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0090-9556/97/2502-0154-0162$02.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
Vol. 25, No. 2

Comparative Metabolism of the Tobacco-Related Carcinogens Benzo[a]pyrene, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol, and N'-Nitrosonornicotine in Human Hepatic Microsomes

Marianne E. Staretz, Sharon E. Murphy, Christopher J. Patten, Maria G. Nunes, Werner Koehl, Shantu Amin, Leeann A. Koenig, F. Peter Guengerich, and Stephen S. Hecht

American Health Foundation (M.E.S., S.E.M., C.J.P., M.G.N., S.A., L.A.K., S.S.H.); Department of Food Chemistry and Environmental Toxicology (W.K.), University of Kaiserslautern; and Department of Biochemistry and Center in Molecular Toxicology (F.P.G.), Vanderbilt University School of Medicine

    Abstract
Abstract
Introduction
Results
Discussion
References

We compared the metabolism in human hepatic microsomes of three tobacco smoke carcinogens believed to be involved in the induction of cancer in humans: benzo[a]pyrene (BaP),4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and N'-nitrosonornicotine (NNN). The metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of NNK, was also investigated. Although the metabolism of some of these compounds by human enzymes or tissue preparations has been previously examined in some studies, they have never been compared in the same human hepatic samples. Moreover, there have been no previous reports of NNAL metabolism by human tissues or enzymes. The tritium-labeled carcinogens (3 µM) were incubated with 10 different human hepatic microsomal preparations and cofactors for 10-20 min, and the products were analyzed by radioflow HPLC. NNN was the best substrate for oxidative metabolism, with the 5'-hydroxylation pathway being the predominant one observed (mean ± SD = 31 ± 17 pmol/min/mg protein). alpha -Hydroxylation of NNK by the methylene and methyl hydroxylation metabolic activation pathways was the next fastest reaction, with rates of 3.1 ± 1.9 and 3.3 ± 1.1 pmol/min/mg protein, respectively. Metabolism of BaP resulted in the formation of dihydrodiols and phenols; trans-7,8-dihydro-7,8-dihydroxy-BaP, its major proximate carcinogen, was formed at a rate of 1.1 ± 0.61 pmol/min/mg protein. alpha -Hydroxylation of NNAL proceeded at a rate of 0.53 ± 0.26 pmol/min/mg protein. The results of this study demonstrate that human hepatic microsomes metabolize all of these tobacco carcinogens resulting in a substantial stream of electrophilic intermediates capable of binding to DNA. The relative rates of oxidative metabolism to electrophiles or their precursors were NNN > NNK > BaP > NNAL. Correlation studies indicated involvement of cytochrome P4502A6 in the 5'-hydroxylation of NNN and cytochrome P4503A4 in the alpha -methylene hydroxylation and pyridine-N-oxidation of NNK and NNAL. The results of this study provide the first data on the comparative metabolism of these important carcinogens in human hepatic microsomes.

    Introduction
Abstract
Introduction
Results
Discussion
References

BaP,2 NNK, and NNN are three important carcinogens found in tobacco products. BaP and related PAHs, as well as NNK and its major metabolite NNAL, are believed to play a significant role in the induction of lung cancer in smokers (1). NNK and NNAL are also likely to be involved in pancreatic cancer etiology in smokers (2, 3). NNN is the most prevalent esophageal carcinogen in tobacco products and may be important as a causative agent for esophageal cancer in smokers (3). A mixture of NNN and NNK induced oral tumors in rats; these two carcinogens, as well as BaP and other PAHs, are probably involved in causing oral cavity cancer in tobacco consumers (1, 3, 4). The structures of these compounds are illustrated in fig. 1.


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  		Fig. 1.   Structures of the compounds examined in this study.

BaP, NNK, NNAL, and NNN all require metabolic activation for expression of their carcinogenic activities. There are competing detoxification pathways. A major microsomal metabolic activation pathway of BaP is conversion to BaP-7,8-diol, catalyzed by cytochrome P450 and epoxide hydrolase, followed by oxidation to BaP-7,8-diol-9,10-epoxide (5, 6; fig. 2). Other metabolites (such as BaP-4,5-diol, BaP-9,10-diol, 1-OH-BaP, 3-OH-BaP, and 9-OH-BaP) result from detoxification pathways (5, 6). Primary microsomal metabolites of NNK and NNAL are summarized in fig. 3 (7, 8). NNK-N-oxide and NNAL-N-oxide are considered to be detoxification products. The metabolic activation of NNK proceeds by hydroxylation of the methylene and methyl carbons adjacent to the N-nitroso group yielding diazohydroxides that alkylate DNA and are critical in the initiation of carcinogenesis. Keto aldehyde and keto alcohol are the stable metabolites resulting from these NNK activation pathways. Similarly, metabolic activation of NNAL produces lactol and diol (9). Electrophilic intermediates are also produced by hydroxylation of NNN at the 2'- and 5'-positions; the corresponding metabolites are keto alcohol and lactol (10).


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  		Fig. 2.   Metabolic activation of BaP to DNA adducts.


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  		Fig. 3.   Overview of NNK and NNAL metabolism.

The metabolism of BaP by human tissue microsomes has been fairly extensively studied, whereas there are only scattered reports on the metabolism of NNK and NNN in human tissue microsomes and related systems (8, 11-21, and references therein). There have been no direct comparisons of metabolism of these important tobacco carcinogens in human tissue preparations. In addition, there have been no previous studies on human hepatic microsomal metabolism of NNAL. NNAL is the predominant metabolite of NNK in human tissue (8, 16-18). Its metabolic activation and detoxification pathways will be critical in determining susceptibility to cancer upon exposure to NNK. Therefore, in this study, we compared the metabolism of BaP, NNK, NNAL, and NNN in human hepatic microsomes to provide insight into the levels of potentially DNA-reactive intermediates that might be produced upon human exposure to these carcinogens.

Materials and Methods

Human Hepatic Microsomes. Microsomes were prepared from 10 human liver samples obtained from organ donors through the Tennessee Donor Services (Nashville, TN) and the Veterans Administration Medical Center (Little Rock, AR) as previously described (22). Protein concentrations were determined using Coomassie Blue G-250 (Pierce Coomassie Plus protein assay; Pierce Chemical Co., Rockford, IL).

BaP Metabolism. [G-3H]BaP (30 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). It was purified the day of use by passage through a short column of silica, with hexane as the eluent. Hexane was removed under a stream of N2. HPLC analysis using the conditions for analysis of microsomal metabolites indicated a radiochemical purity of >= 99.4%. Purified [3H]BaP was diluted with unlabeled BaP (Aldrich Chemical Co., Milwaukee, WI) in DMSO to the desired specific activity (1.3 Ci/mmol). All procedures involving BaP were performed under subdued light. BaP metabolite standards were obtained from the National Cancer Institute Chemical Carcinogen Reference Standard Repository.

Incubation mixtures (total volume 0.5 ml) contained 100 mM potassium phosphate (pH 7.4), 3 mM MgCl2, 1 mM EDTA, an NADPH-generating system (5 mM glucose-6-phosphate, 1 mM NADP+, and 1.5 units of glucose-6-phosphate dehydrogenase), 3 µM BaP, and human liver microsomal protein (0.5 mg/ml). BaP was added in 10 µl of DMSO, and the mixture was incubated at 37°C for 20 min. Reactions were terminated by placing the samples on ice and adding an equal volume of acetone. Two volumes of ethyl acetate and the BaP metabolite standards were then added, and the mixture was vortexed for 2 min. The organic layer was removed and the aqueous layer was extracted two more times with 2 volumes of ethyl acetate. The combined organic layers were dried in vacuo. Samples were redissolved in 70/30 methanol/H2O, sonicated for 10 min, and analyzed by HPLC with radioflow detection. A Waters Associates system was used (Millipore; Waters Division, Milford, MA). It was equipped with a model 440 UV detector (254 nm); a beta -Ram radioflow detector (IN/US Systems, Inc., Fairfield, NJ); and a 4.6 × 250 mm Vydac Specialty Reverse Phase C18, 10-µm column (Alltech Associates, Inc., Deerfield, IL). Solvent A was 20 mM sodium phosphate buffer (pH 7), and solvent B was 95:5 methanol:H2O (v/v). The gradient was 40-55% B in 10 min, 55-70% B in 20 min, 70-80% B in 15 min, 80-100% B in 40 min, and then 100% B for 20 min. Metabolites were identified by coelution with standards. Picofluor 40 (Packard Instruments, Meridan, CT) liquid scintillation cocktail was used in the radioflow detector and was pumped at a flow rate of 3 ml/min.

NNK Metabolism. [5-3H]NNK (2.4 Ci/mmol), which has tritium at the 5-position of the pyridine ring, was purchased from Chemsyn Science Laboratories (Lenexa, KS). It was purified by normal phase HPLC on a 7.8 × 250 mm Alltech Econosil Silica 10-µm column with a hexane/ethanol gradient increasing from 0 to 45% ethanol in 40 min at a flow rate of 2 ml/min. HPLC analysis using the conditions for analysis of microsomal metabolites indicated a radiochemical purity of 99.7%. NNK metabolite standards were synthesized (23-25).

Incubations (total volume 0.5 ml) were conducted in 100 mM sodium phosphate buffer (pH 7.4) in the presence of 3 mM MgCl2, an NADPH-generating system (5 mM glucose-6-phosphate, 5 mM NADP+, and 3.8 units of glucose-6-phosphate dehydrogenase), 3 µM NNK, human liver microsomal protein (0.5 mg/ml), and 1 mM sodium bisulfite. NNK was added to the incubation in 10 µl H2O. Some incubations were performed with 1 mM EDTA, but this had no effect on the results. Mixtures were incubated at 37°C for 10 min. Reactions were terminated by adding 0.3 N barium hydroxide and 0.3 N zinc sulfate (50 µl/incubation), cooled in ice, filtered (0.4 µm), and analyzed by HPLC with radioflow detection. Analyses were conducted on a 3.9 × 300 mm Bondaclone C18, 10-µm column (Phenomenex, Torrance, CA). Solvent A was 20 mM sodium phosphate buffer (pH 6.0) containing 1.0 mM sodium bisulfite, and solvent B was 95:5 methanol:H2O. The gradient was 0-30% B in 60 min, followed by 30-50% B for 10 min. The flow rate was 1 ml/min. Metabolites were identified by coelution with standards.

NNAL Metabolism. [5-3H]NNAL was prepared from purified [5-3H]NNK by NaBH4 reduction (16). It was purified by reversed-phase HPLC using the conditions employed for metabolite analyses and diluted with unlabeled NNAL to 2.0 Ci/mmol. NNAL metabolite standards were prepared as previously described (10, 23, 25, 26).

Incubations were performed with 3 µM [5-3H]NNAL under the same conditions used for BaP metabolism. The mixture was incubated at 37°C for 20 min. Reactions were terminated by addition of 100 µl of 0.3 N zinc sulfate and 100 µl of 0.3 N barium hydroxide. Samples were centrifuged to pellet the precipitated protein, and the supernatants were removed and analyzed by reversed-phase HPLC with radioflow detection. Metabolite separation was accomplished on a 4.6 × 250 mm Rainin Microsorb-MV C18 column (Rainin Instrument Co., Woburn, MA) with a 20 mM sodium phosphate buffer (pH 7):methanol solvent system. The following gradient was used: 0-8% methanol in 15 min, hold at 8% methanol for 15 min, 8-23% methanol in 30 min, hold at 23% methanol for 20 min, and 23-35% methanol in 15 min. Metabolites were identified by coelution with standards. To confirm the identity and purity of the lactol peak, it was collected and reanalyzed under normal phase HPLC conditions. A 4.6 × 250 mm Rainin Microsorb-MV silica column (5 µm, 100 A) was used with elution by solvent A (hexane) and B (2:1, isopropanol:ethanol). The gradient was 0-20% B in 40 min at a flow rate of 1 ml/min. Radioactive peaks were compared with UV standards. The collected lactol region consisted of >= 95% lactol.

NNN Metabolism. [5-3H]NNN (3.29 Ci/mmol), which has tritium of the 5-position of the pyridine ring, was purchased from Chemsyn Science Laboratories. It was >98% pure by HPLC under conditions used for metabolite analysis. It was diluted with unlabeled NNN to a specific activity of 0.65 Ci/mmol. Metabolite standards were synthesized (10, 24).

Incubations were conducted with 3 µM NNN in a total volume of 1 ml, as described (27). Reactions were initiated by the addition of NADP+. Reactions were terminated at 5 or 10 min by addition of barium hydroxide and zinc sulfate, 50 µl of each, as previously described. The precipitated protein was removed by centrifugation, and the supernatant was passed through a 0.4 µm filter and analyzed by reversed-phase HPLC as previously described (27). Lactol and keto alcohol coelute in this system. They were collected and reanalyzed by normal phase HPLC (27). Recoveries of lactol and keto alcohol were determined using the unlabeled standards.

Other Assays. The following assays were previously conducted on these samples to determine cytochrome P450-linked activities in the microsomal samples: EROD (cytochrome P4501A2), MP (P4502C19), BUF (P4502D6); CZ (P4502E1), NF (P4503A4), and COUM (P4502A6) (28).

Statistical Analysis. Student's t test was used to analyze the data in table 1. Pearson and/or Spearman correlations were used to evaluate data in tables 2 and 3.

                              
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TABLE 1
Metabolism of BaP, NNK, NNAL, and NNN in 10 human hepatic microsomal samplesa

                              
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TABLE 2
Significant correlations among human hepatic microsomal metabolites of BaP, NNK, NNAL, and NNN

                              
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TABLE 3
Correlations of human hepatic microsomal metabolites of BaP, NNK, NNAL, and NNN with cytochrome P450-dependent activities

    Results
Abstract
Introduction
Results
Discussion
References

Initially, we investigated the dependence of BaP metabolite formation on protein concentration and time. Based on the results of these experiments, we chose 0.5 mg/ml for these studies, because product formation increased between 0.25 and 0.75 mg/ml. Similarly, we chose 20 min as the incubation time. For NNK, NNAL, and NNN metabolism, conditions were determined as 0.5 mg/ml protein and 10 min (NNK and NNN) or 20 min (NNAL).

All reactions were conducted at a substrate concentration of 3 µM. This concentration was chosen because it was the lowest that could be used considering the extent of product formation and the specific activities of the substrates. Low substrate concentrations would presumably be most relevant to human exposure conditions, although the 3 µM concentration used herein is probably still considerably higher than actual concentrations of the substrates in the livers of smokers.

NNK metabolism was performed in the presence of sodium bisulfite to allow trapping and quantitation of keto aldehyde (29). The effects of sodium bisulfite concentration on metabolite production were investigated in a preliminary study. Metabolite production was not inhibited by 1 mM sodium bisulfite but was inhibited at bisulfite concentrations of 2.5 and 5 mM. For example, the rate of keto alcohol formation was 6.6 pmol/min/mg protein with no bisulfite added, 6.4 at 1 mM, 4.8 at 2.5 mM, and 2.9 at 5 mM. Therefore, we used 1 mM sodium bisulfite.

Typical HPLC traces obtained upon analysis of the incubation mixtures are illustrated in fig. 4 (A-D). Metabolites were identified by coelution with standards. In the case of NNN, keto alcohol and lactol coeluted under the conditions illustrated. Normal phase HPLC was used to separate and quantify these metabolites. Levels of metabolites formed in the incubations with each hepatic microsomal sample are summarized in table 4. The means and ranges for each metabolite are presented in table 1.


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  		Fig. 4.   HPLC chromatograms obtained upon analysis of incubation mixtures of human hepatic microsomal sample 115 with (A) [G-3H]BaP, (B) [5-3H]NNK, (C) [5-3H]NNAL, and (D) [5-3H]NNN.

For structures of metabolites, see figs. 2 and 3.   

                              
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TABLE 4
Metabolism of BaP, NNK, NNAL, and NNN in 10 human hepatic microsomal samplesa,b

The major products of BaP metabolism were dihydrodiols and phenols, as previously reported (5, 6, 11-14). The proximate carcinogen BaP-7,8-diol was formed in all samples. Substantial amounts of polar metabolites, eluting prior to BaP-9,10-diol, were also produced.

The major metabolite of NNK was NNAL (the product of carbonyl reduction), consistent with other studies (16-18). Levels of products resulting from the alpha -hydroxylation metabolic activation pathways---keto aldehyde and keto alcohol---were higher than previously reported (17). In that study, 5 mM sodium bisulfite was used; consequently, these alpha -hydroxylation pathways of NNK would have been inhibited. In most samples, both alpha -hydroxylation pathways occurred at approximately equal rates. Metabolic activation of NNK by alpha -hydroxylation was ~6 times greater than activation of BaP through BaP-7,8-diol (table 1, p < 0.00001).

Oxidation of NNAL to NNK was its major metabolic pathway, although this occurred less extensively than reduction of NNK to NNAL. Lactol and diol, the products of alpha -hydroxylation, were formed at approximately equal rates. These were ~15-fold less than observed for NNK. Total alpha -hydroxylation of NNAL was ~50% as great as formation of BaP-7,8-diol (p = 0.023).

In the alpha -hydroxylation of NNN, levels of lactol were >10-fold greater than those of keto alcohol (table 1). Total alpha -hydroxylation of NNN was significantly greater than alpha -hydroxylation of NNK and NNAL (p < 0.001). Overall, rates of metabolic activation of the four substrates proceeded in the order NNN > NNK > BaP > NNAL.

Statistically significant correlations among the metabolites of BaP, NNK, NNAL, and NNN in the 10 human hepatic microsomal preparations are summarized in table 2. The strongest correlations were observed for metabolites of NNK and NNAL. Keto aldehyde, resulting from alpha -methylene hydroxylation of NNK, was strongly correlated with lactol, resulting from alpha -methylene hydroxylation of NNAL. However, the alpha -methyl hydroxylation products of NNK and NNAL, keto alcohol and diol, were not correlated. Correlations were also observed between keto aldehyde and NNAL-N-oxide, NNK-N-oxide and lactol, and NNK-N-oxide and NNAL-N-oxide. These results indicate that the same human hepatic enzymes conduct the alpha -methylene hydroxylation and pyridine-N-oxidation of NNK and NNAL.

Correlations were also observed among several metabolites of BaP and the nitrosamines. Rates of formation of BaP-4,5-diol, 3-OH-BaP, and 1-OH-BaP were correlated with keto alcohol formation from NNK, thus suggesting common enzyme involvement. Rates of 3-OH-BaP production were also correlated with formation of diol and lactol from NNAL, and with lactol from NNN.

Catalytic activities known to be associated with specific cytochrome P450s have previously been determined in this same set of human hepatic microsomal samples (28). These activities varied 10- to 20-fold, whereas total cytochrome P450 content varied 2- to 3-fold. Correlations among the cytochrome P450 activities and the metabolites of BaP, NNK, NNAL, and NNN are summarized in table 3. The strongest correlations were observed between the NNK metabolites keto aldehyde and NNK-N-oxide and NF, indicating involvement of cytochrome P4503A4 in the formation of these metabolites. NF activity was also correlated with formation of lactol, diol, NNAL-N-oxide, and NNK from NNAL. Several BaP metabolites were correlated with EROD, indicating involvement of cytochrome P4501A2. Formation of lactol from NNN was correlated with COUM activity, indicating a role for cytochrome P4502A6. In addition, when coumarin (250 µM) was incubated with NNN and hepatic microsomal sample HL 115, complete inhibition of lactol formation was observed.

    Discussion
Abstract
Introduction
Results
Discussion
References

The major purpose of this study was to assess the metabolism of different tobacco-related carcinogens in the same human hepatic microsomal samples. Results clearly demonstrate that all four carcinogens were effectively metabolized by human liver microsomes. 5'-Hydroxylation of NNN occurred at the greatest rate. The next fastest reactions were alpha -hydroxylation of NNK at the methylene and methyl carbons. These occurred at greater rates than oxidative pathways of BaP metabolism, which in turn were generally faster than the alpha -hydroxylation reactions of NNAL and pyridine-N-oxidation of NNK or NNAL. Comparative studies of these important carcinogens in the same human microsomal preparations have not been previously reported, and our data thus provide an index of their relative rates of metabolic activation. The direct relationship of hepatic metabolism of these carcinogens to the induction of tobacco-related cancers in extrahepatic tissues, such as lung and esophagus, is not clear at present, although evidence does exist for the transport of activated metabolites of both NNK and BaP (30, 31). Future studies will examine the comparative metabolism of these compounds in human extrahepatic microsomes and in mixtures as present in tobacco products.

There has been only one previous report of NNN metabolism in human hepatic microsomes (15). In that study, the substrate concentration was 10 mM. Rates of 5'-hydroxylation exceeded those of 2'-hydroxylation as in the present study, although the difference was not as great as seen herein. In cultured human extrahepatic tissues, 5'-hydroxylation also exceeded 2'-hydroxylation (16). The significance of NNN 5'-hydroxylation with respect to carcinogenesis is uncertain. 5'-Acetoxy-NNN, a stable precursor to 5'-hydroxy-NNN, is mutagenic toward Salmonella typhimurium, indicating that intermediates formed by this process bind to DNA (10). However, the resulting DNA adducts have not been characterized. A stable precursor to the diazohydroxide resulting from 2'-hydroxylation of NNN was more mutagenic toward S. typhimurium than the analogous precursor to that resulting from 5'-hydroxylation (32). Moreover, DNA adducts resulting from 2'-hydroxylation of NNN have been detected in rat nasal mucosa and esophagus, which are the principal target tissues for NNN carcinogenicity (33, 34). Therefore, 2'-hydroxylation of NNN seems to be more important than 5'-hydroxylation as a metabolic activation pathway.

Lactol formation via 5'-hydroxylation of NNN was correlated with COUM activity in these microsomal samples and was inhibited by coumarin. No other correlations were observed between NNN metabolism and cytochrome P450 activities. This indicates involvement of cytochrome P4502A6 in 5'-hydroxylation of NNN, which is consistent with data recently obtained by Patten et al. (21).

The present results demonstrate that substantial amounts of intermediates resulting from both alpha -hydroxylation pathways of NNK are formed in human hepatic microsomes. Although liver is not considered to be an important target tissue for NNK, alpha -hydroxymethyl-NNK, the precursor to keto alcohol, can be transported as its glucuronide (30). Both alpha -hydroxylation pathways of NNK result in DNA damaging intermediates that have been detected in tissues of animals treated with this nitrosamine, as well as in human tissues (7, 8). In rats, both the DNA methylation pathway, resulting from alpha -methylene hydroxylation (keto aldehyde formation), and the DNA pyridyloxobutylation pathway, resulting from alpha -methyl hydroxylation (keto alcohol formation), are involved in lung carcinogenesis by NNK, whereas in A/J mice the DNA methylation pathway seems to be more important (35-37).

Using different expression systems, human cytochromes P4501A2, P4502A6, and P4503A4 have all been shown to activate NNK metabolically. Cytochrome P4501A2, purified from Escherichia coli or expressed in Hep G2 cells, preferentially catalyzed methyl hydroxylation (e.g. keto alcohol formation) (17, 19). Tiano et al. (38) demonstrated that relatively high concentrations of NNK transform C3H/10T1/2 cells expressing cytochrome P4502A6 (38). Both cytochromes P4503A4 and P4502A6 catalyzed the alpha -hydroxylation of NNK, but the KM for cytochrome P4503A4 was high (20, 38). In the present study, strong correlations were observed between rates of formation of keto aldehyde and NF activity, supporting a role for cytochrome P4503A4 in production of this metabolite in human liver microsomes. NNK-N-oxide formation was also strongly correlated with NF activity. Moreover, the corresponding pathways of NNAL metabolism leading to lactol and NNAL-N-oxide were also correlated with NF activity. Thus, our results suggest a role for cytochrome P4503A4 in the metabolic activation of NNK and NNAL by alpha -methylene hydroxylation, leading to DNA methylation, as well as in the corresponding pyridine-N-oxidation reactions. The strong correlations between NNK and NNAL metabolites formed by these pathways, as shown in table 2, are also consistent with this conclusion. Cytochrome P4503A4 may also be involved in the methyl hydroxylation of NNAL (R = 0.82 for diol production) and in its oxidation to NNK (R = 0.71). Correlations between EROD activity and keto alcohol production from NNK and between COUM activity and keto aldehyde formation were weak, despite the known abilities of cytochromes P4501A2 and P4502A6 to catalyze these reactions.

As previously reported and observed herein, NNK is rapidly converted to NNAL in human tissues (16-18). In laboratory animals, this conversion is also rapid and extensive (39-41). Therefore, substantial amounts of NNAL will reach the liver upon exposure to NNK. Its metabolism by alpha -hydroxylation occurs at a significantly lower rate than does alpha -hydroxylation of NNK. NNAL, like NNK, methylates and pyridyloxobutylates DNA and has similar carcinogenic activity to that of NNK (2, 42). The actual rate of formation of DNA damaging intermediates in human liver upon exposure to NNK will partially depend on the rate of conversion of NNK to NNAL in vivo.

The ability of human tissues to metabolize BaP is well established, and this study confirmed previous results obtained with hepatic microsomes (11, 12). DNA adducts of BaP have been detected in human tissues in many studies (43). BaP-7,8-diol is the major proximate carcinogen among the metabolites detected; but, unlike the alpha -hydroxylation products of NNN and NNK that spontaneously yield diazohydroxides that bind to DNA, it requires further enzymatic oxidation to BaP-7,8-diol-9,10-epoxide before DNA binding can occur (5, 6). Therefore, overall production of DNA damaging intermediates from BaP by this pathway will be lower than indicated by rates of BaP-7,8-diol formation. Human cytochrome P4501A2 has appreciable activity for production of BaP-7,8-diol and other diols and phenols of BaP, according to studies with recombinant enzymes and in expression systems (13, 14). Our results are consistent with this conclusion, because EROD activity was correlated with production of most BaP metabolites (table 3). Production of 3-OH-BaP was weakly correlated with both EROD and NF, which is also consistent with previous studies that showed activity for cytochromes P4501A2 and P4503A4 (12, 13). Reasonably strong correlations were observed between certain metabolites of BaP and NNK (e.g. 3-OH-BaP and keto alcohol, 1-OH-BaP and keto alcohol, and BaP-4,5-diol and keto alcohol). Although cytochrome P4501A2 has good activity for production of all these metabolites in recombinant systems, keto alcohol production was not strongly correlated with EROD activity, suggesting the presence of another human hepatic enzyme that oxidizes BaP and alpha -hydroxylates NNK at its methyl carbon.

A possible limitation of this study is the use of only one substrate concentration (3 µM) that was chosen because it was the lowest concentration that was practical considering the methods used. The 3 µM concentration is close to the observed KM for NNN 5'-hydroxylation in some human liver microsomes (21), but lower than those reported for NNK (53-4500 µM) (20) or BaP (12-33 µM) (44). Multiple cytochrome P450 enzymes are involved in the metabolism of these substrates. The enzymes with low KM values are potentially the most relevant to human metabolism of these carcinogens. Therefore, enzyme activity correlations observed at relatively low substrate concentrations may be most informative. This is illustrated by comparing two studies on the correlation of NNN activation with cytochrome P450 enzymes (21, 45). In the earlier study, using 2 mM NNN, both cytochrome P4502E1 and P4502A6 correlated with NNN metabolic activation (45). In the results reported herein, as well as those of Patten et al. (21), in which 1-3 µM NNN was used, no correlation with cytochrome P4502E1 was observed, probably because cytochrome P4502E1 has a high KM for alpha -hydroxylation of NNN. Nevertheless, comparing the metabolism of four different carcinogens at a single low concentration may not be optimal and could affect our conclusions with respect to relative rates of metabolism.

In summary, this study has demonstrated that the tobacco-specific nitrosamines---NNK, NNAL, and NNN---as well as PAH and BaP, are good substrates for metabolic activation by human hepatic microsomes. The overall rates of metabolic activation to DNA damaging intermediates or their precursors under the conditions used herein occurs in the order NNN > NNK > BaP > NNAL. Cytochrome P4502A6 may play an important role in 5'-hydroxylation of NNN. Cytochrome P4503A4 seems to be an important catalyst of the methylene hydroxylation and pyridine-N-oxidation of NNK and NNAL.

    Footnotes

Received July 9, 1996; accepted October 31, 1996.

   This study was supported by Grants CA-44377, CA-46535, CA-44353, and ES-00267 from the National Institutes of Health.

1   Present address: University of Minnesota Cancer Center, Box 806-UMHC, 420 Delaware Street, S.E., Minneapolis, MN 55455.

Send reprint requests to: Dr. Stephen S. Hecht, University of Minnesota Cancer Center, Box 806 UMHC, 420 Delaware St. S. E., Minneapolis, MN 55455. 

    Abbreviations

Abbreviations used are: BaP, benzo[a]pyrene; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N'-nitrosonornicotine; PAH, polycyclic aromatic hydrocarbon; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; BaP-7, 8-diol, trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene; BaP-7, 8-diol-9,10-epoxide, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BaP-4, 5-diol, trans-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene; 1-OH-BaP, 1-hydroxybenzo[a]pyrene; 3-OH-BaP, 3-hydroxybenzo[a]pyrene; 9-OH-BAP, 9-hydroxybenzo[a]pyrene; NNK-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone; NNAL-N-oxide, 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanol; keto aldehyde, 4-oxo-4-(3-pyridyl)butanal; keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone; lactol, 2-hydroxy-5-(3-pyridyl)tetrahydrofuran; diol, 4-hydroxy-4-(3-pyridyl)-1-butanol; keto acid, 4-oxo-4-(3-pyridyl)butyric acid; hydroxy acid, 4-hydroxy-4-(3-pyridyl)butyric acid; EROD, ethoxyresorufin dealkylase; MP (S)-mephenytoin-4'-hydroxylation, BUF, bufuralol-1'-hydroxylation; CZ, chlorzoxazone-6-hydroxylation; NF, nifedipine oxidase; COUM, coumarin-7-hydroxylation.

    References
Abstract
Introduction
Results
Discussion
References

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Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics


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