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CHARACTERIZATION OF NOVEL CYP2A6 POLYMORPHIC ALLELES (CYP2A6^*18 AND CYP2A6^*19) THAT AFFECT ENZYMATIC ACTIVITY

Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan (T.F., M.N., E.H., H.Y., H.S., T.Y.); and Department of Medicine, Washington University School of Medicine, St. Louis, Missouri (H.L.M.)

(Received April 7, 2005; accepted May 10, 2005)

	Abstract

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Genetic polymorphisms of CYP2A6 gene are known as a causal factor of the interindividual differences in nicotine metabolism. We found three novel CYP2A6 alleles. The CYP2A6^*18A allele has a single nucleotide polymorphism (SNP) of A5668T (A1175T, Y392F) in exon 8. The CYP2A6^*18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) in addition to A5668T (A1175T, Y392F). The CYP2A6^*19 allele has the SNPs of A5668T (A1175T, Y392F), T6354C (intron 8), and T6558C (T1412C, I471T) as well as the conversion with the CYP2A7 sequence in the 3'-untranslated region, in which the latter two changes correspond to CYP2A6^*7. Ethnic differences in the frequencies of these alleles were observed between whites, African-Americans, Japanese, and Koreans. Wild or variant CYP2A6 (CYP2A6^*18, CYP2A6^*19, and CYP2A6^*7) were expressed in Escherichia coli. For coumarin 7-hydroxylation and 5-fluorouracil formation from tegafur, the K_m values were increased, and V_max values were decreased in CYP2A6.18 compared with those in CYP2A6.1, resulting in decreased clearance to 50 and 35% of that of the wild type, respectively. The K_m and V_max values for nicotine C-oxidation were both increased, resulting in no change of clearance. In CYP2A6.19, the effects on the coumarin 7-hydroxylation and 5-fluorouracil formation (increased K_m and decreased V_max) were prominent, resulting in decreased clearance to 8% of those of the wild type. For nicotine C-oxidation, the K_m and V_max values were both decreased, resulting in decreased clearance to 30% of that of the wild type. The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. In vivo nicotine metabolism was evaluated in whites (n = 56) and Koreans (n = 40). Although the CYP2A6^*18 and CYP2A6^*19 alleles were found only heterozygously, a subject with CYP2A6^*7/CYP2A6^*19 showed a lower cotinine/nicotine ratio of the plasma concentration compared with homozygotes of the CYP2A6^*1A, supporting the in vitro results that the CYP2A6^*19 allele leads to decreased enzymatic activity.

There are genetic polymorphisms in the CYP2A6 gene. Several CYP2A6 alleles, such as CYP2A6^*2 (Yamano et al., 1990), CYP2A6^*4 (Oscarson et al., 1999b; Nunoya et al., 1999a,b), CYP2A6^*5 (Oscarson et al., 1999a), CYP2A6^*6 (Kitagawa et al., 2001), CYP2A6^*7 (Ariyoshi et al., 2001), CYP2A6^*9 (Pitarque et al., 2001), CYP2A6^*10 (Yoshida et al., 2002), CYP2A6^*11 (Daigo et al., 2002), CYP2A6^*12 (Oscarson et al., 2002), and CYP2A6^*17 (Fukami et al., 2004) have been reported to show decreased enzymatic activity. We have developed a simple phenotyping method of nicotine metabolism using nicotine gum (Nakajima et al., 2000a, 2001) and have demonstrated that the genetic polymorphisms of the CYP2A6 gene could account for most of the large interindividual differences in nicotine metabolism. In addition, some research groups have reported an association between genetic polymorphisms of CYP2A6 and smoking behavior or lung cancer risk (Miyamoto et al., 1999; Minematsu et al., 2003; Fujieda et al., 2004). These findings would reflect the relatively high frequencies (10–20%) of CYP2A6^*4, CYP2A6^*7, and CYP2A6^*9 in Asian populations (Nakajima et al., 2002; Yoshida et al., 2003). However, the results from such studies in white Europeans have been inconclusive and controversial (Loriot et al., 2001; Schulz et al., 2001) because of the low frequencies (1–7%) of defective alleles (Schoedel et al., 2004).

In the present study, we found two novel CYP2A6 alleles (CYP2A6^*18A and CYP2A6^*18B) containing a single nucleotide polymorphism (SNP) that leads to an amino acid change of Y392F (Fig. 1) from Caucasians. From Korean subjects, the CYP2A6^*18A allele and another novel allele, CYP2A6^*19, possessing SNPs that cause amino acid changes of Y392F as well as I471T, were found (Fig. 1). The amino acid change of I471T in CYP2A6.7 is already known to decrease the enzymatic activities (coumarin 7-hydroxylation and nicotine C-oxidation) in vitro or in vivo (Ariyoshi et al., 2001; Yoshida et al., 2002). We examined the effects of the amino acid changes in CYP2A6^*18 and CYP2A6^*19 alleles on the enzymatic activities in vitro and in vivo.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Schematic representation of the CYP2A6*18A, CYP2A6*18B, CYP2A6*19, and CYP2A6*7 alleles. The nucleotide numbers on exons and introns refer to the reference genomic sequence of NG000008. The corresponding numbers on cDNA (reference NM000762) and amino acids are in parentheses. The nucleotide numbering refers to the ATG in translation starting with A as 1. Shaded boxes in the CYP2A6*19 and CYP2A6*7 allele are the site of gene conversion with the CYP2A7 gene in the 3'-untranslated region.

	Materials and Methods

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Chemicals. Taq polymerase was obtained from Greiner Japan (Tokyo, Japan). Primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Restriction enzymes were purchased from Takara (Shiga, Japan), Toyobo (Osaka, Japan), New England Biolabs (Beverly, MA), and Fermentas (Hanover, MD). Nicotine, cotinine, coumarin, and 7-hydroxycoumarin were from Sigma-Aldrich (St. Louis, MO). 5-Fluorouracil (5-FU) and 5-bromouracil were obtained from Wako Pure Chemicals (Osaka, Japan). Tegafur was kindly provided by Taiho Pharmaceutical (Tokushima, Japan). All other chemicals and solvents were of the highest grade commercially available.

Genomic DNA. This study was approved by the Human Studies Committee of Washington University School of Medicine (St. Louis, MO) and the Ethics Committees of Kanazawa University (Kanazawa, Japan) and Soonchunhyang University Hospital (Chonan, Korea). Written informed consent was obtained from 185 white, 175 African-American, 184 Japanese, and 209 Korean subjects. Blood samples were collected from a cubital vein. Genomic DNA was extracted from peripheral lymphocytes using a Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN).

Genotyping of CYP2A6 Alleles. The genotyping of CYP2A6^*1B (Nakajima et al., 2004), CYP2A6^*1F (Nakajima et al., 2004), CYP2A6^*1G (Nakajima et al., 2004), CYP2A6^*1x2 (Yoshida et al., 2002), CYP2A6^*2 (Nakajima et al., 2000a), CYP2A6^*3 (Nakajima et al., 2000a), CYP2A6^*4A (Nakajima et al., 2004), CYP2A6^*4D (Nakajima et al., 2004), CYP2A6^*5 (Nakajima et al., 2001), CYP2A6^*6 (Yoshida et al., 2002), CYP2A6^*7 (Yoshida et al., 2002), CYP2A6^*8 (Yoshida et al., 2002), CYP2A6^*9 (Yoshida et al., 2003), CYP2A6^*10 (Yoshida et al., 2002), CYP2A6^*11 (Yoshida et al., 2002), CYP2A6^*12 (Nakajima et al., 2004), and CYP2A6^*17 (Fukami et al., 2004) were performed as described previously.

The genotyping methods for CYP2A6^*13, CYP2A6^*14, CYP2A6^*15, and CYP2A6^*16 alleles were established in the present study as follows. A PCR-RFLP method was developed for the genotyping of the CYP2A6^*13 allele concerning G13A. The primers were 2A6^*9S (Yoshida et al., 2003) (Table 1) and 2A6int1AS (Fukami et al., 2004) (Table 1). The reaction mixture contained genomic DNA (100 ng), 1x PCR buffer [67 mM Tris-HCl, pH 8.8, 16.6 mM (NH₄)₂SO₄, 0.45% Triton X-100, 0.02% gelatin], 1.5 mM MgCl₂, 0.25 mM dNTPs, 0.4 µM of each primer, and 1 U of TaqDNA polymerase in a final volume of 25 µl. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 25 s, annealing at 54°C for 25 s, and extension at 72°C for 40 s for 30 cycles. The PCR product was digested with Eco 81 I at 37°C for 3 h. The digestion patterns were determined by electrophoresis in a 2% agarose gel. The CYP2A6^*1 allele yields a 712-bp fragment and the CYP2A6^*13 allele yields 471- and 241-bp fragments.

A PCR-RFLP method was developed for the genotyping of the CYP2A6^*14 allele (G86A). The PCR product as described above (712 bp) was digested with Ear I at 37°C for 3 h. The digestion patterns were determined by electrophoresis in a 2% agarose gel. The CYP2A6^*1 allele yields a 712-bp fragment and the CYP2A6^*14 allele yields 404- and 308-bp fragments.

A PCR-RFLP method was developed for the genotyping of the CYP2A6^*15 allele concerning A580G. Primers were 2A6^*15 (MseI) with a nucleotide modification based on the genomic DNA (accession no. NG000008) and 2A6int4R2 (Table 1). The reaction mixture was the same as the CYP2A6^*13 genotyping method without the primers. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 20 s for 30 cycles. The PCR product was digested with MseI at 37°C for 3 h. The digestion patterns were determined by electrophoresis in a 3% agarose gel. The CYP2A6^*1 allele yields 263- and 21-bp fragments, and the CYP2A6^*15 allele yields 284-bp fragments.

A PCR-RFLP method was developed for the genotyping of the CYP2A6^*16 allele (C607A). Primers were 2A6ex3 and 2A6int4R (Fukami et al., 2004). The reaction mixture was the same as the CYP2A6^*13 genotyping method without the primers. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 25 s, annealing at 59°C for 25 s, and extension at 72°C for 40 s for 30 cycles. The PCR product was digested with HhaI at 37°C for 3 h. The digestion patterns were determined by electrophoresis in a 2% agarose gel. The CYP2A6^*1 allele yields 231-, 113-, 112-, 106-, 53-, 38-, and 28-bp fragments, and the CYP2A6^*16 allele yields 343-, 113-, 106-, 53-, 38-, and 28-bp fragments.

Sequence Analyses of All Exons and Exon-Intron Junctions of the CYP2A6 Gene. According to the method reported previously (Fukami et al., 2004), we performed direct sequence analyses of all exons and exon-intron junctions of the CYP2A6 gene in whites. A PCR product with the primer set of 2A6int7F and 2A6R2 (Nakajima et al., 2004) was subcloned into pT7Blue T-Vector (Novagen, Madison, WI). The plasmid DNA was purified by a QIAGEN plasmid midi kit (QIAGEN, Valencia, CA) and submitted to DNA sequencing using a Thermo Sequenase Cy5.5 dye terminator cycle sequencing kit (Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) with T7F and T7R (Amersham Biosciences UK, Ltd.). DNA sequences were analyzed on a Long-Read Tower DNA sequencer (Amersham Biosciences UK).

Genotyping of CYP2A6^*18 and CYP2A6^*19 Alleles. Allele-specific (AS)-PCR was applied for the genotyping of the CYP2A6^*18 and CYP2A6^*19 alleles. Sense primers were 2A6E8F-A and 2A6E8F-T (Table 1). The antisense primer was 2A6int8R (Table 1). The reaction mixture contained genomic DNA (100 ng), 1x PCR buffer, 1.5 mM MgCl₂, 0.25 mM dNTPs, 0.4 µM each primer, and 1 U of TaqDNA polymerase in a final volume of 25 µl. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s for 30 cycles, followed by a final extension at 72°C for 5 min. An aliquot (10 µl) of the PCR product was analyzed by electrophoresis with 2% agarose gel (Fig. 2). The CYP2A6^*1 allele was amplified with the primer set of 2A6E8F-A and 2A6int8R (467 bp) and the CYP2A6^*18 and CYP2A6^*19 alleles were amplified with the primer set of 2A6E8F-T and 2A6int8R (467 bp).

View larger version (23K): [in this window] [in a new window]   FIG. 2. Genotyping of A1175T in the CYP2A6*18 and CYP2A6*19 alleles by AS-PCR using genomic DNA. A, schematic structure of CYP2A6 gene. Boxes represent exon 8 and lines represent introns. PCR amplification was performed with the primer pairs indicated by horizontal arrows. The primer 2A6E8F-A specifically anneals to the CYP2A6*1 allele, whereas the primer 2A6E8F-T specifically anneals to the CYP2A6*18 and CYP2A6*19 alleles. B, schematic AS-PCR patterns for different CYP2A6 genotypes. The amplified PCR product is 467 bp. C, representative photograph of AS-PCR patterns for different CYP2A6 genotypes of CYP2A6*1/CYP2A6*1 and CYP2A6*1/CYP2A6*18 or CYP2A6*1/CYP2A6*19. In this genotyping method, the CYP2A6*18 and CYP2A6*19 alleles could not be distinguished.

View larger version (36K): [in this window] [in a new window]   FIG. 3. Genotyping for distinguishing between CYP2A6*7/CYP2A6*18 and CYP2A6*1A/CYP2A6*19. A, schematic structure of the CYP2A6 gene. Boxes represent exons and lines represent introns. PCR amplification was performed with the primer pairs indicated by horizontal arrows. The amplification DNA was digested by AccII. The restriction sites of AccII are indicated by vertical arrows. B, schematic PCR-RFLP patterns for CYP2A6*7/CYP2A6*18 and CYP2A6*1A/CYP2A6*19. After the digestion with AccII, CYP2A6*7/CYP2A6*18 yields 1047 and 278 bp in the primer pair of 2A6E8F-A and 2A6R2, and 1325 bp in the primer pair of 2A6E8F-T and 2A6R2. CYP2A6*1A/CYP2A6*19 yields 1325 bp in the primer pair of 2A6E8F-A and 2A6R2, and 1047 and 278 bp in the primer pair of 2A6E8F-T and 2A6R2. C, representative photograph of PCR-RFLP patterns for CYP2A6*1B/CYP2A6*18 and CYP2A6*1A/CYP2A6*19. In the present study, there were no subjects with CYP2A6*7/CYP2A6*18.

Expression in Escherichia coli and Membrane Preparations. The wild-type or variant plasmids were transformed to E. coli JM109. E. coli JM109 cells transformed with plasmid DNA were grown overnight at 37°C with shaking at 170 rpm in LB medium containing 25 µg/ml ampicillin. The culture was seeded at 1% into TB medium containing 100 µg/ml ampicillin and additives (0.5 mM -aminolevulinic acid, 1.0 mM isopropyl ß-D-thiogalacto-side, trace salts, and 1.0 mM thiamine). The expression cultures (100 ml) were grown at 30°C with shaking at 120 rpm for 30 h in 500-ml triple-baffled flasks. E. coli membranes expressing wild or variant CYP2A6/NPR were prepared as described previously (Fukami et al., 2004). The P450 content and protein concentration were determined according to a method described previously (Omura and Sato, 1964; Bradford, 1976). NADPH-cytochrome c reduction activity was determined as described previously (Williams and Kamin, 1962; Yasukochi and Masters, 1976) using ₅₅₀ = 21.1 mM^-1 cm^-1, and the content was calculated using a specific activity of 3.0 µmol of 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. Nicotine C-oxidation was determined as described previously with slight modifications (Nakajima et al., 1996). A typical incubation mixture (final volume of 0.5 ml) contained E. coli membrane preparations (4 pmol of P450), 50 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), 3 mg/ml human liver cytosolic protein, and 5 to 100 µM nicotine. The reaction was initiated by the addition of the NADPH-generating system after a 2-min preincubation at 37°C. After 15-min incubation at 37°C, the reaction was terminated by the addition of 100 µl of 1 N sodium hydroxide, and caffeine (50 ng) was added as an internal standard. The reaction mixtures were extracted by 4 ml of CH₂Cl₂ and 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 40-µl portion of the sample was subjected to 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 Capcell Pak C18 UG120 (4.6 x 250 mm; 5 µm) column (Shiseido, Tokyo, Japan). The eluent was monitored at 260 nm using an L-7405 UV detector (Hitachi). The mobile phase was 8% acetonitrile containing 0.01% acetic acid and 1 mM sodium heptane sulfonate. The flow rate was 1.0 ml/min, and the column temperature was 40°C. Quantification of the metabolite was performed by comparing the HPLC peak heights to those of an authentic standard with reference to an internal standard. Since cotinine contaminants exist in the commercially available nicotine to the extent of 0.15%, the content of cotinine in the mixture incubated without the E. coli membrane preparation was subtracted from that with the E. coli membrane preparation to correct the activity.

5-FU formation from tegafur was determined as described previously with slight modifications (Komatsu et al., 2000). A typical incubation mixture (final volume of 0.25 ml) contained the E. coli membrane preparation (5 pmol of P450), 100 mM Tris-HCl buffer, pH 7.4, the NADPH-generating system, and 0.1 to 2 mM tegafur. The reaction was initiated by the addition of the NADPH-generating system after a 2-min preincubation at 37°C. After the 15-min incubation at 37°C, the reaction was terminated by the addition of 1.5 ml of ethyl acetate. 5-Bromouracil (1 nmol) was added as an internal standard. The reaction mixture was extracted twice with ethyl acetate, 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 40-µl portion of the sample was subjected to HPLC. The HPLC apparatus was the same as described above except that a Mightysil RP-18 C18 GP (4.6 x 150 mm; 5 µm) column (Kanto Chemical, Tokyo, Japan) was used. The eluent was monitored at 270 nm. The mobile phase was 1% methanol containing 20 mM sodium perchlorate. The flow rate was 1.0 ml/min (0–6 min) and 1.3 ml/min (7–25 min). The column temperature was 35°C. Quantification of the metabolite was performed by comparing the HPLC peak heights to those of an authentic standard with reference to an internal standard. Since tegafur is nonenzymatically converted to 5-FU, the content of 5-FU in the mixture incubated without the E. coli membrane preparation was subtracted from that with the E. coli membrane preparation to correct the activity.

Kinetic parameters were estimated from the fitted curve using a computer program (KaleidaGraph; Synergy Software, Reading, PA) designed for nonlinear regression analysis. All data were analyzed using the mean ± S.D. of three independent determinations.

Phenotyping of in Vivo Nicotine Metabolism. This study was approved by the Human Studies Committee of Washington University School of Medicine and the Ethics Committee of Soonchunhyang University Hospital (Chonan, Korea). The phenotyping of in vivo nicotine metabolism was performed according to the method established in our previous study (Nakajima et al., 2001). Briefly, the subjects chewed one piece of nicotine gum (Nicorette, containing 2 mg of nicotine; Pfizer Japan Inc., Tokyo, Japan) for 30 min, chewing for 10 s per 30 s. Blood samples were collected from a cubital vein just before and 2 h after the start of chewing. The concentrations of nicotine and cotinine in the plasma samples were determined by HPLC (Nakajima et al., 2000b). The cotinine/nicotine ratio of the plasma concentration was calculated as an index of nicotine metabolism. Data from 56 white and 40 Korean subjects (they were all nonsmokers) were analyzed in the present study.

Statistical Analyses. Statistical analyses of the kinetic parameters were performed by two-tailed Student's t test. Statistical analysis of the cotinine/nicotine ratios between the different genotypes was performed by Mann-Whitney U test. A value of P < 0.05 was considered statistically significant.

	Results

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Identification of the Novel Alleles of CYP2A6 Gene. With the direct sequence analyses of the CYP2A6 gene in white subjects with low nicotine metabolic potency, we found two novel alleles of CYP2A6^*18A and CYP2A6^*18B (Fig. 1). The CYP2A6^*18A allele has an SNP of A5668T, in which the nucleotide numbering refers to the ATG in translation, starting with A as 1 with the reference genomic sequences of NG000008. The SNP of A5886T corresponds to A1175T on cDNA (NM000762), leading to an amino acid change of Y392F. The CYP2A6^*18B allele has synonymous SNPs of G51A (G51A), T5684C (T1191C), and T5702C (T1209C) as well as the A5668T (A1175T). Yamano et al. (1990) originally reported the CYP2A6 cDNA sequence with the nucleotide 1175A (NM000762). However, after the genomic DNA sequence (NG000008) had been reported with the corresponding nucleotide of 5668T, the reference sequence of NM000762 was modified to 1175T. In the present study, it has been confirmed that nucleotide 1175 was A in most subjects. Therefore, it has been approved that the reference sequence should be 1175A in CYP2A6 cDNA by the Human Cytochrome P450 Allele Nomenclature Committee (http://www.imm.ki.se/CYPalleles/).

With the sequence analyses of the CYP2A6^*18A allele in Korean subjects, we found another allele, CYP2A6^*19 (Fig. 1). The CYP2A6^*19 allele has SNPs of A5668T (A1175T, Y392F) and T6354C (intron 8) in addition to the SNP of T6558C (T1412C, I471T) and conversion with the CYP2A7 sequence in the 3'-untranslated region, in which the latter two changes correspond to the CYP2A6^*7 allele.

Allele Frequencies of CYP2A6^*7, CYP2A6^*18, and CYP2A6^*19 Alleles. We determined the allele frequencies of CYP2A6^*18A, CYP2A6^*18B, and CYP2A6^*19 as well as CYP2A6^*7 in 185 whites, 175 African-Americans, 184 Japanese, and 209 Koreans (Table 2). To distinguish between the CYP2A6^*18A and CYP2A6^*18B alleles, sequence analyses were performed. The CYP2A6^*18A allele was found in whites and Koreans at frequencies of 1.1 and 0.5%, respectively. The CYP2A6^*18B allele was found only in Caucasians at a frequency of 1.1%. The CYP2A6^*7 and CYP2A6^*19 alleles were found in Japanese at frequencies of 13.0 and 0.5%, respectively. These alleles were also found in Koreans at frequencies of 10.0 and 1.0%, respectively. In African-Americans, the CYP2A6^*7, CYP2A6^*18, and CYP2A6^*19 alleles were not found.

Expression of Three Variants of CYP2A6 in E. coli. To investigate the functional alteration by the amino acid changes of Y392F, I471T, and in combination, we established genetically engineered E. coli cells coexpressing the wild or variant CYP2A6 together with NPR. The specific contents of P450, the activities of NPR, and the molar ratio of NPR to P450 in each membrane lysate of the E. coli cells are summarized in Table 3. The molar ratios were calculated according to the specific activity of 3.0 µmol of reduced cytochrome c min/nmol/NADPH-P450 reductase. It was confirmed that the P450 and NPR activities could not be detected with the untransformed E. coli membrane. The reproducibility of the data was confirmed using three independent membrane preparations.

Enzymatic Activities of Three Variants of CYP2A6. Membrane preparations from genetically engineered E. coli cells expressing each CYP2A6 together with NPR were used for the determination of the catalytic activity for coumarin, nicotine, and tegafur. The kinetics of the coumarin 7-hydroxylation, nicotine C-oxidation, and 5-FU formation from tegafur in the membrane preparations were fitted to the Michaelis-Menten equation (Fig. 4).

View larger version (16K): [in this window] [in a new window]   FIG. 4. Kinetic analyses of enzymatic activities catalyzed by heterologously expressed CYP2A6.1, CYP2A6.7, CYP2A6.18, and CYP2A6.19. A, coumarin 7-hydroxylation. B, nicotine C-oxidation. C, 5-FU formation from tegafur. Each data point is the average of duplicated experiments. Kinetic parameters were estimated from the fitted curve using a computer program KaleidaGraph designed for nonlinear regression analysis.

For the nicotine C-oxidation (Fig. 4B), the apparent K_m and V_max values in CYP2A6.1 were 37.0 ± 2.5 µM and 13.4 ± 0.6 pmol/min/pmol P450, respectively. The K_m values of CYP2A6.7 and CYP2A6.19 were significantly (P < 0.05) lower than that of the wild type. In contrast, the K_m value of CYP2A6.18 was significantly (P < 0.05) higher than that of the wild type. The V_max values of CYP2A6.7 and CYP2A6.19 were significantly (P < 0.005) lower than that of the wild type. In contrast, the V_max value of CYP2A6.18 was significantly (P < 0.005) higher than that of the wild type. The V_max/K_m values of CYP2A6.7 and CYP2A6.19 were significantly (P < 0.005) lower than that of the wild type (363.3 ± 30.6 nl/min/pmol P450), showing approximately 30% of that of the wild type. In contrast, the V_max/K_m value of CYP2A6.18 was close to that of the wild type.

For the 5-FU formation from tegafur (Fig. 4C), the apparent K_m and V_max values in CYP2A6.1 were 0.3 ± 0.0 mM and 6.4 ± 0.4 pmol/min/pmol P450, respectively. The K_m values of CYP2A6.7, CYP2A6.18, and CYP2A6.19 were significantly (P < 0.05 or P < 0.005) higher than that of the wild type. The V_max values of CYP2A6.7, CYP2A6.18, and CYP2A6.19 were significantly (P < 0.05 or P < 0.005) lower than that of the wild type. The V_max/K_m values of CYP2A6.7 and CYP2A6.19 were prominently decreased compared with that of the wild type (22.9 ± 3.1 nl/min/pmol P450), showing approximately 8% of that of the wild type (P < 0.005). However, the V_max/K_m value of CYP2A6.18 was 35% of that of the wild type (P < 0.05).

In Vivo Nicotine Metabolism in Subjects with the CYP2A6^*18 and CYP2A6^*19 Alleles. The cotinine/nicotine ratios in plasma of the different CYP2A6 genotype groups in 56 whites and 40 Koreans were determined as an index of nicotine metabolism (Fig. 5). In whites, the cotinine/nicotine ratios in the four subjects with CYP2A6^*1A/CYP2A6^*18B (5.9 ± 3.8) were not significantly different compared with those in the subjects with CYP2A6^*1A/CYP2A6^*1A (7.5 ± 5.2). Three heterozygotes of the CYP2A6^*18A allele (CYP2A6^*1B/CYP2A6^*18A and CYP2A6^*9/CYP2A6^*18A) showed similar metabolic ratios as the subjects with CYP2A6^*1A/CYP2A6^*1A. In Koreans, the heterozygotes of CYP2A6^*1A/CYP2A6^*7 (5.1 ± 2.8) showed significantly decreased cotinine/nicotine ratios compared with those in the subjects with CYP2A6^*1A/CYP2A6^*1A (10.4 ± 9.2). However, the heterozygotes of CYP2A6^*1B/CYP2A6^*7, CYP2A6^*1B/CYP2A6^*18A, and CYP2A6^*1A/CYP2A6^*19 showed similar cotinine/nicotine ratios to those in the subjects with CYP2A6^*1A/CYP2A6^*1A. Subjects with CYP2A6^*7/CYP2A6^*7 (1.0), CYP2A6^*7/CYP2A6^*10 (1.8), and CYP2A6^*7/CYP2A6^*19 (0.8) showed prominently lower cotinine/nicotine ratios compared with that of subjects with CYP2A6^*1A/CYP2A6^*1A.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Cotinine/nicotine ratios in plasma 2 h after chewing one piece of nicotine gum in 56 whites and 40 Koreans who were genotyped for CYP2A6 alleles. All subjects were nonsmokers. The numbers of subjects are shown in parentheses. Data are expressed as mean ± S.D.

	Discussion

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In the present study, we found novel CYP2A6 alleles, CYP2A6^*18, and CYP2A6^*19. The CYP2A6^*18A allele was found in whites and Koreans, whereas CYP2A6^*18B was found only in whites. The absence of the CYP2A6^*19 allele in whites would be due to the lack of the CYP2A6^*7 allele in the population. In contrast, the fact that the CYP2A6^*19 allele was found in Japanese and Koreans would be due to the high frequencies of the CYP2A6^*7 allele in these populations. In African-Americans, the CYP2A6^*7, CYP2A6^*18, and CYP2A6^*19 alleles were not found. However, we recently found CYP2A6 alleles that are specific for African-Americans, CYP2A6^*17 (Fukami et al., 2004), and CYP2A6^*20 (Fukami et al., 2005). Thus, ethnic differences in the genetic polymorphisms of CYP2A6 are noteworthy.

Previously, Ariyoshi et al. (2001) reported that coumarin 7-hydroxylation in the CYP2A6.7 was decreased by 63%, whereas nicotine C-oxidation was not detected at a certain substrate concentration determined by an E. coli expression system. The present study revealed that the K_m was increased and V_max was decreased for coumarin 7-hydroxylation in CYP2A6.7. For nicotine C-oxidation, the K_m and V_max were both decreased in CYP2A6.7. Thus, the effects of the amino acid change of I471T on the kinetics were different between the substrates, although the clearances of both activities in CYP2A6.7 were decreased compared with CYP2A6.1. In addition, we first demonstrated that 5-FU formation from tegafur was also decreased with the CYP2A6^*7 allele to 8% of that of the wild type. The effects of the amino acid change on the kinetics of the 5-FU formation were similar to that of coumarin 7-hydroxylation.

In the analysis of CYP2A6.18 (Y392F) for the coumarin 7-hydroxylation and 5-FU formation from tegafur, the K_m values were increased and the V_max values were decreased. For the nicotine C-oxidation, the K_m and V_max values were both increased in CYP2A6.18. Interestingly, the other CYP2A isoforms (CYP2A1, CYP2A2, Cyp2a4, Cyp2a5, CYP2A7, CYP2A8, and CYP2A13) have phenylalanine at residue 392, but only CYP2A6 has tyrosine (Lewis et al., 1999). Although the tyrosine at residue 392 is located within the ß₁(3) strand (Lewis, 1998) but not in the substrate recognition site, the change may affect the substrate recognition, affinity toward substrates, or turnover number.

The changes of the kinetics in CYP2A6.19 were similar to those in CYP2A6.7. It is reminiscent of CYP2A6.10 possessing amino acid changes of R485L (CYP2A6.8) and I471T (CYP2A6.7). Although there is no in vitro study to determine the effects of the amino acid changes in CYP2A6.8 and CYP2A6.10, our previous in vivo study revealed that the enzymatic activity was decreased in CYP2A6.10 (Yoshida et al., 2002). Taking these findings into consideration, it is conceivable that the amino acid change of I471T is critical for the enzymatic activity of CYP2A6.

The only available information on the 5-FU formation from tegafur pertains to the CYP2A6^*11 allele (Daigo et al., 2002). The CYP2A6^*11 allele was found in a Japanese patient who showed a higher value of the area under the plasma concentration-time curve for tegafur than normal. In the kinetic analysis of the formation of 5-FU from tegafur by CYP2A6.11 expressed in E. coli, the V_max/K_m value was decreased by approximately 50%. In the present study, CYP2A6.7, CYP2A6.18, and CYP2A6.19 showed decreased in vitro clearance of the 5-FU formation. However, concerning the effects of the CYP2A6 alleles on the in vivo pharmacokinetics of tegafur, it should be noted that the 5-FU formation from tegafur is catalyzed not only by CYP2A6 but also by CYP1A2, CYP2C8 (Komatsu et al., 2000), and cytosolic thymidine phosphorylase (Komatsu et al., 2001).

The potencies of in vivo nicotine metabolism in white and Korean subjects who were genotyped for CYP2A6 alleles were determined by the cotinine/nicotine ratios in plasma (Nakajima et al., 2001). CYP2A6^*4 is known as an entire deleted allele lacking enzymatic activity (Oscarson et al., 1999b; Nunoya et al., 1999a,b). Previously, we found that heterozygotes of the CYP2A6^*4 allele can metabolize nicotine at levels similar to homozygotes of CYP2A6^*1A (Kwon et al., 2001; Nakajima et al., 2001). Thus, the evaluation of the in vivo metabolic potency in heterozygotes is difficult because of the effects of the other allele. Due to the low allele frequency, the CYP2A6^*18 allele was found only heterozygously. Therefore, we could not evaluate the effects of the CYP2A6^*18 allele on the in vivo enzymatic activity. The CYP2A6^*19 allele was also found only heterozygously. However, a subject with CYP2A6^*7/CYP2A6^*19 showed a lower cotinine/nicotine ratio compared with homozygotes of the CYP2A6^*1A. The value was similar to those in the subjects with CYP2A6^*7/CYP2A6^*7 or CYP2A6^*7/CYP2A6^*10. These results might support the in vitro finding that the CYP2A6^*19 allele leads to decreased enzymatic activity.

In conclusion, we found novel alleles of CYP2A6^*18 and CYP2A6^*19. The amino acid change of Y392F in the CYP2A6^*18 decreased the clearance of coumarin 7-hydroxylation and 5-FU formation from tegafur, but not nicotine C-oxidation, suggesting differences between substrates. However, the effects of the amino acid changes of Y392F and I471T in CYP2A6^*19 resembled those in CYP2A6^*7 (I471T) with a decrease in the clearance of all three activities. These alleles should be considered in studies of CYP2A6 genotyping in relation to the clearance of CYP2A6 substrates, smoking behavior, and the incidence of lung cancer.

	Acknowledgments

 
The enthusiasm and research support of Tracy Jones, Arnita Pitts, Phyllis Klein, and Ladonna Gaines (Washington University Center for Clinical Studies), and Margaret Ameyaw are greatly appreciated. We acknowledge Brent Bell for reviewing the manuscript.

	Footnotes

 
This study was supported in part by a grant from Japan Health Sciences Foundation with Research on Health Science Focusing on Drug Innovation, by an SRF grant for biomedical research in Japan, and by Philip Morris Incorporated.

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

doi:10.1124/dmd.105.004994.

ABBREVIATIONS: P450, cytochrome P450 enzyme; SNP, single nucleotide polymorphism; 5-FU, 5-fluorouracil; PCR-RFLP, polymerase chain reaction-restriction fragment length polymorphism; PCR, polymerase chain reaction; bp, base pair(s); AS-PCR, allele specific-polymerase chain reaction; NPR, NADPH-P450 reductase; HPLC, high-performance liquid chromatography.

Address correspondence to: Dr. 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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