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[Cancer Research 63, 2752-2761, June 1, 2003]
© 2003 American Association for Cancer Research

Carcinogenesis

Human Enzymes Involved in the Metabolic Activation of the Environmental Contaminant 3-Nitrobenzanthrone: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase1 ,2

Volker M. Arlt3, Marie Stiborova, Alan Hewer, Heinz H. Schmeiser and David H. Phillips

Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, Surrey SM2 5NG, United Kingdom [V. M. A., A. H., D. H. P.]; Department of Biochemistry, Faculty of Science, Charles University, 128 40 Prague 2, The Czech Republic [M. S.]; and Division of Molecular Toxicology, German Cancer Research Center, D-69120 Heidelberg, Germany [H. H. S.].


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determining the capability of humans to metabolize the suspected carcinogen 3-nitrobenzanthrone (3-NBA) and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility to this environmental contaminant found in diesel exhaust and ambient air pollution. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-NBA. Using two enrichment procedures of the 32P-postlabeling method, nuclease P1 digestion and butanol extraction, we found that all hepatic microsomes were competent to activate 3-NBA. DNA adduct patterns with multiple adducts, qualitatively similar to those found recently in vivo in rats, were observed. Additionally one major DNA adduct generated by human microsomes was detected. The role of specific cytochromes P450 (P450) and NADPH:P450 reductase in the human hepatic microsomal samples in 3-NBA activation was investigated by correlating the P450- and NADPH:P450 reductase-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-NBA was attributed to NADPH:P450 reductase. Inhibition of DNA adduct formation in human liver microsomes by {alpha}-lipoic acid, an inhibitor of NADPH:P450 reductase, supported this finding. Using the purified rabbit enzyme and recombinant human NADPH:P450 reductase expressed in Chinese hamster V79 cells, we confirmed the participation of this enzyme in the formation of 3-NBA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells. The role of individual human recombinant P450s 1A1, 1A2, 1B1, 2A6, 2B6, 2D6, 2C9, 2E1, and 3A4 and of NADPH:P450 reductase in the metabolic activation of 3-NBA, catalyzing DNA adduct formation, was also examined using microsomes of baculovirus-transfected insect cells containing the recombinant enzymes (Supersomes). DNA adducts were observed in all Supersomes preparations, essentially similar to those found with human hepatic microsomes and in human cells. Of all of the recombinant human P450s, P450 2B6 and -2D6 were the most efficient to activate 3-NBA, followed by P450 1A1 and -1A2. These results demonstrate for the first time the potential of human NADPH:P450 reductase and recombinant P450s to contribute to the metabolic activation of 3-NBA by nitroreduction.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Environmental factors and individual genetic susceptibility play an important role in many human cancers (1) . nitro-PAHs4 are widely distributed environmental pollutants found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter (2) . The increased lung cancer risk after exposure to these environmental sources (3 , 4) and their detection in tissues of lung cancer patients (5) has led to considerable interest in assessing their potential cancer risk to humans.

Recently a new member of this group of compounds, 3-NBA (3-nitro-7H-benz[de]anthracen-7-one; Fig. 1Citation ), was discovered in diesel exhaust and bound to the surface of airborne particulate matter (6) . 3-NBA was shown to be one of the most potent mutagens in the Ames Salmonella typhimurium assay reported thus far, scoring numbers of revertants comparable with 1,8-dinitropyrene in strain TA98 and YG1024 (6) . Preliminary data also suggest that 3-NBA is carcinogenic in rats (7) . Furthermore, 3-NBA induces micronuclei in mouse and human cells as well as mutations in human cells (6 , 8) . The genotoxicity of this suspected carcinogen was further documented by the detection of specific DNA adducts in vitro and in vivo in rats (9, 10, 11, 12) . Human exposure to 3-NBA has been demonstrated by the fact that 3-ABA, a major metabolite of 3-NBA, was recently found in urine samples of salt mining workers occupationally exposed to diesel exhaust (13) .



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  		Fig. 1. Structure of 3-NBA (3-nitro-7H-benz[de]anthracene-7-one).

 
nitro-PAHs require metabolism to reactive electrophilic species to exert their genotoxic activity. The activation of nitroaromatic hydrocarbons to reactive N-hydroxy arylamine intermediates is through nitroreduction catalyzed primarily by cytosolic reductases, such as xanthine oxidase, DT-diaphorase, and aldehyde oxidase, whereas P450 enzymes are primarily responsible for the oxidative metabolism of these compounds (14) . It was shown that reduction of 3-NBA to 3-ABA in rat lung alveolar type II cells is mediated, at least in part, by xanthine oxidase (10) , and xanthine oxidase-mediated DNA adduct formation occurs in vitro (9 , 12) . The major DNA adducts of 3-NBA formed in vitro after activation with xanthine oxidase are products derived from reductive metabolites bound to purine bases (12) . Although the structures of the DNA adducts remain to be characterized, we have shown that the nitroreduction pathway is responsible for the formation of these adducts in various tissues of Sprague-Dawley rats treated p.o. with 3-NBA (12) . N-Hydroxy arylamine intermediates formed by nitroreduction can be further metabolized by phase II enzymes, such as NATs or SULTs, leading to the formation of reactive esters capable of reacting with DNA to form DNA adducts (15 , 16) . Recently we showed that O-acetylation by human NAT1 and NAT2 as well as O-sulfonation by human SULT1A1 and SULT1A2 strongly contribute to the high genotoxic and mutagenic potential of 3-NBA (17 , 18) .

Determining the capability of humans to metabolize 3-NBA and understanding which human enzymes are involved in 3-NBA activation are important in the assessment of individual susceptibility. Many genes of enzymes that metabolize carcinogens are known to exist in variant forms resulting in differing activities of the gene products. These genetic variations appear to be important determinants of cancer risk (1 , 19) . Whereas the involvement of human microsomal enzymes such as NADPH:P450 reductase and P450 enzymes in the reductive activation of several nitroaromatic hydrocarbons was demonstrated (20 , 21) , their participation in the reductive activation of 3-NBA leading to the formation of covalent DNA adducts is not known.

Therefore, the present study was undertaken to determine the capability of humans to activate 3-NBA and to identify whether human microsomal enzymes are involved in DNA adduct formation by 3-NBA.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Synthesis of 3-NBA.
3-NBA was synthesized as described recently (17) . The authenticity of 3-NBA was confirmed by UV, electrospray mass spectra, and high-field proton NMR spectroscopy.

Cell Culture of MCL-5 Cells and Treatment with 3-NBA.
Human lymphoblastoid MCL-5 cells (22) were obtained under license from Gentest Corp. (Woburn, MA). MCL-5 cells were cultivated as described previously (23) . For treatment, aliquots (10 ml) of suspensions of MCL-5 cells (~8.0 x 105 cells/ml) were incubated at 37°C for 24 h with 0.1, 1, or 10 µM 3-NBA (dissolved in 16.6 µl DMSO). Controls were treated with DMSO only. Cell viability was determined by the trypan blue exclusion assay as described recently (17) . DNA from cells was isolated by the phenol extraction method as described previously (24) .

Preparation of Microsomes and Assays.
Microsomes were isolated from the livers of 10 male Wistar rats, each either untreated or pretreated with ß-NF (Sigma) inducing P450 1A1/2 as described previously (25) , those pretreated with PB-inducing P450 2B1/2 and NADPH:P450 reductase as reported by Hodek et al. (26) , and those pretreated with PCN inducing P450 3A1/2 and NADPH:P450 reductase as reported by Gut et al. (27) . Microsomes from livers of eight human donors who died after traffic accidents were isolated as described (28) and were a gift of Dr. B. Szotakova (Faculty of Pharmacy, Charles University, Hradec Kralove, Czech Republic). The donors ranged in age from 24 to 70 years and included five men and three women (samples no. 2, 5 and 8). None of the donors had any known drug history. None of the donors had a history of alcohol abuse. For certain incubations, commercially available pooled human microsomes from Gentest Corp. (cat. no. H161) were used. Supersomes, microsomes isolated from insect cells transfected with baculovirus constructs containing cDNA of one of the following P450s, P450 1A1, -1A2, -1B1, -2A6, -2B6, -2D6, -2C9, -2E1, or -3A4, and expressing NADPH:P450 reductase were also obtained from Gentest Corp. Protein concentrations in the microsomal fractions were assessed using the bicinchoninic acid protein assay (Pierce Rockford, IL, USA) with serum albumin as a standard (29) . The concentration of P450 was estimated according to Omura and Sato (30) . The content of P450 in rat and human hepatic microsomes is shown in Table 3Citation . Each human microsomal sample was analyzed for specific P450 activities by monitoring the following reactions: ethoxyresorufin O-deethylation (P450 1A1/2), coumarin 7-hydroxylation (P450 2A6), benzyloxyresorufin O-debenzylation (P450 2B6), bufuralol 1'-hydroxylation (P450 2D6), tolbutamide methyl hydroxylation (P450 2C9), chlorzoxazone 6-hydroxylation (P450 2E1), and testosterone 6ß-hydroxylation (P450 3A; (31) ; and references therein). These activities are shown in Table 3Citation . P450 2B6 activities are not shown in the table, because only three microsomal samples (samples no. 4, 5, and 7) exhibited this activity. The activity of NADPH:P450 reductase in rat and human hepatic microsomes was measured according to Sottocasa et al. (32) using cytochrome c as substrate (i.e., as NADPH:cytochrome c reductase). The concentration of NADPH:P450 reductase was estimated as described earlier (33) .


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  		Table 3 P450- and NADPH:P450 reductase-dependent catalytic activities and DNA adduct formation by 3-NBA in human liver microsomes

 
Microsomal Incubations.
The deaerated and argon-purged incubation mixtures, in a final volume of 750 µl, consisted of 50 mM potassium phosphate buffer (pH 7.4), 1 mM NADPH (Sigma), 0.1–1 mg of microsomal protein, 0.3 mM 3-NBA (dissolved in 12.5 µl of DMSO), and 0.5 mg of calf thymus DNA (4 mM; Sigma). The reaction was initiated by adding 3-NBA. Incubations with rat and human microsomes were carried out at 37°C for 2 h. Control incubations were carried out in one of the three following ways: (a) without activating system (microsomes); or (b) with activating system and 3-NBA, but without DNA; or (c) with activating system and DNA but without 3-NBA. To test the effect of enzyme cofactors (e.g., NADH, NADPH) on microsomal activation of 3-NBA, pooled human microsomes (1 mg of protein) from Gentest Corp. were used. Incubation mixtures, in which microsomes containing human recombinant P450s and NADPH:P450 reductase (Supersomes) were used to activate 3-NBA, were of the same composition except that 10 or 25 pmol of P450s were added instead of hepatic microsomes. Supersomes containing human recombinant NADPH:P450 reductase alone were used for comparison. 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 previously (21) .

Enzyme Preparations and Inhibition Studies.
Rabbit liver NADPH:P450 reductase was purified as described previously (34) . In incubations testing the activity, 6.5–250 pmol of pure NADPH:P450 reductase were added to the incubation mixture. In control incubations, NADPH:P450 reductase was omitted from the mixtures. The effect of {alpha}-lipoic acid (dissolved in 7.5 µl of methanol; Sigma), a selective inhibitor of NADPH:P450 reductase (35) , was tested using 1.6–5.0 mM {alpha}-lipoic acid. The effect of sulfaphenazole (dissolved in 7.5 µl of methanol; Sigma), a selective inhibitor of P450 2C9 (36) , was tested using 100 µM sulfaphenazole as reported previously (37 , 38) . An equal volume of methanol alone was added to the control incubations. The mixtures were then incubated at 37°C for 10 min with NADPH before adding 3-NBA. The reaction mixtures were further incubated at 37°C for 60 min. After incubation, DNA was isolated as described above.

Cell Culture of V79 Cells and Treatment with 3-NBA.
The parental V79 Chinese hamster lung fibroblast subclone V79MZ (39) and the recombinant V79 cells V79MZ-hOR expressing human NADPH:P450 reductase (40) were kindly provided by Prof. H. R. Glatt (German Institute of Human Nutrition, Potsdam, Germany) and Prof. J. Doehmer (GenPharmTox BioTech AG, Martinsried, Germany), respectively. All of the V79 cells were cultivated and treated with 3-NBA as described recently (17 , 18) . Cell viability was determined by the trypan blue exclusion assay as described above. DNA from cells was isolated as described above.

Preparation of Reference Compounds.
Wistar rats were treated with a single dose of 3-NBA (2 mg/kg body weight; i.p.) and analyzed as described recently (41) . dA and dG 3'-monophosphates (4 µmol/ml; Sigma) were incubated with 3-NBA (0.3 mM) either enzymatically activated by xanthine oxidase (1 units/ml; Sigma) in 50 mM potassium phosphate buffer (pH 7.0) in the presence of 1 mM hypoxanthine (Sigma) as described previously (12) or chemically activated by zinc dust (20 mg) in 50 mM potassium phosphate buffer (pH 5.8) as described previously (9) . Aliquots of the incubation were used directly for the butanol extraction-mediated 32P-postlabeling procedure.

32P-Postlabeling Analysis and HPLC Analysis of 32P-Labeled 3',5'-Deoxyribonucleoside Bisphosphate Adducts.
32P-postlabeling analysis using nuclease P1 digestion and butanol extraction, and TLC and HPLC were performed as described recently (17 , 41) .

Statistical Analysis.
Statistical associations between total P450 levels and P450- and NADPH:P450 reductase-linked catalytic activities in human hepatic microsomal samples and levels of total 3-NBA-DNA adducts formed by the same microsomes were determined by the linear regression and Spearman correlation coefficients using Statistical Analysis System software, version 6.12. Both types of correlation coefficients were based on a sample size of eight. All of the Ps are two-tailed and considered significant at the 0.05 level. DNA adduct levels in Supersomes incubations were compared by t test analysis and considered significant at the 0.01 level.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Comparison of DNA Adduct Formation of 3-NBA in Human Lymphoblastoid MCL-5 Cells and In Vivo in Rats.
In MCL-5 cells, when 32P-postlabeling with butanol enhancement was used, the DNA adduct pattern induced by 3-NBA on TLC consisted of a cluster of four major adducts (spots 1, 2, 3, and 4; Fig. 2BCitation ). Enhancement by nuclease P1 digestion resulted in a cluster of three major adducts (spots 1, 2, and 3) and one minor adduct (spot 6; Fig. 2BCitation ). Similarly the major DNA adducts (adducts 1, 2, 3, and 4) were detected recently in vivo in rats treated with 3-NBA (compare Fig. 2ACitation ; Ref. 41 ). In vivo in rats, one additional adduct (spot 5) was detected (41) . No DNA adducts were observed in DNA isolated from cells or rat tissue treated with vehicle only (data not shown). DNA adduct formation was concentration-dependent (Table 1)Citation , however, no DNA adducts were detected at the lowest 3-NBA concentration (0.1 µM) tested. Using the same approach as reported by us recently (12) , we found that all of the major 3-NBA-DNA adducts detected in human MCL-5 cells are formed by nitroreduction derived from reaction with either dA (adduct spot 1 and 2) or dG (adduct spot 3 and 4; Fig. 3BCitation ). Although adduct spots 5 (found in vivo in rats) and 6 were not detected as distinct peaks on HPLC after elution of the TLC origin after D1 only (Fig. 3, A and B)Citation , cochromatographic analysis on HPLC showed that adduct spot 5 formed by 3-NBA in vivo in rats eluted with the same retention time as adduct spot 5 observed in incubations of 3-NBA with dG activated by xanthine oxidase (data not shown).



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  		Fig. 2. Autoradiographic profiles of 3-NBA-DNA adducts by using the nuclease P1 (upper panels) or butanol (lower panels) enrichment version of the 32P-postlabeling assay. Aa and Ab, adduct profiles obtained from lung DNA of rats treated with 2 mg of 3-NBA per kg body weight [these profiles are representative of adduct profiles obtained with DNA from other rat tissues including liver, kidney, spleen, heart, and colon (41) ]. Ba and Bb, adduct profiles obtained in human MCL-5 cells treated with 10 µM 3-NBA. Ca and Cb, adduct profiles obtained from calf thymus DNA treated with 3-NBA (300 µM) after activation by uninduced hepatic microsomes from rat. Da and Db, adduct profiles obtained from calf thymus DNA treated with 3-NBA (300 µM) after activation by human hepatic microsomes (sample no. H1). Ea and Eb, adduct profiles obtained from calf thymus DNA treated with 3-NBA (300 µM) after activation with 100 pmol purified rabbit NADPH:P450 reductase. Fa and Fb, adduct profiles in V79MZ-hOR cells expressing human recombinant NADPH:P450 reductase after exposure to 0.1 µM 3-NBA. Ga and Gb, adduct profiles from calf thymus DNA treated with 3-NBA (300 µM) after activation with supersomes containing NADPH:P450 reductase alone (control). Ha and Hb, adduct profiles from calf thymus DNA treated with 3-NBA (300 µM) after activation with Supersomes containing 25 pmol human P450 1A2 and NADPH:P450 reductase.

 

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  		Table 1 DNA adduct formation by 3-NBA in human MCL-5 cells

Results are presented as mean ± SD of triplicate separate cell incubations; each DNA sample was determined by two independent postlabeled analyses.

 


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  		Fig. 3. Separation of 32P-labeled nucleoside 3',5'-bisphosphate adducts derived from 3-NBA on a phenyl-modified reversed-phase column (41) by using the nuclease P1 (right panels) or butanol (left panels) enrichment of the 32P-postlabeling assay. The origins of TLC after D1 only were excised and extracted from the plates, dissolved and injected on HPLC. Aa and Ab, origins were from DNA digests of lung DNA from 3-NBA-treated rats [these profiles are representative of adduct profiles obtained with DNA from other rat tissues including liver, kidney, spleen, heart and colon (41) ]. Ba and Bb, origins were from DNA digests of human MCL-5 cells treated with 3-NBA. Ca and Cb, origins were from DNA digests of calf thymus DNA treated with 3-NBA after activation with human hepatic microsomes (sample no. H1).

 
Activation of 3-NBA by Human and Rat Hepatic Microsomes.
We determined the formation of DNA adducts by 3-NBA in calf thymus DNA in the presence of microsomes isolated from eight different human livers and from livers of rats, uninduced or pretreated with ß-NF, PB, and PCN. Hepatic microsomes from both rats (Fig. 2C)Citation and humans (Fig. 2D)Citation were capable of activating 3-NBA to form DNA adducts. 3-NBA, activated by both species, induced essentially the same major DNA adducts consisting of a cluster of four major adducts (spots 1, 2, 3, and 4) after enhancement by butanol extraction, and of a cluster of three major adducts (spots 1, 2, and 3) after enhancement by nuclease P1 digestion. Similarly, these adducts were observed in vivo in rats (Fig. 2A)Citation and in human MCL-5 cells (Fig. 2B)Citation . In contrast to incubations with rat liver microsomes, one additional adduct spot (spot 7) was detected in incubations with human microsomes (Fig. 2D)Citation . A similar adduct spot was observed previously in calf thymus DNA after activation of 3-NBA by reduction with zinc (9) . Control incubations carried out in parallel without microsomes, without DNA, or without 3-NBA were free of adduct spots in the region of interest even after prolonged exposure times (data not shown). To further characterize adduct spot 7, dA and dG were incubated with 3-NBA in the presence of zinc as the reductive agent according to a procedure described previously (9) . Two major adduct spots (spots 1 and 2) were detected in incubations with dA; one major (spot 3) and two minor adduct spots (assigned spots X1 and X2) were observed in incubations with dG (data not shown). These minor adduct spots (spots X1 and X2) seemed to be migrating similarly to adduct spot 7 detected in calf thymus DNA after activation of 3-NBA with zinc or human liver microsomes, but the low adduct levels prevented HPLC cochromatographic analysis.

Cochromatographic analysis by HPLC confirmed that adduct spots 1, 2, 3, and 4 that are formed with human microsomes are derived from 3-NBA by nitroreduction (Fig. 3C)Citation . Therefore, the microsomes tested in this study contain enzymatic systems capable of catalyzing the reductive activation of 3-NBA leading to the formation of these DNA adducts. NADPH:P450 reductase, NADH:cytochrome b5 reductase and P450s present in microsomes are candidates for the reductive activation of 3-NBA. To investigate these possibilities, the influence of various cofactors on DNA adduct formation by 3-NBA catalyzed by human microsomes were examined. As shown in Table 2Citation , the formation of DNA adducts by 3-NBA had a requirement for NADPH, a known cofactor of NADPH:P450 reductase and P450-dependent enzyme systems (42) . DNA adduct levels were only marginal when NADPH was omitted from the incubation mixture. DNA adduct formation by 3-NBA was also observed using NADH, a cofactor of the microsomal NADH:cytochrome b5 reductase. NADH was less efficient as a cofactor than was NADPH. These results suggest a minor, but detectable, role of NADH:cytochrome b5 reductase in 3-NBA activation, whereas NADPH:P450 reductase or P450 enzymes might be more important for this activation. Although adduct 7 is formed both by NADPH- and NADH-mediated reactions, minor participation of the NADH-mediated reaction in the formation of other adducts (adducts 1, 2, 3, and 4) was detected (data not shown).


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  		Table 2 Effect of enzyme cofactors on DNA adduct formation by 3-NBA in human liver microsomesa

Results are presented as mean ± SD of triplicate in vitro incubations, each DNA sample was determined by two independent postlabeled analyses.

 
To resolve which microsomal enzymes are mainly responsible for the activation of 3-NBA, different experimental approaches were used: (a) correlation of P450- and NADPH:P450 reductase-linked enzymes activities in human hepatic microsomes with DNA adduct formation by 3-NBA with human microsomes; (b) selective enzyme inhibition in human microsomes; (c) purified NADPH:P450 reductase; (d) genetically engineered V79 cells expressing human NADPH:P450 reductase; and (e) use of heterologous baculovirus expression systems of human P450s and NADPH:P450 reductase (Supersomes).

Correlation of P450- and NADPH:450 Reductase-linked Enzyme Activities in Human Microsomes with DNA Adduct Formation by 3-NBA.
Catalytic activities known to be associated with specific P450 enzymes (P450 1A1/2, -2A6, -2C9, -2D6, -2E1, and -3A4) were analyzed in all eight of the human hepatic microsomal preparations (Table 3)Citation . P450 2B6 activities were also analyzed but not included in the correlation analysis, because only three microsomal samples exhibited this activity. NADPH:P450 reductase enzyme activities were also determined. Large individual variations in catalytic activities were evident among these different hepatic microsomal samples (Table 3)Citation . Quantitative 32P-postlabeling analysis, as shown in Table 3Citation , also showed wide individual variations in DNA binding by 3-NBA in the human microsomal incubations, ranging from 1.9 to 31.8 and from 6.7 to 53.8 adducts per 108 nucleotides after nuclease P1 and butanol enrichment, respectively. Total DNA binding by 3-NBA was highly significantly correlated with activities of NADPH:P450 reductase (Table 4)Citation . A significant correlation was also found between DNA adduct formation by 3-NBA and the enzyme activities of P450 2C9 (Table 4)Citation . However, there was also a cross-correlation between NADPH:P450 reductase and P450 2C9 in these human microsomal samples (r = 0.942, P < 0.001). To further clarify this correlation, multivariate analysis was used to investigate the dependence of the 3-NBA activation on the above two enzyme activities. Each of the two activities (NADPH:P450 reductase and P450 2C9) were combined in pairs to see if a combination of two activities gave an improvement in the correlation with levels of 3-NBA-DNA adducts, i.e., an increase in the correlation coefficient when compared with the correlation with the individual activities. The inclusion of the P450 2C9 activity produced no improvement in the correlation coefficient (data not shown). No significant correlation was determined between any other examined P450 activities (P450 1A1/2, -2A6, -2D6, -2E1, and -3A4) and the formation of DNA adducts by 3-NBA.


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  		Table 4 Linear regression correlation coefficients (r1) and Spearman correlation coefficients (r2) among P450- and NADPH:P450 reductase-linked activities and total levels of 3-NBA-DNA adducts formed in human liver microsomes

 
In the species comparison, microsomes from human and rat exhibited comparable efficiencies to activate 3-NBA. Similarly to human hepatic microsomes, the efficiency of rat hepatic microsomes to activate 3-NBA correlated with the NADPH:P450 reductase activity (Table 3)Citation . Incubations of 3-NBA and DNA with hepatic microsomes of rats pretreated with inducers of NADPH:P450 reductase, PCN and PB, led to a 4- and 3-fold increase, respectively, in the formation of 3-NBA-DNA adducts (Table 3)Citation .

Effect of Inhibitors of NADPH:P450 Reductase and P450 2C9 on Activation of 3-NBA in Human Microsomes.
Inhibition experiments further supported the role of NADPH:P450 reductase in the activation of 3-NBA in human hepatic microsomes. {alpha}-Lipoic acid, a selective inhibitor of NADPH:P450 reductase (35) , was effective in inhibiting DNA adduct formation by 3-NBA when a 15-fold molar excess of this inhibitor over 3-NBA was used (Table 5)Citation . To further investigate the role of the human hepatic P450 2C9 enzyme in 3-NBA activation, one human microsomal sample (sample no. 3) with high P450 2C9 activity was selected, and incubations were carried out in the absence and presence of a specific inhibitor of P450 2C9, sulfaphenazole. No inhibition of DNA binding was observed (Table 5)Citation , indicating that P450 2C9 is not involved in the activation of 3-NBA in human hepatic microsomes.


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  		Table 5 The effect of NADPH:P450 reductase inhibitor, {alpha}-lipoic acid, and P450 2C9 inhibitor, sulfaphenazole, on DNA adduct formation by 3-NBA in human liver microsomesa

 
Activation of 3-NBA by Purified NADPH:P450 Reductase.
To confirm the role of NADPH:P450 reductase in the activation of 3-NBA, this enzyme was purified from rabbit livers and used in additional experiments. Fig. 2ECitation shows that incubations of 3-NBA with DNA and with purified NADPH:P450 reductase together with its cofactor, NADPH, resulted in the formation of the same DNA adduct pattern as with other activating systems and in vivo in rats. Adduct spot 7 detected in incubations with human hepatic microsomes was not detected in incubations with purified NADPH:P450 reductase. NADPH:P450 reductase-mediated DNA adduct formation was concentration-dependent (Fig. 4)Citation .



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  		Fig. 4. DNA binding of 3-NBA in calf thymus DNA after activation with purified rabbit NADPH:P450 reductase. Values represent mean ± SD of four determinations (duplicate analyses of two independent in vitro incubations). RAL, relative adduct labeling.

 
DNA Adduct Formation of 3-NBA in V79 Cells Expressing Recombinant Human NADPH:P450 Reductase.
Chinese hamster lung V79 cells completely lack P450-dependent enzyme activities (39) , but contain detectable amounts of native NADPH:P450 reductase. To further investigate the involvement of human NADPH:P450 reductase in the metabolic activation of 3-NBA, we used a V79 cell line (V79MZ-hOR) expressing human recombinant NADPH:P450 reductase (40) . Parental (V79MZ) and transfected V79 cells were treated with 3-NBA. To compare DNA adduct formation, DNA binding for 3-NBA was determined in both V79 cells after treatment with 0.01 and 0.1 µM 3-NBA. As shown in Fig. 2FCitation , the DNA adduct pattern induced by 3-NBA was essentially similar to those observed in vivo in rats (Fig. 2A)Citation , in human MCL-5 cells (Fig. 2B)Citation , and by using human hepatic microsomes (Fig. 2D)Citation . In contrast, adduct spot 7 detected in incubations with human microsomes was not detectable in V79 cells expressing human NADPH:P450 reductase. No DNA adducts were observed in DNA isolated from cells treated with vehicle (DMSO) only (data not shown). DNA binding was concentration dependent, yielding remarkably high levels of up to 213 and 278 adducts in 108 nucleotides after nuclease P1 digestion and butanol extraction, respectively (Fig. 5B)Citation . Compared with the parental cells, V79MZ, total DNA binding by 3-NBA was significantly higher in V79 cells expressing human NADPH:P450 reductase (12–34-fold and 45–78-fold after treatment with 0.01 µM and 0.1 µM 3-NBA, respectively; P < 0.01 in both cases), demonstrating that human NADPH:P450 reductase strongly contributes to the metabolic activation of 3-NBA to form DNA adducts. The formation of 3-NBA-DNA adducts correlated with its cytotoxicity in V79 cells (Fig. 5A)Citation .



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  		Fig. 5. A, cell viability in parental V79MZ and V79MZ-hOR cells expressing human recombinant NADPH:P450 reductase after treatment with 3-NBA. B, DNA adduct formation in V79MZ and V79MZ-hOR cells. Values represent mean ± SD of three separate cell incubations; each DNA sample was determined by two independent postlabeled analyses. Comparison was performed by t test analysis: *, P < 0.01 by comparison with parental V79MZ. RAL, relative adduct labeling.

 
Activation of 3-NBA by Recombinant Human P450s and NADPH:P450 Reductase 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, even though the 3-NBA-DNA adduct formation did not correlate with activities of P450s in human hepatic microsomes, we tested in more detail whether individual P450 enzymes might activate 3-NBA. For such studies, we used human recombinant P450s in microsomes of baculovirus-transfected insect cells (Supersomes) containing recombinantly expressed human P450s (-1A1, -1A2, -1B1, -2A6, -2B6, -2D6, -2C9, -2E1, and -3A4) and/or human NADPH:P450 reductase.

DNA adduct formation was observed in all Supersomes incubations (Fig. 2, G and H)Citation . 3-NBA induced practically the same DNA adduct pattern as those obtained in vivo in rats (Fig. 2A)Citation , in human MCL-5 cells (Fig. 2B)Citation , and by using human liver microsomes (Fig. 2D)Citation . However, adduct spot 7, which was detected in incubations with human microsomes, was not detected. We compared total DNA binding in Supersomes incubations that contained the respective human P450 enzyme and human NADPH:P450 reductase with DNA binding in Supersomes incubations that contained human NADPH:450 reductase alone (controls; Fig. 6Citation ). Using 25 pmol of human P450s, we found that P450 2B6 and -2D6 were the most active in 3-NBA activation, followed by P450 1A1 and -1A2 (P < 0.01; Fig. 6Citation ). Similar results were obtained using 10 pmol of human P450s (data not shown). In incubations using 25 pmol of P450 enzyme, partially increased DNA adduct levels by 3-NBA were also determined by P450 2C9 and -2E1, whereas a decrease in DNA binding was observed by P450 1B1 (P < 0.01; Fig. 6Citation ).



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  		Fig. 6. DNA binding of 3-NBA after activation with Supersomes containing different human recombinant P450s (25 pmol) and NADPH:P450 reductase or NADPH:P450 reductase alone (control). The nuclease P1 (A) and the butanol enrichment (B) versions of the 32P-postlabeling assay were used. Values represent mean ± SD of three separate incubations each determined by two independent postlabeling analyses. F = fold increase in DNA binding by P450-mediated activation. Comparison was performed by t test analysis: *, P < 0.01 by comparison with NADPH:P450 reductase (control) incubations. a RAL, relative adduct labeling.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Some limited evidence suggests that diesel exhaust and airborne particulates, which contain significant amounts of nitro-PAHs, may be carcinogenic in humans (2 , 4) . 3-NBA is a very potent direct acting mutagen and suspected carcinogen that has been identified recently in these environmental sources (6 , 7 , 13) . 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 ambient air pollution and diesel exhaust (43 , 44) . This may be predictive of cancer risk. Determining the capability of humans to activate 3-NBA and form DNA adducts and understanding which human enzymes are involved in its metabolic activation are important in the assessment of individual susceptibility to this environmental contaminant. The present article reports on the identification of microsomal enzymes participating in the bioactivation of 3-NBA.

Previously, we demonstrated the formation of specific 3-NBA-DNA adducts derived from nitroreduction in vitro and in vivo in rats (9 , 12 , 41) . Using the 32P-postlabeling assay, we observed an essentially similar adduct pattern in human lymphoblastoid MCL-5 cells. Therefore, MCL-5 cells contain enzyme systems capable of catalyzing the reductive activation of 3-NBA leading to DNA adducts. Besides cytosolic nitroreductases, MCL-5 cells contain native NADPH:P450 reductase and express high levels of native P450 1A1 and human P450 1A2, -2A6, -2E1, and -3A4 (22) , all of which are enzymes that might participate in the reductive activation of 3-NBA. Comparative analyses of the major adducts obtained in cell culture with those detected in vitro and in vivo in rats revealed that these 3-NBA-DNA adducts were chromatographically indistinguishable. Previous work has shown that the four major adducts are products derived from reductive metabolites bound to dA (adducts 1 and 2) or dG (adducts 3 and 4; Ref. 12 ). Here, we show that minor adduct 5 is derived from dG. Moreover, because xanthine oxidase has not yet been shown to be capable of reducing keto-groups, we assume that all 3-NBA-DNA adducts are formed by simple nitroreduction. This is consistent with a previous observation reported by others (45) when investigating the xanthine oxidase-catalyzed nitroreduction of another aromatic nitroketone, 9-oxo-2-nitrofluorene; reduction of only the nitro group to the corresponding amine was observed. Further structural characterization of these 3-NBA-DNA adducts is currently being undertaken.

We found previously that rat liver S9 mix generates 3-NBA-DNA adduct profiles identical to the profiles found in rat tissues in vivo (9 , 12 , 41) . The results of the present study show that 3-NBA is activated also by rat liver microsomes, forming identical adducts to those obtained in human MCL-5 cells and also identical to those formed in vivo in rats (12 , 41) . Here, we clearly demonstrate that different human hepatic microsomes are capable of activating 3-NBA leading to a DNA adduct pattern that is essentially similar to that formed by rat liver microsomes. One additional major 3-NBA-DNA adduct (adduct 7) was detected in incubations with human hepatic microsomes. A chromatographically similar adduct was observed previously in DNA after activation of 3-NBA by reduction with zinc (9) . However, synthetic adduct standards prepared by reacting dG and dA with 3-NBA in the presence of zinc did not allow us to clearly characterize the nature of this adduct, and its identity requires further investigation. Formation of this adduct suggests that human hepatic microsomes contain additional potent reductase(s) that activate 3-NBA. Because NADH (besides NADPH) is another cofactor mediating the formation of adduct 7 efficiently, human NADH:cytochrome b5 reductase seems to be one such enzyme. The question as to why this hepatic enzyme in humans is more efficient in activating 3-NBA than the enzyme in rats remains to be resolved.

As shown in Table 4Citation , the formation of 3-NBA-DNA adducts was strongly dependent on the catalytic activities of NADPH:P450 reductase present in all eight of the human hepatic microsomal samples assayed. This conclusion was further supported by the inhibition of 3-NBA-DNA adduct formation with {alpha}-lipoic acid, a specific inhibitor of NADPH:P450 reductase, in one human hepatic microsomal sample that exhibited high activity of NADPH:P450 reductase. Utilization of recombinant human NADPH:P450 reductase expressed in microsomes of baculovirus-transfected insect cells (Supersomes) and of recombinant human NADPH:P450 reductase expressed in Chinese hamster lung V79 cells fully confirmed the major role of this enzyme in the bioactivation of 3-NBA. Moreover, purified rabbit NADPH:P450 reductase alone also catalyzed 3-NBA-DNA adduct formation. The identification of human NADPH:P450 reductase in the reductive activation of the nitroaromatic compound 3-NBA is consistent with results reported by Shimada and Guengerich (46) demonstrating the reductive metabolism of 1,8-dinitropyrene by NADPH:P450 reductase in human liver microsomes. NADPH:P450 reductase is expressed in human bronchial epithelial cells and alveolar macrophages, a primary defense system against inhaled material (47) . Therefore NADPH:P450 reductase expression in the human respiratory tract may contribute substantially to the metabolic activation of 3-NBA.

In comparison with NADPH:P450 reductase, none of the human P450 enzymes examined (P450 1A1/2, -2A6, -2C9, -2D6, -2E1, and -3A4) had a measurable impact on the capacity to activate 3-NBA to form DNA adducts in the human samples assayed. In contrast to this finding, we found that several human recombinant P450s present (overexpressed) in microsomes of baculovirus-transfected insect cells (Supersomes) were effective activators of this compound. Of the human recombinant P450s tested, P450 2B6 and -2D6 were the most efficient to activate 3-NBA, followed by P450 1A1 and -1A2. The involvement of human recombinant P450s in reductive activation of nitroaromatics like 3-NBA is consistent with previous reports by different groups including ours (20 , 21) . Chae et al. (20) showed that human recombinant P450 1A2, reconstituted with NADPH:P450 reductase, was able to reduce 4-nitropyrene and to a lesser extent 1- and 2-nitropyrene. Moreover, the reductive activation of the nitro-phenanthrene carboxylic acid aristolochic acid, a plant carcinogen, by P450 1A1 and -1A2 in human hepatic microsomal samples leading to DNA adduct formation, as well as by the pure recombinant enzymes in a reconstituted system and Supersomes, is consistent with our results (21) . Nevertheless, the discrepancy between analysis using human hepatic microsomes and analysis using human recombinant P450s present in Supersomes remains to be explained. The lack of measurable participation of P450 2B6 in 3-NBA activation may be attributable to its negligible activity in human hepatic microsomes used in the experiments, which, in turn, is attributable to its low expression in human livers [P450 2B6 represent ~0.2% of the total hepatic P450 complement (36) ]. Likewise, P450 2D6 expression in human liver is low [~ 2.5% of the total P450 content (36) ]. However, the negative results on participation of P450 1A2 enzyme that is expressed in human liver in larger quantities [more than 10% of the total hepatic P450 complement (36 , 38) ] are rather surprising. Although ethoxyresorufin O-deethylation activity is detectable in all of the hepatic microsomal samples used in our study, this activity is quite low in several of them [10 times lower than one would expect in the human liver (48) ]. Therefore, the negligible P450 1A2 impact on the capacity to activate 3-NBA compared with the capacity of NADPH:P450 reductase may be attributable mainly to this reason. Indeed, using Supersomes containing high levels of the P450 1A2 enzyme (overexpression of P450 1A2 in Supersomes) in comparison with human hepatic microsomes showed the efficiency of P450 1A2 in 3-NBA activation. Furthermore, we found recently the metabolic activation of 3-NBA by P450 1A2 in Chinese hamster V79 cells expressing the recombinant human enzyme (18) . Another reason for observed discrepancies might be the different activities of recombinant and authentic human P450 enzymes.

Even though we did not observe that P450s in human hepatic microsomes are enzymes activating 3-NBA, the finding that human recombinant P450 1A1/2, -2B6, and -2D6 are efficient in such activation may be of great significance. Human exposure to 3-NBA is thought to occur primarily via the respiratory tract. Although the total P450 content of the lungs is low compared with that in the liver, because of the high rate of blood circulation through the lungs and the possible exposure to 3-NBA through respiration, P450 enzymes present in lungs may play an important role in extrahepatic bioactivation. Human P450 1A1 is an extrahepatic enzyme expressed in the gastrointestinal and urinary tract, and in lungs on induction (49) . Moreover, human P450 2B6 is expressed in several extrahepatic tissues including lungs (50) . The results of several studies on CYP2D6 expression in human lung tissue have yielded contradictory results. Whereas some studies reported the expression of CYP2D6 in human lung (51) , others did not find CYP2D6 to be present at a detectable level in this tissue (50 , 52) . Recently, we found that 3-NBA is activated by human recombinant NAT1 and NAT2 as well as by SULT1A1 and SULT1A2 expressed in Chinese hamster lung V79 cells (17 , 18) , enzymes that are also expressed in human lung; this suggests that the expression of CYPs, NATs, and SULTs in the human respiratory tract could contribute significantly and specifically to the metabolic activation of 3-NBA. In these V79 cells, 3-NBA induced a dose-dependent increase in the mutation frequency at the hrpt locus5 and formed the same pattern of DNA adducts as that found in vivo in rats treated with 3-NBA (12 , 41) . These results support the conclusion that some or all of the major 3-NBA-DNA adducts (adducts 1, 2, 3, 4, and 5) detected in the present study represent premutagenic lesions involved in the mutagenic process, at least in V79 cells. Preliminary data indicate that 3-NBA is carcinogenic in F344 rats after intratracheal administration of 3-NBA (7) . Whereas 3-NBA has been shown to induce specific 3-NBA-DNA adducts in various tissues of Sprague Dawley (oral treatment) or Wistar (i.p. treatment) rats treated with 3-NBA (12 , 41) , its potential to induce DNA adducts in F344 rats has not yet been examined. Nevertheless, we suggest that 3-NBA-DNA adduct formation is critical to the mechanism of 3-NBA carcinogenicity in F344 rats, and experiments to investigate adduct formation in this strain after intratracheal administration of 3-NBA are planned.

Human P450 1A2 protein, which is constitutively expressed in liver (49) , may participate by another pathway in DNA adduct formation by 3-NBA also. It has been shown that this P450 oxidizes aromatic and heterocyclic amines to reactive intermediates binding to DNA via N-hydroxylation (53) . The aromatic amine 3-ABA is a final metabolite of 3-NBA reduction and was detected in the urine of salt mining workers exposed to 3-NBA at concentrations similar to that of 1-aminopyrene, a biomarker for exposure to 1-nitropyrene attributable to diesel emission (13) . Recent data show that human recombinant P450 1A2-mediated activation of 3-ABA leads to the same DNA adducts as formed by 3-NBA (18) . Collectively, these results may suggest that although human P450 1A2 could contribute to the metabolic activation of 3-NBA by nitroreduction only to a minor extent (if any), its participation in the N-hydroxylation of 3-ABA may be of major importance.

Levels of expression and activities of NADPH:P450 reductase and P450s (e.g., -1A1/2, -2B6, and -2D6) in humans are influenced by several factors (nutrition, smoking, drugs, environmental chemicals, and genetic polymorphisms) and differ considerably among individuals (1 , 36 , 54 , 55) . Another factor causing variability of the activities and levels of NADPH:P450 reductase is a variation in hormonal levels (56) . Consequently, the variability of expression and activities of NADPH:P450 reductase and P450s could be important determinants of a possible cancer risk with 3-NBA. However, the precise kinetics and activation of 3-NBA and its metabolites by phase I and phase II enzymes in human tissue await further investigation.

In summary, we present for the first time that human hepatic microsomes activate the strong mutagen and suspected carcinogen 3-NBA to species forming DNA adducts identical to those formed in vivo in rats. This is important for the estimation of the 3-NBA genotoxicity (carcinogenicity) for humans. The results of the present study strongly suggest a genotoxic potential of 3-NBA for humans. Because of its presence in diesel exhaust and ambient air pollution, exposure to 3-NBA may represent a health hazard for large sections of the population.


   ACKNOWLEDGMENTS
 
We thank Dr. C. A. Bieler (German Cancer Research Center) for continuous support, K. J. Cole (Institute of Cancer Research) for assistance with MCL-5 cell culture, and M. Sulc (Charles University) for rabbit liver NADPH:P450 reductase enzyme purification.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Cancer Research United Kingdom, Ministry of Education of the Czech Republic (Grant MSM 1131 00001), and Baden-Württemberg (BWPLUS, BWB 20003). Back

2 Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). Back

3 To whom requests for reprints should be addressed, at Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom. Phone: 44-208-722-4405; Fax: 44-208-722-4052; E-mail: v.arlt{at}icr.ac.uk Back

4 The abbreviations used are: 3-NBA, 3-nitrobenzanthrone; P450, cytochrome P450; nitro-PAH, nitropolycyclic aromatic hydrocarbon; 3-ABA, 3-aminobenzanthrone; NAT, N,O-acetyltransferase; SULT, sulfotransferase; ß-NF, ß-naphthoflavone; PB, phenobarbital; PCN, pregnenolone-16{alpha}-carbonitrile; TLC, thin-layer chromatography; dA, deoxyadenosine; dG, deoxyguanosine; HPLC, high-performance liquid chromatography. Back

5 H. R. Glatt, personal communication. Back

Received 11/ 1/02. Accepted 3/27/03.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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