34

HG56

HK34

HG56

4.8 (100%) (5.1, 4.5) NDb 2.3 (48%) (2.3, 2.3)

6.1 (100%) (5.6, 6.5) 2.6 (42%) (3.1, 2.1) 4.9 (80%) (5.3, 4.5)

8.6 (100%) (8.8, 8.4)

18.8 (100%) (19.4, 18.4) 7.5 (40%) (7.4, 7.6) 19.0 (101%) (23.5, 14.6)

5.1 (59%) (5.0, 5.2)

Mean RAL of four determinations (duplicate analyses of two independent in vitro incubations). b ND ) not determined.

DNA in the presence of microsomes isolated from eight different human livers and from livers of rats, uninduced or pretreated with β-NF, Sudan I, PB, and PCN. All rat (Figure 2C) and human hepatic microsomes (Figure 2D) were capable of activating 3-ABA to form DNA adducts. 3-ABA, activated by both species, induced essentially the same DNA adducts consisting of a cluster of four adducts (spots 1, 2, 3, and 4) and of a cluster of four adducts (spots 1, 2, 3, and 6) after butanol and nuclease P1 enrichment, respectively. Similarly, human hepatic microsome-mediated activation of 3-ABA essentially leads to the same DNA adducts as formed by its nitroaromatic counterpart, 3-NBA, in vitro and in vivo in rats (25, 27, 29). Moreover, the same adducts were observed in vivo in rats treated with 3-ABA (compare Figure 2A) and human MCL-5 cells (compare Figure 2B). In contrast to incubations with 3-NBA and human liver microsomes (29), adduct spot 7 was not detected in incubations using 3-ABA (Table S2 in Supporting Information). Chromatograms of DNA digests from control incubations carried out in parallel without microsomes, without DNA, or without 3-ABA were free of adduct spots in the region of interest (data not shown). Cochromatographic analysis by HPLC confirmed that all 3-ABA-derived adducts (spots 1, 2, 3, and 4) that are formed with human microsomes are identical to those derived from 3-NBA by nitroreduction (Figure S1 in Supporting Information). As reported before (25), we showed that all major 3-NBA-derived adducts are products derived from simple nitroreduction bound to dA (spots 1 and 2) or dG (spots 3 and 4) (Figure S1 in Supporting Information). When equal amounts of radioactivity of adduct spots found in vivo in rats treated with 3-ABA or formed with human hepatic microsomes incubated with 3-ABA and the corresponding purine 3-NBAderived adducts spots obtained in vitro were mixed prior to analysis, a single peak was found (data not shown). To resolve which microsomal enzymes are mainly responsible for the activation of 3-ABA, different experimental approaches were used as follows: (i) correlation of P450-linked enzyme activities in human and rat hepatic microsomes with DNA adduct formation by 3-ABA with human and rat microsomes, (ii) selective enzyme inhibition in human microsomes, (iii) genetically engineered V79 cells expressing various human P450 enzymes, (iv) purified P450 enzymes, and (v) use of heterologous baculovirus expression system of human P450s and NADPH:P450 reductase (Supersomes). Correlation of P450-Linked Enzyme Activities in Human and Rat Microsomes with DNA Adduct Formation by 3-ABA. Human hepatic microsomes used in the present study are able to catalyze reactions known to be associated with specific P450 enzymes (P450 1A1 and 1A2, 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A) (Table 1). Large individual variations in catalytic activities were evident among these different

hepatic microsomal samples (Table 1). Quantitative 32Ppostlabeling analysis, as shown in Table 1, also showed wide individual variations in DNA binding by 3-ABA in the microsomal incubations, ranging from 2.4 to 10.4 and from 5.3 to 14.5 adducts per 108 nucleotides after nuclease P1 and butanol enrichment, respectively. The total DNA binding by 3-ABA was significantly correlated with activities of Sudan I oxidation, a marker for P450 1A1 (r ) 0.858 and r ) 0. 898, P < 0.01, using the nuclease P1 and butanol enhancement versions of the assay, respectively), and with activities of phenacetin O-deethylation, a marker for P450 1A2 (r ) 0.963 and r ) 0.891, P < 0.01, using the nuclease P1 and butanol enhancement versions of the assay, respectively) (Table S3 in Supporting Information). A weaker, but significant, correlation was determined between ethoxyresorufin O-deethylation, a marker for P450 1A1 and 1A2, and 3-ABA-derived adduct levels using enhancement by nuclease P1 (r ) 0.746, P < 0.05) (Table S3 in Supporting Information). No significant correlation was determined between any other examined P450 activities (P450 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, and 4A) and formation of DNA adducts by 3-ABA. In the species comparison, microsomes from human and rat either uninduced or induced with PB and PCN exhibited comparable efficiencies to activate 3-ABA, whereas rat microsomes induced with β-NF and Sudan I were more efficient than human microsomes (Table 1). Similarly to human hepatic microsomes, the efficiency of rat microsomes to activate 3-ABA highly correlated with the P450 1A1 activity (r ) 0.962 and r ) 0.991, P < 0.001, using the nuclease P1 and butanol enhancement versions of the assay, respectively). Hepatic microsomes of rats pretreated with inducers of P450 1A1 and 1A2, and P450 1A1 alone, β-NF and Sudan I, led to a 11.316.7-fold and 11.5-26.1-fold increase, respectively, in the formation of 3-ABA-derived DNA adducts. Levels of individual DNA adducts formed by 3-ABA in rat microsomes are given in Table S4 of the Supporting Information. Effect of Inhibitors of P450 1A1 and P450 1A2 on Activation of 3-ABA in Human Microsomes. Inhibition experiments supported the role of P450 1A1 and 1A2 in the activation of 3-ABA in human hepatic microsomes. R-NF, a selective inhibitor of P450 1A1 and 1A2 (43), was effective in inhibiting DNA adduct formation by 3-ABA in one human microsomal sample with high activities of all P450s tested (HG56) when used in equal molar amounts as 3-ABA (100 µM) (Table 2). To further investigate the role of P450 1A2 in 3-ABA activation, two human microsomal samples (HK34 and HG56) were selected, and incubations were carried out in the absence and presence of a specific inhibitor of P450 1A2, furafylline. In microsomal sample HK34, furafylline was an efficient inhibitor of DNA binding (Table 2). However,


Chem. Res. Toxicol., Vol. 17, No. 8, 2004 1097 Table 3. DNA Adduct Formation by 3-ABA Activated with Purified Rat Recombinant P450 1A1 RALa (mean ( SD/108 nucleotides) P450 1A1 (pmol)

nuclease P1

butanol

0 (control) 50 100 250

NDb 8.9 ( 3.0 15.6 ( 3.0 29.8 ( 4.6

ND 16.8 ( 2.1 31.9 ( 1.5 53.7 ( 16.5

a Mean RAL ( SD of three determinations of one in vitro incubation. b ND ) not detected.

Figure 4. Total DNA binding in parental (V79MZ) and recombinant (V79MZ-h1A1, V79MZ-h1A2, and V79MZ-3A4) V79 cells treated with 3-ABA. The nuclease P1 and the butanol enrichment versions of the 32P-postlabeling assay were used. Values represent means ( SD of triplicate separate cell incubations; each DNA sample was determined by two postlabeled analyses. Cell viability (% of control) was always greater than 80%. DNA adduct levels in V79MZ-h1A2 cells treated with 3-ABA have been published before (28). N.d. ) not detected.

no inhibition of DNA binding was observed in sample HG56 under the conditions used (Table 2). The effects of inhibitors on levels of individual DNA adducts formed by 3-ABA in human hepatic microsomes are shown in Table S5 of the Supporting Information. 3-ABA Is Activated in V79 Cells Expressing Recombinant Human P450 1A1 and P450 1A2 but Not in V79 Cells Expressing Recombinant Human P450 3A4. Chinese hamster lung V79 cells completely lack P450-dependent enzyme activities but contain detectable amounts of native NADPH:P450 reductase (44). Using V79 cells (V79MZ-h1A2) expressing human recombinant P450 1A2 (45), we recently demonstrated that 3-ABA is activated by this P450 isoform (see Figure 4) (28). To further investigate the involvement of human P450 1A1 and 3A4, the latter P450 isoform is highly expressed in human livers, in the metabolic activation of 3-ABA, and we used V79 cell lines (V79MZ-h1A1 and V79MZ-h3A4) expressing human recombinant P450 1A1 or 3A4 (46, 47). As shown in Figure 2E, the DNA adduct patterns induced by 3-ABA in V79MZ-h1A1 cells were essentially similar to those observed in vivo in rats, in human MCL-5 cells, and by using human hepatic microsomes (present study). As found in MCL-5 cells, adduct spot 7 was also detected in V79MZ-h1A1 cells (compare Figure 2B). Interestingly, adduct spot 7 was detectable at the two lowest concentrations used only (0.01 and 0.1 µM) but not at the highest concentration (1 µM) (Table S6 in Supporting Information). No DNA adduct formation was observed in parental cells, V79MZ, and V79 cells expressing human P450 3A4 (Figure 4). No DNA adducts were observed in DNA isolated from cells treated with vehicle (DMSO) only (data not shown). In V79 cells expressing human P450 1A1, one major adduct (9.7 ( 3.9 adducts per 108 nucleotides) was detected after treatment with benzo[a]pyrene (positive control) whereas in the parental cells, V79MZ, no such adduct was found (data not shown). As reported previously (52), high levels of ellipticinederived DNA adducts (41.2 ( 15.6 adducts per 108 nucleotides) were observed in V79 cells expressing human P450 3A4 after exposure to ellipticine (data not shown). DNA adduct formation for 3-ABA was concentration-

dependent in V79MZ-h1A1 cells (Figure 4), ranging from 1.8 to 12.3 and from 4.1 to 21.4 adducts per 108 nucleotides for total DNA binding after nuclease P1 digestion and butanol extraction, respectively. However, the formation of 3-ABA-derived DNA adducts was not associated with an increase in cytotoxicity in V79 cells. Activation of 3-ABA by Purified Rat P450 1A1. To confirm the role of P450 1A1 in the activation of 3-ABA in rat microsomes, purified rat recombinant P450 1A1 was used in additional experiments. P450 1A1 reconstituted with NADPH:P450 reductase was active with its typical substrates (e.g., Sudan I, 7-ethoxyresorufin) (data not shown). Figure 2F shows that incubations of 3-ABA with DNA and purified P450 1A1 reconstituted with NADPH:P450 reductase together with its cofactor, NADPH, resulted in the formation of the same DNA pattern as with rat hepatic microsomes (compare Figure 2C) and in vivo in rats (compare Figure 2A). P450 1A1-mediated DNA adduct formation was concentration-dependent (Table 3). Levels of individual DNA adducts formed by 3-ABA with purified rat recombinant P450 1A1 are shown in Table S7 of the Supporting Information. Activation of 3-ABA by Recombinant Human P450s in Supersomes. Although human livers are rich in many P450s, some of these enzymes are missing or are expressed in very low levels in liver tissue (e.g., P450 1A1, 1B1, 2B6, and 2D6). Therefore, we tested in more detail whether individual P450 enzymes might activate 3-ABA. For this purpose, we used human recombinant P450s in microsomes of baculovirus-transfected insect cells (Supersomes) containing recombinantly expressed human P450s (P450 1A1, 1A2, 2A6, 1B1, 2B6, 2C9, 2D6, 2E1, and 3A4) and/or NADPH:P450 reductase. The recombinant human P450s used in the experiments efficiently oxidized their typical substrates (data not shown). Of all P450s tested, P450 1A1, 1A2, 2A6, 1B1, and 2B6 were capable to activate 3-ABA (Figure 5). P450 1A1 and 2A6 were the most active in 3-ABA activation. As exemplified in Figure 2G, 3-ABA induced essentially the same DNA adduct pattern as those obtained in vivo in rats, in human MCL-5 cells, and by using human liver microsomes (present study). In contrast, no DNA adducts were observed in Supersomes incubations that contained NADPH:P450 reductase alone and served as controls (data not shown). Using 10, 25, and 50 pmol of human P450s, a concentration-dependent activation of 3-ABA was observed (Figure 5). The only exceptions were P450 1A2 and 2B6 using nuclease 1 enrichment; in these cases, DNA adduct formation was only observed after using butanol enrichment (Figure 5). In general, DNA binding after butanol enrichment was much higher than after enhancement with nuclease P1 (Figure 5). Levels of DNA adducts formed by 3-ABA using P450-mediated metabolic activation with Supersomes are given in Table S8 of the Supporting Information.


Arlt et al.

Figure 5. DNA adduct formation by 3-ABA after metabolic activation with Supersomes containing different recombinant human P450s and using different P450 concentrations (10, 25, and 50 pmol of P450). The nuclease P1 and the butanol enrichment versions of the 32P-postlabeling assay were used. Values represent means of two in vitro incubations; each DNA sample was determined by two postlabeled analyses within a maximal error range of 25%. N.d. ) not detected.

Discussion 3-ABA, the major metabolite of the ubiquitous environmental pollutant 3-NBA, was recently detected in the urine of smoking and nonsmoking salt mining workers occupationally exposed to diesel emissions, at a similar concentration (1-143 ng/24 h urine) to 1-aminopyrene (2-200 ng/24 h urine), the corresponding amine of the most abundant nitro-PAH detected in diesel exhaust matter (12). The results of our study clearly demonstrate that human hepatic microsomes are capable of activating 3-ABA to DNA binding species. The DNA adduct patterns generated by 3-ABA activated by human microsomes are identical to that formed by its nitroaromatic counterpart, 3-NBA (29), indicating that N-OH-ABA is the critical intermediate for the formation of electrophilic arylnitrenium ions capable of reacting with DNA (27, 28). An important finding of our study is that metabolic activation of 3-ABA by the human enzymatic system is analogous to that observed in rats (27). Human microsomes generated the same pattern of 3-ABA-derived DNA adducts as hepatic microsomes of rats. In addition, the present study shows the role of specific human P450 enzymes in activating pathways of 3-ABA. P450 1A1 and, to a lesser extent, P450 1A2 were the principal enzymes responsible for the microsomal activation of 3-ABA to DNA binding species. Whereas P450 1A2 is constitutively expressed in human livers (53), there is still conflicting evidence for the expression or inducibility of P450 1A1 protein in human livers (54). The range of P450 1A1 expression levels in our eight human livers is comparable with values reported recently (36, 55). The formation of 3-ABA-derived DNA adducts catalyzed by human liver microsomes was dependent on the catalytic activities of P450 1A1 and 1A2 present in all eight of the human hepatic microsomal samples assayed. The participation of P450 1A1 and 1A2 in the metabolic activation of 3-ABA in human liver microsomes was confirmed by the inhibition of 3-ABA-derived DNA adduct formation with R-NF, a specific inhibitor of P450 1A1 and 1A2 enzyme activity, in one human hepatic microsomal sample (HG56) that exhibited high activity of P450 1A1 and 1A2, whereas furafylline, a specific inhibitor of P450

1A2, did not inhibit DNA adduct formation in the same microsomal sample. In contrast, in another human hepatic microsomal sample (HK34) with low P450 1A1 and higher P450 1A2 enzymatic activity, furafylline was an efficient inhibitor of DNA binding. However, it should be noted that the interpretation of inhibitions studies is sometimes difficult, because one inhibitor may be more effective than another. Nevertheless, the results with recombinant human P450 1A1 and 1A2 expressed in microsomes of baculovirus-transfected insect cells (Supersomes) and recombinant human P450 1A1 and 1A2 expressed in Chinese hamster lung V79 cells fully corroborated the role of P450 1A1 and 1A2 in the metabolic activation of 3-ABA. Using human B-lymphoblastoid MCL-5 cells, we found an essentially similar DNA adduct pattern as observed with human liver microsomes. Therefore, MCL-5 cells contain enzyme systems capable of catalyzing the oxidative activation of 3-ABA leading to DNA adducts. MCL-5 cells express high levels of native P450 1A1 and are transfected with plasmids carrying cDNAs of the human P450 1A2, 2A6, 2E1, and 3A4 gene (32), P450 enzymes, which might participate in the bioactivation of 3-ABA in these cells. Although P450 2A6, 2E1, and 3A4 enzyme activities did not correlate with the metabolic activation of 3-ABA to form DNA adducts in human samples assayed, we found that recombinant human P450 2A6 present (overexpressed) in Supersomes was an effective activator of 3-ABA. In addition, we found that recombinant human P450 1B1 and 2B6 in Supersomes were able to activate 3-ABA leading to DNA adducts. The discrepancy between analysis using human hepatic microsomes and analysis using recombinant human P450s may be attributed to low enzyme expression in human livers, higher expression levels in Supersomes, and/or different activities of recombinant and authentic human P450 enzymes. Interestingly, DNA binding of 3-ABA using Supersomes was substantially higher after butanol as compared to nuclease P1 enrichment (in some cases over 10-fold). Similary, DNA binding of 3-NBA utilizing Supersomes was also much higher using butanol enrichment (29). It is noteworthy that these strong differences were not observed in vivo in rats,


in cell culture, or other in vitro activation systems used in the present study; however, this phenomenon requires further study to clarify it. Comparative analyses of the major 3-ABA-derived DNA adducts obtained in cell culture, generated using hepatic microsomes, and in rats treated with 3-ABA with those prepared in vitro with 3-NBA and in rats treated with 3-NBA were chromatographically indistinguishable. Previous work has shown that the major 3-NBA-DNA adducts (spots 1-4) are products derived from reductive metabolites bound to deoxyadenosine (adducts 1 and 2) or deoxyguanosine (adducts 3 and 4) (25). On the other hand, N-oxidation by P450, namely, P450 1A1 and 1A2 in human hepatic microsomes, seems crucial for the metabolic activation of 3-ABA; on the basis of the detected 3-ABA-derived DNA adducts, no indication for ring oxidation was observed. Thus, N-OH-ABA seems to be the critical intermediate for the formation of the electrophilic arylnitrenium ions capable of reacting with DNA. Nevertheless, studies on the metabolism of 3-ABA generated by P450-mediated activation might confirm or exclude this suggestion. Therefore, such a study is planned in our laboratory. The proposed metabolic pathways of 3-NBA and 3-ABA in DNA adduct formation are summarized in Figure 1. Further structural characterization of these 3-NBA-DNA adducts is currently being undertaken. Interindividual variations in susceptibility and variations in drug-metabolizing enzymes in target tissues appear to be important determinants of cancer risk (2, 56). Human exposure to 3-NBA is thought to occur primarily via the respiratory tract. Although the total P450 content of the lungs is low as compared with that in the liver, P450 enzymes present in lungs may play an important role in extrahepatic bioactivation because of the high rate of blood circulation through the lungs and the possible exposure to 3-NBA through respiration. In this context, it is noteworthy that 3-ABA is the major metabolite in human fetal bronchial, rat alveolar type II, rat epithelial bronchial, and rat mesenchymal lung cells treated with 3-NBA (23), indicating that 3-NBA is metabolized in lung cells. P450 1A1 is the most studied human pulmonary enzyme due to its importance in PAH metabolism (57). P450 1A2 mRNA was also found in lung peripheral tissues but not in the bronchial epithelium (58). Levels of expression and activities of P450 1A1 and 1A2 in humans are influenced by several factors (nutrition, smoking, drugs, environmental chemicals, and genetic polymorphisms) (53, 57, 59, 60) and differ considerably between individuals. The polymorphic expression of P450 1A1 and 1A2 has been attributed to altered expression of the transcription factor that modulates its regulation, that is, the aryl hydrocarbon (Ah) receptor, or its associated transcription factor, the Ah receptor nuclear translocator protein (53, 57). Moreover, the P450 1A1 gene is genetically polymorphic (53, 57, 59). Thus, variability of P450 1A1 and 1A2 levels and activities might play an important role in the metabolic capability to activate 3-ABA leading to DNA adduct formation, thereby enhancing the genotoxic potential of 3-NBA. Human P450 1A1 seems to be induced by planar aromatic compounds binding to the Ah receptor, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (55), and by polycyclic hydrocarbons present in cigarette smoke (53, 57). Similarly to other nitro-PAHs, such as 1-nitropyrene (61) and 6-nitrochrys-


ene (62), 3-NBA is able to induce P450 1A1 enzyme activity (Stiborova, unpublished data). Hence, long-term exposure to cigarette smoke and/or occupational exposure to 3-NBA might be an important risk factor for human individuals, increasing 3-ABA activation and its binding to DNA, thereby enhancing the genotoxic potential of 3-NBA. Even though we did not observe that P450 2A6, 1B1, and 2B6 in human hepatic microsomes are enzymes activating 3-ABA in human hepatic microsomes, the finding that recombinant human enzymes are efficient in such activation may still be of significance. All of these P450 enzymes are present in lung (57, 58, 63) and may play an important role in extrahepatic bioactivation. For instance, P450 1B1 expression levels are induced in lungs of rats exposed to diesel exhaust or nitro-PAHs such as 1-nitropyrene (61, 64), and recombinant human P450 1B1 coexpressed with NADPH:P450 reductase in Salmonella typhimurium strongly activates 1-nitropyrene and its metabolite 1-aminopyrene (64). Comparison between experimental animals and human enzyme systems is essential for the extrapolation of animal mutagenicity and carcinogenicity data to assess human health risk, and consideration of species differences in catalytic activities of enzymes is important (36, 42, 43). Previously, we showed that rats are a suitable model to mimic exposure to 3-NBA (25, 27, 29). The results of our present study suggest that rats may predict human susceptibility to 3-ABA. We have shown that human and rat hepatic microsomes are capable of activating 3-ABA to the same species binding covalently to DNA. P450 1A1 and 1A2 were the principal enzymes responsible for the activation of 3-ABA in rat microsomes. This conclusion is supported by a strong increase in DNA adduct formation in rat hepatic microsomes induced with β-NF, an inducer for P450 1A1 and 1A2, and with Sudan I, a specific inducer for P450 1A1 enzyme activity. Using purified recombinant rat P450 1A1 reconstituted with NADPH:P450 reductase confirmed the role of P450 1A1 in the bioactivation of 3-ABA. 3-ABA also forms the same DNA adducts in vivo in rats (27) as those observed after bioactivation with human and rat hepatic microsomes. Although the potential of 3-ABA to induce mutations in vivo and/or in vitro has not yet been examined, our data show that exposure to 3-NBA and 3-ABA leads to the same DNA adducts, in different in vitro systems, in cell culture and in vivo in rats (27-29). Preliminary data indicate that 3-NBA is carcinogenic in F344 rats after intratracheal administration of 3-NBA (11). In preliminary findings on moribund rats, the authors describe squamous metaplasia from trachea to brochial epithelium. In lung parenchyma, advanced squamous metaplasia from brochiole to the aveolar region was observed. As discussed recently (29), we suggest that 3-NBAderived DNA adduct formation is critical to the mechanism of 3-NBA carcinogenicity and that some or all of the DNA adducts (adducts 1-7) detected in the present study, either after treatment with 3-NBA or 3-ABA, represent premutagenic lesions involved in the mutagenic process triggering tumor development. Recent results suggest that G:C to T:A transversion mutations induced by 3-NBA in the liver cII gene of MutaMouse after intraperitoneal treatment with 3-NBA are caused by misreplication of adducted guanine residues in liver DNA through incorporation of adenine opposite the adduct (“A”-rule) (65). Although nothing is reported yet regarding the mutagenic and carcinogenic potential of 3-ABA,


3-NBA-induced DNA adduct formation is manifoldly higher as compared to 3-ABA, either in vivo in rats or in vitro (e.g., MCL-5 cells). This higher capability of 3-NBA to induce DNA adducts is predicted to be reflected in a much higher mutagenic and carcinogenic potency of 3-NBA as compared to 3-ABA. Collectively, 3-ABA is not only a suitable biomarker of exposure to 3-NBA but may also directly contribute to the high genotoxic potential of 3-NBA. Our results show the analogy in DNA adduct formation by 3-NBA and 3-ABA after metabolic activation with human and rat enzyme systems, strongly suggesting a carcinogenic potential for both compounds in humans. P450 1A1 and 1A2 expression in human respiratory tract could contribute significantly and specifically to the metabolic activation of 3-ABA and could be important determinants of a possible lung cancer risk from 3-NBA exposure.

Acknowledgment. This work was supported by Cancer Research UK, Ministry of Education of the Czech Republic (Grant MSM 1131 00001), and Baden-Wu¨rttemberg (BWPLUS, BWB 20003). Supporting Information Available: Levels of individual DNA adducts in human MCL-5 cells after exposure to 3-NBA and 3-ABA. Levels of individual DNA adducts formed by 3-ABA in human hepatic microsomes. Correlation coefficients (r) among P450-linked activities and total levels of 3-ABA-derived DNA adducts in human hepatic microsomes. HPLC cochromatographic analysis of 3-ABA-derived DNA adducts after activation in rats treated with 3-ABA and DNA treated with 3-ABA after activation by human hepatic microsomes (HG56). Levels of individual DNA adducts formed by 3-ABA in rat hepatic microsomes. The effect of P450 1A1 and P450 1A2 inhibitor, R-NF, and P450 1A2 inhibitor, furafylline, on individual DNA adduct levels formed by 3-ABA in human hepatic microsomes. Levels of individual DNA adducts formed by 3-ABA activated with purified rat recombinant P450 1A1. Levels of individual DNA adducts in parental and recombinant V79MZ-derived cells after exposure to 3-ABA. Levels of DNA adducts formed by 3-ABA using P450-mediated metabolic activation with Supersomes. This material is available free of charge via the Internet at http://pubs.acs.org.

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