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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION
Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
Received October 19, 2003; accepted March 4, 2004.
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Abstract |
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; Nelson et al., 1996). Human CYP3A forms metabolize more than about half of therapeutic drugs (Cholerton et al., 1992; Li et al., 1995) and are also involved in the metabolism of endogenous chemicals such as bile acids (Araya and Wikvall, 1999), steroid hormones (Waxman et al., 1988), and retinoic acid (Marill et al., 2000). CYP3A forms are expressed predominantly in the liver but are also found in other organs such as the gut (Kolars et al., 1994), white blood cells (Janardan et al., 1996; Sempoux et al., 1999), and brain (Wang et al., 1996). The level of CYP3A4 in the intestine is reported to share more than 50% of the total cytochrome P450 (P450) content (Zhang et al., 1999).
In rats, CYP3A1 (Gonzalez et al., 1985), CYP3A2 (Gonzalez et al., 1986), CYP3A9 (Wang et al., 1996), CYP3A18 (Strotkamp et al., 1995; Nagata et al., 1996), and CYP3A23 (Kirita and Matsubara, 1993; Komori and Oda, 1994) have been reported as rat CYP3A forms. CYP3A23 was, however, identified to be the same form as CYP3A1 by analysis of the CYP3A1 gene (Nagata et al., 1999). These CYP3A forms appear in a sex-dependent manner in rats. For example, CYP3A2 (Yamazoe et al., 1988; Cooper et al., 1993) and CYP3A18 (Nagata et al., 1996; Robertson et al., 1998) are male-specific forms, whereas CYP3A9 is a female-dominant form (Wang and Strobel, 1997; Robertson et al., 1998). The expression profiles in the intestinal tract, however, are obscure with all of the forms.
Levels of CYP3A forms are enhanced after treatment with structurally diverse compounds such as dexamethasone, clotrimazole, and rifampicin (Hostetler et al., 1989; Daujat et al., 1991; Burger et al., 1992; Kocarek et al., 1995). Intestinal CYP3A forms play important roles on the first-pass effect of drugs. In humans, however, rather distinct controls of hepatic and small intestinal CYP3A4s were suggested from experiments using CYP3A4 probe drugs and also from the protein levels. Thus, an understanding of their enzymatic and molecular biological properties is necessary before predicting drug-drug interaction.
As the results of the genome sequencing in various experimental animal species, a number of unidentified genes have been found to provide the information of a novel protein. We have previously isolated six different CYP3A-related DNA clones from a rat genomic library (K. Nagata, T. Matsubara, and Y. Yamazoe, unpublished data). The four DNA clones contained information on a part of the first exon boundary of CYP3A1, CYP3A2, CYP3A9, and CYP3A18 genes, whereas the other two remained unidentified.
In the present study, we have isolated a novel CYP3A cDNA encoding CYP3A62 from rat liver cDNAs. We have also characterized enzymatic and molecular biological properties of this new form in comparison with the other four rat CYP3A forms.
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Materials and Methods |
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Isolation and Sequencing of CYP3A62 cDNA. Oligonucleotide primers used for isolation of CYP3A62 cDNA were 5'-GCAGCACACACAAGCTAAGAA-3' (fragment 1), 5'-CTGTGACCTATGATGTCCTG-3' (fragment 2), and 5'-AGCAGCAATGGACCTGATCC-3' (fragment 3) for the forward primers, and 5'-GAGAGCAAACCTCATGCC-3' (fragment 1), 5'-TTTTTTTTTTTTTTTTTT-3' (fragment 2), and 5'-CCACTCATGGTTCAATC-3' (fragment 3) for the reverse primers, respectively. DNA fragments from the liver cDNAs of a male adult rat were amplified by the use of Takara Taq (Takara, Kyoto, Japan). The reaction mixture (30 µl) contained 1 µl of the template DNA solution, 20 pmol of each of the forward and reverse primers, 250 µM dATP, dCTP, dTTP, and dGTP each, and 1 unit of Taq polymerase. After initial denaturation at 94°C for 5 min, the amplification was carried out for 30 cycles with 0.5 min at 94°C for denaturation, 1 min at 55°C for annealing, 1.5 min at 72°C for extension, and a final extension period of 7 min at 72°C.
Transfection of CYP3As into COS-1 Cells and Expression of CYP3A Forms. Constructions of plasmids for CYP3A62, CYP3A9, and CYP3A18 cDNAs were carried out by insertion between the MluI and BglII sites of pCMV4; constructions of plasmids for CYP3A1 and CYP3A2 cDNAs were carried out by insertion into the EcoRI sites of p91023(B) as reported previously under Methods (Miyata et al., 1994; Nagata et al., 1999). These cDNAs were isolated from rat male DNA libraries using a PCR method. These plasmid constructs (50 µg) were transfected into COS-1 cells (2.0 x 106 cells) using an electroporation method. The COS-1 cells cultured at 37°C for 72 h were collected in 2 ml of phosphate-buffered saline. The precipitated cells were resuspended in 100 µl of 75 mM potassium phosphate buffer (pH 7.4) after centrifugation at 2000g for 5 min and then homogenized. The homogenate was centrifuged at 9000g for 20 min. The supernatant was further centrifuged at 105,000g for 60 min, and the microsomal pellet was resuspended in 50 µl of buffer (20% glycerol in 0.1 M potassium phosphate buffer; pH 7.4). Cytochrome P450 content was estimated by the method of Omura and Sato (1964).
Treatment of Animals and Preparation of Microsomes. Male and female Sprague-Dawley rats (10 weeks old) purchased from Japan SLC (Shizuoka, Japan) were acclimated for 3 days. They were divided into three groups (control, dexamethasone-treated, and lithocholic acid-treated). Dexamethasone suspended in corn oil was given intraperitoneally to rats at a dose of 100 mg/kg/day for 3 consecutive days. Lithocholic acid was given orally at a dose of 100 mg/kg/day for 3 consecutive days. Corn oil (1 ml/head) was given to the controls. Microsomes and total RNAs were prepared 20 h after the last dose. Microsomes were prepared as previously described (Yamazoe et al., 1986). Intestinal mucosa microsomes were prepared as follows. The small intestine removed was immediately placed in liquid nitrogen. The tissue cut into small pieces was added to ice-cold buffer (75 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml trypsin inhibitor, and 19 µg/ml aprotinin). The microsomal fraction was isolated using the procedure described for liver microsomes. Microsomal protein was determined by the method of Lowry et al. (1951).
Immunoblot Analysis. Microsomal proteins were electrophoresed in 16 cm of a 7.5% SDS-PAGE for separation and 1 cm of a 2.0% SDS-PAGE for stacking and transferred to a nitrocellulose membrane. The sheet was immunostained with human anti-CYP3A antibody prepared as described previously (Kawano et al., 1987), alkaline phosphatase-conjugated goat anti-rabbit IgG, 5-bromo-4-chloro-3-indolylphosphate, and nitro blue tetrazonium as described previously (Blake et al., 1984).
Testosterone Hydroxylation. The reaction mixture for measurement of testosterone 6
-hydroxylase activities consists of 50 µg of protein of COS-1 microsomes expressing a CYP3A form, 100 mM potassium phosphate buffer (pH 7.4), 5 pmol of cytochrome b5, 0.1 unit (0.1 mmol of cytochrome c per minute) of NADPH-P450 reductase, and 5 µg of sodium cholate in a final volume of 100 µl. The reaction was started by the addition of NADPH (final concentration, 0.5 mM) and terminated by adding ethyl acetate after 40 min of incubation at 37°C. Testosterone hydroxylation was quantified by the method described previously (Yamazoe et al., 1988; Guo et al., 2000).
Amiodarone and Lidocaine De-ethylations. The reaction mixtures for amiodarone and lidocaine N-de-ethylase activities consisted of 50 µg of protein of COS-1 microsomes expressing a CYP3A form, 100 mM potassium phosphate buffer (pH 7.4), 5 pmol of cytochrome b5, 0.1 unit of NADPH-P450 reductase, 5 µg of sodium cholate, and 200 nmol of amiodarone or lidocaine in a final volume of 100 µl. The reaction was started by the addition of NADPH (final concentration, 0.5 mM) and then terminated by the addition of zinc sulfate and barium hydroxide after 40 min of incubation at 37°C. The acetaldehyde thus formed was converted to a decahydroacridine derivative using the reaction with 4 µg of 1,3-cyclohexandione, 4 mg of ammonium acetate, and 2 mg of acetic acid at 80°C for 30 min in a final volume of 300 µl. The derived product was quantified using a high-performance liquid chromatography system equipped with a C18 reversed-phase analytical column (particle size, 7 µm, 4.6 x 150 mm). The metabolites were detected with the fluorescence at excitation and fluorescence wavelengths of 390 and 457 nm, respectively. The sample was eluted using acetonitrile/0.5% acetic acid in distilled water (1:4) at a flow rate of 1 ml/min.
Analysis of Rat CYP3A mRNAs. Total RNA was extracted from the following tissues: liver, kidney, spleen, lung, heart, adrenal, brain, stomach, duodenum, jejunum, ileum, colon, testis, prostate, ovary, and uterus of male and female rats using an acid guanidinium thiocyanate-phenol-chloroform method. Total RNAs of each tissue were combined for a pool of four individuals. The cDNA was reverse-transcripted from those total RNAs with Ready-To-Go (Amersham Biosciences Inc., Piscataway, NJ). The nucleotide sequences of CYP3A62-, CYP3A1-, CYP3A2-, CYP3A9-, CYP3A18-, and G6PDH-selective oligonucleotide primers are shown in Table 1. cDNA fragments for CYP3A62, CYP3A1, CYP3A2, CYP3A9, CYP3A18, and G6PDH were amplified by use of Takara Taq. The reaction mixture (30 µl) contained 1 µl of the cDNA solution as a template DNA, 20 pmol of each forward and reverse primer as described above, 250 µM dATP, dCTP, dTTP, and dGTP each, 1 unit each of the enzyme and the buffer. After initial denaturation at 94°C for 5 min, the targeted nucleotides were amplified for 35 cycles (RT-PCR) or 40 cycles (real-time PCR), with 30 s at 94°C for denaturation, 15 s at 55°C (CYP3A9 and G6PDH), 60°C (CYP3A62 and CYP3A2), or 63°C (CYP3A1 and CYP3A18) for annealing, 30 s at 72°C for extension, and a final extension period of 7 min at 72°C. The quantification of mRNAs was carried out with SYBR Green by using ABI PRISM 7000 (Applied Biosystems, Foster City, CA). A real-time PCR method was used to determine the expression amounts of CYP3A mRNAs in various rat tissues. In these experiments, levels of CYP3A mRNAs were normalized from the amount of total RNA.
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Results |
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), CYP3A2 (Gonzalez et al., 1986), CYP3A9 (Wang et al., 1996), and CYP3A18 (Nagata et al., 1996), respectively] that were identified previously. The other two clones seemed to encode unknown CYP3A genes. Based on this information, a novel CYP3A form (CYP3A62) cDNA has been isolated using RT-PCR. A fragment 1 of CYP3A62 cDNA was at first amplified from the liver cDNA of a male adult rat with the forward primer 1 and reverse primer 1 (Fig. 1), and the nucleotide sequence was determined. The reverse primer 1 was constructed from a region of highly conserved nucleotide sequences among CYP3A cDNAs. Second, a fragment 2 was amplified with the forward primer 2 and reverse primer 2 to determine the nucleotide sequence. Finally, fragment 3 of the CYP3A62 cDNA, including an entire open reading frame, was amplified from the rat liver cDNAs with the forward primer 3 and reverse primer 3. The nucleotide sequence of fragment 3 was completely identical with those of fragments 1 and 2 (Fig. 1). In this strategy, the identified cDNA was 1746 bp in length, which had an open reading frame of 1491 bp (corresponding to 497 amino acids), 93 bp and 209 bp of the 5'- and 3'-noncoding regions, respectively. This nucleotide sequence was deposited with the DDBJ nucleotide sequence database (Accession no. AB084894
[GenBank]
). CYP3A62 cDNA showed the highest similarity in the nucleotide sequence with rat CYP3A9 and mouse Cyp3a13 cDNAs (both 84.4%). CYP3A62 showed the highest similarity with CYP3A9 (79.9%) and also showed 67.0 to 73.4% similarity in amino acid sequence with other rat and human CYP3A forms (Table 2). A unique property of this new form is in the number of cording residues. A nucleotide change (change A to T) at 1584 bp of CYP3A62 cDNA to form a termination codon resulted in 3- or 6-amino acid shorter sequences as compared with those of other CYP3A forms except for CYP3A18.
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Quantification of CYP3A mRNAs in Liver and Intestinal Tract. The quantification of individual CYP3A mRNAs was carried out by the use of a real-time PCR method. As shown in Table 3, predominant expression of CYP3A62 mRNA and CYP3A9 mRNA in the female over the male was observed in the liver. The level of CYP3A62 mRNA was about 5 times higher in the female than in the male in liver (5.73 and 1.05 attomole/µg total RNA, respectively). CYP3A62 mRNA was also detected in the intestinal tract of both sexes. The level was rather higher in the male intestinal tract than in the liver (duodenum, jejunum, ileum, and colon were 9.61, 7.88, 5.50, and 3.01 attomole/µg total RNA, respectively). In female rats, the level was roughly equivalent between the liver and duodenum (5.73 and 7.27 attomole/µg total RNA, respectively), and lower in the jejunum, ileum, and colon than in the duodenum (jejunum, ileum, and colon were 2.54, 1.09, and 0.24 attomole/µg total RNA, respectively). Their tissue distribution profiles were similar to those of CYP3A9 mRNA. CYP3A2 and CYP3A18 mRNAs were predominantly expressed in male rat livers as previously reported (Cooper et al., 1993; Robertson et al., 1998). The amount of CYP3A2 mRNA was highest among rat CYP3A forms (382.69 attomole/µg total RNA). CYP3A1 mRNA was also observed in livers of both sexes (male and female were 52.48 and 39.09 attomole/µg total RNA, respectively), although the levels were lower than that of CYP3A2 mRNA in male rats. An interesting thing is that CYP3A1 and CYP3A2 mRNAs were not detected in the intestinal tract using real-time PCR. CYP3A18 mRNA was detected as a male-predominant form in the liver and intestinal tract, although the level was very low in the intestinal tract.
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After treatment of rats with dexamethasone intraperitoneally, both CYP3A1 and CYP3A2 mRNAs were clearly increased (25 to 200 times) in the liver but not at all in the intestinal tracts as shown in Fig. 2. CYP3A18 mRNA was also enhanced in the liver (about 20 times) and to a lesser extent in the ileum (2-4 times). The expression profile of CYP3A62 mRNA differed from those of CYP3A1, CYP3A2, and CYP3A18 mRNAs. Levels of CYP3A62 mRNA were increased in both liver and intestinal tracts of both sexes (2-30 times) by the treatment. Similar profiles were also detected in the level of CYP3A9 mRNA. These results were confirmed by repeated experiments (data not shown). On the other hand, only CYP3A2 mRNA was strongly increased in the liver of both sexes after treatment with lithocholic acid. In the jejunum of both sexes, CYP3A62 mRNA was increased (2-5 times) by the treatment.
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Tissue Distribution of CYP3A62 and Other Rat CYP3A Forms. To assess the tissue distribution of rat CYP3A forms other than liver and intestine, selectively amplified mRNA levels were detected in various tissues by RTPCR with specific primers as shown in Table 1. The band for CYP3A62 mRNA was found in the stomach of both sexes (Fig. 3). CYP3A9 mRNA was detected in the stomach, lung, and brain of both sexes. CYP3A1 and CYP3A2 mRNAs were also not detected in these tissues. CYP3A18 mRNA was detected in the lungs of both sexes and in the kidney and spleen of the male.
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Detection of the CYP3A62 Protein. To characterize the enzymatic properties of a protein derived from CYP3A62 cDNA, all rat CYP3A forms identified were expressed in COS-1 cells as described under Materials and Methods. Microsomal proteins in COS-1 cells were immunoblotted by the use of anti-CYP3A antibodies. As shown in Fig. 4A, individual recombinant CYP3A forms expressed in COS-1 cells were clearly separated and detected at different electrophoretic mobilities. The order of those electrophoretic mobilities was CYP3A18, CYP3A62, CYP3A2, CYP3A1, and CYP3A9 from lower dalton registers. The band corresponding to CYP3A62 was detected in the female liver but not clearly in the male liver (Fig. 4B). Bands to CYP3A2 and/or CYP3A1 and CYP3A18 were clearly detected in the male liver, and a band corresponding to CYP3A9 was also detected (Fig. 4B). In livers of female rats, bands of CYP3A1 and CYP3A9 were clearly detected, but not those of CYP3A2 and CYP3A18 (Fig. 4B). In small intestines of both sexes, the bands corresponding to CYP3A62 and CYP3A9 were detected. CYP3A18 was also detected, but the expressed level varied clearly among individuals. An unidentified band was detected in small intestines of both sexes as indicated by the arrow.
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Microsomal levels of individual CYP3A forms are summarized in Table 4. Due to overlapping mobilities of CYP3A2 and CYP3A1 in SDS-PAGE, combined amounts are shown for livers of male rats. CYP3A2/CYP3A1 and CYP3A18 had 53.95 and 28.24 pmol/mg protein in male rat livers, respectively. CYP3A9 was 5.12 pmol/mg protein in male rat livers, but CYP3A62 was not clearly quantified (<0.1 pmol/mg protein). On the other hand, CYP3A1 and CYP3A9 had 19.07 and 11.24 pmol/mg protein in female rat livers. CYP3A62 was predominantly detected in female livers (4.81 pmol/mg protein), and CYP3A2 and CYP3A18 in female livers were not detected (<0.1 pmol/mg protein). In small intestines of both sexes, the expressed level of the CYP3A62 was highest among CYP3A forms. The levels of CYP3A62, CYP3A9, and CYP3A18 were quantified at 2.31, 0.78, and 0.84 pmol/mg protein in males, respectively. On the other hand, in females the levels of CYP3A62, CYP3A9, and CYP3A18 were 2.01, 0.73 and 0.89 pmol/mg protein, respectively. Neither CYP3A1 nor CYP3A2 was detected in small intestines of both sexes.
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Comparison of Catalytic Activities among Recombinant CYP3A Forms. Testosterone 6-hydroxylation is known as a typical catalytic activity for CYP3A forms. Some of the members are also known to catalyze 2- and 15-hydroxylations of testosterone, although the extent of those activities is lower than that of the 6-hydroxylation. In the present study, the catalytic property of CYP3A62 was compared with other forms using testosterone hydroxylation. As shown in Table 5B, recombinant CYP3A62 mediated testosterone 6- and 2-hydroxylations at the lowest rate (1.14 and 0.06 nmol/min/nmol P450, respectively) among recombinant rat CYP3A forms examined. A catalytic activity of testosterone 16
-hydroxylation (0.76 nmol/min/nmol P450), which could not be detected in CYP3A1, CYP3A2, and CYP3A9, was observed in CYP3A62 as well as in CYP3A18. As shown in Table 5B, CYP3A62 activity was only slightly increased (about 1.3 times) by addition of cytochrome b5, despite the clear changes in other forms.
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To further characterize the drug-metabolizing activity in CYP3A62, catalytic activities of amiodarone and lidocaine N-de-ethylations were tested with recombinant rat CYP3A forms. As shown in Table 6, CYP3A62 showed low but clear N-de-ethylating activities of both amiodarone and lidocaine (0.007 and 0.054 nmol/min/nmol P450, respectively). In addition, CYP3A9 showed the highest activity of both N-de-ethylations among rat CYP3A forms (0.156 and 0.178 nmol/min/nmol P450).
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Discussion |
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CYP3A62 mRNA and CYP3A9 mRNA were detected in the liver and intestinal tract using real-time PCR, and their profiles were similar to one another (Table 3). These profiles were also supported by the quantification of both proteins detected by immunoblot analyses (Fig. 4; Table 4). Another form, CYP3A18, was also detected, but major hepatic forms, CYP3A1 and CYP3A2, were not detected in the intestinal tracts of both sexes (Table 3). This may be related to the difference of catabolic and metabolic activities between livers and small intestines in rats. A large individual variation was observed on the expression level of CYP3A18 protein in the small intestine. The extent of transcriptional activation after the treatment with dexamethasone or lithocholic acid also differs among rat CYP3A genes. As shown in Fig. 2, all the CYP3A mRNAs in livers were increased after the treatment of both sexes of rats with dexamethasone. CYP3A1 and CYP3A2 mRNAs were not detected even with real-time PCR in intestines of rats treated with dexamethasone. In contrast, CYP3A62 and CYP3A9 mRNAs were readily detectable and enhanced after dexamethasone treatment in the small intestine and liver. These results clearly indicate the liver-selective expression of CYP3A1 and CYP3A2 and the intestinal-dominant expression of CYP3A62 and CYP3A9. Human CYP3A4 was detected mainly in the liver and intestinal tract and also increased in both tissues after treatment with chemical inducers (Kolars et al., 1992; Goodwin et al., 1999; Schmiedlin-Ren et al., 2001). These expression profiles are more similar to those of CYP3A62 and CYP3A9 than to those of CYP3A1 and CYP3A2. In addition to the expression profile, the nucleotide sequence of the CYP3A62 proximal promoter region shows higher similarity with that of the CYP3A4 genes than that of CYP3A1 and CYP3A2 genes (Fig. 5). It has been reported that CYP3A1 and CYP3A2 genes have hepatocyte nuclear factor-4 (HNF-4) binding element in their proximal promoter regions (Miyata et al., 1995; Nagata et al., 1999) as shown in Fig. 5. On the other hand, CYP3A62 as well as CYP3A4 do not contain HNF-4 binding element in their proximal promoter regions. Localization of HNF-4 binding site at the proximal promoter region may be associated with the strict liver-specific expression of CYP3A1 and CYP3A2.
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Recombinant CYP3A62 mediated testosterone 6-hydroxylation, but the rate was the lowest among recombinant rat CYP3A forms examined. Microsomal testosterone 6-hydroxylation of CYP3A62 was slightly enhanced after addition of cytochrome b5, unlike CYP3A1 and CYP3A2. A profile similar to CYP3A62 on testosterone hydroxylation was observed in that of CYP3A18. As reported, requirement of cytochrome b5 was dependent on the combination of P450 and substrate (Guengerich, 1983; Schenkman and Jansson, 2003). It may imply that the energy to transfer a second electron from cytochrome b5 to P450 is different among P450 and/or substrate, but no clear evidence can be provided.
In conclusion, we have isolated a new rat CYP3A form and identified it as CYP3A62 in the present study. Nucleotide sequences of the promoter region and CYP3A62 cDNA exhibited high similarity with the nucleotide sequences of CYP3A4 and CYP3A9 compared with the nucleotide sequences of CYP3A1 and CYP3A2. CYP3A62 was a predominant form in the intestinal tract, whereas CYP3A1 and CYP3A2 were detected only in the liver. In addition, the expression profile of CYP3A62 was also similar to that of CYP3A4 and CYP3A9. Judging from the absence of CYP3A1 and CYP3A2 in the gastrointestinal tract, CYP3A62, as well as CYP3A9 and CYP3A18, may play an important role in endogenous or exogenous detoxification at absorption in the small intestine.
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Footnotes |
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ABBREVIATIONS: P450, cytochrome P450; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; G6PDH, glucose-6-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair; HNF-4, hepatocyte nuclear factor-4.
Address correspondence to: Kiyoshi Nagata, Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, 980-8578, Japan. E-mail: nagataki{at}mail.tains.tohoku.ac.jp
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