Short Communication Specificity of Cytochrome P450 2A3-Catalyzed a-Hydroxylation of N*-Nitrosonornicotine Enantiomers

N*-nitrosonornicotine (NNN) induces tumors in the rat nasal cavity and esophagus and is believed to be a causative agent for esophageal cancer in tobacco users. To exert its carcinogenic potential, NNN must be metabolically activated by a-hydroxylation at either the 2*or 5*-carbon. We previously reported that the human cytochrome P450 (P450), 2A6, efficiently and specifically catalyzed NNN 5*-hydroxylation. P450 2A3, which is expressed in the rat nasal cavity and to a small extent in the esophagus, is closely related to P450 2A6. P450 2A3, like 2A6, is a good catalyst of NNN a-hydroxylation (Km 7 mM; Vmax 17 nmol/min/nmol). However, in contrast to P450 2A6, 2A3 catalyzed both 5*and 2*-hydroxylation of NNN. The ratio of 2*to 5*-hydroxylation was 1:3. These data, both with P450 2A6 and 2A3, were obtained using racemic NNN. P450 2A3 catalyzed metabolism of (S)-NNN occurred exclusively at the 5*-position. The predominant pathway of (R)-NNN metabolism was 2*-hydroxylation, and occurred to a 3-fold greater extent than did 5*-hydroxylation. These data are in contrast to those obtained from a recent study of (R)and (S)-NNN metabolism by cultured rat esophagus. In that study, (S)-NNN was metabolized predominantly by 2*-hydroxylation and (R)-NNN equally by 2*and 5*-hydroxylation. Taken together, these data provide strong evidence that P450 2A3 is not the rat esophageal P450 that catalyzes the metabolic activation of NNN. P450 2A3 may be an important catalyst of NNN activation in rat nasal mucosa. N9-Nitrosonornicotine (NNN) is an esophageal and nasal carcinogen in the rat and is believed to be a causative agent for esophageal and oral cancer in tobacco users (Hecht, 1998). This tobacco-specific nitrosamine requires metabolic activation to exert its carcinogenic potential. Activation is believed to occur through P450-catalyzed a-hydroxylation in the target tissue. It has been proposed by us and others that this tissue-specific activation contributes to the tissuespecific induction of tumors by nitrosamines (Bartsch et al., 1977; Hodgson et al., 1980; Murphy and Spina, 1994). However, what P450s catalyze the a-hydroxylation of NNN in either the esophagus or nasal mucosa have yet to be determined. Previously, we reported that the human P450 2A6, a coumarin 7-hydroxylase, is an efficient and specific catalyst of NNN a-hydroxylation at the 59-carbon (Patten et al., 1997). In the study presented here, we characterized the metabolism of NNN by a rat coumarin 7-hydroxylase, P450 2A3, which is 85% homologous to P450 2A6 (Kimura et al., 1989). a-Hydroxylation of NNN may occur at either the 29or 59-carbon [Fig. 1; (Hecht, 1998)]. Both hydroxy NNN products formed are unstable. They decompose to the corresponding diazohydroxides, which then react with H2O to produce HPB and lactol respectively (Fig. 1). The pyridyloxobutyl diazohydroxide formed by 29-hydroxylation of NNN can alkylate DNA, generating adducts that release HPB upon hydrolysis (Hecht, 1998). DNA adduct formation by the 59hydroxylation pathway has not been detected to date. The 29-hydroxylation pathway is the predominant one in both the rat esophagus and nasal cavity (Hecht et al., 1982, 1998; Brittebo et al., 1983; Murphy et al., 1990). Preferential a-hydroxylation at the 29-position and the detection of HPB releasing DNA adducts in these tissues, which are susceptible to NNN tumorigenesis, has led to the view that 29hydroxylation is the more important pathway of NNN activation (Murphy et al., 1990; Trushin et al., 1994; Hecht, 1998). Rat esophageal microsomes catalyze both the 29and 59-hydroxylation of NNN (Murphy and Spina, 1994). This activity is NADPHdependent and inhibited by carbon monoxide, consistent with the role of a P450 enzyme as the catalyst of these reactions. The total a-hydroxylation of NNN has an apparent Km of 49 mM and the ratio of 29to 59-hydroxylation is 3:1 for all concentrations of NNN (Murphy and Spina, 1994). There are few data on what P450s are present in the esophagus. P450 1A1 and P450 17 mRNA have been detected by RT-PCR (Traber et al., 1992; Valle et al., 1995). P450 1A1 protein was detected by Western blot analysis, whereas P450 2E1 and 2B1 were not (Ahn et al., 1996). Using RT-PCR, we recently detected low levels of P450 2A3 expression in the esophagus (Gopalakrishnan et al., 1999). The mRNA level was 1/60th of that detected in the lung, the tissue from which the cDNA for P450 2A3 was originally isolated (Kimura et al., 1989). We did detect small amounts of P450 2A protein by Western blot analysis with an antibody to the related mouse P450 2A5 (Gopalakrishnan et al., 1999). Rat nasal mucosa microsomes also catalyze both the 29and 59This study was supported by Grant CA-74913 (to S.E.M.) from the National Cancer Institute and Grant ES-07462 (to X.D.) from the National Institute of Environmental Health Sciences. 1 Abbreviations used are: NNN, N9-nitrosonornicotine; HPB, 4-hydroxy-4-(3pyridyl)-1-butanone; lactol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran; P450, cytochrome P450; RT-PCR, reverse transcription-polymerase chain reaction. Send reprint requests to: Sharon E. Murphy, University of Minnesota Cancer Center, 420 Delaware St. SE, Minneapolis, MN. E-mail: [email protected] 0090-9556/00/2811-1263–1266$03.00/0 DRUG METABOLISM AND DISPOSITION Vol. 28, No. 11 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics 150/859393 DMD 28:1263–1266, 2000 Printed in U.S.A. 1263 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from