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SHORT COMMUNICATION

CYP2A13 Metabolizes the Substrates of Human CYP1A2, Phenacetin, and Theophylline

Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan

(Received October 24, 2006; accepted December 15, 2006)

	Abstract

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Human cytochrome CYP2A13 shows overlapping substrate specificity with CYP2A6, catalyzing the metabolism of coumarin, nicotine, cotinine, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Recently, it was found that CYP2A13 could catalyze the metabolic activations of 4-aminobiphenyl and aflatoxin B₁, which are known to be catalyzed by human CYP1A2. In the present study, we investigated the substrate specificity of CYP2A13. It was shown that CYP2A13 could catalyze ethoxyresorufin O-deethylation, methoxyresorufin O-demethylation, and phenacetin O-deethylation, which are used as marker activities for human CYP1A2. Although the intrinsic clearances (V_max/K_m) of the two former reactions by CYP2A13 were much lower than that of CYP1A2, the value of the last reaction by CYP2A13 was 2-fold higher than that of CYP1A2. Of particular interest was that CYP2A13 has higher affinity toward phenacetin than CYP1A2. In contrast, CYP2A6 hardly catalyzed these reactions, although the amino acid identity with CYP2A13 is as high as 93.5%. Furthermore, we found that CYP2A13 can catalyze theophylline 8-hydroxylation and 3-demethylation, which are known to be mainly catalyzed by human CYP1A2, although the intrinsic clearances were approximately one-tenth that of CYP1A2. CYP2A13 would not contribute to the systemic clearance of these drugs because CYP2A13 is hardly expressed in human liver. However, it may play a role in metabolism in local tissues such as lung or trachea. In conclusion, the results of the present study could extend our understanding of the substrate specificity of CYP2A13.

Recently, we found that CYP2A13 could catalyze the metabolic activation of 4-aminobiphenyl (Nakajima et al., 2006). Furthermore, it has been reported that CYP2A13 is active for the metabolism of aflatoxin B₁ (He et al., 2006). In contrast, CYP2A6 is not active for these substrates. These activities had been known to be catalyzed by CYP1A2 (Gallagher et al., 1996; Hammons et al., 1997). In the present study, we investigated whether CYP2A13 has the ability to metabolize the compounds that are representative substrates of CYP1A2.

	Materials and Methods

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Chemicals. Coumarin, 7-hydroxycoumarin, acetaminophen, theophylline, 1-methylxanthine (1-MX), 3-methylxanthine (3-MX), 1,3-dimethyluric acid (1,3-DMU), and theobromine were obtained from Wako Pure Chemicals (Osaka, Japan). Phenacetin, ethoxyresorufin, methoxyresorufin, and resorufin were from Sigma-Aldrich (St. Louis, MO). All the other chemicals and solvents were of analytical grade or the highest grade commercially available.

Enzyme Preparations. Escherichia coli membranes expressing recombinant human CYP1A1/NPR (Yamazaki et al., 2002), CYP1A2/NPR (Yamazaki et al., 2002), CYP2A6/NPR (Fukami et al., 2004), and CYP2A13/NPR (Yamanaka et al., 2005) were previously prepared in our laboratory. The cytochrome P450 (P450) content and protein concentration were determined according to a method described previously (Omura and Sato, 1964; Bradford, 1976). NADPH-cytochrome c reductase activity was determined as described previously (Williams and Kamin, 1962; Yasukochi and Masters, 1976) using ₅₅₀ = 21.1 mM/cm, and the content was calculated using the specific activity of 3.0 µmol reduced cytochrome c/min/nmol NPR based on purified rabbit NPR preparations (Parikh et al., 1997).

Enzyme Assays. Coumarin 7-hydroxylation was determined as described previously (Ohyama et al., 2000). The substrate concentration was 0.1 to 5 µM. Ethoxyresorufin O-deethylation and methoxyresorufin O-demethylation were determined as described previously (Nakajima et al., 1998). The concentrations of ethoxyresorufin and methoxyresorufin were 0.1 to 2.5 µM for CYP1A1 and CYP1A2 or 0.5 to 7.5 µM for CYP2A6 and CYP2A13.

Phenacetin O-deethylation was determined as follows: a typical incubation mixture (final volume of 0.2 ml) contained the E. coli membrane preparation (5 pmol of P450), 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.5 mM NADP⁺, 5 mM glucose 6-phosphate, 5 mM MgCl₂, and 1 U/ml glucose-6-phosphate dehydrogenase), and 10 to 250 µM phenacetin. The reaction was initiated by the addition of the NADPH-generating system after 2-min preincubation at 37°C. After the 20-min incubation at 37°C, the reaction was terminated by the addition of 10 µl of 60% perchloric acid. After the removal of protein by centrifugation at 10,000 rpm for 5 min, a 20-µl portion of the supernatant was subjected to high-performance liquid chromatography (HPLC). HPLC analyses were performed using an L-7100 pump (Hitachi, Tokyo, Japan), L-7200 autosampler (Hitachi), and a D-2500 integrator (Hitachi) equipped with a Mightysil RP-18 C18 GP (4.6 x 150 mm, 5 µm) column (Kanto Chemical, Tokyo, Japan). The eluent was monitored at 245 nm. The mobile phase was 8% acetonitrile containing 50 mM potassium phosphate (pH 4.2). The flow rate was 1.0 ml/min. The column temperature was 35°C. The quantification of acetaminophen was performed by comparing the HPLC peak height with that of an authentic standard.

Theophylline demethylation and hydroxylation were determined as follows: a typical incubation mixture (final volume of 0.5 ml) contained an E. coli membrane preparation (5 pmol of P450), 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system, and 0.1 to 125 mM theophylline. The reaction was initiated by the addition of the NADPH-generating system after 2-min preincubation at 37°C. After the 30-min incubation at 37°C, the reaction was terminated by the addition of 25 µl of 1 M hydroxychloride. Theobromine (250 pmol) was added as an internal standard. The reaction mixture was extracted with 5 ml of dichloromethane/isopropyl alcohol (75:25 v/v) and then centrifuged at 2000 rpm for 10 min to separate the aqueous and organic fractions. The organic fraction was evaporated under a gentle stream of nitrogen at 40°C. The residue was redissolved in 100 µl of mobile phase, and then the 50-µl portion of the sample was subjected to HPLC. The HPLC apparatus was the same as described above except for a Mightysil RP-18 C18 GP Aqua (4.6 x 150 mm, 5 µm) column (Kanto Chemical). The eluent was monitored at 274 nm with a noise-base clean Uni-3 (Union, Gunma, Japan). The mobile phase was 2.5% methanol containing 10 mM sodium acetate (pH 4.5). The flow rate was 1.0 ml/min (0–23 min) and 1.5 ml/min (24–39 min). The column temperature was 35°C. Quantification of the metabolites was performed by comparing the HPLC peak height ratios with that of authentic standards with reference to an internal standard.

Data Analysis. Kinetic parameters were estimated from the fitted curve using a computer program designed for nonlinear regression analysis (KaleidaGraph, Synergy Software, Reading, PA). All the data were analyzed using the mean of duplicated determinations. Data are mean ± S.D. of three independent experiments. Statistical analyses of the kinetic parameters were performed using the two-tailed Student's t test. A value of P < 0.05 was considered statistically significant.

	Results

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Coumarin 7-Hydroxylation. To confirm that recombinant CYP2A13 expressed in E. coli, which we constructed, is enzymatically active, the coumarin 7-hydroxylase activity was measured. The K_m and V_max values by CYP2A13 were 0.7 ± 0.0 µM and 1.7 ± 0.1 pmol/min/pmol P450, respectively. The K_m and V_max values in CYP2A6 were 1.3 ± 0.2 µM and 5.0 ± 0.8 pmol/min/pmol P450, respectively. The intrinsic clearance (V_max/K_m) of CYP2A13 (2.6 ± 0.1 µl/min/pmol P450) was lower than that of CYP2A6 (3.8 ± 0.7 µl/min/pmol P450).

Ethoxyresorufin O-Deethylation and Methoxyresorufin O-Demethylation. Using the recombinant CYP2A13, the ethoxyresorufin O-deethylase activity and methoxyresorufin O-demethylase activity were measured. CYP2A13 exhibited both activities, although the activities were inconsiderable compared with CYP1A1 or CYP1A2. In contrast, CYP2A6 did not show detectable activities. The kinetics by CYP2A13, CYP1A1, and CYP1A2 were fitted to the Michaelis-Menten equation (Fig. 1, A and B). For the ethoxyresorufin O-deethylation, the K_m and V_max values of CYP2A13 were higher and lower, respectively, than those of CYP1A1 and CYP1A2, resulting in conspicuously lower intrinsic clearance of CYP2A13 than those of CYP1A1 and CYP1A2 (Table 1). Similarly, the intrinsic clearance of CYP2A13 for the methoxyresorufin O-demethylation was also lower than those of CYP1A1 and CYP1A2 (Table 1). Thus, CYP2A13 showed slight activities for ethoxyresorufin O-deethylation and methoxyresorufin O-demethylation.

View larger version (28K): [in this window] [in a new window]   FIG. 1. Kinetic analyses of ethoxyresorufin O-deethylation (A), methoxyresorufin O-demethylation (B), phenacetin O-deethylation (C), theophylline metabolism (D, 8-hydroxylation; E, 3-demethylation; F, 1-demethylation) catalyzed by recombinant CYP1A1, CYP1A2, CYP2A6, and CYP2A13 expressed in E. coli. The kinetic parameters were estimated from the fitted curve using the computer program KaleidaGraph designed for nonlinear regression analysis. Each point represents the mean of duplicate determinations.

Phenacetin O-Deethylation. We determined whether CYP2A13 catalyzes phenacetin O-deethylation, which is known as a typical activity for CYP1A2 (Sesardic et al., 1988). Surprisingly, CYP2A13 showed higher activity for phenacetin O-deethylation than CYP1A2 (Fig. 1C). In contrast, CYP2A6 showed scarce activity (0.03 pmol/min/pmol P450) only at a high substrate concentration (250 µM); therefore, the kinetic parameters could not be calculated. The K_m and V_max values of CYP2A13 were lower and higher, respectively, than those of CYP1A1 and CYP1A2 (Table 2), resulting in higher intrinsic clearance of CYP2A13 than that of CYP1A2 (2-fold) and CYP1A1 (5-fold).

Theophylline Metabolism. Theophylline is mainly metabolized to three metabolites, 1,3-DMU, 1-MX, and 3-MX by P450s, mainly CYP1A2 in human liver. It has been reported that human lung microsomes can also convert theophylline to 1,3-DMU (Bowen et al., 1991). Because CYP2A13 is highly expressed in the human respiratory tract, we determined whether CYP2A13 is able to metabolize theophylline. As shown in Fig. 1, D and E, CYP2A13 showed metabolic activities for theophylline 8-hydroxylation (1,3-DMU formation) and 3-demethylation (1-MX formation). Interestingly, CYP2A6 also showed activity for theophylline 8-hydroxylation. CYP2A13 showed moderate intrinsic clearance for 8-hydroxylation, following CYP1A2 (Table 3). Although the intrinsic clearance of CYP2A13 was one-tenth that of CYP1A2, it was higher than that of CYP2A6 and CYP1A1. The intrinsic clearance of CYP2A13 for theophylline 3-demethylation was one-sixth that of CYP1A2. Theophylline 1-demethylation (3-MX formation) was detected only by CYP1A2.

	Discussion

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The CYP2A13 gene was first cloned in 1995 (Fernandez-Salguero et al., 1995), and the expression of CYP2A13 mRNA in human tissues was determined in 1999 (Koskela et al., 1999). Before 2000, the CYP2A13 protein was assumed to be nonfunctional based on its sequence features of CYP2A13 that resemble the nonfunctional CYP2A7. However, the fact that CYP2A13 is a functional enzyme was first shown by constructing heterologously expressed CYP2A13 in baculovirus-infected insect cells (Su et al., 2000). They reported that CYP2A13 could catalyze coumarin 7-hydroxylation but was much less active than CYP2A6 (0.26 versus 2.2 pmol/min/pmol P450 at 100 µM coumarin concentration). von Weymarn and Murphy (2003) subsequently reported that the K_m and V_max values of coumarin 7-hydroxylation by recombinant CYP2A13 in baculovirus-infected insect cells were 0.48 µM and 0.15 pmol/min/pmol P450, respectively. Similarly, He et al. (2004) reported that K_m and V_max values by the recombinant CYP2A13 in baculovirus-infected insect cells were 2.2 µM and 0.69 pmol/min/pmol P450, respectively. Thus, the kinetic parameters for coumarin 7-hydroxylation by CYP2A13 obtained in the present study were similar to those of previous results.

Ethoxyresorufin O-deethylation, methoxyresorufin O-demethylation, and phenacetin O-deethylation are used as marker activities for CYP1A2 (Eugster et al., 1993; Tassaneeyakul et al., 1993). Interestingly, the data presented here showed that CYP2A13 could catalyze these reactions. Of particular interest is that CYP2A13 showed higher activity for phenacetin O-deethylation than CYP1A2. In contrast to CYP2A13, CYP2A6 hardly catalyzes phenacetin O-deethylation. Previously, Venkatakrishnan et al. (1998) reported that the K_m value of CYP2A6 for phenacetin O-deethylation was 4098 µM. Thus, phenacetin is a substrate of CYP2A13, but not CYP2A6, even though the amino acid identity between the two enzymes is as high as 93.5%. Rabbit CYP2A isoforms, CYP2A10 and CYP2A11, have been reported to catalyze phenacetin O-deethylation (Peng et al., 1993). These isoforms show higher amino acid identity to CYP2A13 rather than CYP2A6, as regards 32 amino acids showing differences between CYP2A6 and CYP2A13 (22 of 32 amino acids correspond to CYP2A13, whereas only 5 of 32 amino acids correspond to CYP2A6). Therefore, these subtle changes in amino acids may contribute to determining the substrate specificity toward phenacetin.

Theophylline is used to manage bronchial asthma and chronic obstructive pulmonary disease. Although it is well known that theophylline is mainly metabolized to 1,3-DMU, 1-MX, and 3-MX by CYP1A2, we first found that CYP2A13 can metabolize theophylline. The clinical significance of CYP2A13 in the metabolism of phenacetin and theophylline in human liver would be limited because systemic clearance of drugs is caused by the metabolism in liver where CYP2A13 is hardly expressed. However, CYP2A13 is highly expressed in the respiratory tract. It has been shown that human lung is one of the tissues where local metabolism of xenobiotics may take place. Indeed, theophylline is distributed in the lung at the same concentration as in blood (Schack and Waxler, 1949); human lung microsomes can convert theophylline to 1,3-DMU (Bowen et al., 1991). Therefore, CYP2A13 possibly contributes to the theophylline metabolism in human lung and may cause a change in therapeutic efficacy, although other isoforms such as CYP1A1, CYP2E1, CYP2D6, and CYP3A4 expressing in human lung and having capability for theophylline metabolism (Zhang and Kaminsky, 1995; Bernauer et al., 2006) may also contribute to the extrahepatic metabolism.

We found that CYP2A13 shares substrates with CYP1A2. The amino acid homology between CYP2A13 and CYP1A2 is less than 40%. Even if we compared the amino acids within site recognition sites of CYP2A13 and CYP1A2, the amino acid homology is extremely low. In addition, three-dimensional quantitative structure-activity relationship analysis of CYP2A13 is not accomplished yet. Thus, although available information is limited, our data potentially suggested the structural similarity of the substrate binding site of CYP2A13 with that of CYP1A2. Recently, we found that CYP2A13 can catalyze the metabolic activation of 4-aminobiphenyl that is also known to be metabolized by CYP1A2 (Nakajima et al., 2006). Therefore, we investigated whether CYP2A13 is involved in the activation of other arylamines such as 2-aminofluorene, 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine that were reported to be activated by CYP1A by umu assay. However, the metabolic activation of these compounds by CYP2A13 was not detected (data not shown). Thus, CYP2A13 may not necessarily be involved in the metabolism of substrates of human CYP1A.

In the present study, we found that CYP2A13 can metabolize several compounds that are known as the substrates of CYP1A2. CYP2A13 may play roles in the local metabolism of drugs in the human respiratory tract. This study significantly increased our understanding of the substrate specificity of human CYP2A13.

	Acknowledgments

 
We thank Brent Bell for reviewing the manuscript.

	Footnotes

 
T.F. was supported as a Research Fellow of the Japan Society for the Promotion of Science.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.011064.

ABBREVIATIONS: 1-MX, 1-methylxantine; 3-MX, 3-methylxantine; 1,3-DMU, 1,3-dimethyluric acid; P450, cytochrome P450; HPLC, high-performance liquid chromatography.

Address correspondence to: Miki Nakajima, Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail: nmiki{at}kenroku.kanazawa-u.ac.jp

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