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 in the microsomal metabolism of NNK. Our findings indicate that ~12% Downloaded from of lung microsomes studied had relatively high CYP2A13 protein expression, and that these microsomes also had higher activities in NNK metabolic activation. jpet.aspetjournals.org at ASPET Journals on September 28, 2021
6 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
Materials and Methods
Chemicals and Enzymes. [5-3H]NNK (10.0-10.9 Ci/mmol; > 98% pure) was purchased from Moravek Biochemicals (Brea, CA) and was further purified by reverse-phase high performance liquid chromatography (HPLC) prior to use. NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and 8-MOP were purchased from Sigma-Aldrich (St.
Louis, MO). Rat NADPH-cytochrome P450 reductase was obtained as described elsewhere
(Zhang et al., 1998). Flo-Scint II scintillation fluid was from Perkin Elmer (Waltham, MA). Downloaded from
Microsomes containing heterologously expressed CYP2A6 (Liu et al., 1996) or CYP2A13 (Su et al., 2000) and polyclonal antibodies against mouse CYP2A5 (Gu et al., 1998) were prepared as jpet.aspetjournals.org described. All other chemicals and enzymes were purchased from Sigma-Aldrich or Bio-Rad
(Hercules, CA).
Preparation of Human Lung Microsomes. The Institutional Review Board of New at ASPET Journals on September 28, 2021
York State Department of Health approved the present study with human tissues. Adult human peripheral lung surgical resection specimens (noninvolved, adjacent tissue) were provided by the
National Cancer Institute Cooperative Human Tissue Network (CHTN); tissues were obtained within 1 h after surgical removal and stored at -80ºC until use. All tissues were confirmed by
CHTN to be histologically normal. The 116 tissue samples were obtained from 64 males and 52 females, ages 21-82 (~73% were between the ages of 51 and 70 years), comprising 93
Caucasians, 12 African Americans, and 11 of unknown ethnic origin. Clinical diagnosis for these patients included adenocarcinoma (42), squamous cell carcinoma (31), large cell carcinoma (2), undetermined non-small cell carcinoma (5), other types of lung carcinoma (9), secondary carcinoma (6), benign lung tumors (7), non-tumor diseases (6), or unknown/undetermined
7 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 diseases (8). Microsomes were prepared as described previously (Ding and Coon, 1990) and stored at -80°C.
Immunopurification of CYP2A proteins. Magnetic Dynabeads® M-280 Tosylactivated
(Invitrogen, Carlsbad, CA) were coated with rabbit anti-CYP2A5 immunoglobulin G (IgG) according to a protocol provided by the manufacturer. Briefly, beads (2 x 109/ml) were washed once with 0.1 M borate buffer (pH 9.5), before they were incubated in the borate buffer with ~3
7 µg of IgG per10 magnetic beads for ~20 h at 37°C. The beads were then washed sequentially as Downloaded from follows: two washings in phosphate-buffered saline (PBS) containing 0.1% (w/v) bovine serum albumin (BSA) for 5 min at 4°C, one washing in blocking buffer (0.2 M Tris-chloride, pH 8.5, jpet.aspetjournals.org 0.1% BSA) for 4 h at 37°C, one washing in PBS containing 0.1% BSA for 5 min at 4°C, one washing in 1% Tween-20 for 10 min at room temperature, and one washing in PBS containing
0.1% BSA for 5 min at 4°C. The final preparation of coated beads was stored at 4°C in PBS at ASPET Journals on September 28, 2021 containing 0.1% BSA and 0.02% sodium azide at a concentration of ~2 x 109 beads/ml prior to use.
Human lung microsomal samples (0.5 mg protein each) in 150 µl microsome storage buffer (50 mM Tris-acetate, pH 7.4, 1.0 mM EDTA, and 20% glycerol) were mixed with 150 µl of a solubilization buffer (100 mM Tris-chloride, pH 7.4, 300 mM NaCl, 2.0% Triton X-100,
2.0% deoxycholate, 10 mM EDTA, and the Complete Mini protease inhibitor cocktail (Roche
Applied Science, Indianapolis, IN)). The mixtures were incubated, in a head-to-tail rotator, for 1 h at 4°C, followed by centrifugation at 4°C for 10 min, at 14,800 g. The resultant supernatant fraction was removed and added to ~6.6 x 107 pelleted, antibody-coated beads, which were washed once with PBS containing 0.1% BSA before use. The coated beads were incubated with the solubilized microsomal proteins overnight at 4°C, in a head-to-tail rotator. Subsequently, the
8 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 beads were washed, at 4°C, once in PBS containing 0.05% Tween-20, and once in PBS alone, and the captured proteins were eluted by mixing the pelleted beads with 16 µl of the Laemmli sample buffer (Bio-Rad), followed by centrifugation at 4°C for 5 min at 14,800 g. The beads were applied to a magnetic concentrator (Invitrogen, Carlsbad, CA) and the immunopurified proteins were stored at –20°C before immunoblot analysis.
Immunoblot analysis. High-resolution sodium dodecyl sulfate (SDS)-polyacrylamide
gel electrophoresis was performed using a DNA sequencing apparatus (Bio-Rad) as previously Downloaded from described (Wong, 2005), with minor modifications. The immunopurification products (16 µl), mixed with 0.8 µl of β-mercaptoethanol and 20 µg Sf9 insect cell microsomal protein (as carrier jpet.aspetjournals.org protein), were boiled for 5 min before loading onto the gel. All samples were analyzed on 10%
SDS-polyacrylamide (37.5:1, acrylamide:bis-acrylamide) gels (0.75-mm thick), with the use of standard Laemmli buffers (Laemmli, 1970). Electrophoresis was carried out for ~24 h, at room at ASPET Journals on September 28, 2021 temperature, in the constant-current mode, at 15 mA for stacking and 11-18 mA for separation. A cooling fan was used, and the electrophoresis buffer was replenished at least three times during the separation. After electrophoresis, proteins were transferred to a nitrocellulose membrane
(0.45 µm; Bio-Rad).
Immunoblot analysis was performed essentially as described previously (Ding and Coon,
1990), with an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) and a rabbit anti-CYP2A5 antibody (Gu et al., 1998). Heterologously expressed CYP2A6 and CYP2A13, contained in Sf9 microsomes, were used as positive controls. The nitrocellulose membranes were incubated with the CYP2A5 antiserum (1:2000) overnight at 4°C and then for an additional 60 min at room temperature. Densitometric analysis of scanned gel images was carried out with use of the LabWorks, Version 4.5 (UVP, Upland, CA). Estimates of the amounts of CYP2A6 and
9 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
CYP2A13 proteins were obtained by comparing the signal intensity of a given sample to the intensities of CYP2A6 and CYP2A13 protein standards that were analyzed on the same gel.
Quantitative Analysis of CYP2A6 and CYP2A13 mRNA Expression. Total RNA was isolated from lung tissues with use of the RNeasy Plus Mini kit (QIAGEN, Valencia, CA). First- strand cDNA was synthesized from 1.0 µg of total RNA in a total volume of 20 µl, using the
SuperScript III first-strand synthesis system and an Oligo(dT)20 primer (Invitrogen, Carlsbad,
CA). Real-time PCR was carried out using the model 7500 Fast real-time PCR system and the Downloaded from
SYBR Green PCR core reagents (Applied Biosystems, Foster City, CA). The mRNA levels were determined for CYP2A6, CYP2A13, and β-actin. The primers used for CYP2A6 were 2A6-F jpet.aspetjournals.org (5’-gggccaagatgccct-3’, located in exon 7) and 2A6-R (5’-aatgtccttaggtgactggga-3’, located in exon 9). The size of the expected PCR product was 377 base pairs. The primers used for
CYP2A13 and β-actin have been described in our previous studies (Zhang et al., 2004). at ASPET Journals on September 28, 2021
PCR was carried out in a reaction volume of 20 µl. Reaction mixtures for quantification of CYP2A6 or CYP2A13 mRNA contained 5 µl of 10-fold-diluted RT product, 2.0 µl 10X
SYBR Green PCR buffer, 1.6 µl dNTP mix (containing 2.5 mM each of dATP, dCTP, and dGTP, and 5.0 mM dUTP), 3.0 mM MgCl2, 0.25 µM (for CYP2A6) or 0.3 µM (for CYP2A13) of each primer, 0.2 unit of AmpErase UNG, and 0.5 unit of AmpliTaq Gold DNA polymerase.
The reaction mixture for β-actin mRNA quantification was the same as that for CYP2A13, except that 2 µl of 10-fold-diluted RT product and 2.5 mM MgCl2 were used, and the AmpErase
UNG was omitted. Reactions for CYP2A6 and CYP2A13 were initiated by incubation at 50°C for 2 min (to allow degradation of any contaminating PCR products by the AmpErase UNG) and then denaturation at 95°C for 10 min (to inactivate the AmpErase UNG), followed by 50 cycles of amplification (95°C for 15 s, 64°C for 15 s, and 72°C for 30 s). For β-actin, the initial
10 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 incubation at 50°C was omitted, and the PCR was performed for 40 cycles (95°C for 15 s, 63°C for 10 s, and 72°C for 30 s). Data collection was performed at 72°C. All reactions were performed in duplicate. Each PCR run included reactions using standard templates (10-fold serial dilutions of cloned cDNA for CYP2A6 and CYP2A13, or 10-fold serial dilutions of gel-purified
RT-PCR product of β-actin) and a no-template control, in addition to reactions using first-strand lung cDNA. Standard curves were generated by plotting the values of the threshold-crossing
points against log-transformed copy numbers of standard templates. The levels of CYP2A6 and Downloaded from
CYP2A13 mRNAs in various total RNA preparations were normalized by the level of β-actin mRNA in a given sample. jpet.aspetjournals.org Detection of the occurrence of variant CYP2A genes and transcripts. Sequence analysis of the CYP2A13 exons and exon-intron junctions was performed as described previously
(Zhang et al., 2002; 2003), in order to detect any occurrence of genetic polymorphisms. Potential at ASPET Journals on September 28, 2021 variations in CYP2A13 mRNA sequence, resulting from alternative splicing, were detected via
RNA-PCR; the CYP2A13 mRNA was amplified in four fragments, spanning exons 1 to 3, 3 to 6,
6 to 7, and 7 to 9, with the use of primers 5’-tgtgatggtcttgatgtcagtc-3’ and 5’-cccacgccaaaacccctt-
3’, 5’-aaggagctacggcgcgaa-3’ and 5’-catgagcagcaggaaaccg-3’, 5’-acctggtgatgaccaccc-3’ and 5’- cgtggatcactgcctctg-3’, and 5’-gggccaagatgccctacaca-3’ and 5’-gatatccttaggcgactgagg-3’, respectively. The sizes of the PCR products were compared to those amplified from lung samples that did not contain the CYP2A-X protein. To detect potential occurrence of mRNAs corresponding to CYP2A7 or CYP2A6/CYP2A7 hybrid (CYP2A6*3), RNA-PCR analysis was performed using PCR primers complementary to CYP2A6 and CYP2A7 exons 7 (5’- gggccaagatgccctacatg-3’) and 9 (5’-aatgtccttaggtgactggga-3’).
11 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
Assay for NNK Metabolism. The contents of the reaction mixtures are described in the notes to tables. The reaction mixtures were pre-incubated for 1 min at 37ºC, prior to the addition of NADPH. Reactions were terminated by the addition of 25 µl each of 25% zinc sulfate and saturated barium hydroxide. The samples were then spun, and 50 µl of the supernatant fraction were analyzed on a reverse-phase HPLC system equipped with a Radiomatic series A-500 on- line radioactivity detector (Perkin Elmer), and a Waters µBondaPak C18 column (3.9 x 300 mm;
10 µm). The analytes were eluted, following an initial wash with 100%A (20 mM Tris-chloride, Downloaded from pH 6.0) for 1 min, by a linear gradient from 100%A to 70%A and 30%B (95% methanol) over
74 min, at a flow rate of 1 ml/min. Sodium bisulphite, a trapping agent for OPB, was found to jpet.aspetjournals.org interfere with detection of radiolabeled OPB under conditions that permit separation of 4-
(methyl-nitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and HPB, as previously described by others (Smith et al., 1995). Therefore, sodium bisulphite was not included in the reaction mixture at ASPET Journals on September 28, 2021 or the mobile phase. Under these conditions, OPB and HPB were together detected as a single peak (Smith et al., 1995), which represented abundance of total metabolites formed through the two differing α-hydroxylation pathways (Peterson et al., 1991).
Other methods. Statistical significance of differences between two groups was analyzed using Student’s t-test or Mann-Whitney Rank Sum test (for data that failed the normality test or equal-variance test), and statistical significance of differences among multiple groups was analyzed using one-way ANOVA. Protein concentration was determined using the BCATM
Protein Assay Kit (Pierce, Rockford, IL).
12 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
Results
Detection of CYP2A6 and CYP2A13 Proteins in Adult Human Lung Microsomes.
Expression of CYP2A6 and CYP2A13 proteins was investigated by immunoblot analysis with the use of anti-CYP2A5 antibody. This antibody is known to cross-react with both human
CYP2A6 and CYP2A13, but not with 12 other human CYP enzymes examined on immunoblots, including CYP1A1, CYP1A2, CYP2B6, CYP2E1, CYP3A4, CYP1B1, CYP2C8, CYP2C9-Arg,
CYP2C19, CYP2D6-Val, CYP3A5, and CYP4A11 (Zhuo et al., 1999; Su et al., 2000). The Downloaded from
CYP2A6 and CYP2A13 proteins, which could not be resolved on a conventional SDS- polyacrylamide gel (Gu et al., 2000), were separated using a 40-cm long gel; the electrophoresis jpet.aspetjournals.org was carried out for 24 h in a DNA sequencing apparatus. The sensitivity of CYP2A detection was increased by immunopurification of CYP2A proteins using an anti-CYP2A5 antibody, prior to electrophoresis. CYP2A6 and CYP2A13 proteins were identified on the basis of co-migration at ASPET Journals on September 28, 2021 with recombinant CYP2A proteins, while the levels of the CYP2A proteins were estimated through comparisons with known amounts of CYP2A protein standards.
CYP2A6 and CYP2A13 were both detected in adult human lung microsomes (Fig. 1, panels A and B). A CYP2A-immunoreactive unknown protein (designated CYP2A-X), which migrated more slowly than did either CYP2A6 or CYP2A13, was also detected in a small number of samples (Fig. 1, panel C). The results from all immunoblot experiments are summarized in Table 1. Of the 116 human adult lung microsomal samples analyzed, CYP2A6 was detected in 104 (89.7%) samples (87 with CYP2A6 alone, 13 with both CYP2A6 and
CYP2A13, and four with CYP2A6 and CYP2A-X), whereas CYP2A13 was detected in 14
(12.1%) samples (one with CYP2A13 alone, and 13 with both CYP2A13 and CYP2A6).
CYP2A-X was detected in five of the 116 (4.3%) samples (one with the unknown protein alone,
13 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 and four with both CYP2A-X and CYP2A6). No CYP2A-immunoreactive protein was detected in 10 of the 116 (8.6%) samples (Table 1). Notably, for samples with relatively high levels of
CYP2A6 protein, it was not necessary to perform immunopurification, in order to detect
CYP2A6 on immunoblots (Fig. 1, panel D).
Remarkable inter-individual variations were found for levels of the CYP2A proteins detected in human lung microsomes (Fig. 1). Recovery of recombinant CYP2A6 and CYP2A13
in the immunopurification step was estimated to be ~70-80% for both proteins (data not shown). Downloaded from
By assuming that the recovery of CYP2A proteins from various lung microsomal preparations was constant, we estimate that the amounts of CYP2A6 protein detected in human lung jpet.aspetjournals.org microsomes ranged from ≤2 fmol (detection limit) to ~220 fmol/mg microsomal protein, and that the amounts of CYP2A13 protein ranged from ≤2 fmol (detection limit) to ~20 fmol/mg microsomal proteins. The detection limit was estimated by immunoblot analysis of varying at ASPET Journals on September 28, 2021 amounts of recombinant CYP2A proteins.
In experiments not shown, we attempted to identify the CYP2A-X protein. We hypothesized that CYP2A-X corresponds to a splice variant of CYP2A13, or less likely,
CYP2A6, given the cross-reactivity of CYP2A-X with the anti-CYP2A antibody and the size difference between CYP2A-X and either CYP2A6 or CYP2A13; the variant is more likely related to CYP2A13 than it is related to CYP2A6, given that CYP2A13, but not CYP2A6, was never detected in CYP2A-X-containing samples (Table 1). Alternatively, we reasoned that
CYP2A-X might correspond to CYP2A7, or CYP2A6*3, the gene conversion product of CYP2A6 and CYP2A7 (Fernandez-Salguero et al., 1995); neither CYP2A7 nor CYP2A6.3 protein had been detected in human lung before. We found, through sequence analysis of the CYP2A13 exons and exon-intron junctions for one of the CYP2A-X-containing tissue samples, that the
14 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
DNA sample had only sequences corresponding to the CYP2A13*1 allele; therefore, CYP2A-X is unlikely to be a CYP2A13 allelic variant. An analysis of the sizes of CYP2A13 RNA-PCR products from three CYP2A-X-containing samples did not reveal any CYP2A13 splicing variants. Further RNA-PCR analysis of the three samples detected cDNA sequence corresponding to the exon 8 of CYP2A6, but not that of CYP2A7, a finding indicative of the absence of either CYP2A7 or the CYP2A6/CYP2A7 hybrid (CYP2A6*3) mRNA. Thus,
although the identity of CYP2A-X remains to be determined, this unknown, CYP2A-related Downloaded from protein appears not to be encoded by CYP2A7 or CYP2A6*3, nor does it correspond to a
CYP2A13 splicing variant. jpet.aspetjournals.org Correlation of CYP2A mRNA Expression with CYP2A Protein Expression in
Human Lung Tissues. To determine whether the inter-individual differences in the expression of CYP2A proteins were concordant with inter-individual differences in CYP2A mRNA levels in at ASPET Journals on September 28, 2021 adult lung tissues, we selected tissue samples having high or low CYP2A protein levels for
CYP2A mRNA quantification. For the analysis of CYP2A6 expression, we selected 7 samples with relatively high levels of microsomal CYP2A6 protein (CYP2A6-High) and 9 samples with low or undetectable levels of CYP2A6 protein (CYP2A6-Low). We found (Table 2) that the abundance of CYP2A6 mRNA, normalized by the abundance of β-actin mRNA, was significantly higher in the CYP2A6-High group than in the CYP2A6-Low group (84±71 vs.
24±29 copies/106 copies of β-actin, mean ± S.D.; P<0.05), a result indicative of a positive correlation between levels of CYP2A6 protein and CYP2A6 mRNA in the lung.
For the analysis of CYP2A13 expression, we selected 8 samples with detectable levels of
CYP2A13 protein (CYP2A13-High) and 16 samples with no detectable CYP2A13 protein
(CYP2A13-Low). We found (Table 2) that samples of the CYP2A13-High group contained
15 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 higher levels of CYP2A13 mRNA, than did samples of the CYP2A13-Low group (median: 18 vs. 3.0 copies of CYP2A13/106 copies of β-actin; P<0.05), a result supportive of a positive correlation between levels of CYP2A13 protein and CYP2A13 mRNA in the lung.
Correlation of Lung Microsomal CYP2A13, but not CYP2A6, Protein Level with
Activity of NNK α-Hydroxylation. Metabolism of NNK by human lung microsomes, assayed in the absence of sodium bisulphite, resulted in the detection of two NADPH-dependent
metabolite peaks: one corresponding to NNK α-hydroxylation products (HPB and OPB), and the Downloaded from other (a doublet) corresponding to NNAL stereoisomers (Fig. 2). NNAL was identified on the basis of co-elution with authentic compounds, whereas HPB and OPB were identified on the jpet.aspetjournals.org basis of co-elution with NNK metabolites generated by recombinant CYP2A13, which is known to produce both HPB and OPB (Su et al., 2000), in a reconstituted system (data not shown). No metabolite was detected in complete reaction mixtures that contained boiled human lung at ASPET Journals on September 28, 2021 microsome; small peaks corresponding to substrate impurities or breakdown products were seen at ~20, 46, and 60 min (Fig. 2A). A small peak corresponding to OPB/HPB was detected in no-
NADPH control reactions; this peak appeared superimposed on the broad, background peak at
~46 min (Fig. 2B). A peak corresponding to OPB/HPB, as well as the NNAL peaks, was clearly detected in complete reaction mixtures with lung microsomes that contained CYP2A13 (Fig.
2C). While NNAL formation was completely NADPH-dependent, formation of HPB/OPB was observed in incubations with NADPH as well as in those without NADPH (albeit at lower levels in incubations without NADPH). The lung microsomal preparations did not appear to contain endogenous NADPH, given that an additional cycle of resuspension and pelleting of a microsomal sample did not lead to any reduction in the rate of NADPH-independent formation of HPB/OPB (data not shown).
16 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
Human lung microsomes were grouped into three categories, for determination of in vitro
NNK metabolism: samples containing no detectable CYP2A13 protein, but either high (Group
1), or low (Group 2) levels of CYP2A6 protein, and samples containing detectable levels of
CYP2A13 protein, and varying levels of CYP2A6 (Group 3). As shown in Table 3, Group-1 and
Group-2 microsomal samples did not differ significantly in their rates of NADPH-dependent
NNK α-hydroxylation (0.61 vs. 0.64 pmol/min/mg protein; P > 0.05), indicating that, with NNK
at 10 µM, CYP2A6 did not contribute substantially to NNK metabolic activation in human lung Downloaded from microsomal reactions. In contrast, Group-3 microsomes had significantly higher rates of NNK α- hydroxylation than did microsomes in Groups 1 and 2 (combined) (0.87 vs 0.62 pmol/min/mg jpet.aspetjournals.org protein), suggesting that CYP2A13 protein does contribute to NNK metabolic activation in human lung microsomes that have relatively high CYP2A13 expression. On the other hand, the three groups did not differ significantly in their rates of NNAL formation (Table 3), a finding at ASPET Journals on September 28, 2021 consistent with the lack of involvement of either CYP2A in the carbonyl reduction of NNK. In experiments not presented, coumarin 7-hydroxylase activity (a representative activity for
CYP2A6) was detected in Group-1 microsomes, but not in Group-2 microsomes. This observation supports the notion that CYP2A6 protein is functional in human lung microsomes, despite their apparent lack of significant contribution to NNK α-hydroxylation.
Effect of 8-MOP on NNK α-Hydroxylation in Adult Human Lung Microsomes. The role of CYP2A13 in lung microsomal NNK metabolic activation was further indicated by the effects of 8-MOP, a known inhibitor of CYP2A6 and CYP2A13 (von Weymarn et al., 2005). As shown in Table 4, microsomal preparations with relatively high CYP2A13 protein levels showed not only higher rates of total HPB/OPB formation, but also greater extent of inhibition (46%) by
8-MOP, than did microsomes with low CYP2A13 protein expression (31% inhibition). A
17 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 relatively low concentration of 8-MOP was chosen so as to minimize inhibition of P450 enzymes other than CYP2A6 or CYP2A13 (Koenigs et al., 1997; Zhang et al., 2001); the amount used
(2.5 µM) was found to be sufficient to inhibit >90% of the activity of recombinant CYP2A13 toward 10 µM NNK (data not shown). In contrast, the NNK α-hydroxylation activity of
CYP2A13-Low microsomes with either high or low CYP2A6 protein showed not only similar rates of total HPB/OPB formation, but also similar extent of inhibition by 8-MOP (data not
shown), consistent with the lack of a significant role for CYP2A6 in this reaction. As an internal Downloaded from control, NADPH-dependent formation of NNAL was not affected by the addition of 8-MOP
(Table 4). In other studies not presented, addition of IgG to reaction mixtures increased the rates jpet.aspetjournals.org of NADPH-dependent product formation, a phenomenon that made it impossible to determine the specific effects of anti-2A antibody on NNK metabolism in these lung microsomes. at ASPET Journals on September 28, 2021
18 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
Discussion
Critical to the hypothesis that CYP2A13 plays an important role in tobacco-related human lung tumorigenesis is the assumption that CYP2A13 protein is expressed in human lung, where it is active in the metabolic activation of NNK. However, although CYP2A13 protein has been detected in human fetal nasal mucosa, through the use of a high-resolution immunoblot method (Wong et al., 2005), and in human lung, through immunohistochemical analysis of tissue
sections using an anti-CYP2A13 peptide antibody (Zhu et al., 2006), a quantitative assessment of Downloaded from both the levels of CYP2A13 protein expression, and the extent of the enzyme’s inter-individual variations, was not possible until now. jpet.aspetjournals.org In the present study, we have, for the first time, successfully detected CYP2A13 protein in human lung microsomal preparations. Our ability to resolve and detect CYP2A6 and
CYP2A13 proteins in human lung microsomes, through the use of our recently developed high- at ASPET Journals on September 28, 2021 resolution immunoblot method (Wong et al., 2005), combined with the newly adopted use of immunopurification for enrichment of CYP2A proteins prior to immunoblot analysis, allowed us to estimate the expression levels for the two highly similar P450 proteins in 116 human lung microsomal samples. Our results demonstrate that both CYP2A6 and CYP2A13 proteins are expressed in human lung, but their expression levels varied dramatically among the samples analyzed. We further confirmed that, overall, the expression of CYP2A6 and CYP2A13 proteins correlated with the expression of the corresponding mRNAs in the same tissue samples.
CYP2A6 and CYP2A13 proteins were detected in ~90% and ~12%, respectively, of lung microsome samples examined. The notion that those samples that failed to show detectable
CYP2A proteins do nevertheless contain the CYP2A proteins, albeit at levels below our detection limit (~2 fmol/mg microsomal protein), is supported by 1) the positive detection of
19 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 corresponding CYP2A mRNAs in most of the specimens analyzed; 2) the greater sensitivity of
PCR-based mRNA detection, over the sensitivity of protein detection on immunoblot; and 3) the recent report of localized expression of CYP2A13 protein in airway epithelial cells (Zhu et al.,
2006).
For the majority of microsomal samples analyzed, the level of CYP2A13 was found to be lower than the level of CYP2A6. The content of CYP2A13 protein in the lung microsomal
preparations examined was estimated to be not higher than 20 fmol per mg protein, a level ~10- Downloaded from fold lower than the highest level of CYP2A6 protein detected. Notably, we had previously found
(in a much smaller set of human lung samples) that CYP2A13 mRNA was present at higher jpet.aspetjournals.org levels than was CYP2A6 mRNA (Su et al., 2000). Given the large inter-individual variations in the expression levels of the two CYP2A genes, it is possible that the number of samples analyzed in the previous study was too small to be representative of the general population. Remarkably, at ASPET Journals on September 28, 2021 we found in the present study that the level of CYP2A13 protein, but not CYP2A6 protein, was correlated with lung microsomal NNK metabolic activation activity. Furthermore, addition of 8-
MOP, a potent CYP2A inhibitor, led to greater inhibition of NNK metabolic activation in
CYP2A13-High microsomes than in CYP2A13-Low samples. Taken together, these data indicate that human lung microsomal CYP2A13 is active in NNK metabolic activation; accordingly, individuals who have relatively high levels of pulmonary CYP2A13 expression will likely have an increased risk of developing smoking-related lung cancer. Similarly, for lung toxicants that are preferentially activated by CYP2A6, individuals with high CYP2A6 expression in the lung will likely have greater risk of chemical-induced lung toxicity than will those individuals with little CYP2A6 expression.
20 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 observed large variation in pulmonary CYP2A6 and CYP2A13 expression is unlikely to be due to variations in the quality of tissue specimens used, given that many samples having no detectable CYP2A13 did have relatively high CYP2A6 levels, whereas samples with a level of CYP2A13 higher than the level of CYP2A6 were also observed. On the other hand, given the reported higher expression levels of CYP2A13 protein in lung airway epithelial cells than in other lung cells (Zhu et al., 2006), the extent of variation in CYP2A13 expression might
have been confounded to some extent by variations in the abundance of epithelial cells in each Downloaded from biopsy samples. However, the extent of variation in cellular composition among the biopsy samples studied is expected to be small, given that all tissue samples are from peripheral lung jpet.aspetjournals.org and that we did not notice any obvious, large airway structures during tissue processing. The genetic, epigenetic, and environmental factors that influence CYP2A expression in the lung are subjects of on-going studies in the Ding laboratory. In this regard, we have identified a at ASPET Journals on September 28, 2021
CYP2A13 7520C>G variation that is associated with decreased CYP2A13 expression in human lung (Zhang et al., 2004), and we have also obtained evidence supporting the involvement of epigenetic factors in CYP2A13 regulation (Ling et al., 2007).
Metabolism of NNK by human lung microsomes resulted in the formation of products from both α-hydroxylation and carbonyl reduction, as previously reported (Smith et al. 1992;
1995; 2003), with NNAL being the major product when the substrate is at µM concentrations. It was striking that much higher rates of NNK α-hydroxylation were found in our study than those reported in three previous studies (Smith et al., 1992, 1995, and 2003). One difference between our study and those reported previously was that the time between tissue excision and tissue freezing was 1 h or less in the present study, while in the others it was reportedly 6-11 h (Smith et al., 1992), 0.25-5 h (Smith et al., 1995), or not specified (Smith et al., 2003).
21 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 level of CYP2A13 protein detected here in human lung microsomes was much lower than that found previously in human fetal nasal mucosal microsomes (~1.6 pmol/mg protein); in the latter, CYP2A13 protein was readily detected, without the need for immunopurification
(Wong et al., 2005). The lower expression of CYP2A13 protein in human lung than in human nasal mucosa is consistent with previous findings for CYP2A13 mRNA expression (Su et al.,
2000). However, given that CYP2A13 is preferentially expressed in airway epithelial cells (Zhu
et al., 2006), the concentration of CYP2A13 in these cells would be much higher than was Downloaded from detected in whole lung microsomes, in which the cells containing relatively high levels of
CYP2A13 protein are admixed with a much larger pool of cells with little CYP2A13 expression. jpet.aspetjournals.org By the same token, the rates of NNK α-hydroxylation were, on average, only ~40% higher in microsomes containing 3-20 fmol/mg of CYP2A13 than in microsomes containing <2 fmol/mg of CYP2A13; the rate difference would be much greater if the rates were normalized by total at ASPET Journals on September 28, 2021 protein in airway epithelial cells, rather than by proteins of all lung cells. In support of this idea, a previous study had reported increased formation of NNK α-hydroxylation products in an enriched human alveolar type II cell preparation, as compared to the amounts formed by a mixed population of lung cells or an enriched alveolar macrophage preparation (Smith et al., 1999).
Accordingly, even in individuals with <2 fmol CYP2A13/mg microsomal protein, CYP2A13 may still play an important role in NNK metabolic activation in airway epithelial cells, a notion supported by the 30% reduction in activity rendered by the addition of 8-MOP to CYP2A13-Low samples.
The enzymes responsible for the remaining microsomal activities seen in the presence of
8-MOP have yet to be identified. In previous studies, several P450 enzymes were found, on the basis of inhibition by antibodies or chemical inhibitors, to contribute to human lung microsomal
22 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
NNK α-hydroxylation (Smith et al., 1995 and 2003); these included CYP2A, CYP2B6, CYP2E1, and CYP3A4/5, of which only CYP2B6 has a Km value for NNK that is close to that of
CYP2A13 (Jalas et al., 2005). Although the catalytic efficiency of CYP2B6 toward NNK is much lower than that of CYP2A13, CYP2B6 appeared to be more efficient than CYP2A6 or the other human P450 enzymes that are known to be active toward NNK (Jalas et al., 2005).
Notably, the finding that addition of antibody preparations (e.g., anti-CYP2A6 or control IgG) to
incubation mixtures can lead to increased NNK metabolism (Smith et al., 1995, 2003), an Downloaded from observation confirmed in the present study (data not shown), suggests that the antibody inhibition approach is not effective for lung microsomal preparations with very low activity jpet.aspetjournals.org toward NNK α-hydroxylation. A similar lack of antibody inhibition of CYP2A activity was reported for human bladder microsomes, in which CYP2A13 is believed to catalyze the metabolic activation of 4-aminobiphenyl (Nakajima et al., 2006). at ASPET Journals on September 28, 2021
NADPH-independent formation of NNK α-hydroxylation products by human lung microsomes was observed in this study, as well as in previous studies (Smith et al., 1995 and
2003). This reaction was believed to involve lipoxygenases, free radicals, peroxidases, and P450s
(Smith et al., 1995), although a more recent study indicated that human lung lipoxygenases could not metabolize NNK (Bedard et al., 2002). The significance of an NADPH-independent pathway of NNK α-hydroxylation remains to be determined.
In conclusion, both CYP2A6 and CYP2A13 proteins are expressed in adult human lung, with tremendous inter-individual variations in the levels of their expression. Although CYP2A6 protein is expressed at higher levels than CYP2A13 protein in most individuals examined, it is
CYP2A13, rather than CYP2A6, that is involved in NNK metabolic activation in human lung microsomes. The high inter-individual variations in CYP2A protein expression could result in
23 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 differing susceptibilities to chemical-induced lung toxicity among individuals. In particular, individuals with relatively high expression of CYP2A13 will likely be at a higher risk of developing smoking related lung cancers. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
24 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
Acknowledgments
We gratefully acknowledge the use of the Biochemistry and Molecular Genetics Core
Facilities of the Wadsworth Center. We thank Dr. Adriana Verschoor for reading the manuscript and CHTN for providing human lung tissues. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
25 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
References
Bedard LL, Smith GB, Reid KR, Petsikas D, and Massey TE (2002) Investigation of the role of
lipoxygenase in bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in
human lung. Chem.Res.Toxicol. 15:1267-1273.
Ding X and Coon MJ (1990) Immunochemical characterization of multiple forms of cytochrome
P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa
and NMb. Mol.Pharmacol. 37:489-496. Downloaded from
Fernandez-Salguero P, Hoffman SM, Cholerton S, Mohrenweiser H, Raunio H, Rautio A,
Pelkonen O, Huang JD, Evans WE, Idle JR, and . (1995) A genetic polymorphism in jpet.aspetjournals.org coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant
CYP2A6 alleles. Am.J.Hum.Genet. 57:651-660.
Gu J, Zhang QY, Genter MB, Lipinskas TW, Negishi M, Nebert DW, and Ding X (1998) at ASPET Journals on September 28, 2021
Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1:
role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory
mucosal microsomes. J.Pharmacol.Exp.Ther. 285:1287-1295.
Gu J, Su T, Chen Y, Zhang QY, and Ding XX (2000) Expression of biotransformation enzymes
in human fetal olfactory mucosa: Potential roles in developmental toxicity. Toxicology and
Applied Pharmacology 165:158-162.
Hecht SS (1998) Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines.
Chem.Res.Toxicol. 11:559-603.
Hecht SS, Hochalter JB, Villalta PW, and Murphy SE (2000) 2'-Hydroxylation of nicotine by
cytochrome P450 2A6 and human liver microsomes: formation of a lung carcinogen
precursor. Proc.Natl.Acad.Sci.U.S.A 97:12493-12497.
26 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
Jalas JR, Hecht SS, and Murphy SE (2005) Cytochrome P450 enzymes as catalysts of
metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco specific
carcinogen. Chem.Res.Toxicol. 18:95-110.
Koenigs LL, Peter RM, Thompson SJ, Rettie AE, and Trager WF (1997) Mechanism-based
inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen. Drug Metab
Dispos. 25:1407-1415.
Koskela S, Hakkola J, Hukkanen J, Pelkonen O, Sorri M, Saranen A, Anttila S, Fernandez- Downloaded from
Salguero P, Gonzalez F, and Raunio H (1999) Expression of CYP2A genes in human liver
and extrahepatic tissues. Biochem.Pharmacol. 57:1407-1413. jpet.aspetjournals.org Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227:680-685.
Ling G, Wei Y, and Ding X (2007) Transcriptional regulation of human CYP2A13 expression in at ASPET Journals on September 28, 2021
the respiratory tract by CCAAT/enhancer binding protein and epigenetic modulation.
Mol.Pharmacol. 71:807-816.
Liu C, Zhuo X, Gonzalez FJ, and Ding X (1996) Baculovirus-mediated expression and
characterization of rat CYP2A3 and human CYP2a6: role in metabolic activation of nasal
toxicants. Mol.Pharmacol. 50:781-788.
Nakajima M, Itoh M, Sakai H, Fukami T, Katoh M, Yamazaki H, Kadlubar FF, Imaoka S, Funae
Y, and Yokoi T (2006) CYP2A13 expressed in human bladder metabolically activates 4-
aminobiphenyl. Int.J.Cancer 119:2520-2526.
Peterson LA, Mathew R, and Hecht SS (1991) Quantitation of microsomal alpha-hydroxylation
of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer
Res. 51:5495-5500.
27 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
Shimada T, Yamazaki H, Mimura M, Wakamiya N, Ueng YF, Guengerich FP, and Inui Y (1996)
Characterization of microsomal cytochrome P450 enzymes involved in the oxidation of
xenobiotic chemicals in human fetal liver and adult lungs. Drug Metab Dispos. 24:515-522.
Smith GB, Castonguay A, Donnelly PJ, Reid KR, Petsikas D, and Massey TE (1999)
Biotransformation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) in freshly isolated human lung cells. Carcinogenesis 20:1809-1818.
Smith GB, Bend JR, Bedard LL, Reid KR, Petsikas D, and Massey TE (2003) Biotransformation Downloaded from
of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in peripheral human lung
microsomes. Drug Metab Dispos. 31:1134-1141. jpet.aspetjournals.org Smith TJ, Guo Z, Gonzalez FJ, Guengerich FP, Stoner GD, and Yang CS (1992) Metabolism of
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human lung and liver microsomes and
cytochromes P-450 expressed in hepatoma cells. Cancer Res. 52:1757-1763. at ASPET Journals on September 28, 2021
Smith TJ, Stoner GD, and Yang CS (1995) Activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) in human lung microsomes by cytochromes P450, lipoxygenase, and
hydroperoxides. Cancer Res. 55:5566-5573.
Su T, Bao Z, Zhang QY, Smith TJ, Hong JY, 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.
Ulrike B, Barbara HH, Mario T, Peter-Matthias W, and Ursula GR (2006) Characterisation of the
xenobiotic-metabolizing Cytochrome P450 expression pattern in human lung tissue by
immunochemical and activity determination. Toxicology Letters 164:278-288.
28 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
Wang H, Tan W, Hao B, Miao X, Zhou G, He F, and Lin D (2003) Substantial reduction in risk
of lung adenocarcinoma associated with genetic polymorphism in CYP2A13, the most active
cytochrome P450 for the metabolic activation of tobacco-specific carcinogen NNK. Cancer
Res. 63:8057-8061. von Weymarn LB, Zhang QY, Ding X, and Hollenberg PF (2005) Effects of 8-methoxypsoralen
on cytochrome P450 2A13. Carcinogenesis 26:621-629.
Wong HL, Zhang X, Zhang QY, Gu J, Ding X, Hecht SS, and Murphy SE (2005) Metabolic Downloaded from
activation of the tobacco carcinogen 4-(methylnitrosamino)-(3-pyridyl)-1-butanone by
cytochrome P450 2A13 in human fetal nasal microsomes. Chem.Res.Toxicol. 18:913-918. jpet.aspetjournals.org Zhang QY, Raner G, Ding X, Dunbar D, Coon MJ, and Kaminsky LS (1998) Characterization of
the cytochrome P450 CYP2J4: expression in rat small intestine and role in retinoic acid
biotransformation from retinal. Arch.Biochem.Biophys. 353:257-264. at ASPET Journals on September 28, 2021
Zhang W, Kilicarslan T, Tyndale RF, and Sellers EM (2001) Evaluation of methoxsalen,
tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro. Drug
Metab Dispos. 29:897-902.
Zhang X, Su T, Zhang, Q-Y, Gu J, Caggana M, Li H, and Ding X (2002) Genetic
polymorphisms of the human CYP2A13 gene: Identification of single-nucleotide
polymorphisms and functional characterization of an Arg257Cys variant. J Pharmacol Exp
Ther 302:416-423.
Zhang X, Chen Y, Liu YQ, Ren X, Zhang Q-Y, Caggana M, and Ding X (2003) Single
nucleotide polymorphisms of the human CYP2A13 gene: Evidence for a null allele. Drug
Metab Dipos 31:1081-1085.
29 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
Zhang X, Caggana M, Cutler TL, and Ding X (2004) Development of a real-time polymerase
chain reaction-based method for the measurement of relative allelic expression and
identification of CYP2A13 alleles with decreased expression in human lung.
J.Pharmacol.Exp.Ther. 311:373-381.
Zhu LR, Thomas PE, Lu G, Reuhl KR, Yang GY, Wang LD, Wang SL, Yang CS, He XY, and
Hong JY (2006) CYP2A13 in human respiratory tissues and lung cancers: an
immunohistochemical study with a new peptide-specific antibody. Drug Metab Dispos. Downloaded from
34:1672-1676.
Zhuo X, Gu J, Zhang QY, Spink DC, Kaminsky LS, and Ding X (1999) Biotransformation of jpet.aspetjournals.org coumarin by rodent and human cytochromes P-450: metabolic basis of tissue-selective
toxicity in olfactory mucosa of rats and mice. J.Pharmacol.Exp.Ther. 288:463-471.
at ASPET Journals on September 28, 2021
30 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
Footnotes
This work was supported in part by Public Health Service grants CA092596 (to XD) and
CA084529 (to SM) from the National Institutes of Health. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
31 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
Legends for Figures
Fig. 1. Inter-individual differences in the levels of lung microsomal CYP2A6 and CYP2A13 proteins. Immunopurified proteins, pulled down with anti-CYP2A5 from 500 µg of lung microsomes, or 20~25 fmol of recombinant CYP2A6 or CYP2A13 (both in insect Sf9-cell microsomes), were analyzed on immunoblots with the anti-CYP2A5 antibody. A. Representative samples with high or low CYP2A6 expression, but no detectable CYP2A13 expression. Lanes 1-
5, immunopurified CYP2A from various lung microsomal samples; lane 6, immunopurified Downloaded from
CYP2A from a mixture of recombinant CYP2A6 and CYP2A13, 25 fmol each; lane 7, 20 fmol recombinant CYP2A13 (no immunopurification). B. Representative samples with positive jpet.aspetjournals.org CYP2A13 expression, and varying levels of CYP2A6 expression. Lanes 1-6, immunopurified
CYP2A from various lung microsomal samples; lane 7, immunopurified CYP2A from a mixture of recombinant CYP2A6 and CYP2A13, 25 fmol each. The images were compiled from five at ASPET Journals on September 28, 2021 separate gels. C. An example of the unknown, CYP2A-immunoreactive protein (CYP2A-X), detected in selected lung microsomal samples. Lane 1, 20 fmol recombinant CYP2A13 (no immunopurification); lanes 2 and 3, immunopurified, CYP2A-related proteins from two individual lung microsomal samples, one containing CYP2A-X, the other containing CYP2A6.
D. Immunoblot detection of CYP2A6, without immunopurification, in selected human lung microsomal samples (Lanes 1, 2, and 5; 50 µg protein per lane), but not others (Lanes 3 and 4; 50
µg protein per lane). Lanes 6 and 7, recombinant CYP2A6 and CYP2A13, respectively; 10 fmol each. * indicates lanes containing recombinant CYP2A protein standards.
Fig. 2. HPLC analysis of NNK metabolites formed by human lung microsomes. Assays for NNK metabolism were performed as described in Materials and Methods, with the use of 10 µM [5-
32 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
3H]NNK. Contents of incubation mixtures are described in the notes to Table 3. Metabolites were analyzed using an HPLC system equipped with an on-line radioactivity detector.
Representative chromatograms for three different types of incubation mixtures are shown. A.
Complete reaction mixtures that contained boiled human lung microsome. B. no-NADPH control reactions. C. Complete reaction mixtures, with lung microsomes that contained CYP2A13. The same microsomal preparation was used for the reactions shown in B and C. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 28, 2021
33 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
TABLE 1
Summary of results from immunoblot analysis of CYP2A protein expression in 116 adult human lung microsomal samples
Immunoblot analysis of human lung microsomal CYP2A expression was performed, following immunopurification, as described in Materials and Methods. Examples are shown in
Figure 1. Microsomes were prepared from normal lung biopsy tissues from a total of 116
patients. Details of tissue procurement and clinical diagnosis are described in Materials and Downloaded from
Methods.
Number of samples Subtotal (% of jpet.aspetjournals.org total) Proteins detected CYP2A6 CYP2A13 CYP2A-X Nonea
CYP2A6 87 13 4 0 104 (89.7%)
CYP2A13 13 1 0 0 14 (12.1%) at ASPET Journals on September 28, 2021
CYP2A-X 4 0 1 0 5 (4.3%)
Nonea 0 0 0 10 10 (8.6%) aBelow detection limit (2 fmol/mg microsomal protein)
34 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
TABLE 2
CYP2A6 and CYP2A13 mRNA levels in human lung tissues with differing levels of CYP2A proteins
CYP2A6 and CYP2A13 mRNA levels were determined, using real-time RNA-PCR.
Lung tissues that were found to have either relatively high (~30-110 fmol/mg microsomal protein;
CYP2A6-High) or low (<2 to ~8 fmol/mg microsomal protein; CYP2A6-Low) levels of CYP2A6
were analyzed for CYP2A6 mRNA expression, whereas lung tissues that were found to have Downloaded from either relatively high (~3-20 fmol/mg microsomal protein; CYP2A13-High) or low (<2 fmol/mg microsomal protein; CYP2A13-Low) levels of CYP2A13 were analyzed for CYP2A13 mRNA jpet.aspetjournals.org expression. RNA samples that failed to produce a positive signal after 50 cycles of real-time
PCR were assigned an abundance value of 0 copies, for calculation of means and medians.
Tissue samples n Relative mRNA Abundance (copies/106 copies of β-actin mRNA) at ASPET Journals on September 28, 2021
CYP2A6 CYP2A13
Mean ± S.D. Median (25%, 75%) Mean ± S.D. Median (25%, 75%)
CYP2A6-High 7 84 ± 71 88 (9, 130) NDa ND CYP2A6-Low 9 24 ± 29b 15 (5, 34) ND ND CYP2A13-High 8 ND ND 53 ± 71 18 (4, 130) CYP2A13-Low 16 ND ND 10 ± 16c 3.0 (0.3, 14)d aND, not determined bSignificantly lower than CYP2A6-High; P <0.05, Student’s t-test cComparisons between CYP2A13-High and CYP2A13-Low groups failed equal-variance and normality tests dSignificantly lower than CYP2A13-High; P <0.05, Mann-Whitney Rank Sum test
35 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
TABLE 3
NADPH-dependent NNK α-hydroxylation and NNAL formation by human lung microsomes with varying expression levels of CYP2A6 and CYP2A13 proteins
Complete reaction mixtures contained 100 mM phosphate buffer (pH 7.4), 10 µM [5- 3 H]NNK, 1.0 mM EDTA, 3.0 mM MgCl2, 5.0 mM glucose-6-phosphate, 1.0 mM NADPH, 0.15 unit glucose-6-phosphate dehydrogenase, and 62.5 µg human lung microsomal protein, in a final volume of 50 µl. The NADPH and glucose-6-phosphate dehydrogenase were omitted in no-
NADPH controls. Reactions were carried out for 20 min, a length of time that afforded a Downloaded from determination of initial rates. NNK metabolites were detected as described in Materials and Methods. All experiments were performed in duplicate. The rates of NADPH-dependent NNK α-
hydroxylation were calculated by subtracting the values for no-NADPH controls from the values jpet.aspetjournals.org for the corresponding complete reactions. Values presented are means ± S.D. Available microsomal samples were assayed in groups according to their levels of CYP2A6 and CYP2A13 proteins (fmol/mg microsomal protein): CYP2A6-High, 30-220; CYP2A6-Low, <2 to 10; CYP2A13-
High, 3-20; CYP2A13-Low, <2. at ASPET Journals on September 28, 2021
Group Rates of metabolite formation HPB and OPBa NNAL (pmol/min/mg microsomal protein) 1 – CYP2A6-High/CYP2A13-Low (n=10) 0.61 ± 0.29 49 ± 16 2 – CYP2A6-Low/CYP2A13-Low (n=12) 0.64 ± 0.20b 41 ± 7b 1+2 – CYP2A6-High or Low/CYP2A13-Low (n=22)c 0.62 ± 0.24 45 ± 13 3 - CYP2A6-High or Low/CYP2A13-High (n=10) 0.87 ± 0.15d 49 ± 21e aThe two products of NNK α-hydroxylation were not resolved under the HPLC conditions used. bNot significantly different from Group 1; P > 0.05, Student’s t-test cGroups 1 and 2, both having no detectable CYP2A13 protein, are combined, for comparisons with Group 3, which has detectable CYP2A13 dSignificantly different from Group 1+2; P < 0.05, Student’s t-test eNot significantly different from Group 1+2; P > 0.05, Student’s t-test
36 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
TABLE 4
Inhibition of human lung microsomal NNK α-hydroxylation by 8-methoxypsoralen
NNK metabolism assays were performed as described in the notes to Table 3, except that
8-methoxypsoralen (dissolved in methanol) was added to the reaction mixture at a final concentration of 2.5 µM. At the amount used (1%, final concentration), methanol had no effect on the rate of NNK metabolism. Microsomes containing 5-20 fmol/mg (CYP2A13-High) or <2
fmol/mg (CYP2A13-Low) CYP2A13 protein were analyzed. Values presented are means ± S.D. Downloaded from
“CYP2A-dependent rate” represents the difference between rate determined without 8-MOP addition and rate determined with 8-MOP addition. There was no significant difference among jpet.aspetjournals.org the four rates of NNAL formation (One-way ANOVA, p>0.05).
Group n 8-MOP Rates of metabolite formation (pmol/min/mg) (µM) HPB and OPB CYP2A-dependent NNAL CYP2A13-High 4 0 0.93 ± 0.08 52 ± 25 at ASPET Journals on September 28, 2021 CYP2A13-High 4 2.5 0.51 ± 0.20 0.43 ± 0.16 53 ± 25 CYP2A13-Low 5 0 0.49 ± 0.08a 37 ± 8 CYP2A13-Low 5 2.5 0.34 ± 0.08b 0.15 ± 0.04c 34 ± 8 aCYP2A13-High vs. CYP2A13-Low, in the absence of 8-MOP, P< 0.05, Student’s t-test bCYP2A13-High vs. CYP2A13-Low, in the presence of 8-MOP, P> 0.05, Student’s t-test cCYP2A13-High vs. CYP2A13-Low, P< 0.05, Mann-Whitney Rank Sum test
37 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.
Fig. 1
A ** CYP2A13 CYP2A6 1 2 3 4 5 6 7 B * CYP2A13 CYP2A6 Downloaded from 1 2 3 4 5 6 7
C * CYP2A-X CYP2A13
CYP2A6 jpet.aspetjournals.org 1 2 3 D ** CYP2A13 CYP2A6 1 2 3 4 5 6 7 at ASPET Journals on September 28, 2021 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.
Fig. 2
3,000 A NNK 1,500
0 Downloaded from 3,000 B NNK 1,500 jpet.aspetjournals.org
Radioactivity, cpm Radioactivity, 0 3,000 C NNK OPB/HPB
1,500 at ASPET Journals on September 28, 2021
0 NNAL 02040 60 Retention time, min