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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Six Novel Nonsynonymous CYP1A2 Gene Polymorphisms: Catalytic Activities of the Naturally Occurring Variant Enzymes

Project Team for Pharmacogenetics (N.Mu., A.S., Y.S., Y.N., S.O., J.-i.S.), Division of Biochemistry and Immunochemistry (Y.S., J.-i.S.), Division of Pharmacology (S.O.), National Institute of Health Sciences, Tokyo, Japan; Departments of Cardiology (K.K., S.K., Ma.Ki.), Cardiovascular Dynamics Research Institute (K.K.), and Pharmacy (K.U.), National Cardiovascular Center, Osaka, Japan; National Institute of Neuroscience (H.K., Y.-i.G., N.Mi.); and National Center Hospital for Mental Nervous and Muscular Disorders (O.S., Ma.Ka., Te.O., Mi.Ka., K.S., Ta.O., C.S., N.Mi.), National Center for Neurology and Psychiatry, Tokyo, Japan

Received June 20, 2003; accepted October 9, 2003.

	Abstract

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Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the V_max of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower K_m value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

	Materials and Methods

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Human Genomic DNA Samples and Polymerase Chain Reaction (PCR) Conditions for DNA Sequencing for Single Nucleotide Polymorphism (SNP) Discovery. A total of 206 arrythmic or epileptic patients were analyzed in this study. No significant differences in the frequencies of the SNPs were found in the two populations. Total genomic DNA was extracted from blood leukocytes and was used for DNA sequencing. The ethics committees of the National Cardiovascular Center (Osaka, Japan), National Center for Neurology and Psychiatry (Tokyo, Japan), and the National Institute of Health Sciences (Tokyo, Japan) approved this study. Written informed consent was obtained from all participating subjects.

Before the analysis of each CYP1A2 exon and intron, a DNA fragment (8.3 kb), including almost the entire CYP1A2 gene, was amplified using the following primers: (5'-tctaatctccagtccgtgctt-3', 1A2F1; 5'-ggagggactgctaatgggtg-3', 1A2R1). PCR was conducted in a reaction mixture (100 µl) containing 1x Z Taq buffer, 2.5 mM dNTPs, 1 unit of Z Taq polymerase (TaKaRa shuzo, Kyoto, Japan), 200 ng of genomic DNA, and 0.5 µM of each primer. The first-round PCR conditions were 30 cycles of 98°C for 5 s, 55°C for 5 s, and 72°C for 190 s. Next, each exon was amplified by Ex Taq polymerase (TaKaRa shuzo) with the appropriate set of CYP1A2-specific primers, the sequences of which are described in Table 1. The second round of PCR was done at 94°C for 5 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 2 min. PCR was conducted in a reaction mixture (50 µl) containing 1x Ex Taq buffer, 3 mM MgCl₂, 2.5 mM dNTPs, 1 unit of Ex Taq polymerase, 2 µl of first-round PCR product, and 0.5 µM of each primer. The PCR product (5 µl) was then incubated with exonuclease I (1 µl) and shrimp alkaline phosphatase (1 µl) contained in the PCR product presequencing kit (USB, Cleveland, OH) for 15 min at 37°C, and then inactivated for 15 min at 80°C, and finally incubated at 4°C. Exonuclease I and shrimp alkaline phosphatase included in the PCR product presequencing kit were used to effectively remove the excess deoxynucleoside triphosphates and primers from DNA produced by PCR amplification (Matter et al., 1999). For cycle sequencing, the reaction mixture (20 µl) consisted of 2 µl of BigDye Terminator Version 3 (Applied Biosystems, Uppsala, Sweden), 1.6 pmol of a sequencing primer (Table 2), 3 µlof(1x) sequencing buffer (Applied Biosystems), and 4.4 µlofthe PCR products (10–40 ng of the PCR-amplified DNA). The sequencing reaction was done as follows: 10 s at 96°C, 5 s at 50°C, and 4 min at 60°C for 25 cycles. After the cycling, the products were purified and analyzed on the ABI PRISM 3700 DNA autosequencer (Applied Biosystems, Foster City, CA). After performing the first long PCR using Z Taq polymerase, at least two separate runs of the second-round PCR step and subsequent sequencing in both directions gave the consistent sequencing results. Furthermore, to verify the six novel single nucleotide polymorphisms studied, we repeated the first Z Taq polymerase step, followed by second-round PCR and subsequent sequencing. These second-round sequencing analysis confirmed these nucleotide variations.

Cell Culture and Protein Analysis. The V79 Chinese hamster lung fibroblast cells were cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO), supplemented with 5% fetal calf serum (Sigma-Aldrich), 100 U/ml penicillin G, and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.

Site-Directed Mutagenesis of CYP1A2 cDNA. We introduced mutations into the transfection vectors carrying a CYP1A2 cDNA (pcDNA3.1/CYP1A2 wild type) using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Procedure for the mutagenesis includes denaturation of the DNA template, annealing mutagenic primers containing mutation, and extension and incorporation of primers with PfuTurbo DNA polymerase. We sequenced not only around the mutated nucleotide but also the entire coding region of each mutant after the introduction of the mutations into the pcDNA3.1/CYP1A2 wild type.

Transient Transfection of CYP1A2 and Its Variants. V79 cells were seeded in 100-mm culture dishes. After reaching 70 to 80% confluence, the cells were rinsed with serum-free OPTI-MEM (Invitrogen) before transfection. The pcDNA3.1/CYP1A2 wild-type, pcDNA3.1/E168Q, pcDNA3.1/F186L, pcDNA3.1/S212C, pcDNA3.1/T83M, pcDNA3.1/G299S, and pcDNA3.1/T438I plasmids (6 µg each) were transfected separately using the LipofectAMINE PLUS reagents (Invitrogen) as described previously (Murayama et al., 2002). The cells were harvested and homogenized after 24 h in 100 mM potassium phosphate buffer (pH 7.4). Cell homogenates were centrifuged at 9000g for 20 min, and the supernatants were centrifuged at 105,000g for 1 h. The resultant pellets were used as microsomes.

Immunoblotting of CYP1A2 Protein. The microsomal protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA). To determine the expressed levels of the wild-type and variant CYP1A2 proteins in V79 cells, 20 µg of microsomal proteins was dissolved in SDS-sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred onto nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany). For immunostaining, antiserum for human CYP1A1/1A2 (Daiichi Pure Chemicals Co., Tokyo, Japan) was used as the first antibody (Code et al., 1997). The second antibody, rabbit anti-goat IgG (Medical and Biological Laboratories Co., Ltd., Nagoya City, Japan) conjugated with horseradish peroxidase, was visualized with the enhanced chemiluminescence kit (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK). The signal intensities were analyzed by the aid of ZERO-Dscan software (Scanalytics, Billerica, MA) in a chemiluminescence detection system, DIANAII (Raytest, Straubenhardt, Germany).

Determination of Microsomal 7-Ethoxyresorufin O-Deethylase Activity. 7-Ethoxyresorufin O-deethylation was measured fluorometrically at a 100 µM substrate concentration as described previously (Leclercq et al., 1996) with some modification. The incubation mixtures (0.2 ml) contained 0.1 mg microsomal protein in 100 mM Tris-HCl buffer (pH 7.4), containing an NADPH-generating system (0.5 mM NADP⁺, 5 mM glucose 6-phosphate, and 0.5 unit glucose-6-phosphate dehydrogenase) and 100 µM substrate. The generating system was added to the mixture to initiate the reaction. After a-30 min incubation, the reaction was terminated by the addition of 0.05 ml of cold methanol. p-Acetanisidine (0.8 µg) was added to the incubation mixture as an internal standard. The product fluorescence was determined by high-performance liquid chromatography (HPLC) using a Shimadzu fluorescence detector (RF-10AXL) with excitation at 530 nm and emission at 585 nm. For kinetic studies, wild-type and six CYP1A2 variant microsomal proteins were incubated at a 1 mg/ml protein concentration with 0.25 to 4 µMofthe substrate.

Determination of Microsomal Phenacetin O-Deethylase Activity. The phenacetin O-deethylation activity was determined by detecting acetaminophen, the enzymatically O-deethylated phenacetin, the amount of which was determined by HPLC. The incubation mixtures (0.2 ml) contained 0.1 mg of microsomal protein in 50 mM potassium phosphate buffer (pH 7.4), containing an NADPH-generating system (0.5 mM NADP⁺, 5 mM glucose 6-phosphate, 0.5 unit glucose-6-phosphate dehydrogenase) and 100 µM substrate. The generating system was added to the mixture to initiate the reaction. After a 60 min incubation, the reaction was terminated by the addition of 0.05 ml cold CH₃CN. Caffeine (0.1 mg/ml, 4 µg total) was added to the incubation mixture as an internal standard. After centrifugation for 5 min, the supernatant was injected onto the HPLC. The mobile phase was 10% acetonitrile, containing 50 mM ammonium acetate buffer (pH 4.6). The flow rate was 1 ml/min. The acetaminophen product was monitored at 245 nm.

Statistical Analyses. Differences in the O-deethylation of phenacetin and 7-ethoxyresorufin between the wild-type CYP1A2 and its six variants were analyzed by one-way analysis of variance with Tukey's post hoc test.

	Results

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Nonsynonymous SNPs Found in This Study. From the sequence analysis of 206 Japanese DNA samples, six nonsynonymous SNPs in the CYP1A2 coding region were identified. The frequencies of the g502c (E168Q) and c558a (F186L) alleles were 0.002; those of the c248t (T83M), a634t (S212C), and c1313t (T438I) alleles were 0.004, and that of the g895a (G299A) allele was 0.006 (Table 3). Each of the six SNPs was independently identified. The patients were heterozygous for the variations reported. Electropherograms for all six SNPs are shown in Fig. 1.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Electropherograms of six nonsynonymous SNPs identified in this study. The arrows indicate the SNP sites. Bases colored with green, blue, black, and red represent adenine, cytosine, guanine, and thymine, respectively. A and B are sequence electropherograms of the homozygous wild type (WT) and a T83M heterozygote; C and D represent those of the homozygous WT and a heterozygous E168Q; E and F represent those of the homozygous WT and a heterozygous F186L; G and H represent those of the homozygous WT and a heterozygous S212C; I and J represent those of the homozygous WT and a heterozygous G299S; and K and L represent those of the homozygous WT and a heterozygous T438I, respectively.

Expression of CYP1A2 in a V79 Cell Expression System. To examine any functional alterations due to amino acid changes from the six SNPs, pcDNA3.1 plasmids were prepared that contained full-length cDNA inserts encoding the wild-type human CYP1A2 and the variants T83M, F168Q, F186L, S212C, G299A, and T438I. These plasmid DNAs were transiently transfected into Chinese hamster V79 cells. V79 cells have been shown to lack at least CYP1A and CYP2B (Fuhr et al., 1992; Rodrigues et al., 1994). V79 cells express NADPH-dependent cytochrome P450-reductase, an enzyme required for P450 monooxygenase activity (Schmalix et al., 1996). This transient transfection system, expression of CYP1A2 in the cells was highest 24 h after transfection of the cDNA. Thus, we prepared microsomes from the cells after 24-h cDNA transfection. The expressed levels of the wild type and the six variant CYP1A2 proteins were determined by comparisons with known CYP1A2 levels in human liver microsomes (45 pmol of CYP1A2/mg; BD Gentest, Woburn, MA). Microsomal CYP1A2 contents were 12 ± 2 pmol/mg protein for the wild type, 9 ± 2 pmol/mg protein for T83M, 7 ± 1 pmol/mg protein for E168Q, 11 ± 2 pmol/mg protein for F186L, 12 ± 1 pmol/mg protein for S212C, 11 ± 2 pmol/mg protein for G299S, and 7 ± 2 pmol/mg protein for T438I (n = 3). There were no significant differences in the expression levels of CYP1A2 protein between the wild type and the six variants (Fig. 2). The amino acid substitutions in the present study might not affect expression or degradation rate of the wild-type and the variant CYP1A2 protein on the assumption that transfection efficiencies were similar for the wild-type and variant CYP1A2 cDNAs.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Expression of the vector only, the wild type, and the six variant CYP1A2 cDNA constructs. No expression was observed in the vector-transfected cells (Null). The amounts of immunoreactive proteins of the wild-type CYP1A2 and the variants were densitometrically determined as described under Materials and Methods. HM, human liver microsome.

Metabolism and Kinetic Characterization of 7-Ethoxyresorufin O-Deethylation by CYP1A2 Variant Enzymes. Metabolism of 7-ethoxyresorufin by the wild-type CYP1A2 and six variants were compared using microsomes from cells cultured for 24 h after transfection. On the basis of picomoles of CYP1A2 expressed, 7-ethoxyresorufin O-deethylase activity of the wild-type CYP1A2 cells was 0.088 ± 0.001 pmol/pmol CYP1A2/min (Fig. 3A). On the assumption that some variant might show high K_m values, we used a substrate concentration of 100 µM. The F186L variant also exhibited the lowest 7-ethoxyresorufin O-deethylating activity among the wild type and six CYP1A2 variants. The variant catalyzed ethoxyresorufin O-deethylation at a rate of 0.025 ± 0.003 pmol/min/pmol CYP1A2, with about 28% of the wild-type activity. The catalytic activity of the T83M, E168Q, S212C, and G299S variants was 76.6, 80.1, 83.2, and 72.2% of that of the wild type, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Enzymatic activities of the wild-type CYP1A2 and the variants with 7-ethoxyresorufin (A) and phenacetin (B). O-Deethylation of 7-ethoxyresorufin and phenacetin was determined at a 100 µM substrate concentration on the basis of picomoles of expressed CYP1A2 (i.e., y-axes represent pmol substrate hydroxylated per picomoles of CYP1A2 expressed per minute) as described under Materials and Methods, which enabled direct comparison of the turnover rate for these substrates. Data are shown as mean (height of bars) ± S.D.

Kinetic analyses of ethoxyresorufin O-deethylation were also performed. Approximately, 1.9- and 3.7-fold lower K_m values compared with the wild type were obtained with the E168Q (p < 0.05) and F186L (p < 0.001) variants, respectively (Table 4). The F186L V_max value was 16-fold lower (p < 0.05) than that of the wild type, which resulted in a 5.1-fold lower V_max/K_m (p < 0.05). Despite the lower K_m value, the V_max/K_m of the E168Q variant for 7-ethoxyresorufin O-deethylation was similar to that of the wild type (0.051 versus 0.030; Table 4). Differences in the V_max/K_m values of the T83M, S212C, and G299A variants were marginal, although the S212C K_m value was approximately 1.5-fold higher (p < 0.01) than that of the wild type. These results showed the remarkable reduction in the enzymatic activity of the F186L variant for the O-deethylation of phenacetin and 7-ethoxyresorufin among the naturally occurring CYP1A2 variants studied.

Phenacetin O-Deethylation by CYP1A2 Variant Enzymes. Metabolism of phenacetin by the wild-type CYP1A2 and six variants were compared using microsomes from cells cultured for 24 h after transfection. On the basis of picomoles of CYP1A2 expressed, phenacetin O-deethylase activity of the wild type was 0.275 ± 0.072 pmol/pmol CYP1A2/min at a 100 µM substrate concentration (Fig. 3B). The lowest phenacetin O-deethylation was observed in the F186L variant-expressing cells, which was about 12.5% of the wild-type enzymatic activity (0.033 ± 0.0079 pmol/pmol CYP1A2/min). No statistically significant changes in activity were obtained for the T83M, E168Q, S212C, G299A, and T438I variants. The percentages of the wild-type enzymatic activities were 82, 86, 88, 80, and 116% for each variant, respectively.

	Discussion

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CYP1A2 encodes an enzyme that carries out the oxidative metabolism of a variety of therapeutic drugs, converting them to inactive metabolites. CYP1A2 has been shown to be involved in the metabolic activation of carcinogenic arylamines and acetaminophen, producing reactive intermediates (Guengerich, 1995; Kivisto and Kroemer, 1997; Hines and McCarver, 2002; Lewis, 2002; Rendic, 2002; Scordo and Spina, 2002).

Constitutive CYP1A2 mRNA expression levels in human liver samples can vary by as much as 15-fold (Hammons et al., 2001). Interindividual difference in CYP1A2 protein was reported to be up to 60-fold (Shimada et al., 1994). It has been documented that genetic differences play an important role in the individual variation in constitutive and/or inducible CYP1A2 levels, which influences the effectiveness of prescribed medications, and susceptibility to environmental mutagens and carcinogens.

In this study, the CYP1A2 gene from 206 Japanese individuals was sequenced and six nonsynonymous SNPs were found. Next, we investigated whether any CYP1A2 functional change accompanied these six polymorphisms. Site-directed mutagenesis was used to introduce these mutations into the wild-type CYP1A2 cDNA. These mutated cDNAs inserted into the pcDNA3.1 expression vector were subsequently expressed in V79 cells. Catalytic activities of the CYP1A2 wild type and six CYP1A2 variant forms for 7-ethoxyresorufin O-deethylation and phenacetin O-deethylation are described in Fig. 3. Among the six variants, the most remarkable reduction in enzymatic activity was found in the F186L substitution. Catalytic activities of this variant for phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were approximately 12 and 28% of the wild type, respectively (Fig. 3).

In regard to the CYP1A2 amino acid sequences affecting its enzyme activity, several investigators have documented the critical amino acids. The Guengerich research group defined six substrate recognition sequence (SRS) regions of CYP1A2 based on the amino acid sequence alignment of CYP2 family members (Gotoh, 1992): SRS1, GRPDLYTSTLITDGQSLTF¹²⁶STDSG; SRS2, VKNTH²²⁵EF; SRS3, FK²⁵⁵AFNQR; SRS4, KIVNLVNDIFGAGF³²⁰DTVTTA; SRS5, SSFLPF³⁸⁵TIPHS; and SRS6, TPI⁴⁹⁵YGLTM (Parikh et al., 1999). Parikh et al. (1999) performed a large random mutagenesis study in the SRS regions. An acidic residue, 320D, was thought to stabilize the I-helix in the heme distal region and to be required for oxygen activation. Human ³²⁰D corresponds to ³¹⁸E in rat CYP1A2. Shimizu et al. (1994) suggested that rat 318E and 319T, present in the distal site, serve as a proton source for the heterolytic scission of hydroperoxides as in the wild-type CYP1A2. Parikh et al. (1999) also discussed that 226F was a critical amino acid because the k_cat/K_m was extremely reduced by the F226Y substitution, despite a relatively small modification. The authors pointed out that the CYP1A2 active site-forming sequences were not limited to the six SRS regions. The F186L substitution, which is not located within any SRS region, indeed resulted in the most remarkable reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Sequence analysis indicated that F186 is a characteristic and common amino acid of the CYP1A subfamily throughout human and other animal species (Fig. 4). It is possible that this amino acid is critical for the typical CYP1A-catalyzing reactions, because phenylalanine at residue 186 might interact with CYP1A2 substrates through - stacking and/or hydrogen bond considering a recent study by Lewis et al. (2003).

View larger version (64K): [in this window] [in a new window]   Fig. 4. Alignments of the deduced amino acid sequences of human P450 forms in a region surrounding CYP1A2 codon 186. The term "CYP" should be added before each sequence designation (e.g., "1A2" should read "CYP1A2", and so on.). The alignments were done with the aid of Dr. Nelson's alignment (http://drnelson.utmem.edu/humP450.aln.html) and references from Gotoh and Graham-Lorence and Peterson (Gotoh, 1992; Graham-Lorence and Peterson, 1996). Phenylalanine at CYP1A2 codon 186 and the corresponding phenylalanine in CYP1A1 are emphasized, respectively, by a rectangle. hum, human; ham, hamster; mou, mouse; rab, rabbit; tro, trout.

Lewis reported that the homology modeling of P450 family members CYP1, CYP2, CYP3, and CYP4 demonstrated the active site of P450 enzymes and indicated the consistency of the predicted structures with reported experimental metabolic data and results obtained from site-directed mutagenesis studies (Lewis et al., 1999, 2003; Lewis, 2002). Lewis also reported in his model that the three methyl groups of caffeine, a typical CYP1A2 marker substrate, could be nearly equally distant from the heme iron, with a slight preference for the N-3 position and that caffeine was positioned on average parallel to the heme (Lewis et al., 1999; Lewis, 2002). This result is consistent with the primary N-3 demethylation of the substrate to form paraxanthine (Regal and Nelson, 2000). In particular, three hydrogen bonds are postulated to be formed from ⁷⁸Thr, ⁸⁷Thr, and ⁴³⁸Thr to the two carbonyl oxygen atoms and ring nitrogen of caffeine, such that the N-3-methyl group is positioned directly above the heme iron for oxidation (Lewis et al., 1999; Lewis, 2002). In this study, however, the T438I substitution failed to result in a remarkable change in the enzymatic function for the O-deethylation of phenacetin and 7-ethoxyresorufin. Our results indicated that the threonine hydroxyl group at codon 438 is not critical for the O-deethylating activities of these substrates. By comparison of ⁴³⁸T in human CYP1A2 with that of other animal species, threonine is not necessarily conserved: Thr in hamster, Ser in mouse, Thr in rat, and Ala in rabbit. This may suggest threonine at residue 438 is not necessarily critical.

In human CYP1A1, residues 201 to 214 correspond to the loop connecting helix E to helix F in bacterial CYP102 (Urban et al., 2001). This segment is highly variable, because 9 of 11 residues are different between human CYP1A1 and 1A2. Urban et al. (2001) reported that despite mutations in the 204 to 214 amino acid segment of CYP1A1 corresponding to CYP1A2 204 to 214, there was no shift in its substrate specificity to CYP1A2. Consistently, S212C in this study did not remarkably alter the O-deethylating activities of phenacetin and 7-ethoxyresorufin. It should be noted, however, that a significantly higher K_m value was observed for ethoxyresorufin O-deethylation (Table 4).

In conclusion, this study clearly indicates that among the newly found six naturally occurring amino acid substitutions, the F186L substitution remarkably reduced the catalytic activity for phenacetin and 7-ethoxyresorufin. This is the first report on six novel nonsynonymous SNPs found in a Japanese population. Furthermore, it is worthwhile to mechanistically investigate the role of ¹⁸⁶F in the catalytic function of CYP1A2.

Note Added in Proof. Designations for the novel alleles reported in the present study have been made by the Human CYP Allele Nomenclature Committee and posted at the website (http://www.imm.ki.se/CYPalleles/cyp1a2.htm): T83M as CYP1A2^*9; E168Q as CYP1A2^*10; F186L as CYP1A2^*11; S212C as CYP1A2^*12; G299A as CYP1A2^*13; T438I as CYP1A2^*14.

	Acknowledgements

 
We thank Chie Knudsen for secretarial assistance.

	Footnotes

 
This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-2, MPJ-3, and MPJ-6) of the Organization for Pharmaceutical Safety and Research of Japan.

DOI: 10.1124/jpet.103.055798.

ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; HPLC, high-performance liquid chromatography; SNP, single nucleotide polymorphism; HPLC, high-performance liquid chromatography; SRS, substrate recognition sequence.

¹ These authors contributed equally to this work.

Address correspondence to: Dr. Shogo Ozawa, Division of Pharmacology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan. E-mail: sozawa{at}nihs.go.jp

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