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[Cancer Research 59, 1473-1480, April 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 1473-1480, April 1, 1999]
© 1999 American Association for Cancer Research

Carcinogenesis

Comparative Metabolism of 1-, 2-, and 4-Nitropyrene by Human Hepatic and Pulmonary Microsomes1

Young-Heum Chae, Tom Thomas, F. Peter Guengerich, Peter P. Fu and Karam El-Bayoumy2

Division of Cancer Etiology and Prevention, American Health Foundation, Valhalla, New York 10595 [Y-H. C., T. T., K. E-B.]; Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 [F. P. G.]; and Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 [P. P. F.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Determining the capability of humans to metabolize the mononitropyrene (mono-NP) isomers 1-, 2-, and 4-NP and understanding which human cytochrome P450 (P450) enzymes are involved in their activation and/or detoxification is important in the assessment of individual susceptibility to these environmental carcinogens. We compared the ability of 15 human hepatic and 8 pulmonary microsomal samples to metabolize each of the three isomers. Human hepatic microsomes were competent in metabolizing all three isomers. Qualitatively similar metabolic patterns were observed, although at much lower levels, upon incubating mono-NP with pulmonary microsomes. Ring-oxidized metabolites (phenols and trans-dihydrodiols) were produced from all three isomers. However, the nitroreductive metabolism leading to the formation of aminopyrene was evident only with 4-NP. The role of specific P450 enzymes in the human hepatic microsomal metabolism of mono-NP was investigated by correlating the P450-dependent catalytic activities in each microsomal sample with the levels of individual metabolites formed by the same microsomes and by examining the effects of agents that can either inhibit or stimulate specific P450 enzymes in mono-NP metabolism. On the basis of these studies, we attribute most of the hepatic microsomal metabolism of 1- and 4-NP to P450 3A4, although a minor role for P450 1A2 cannot be ruled out. Specifically, P450 3A4 was responsible for the formation of 3-hydroxy-1-nitropyrene from 1-NP and the formation of trans-9,10-dihydro-9,10-dihydroxy-4-nitropyrene, 9(10)-hydroxy-4-nitropyrene, and 4-aminopyrene from 4-NP. None of the P450 enzymes examined (P450s 3A4, 1A2, 2E1, 2A6, 2D6, and 2C9) appeared to be involved in catalyzing the formation of trans-4,5-dihydro-4,5-dihydroxy-2-nitropyrene and 6-hydroxy-2-nitropyrene from 2-NP in human hepatic microsomes. These results, the first report on the comparative metabolism of mono-NP in humans, clearly demonstrate that the role of specific human P450 enzymes in catalyzing oxidative and reductive pathways of mono-NP is dependent upon the position of the nitro group.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mono-NPs3 (Fig. 1)Citation have been found in various environmental matrices (1 , 2) . Among the three mono-NP isomers, 1-NP is the most prevalent. In urban air, the levels of 2- and 4-NP are comparable but much lower than that of 1-NP (3) . Both 1-NP and 4-NP have been detected in diesel engine exhaust, where the level of 1-NP is much higher than that of 4-NP (4) . Kinouchi et al. (5) reported detection of 1-NP in grilled chicken. The presence of 1-NP in lung specimens of lung cancer patients has been documented as a result of inhaling soot from the combustion of coal and heavy oil used for cooking and indoor heating (6) .



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  		Fig. 1. Structures of 1-NP, 2-NP, and 4-NP.

 
Many nitropolycyclic aromatic hydrocarbons are mutagenic in bacterial and mammalian systems and also carcinogenic in rodents (1 , 2) ; they are implicated to play a role in the etiology of some human cancers (7 , 8) . The position of the nitro group determines the mutagenic and carcinogenic activities of mono-NP. In bacterial systems, 2-NP is more mutagenic than 1-NP; in turn, the mutagenic activity of 4-NP exceeds that of 2-NP (9, 10, 11) . The same trends have been observed for transforming activities of 1-, 2-, and 4-NP in the BALB/3T3 mouse embryo cells (12) . However, a mutation assay using a human lymphoblastoid cell line (MCL-5), which contains P450 enzymes and microsomal epoxide hydrolase, revealed that 4-NP was almost three times as potent as 1-NP, whereas 2-NP was not mutagenic (13) . All three mono-NP isomers are tumorigenic in mouse lung and liver (14 , 15) . 1-NP induces tumors in the lungs and trachea of hamsters (16) . Both 1-NP and 4-NP have been shown to be mammary carcinogens in rats (17 , 18) . Among the three isomers, 4-NP exhibits the strongest carcinogenic activity in mouse lung and liver as well as in the mammary glands of rats (9 , 15 , 19 , 20) .

Metabolism studies in vivo in laboratory animals as well as in vitro, using rodent hepatic microsomes or 9000 x g supernatant, demonstrated that all three mono-NP isomers undergo both reductive and oxidative reactions (21, 22, 23, 24, 25, 26) . However, nitroreduction appears to be a principal pathway for the activation of mono-NP, leading to the formation of DNA adducts. The major DNA adduct in Salmonella typhimurium (27) , cells in culture (28 , 29) , and in some but not all rodents treated with 1-NP (30, 31, 32) has been unequivocally identified as N-(deoxyguanosin-8-yl)-1-aminopyrene. In the case of 2-NP, both deoxyguanosine and deoxyadenosine adducts derived from nitroreduction were formed in S. typhimurium (9) and upon incubation of 2-NP with DNA in the presence of rat hepatic microsomes (33) . 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 the rat mammary glands after treatment with 4-NP (22 , 34) . Moreover, a greater extent of DNA binding in the mammary glands was observed with 4-NP compared with 1- or 2-NP, which may, in part, account for the greater carcinogenicity of 4-NP.

Human metabolism of 1-NP has been described in several studies. Metabolites derived from nitroreduction (1-AP) as well as ring oxidation (trans-dihydrodiols and phenols) were detected in the human hepatoma cell line HepG2 treated with 1-NP (28 , 35 , 36) . In contrast, human hepatic microsomal incubations of 1-NP yielded only oxidized products; 1-AP was not formed (37) . In an effort to define the role of human P450 enzymes in the metabolism of 1-NP, Howard et al. (38) examined the ability of 12 human P450 enzymes to metabolize 1-NP using Vaccinia virus expression of P450 cDNAs in HepG2 cells. These investigators reported that P450 3A3 and P450 3A4 are the principal forms responsible for most of the oxidative metabolism. The involvement of the P450 3A subfamily in 1-NP metabolism was further confirmed in a study using human hepatic microsomes (37) . On the other hand, studies aimed at determining which human P450 enzymes are responsible for the metabolism of 2-NP and 4-NP are scarce. In examining the role of human P450 1B1 in the activation of a variety of environmental carcinogens, Shimada et al. (39) demonstrated that P450 1B1 is highly efficient in catalyzing the activation of 2-NP as determined by the umu gene response.

The present study was undertaken to determine the capability of humans to metabolize mono-NP and to understand which human cytochrome P450 enzymes are involved in their metabolic activation and/or detoxification. This knowledge will be useful in evaluating individual susceptibility to these environmental carcinogens.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Enzymes.
[4,5,9,10-3H]1-NP (specific activity, 5.8 Ci/mmol), [G-3H]2-NP (specific activity, 188 mCi/mmol), and [G-3H]4-NP (specific activity, 1.34 Ci/mmol) were purchased from Chemsyn Science Laboratories, Inc. (Lenexa, KS) and purified by HPLC prior to use (>98% radiochemically pure according to HPLC). 1-NP, {alpha}-naphthoflavone, 4-methylpyrazole, diethyldithiocarbamate, and quinidine were acquired from Aldrich Chemical Co. (Milwaukee, WI). 2-NP and 4-NP were synthesized as reported previously (22, 23, 24) . Ketoconazole and sulfaphenazole were kindly provided by Dr. J. Capdevila (Vanderbilt University, Nashville, TN) and Dr. T. Shimada (Osaka Prefectural Institute of Public Health, Osaka, Japan), respectively.

Preparation of Mono-NP Metabolite Standards.
Mono-NP metabolite standards other than trans-9,10-DHD-4-NP were synthesized as described earlier (22, 23, 24, 25, 26) . trans-9,10-DHD-4-NP was prepared by incubating 4-NP with rat liver 9000 x g supernatant. Briefly, 4-NP (10 mg) was incubated with liver 9000 x g supernatant (100 mg of protein) of rats treated with Aroclor 1254 (ICN Biomedicals, Costa Mesa, CA) at 37°C for 1 h and extracted with ethyl acetate. HPLC analysis of the ethyl acetate extract showed the formation of one major and a few minor metabolites. The major metabolite was identified as trans-9,10-DHD-4-NP, based on the following mass spectral data (obtained on a Hewlett-Packard Model HP5988A dual-source mass spectrometer using electron ionization; Hewlett-Packard Co., Palo Alto, CA) and 1H-NMR analysis (performed on a Bruker AM-360 WB spectrometer; USA Bruker Instruments, Inc., Billerica, MA): mass spectrum (relative intensity) m/z 281 (M+, 100), 263 (65), 246 (11), 234 (13); 1H-NMR (360 MHz, CDCl3) {delta} 8.58 (s, 1H, H5), 8.52 (d, 1H, H3, J2,3=9.57 Hz), 8.00–8.08 (m, 3H, H1,6,8), 7.76–7.85 (m, 2H, H2,7), 5.20 (s, 2H, H9,10). These spectral data are identical to those reported previously (24) .

Preparation of Microsomes.
Microsomes were prepared from 15 human liver and 8 human lung specimens obtained through Tennessee Donor Services (Nashville, TN) and the Cooperative Human Tissue Network, respectively. Protein concentrations were assessed using the bicinchoninic acid protein assay (BCA assay for protein quantification; Pierce Chemical Co., Rockford, IL). P450 concentrations in hepatic microsomes were estimated by Fe2+·CO versus Fe2+ difference spectroscopy (40) .

Metabolism of Mono-NP by Human Microsomes.
[3H]1-NP and [3H]4-NP were mixed with the corresponding unlabeled compounds to obtain a specific activity identical to that of [3H]2-NP (188 mCi/mmol). Each of the [3H]mono-NP isomers (7 µM) was incubated at 37°C in 1 ml of 50 mM potassium phosphate buffer (pH 7.4) containing 1 mg of microsomal protein, 3 mM MgCl2, and an NADPH-generating system consisting of 1 mM NADP+, 4 mM glucose 6-phosphate, and 1 unit of glucose 6-phosphate dehydrogenase; levels of substrates and microsomal protein used here were selected based on previous studies (Ref. 41 and references therein). The reaction was initiated by adding the substrate dissolved in 6 µl of DMSO and terminated by adding 1 ml of ice-cold methanol. Time course studies (15, 30, 45, and 60 min) with each isomer revealed that the hepatic microsomal metabolism of mono-NP was linear up to 30 min. Thus, an incubation time of 30 min was selected. Because P450 activities in the lung are much lower than those in the liver, the incubation of [3H]mono-NP with pulmonary microsomes (1 mg protein) was carried out as described above for hepatic metabolism but for 1 h. Control incubations were performed as described above but in the presence of boiled microsomes. The incubations with hepatic microsomes were performed at least in triplicate, unless stated otherwise; however, because of the small amounts of microsomal proteins available, the pulmonary metabolism assays were limited to two determinations per sample.

Inhibition Studies.
The following chemicals were used to inhibit the metabolism of mono-NP in human hepatic microsomes (specific P450 enzymes known to be inhibited): ketoconazole (P450 3A4), {alpha}-naphthoflavone (P450s 1A2 and 1A1), diethyldithiocarbamate (P450s 2A6 and 2E1), sulfaphenazole (P450 2C), 4-methylpyrazole (P450 2E1), and quinidine (P450 2D6). Inhibitors dissolved in 5 µl of DMSO, to yield final concentrations of 2–50 µM, depending on the chemical, were added to the incubation mixtures containing microsomes and mono-NP (42) . The mixtures were then preincubated at 37°C for 3 min prior to adding the NADPH-generating system. An equal volume of DMSO alone was added to the control incubations. In studies using a mechanism-based inactivator, diethyldithiocarbamate, the agent was preincubated at 37°C for 20 min with microsomes in the presence of an NADPH-generating system, after which the reaction was initiated by adding mono-NP isomers. The reaction mixtures were further incubated at 37°C for 30 min.

Metabolism of Mono-NP by Human P450 Recombinant Enzymes.
Each of [3H]mono-NP isomers was incubated with the protein premixes containing recombinant human enzymes, P450 3A4 or P450 1A2, NADPH-P450 reductase, cytochrome b5, and liposomes (RECO System; PanVera Corp., Madison, WI). The P450s were expressed in Escherichia coli and purified according to methods described previously (43 , 44) . It has been demonstrated that these P450 premixes are competent in the oxidation of their known substrates (45) . The buffer mixes for P450 3A4 and P450 1A2 were (a) 200 mM potassium/HEPES (pH 7.4), and (b) 12 mM reduced glutathione, 150 mM MgCl2, and 1 M potassium/sodium phosphate (pH 7.4), respectively. Incubation conditions were identical to those described above for metabolism study with human hepatic microsomes, except that 0.5 nmol of each P450 was added to the incubation mixture in place of microsomal proteins.

Analysis of Mono-NP Metabolites.
Reactions were quenched by adding 1 ml of ice-cold methanol, and the mixture was extracted with an equal volume of a 1:1 mixture of ethyl acetate and chloroform. The organic solvent-extractable metabolites of mono-NP were analyzed by HPLC on a C18 Vydac analytical column (10 µm; 0.46 x 25 cm; Separations Group, Hesperia, CA), using a linear gradient from 30% methanol in water to 100% methanol in 60 min at a flow rate of 1 ml/min. Radioactivity was monitored every 2 s by a ß-Ram Radio-HPLC Detector (IN/US Systems, Inc., Tampa, FL). The identification of metabolites was based on cochromatography with the synthetic standards. In some instances, chemical transformation was performed for further identification of metabolites.

Catalytic Assays.
The following substrates were used to determine P450-linked catalytic activities in human hepatic microsomes: phenacetin (P450 1A2), coumarin (P450 2A6), nifedipine (P450 3A4), tolbutamide (P450 2C9), bufuralol (P450 2D6), and chlorzoxazone (P450 2E1). Details of the experimental protocols are described elsewhere (Ref. 42 and references therein).

Statistical Analyses.
Statistical associations between P450-linked catalytic activities in human hepatic microsomal samples and levels of individual mono-NP metabolites formed by the same microsomes were determined by the Spearman correlation coefficient using version 6.12 Statistical Analysis System software (SAS Institute, Inc., Cary, NC). Values that were below the detection limit were imputed as 0.1 pmol/mg protein/min and were included in the calculation. All Spearman correlation coefficients were based on a sample size of 15. All Ps are two-tailed and considered significant at the 0.05 level.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Metabolism of Mono-NP by Human Hepatic Microsomes.
Human hepatic microsomes were capable of metabolizing all three isomers of mono-NP. Typical HPLC radiochromatograms of the organic solvent-extractable metabolites of mono-NP obtained upon incubating mono-NP with human hepatic microsomes are shown (Fig. 2)Citation . All 15 human hepatic microsomal preparations yielded qualitatively similar metabolic profiles; however, large interindividual variations in levels of each metabolite were evident (Tables 1Citation 2Citation 3)Citation .



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  		Fig. 2. Representative HPLC radiochromatograms of the organic solvent-extractable metabolites of [3H]1-NP (A), [3H]2-NP (B), and [3H]4-NP (C) obtained upon incubating each of the three isomers with human hepatic microsomes in the presence of an NADPH-generating system as described in "Materials and Methods." Asterisks in A and B, retention times of 1-AP and 2-AP, respectively.

 

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  		Table 1 The organic solvent-extractable metabolites of 1-NP formed by human hepatic microsomes

 

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  		Table 2 The organic solvent-extractable metabolites of 2-NP formed by human hepatic microsomes

 

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  		Table 3 The organic solvent-extractable metabolites of 4-NP formed by human hepatic microsomes

 
Human hepatic microsomes metabolized all three mono-NP to form oxidized products phenols and trans-dihydrodiols. On the basis of cochromatography with the synthetic standards, major metabolites of 1-NP formed by human hepatic microsomes were identified as trans-4,5-DHD-1-NP and 3-, 6-, and 8-OH-1-NP (Fig. 2ACitation and Table 1Citation ). The metabolites designated as Products 1–3 were not structurally identified. With the exception of two samples, the amount of phenolic metabolites exceeded that of trans-4,5-dihydrodiol derived from 1-NP. Of 15 hepatic microsomal samples, 11 metabolized 1-NP to preferentially form 3-OH-1-NP over 6- or 8-OH-1-NP. In the case of incubations with 2-NP, two major metabolites, trans-4,5-DHD-2-NP and 6-OH-2-NP, were identified (Fig. 2BCitation and Table 2Citation ). 4-NP was also metabolized to oxidized products, trans-9,10-DHD-4-NP, and phenolic metabolites [6(8)-OH- and 9(10)-OH-4-NP] (Fig. 2CCitation and Table 3Citation ). In addition, nitroreduction of 4-NP to 4-AP was noticed in all 15 human hepatic microsomal samples; however, the formation of 1-AP and 2-AP from 1-NP and 2-NP, respectively, was not detected under the conditions of these incubations.

P450-Linked Catalytic Activities in Human Hepatic Microsomes.
Catalytic activities known to be associated with specific P450 enzymes were analyzed in 15 human hepatic microsomal preparations. The assays used were nifedipine oxidation (P450 3A4), phenacetin O-deethylation (P450 1A2), bufuralol 1'-hydroxylation (P450 2D6), chlorzoxazone 6-hydroxylation (P450 2E1), coumarin 7-hydroxylation (P450 2A6), and tolbutamide methyl hydroxylation (P450 2C9). Wide variations in catalytic activities were evident among these different hepatic microsomal samples (Table 4)Citation , which could not account for the levels of total P450 (per mg protein). Correlations between these catalytic activities and the rates of formation of each of the mono-NP metabolites in the same set of human hepatic microsomes were used to examine the role of specific human P450 enzymes in the metabolism of mono-NP isomers.


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  		Table 4 P450-dependent catalytic activities in human hepatic microsomal samplesa

 
Involvement of Human Hepatic P450 Enzymes in the Metabolism of 1-NP.
Highly significant correlations were found between rates of nifedipine oxidation, a marker for P450 3A4, and the levels of 3-OH-1-NP (Table 5)Citation . The levels of three unknown 1-NP metabolites (Products 1–3) were also well correlated with P450 3A4 activities. A weak but significant correlation was seen between phenacetin O-deethylation activities (P450 1A2) and the formation of 6-OH-1-NP and one of the 1-NP metabolites (Product 1).


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  		Table 5 Spearman correlation coefficients (r) among P450-linked catalytic activities and levels of mono-NP metabolites formed by human hepatic microsomes

 
To confirm the role of P450 3A4 in the metabolism of 1-NP, two hepatic microsomes with high P450 3A4 activity were selected, and incubations were carried out in the absence and presence of ketoconazole, a specific inhibitor of P450 3A4 (Table 6)Citation . The hepatic microsomal 1-NP metabolism assay with ketoconazole resulted in a 65–94% decrease in the levels of 3-OH-1-NP. Ketoconazole also inhibited the formation of Products 1–3 (Table 6)Citation . When {alpha}-naphthoflavone, a strong inhibitor of P450 1A2/1A1 but shown to sometimes stimulate P450 3A4 activity, was added to the incubation mixtures, an 8–15-fold increase in the yields of 3-OH-1-NP was observed (Table 6)Citation . The formation of other 1-NP metabolites including Products 1–3 was also enhanced in the presence of {alpha}-naphtho-flavone.


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  		Table 6 Levels of 1-NP metabolites in the absence and presence of inhibitorsa

 
Involvement of Human Hepatic P450 Enzymes in the Metabolism of 4-NP.
The formation of six of eight 4-NP metabolites, which include trans-9,10-DHD-4-NP, 9(10)-OH-4-NP, and 4-AP, was highly correlated with P450 3A4 activities, measured as rates of nifedipine oxidation (Table 5)Citation . A small but significant correlation was seen between rates of phenacetin O-deethylation, known to be catalyzed by P450 1A2, and levels of trans-9,10-DHD-4-NP.

Inhibition experiments further supported the role of P450 3A4 in the oxidation as well as reduction of 4-NP. A specific P450 3A4 inhibitor, ketoconazole, was highly effective in inhibiting the metabolism of 4-NP in general (Table 7)Citation . Ketoconazole inhibited the formation of trans-9,10-DHD-4-NP and 9(10)-OH-4-NP by 36–66% and 56–92%, respectively. The yields of 4-AP were markedly diminished in the presence of ketoconazole; actually, they were near the detectable level. {alpha}-Naphthoflavone, which is known to inhibit P450 1A2/1A1 but stimulate some reactions catalyzed by P450 3A4, generally enhanced 4-NP metabolism, including the formation of 4-AP (5–10-fold), (9,10)-OH-4-NP (3–4-fold), and trans-9,10-DHD-4-NP (35–94%).


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  		Table 7 Levels of 4-NP metabolites in the absence and presence of inhibitorsa

 
Other Observations in the Metabolism of 1-NP and 4-NP by Human Hepatic Microsomes.
Other catalytic activities examined, i.e., bufuralol 1'-hydroxylation (P450 2D6), coumarin 7-hydroxylation (P450 2A6), and tolbutamide methyl hydroxylation (P450 2C9) did not exhibit significant correlations with the levels of any of the 1-NP or 4-NP metabolites formed by the same human hepatic microsomes (Table 5)Citation . Consistent with these results, no significant effects were found with the inhibitors of P450 2D6 (quinidine), P450 2A6 (diethyldithiocarbamate), and P450 2C (sulfaphenazole) in the metabolism of 1-NP and 4-NP (data not shown).

Involvement of Human Hepatic P450 Enzymes in the Metabolism of 2-NP.
No significant correlations were found between any of the catalytic activities examined in this study and the formation of 2-NP metabolites, trans-4,5-DHD-2-NP and 6-OH-2-NP (Table 5)Citation . Agents known to inhibit specific P450 enzymes, i.e., ketoconazole (P450 3A4), {alpha}-naphthoflavone (P450s 1A2 and 1A1), diethyldithiocarbamate (P450s 2A6 and 2E1), sulfaphenazole (P450 2C), 4-methylpyrazole (P450 2E1), and quinidine (P450 2D6), did not alter the levels of either of these two 2-NP metabolites formed by human hepatic microsomes (data not shown).

Metabolism of Mono-NP by Recombinant Human P450 Enzymes.
Incubations of 1-NP with human P450 3A4 and cofactors resulted in the formation of a number of metabolites that were extractable with a 1:1 mixture of chloroform and ethyl acetate, including all of those seen with hepatic microsomes. However, there was no evidence for the formation of 1-AP by P450 3A4 under the conditions used. The major metabolite detected, 3-OH-1-NP, was formed at a rate of 38 pmol/nmol P450/min (the mean of duplicate assays). Recombinant human P450 1A2 also produced several 1-NP metabolites including trans-4,5-DHD-1-NP, 6-OH-1-NP, and 3-OH-1-NP. Moreover, it was found that P450 1A2 is able to catalyze nitroreduction of 1-NP to 1-AP (8 pmol/nmol P450/min).

Recombinant human P450 3A4 metabolized 2-NP to form trans-4,5-DHD-2-NP and 6-OH-2-NP and also several other unknown metabolites. As was with 1-NP, P450 3A4 did not convert 2-NP to 2-AP. Metabolism of 2-NP by P450 1A2 was more diverse than that observed with P450 3A4; numerous metabolites in addition to those found with P450 3A4 were detected. The most abundant metabolite formed by P450 1A2 was 6-OH-2-NP (23 pmol/nmol P450/min). Unlike P450 3A4, P450 1A2 catalyzed nitroreduction of 2-NP to form 2-AP at a rate of 5 pmol/nmol P450/min.

4-AP, 6(8)-OH-4-NP, and 9(10)-OH-4-NP were among major 4-NP metabolites formed by human P450 3A4, indicating the role of P450 3A4 in both oxidation and nitroreduction of 4-NP. Nitroreduction of 4-NP was even more prominent with P450 1A2. 4-AP was the most abundant metabolite formed by P450 1A2 (41 pmol/nmol P450/min), and the formation of phenols was minimal.

Metabolism of Mono-NP by Human Pulmonary Microsomes.
HPLC analyses revealed that eight human pulmonary microsomes examined yielded metabolic profiles similar to those obtained with human hepatic microsomes (data not shown). However, metabolism of mono-NP by human pulmonary microsomes was at least 10–70 times lower than that observed with human hepatic microsomes. Thus, an accurate quantification of each individual mono-NP metabolite formed by pulmonary microsomes was not possible. Nevertheless, based on cochromatography with the synthetic standards, trans-4,5-DHD-1-NP and/or phenolic metabolites were detected in three of eight pulmonary microsomal incubations of 1-NP. Upon incubating 2-NP, all eight pulmonary microsomes metabolized 2-NP exclusively to 6-OH-2-NP. Following incubations of 4-NP with human pulmonary microsomes, metabolites derived from oxidation (trans-9,10-DHD-4-NP and phenols) and/or nitroreduction (4-AP) were found in all eight samples, as was also seen with hepatic microsomes.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of this study clearly demonstrate that human hepatic microsomes can metabolize all three mono-NP isomers, 1-NP, 2-NP, and 4-NP. Human pulmonary microsomes were also capable of metabolizing the three mono-NP isomers, however, at extremely low levels when compared to hepatic microsomes. As observed in previous studies with other nitropolycyclic aromatic hydrocarbons (41 , 46) , humans demonstrated large interindividual variations in the metabolism of mono-NP. Human hepatic microsomes metabolized all three mono-NP isomers to oxidized products, phenols, and trans-dihydrodiols; however, nitroreduction, considered to be an activation pathway in rodents, was only apparent with 4-NP, not with 1-NP or 2-NP. In addition, the present study documents the role of specific human P450 enzymes in metabolic pathways of the mono-NP isomers. Human hepatic P450 3A4 was the principal enzyme responsible for the metabolism of 1-NP and 4-NP. In particular, oxidation of 1-NP to 3-OH-1-NP and of 4-NP to trans-9,10-DHD-4-NP and 9(10)-OH-4-NP, as well as nitroreduction of 4-NP to 4-AP, were catalyzed by human hepatic P450 3A4. None of the P450 enzymes examined in this study seemed to be responsible for human hepatic microsomal metabolism of 2-NP.

Literature data suggest that nitroreduction is an important pathway in the bioactivation of all three isomers (27, 28, 29, 30, 31, 32, 33, 34) and that the carcinogenic potency of 4-NP in rodents is much greater than that of 1-NP and 2-NP (9 , 15 , 19 , 20) . The present study clearly shows that human hepatic microsomes catalyze metabolic activation of 4-NP (nitroreduction) but not of the other two mono-NP isomers. Collectively, these results suggest that following exposure to mono-NP, humans may be more susceptible to the carcinogenic activity of 4-NP than that of 1-NP or 2-NP. In mammalian systems, both hepatic cytosol and microsomes contain reductive enzymes. Reduction of mono-NP can be catalyzed by hepatic microsomal enzymes, P450 and NADPH-P450 reductase, and also by the cytosolic nitroreductases, including DT-diaphorase, xanthine oxidase, and aldehyde oxidase (21) . Although the contributions of NADPH-P450 reductase and the cytosolic enzymes to the nitroreductive metabolism of mono-NP have not been examined, the present studies clearly indicate a role of P450 enzymes in the nitroreduction of 4-NP. In human hepatic microsomes, nitroreduction of 4-NP to 4-AP was predominantly catalyzed by P450 3A4, as supported by a strong correlation coefficient between the levels of 4-AP in various hepatic microsomes and rates of nifedipine oxidation, a P450 3A4-dependent reaction. This is confirmed also by a drastic inhibition of 4-AP formation by ketoconazole, a specific inhibitor of P450 3A4. An increase in the formation of 4-AP by {alpha}-naphthoflavone, which is known to enhance some reactions catalyzed by P450 3A4 (Ref. 41 and references therein), provides additional evidence for the role of P450 3A4 in the nitroreduction of 4-NP. The involvement of human hepatic P450 in the nitroreductive metabolism is not limited to 4-NP. We have shown that P450 3A4 is mainly responsible for the formation of 6-aminochrysene in the metabolism of 6-nitrochrysene in human hepatic microsomes (41) . We also reported that human P450 3A4 is involved in the nitroreduction of 1,6-dinitropyrene (46) . P450-mediated nitroreduction has been documented in the metabolism of other nitro compounds by laboratory animals, including 1-NP (47) , 1,3-dinitropyrene, 1,6-dinitropyrene, and 1,8-dinitropyrene (46) , nitrobenzene (48) , and p-nitrobenzoate (49) .

The human hepatoma cell line HepG2 has been shown to metabolize 1-NP via both oxidative and nitroreductive pathways (28 , 35 , 36) . In uninduced HepG2 cells, 1-NP was metabolized primarily to 1-AP; phenols and trans-dihydrodiols were also formed at lower rates. However, Silvers et al. (36) and Belisario et al. (50) have demonstrated that pretreatment of HepG2 cells with P450 1A-inducers (3-MC and 2,3,7,8-tetrachlorodibenzo-p-dioxin) results in a dramatic increase in the formation of phenols with a concomitant decrease in 1-AP formation, suggesting the involvement of P450 in the oxidative metabolism but not in nitroreduction of 1-NP. Consistent with this observation, Silvers et al. (37) and Howard et al. (38) showed that only oxidized metabolites were formed upon incubating 1-NP with various human hepatic microsomes or Vaccinia-expressed human P450. Moreover, Silvers et al. (37) established the role of P450 3A4 in human microsomal metabolism of 1-NP, whereas Howard et al. (38) reported that among 12 human P450 examined, only P450s 3A3 and 3A4 demonstrated significant catalytic activity for the oxidation of 1-NP. Thus, our observations that human hepatic microsomes metabolize 1-NP to form only oxidized products and that human P450 3A4 is responsible for its oxidative metabolism are in good agreement with earlier reports on the human metabolism of 1-NP. Furthermore, the present study provides information on the role of specific P450 enzymes in the formation of individual 1-NP metabolites. The formation of four 1-NP metabolites appears to be catalyzed by hepatic P450 3A4. Particularly, P450 3A4 is involved in the formation of 3-OH-1-NP, the major metabolite detected in human hepatic microsomes, as indicated by results obtained with correlation analyses and chemical inhibition and stimulation studies. High catalytic activity of recombinant P450 3A4 for the formation of 3-OH-1-NP further supports this view. A minor role of P450 1A2 in 1-NP metabolism was also suggested by a good correlation coefficient between the levels of two metabolites including 6-OH-1-NP and rates of phenacetin O-deethylation, a marker for P450 1A2 activity. This result is in line with the observation in HepG2 cells, in which P450 1A-inducers (3-MC and 2,3,7,8-tetrachlorodibenzo-p-dioxin) dramatically increased the formation of 6-OH-1-NP and/or 8-OH-1-NP (36 , 48) ; {alpha}-naphthoflavone (1A inhibitor) inhibited levels of the latter metabolites in 3-MC-treated HepG2 cells.

Results from our correlation analyses, together with chemical inhibition and stimulation experiments, strongly suggest that hepatic P450 3A4 is the principal form responsible for the formation of six 4-NP metabolites which include trans-9,10-DHD-4-NP and 9(10)-OH-4-NP as well as 4-AP, as described earlier. On the other hand, none of the P450 enzymes examined in this study appeared to be involved in the human hepatic microsomal metabolism of 2-NP. Shimada et al. (39) have demonstrated that human P450 1B1 catalyzes the activation of 2-NP, measured as umu gene expression. P450 1B1 was not examined in the present study because it is known that 1B1 is not expressed in the liver. Consistent with our findings, 2-NP was not mutagenic in human B-lymphoblastoid cells that contain endogenous P450 1A1 and cDNAs for additional P450 enzymes found in human liver (P450s 1A2, 2A6, 2E1, and 3A4), whereas 4-NP was highly mutagenic and more potent than 1-NP in this system (13) . It is conceivable that minor forms of P450 enzymes in the liver, which were not included in this study or not yet characterized, are responsible for the formation of two 2-NP metabolites (trans-4,5-DHD-2-NP and 6-OH-2-NP) observed in incubations with human hepatic microsomes.

The human hepatic microsomal metabolism of mono-NP appears contrary to that observed in rodents. As reported previously (37) , 1-AP was not detected in this study on human hepatic microsomal metabolism of 1-NP. However, the nitroreductive metabolism of 1-NP to 1-AP was noted following incubations with rat liver microsomes (51 , 52) . Similar to the observation with 1-NP, human hepatic microsomes failed to reduce 2-NP to 2-AP under the conditions used in this study; reports on the metabolism of 2-NP by liver microsomes from rodents are not available. In the case of 4-NP, the situation is reversed. Rat liver microsomal metabolism of 4-NP yielded 4-NP-9,10-dione as the major metabolite; there was no evidence for nitroreduction (24) . In this study, we clearly demonstrated that all of the human hepatic microsomal samples were capable of reducing 4-NP to 4-AP. The differences between human and rodent microsomal metabolism also include regiospecificity in oxidation of 1-NP. As observed previously (37 , 38) , 11 of 15 human hepatic microsomal samples demonstrated a preference for 3-OH-1-NP formation over 6-OH-1-NP or 8-OH-1-NP formation, which is in contrast to findings in rodents (rats, mice, hamsters, and guinea pigs), where the formation of 6-OH-1-NP or 8-OH-1-NP is far more prevalent (52, 53, 54) . This regiospecificity may be due to the fact that specific P450 enzymes that are involved in the metabolism of 1-NP in humans differ from those responsible in rodents. In addition, Kataoka et al. (55) described the differences between humans and rodent species in the formation of the K-region epoxides and dihydrodiol metabolites of 1-NP. They found that human pathways preferentially lead to trans-4,5-DHD-1-NP formation over glutathione conjugation, whereas most rodent species exhibit preferred glutathione conjugation. Thus, our results, together with those reported earlier, suggest that rodents may not accurately predict human susceptibility to mono-NP.

In summary, human hepatic and pulmonary microsomes were capable of metabolizing all three isomers of mono-NP to oxidized products (trans-dihydrodiols and phenols). Human P450 3A4 and, to a lesser extent, P450 1A2 were the principal enzymes involved in oxidative metabolism of 1-NP and 4-NP in hepatic microsomes. None of the P450 enzymes examined in this study (P450s 3A4, 1A2, 2E1, 2A6, 2D6, and 2C9) appeared to be involved in oxidation of 2-NP. Nitroreduction, believed to be a metabolic activation pathway for mono-NP, was observed only with 4-NP, not with 1-NP and 2-NP; P450 3A4 was responsible for nitroreduction of 4-NP to 4-AP. This is highly significant in view of the fact that 4-NP is the most potent carcinogen among mono-NP isomers in rodents.


   ACKNOWLEDGMENTS
 
We are grateful to the staff of the Research Animal Facility for technical support, to Dr. Seth Thompson for statistical expertise, and to Ilse Hoffmann for editorial assistance.


   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 in part by the National Cancer Institute Grants CA 35519 (to K. E-B.) and CA 44353 and ES 00267 (to F. P. G.). Back

2 To whom requests for reprints should be addressed, at American Health Foundation, 1 Dana Road, Valhalla, NY 10595. Back

3 The abbreviations used are: NP, nitropyrene; P450, cytochrome P450; [3H]1-NP, [3H]2-NP, and [3H]4-NP, 1-nitro[4,5,9,10-3H]pyrene, 2-nitro[G-3H]pyrene, and 4-nitro[G-3H]pyrene, respectively; AP, aminopyrene; DHD, dihydro; 3-MC, 3-methylcholanthrene; HPLC, high performance liquid chromatography. Back

Received 9/27/98. Accepted 1/29/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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