Metabolic Activation of the Tobacco Carcinogen 4-(Methylnitrosamino

Publication Date (Web): May 13, 2005 ... P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo...
0 downloads 0 Views 155KB Size
Download PDF
JUNE 2005 VOLUME 18, NUMBER 6 © Copyright 2005 by the American Chemical Society

Communications Metabolic Activation of the Tobacco Carcinogen 4-(Methylnitrosamino)-(3-pyridyl)-1-butanone by Cytochrome P450 2A13 in Human Fetal Nasal Microsomes Hansen L. Wong,†,‡ Xiuling Zhang,‡,§ Qing-Yu Zhang,§ Jun Gu,§ Xinxin Ding,§ Stephen S. Hecht,† and Sharon E. Murphy*,† The Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, and Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York at Albany, Albany, New York 12201 Received March 17, 2005

Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) R-hydroxylation. This reaction is a key bioactivation pathway in NNK-induced carcinogenesis. P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo. Therefore, we studied NNK R-hydroxylation in human fetal nasal mucosal microsomes, which have been shown to contain high levels of P450 2A protein, presumed to be a mixture of P450 2A6 and 2A13. The microsomes efficiently catalyzed NNK R-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited R-hydroxylation by these microsomes greater than 90%. Km and Vmax values for R-methylene hydroxylation were 6.5 ( 1.1 µM and 3.0 ( 0.1 pmol/min/mg; for R-methyl hydroxylation, they were 6.7 ( 0.8 µM and 0.85 ( 0.03 pmol/min/ mg. The Km values agree closely with those for NNK metabolism by P450 2A13. Using a new technique, we separated P450 2A13 from P450 2A6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Quantitative immunoblot analysis indicated that the level of P450 2A13 in the pooled fetal nasal microsome sample used for kinetic analysis was approximately 1.6 pmol/mg protein. In the same sample, P450 2A6 was not detected (detection limit, 67 fmol/mg protein). These kinetic, immunoinhibition, and immunoblot data confirm that P450 2A13 is a functional enzyme and the catalyst of NNK R-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK R-hydroxylation in a human tissue.

Introduction 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK;1 Scheme 1) is believed to play an important role in the * To whom correspondence should be addressed. Tel: 612-624-7633. Fax: 612-626-5135. E-mail: [email protected]. † University of Minnesota. ‡ The first and second authors contributed equally to this work. § Wadsworth Center, New York State Department of Health.

induction of cancer in people who use tobacco products. NNK is a potent systemic lung carcinogen in rats, inducing tumors at total doses approaching those to which smokers are exposed (1). It also readily induces 1Abbreviations: NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; OPB, 4-(3-pyridyl)-4-oxobutanal; SDS, sodium dodecyl sulfate.

10.1021/tx0500777 CCC: $30.25 © 2005 American Chemical Society Published on Web 05/13/2005

914

Chem. Res. Toxicol., Vol. 18, No. 6, 2005

Communications

Scheme 1. r-Hydroxylation of NNK

tumors of the nasal mucosa, liver, and pancreas in rats (1). NNK is in addition a well-established pulmonary carcinogen in mice and hamsters (1). The potent carcinogenicity of NNK, particularly in the rat, and its occurrence in substantial amounts in both unburned tobacco and tobacco smoke, support its role as a cause of lung cancer and other cancers in people who use tobacco products. Metabolism is essential for NNK carcinogenicity in the lung and nasal mucosa of laboratory animals (1, 2). Extensive studies clearly demonstrate that R-hydroxylation is the key metabolic step leading to the formation of DNA adducts that are critical for carcinogenicity in these tissues (1). R-Hydroxylation is catalyzed by P450 enzymes. Hydroxylation of NNK at the R-methylene carbon results in a DNA methylating agent and 4-(3pyridyl)-4-oxobutanal (OPB; Scheme 1). R-Methyl hydroxylation produces DNA pyridyloxobutylating species and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB; Scheme 1). Another NNK transformation pathway is carbonyl reduction, yielding 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), itself a potent rat lung carcinogen; NNAL formation is catalyzed mainly by enzymes other than P450s. On the basis of these data, the role of NNK as a human carcinogen in the lung, nasal mucosa, and perhaps other tissues, would depend on its metabolism by R-hydroxylation in these tissues. Several published studies to date demonstrate NNK R-hydroxylation by microsomal preparations from human liver, lung, and other tissues (2). However, the efficiency of the reaction is generally low and Km values, where reported, are usually high, raising some doubts about parallel mechanisms in humans and laboratory animals (2). Although several human P450s, including P450 1A2 and P450 2A6, catalyze NNK R-hydroxylation, P450 2A13 stands out as the most efficient.

Heterologously expressed P450 2A13 catalyzes NNK metabolism to OPB and HPB with low Km and high Vmax/ Km values similar to those of rat P450 2A3, which is implicated as a key enzyme in rat lung and nasal carcinogenesis by NNK (2-4). In contrast, P450 2A6 is a more than 500-fold less efficient catalyst. P450 2A13 mRNA has been detected at relatively high levels in human nasal mucosa and lung as compared to the liver (3) and has been suggested to play a key role in NNK activation in humans, particularly in the respiratory tract. However, because of the low level of P450 protein expression in extrahepatic tissues and the 94% sequence identity between P450 2A6 and P450 2A13, it has been difficult to demonstrate that P450 2A13 protein is expressed in human tissues. The studies described here identify P450 2A13 as a functional enzyme in vivo and demonstrate that P450 2A13 present in microsomal preparations of human fetal nasal mucosa efficiently catalyze NNK R-hydroxylation.

Materials and Methods Chemicals and Enzymes. [5-3H]NNK (10.3 Ci/mmol) purchased from Moravek Biochemicals (Brea, CA) was purified to >99.9% radiochemical purity by reverse phase HPLC (4). Unlabeled NNK and metabolite standards were prepared as previously described (4, 5). Microsomes containing expressed P450 2A6 or 2A13 and polyclonal antibodies against mouse P450 2A5 were prepared as described (3, 6, 7). This antibody recognizes both P450 2A6 and P450 2A13 but does not recognize a number of other human P450s on immunoblots, including 1A1, 1A2, 1B1, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11 (3, 8). NADPH-P450 oxidoreductase was obtained from Panvera (Madison, WI). Picofluor 40 scintillation fluid was purchased from Perkin-Elmer Life Sciences (Boston, MA). All other chemicals and enzymes were purchased from Sigma-Aldrich Co. or Fisher Scientific Chemical Co. (Pittsburgh, PA). Isolation of Microsomes. Human fetal nasal mucosa and adult human liver tissues were provided by the University of

Communications Washington Birth Defects Research Laboratory and the National Cancer Institute Cooperative Human Tissue Network, respectively. The gestational age of the fetus from which tissue was available ranged from 90 to 120 days. The fetal nasal tissue and microsomes were prepared as described previously (9, 10). Identification of nasal tissues was confirmed by immunohistochemical detection of the olfactory marker protein (9). Between 100 and 200 mg of nasal tissue was obtained from each fetus and about 3 µg microsomal protein per mg tissue. Microsomes were stored at -80 °C prior to use. There were six preparations of fetal nasal microsomes, from single or pooled samples (gestational age listed in parentheses) as follows: #1, five subjects (92, 98, 98, 110, and 120 days), three males, one female, and one of unknown gender, ethnicity unknown; #2, two male subjects (103 and 108 days), ethnicity unknown; #3, one Caucasian male subject, (108 days); #4, one Caucasian male subject (108 days); #5, one Caucasian female subject (105 days); and #6, one Caucasian female subject (92 days). Adult liver microsomes were prepared from tissue from a 61 year old Caucasian male (HLM1) or a 38 year old Caucasian female (HLM2). In Vitro Metabolism of NNK. Kinetic parameters were determined from three independent experiments using microsomes (preparation #1) that had not been thawed prior to use. Reactions were carried out as previously described with some modifications (11). Briefly, 5-60 µM [5-3H]NNK (1.6-10 µCi, 0.6-10.3 Ci/mmol) and 167-250 µg/mL microsomal proteins were incubated in 0.5 mL of 50 mM Tris buffer, pH 7.4, 5 mM MgCl2, 1 mM EDTA, and 5 mM sodium bisulfite (to trap OPB). Reactions were initiated with the addition of an NADPH generating system (0.4 mM NADP+, 0.4 units/mL glucose 6-phosphate dehydrogenase, 10 mM glucose 6-phosphate). After 60 min, reactions were terminated, centrifuged, and analyzed by HPLC with radioflow detection, as previously described (4). To confirm the identity of OPB and HPB, HPLC fractions coeluting with the respective metabolite standards were collected, allowed to react with NaBH4, and then reanalyzed for the reduction products as described previously (5). NNK metabolites were not formed in control incubations without an NADPH generating system, without microsomes, or with heattreated (100 °C) microsomes. Microsomal protein concentrations and incubation times were varied to confirm that kinetic studies were performed under initial rate conditions. Rates of OPB formation were corrected for a small impurity in [5-3H]NNK, which coeluted with OPB. Kinetic parameters were calculated as described (4). Two immunoinhibition studies were performed as described above with the following modifications: One, preparation #1 (167 µg/mL) and [3H]NNK (5 µM; 3.33 Ci/mmol) and cofactors were incubated with or without P450 2A5 antibodies (51.4 µg/ mL). Two, preparation #2 (200 µg/mL), [5-3H]NNK (5 µM, 2.5 Ci/mmol), and cofactors were incubated with 0, 25.7, or 51.4 µg/ mL anti-P450 2A5 IgG. Immunoblot Analysis of P450 2A6 and 2A13. High resolution sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed using a DNA sequencing apparatus (Bio-Rad Laboratories, Hercules, CA) for separating P450 2A6 and P450 2A13 proteins. Microsomal proteins (100 ng to 15 µg) were added to Leammli sample buffer (Bio-Rad Laboratories) containing 5% β-mercaptoethanol, and the mixture was heated (100 °C for 5 min). Samples were analyzed on 10% SDS-polyacrylamide (ratio of acrylamide to bis-acrylamide equals 37.5:1) gels (0.4 or 0.75 mm thick), using the standard Laemmli buffers (12). Electrophoresis was carried out at room temperature and in the constant current mode, at 20 mA for stacking and 7-15 mA for separation for 0.4 mm gels or at 15 mA for stacking and 11-18 mA for separation for 0.75 mm gels. For running 0.75 mm gels, a cooling fan was used, and the electrophoresis buffer was replenished at least three times during separation. After electrophoresis, proteins were transferred to a nitrocellulose membrane (0.45 µm, Bio-Rad Laboratories). For immunoblot quantification of P450 2A13 in the

Chem. Res. Toxicol., Vol. 18, No. 6, 2005 915

Figure 1. Representative HPLC-radioflow chromatographic analyses of reaction mixtures containing [3H]NNK (5 µM; 3.33 Ci/mmol) and human fetal nasal microsomes (167 µg/mL; preparation #1) in the absence (gray trace) or presence (white trace) of antibodies against mouse P450 2A5 (near-maximal inhibitory concentration of 51.4 µg/mL). Total radioactivity injected for HPLC analysis, 7.6-7.8 × 106 DPM. OPB was detected as its bisulfite adduct. pooled human fetal nasal microsomes (preparation #1), electrophoresis was performed using a mini-gel system (Mini PROTEAN 3 cell, Bio-Rad Laboratories). Immunoblot analysis was performed essentially as previously described (10), with an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) and P450 2A5 antibodies. Heterologously expressed P450 2A6 and P450 2A13 were used as positive controls. Standard curves for P450 2A6 and P450 2A13 were generated by loading 1-20 fmol of P450 per lane. The nitrocellulose membranes were incubated with rabbit anti2A5 serum (1:2000) overnight at 4 °C and then for an additional 60 min at room temperature. Immunoblot quantification was carried out with use of the Personal Densitometer SI (Molecular Dynamics, Sunnyvale, CA).

Results Human Fetal Nasal Microsomal Metabolism of NNK and Its Immunoinhibition. Three metabolites of NNK generated by human fetal nasal mucosa microsomes were detected by radioflow HPLC analysis. Two, OPB (13 min) and HPB (29 min), were products of R-hydroxylation; the third was NNAL (35 min; Figure 1). The identities of OPB and HPB were confirmed by reduction of each isolated metabolite to 4-(3-pyridyl)butane-1,4-diol (data not shown). The formation of both HPB and OPB by two separate microsomal preparations was inhibited greater than 90% with antibodies against mouse P450 2A5 (Figures 1 and 2). A 2-fold increase in antibody concentration did not greatly increase the observed inhibition (Figure 2). In contrast, these antibodies did not inhibit NNAL formation (Figures 1 and 2). In control experiments, the P450 2A5 antibodies completely inhibited both P450 2A6- and 2A13- and human liver microsome-mediated coumarin 7-hydroxylation (data not shown). Kinetic parameters of NNK metabolism catalyzed by human fetal nasal microsomes were determined (Table 1). The Km and Vmax values for OPB formation were 6.5 ( 1.1 µM and 3.0 ( 0.1 pmol/min/mg. HPB was formed with Km and Vmax values of 6.7 ( 0.8 µM and 0.85 ( 0.03 pmol/min/mg. The formation of NNAL was not saturated under the conditions used, and estimated Km and Vmax values were calculated to be 400 µM and 2300 pmol/min/ mg.

916

Chem. Res. Toxicol., Vol. 18, No. 6, 2005

Communications

Figure 2. Immunoinhibition of NNK metabolism by human fetal nasal microsomes. [3H]NNK (5 µM; 2.5 Ci/mmol) was incubated with microsomes (200 µg/mL; preparation #2) and cofactors in the presence of 0 (black bars), 25.7 (hatched bars), or 51.4 (white bars) µg/mL of anti-P450 2A5 IgG, which inhibits P450 2A6 and 2A13. Control incubations were performed in duplicate where their rates did not vary by more than 10% from each other. One incubation was performed with each of the two antibody concentrations. Table 1. Kinetic Parameters of NNK Metabolism Catalyzed by Human Fetal Nasal Microsomesa product

Km (µM)

Vmax (pmol/min/mg)

Vmax/Km

OPB HPB NNAL

6.5 ( 1.1 6.7 ( 0.8 400b

3.0 ( 0.1 0.85 ( 0.03 2300b

0.46 ( 0.08 0.13 ( 0.02 5.8b

a NNK concentrations were 0.5, 3.0, 5.0, 15, 30, and 60 µM. Values ( SE for goodness of fit were calculated using the Enzyme Kinetics Module of SigmaPlot (SPSS Inc., San Rafeal, CA). Two independent reactions were carried out. Replicate reactions were analyzed for each concentration except 5 (n ) 1) and 30 (n ) 3) µM. b An estimate was given because saturating conditions were not achieved.

Immunoblot Analyses of P450 2A13 in Fetal Nasal Mucosa Microsomes. Five preparations of human fetal nasal mucosa microsomes and two preparations of adult human liver microsomes were analyzed for P450 2A13 and P450 2A6 expression at the protein level. Previously, P450 2A proteins were detected in human fetal nasal mucosa by immunoblot analysis with the P450 2A5 antibodies (9, 13). These antibodies do not cross-react with P450 1A1, 1A2, 2B6, 2E1, 3A4, 1B1, 2C8, 2C9-Arg, 2C19, 2D6-Val, 3A5, and 4A11 (3, 8); however, they recognize both P450 2A6 and P450 2A13 (3, 8). Until now, it had not been possible to resolve these two proteins on a conventional SDS-polyacrylamide gel (3). In the present study, we developed a new method that allows resolution of P450 2A6 and P450 2A13 proteins. An electrophoresis apparatus designed for DNA sequencing was used for analyzing proteins. Microsomal proteins were separated on a 40 cm long gel in approximately 24 h. As shown in Figure 3A, lanes 1-3, heterologously expressed P450 2A6 and P450 2A13 were well-separated, with relatively lower mobility of P450 2A13 than P450 2A6. As compared with P450 2A6 and P450 2A13 standards, a major P450 2A13 band was detected in fetal nasal microsomal preparation #3 (Figure 3A, lane 4). A very weak P450 2A6 band was also visible in this preparation but only with a longer exposure time. In preparation #4, only a P450 2A13 band was detected (Figure 3A, lane 5). Two adult liver microsomes were also analyzed, and a single P450 2A6 band was detected in both, albeit at very different levels (Figure 3A, lanes 6 and 7). The identity of the weak band below the P450 2A6 band in lane 7 (HLM2) remains to be determined.

Figure 3. Immunoblot detection of P450 2A6 and P450 2A13 proteins in human fetal nasal mucosa and adult human liver. Microsomal proteins from human fetal nasal mucosa and from adult liver were analyzed on immunoblots with a rabbit antiserum to P450 2A5. Heterologously expressed P450 2A6 and P450 2A13 in insect Sf9 cell microsomes were used as standards. (A) Lane 1, P450 2A6, 7.5 fmol; lane 2, P450 2A13, 7.5 fmol; lane 3, P450 2A6 and P450 2A13, 7.5 fmol each; lanes 4 and 5, fetal nasal microsomal preparations #3 and #4, respectively (7.5 µg per lane); lanes 6 and 7, adult liver microsomes HLM1 and HLM2, respectively, (100 ng per lane). (B) Lane 1, fetal nasal microsomal preparation #1, 15 µg (containing approximately 24 fmol of P450 2A13); lane 2, P450 2A13, 20 fmol; lane 3, P450 2A6 and P450 2A13, 20 fmol each; lanes 4-10, 20, 10, 8, 6, 4, 2, and 1 fmol of P450 2A6, respectively. The film was exposed for 1 min. (C) Same as panel B, but the film was exposed for 20 min. P450 2A6 was not detectable in the nasal microsome sample in lane 1, with the detection limit at 1 fmol.

In the pooled nasal microsomal sample used for NNK kinetic analysis (preparation #1), P450 2A13 alone was detected (Figure 3B, lane 1). The level of P450 2A13 in this sample was estimated to be 1.6 pmol/mg in a separate, quantitative immunoblot experiment (not shown), by comparing to a P450 2A13 standard curve. P450 2A6 was not detected in this sample, even after a prolonged exposure of the film, as shown in Figure 3C (limit of detection 67 fmol/mg). P450 2A expression in two additional fetal nasal microsomal samples (preparations #5 and #6) was also analyzed. Both P450 2A6 and P450 2A13 were detected in #6, but neither was detected in #5 (data not shown).

Discussion Among all human P450s examined, P450 2A13 is by far the most efficient catalyst of NNK R-hydroxylation (2). This reaction is believed to be the critical initiation step in NNK-induced carcinogenesis in humans. P450 2A13 also catalyzes the R-hydroxylation of other nitrosamines and the metabolism of several nasal toxins (3, 14). We report here for the first time that P450 2A13, which is likely to be a key catalyst for NNK activation in humans, is a functional enzyme in vivo. P450 2A13 catalysis of NNK metabolism was investigated in the fetal nasal mucosa because several lines of evidence suggested that significant amounts of P450 2A13 protein were present in this tissue. The highest levels of extrahepatic P450 2A protein, adult or fetal, were detected previously in nasal mucosa (9). Also, P450 2A13 mRNA expression in the nasal mucosa is higher than in other tissues including lung, trachea, and liver (3), and on the basis of the 18-41-fold higher levels of 2A13 mRNA as compared to P450 2A6 detected in fetal nasal mucosa, the P450 2A protein present was presumed to be mostly P450 2A13. The results of the present study confirm this assumption. In the pooled nasal microsomal

Communications

samples used for the kinetic studies on NNK metabolism, the protein level of P450 2A13 was at least 20-fold greater than that of P450 2A6. This is the first time that P450 2A13 protein has been unambiguously detected in a human tissue. The kinetic parameters for NNK R-hydroxylation by fetal nasal microsomes clearly and strongly support P450 2A13 as the catalyst of these reactions. The Km values reported for these reactions by P450 2A13 ranged from 2.8 to 14.0 µM (2, 3, 15) and are consistent with the Km values of 6.5 and 6.7 µM reported for methylene and methyl hydroxylation by fetal nasal microsomes (Table 1). These values are in contrast to the much higher Km values reported for P450 2A6 (118-392 µM) (2). In addition, P450 2A13, like the nasal microsomes, catalyzes methylene hydroxylation at about three times the rate of methyl hydroxylation. Our results also characterize fetal nasal mucosa as the first human tissue found to efficiently catalyze NNK R-hydroxylation. Microsomes from other human tissues exhibited none to marginal NNK R-hydroxylation activity (1, 2). As compared to fetal nasal microsomes, adult liver microsomes catalyzed NNK R-hydroxylation with higher Km values (367-1200 µM) but comparable Vmax/Km values (2). However, if the Vmax/Km values for fetal nasal and adult liver microsomes are compared per nmol of total P450 in the respective tissues, fetal nasal microsomes are likely to be the more efficient catalysts of NNK R-hydroxylation. Although not determined, the total P450 concentration in fetal nasal mucosa microsomes is expected to be much lower than that of adult liver microsomes. The average P450 concentration in liver microsomes was about 350 pmol/mg microsomal protein (16). Of interest, the level of P450 2A6 protein was estimated to be around 1-10 pmol/mg, in adult human liver microsomes. Therefore, the relative levels of adult hepatic microsomal P450 2A6 and fetal nasal microsomal P450 2A13 and the kinetic parameters of these two enzymes are consistent with the NNK R-hydroxylation rates observed in the respective microsomal samples. On the basis of our findings in human fetal nasal mucosa and P450 2A13 mRNA levels in the adult lung (3), it is likely that P450 2A13 is the predominant P450 2A enzyme in the human lung, a target tissue for NNKinduced carcinogenesis (3). NNK is a lung carcinogen, and we and others have hypothesized that this is in part due to tissue specific activation. On the basis of the kinetic parameters of P450 2A13 and of the other P450 enzymes expressed in the human lung, P450 2A13 is the probable catalyst of NNK activation in this tissue (2). Quantifying P450 2A13-catalyzed NNK R-hydroxylation in the lung will be challenging due to low total P450 expression in extrahepatic tissues and the recognized difficulty in obtaining active human lung microsomal preparations (2). In addition, P450s are likely to be expressed differentially in different cell types and regions of the human lung (17). In the present study, fetal nasal microsomes catalyzed NNK R-hydroxylation. Previously, we have reported that fetuses of smoking mothers are exposed in utero to NNK and/or NNAL via placental transfer (18). Together, these data suggest that fetal exposure to smoking may result in NNK bioactivation by the fetus. While these data support the biological feasibility of a link between fetal NNK exposure and cancer induction in the nasal mucosa, clearly a number of other variables will influence this

Chem. Res. Toxicol., Vol. 18, No. 6, 2005 917

relationship. A meta-analysis of 12 studies reported a relative risk of 1.1 (95% confidence intervals 1.03-1.19) for the incidence of childhood cancer among individuals whose mothers smoked during pregnancy (19). However, establishing a relationship between maternal smoking and a specific cancer in adults is complicated by a number of factors, including an increased exposure to secondhand smoke in the home and an increase in smoking initiation among these individuals (20). In conclusion, the kinetic, immunoinhibition, and immunodetection experiments presented here clearly demonstrate that human fetal nasal mucosa efficiently catalyzes NNK bioactivation and that P450 2A13 is the catalyst. This is the first study to report that P450 2A13 is a functional enzyme in vivo and that microsomes from a human tissue efficiently catalyze NNK R-hydroxylation. Studies to determine the role of P450 2A13 in NNK bioactivation in the lung are ongoing.

Acknowledgment. We thank the Cooperative Human Tissue Network and Dr. Alan Fantel of the Birth Defect Research Laboratory at the University of Washington (funded by NIH Grant HD00836) for providing human tissues. This study was supported by Grants CA084529, CA-081301, and CA-092596 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) Jalas, J. R., Hecht, S. S., and Murphy, S. E. (2005) Cytochrome P450 enzymes as catalysts of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen. Chem. Res. Toxicol. 18, 95-110. (3) Su, T., Bao, Z., Zhang, Q. Y., Smith, T. J., Hong, J. Y., and Ding, X. (2000) Human cytochrome P 450 CYP2A13: Predominant expression in the respiratory tract and in high efficiency metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone. Cancer Res. 60, 5074-5079. (4) 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 cytochrome P450 2A13. Drug Metab. Dispos. 31, 11991202. (5) Hecht, S. S., Young, R., and Chen, C. B. (1980) Metabolism in the F344 rat of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific carcinogen. Cancer Res. 40, 4144-4150. (6) Liu, C., Zhuo, X., Gonzalez, F. J., and Ding, X. (1996) Baculovirusmediated expression and characterization of rat CYP2A3 and human CYP2A6: Role in metabolic activation of nasal toxicants. Mol. Pharmacol. 50, 781-788. (7) Gu, J., Zhang, Q. Y., Genter, M. B., Lipinskas, T. W., Negishi, M., Nebert, D. W., and Ding, X. (1998) Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: Role in metabolic activation of acetaminophen and 2,6dichlorobenzonitrile in mouse olfactory mucosal microsomes. J. Pharmacol. Exp. Ther. 285, 1287-1295. (8) 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. (9) Chen, Y., Liu, Y. Q., Su, T., Ren, X., Shi, L., Liu, D., Gu, J., Zhang, Q. Y., and Ding, X. (2003) Immunoblot analysis and immunohistochemical characterization of CYP2A expression in human olfactory mucosa. Biochem. Pharmacol. 66, 1245-1251. (10) Ding, X., and Coon, M. J. (1990) Immunochemical characterization of multiple forms of cytochrome P450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb. Mol. Pharmacol. 37, 489-496. (11) Peterson, L. A., Mathew, R., and Hecht, S. S. (1991) Quantitation of microsomal R-hydroxylation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 51, 5495-5500.

918

Chem. Res. Toxicol., Vol. 18, No. 6, 2005

(12) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. (13) Gu, J., Su, T., Chen, Y., Zhang, Q. Y., and Ding, X. (2000) Expression of biotransformation enzymes in human fetal olfactory mucosa: Potential roles in developmental toxicity. Toxicol. Appl. Pharmacol. 165, 158-162. (14) Wong, H. L., Murphy, S. E., and Hecht, S. S. (2005) Cytochrome P450 2A-catalyzed metabolic activation of structurally similar carcinogenic nitrosamines: N′-nitrosonornicotine enantiomers, N-nitrosopiperidine, and N-nitrosopyrrolidine. Chem. Res. Toxicol. 18, 61-69. (15) He, X. Y., Shen, J., Ding, X., Lu, A. Y., and Hong, J. Y. (2004) Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen. Drug Metab. Dispos. 32, 1516-1521. (16) Shimada, T., Yamazaki, H., Mimura, M., Inui, Y., and Guengerich, F. P. (1994) Interindividual variations in human liver cytochrome

Communications

(17)

(18) (19) (20)

P450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: Studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther. 270, 414-423. Boland, B., Lin, C. Y., Morin, D., Miller, L., Plopper, C. G., and Buckpitt, A. (2004) Site specific metabolism of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques. J. Pharmacol. Exp. Ther. Milunsky, A., Carmella, S. G., Ye, M., and Hecht, S. S. (2000) A tobacco-specific carcinogen in the fetus. Prenatal Diagn. 20, 307310. Boffetta, P., Tredaniel, J., and Greco, A. (2000) Risk of childhood cancer and adult lung cancer after childhood exposure to passive smoke: A meta-analysis. Environ. Health Perspect. 108, 73-82. Barman, S. K., Pulkkinen, L., Kaprio, J., and Rose, R. J. (2004) Inattentiveness, parental smoking and adolescent smoking initiation. Addiction 99, 1049-1061.

TX0500777