JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 JPET FastThis article Forward. has not beenPublished copyedited on and Augustformatted. 1,The 2007 final version as DOI:10.1124/jpet.107.127068 may differ from this version. JPET #127068 CYP2A13: Variable Expression and Role in Human Lung Microsomal Metabolic Activation of the Tobacco-Specific Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1- butanone Xiuling Zhang, Jaime D’Agostino, Hong Wu, Qing-Yu Zhang, Linda von Weymarn, Sharon E. Murphy, and Xinxin Ding Wadsworth Center, New York State Department of Health, and School of Public Health, SUNY Downloaded from at Albany, Albany, NY (X.Z., J.D., H.W., Q.-Y.Z., X.D.), and Cancer Center, University of Minnesota, Minneapolis, MN (L.W., S.M.) jpet.aspetjournals.org at ASPET Journals on September 28, 2021 1 Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 This article has not been copyedited and formatted. The final version may differ from this version. JPET #127068 RUNNING TITLE: CYP2A13 protein expression and NNK metabolism in human lung ADDRESS CORRESPONDENCE TO: Dr. Xinxin Ding, Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509. Tel. 518-486-2585; Fax: 518-486-1505; E-mail: [email protected] Number of Text Pages: 24 Downloaded from Number of Tables: 4 Number of Figures: 2 jpet.aspetjournals.org Number of References: 31 Number of words: Abstract 250 Introduction 684 at ASPET Journals on September 28, 2021 Discussion 1473 ABBREVIATIONS: P450, cytochrome P450; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone; HPLC, high performance liquid chromatography; 8-MOP, 8-methoxypsoralen; CHTN, Cooperative Human Tissue Network; IgG, immunoglobulin G; PBS, phosphate buffered saline (10 mM phosphate buffer, pH 7.4, containing 138 mM NaCl and 2.7 mM KCl); BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; Sf, spodoptera frugiperda; PCR, polymerase chain reaction; RT, reverse transcription; OPB, 4-oxo-1-(3-pyridyl)-1-butanone; HPB, 4-hydroxy-1-(3- pyridyl)-1-butanone; NNAL, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol; ANOVA, analysis of variance. SECTION ASSIGNMENT: Metabolism, Transport, and Pharmacogenomics 2 JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 This article has not been copyedited and formatted. The final version may differ from this version. JPET #127068 ABSTRACT CYP2A13 is the most efficient P450 enzyme in the metabolic activation of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific lung carcinogen. The aims of this study were to determine the levels of CYP2A13 protein in human lung microsomes, and to ascertain whether CYP2A13 plays any role in lung microsomal NNK metabolic activation. The expression of CYP2A6 and CYP2A13 was examined using a high-resolution immunoblotting method, following immunopurification with an anti-CYP2A5 antibody. We Downloaded from found that, of 116 human lung microsomal samples analyzed, ~90% had detectable CYP2A6, whereas only 12% had detectable CYP2A13, with a detection limit of ~2 fmol CYP2A/mg jpet.aspetjournals.org protein. For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6; overall, the highest level of CYP2A13 found (~20 fmol/mg protein) was ~10-fold lower than the highest level of CYP2A6 detected. Quantitative RNA-PCR at ASPET Journals on September 28, 2021 analysis confirmed that the highly variable expression of the CYP2A proteins was consistent with variations in the levels of the corresponding CYP2A mRNAs in the same tissue samples. Importantly, the level of CYP2A13, but not CYP2A6, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, addition of 8-methoxypsoralen, a CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in microsomes containing relatively high levels of CYP2A13 than in samples containing no detectable CYP2A13. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation. Therefore, individuals having relatively high levels of CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer. 3 JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 This article has not been copyedited and formatted. The final version may differ from this version. JPET #127068 Introduction The human cytochrome P450 (P450) CYP2A gene subfamily is known to have two functional members, CYP2A6 and CYP2A13 (Fernandez-Salguero et al., 1995, Su et al., 2000). Previous studies on CYP2A mRNA expression in human tissues indicated that CYP2A6 is expressed in the lung at much lower levels than in the liver, whereas CYP2A13 is selectively expressed in the respiratory tract (Koskela et al., 1999; Su et al., 2000). A more recent study indicated that CYP2A13 is also expressed in the bladder (Nakajima et al., 2006). Heterologously Downloaded from expressed CYP2A6 and CYP2A13 enzymes are both active in the metabolic activation of 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a lung procarcinogen, which is found in jpet.aspetjournals.org cigarettes (Hecht, 1998), and which can also be produced endogenously from nicotine metabolites (Hecht et al., 2000). NNK metabolic activation involves P450-catalyzed hydroxylation at one of the two α carbons to the N-nitroso group, leading to the formation of at ASPET Journals on September 28, 2021 reactive intermediates that can form either DNA adducts or else stable metabolites, including 4- hydroxy-1-(3-pyridyl)-1-butanone (HPB) and 4-oxo-1-(3-pyridyl)-1-butanone (OPB) (Hecht, 1998). Although multiple human P450 enzymes, including CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2E1, and CYP3A4, can catalyze NNK α-hydroxylation, CYP2A13 is by far the most efficient, with Km values in the low µM range (Su et al., 2000; Jalas et al., 2005). The catalytic efficiency of CYP2A13 is >100 times higher than that found for CYP2A6 (Jalas et al., 2005). Several lines of evidence, including the high catalytic efficiency toward NNK, the tissue- selective expression of CYP2A13 mRNA in human respiratory tract, and the finding of a significant association between a reduced-activity CYP2A13 allele and decreased incidence of lung adenocarcinoma in light smokers (Wang et al., 2003), strongly suggest that CYP2A13 plays 4 JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 This article has not been copyedited and formatted. The final version may differ from this version. JPET #127068 an important role in the metabolic activation of NNK in the respiratory tract of human smokers. The expression of CYP2A13 protein in a human tissue (nasal mucosa) was not demonstrated until recently, a success made possible by our development of a high-resolution immunoblotting method (Wong et al., 2005). There, we demonstrated that CYP2A13 protein is more abundant than CYP2A6 protein in human fetal nasal mucosa, and that, whereas CYP2A6 is the major enzyme for coumarin hydroxylation, CYP2A13 is the main enzyme for NNK metabolic activation in human fetal nasal microsomes. More recently, CYP2A13 protein was found to be Downloaded from selectively expressed in airway epithelial cells in adult human lung, in an immunohistochemical study using a newly generated anti-CYP2A13 peptide antibody (Zhu et al., 2006). Nevertheless, jpet.aspetjournals.org it remained to be determined (1) whether CYP2A13 protein can be detected in human lung microsomes; (2) whether microsomal levels of CYP2A13 protein differ among individuals; and (3) whether CYP2A13 plays a major role in NNK metabolism in lung microsomes. It should be at ASPET Journals on September 28, 2021 noted that, although CYP2A protein was detected in human lung microsomes in earlier studies (Shimada et al., 1996, Ulrike et al., 2006), it could not be determined whether CYP2A6 or CYP2A13 was detected in those studies, given that the two proteins could not be distinguished under the conditions used. Previous studies have demonstrated that human lung microsomes are active in the metabolic activation of NNK, and that this microsomal reaction is at least partially catalyzed by P450 enzymes (Smith et al., 1992; 1995; 2003). A definitive identification of the specific P450 enzymes responsible for NNK metabolic activation in human lung microsomes was not possible in those studies, but the involvement of multiple P450s, including CYP2A and CYP2B6, was suggested. In the present study, we have estimated the expression levels of CYP2A6 and CYP2A13 proteins for a large number of adult human lung microsomal samples. We found that 5 JPET Fast Forward. Published on August 1, 2007 as DOI: 10.1124/jpet.107.127068 This article has not been copyedited and formatted. The final version may differ from this version. JPET #127068 the levels of CYP2A6 and CYP2A13 proteins varied drastically among the samples analyzed. Subsequently, we performed quantitative analysis of CYP2A6 and CYP2A13 mRNA expression, to establish whether the variable expression of the CYP2A proteins was accompanied by similar variations at the mRNA level. Furthermore, we then analyzed microsomal samples with varying levels of CYP2A protein expression for rates of NNK α-hydroxylation. In addition, a known CYP2A inhibitor, 8-methoxypsoralen (8-MOP), was used to confirm the participation of CYP2A6 or CYP2A13