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N′-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in m...
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Chem. Res. Toxicol. 2005, 18, 61-69

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Cytochrome P450 2A-Catalyzed Metabolic Activation of Structurally Similar Carcinogenic Nitrosamines: N′-Nitrosonornicotine Enantiomers, N-Nitrosopiperidine, and N-Nitrosopyrrolidine Hansen L. Wong, Sharon E. Murphy, and Stephen S. Hecht* University of Minnesota, The Cancer Center, 420 Delaware Street SE, Mayo Mail Code 806, Minneapolis, Minnesota 55455 Received August 17, 2004

N′-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. R-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine R-hydroxylation. Therefore, a structure-activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for R-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, R-hydroxylation can occur at the 2′- or 5′-carbon. P450 2As catalyzed 5′-hydroxylation of (R)- or (S)-NNN with Km values of 0.74-69 µM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2′-hydroxylation with Km values of 0.7366 µM. (S)-NNN 2′-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5′-hydroxylation, respectively. The catalytic efficiencies (kcat/Km) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5′hydroxylation with significantly lower Km and higher kcat/Km values than NPIP or NPYR R-hydroxylation (p < 0.05). Furthermore, P450 2As were better catalysts of NPIP R-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower Km and higher kcat/Km values for NPIP than NPYR R-hydroxylation (p < 0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR R-hydroxylation.

Introduction N′-Nitrosonornicotine (NNN),1 N-nitrosopiperidine (NPIP), and N-nitrosopyrrolidine (NPYR) (Schemes 1-3) are carcinogens in laboratory animals (1-5) and possible causative agents in human cancer. NNN is the most abundant of all of the carcinogenic nitrosamines present in tobacco (6, 7). It has a chiral center at the 2′-carbon, and (S)-NNN constitutes about 75% of the total NNN in unburned tobacco (8). Humans are exposed to NNN when they use tobacco products and exogenously to NPIP and NPYR through the diet and tobacco smoke (6, 7, 9). Significantly higher levels of exposure to NNN, NPIP, and NPYR could come from endogenous nitrosation of their respective amine precursors (10, 11). Because of considerable human exposure to NNN, NPIP, and NPYR, it is imperative to understand the molecular basis for their carcinogenicities. * To whom correspondence should be addressed. Tel: 612-624-7604. Fax: 612-626-5135. E-mail: [email protected]. 1 Abbreviations: 2-OH-THF, 2-hydroxytetrahydrofuran; 2-OH-5MeTHF, 2-hydroxy-5-methyltetrahydrofuran; 2-OH-THP, 2-hydroxytetrahydro-2H-pyran; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; keto alcohol, 4-hydroxy-1-(3-pyridyl)-1-butanone; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNN, N′-nitrosonornicotine; NPIP, N-nitrosopiperidine; NPYR, N-nitrosopyrrolidine; SRS, substrate recognition sequence.

Although NNN, NPIP, and NPYR are structurally related, they have differing carcinogenic activities in rodents. NNN and NPIP are potent esophageal and nasal cavity carcinogens in rats and effective pulmonary carcinogens in mice (1-5). NPYR induces mainly hepatic tumors in rats (4). NPYR is noncarcinogenic in the esophagus and weakly carcinogenic toward the nasal cavity of rats (4, 5). In mice, it is a weak pulmonary carcinogen (2). We seek to understand the biochemical factors that mediate these differences in carcinogenic activities. Cytochrome P450s catalyze the metabolic activation of nitrosamines, a critical step to unleash their carcinogenic potential. The key pathway is believed to be hydroxylation at the carbon R to the nitroso group. For NNN, P450s catalyze R-hydroxylation at the 2′- and 5′-carbons (Scheme 1) (1). The resultant 2′- and 5′-hydroxyNNN are unstable and spontaneously ring open to form the corresponding diazohydroxides. These electrophilic intermediates react with H2O to form 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol) and 5-(3-pyridyl)-2-hydroxytetrahydrofuran (lactol), respectively. The diazohydroxide from 2′-hydroxylation can alkylate DNA generating pyridyloxobutyl adducts (1, 12, 13). dGuo adducts from the diazohydrox-

10.1021/tx0497696 CCC: $30.25 © 2005 American Chemical Society Published on Web 12/21/2004

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Scheme 1. Metabolism of NNN by r-Hydroxylation

Scheme 3. Metabolism of NPYR by r-Hydroxylation

Table 1. Percent Amino Acid Sequence Identity of P450 2Asa P450

Scheme 2. Metabolism of NPIP by r-Hydroxylation

2A3 2A4 2A5 2A6 2A13

2A3 94.7 (26) 96.2 (19) 85.4 (72) 88.9 (55)

2A4

2A5

2A6

2A13

94.7 (26b)

96.2 (19) 97.8 (11)

85.4 (72) 83.8 (80) 85.4 (72)

88.9 (55) 86.4 (67) 88.3 (58) 93.5 (32)

97.8 (11) 83.8 (80) 86.4 (67)

85.4 (72) 88.3 (58)

93.5 (32)

a

Amino acid sequences for P450 2A3 (accession number P20812), 2A4 (P15392), 2A5 (P20852), and 2A6 (P11509) were obtained from the Swiss-Prot database (www.expasy.org). The P450 2A13 sequence was acquired from ref 33. Amino acid sequences were aligned, and the percent identity was determined by BLAST (Basic Local Alignment Search Tool) at the www.ncbi.nlm.nih.gov/blast/ bl2seq/bl2.html website. b Number of amino acids (out of 494) that differ between two P450 sequences. For example, P450 2A3 and 2A4 contain 26 nonconserved residues.

ide resulting from 5′-hydroxylation recently have been identified in our laboratory (unpublished results). 2′-Hydroxylation is believed to be the important pathway for NNN carcinogenesis. This pathway is the predominant one in the rat esophagus and nasal cavity, target tissues of NNN carcinogenesis. In these tissues, the ratios of 2′-to-5′-hydroxylation are 2-4 (1). In contrast to NNN, 2′-hydroxylation is not the predominant pathway for the esophageal metabolism of N′-nitrosoanabasine, the homologous six-membered cyclic nitrosamine and a weak esophageal carcinogen. The major pathway was 6′-hydroxylation (equivalent to 5′-hydroxylation for NNN) (14). NPIP undergoes P450-mediated R-hydroxylation (Scheme 2). The resulting unstable species decomposes to produce a diazohydroxide as well as diazonium and oxonium ions. These electrophilic species can react with DNA to form DNA adducts. Adducts generated from R-acetoxyNPIP, a stable R-hydroxyNPIP precursor, have been detected following reaction with dGuo and calf thymus DNA (15-18). Reaction with H2O yields 2-hy-

droxytetrahydro-2H-pyran (2-OH-THP), 2-hydroxy-5-methyltetrahydrofuran (2-OH-5-MeTHF), and cis-4-oxo-2pentenal (17, 19). The major NPIP R-hydroxylation products are 2-OH-THP and 2-OH-5-MeTHF (20). Metabolic activation of NPYR, like NPIP, proceeds via R-hydroxylation (Scheme 3). Adducts generated from NPYR and R-acetoxyNPYR have been detected in reactions with dGuo and calf thymus DNA and in vivo (15, 16, 2123). The electrophiles can also react with H2O to form 2-hydroxytetrahydrofuran (2-OH-THF), which is the major R-hydroxylation product, and crotonaldehyde (24, 25). Cytochrome P450 2As are important catalysts for the R-hydroxylation of several nitrosamines including NNN, NPIP, and NPYR (26-29). Members of the P450 2A subfamily share a high amino acid sequence identity with each other (Table 1). P450 2A3 mRNA and protein have been detected in the nasal mucosa and lung of rats (30). mRNA and protein of mouse P450 2A4 and 2A5 have been detected in the nasal mucosa, liver, lung, and kidney of mice (31). Human P450 2A6 is principally expressed in the liver; the average concentration was 0.014 ( 0.013 nmol/mg protein (or 4.0 ( 3.2% of total P450) based on microsomes prepared from 60 individuals (32). By comparison, P450 3A made up about 29% of total P450 (32). P450 2A6 is also present, in lesser amounts, in the nasal mucosa, esophagus, trachea, and lung (33, 34). As compared to P450 2A6, human P450 2A13 mRNA is expressed at higher levels in the nasal mucosa, trachea, and lung and at lower levels in the liver (33); P450 2A13 protein was recently identified in fetal nasal mucosa (35). To our knowledge, there are no literature reports identifying P450 2A13 mRNA in the esophagus.

NNN, NPIP, and NPYR Activation by P450 2As

Previous studies have examined the metabolism of (R)NNN, (S)-NNN, NPIP, and NPYR by P450 2A3 (26, 28). However, there has been no systematic comparison of carcinogenic cyclic nitrosamine metabolism by P450 2As. Therefore, in this study, kinetic parameters were determined and compared for R-hydroxylation of (R)-NNN, (S)NNN, NPIP, and NPYR catalyzed by P450 2A3, 2A4, 2A5, 2A6, or 2A13. Structure-R-hydroxylation relationships from these data may give important information on the P450 2A active site residues that affect R-hydroxylation of carcinogenic cyclic nitrosamines.

Experimental Procedures Caution: NNN, NPIP, and NPYR are carcinogenic in laboratory animals. Handle with extreme care with proper personal protective equipment and in a well-ventilated hood. Chemicals and Enzymes. [3H]NPIP (8.29 Ci/mmol) and [3H]NPYR (1.66 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA) and purified to >99.5% radiochemical purity as described previously (28). Unlabeled NPIP and NPYR were obtained from Sigma-Aldrich Co. (St. Louis, MO) and used without further purification. Enantiomers of unlabeled [5-3H]NNN (23 Ci/mmol) were prepared as described previously (8, 36). Tritium-labeled NNN enantiomers were purified by reverse phase HPLC (radiochemical purity, >99.5%). 2-OH-THP, 2-OHTHF, lactol, and keto alcohol were synthesized as described previously (37-40). Microsomes were prepared from Spodoptera frugiptera insect cells that express P450 2A4 or 2A5 as previously described (41). Microsomes containing expressed P450 2A3 or 2A13 from the same insect cell line were generous gifts from Xinxin Ding (Wadsworth Center, Albany, NY). P450 2A6 + P450 reductase and insect cell control Supersome enzymes were purchased from BD Biosciences (San Jose, CA). NADPH-P450 oxidoreductase was obtained from Panvera (Madison, WI). Monoflow 5 scintillation fluid was purchased from National Diagnostics (Atlanta, GA). All other chemicals and enzymes were purchased from Sigma-Aldrich Co. or Fisher Scientific Chemical Co. (Pittsburgh, PA). Apparatus. HPLC analyses for all systems were performed using two Waters Associates pumps (Waters Division, Millipore, Milford, MA), an in-line Gilson UV detector (Gilson Medical Electronics, Middleton, WI) set at 269 nm for NPIP and NPYR analyses or a Waters UV detector model 440 set at 254 nm for NNN analyses, and a β-ram flow-through radioflow detector (model 2B, IN/US Systems, Tampa, FL, scintillation fluid-toHPLC eluant ratio of 3). Analytes were eluted with a 250 mm × 4.6 mm Metasil C18 column (Metachem, Torrance, CA) using the following HPLC systems: (1) isocratically with 4% methanol and 96% 25 mM ammonium acetate, pH 4.5, buffer for 30 min, followed by a linear gradient from 4 to 5% methanol over 1 min, held at 5% methanol for 19 min, and then a linear gradient from 5 to 40% methanol over 10 min; (2) isocratically with 5% methanol and 95% 25 mM ammonium acetate, pH 4.5, for 20 min, followed by a linear gradient from 5 to 15% methanol for 10 min, held at 15% methanol for 15 min, and then a linear gradient from 15 to 40% methanol for 5 min; or (3) isocratically with 100% 25 mM ammonium acetate, pH 4.5, for 10 min followed by a linear gradient from 100% buffer to 20% methanol and 80% buffer for 15 min, held at 20% methanol for 15 min, and then a linear gradient from 20 to 40% methanol. Metabolism of Nitrosamines. Kinetic parameters for (R)and (S)-NNN R-hydroxylation catalyzed by P450 2A3, 2A4, 2A5, and 2A13 were determined in an analogous fashion to NPIP and NPYR metabolism catalyzed by P450 2A3 as previously described (28). Briefly, (R)- or (S)-NNN was incubated with expressed P450 and cofactors. The concentrations of (R)- or (S)NNN were 0.3, 0.5, 1, 3, 5, 10, 25, 50, 100, 200, and 400 µM. The specific activity of [5-3H](R)- and (S)-NNN ranged from 0.03 to 10 Ci/mmol. P450 concentrations are given in Table 2. The

Chem. Res. Toxicol., Vol. 18, No. 1, 2005 63 Table 2. Kinetic Parameters of (R)- and (S)-NNN 5′-Hydroxylation to Lactol by P450 2Asa species P450 substrate rat mouse mouse human human

Kmb (µM)

kcatb (min-1)

kcat/Kmb (min-1 µM-1)

(R)-NNN 0.77 ( 0.38c,e 0.43 ( 0.03 0.56 ( 0.28d,e (S)-NNN 3.3 ( 1.3e 3.2 ( 0.2 0.97 ( 0.39d,e,f 2A4 (R)-NNN 69 ( 20e 1.0 ( 0.1 0.015 ( 0.005c,f (S)-NNN 54 ( 12e 2.5 ( 0.2 0.046 ( 0.011e,f 2A5 (R)-NNN 1.5 ( 0.4c,e 3.7 ( 0.2 2.5 ( 0.6d,e,f (S)-NNN 0.74 ( 0.25e 1.6 ( 0.1 2.1 ( 0.7d,e,f 2A6 (R)-NNN 22 ( 6c,e 1.3 ( 0.1 0.058 ( 0.016c,e,f (S)-NNN 2.3 ( 0.6e 1.0 ( 0.1 0.45 ( 0.12e,f 2A13 (R)-NNN 24 ( 4e 13 ( 1 0.54 ( 0.10e,f (S)-NNN 23 ( 8e 10 ( 1 0.43 ( 0.15e 2A3

a Incubations were performed and analyzed as described in the Experimental Procedures. NNN concentrations were 0.3-400 µM. Concentrations of P450, given in parentheses, were 2-20 (P450 2A3), 10-50 (P450 2A4), 2-20 (P450 2A5), 6-50 (P450 2A6), and 1.5-20 (P450 2A13) pmol/mL. b Values ( SD for goodness of fit were calculated using the Enzyme Kinetics Module of SigmaPlot (SPSS Inc.) from three replicate incubations at each substrate concentration (n ) 3), except for 50 and 400 µM (R)-NNN (n ) 2) for studies with P450 2A13. c p < 0.05 as compared to the respective value for (S)-NNN. d p < 0.05 as compared to the respective value for P450 2A4. e p < 0.05 as compared to the respective value for NPYR. f p < 0.05 as compared to the respective value for NPIP.

total volume of the reaction mixture was 200 µL. After termination of the reaction with 20 µL each of Ba(OH)2 and ZnSO4, metabolite standards were added. Samples were centrifuged at 13000g, and the supernatants were analyzed by HPLC-UV radioflow using system 1. To determine whether the metabolite with an HPLC retention time of 49 min (Figure 1A,B) was myosmine, the product was collected following reaction of [3H](R)- or (S)-NNN with P450 2As and allowed to react with NaBH4 as described previously (42). The reaction mixture was analyzed for the formation of a tritium-labeled product peak coeluting with nornicotine by radioflow HPLC using system 1. Kinetic parameters for NPIP and NPYR R-hydroxylation by P450 2A4, 2A5, and 2A13 were determined as described previously for incubations with P450 2A3 with a few exceptions (28). P450 and cofactors were incubated with NPIP (1, 3, 10, 20, 50, 100, 250, 500, 1000, and 1500 µM) or with NPYR (1, 2, 5, 20, 50, 100, 300, 750, 1500, 3000, and 5000 µM). The specific activities of [3H]NPIP and [3H]NPYR ranged from 0.009 to 2.5 and 0.003 to 1.66 Ci/mmol, respectively. The P450 concentrations are given in Table 4. NPIP and NPYR metabolites were analyzed by HPLC-UV radioflow using systems 2 and 3, respectively. P450 2A6 kinetic studies with NNN enantiomers, NPIP, and NPYR were performed as described above for the expressed P450 2As with the following exception. The Supersomes containing coexpressed P450 2A6 and P450 reductase were used for these studies. Therefore, the reactions were not supplemented with P450 reductase. Each incubation condition was performed in triplicate (three data points per substrate concentration; n ) 3), except 50 and 400 µM (R)-NNN (n ) 2) for studies with P450 2A13. (R)-NNN, (S)-NNN, NPIP, and NPYR metabolites were formed linearly with incubation time and P450 concentration and were not formed in control incubations without the NADPH generating system. Metabolites also were not formed in the absence of P450 reductase for studies using P450 2A3, 2A4, 2A5, or 2A13 and with isolate from control insect cells not expressing P450 for studies with P450 2A6. Data Analysis. Kinetic parameters were calculated by a fit of the velocity data to the Michaelis-Menten equation using the Enzyme Kinetics Module, Version 1.1 of SigmaPlot 2001 for Windows (SPSS Inc., Chicago, IL). Km and kcat/Km values for reactions with different substrates but the same P450 or the same substrate but different P450s were compared using the

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Figure 1. Representative radioflow HPLC analyses of (A) (R)-NNN and (B) (S)-NNN metabolism catalyzed by mouse P450 2A4 and (C) NPIP and (D) NPYR metabolism catalyzed by human P450 2A13. Either [5-3H](R)- or (S)-NNN (15 µM, 0.47 Ci/mmol) was incubated with P450 2A4 (30 pmol/ml) or [3H]NPIP or [3H]NPYR (50 µM, 0.13 Ci/mmol) was incubated with P450 2A13 (25 pmol/ mL) for 30 min as described in the Experimental Procedures. Reaction mixtures of NNN, NPIP, and NPYR were analyzed by radioflow HPLC using systems 1, 2, and 3, respectively. NPIP R-hydroxylation products, 2-OH-THP and 2-OH-5-MeTHF, coeluted in reverse phase HPLC system 2. Table 3. Kinetic Parameters of (R)-NNN 2′-Hydroxylation to Keto Alcohol by P450 2Asa species

P450

Kmb (µM)

kcatb (min-1)

kcat/Kmb (min-1 µM-1)

rat mouse mouse human human

2A3 2A4 2A5 2A6 2A13

1.6 ( 0.5 66 ( 15 0.73 ( 0.43 NDc 21 ( 12

0.50 ( 0.03 0.92 ( 0.07 0.14 ( 0.02 ND 0.63 ( 0.10

0.31 ( 0.11 0.014 ( 0.003 0.19 ( 0.12 ND 0.030 ( 0.017

a Incubations were performed and analyzed as described in the Experimental Procedures. NNN concentrations were 0.3-400 µM. Concentrations of P450, given in parentheses, were 2-20 (P450 2A3), 10-50 (P450 2A4), 2-20 (P450 2A5), 6-50 (P450 2A6), and 1.5-20 (P450 2A13) pmol/mL. b Values ( SD for goodness of fit were calculated using the Enzyme Kinetics Module of SigmaPlot (SPSS Inc.) from three replicate incubations at each substrate concentration (n ) 3), except for 50 and 400 µM (R)-NNN (n ) 2) for studies with P450 2A13. c ND, not detected.

two-sample t-test (SigmaStat Version 3.00 for Windows, SPSS Inc.). P values 0.3). Conversely, P450 2A3 catalyzed (R)-NNN 5′-hydroxylation with a 4.3-fold lower Km value than (S)-NNN (p < 0.05). There were significant differences between catalytic efficiencies of (R)- and (S)-NNN 5′-hydroxylation. P450 2A4 was a 3.3-fold more efficient catalyst for lactol formation from (S)-NNN than (R)-NNN (p < 0.05). The respective kcat/Km values for (R)- and (S)-NNN were 0.015 ( 0.005 and 0.046 ( 0.011 min-1 µM-1 (Table 2). Similarly, P450 2A6 was a 7.8-fold more effective catalyst for (S)-NNN 5′-hydroxylation (p < 0.05). kcat/Km values for (R)- and (S)-NNN were 0.058 ( 0.016 and 0.45 ( 0.12 min-1 µM-1, respectively (Table 2). P450 2A5 was the most efficient catalyst of lactol formation from both NNN enantiomers among the five P450s examined. kcat/Km values were 2.5 ( 0.6 and 2.1 ( 0.7 µM min-1 µM-1, respectively, for (R)- and (S)-NNN (Table 2). In contrast, P450 2A4 was the least efficient 5′-hydroxylase of the P450 2As evaluated in this study; the kcat/Km values for (R)- and (S)-NNN were 170- and 46-fold lower, respectively, than P450 2A5 (p < 0.05; Table 2). P450 2A3 and 2A13 were also more efficient enzymes than P450 2A4. The kcat/Km values for P450 2A3catalyzed (R)- and (S)-NNN 5′-hydroxylation were 37and 21-fold higher, respectively (p < 0.05; Table 2). P450 2A13 catalyzed these reactions with 36- and 9.3-fold higher kcat/Km values, respectively (p < 0.05; Table 2). Keto alcohol formation, from 2′-hydroxylation of (R)NNN, was observed for the P450 2As in this study except P450 2A6. Km values ranged from 0.73 ( 0.43 µM for P450 2A5 to 66 ( 15 µM for P450 2A4 (Table 3). P450 2A3 and 2A5 were the most efficient catalysts with kcat/ Km values of 0.31 ( 0.11 and 0.19 ( 0.12 µM min-1 µM-1, respectively (Table 3). None of the P450 2As catalyzed 2′-hydroxylation of (S)-NNN to keto alcohol. Metabolism of NPIP and NPYR. NPIP and NPYR metabolites were quantified by HPLC with radioflow detection. NPIP R-hydroxylation was monitored by formation of 2-OH-THP plus 2-OH-5-MeTHF; the ratio of 2-OH-THP to 2-OH-5-MeTHF was approximately 3:1, as previously reported (20). A representative radioflow HPLC chromatogram for the reaction of [3H]NPIP (53 min) and P450 2A13, yielding a mixture (18 min) of the two tritiated R-hydroxylation products, is shown in Figure 1C. NPYR R-hydroxylation was quantitated by 2-OH-THF formation. A representative radioflow HPLC chromatogram for P450 2A13-catalyzed metabolism of [3H]NPYR (27 min) to [3H]2-OH-THF (10 min) is illustrated in Figure 1D. The kinetics of NPIP and NPYR R-hydroxylation catalyzed by P450 2A4, 2A5, 2A6, and 2A13 were studied. Kinetic parameters are given in Table 4. P450 2A5, 2A6, and 2A13 catalyzed NPIP R-hydroxylation with Km values of 39 ( 11, 82 ( 15, and 69 ( 15 µM, respectively (Table 4). In contrast, NPIP was a poor substrate for P450 2A4 with a Km of 850 ( 100 µM. P450 2As catalyzed NPIP R-hydroxylation with significantly lower Km values than

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NPYR. For P450 2A4, 2A5, 2A6, and 2A13, Km values for NPIP were 2.8-, 7.7-, 230-, and 7.1-fold, respectively, lower than those for NPYR (p < 0.05; Table 4). Furthermore, these P450 2A enzymes exhibited higher kcat/Km values for NPIP than NPYR (Table 4). There were 2.7and 10-fold differences in catalytic efficiencies (kcat/Km) for mouse P450 2A4 and 2A5, respectively (p < 0.05). Furthermore, human P450 2A6 and 2A13 were 9.5- and 12-fold more efficient, respectively, in NPIP R-hydroxylation relative to NPYR (p < 0.05). Comparison of NNN vs NPIP or NPYR Metabolism. There were some tremendous differences in P450catalyzed R-hydroxylation of the NNN enantiomers as compared to NPYR. P450 2A4, 2A5, 2A6, and 2A13 catalyzed (R)- and (S)-NNN 5′-hydroxylation with 208300-fold lower Km values than those of NPYR R-hydroxylation, respectively (p < 0.05; Tables 2 and 4). P450 2A5, 2A6, and 2A13 were significantly better catalysts of (R)- or (S)-NNN 5′-hydroxylation than NPYR R-hydroxylation; kcat/Km values for (R)- or (S)-NNN were 291100-fold higher (p < 0.05). For P450 2A4, only the kcat/ Km value for NPYR R-hydroxylation was significantly lower than that of (S)-NNN 5′-hydroxylation (p < 0.05); there was no statistical difference as compared to (R)NNN 5′-hydroxylation (p > 0.2). Similar to the comparison between NNN and NPYR, P450 2A4, 2A5, 2A6, and 2A13 generally catalyzed 5′hydroxylation of the NNN enantiomers more effectively than NPIP R-hydroxylation. There were 3.0-53-fold lower Km values for (R)- and (S)-NNN 5′-hydroxylation relative to NPIP R-hydroxylation (Tables 2 and 4). P450 2A5, 2A6, and 2A13 exhibited 2.4-120-fold higher kcat/ Km values for (R)- or (S)-NNN 5′-hydroxylation relative to NPIP R-hydroxylation (Tables 2 and 4). All of these kcat/Km comparisons were significant (p < 0.05) except for NPIP R-hydroxylation as compared to (S)-NNN 5′-hydroxylation catalyzed by P450 2A13. In contrast, P450 2A4 catalyzed (R)-NNN 5′-hydroxylation with a 1.8-fold lower kcat/Km value than NPIP R-hydroxylation (p < 0.05) (Tables 2 and 4).

Discussion Cytochrome P450 2As catalyze the metabolic activation of several carcinogenic nitrosamines (26-29, 33, 44-51). Previous reports demonstrated that there were differences in R-hydroxylation of (R)- and (S)-NNN and of the structural homologues, NPIP and NPYR, by P450 2A3 (26, 28). The major purpose of this study was to compare the R-hydroxylation of these cyclic nitrosamines by a series of closely related P450 2As to gain further insight into enzyme-catalyzed mechanisms of their metabolic activation. P450 2A3, 2A4, 2A5, and 2A13 catalyzed 2′- and 5′hydroxylation of (R)-NNN, whereas P450 2A6 mediated only 5′-hydroxylation (Table 2). All five P450 2As catalyzed 5′-, but not 2′-hydroxylation of (S)-NNN (Table 2). It has been reported previously that P450 2A3 catalyzed 2′- and 5′-hydroxylation of (R)-NNN with Km values of 18.7 and 11.3 µM, respectively (26). In the present study, Km values were approximately an order of magnitude lower. Differences in the concentration of P450 reductase between the previous and the current study may account for the differing results. In the previous study, there was a greater than 100-fold molar excess of P450 reductase as compared to 25-fold for the present study.

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Our results demonstrate that P450 2A5 is an excellent catalyst for (R)- and (S)-NNN R-hydroxylation whereas P450 2A4 is a poor one (Tables 2 and 3). Likewise, P450 2A5 is a significantly better catalyst for NPIP R-hydroxylation than P450 2A4 (Table 4). These differences between the catalytic activities of P450 2A4 and 2A5 have been previously reported with other P450 2A substrates. P450 2A5 is the most efficient catalyst of coumarin 7-hydroxylation (probe reaction for human P450 2A6) among the five P450 2As in this study. On the basis of kcat/Km values, P450 2A5 (7.31 min-1 µM-1) was at least 7.3-fold more efficient than P450 2A3 (1.0 min-1 µM-1), 2A6 (0.35 min-1 µM-1), or 2A13 (0.31 min-1 µM-1) (47, 52, 53). In contrast to P450 2A5, P450 2A4 does not catalyze coumarin 7-hydroxylation but is a steroid 15R-hydroxylase (54). Interestingly, Negishi and co-workers demonstrated that mutation of Phe209 in P450 2A5 to Leu (as found in P450 2A4) was sufficient to confer steroid 15R-hydroxylase activity (54). There were also marked differences in R-hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK), a powerful pulmonary lung carcinogen in mice and a putative causative agent in human lung cancer. For NNK, metabolic activation proceeds via R-hydroxylation. The Km values of P450 2A5-mediated R-hydroxylation were 4-5 µM, as compared to 67-97 µM for P450 2A4 (45), and P450 2A5 was a 52-fold more efficient NNK R-methyl hydroxylase than P450 2A4 (45). Five of 11 nonconserved amino acid residues between P450 2A4 and 2A5 lie with the substrate recognition sequence (SRS) regions, mapped by Gotoh (55). They are residues 116 (Ile for 2A4, Val for 2A5), 117 (Ala for 2A4, Val for 2A5), 209 (Leu for 2A4, Phe for 2A5), 365 (Leu for 2A4, Met for 2A5), and 481 (Val for 2A4, Ala for 2A5) (56, 57). Numerous studies with coumarin and ∆4 3-keto steroids have determined that all of these critical residues except residue 116 may play a role in P450 2A4 and 2A5 substrate specificity (57). These residues may be contributing factors in (R)-NNN, (S)-NNN, and NPIP R-hydroxylation. In particular, molecular modeling studies with NNK docked in the active site of P450 2A5 suggested that Phe209 is positioned above the heme and could bind with substrates via π-stacking (58). This interaction may align substrates for oxidation and would provide an explanation for the low Km values observed for 5′-hydroxylation of (R)- and (S)-NNN (Table 2). Molecular models of NNK in P450 2A4 suggest that the loss of the π-π interaction with the pyridine ring of NNK due to Leu at position 209 explains its higher Km values (58). This phenomenon may also explain the kinetic parameters observed for NNN 5′-hydroxylation catalyzed by P450 2A4 (Table 2). For each substrate, Km and kcat/Km values for R-hydroxylation catalyzed by P450 2A3 were similar to those of P450 2A5 (Tables 2-4). These P450s differ by 19 amino acids (Table 1), but they contain identical SRS residues except position 117 where P450 2A3 and 2A5 contain Ala and Val, respectively (55, 56, 59). The near-identical SRS regions may account for the expectedly similar Km values. Our studies are consistent with previous reports demonstrating that P450 2A3 and 2A5 catalyzed coumarin 7-hydroxylation with similar Km values (1.3 vs 1.9 µM) (41, 47). Comparable Km values (4-5 µM) were also reported for NNK R-hydroxylation catalyzed by P450 2A3 and 2A5 (44, 45). P450 2A6 and 2A13 catalyzed NNK R-hydroxylation with major differences in Km values. Although these

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Figure 2. (R)-NNN, (S)-NNN, NPIP, and NPYR metabolism: Comparison of catalytic efficiencies. Nitrosamines were ordered according to kcat/Km values of (R)- or (S)-NNN 5′-hydroxylation or NPIP or NPYR R-hydroxylation for the given P450 (Tables 2-4). Greater than (>) and equal ()) symbols were placed between kcat/Km values of substrates that were significantly different (p < 0.05) and not significantly different (p g 0.05), respectively. Boldface indicates the most efficient catalysts of R-hydroxylation of a given nitrosamine among P450 2A3, 2A4, 2A5, 2A6, and 2A13. akcat/Km values of both (R)- and (S)-NNN are greater than that of NPIP. bOnly the kcat/Km value of (R)NNN is greater than that of NPIP.

enzymes share 93.5% sequence identity (Table 1), the Km values of P450 2A13 were 33-44-fold lower than those of P450 2A6 (44, 46). Likewise, P450 2A13 had a 13-fold lower Km value for coumarin 7-hydroxylation (0.48 vs 6.0 µM) (52, 53). Ten of the 32 nonconserved residues lie within the SRS regions and may account for the dramatic differences in Km values (33, 55, 60). Consistent with these studies, we observed a 37-fold lower Km value for NPYR R-hydroxylation by P450 2A13 than 2A6 (Table 4). Furthermore, P450 2A13 mediated 2′-hydroxylation of (R)-NNN whereas P450 2A6 did not (Table 3). However, this relationship was not universally observed. There were no significant differences in Km values for (R)NNN 5′-hydroxylation and NPIP R-hydroxylation by P450 2A6 vs those by P450 2A13 (Tables 2 and 4). If the differences in Km values for P450 2A6 and 2A13 are reflective of their relative enzyme-substrate affinities, then these kinetic data may prove useful in understanding the profound effects of the respective SRS residues on NNN, NPIP, and NPYR activation. There were striking differences between R-hydroxylation activity of NNN and that of NPIP or NPYR. A summary of the relative catalytic efficiencies for (R)- and (S)-NNN 5′-hydroxylation and NPIP and NPYR R-hydroxylation by P450 2As is presented in Figure 2. P450 2A3, 2A5, 2A6, and 2A13 were more efficient catalysts of NNN 5′-hydroxylation than NPIP or NPYR R-hydroxylation (Tables 2 and 4). One possible contributing factor for these differences is the presence of NNN’s pyridine substituent. As mentioned earlier, Phe209 of P450 2A5 may aid to coordinate NNN over the heme via π-π interaction. This interaction with NNN also may occur with P450 2A3, 2A6, and 2A13, which contain Phe at position 209 (33, 59, 60). P450 2A4, 2A5, 2A6, and 2A13 were more efficient R-hydroxylases of NPIP relative to NPYR (Figure 2 and Table 4). As previously reported, P450 2A3 also preferentially catalyzed NPIP R-hydroxylation (28). There are several possible explanations for these differences in catalytic activities. First, the conserved structural features found in this P450 subfamily may favor the substrate binding of NPIP relative to NPYR. Second, NPIP may sit in a better position than NPYR in the substrate binding site to permit hydrogen abstraction, a critical step in P450 catalysis. One possibility is that the C-2 hydrogen atom of NPIP, when bound, may be closer

NNN, NPIP, and NPYR Activation by P450 2As

to the P450-bound heme for its abstraction than that of NPYR. Third, the chair conformation of NPIP may provide easier access for the heme than the puckered one of NPYR. We and others have previously suggested that the efficient metabolic activation of NNN and NPIP in target tissues leads to tissue selective tumor induction. In the mouse, previous studies have shown that P450 2A5 is an important catalyst of NNK activation in the lung, the target tissue of NNK-induced carcinogenesis (45). P450 2A5 protein is expressed in the mouse lung (31). This P450 is a good catalyst of NNN and NPIP R-hydroxylation (Tables 2-4) and therefore may be important for NNN and NPIP activation in this tissue. NNN and NPIP are effective lung carcinogens in mice (2, 3). In contrast, P450 2A4 may not contribute significantly to NNN and NPIP activation relative to P450 2A5. P450 2A4 is a poor catalyst for their activation (Tables 2-4) and its protein levels in the lung are lower than those of P450 2A5 (31). Further studies are necessary to investigate the individual contributions of mouse lung P450 2A4 and P450 2A5 toward NNN and NPIP metabolic activation. In rats, P450 2A3 is an important catalyst of NPIP activation and probably NNN activation in the nasal cavity, a target tissue for their carcinogenicity. P450 2A3 is a major P450 in the rat nose (30). It has been previously shown that this P450, which mediated NPIP R-hydroxylation with low Km and high kcat/Km values, is the principal catalyst of NPIP activation in the nasal cavity (20, 28). For NNN, P450 2A3 has been shown previously to exhibit high R-hydroxylase activity, suggesting that this P450 plays a significant role in its activation (26). The results of the present study (Tables 2 and 3) are consistent with this hypothesis. Conversely, poor NPYR R-hydroxylase activity for P450 2A3 is consistent with lower nasal tumorigenicity (20). Our findings represent the first kinetic data identifying human enzymes, P450 2A6 and 2A13, as catalysts of NPIP activation. The Km values were 82 ( 15 and 69 ( 15 µM, respectively (Table 4). P450 2A6 and 2A13 both may contribute to NPIP activation in the esophagus; this nitrosamine may be a causative agent in human esophageal cancer. P450 2A6 mRNA has been detected in the esophagus (34) whereas P450 2A13 mRNA has not been identified. P450 2A protein, which presumably consists of P450 2A6 and possibly P450 2A13, was detected in human esophageal mucosa by immunoblot analysis (61). P450 2E1 may also catalyze NPIP activation in humans. NPIP was strongly mutagenic in Salmonella strains coexpressing human P450 2E1 (62). Yet, kinetic parameters of NPIP activation by this P450 have not been reported. For NNN, a target tissue in humans is also believed to be the esophagus. 2′-Hydroxylation is thought to be an important bioactivation pathway of NNN. Our studies demonstrate that P450 2A13 catalyzes 2′-hydroxylation of (R)-NNN, which is present at levels up to 25% of total NNN in tobacco products, with a low Km value of 21 µM (Table 3). If P450 2A13 is present in the esophagus, then it may be involved in NNN activation. Other P450s that may have some role in NNN activation in the esophagus are P450 1A1 and 1A2 (63). P450 1A2 exhibited low 2′hydroxylation activity (64). NNN was mutagenic in Salmonella strains coexpressing P450 1A1 (62). Human P450 3A4, which is the main catalyst of NNN 2′hydroxylation in liver microsomes, is not expressed in

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the esophagus (27, 61). 5′-Hydroxylation also may be an NNN activation pathway; recently, dGuo adducts generated from this reaction were identified in our laboratory (unpublished results). P450 2A6 and 2A13 are good catalysts of this reaction (Table 2). The metabolism of (R)- and (S)-NNN by individual P450 2As may be useful to predict the metabolism of other important xenobiotics such as nicotine. Nicotine, an addictive agent in tobacco products, is an analogue of NNN with an N-methyl group substituted for the Nnitroso moiety. (S)-(-)-Nicotine is the predominant form in tobacco and nicotine replacement therapy products, with up to 1.2% of total nicotine present as the (R)enantiomer (65). Nicotine metabolism proceeds predominantly via 5′-hydroxylation to cotinine and its metabolites (66). As previously described, P450 2A6 catalyzes predominantly 5′-hydroxylation of (S)-(-)-nicotine with small levels of 2′-hydroxylation (67). 2′-Hydroxylation is of particular toxicological importance because this pathway has been shown to yield 4-(methylamino)-1-(3-pyridyl)1-butanone, an NNK precursor (67). As stated above, NNK is a potent pulmonary carcinogen in mice. It has been suggested previously that the metabolism of nicotine catalyzed by P450 2As would be similar to that of NNN (68). It is plausible that NNN and nicotine bind to the active site in the same orientation. Analogous to NNN metabolism, P450 2A3, 2A4, 2A5, and 2A13 may catalyze the 2′-hydroxylation of (R)-(+)-nicotine. Our findings suggest that these enzymes may generate an NNK precursor from (R)-(+)-nicotine. In summary, the results of this study demonstrate that there are striking differences in P450 2A-mediated metabolic activation between (R)- and (S)-NNN, NPIP, and NPYR. Our findings may prove to be important in assessing the risk of human exposure to these carcinogenic nitrosamines. Molecular modeling studies of P450 2As may provide further insight on the mechanisms mediating the metabolic activation of (R)-NNN, (S)-NNN, NPIP, and NPYR.

Acknowledgment. We thank Pramod Upadhyaya for his assistance in purifying the tritium-labeled NNN enantiomers. We are grateful to Xinxin Ding for the generous gifts of P450 2A3 and 2A13. This study was supported by Grant CA-85702 from the National Cancer Institute.

References (1) Hecht, S. S. (1998) Biochemistry, biology, and carcinogenicity of tobacco specific N-nitrosamines. Chem. Res. Toxicol. 11, 559-603. (2) Greenblatt, M., and Lijinsky, W. (1972) Failure to induce tumors in Swiss mice after concurrent administration of amino acids and sodium nitrite. J. Natl. Cancer Inst. 48, 1389-1392. (3) Takayama, S. (1969) Induction of tumors in ICR mice with N-nitrosopiperidine, especially in forestomach. Naturwissenschaften 56, 142. (4) Gray, R., Peto, R., Brantom, P., and Grasso, P. (1991) Chronic nitrosamine ingestion in 1040 rodents: The effect of the choice of nitrosamine, the species studied, and the age of starting exposure. Cancer Res. 51, 6470-6491. (5) Lijinsky, W., and Taylor, H. W. (1976) The effect of substituents on the carcinogenicity of N-nitrosopyrrolidine in Sprague-Dawley rats. Cancer Res. 36, 1988-1990. (6) Spiegelhalder, B., and Bartsch, H. (1996) Tobacco-specific nitrosamines. Eur. J. Cancer Prev. 5, 33-38. (7) Hecht, S. S., and Hoffmann, D. (1988) Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis 9, 875-884.

68

Chem. Res. Toxicol., Vol. 18, No. 1, 2005

(8) Carmella, S. G., McIntee, E. J., Chen, M., and Hecht, S. S. (2000) Enantiomeric composition of N′-nitrosonornicotine and N′-nitrosoanatabine in tobacco. Carcinogenesis 21, 839-843. (9) Gough, T. A., Webb, K. S., and Coleman, R. F. (1978) Estimate of the volatile nitrosamine content of UK food. Nature 272, 161163. (10) Carmella, S. G., Borukhova, A., Desai, D., and Hecht, S. S. (1997) Evidence for endogenous formation of tobacco-specific nitrosamines in rats treated with tobacco alkaloids and sodium nitrite. Carcinogenesis 18, 587-592. (11) Tricker, A. R., Pfundstein, B., Kalble, T., and Preussmann, R. (1992) Secondary amine precursors to nitrosamines in human saliva, gastric juice, blood, urine, and faeces. Carcinogenesis 13, 563-568. (12) Hecht, S. S., Villalta, P. W., Sturla, S. J., Cheng, G., Yu, N., Upadhyaya, P., and Wang, M. (2004) Identification of O2substituted pyrimidine adducts formed in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol with DNA. Chem. Res. Toxicol. 17, 588-597. (13) Wang, M., Cheng, G., Sturla, S. J., Shi, Y., McIntee, E. J., Villalta, P. W., Upadhyaya, P., and Hecht, S. S. (2003) Identification of adducts formed by pyridyloxobutylation of deoxyguanosine and DNA by 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a chemically activated form of tobacco specific carcinogens. Chem. Res. Toxicol. 16, 616-626. (14) Hecht, S. S., and Young, R. (1982) Regiospecificity in the metabolism of the homologous cyclic nitrosamines, N′-nitrosonornicotine and N′-nitrosoanabasine. Carcinogenesis 3, 11951199. (15) Young-Sciame, R., Wang, M., Chung, F. L., and Hecht, S. S. (1995) Reactions of R-acetoxy-N-nitrosopyrrolidine and R-acetoxy-Nnitrosopiperidine with deoxyguanosine: Formation of N2-tetrahydrofuranyl and N2-tetrahydropyranyl adducts. Chem. Res. Toxicol. 8, 607-616. (16) Wang, M., Young-Sciame, R., Chung, F. L., and Hecht, S. S. (1995) Formation of N2-tetrahydrofuranyl and N2-tetrahydropyranyl adducts in the reactions of R-acetoxy-N-nitrosopyrrolidine and R-acetoxy-N-nitrosopiperidine with DNA. Chem. Res. Toxicol. 8, 617-624. (17) Hecht, S. S., Young-Sciame, R., and Chung, F. L. (1992) Reaction of R-acetoxy-N-nitrosopiperidine with deoxyguanosine: Oxygendependent formation of 4-oxo-2-pentenal and a 1,N2-ethenodeoxyguanosine adduct. Chem. Res. Toxicol. 5, 706-712. (18) Liu, Z., Young-Sciame, R., and Hecht, S. S. (1996) Liquid chromatography-electrospray ionization mass spectrometric detection of an ethenodeoxyguanosine adduct and its hemiaminal precursors in DNA reacted with R-acetoxy-N-nitrosopiperidine and cis-4-oxo-2-pentenal. Chem. Res. Toxicol. 9, 774-780. (19) Leung, K. H., Park, K. K., and Archer, M. C. (1978) R-Hydroxylation in the metabolism of N-nitrosopiperidine by rat liver microsomes: Formation of 5-hydroxypentanal. Res. Commun. Chem. Pathol. Pharmacol. 19, 201-211. (20) Wong, H. L., Murphy, S. E., and Hecht, S. S. (2003) Preferential metabolic activation of N-nitrosopiperidine compared to its structural homologue N-nitrosopyrrolidine by rat nasal microsomes. Chem. Res. Toxicol. 16, 1298-1305. (21) Wang, M., McIntee, E. J., Shi, Y., Cheng, G., Upadhyaya, P., Villalta, P. W., and Hecht, S. S. (2001) Reactions of R-acetoxyN-nitrosopyrrolidine with deoxyguanosine and DNA. Chem. Res. Toxicol. 14, 1435-1445. (22) Wang, M., and Hecht, S. S. (1997) A cyclic N7,C-8 guanine adduct of N-nitrosopyrrolidine (NPYR): Formation in nucleic acids and excretion in the urine of NPYR-treated rats. Chem. Res. Toxicol. 10, 772-778. (23) Wang, M., Chung, F. L., and Hecht, S. S. (1992) Formation of 7-(4-oxobutyl)guanine in hepatic DNA of rats treated with Nnitrosopyrrolidine. Carcinogenesis 13, 1909-1911. (24) Hecht, S. S., McCoy, G. D., Chen, C. H., and Hoffmann, D. (1981) The metabolism of cyclic nitrosamines. In N-Nitroso Compounds (Scanlan, R. A., and Tannenbaum, S. R., Eds.) pp 49-75, American Chemical Society, Washington, DC. (25) Wang, M. Y., Chung, F. L., and Hecht, S. S. (1988) Identification of crotonaldehyde as a hepatic microsomal metabolite formed by R-hydroxylation of the carcinogen N-nitrosopyrrolidine. Chem. Res. Toxicol. 1, 28-31. (26) Murphy, S. E., Issa, I. S., Ding, X., and McIntee, E. J. (2000) Specificity of cytochrome P450 2A3-catalyzed R-hydroxylation of N′-nitrosonornicotine enantiomers. Drug Metab. Dispos. 28, 12631266. (27) Patten, C. J., Smith, T. J., Friesen, M. J., Tynes, R. E., Yang, C. S., and Murphy, S. E. (1997) Evidence for cytochrome P450 2A6

Wong et al.

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39) (40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

and 3A4 as major catalysts for N′-nitrosonornicotine alphahydroxylation by human liver microsomes. Carcinogenesis 18, 1623-1630. Wong, H. L., Murphy, S. E., Wang, M., and Hecht, S. S. (2003) Comparative metabolism of N-nitrosopiperidine and N-nitrosopyrrolidine by rat liver and esophageal microsomes and cytochrome P450 2A3. Carcinogenesis 24, 291-300. Flammang, A. M., Gelboin, H. V., Aoyama, T., Gonzalez, F. J., and McCoy, G. D. (1993) N-Nitrosopyrrolidine metabolism by cDNA-expressed human cytochrome P450s. Biochem. Arch. 9, 197-204. Gopalakrishnan, R., Morse, M. A., Lu, J., Weghorst, C. M., Sabourin, C. L., Stoner, G. D., and Murphy, S. E. (1999) Expression of cytochrome P450 2A3 in rat esophagus: Relevance to N-nitrosobenzylmethylamine. Carcinogenesis 20, 885-891. Su, T., Sheng, J. J., Lipinskas, T. W., and Ding, X. (1996) Expression of CYP2A genes in rodent and human nasal mucosa. Drug Metab. Dispos. 24, 884-890. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Causasians. J. Pharmacol. Exp. Ther. 270, 414-423. Su, T., Bao, Z., Zhang, Q., Smith, T. J., Hong, J., and Ding, X. (2000) Human cytochrome P450 CYP2A13: Predominant expression in the respiratory tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone. Cancer Res. 60, 5074-5079. Godoy, W., Albano, R. M., Moraes, E. G., Pinho, P. R. A., Nunes, R. A., Saito, E. H., Higa, C., Filho, I. M., Krue, C. D. P., Schirmer, C. C., Gurski, R., Lang, M. A., and Pinto, L. F. R. (2002) CYP2A6/ 2A7 and CYP2E1 expression in human oesophageal mucosa: Regional and inter-individual variation in expression and relevance to nitrosamine metabolism. Carcinogenesis 23, 611-616. Wong, H. L., Zhang, X., Zhang, Q.-Y., Gu, J., Ding, X., Hecht, S. S., and Murphy, S. E. (2004) Metabolic activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cytochrome P450 ZA13 in human fetal nasal microsomes, submitted for publication. McIntee, E. J., and Hecht, S. S. (2000) Metabolism of N′nitrosonornicotine enantiomers by cultured rat esophagus and in vivo in rats. Chem. Res. Toxicol. 13, 192-199. Hecht, S. S., Chen, C. B., Dong, M., Ornaf, R. M., Hoffmann, D., and Tso, T. C. (1977) Studies on nonvolatile nitrosamines in tobacco. Beitr. Tabakforsch. 9, 1-6. Chen, C. B., Hecht, S. S., and Hoffmann, D. (1978) Metabolic R-hydroxylation of the tobacco-specific carcinogen, N-nitrosonornicotine. Cancer Res. 38, 3639-3645. Normant, H. (1949) Preparation and reactions of 2,3-dihydrofuran. C. R. Hebd. Seances Acad. Sci. 228, 102-104. Schniepp, L. E., and Geller, H. H. (1946) Preparation of dihydropyran, δ-hydroxyvaleraldehyde and 1,5-pentanediol from tetrahydrofurfuryl alcohol. J. Am. Chem. Soc. 68, 1646-1648. Felicia, N. D., Rekha, G. K., and Murphy, S. E. (2000) Characterization of cytochrome P450 2A4 and 2A5 catalyzed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism. Arch. Biochem. Biophys. 384, 418-424. Hu, M. W., Bondinell, W. E., and Hoffmann, D. (1973) Chemical studies on tobacco smoke XXIII. Synthesis of carbon-14 labeled myosamine, nornicotine, and N′-nitrosonornicotine. J. Labelled Compd. Radiopharm. 10, 79-88. Hecht, S. S., Lin, D., and Chen, C. B. (1981) Comprehensive analysis of urinary metabolites of N′-nitrosonornicotine. Carcinogenesis 2, 833-838. Jalas, J. R., Ding, X., and Murphy, S. E. (2003) Comparative metabolism of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL) by rat cytochrome P450 2A3 and human P450 2A13. Drug Metab. Dispos. 31, 1199-1202. Jalas, J. R., McIntee, E. J., Kenney, P. M. J., Upadhyaya, P., Peterson, L. A., and Hecht, S. S. (2003) Stereospecific deuterium substitution attenuates the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone (NNK). Chem. Res. Toxicol. 16, 794-806. Patten, C. J., Smith, T. J., Murphy, S. E., Wang, M. H., Lee, J., Tynes, R. E., Koch, P., and Yang, C. S. (1996) Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes. Arch. Biochem. Biophys. 333, 127-138. von Weymarn, L. B., Felicia, N. D., Ding, X., and Murphy, S. E. (1999) N-Nitrosobenzylmethylamine hydroxylation and coumarin

NNN, NPIP, and NPYR Activation by P450 2As

(48)

(49)

(50)

(51)

(52) (53)

(54) (55)

(56)

(57)

7-hydroxylation: Catalysis by rat esophageal microsomes and cytochrome P450 2A3 and 2A6 enzymes. Chem. Res. Toxicol. 12, 1254-1261. Chen, S. C., Wang, X., Xu, G., Zhou, L., Vennerstrom, J. L., Gonzalez, F., Gelboin, H. V., and Mirvish, S. S. (1999) Depentylation of [3H-pentyl]methyl-n-amylnitrosamine by rat esophageal and liver microsomes and by rat and human cytochrome P450 isoforms. Cancer Res. 59, 91-98. Camus, A.-M., Geneste, O., Hankakoski, P., Be´re´ziat, J.-C., Henderson, C. J., Wolf, C. R., Bartsch, H., and Lang, M. A. (1993) High variability of nitrosamine metabolism among individuals: Role of cytochromes P450 2A6 and 2E1 in the dealkylation of N-nitrosodimethylamine and N-nitrosodiethylamine in mice and humans. Mol. Carcinog. 7, 268-275. Hong, J.-Y., Ding, X., Smith, T. J., Coon, M. J., and Yang, C. S. (1992) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, by rabbit nasal microsomes and cytochrome P450s NMa and NMb. Carcinogenesis 13, 2141-2144. Bellec, G., Dreano, Y., Lozach, P., Menez, J. F., and Berthou, F. (1996) Cytochrome P450 metabolic dealkylation of nine Nnitrosodialkylamines by human liver microsomes. Carcinogenesis 17, 2029-2034. von Weymarn, L. B., and Murphy, S. E. (2003) CYP2A13-catalysed coumarin metabolism: Comparison with CYP2A5 and CYP2A6. Xenobiotica 33, 73-81. Zhuo, X., Gu, J., Zhang, Q.-Y., Spink, D. C., Kaminsky, L. S., and Ding, X. (1999) Biotransformation of coumarin by rodent and human cytochrome P-450: Metabolic basis of tissue-selective toxicity in olfactory mucosa of rats and mice. J. Pharmacol. Exp. Ther. 288, 463-471. Lindberg, R. L. P., and Negishi, M. (1989) Alteration of mouse cytochrome P450coh substrate specificity by mutation of a single amino acid residue. Nature 339, 632-634. Gotoh, O. (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J. Biol. Chem. 267, 83-90. Lindberg, R., Burkhart, B., Ichikawa, T., and Negishi M. (1989) The structure and characterization of type I P-450(15) alpha gene as major steroid 15 alpha-hydroxylase and its comparison with type II P-450(15) alpha gene. J. Biol. Chem. 264, 6465-6471. Negishi, M., Uno, T., Honkakoski, P., Sueyoshi, T., Darden, T. A., and Pedersen, L. P. (1996) The roles of individual amino acids in altering substrate specificity of the P450 2a4/2a5 enzymes. Biochimie 78, 685-694.

Chem. Res. Toxicol., Vol. 18, No. 1, 2005 69 (58) Jalas, J. R., Seetharaman, M., Hecht, S. S., and Murphy, S. E. (2004) Molecular modeling of CYP2A enzymes: Application to metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Xenobiotica 34, 515533. (59) Ueno, T., and Gonzalez, F. (1990) Complete sequence of the rat CYP2A3 gene specifically transcribed in lung. Nucleic Acids Res. 18, 4623-4624. (60) Yamano, S., Tatsuno, J., and Gonzalez, F. J. (1990) The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29, 1322-1329. (61) Lechevrel, M., Casson, A. G., Wolf, C. R., Hardie, L. J., Flinterman, M. B., Montesano, R., and Wild, C. P. (1999) Characterization of cytochrome P450 expression in human oesophageal mucosa. Carcinogenesis 20, 243-248. (62) Fujita, K. K., and Kamataki, T. (2001) Predicting the mutagenicity of tobacco-related N-nitrosamines in humans using 11 strains of Salmonella typhimurium YG7108, each coexpressing a form of human cytochrome P450 along with NADPH-cytochrome P450 reductase. Environ. Mol. Mutagen. 38, 339-346. (63) Wei, C., Caccavale, R. J., Kehoe, J. J., Thomas, P. E., and Iba, M. M. (2001) CYP1A2 is expressed along with CYP1A1 in the human lung. Cancer Lett. 171, 113-120. (64) Smith, T. J., Guo, Z., Guengerich, F. P., and Yang, C. S. (1996) Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by human cytochrome P450 1A2 and its inhibition by phenethyl isothiocyanate. Carcinogenesis 17, 809-813. (65) Armstrong, D. W., Wang, X., Lee, J., and Liu, Y. (1999) Enantiomeric composition of nornicotine, anatabine, and anabasine in tobacco. Chirality 11, 82-84. (66) Gorrod, J. W., and Schepers, G. (1999) Biotransformation of nicotine in mammalian systems. In Analytical Determination of Nicotine and Related Compounds and Their Metabolites (Gorrod, J. W., and Jacob, P., III, Eds.) pp 45-67, Elsevier, Amsterdam. (67) Hecht, S. S., Hochalter, J. B., Villalta, P. W., and Murphy, S. E. (2000) 2′-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: Formation of a lung carcinogen precursor. Proc. Natl. Acad. Soc. 97, 12493-12497. (68) Murphy, S. E., Johnson, L. M., and Pullo, D. A. (1999) Characterization of multiple products of cytochrome P450 2A6-catalyzed cotinine metabolism. Chem. Res. Toxicol. 12, 639-645.

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