Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2014 May 13.
Published in final edited form as: J Toxicol Sci. 2013;38(3):349–354. doi: 10.2131/jts.38.349

The CYP3A4 intron 6 C>T polymorphism (CYP3A4*22) is associated with reduced CYP3A4 protein level and function in human liver microsomes

Maho Okubo 1, Norie Murayama 1, Makiko Shimizu 1, Tsutomu Shimada 2, F Peter Guengerich 3, Hiroshi Yamazaki 1,*
PMCID: PMC4018728  NIHMSID: NIHMS578520  PMID: 23665933

Abstract

Effects of the CYP3A4 intron 6 C>T (CYP3A4*22) polymorphism, which has recently been reported to have a critical role in vivo, were investigated by measuring CYP3A4 protein expression levels and CYP3A4-dependent drug oxidation activities in individual human liver microsomes in vitro. Prior to protein analysis, analysis of DNA samples indicated that 36 Caucasian subjects were genotyped as CYP3A4*1/*1 and five subjects were CYP3A4*1/*22, with a CYP3A4*22 allelic frequency of 6.1%. No CYP3A4*22 alleles were found in the Japanese samples (106 alleles). Individual differences in CYP2D6-dependent dextromethorphan O-demethylation activities in liver microsomes from Caucasians were not affected by either the CYP3A4*1/*22 or CYP3A5*1/*3 genotype. Liver microsomes genotyped as CYP3A4*1/*22 (n=4) showed significantly lower CYP3A-dependent dextromethorphan N-demethylation, midazolam 1′-hydroxylation, and testosterone 6β-hydroxylation activities, as well as lower expression levels of CYP3A protein (28% of control), compared with those of the CYP3A4*1/*1 group (n=19). The other polymorphism, CYP3A5*1/*3, did not show these differences (n=4). The CYP3A4*22 polymorphism was associated with reduced CYP3A4 protein expression levels and resulted in decreased CYP3A4-dependent activities in human livers. The present results suggest an important role of low expression of CYP3A4 protein associated with the CYP3A4*22 allele in the individual differences in drug clearance.

Keywords: P450 3A4, P450 3A5, protein expression, ethnic difference, impaired polymorphism

Introduction

Cytochrome P450 (P450 or CYP) comprises a superfamily of enzymes and plays an important role in the oxidative metabolism of a large number of endogenous and exogenous compounds (Guengerich 2008). CYP3A4 is the most abundant P450 enzyme in human livers and is involved in metabolism of > 50% of marketed drugs (Rendic 2002; Shimada et al., 1994). CYP3A activity has been shown to display 10- to 100-fold variation among individuals (Lamba et al., 2002; Shimada et al., 1994; Westlind et al., 1999; Westlind-Johnsson et al., 2003), which may influence drug response and toxicity. Low drug clearances mediated by impaired CYP3A4 would invoke unexpected side effects or drug interactions with co-administered food or medicines. It has been suggested that ~85% of the inter-individual variability in hepatic CYP3A4 expression and activity is attributable to genetic factors (Ozdemir et al., 2000). However, most of the CYP3A4 polymorphisms examined to date have low frequencies (http://www.cypalleles.ki.se/cyp3a4.htm). The most common CYP3A4 variant has been a promoter variant (CYP3A4*1B) leading to modified CYP3A4 activity (Garcia-Martin et al., 2002), but subsequent work on this has not been clear regarding its significance (Ball et al., 1999; Garcia-Martin et al., 2002).

A recent study identified a functional SNP in CYP3A4 intron 6 (rs35599367C>T; CYP3A4*22) (Wang et al., 2011). The CYP3A4*22 genotype is associated with a good lipid-lowering response to the drug simvastatin (Elens et al., 2011a). In addition, renal transplant patients with the CYP3A4*22 allele show reduced clearance of a calcineurin inhibitor and therefore might be associated with increased risk of drug overexposure (Elens et al., 2011b; Elens et al., 2011c; Elens et al., 2012). However, there are no clear in vitro reports regarding any effects of the CYP3A4*22 allele on protein levels. On the other hand, CYP3A5, demonstrating 84% amino acid sequence identity with CYP3A4, has been associated with the metabolism of a variety of CYP3A4-probe drugs (Wrighton et al., 1992). The most frequent variant of functional importance in the CYP3A5 gene has been CYP3A5*3, leading to alternative splicing with impaired protein expression (Kuehl et al., 2001; van Schaik et al., 2002). Therefore, CYP3A5 polymorphisms should also have potential effects on CYP3A-dependent activities in human livers. However, there are no available studies to clarify the CYP3A4*22 and CYP3A5*3 polymorphisms, in combination, on the catalytic function in human liver microsomes.

In the present study, effects of the CYP3A4*22 and CYP3A5*3 polymorphisms were investigated by measuring CYP3A protein expression levels and CYP3A-dependent drug oxidation in individual human livers. Ethnic differences of the frequency of the CYP3A4*22 allele were seen between Caucasian and Japanese subjects. We report, for the first time, a clear association between the CYP3A4*22 polymorphism, CYP3A4 protein levels, and catalytic function in human liver microsomes.

Materials and Methods

Chemicals

Dextromethorphan was obtained from Sigma-Aldrich (St. Louis, MO). Midazolam and testosterone were obtained from Wako Pure Chemicals (Osaka, Japan). Other chemicals and reagents used in this study were obtained from the sources described previously and were of the highest quality commercially available (Yamazaki et al., 2002; Yamazaki et al., 2006).

Enzyme preparations

The use of the human livers for this study was approved by the Ethics Committees of Vanderbilt University School of Medicine and Showa Pharmaceutical University. The human livers (5– to 74-year-old males and females) were obtained from patients after pathological examination of specimens isolated during hepatic surgery or after death (Inoue et al., 1997; Shimada et al., 2001; Yamaori et al., 2004; Yamaori et al., 2005; Yamazaki et al., 2003). Human liver microsomes were prepared in 10 mM Tris-HCl buffer (pH 7.4) containing 0.10 mM EDTA and 20% (v/v) glycerol as described previously (Yamaori et al., 2005).

Genotyping

Genomic DNA was isolated from human livers as previously described (Inoue et al., 1997; Shimada et al., 2001; Yamaori et al., 2004; Yamaori et al., 2005; Yamazaki et al., 2003). The CYP3A4*22 genotype was determined with 10 ng genomic of DNA in an allelic discrimination reaction performed with TaqMan® (Applied Biosystems, Foster City, CA, USA) genotyping assays (C_59013445_10) using a 7300 Real Time PCR System® (Applied Biosystems). The CYP3A5*3 genotype was determined according to a previously published method (Adler et al., 2009).

Enzyme assays

Microsomal protein concentrations were estimated using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Activities for O- and N-demethylation of dextromethorphan, 1′-hydroxylation of midazolam, and 6β-hydroxylation of testosterone were assayed according to described methods (Kronbach et al., 1989; Uno et al., 2010; Yamazaki et al., 1997). Briefly, the standard incubation mixtures consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate, and 0.25 unit/ml glucose 6-phosphate dehydrogenase), a substrate (400 μM dextromethorphan, 100 μM midazolam, or 50 μM testosterone), and liver microsomes (0.20–0.50 mg protein/ml) in a final volume of 0.25 ml. Dextromethorphan and midazolam were incubated with microsomes at 37°C for 15 min and terminated by the addition of 10 μl of 60% perchloric acid (w/v) or 0.25 ml of ice-cold methanol. Testosterone oxidation reactions were incubated at 37 °C for 10 min and terminated by the addition of 1.5 ml of ethyl acetate and 25 μl of 3 M sodium chloride. After extraction, the organic phase of each sample was evaporated under a nitrogen stream. Product formation was determined by high-performance liquid chromatography with an analytical octadecylsilane (C18) column (4.6 mm × 150 mm, 5 μm) according to described methods (Kronbach et al., 1989; Uno et al., 2010; Yamazaki et al., 1997).

Western blot analysis

SDS-PAGE was performed using 7.5% (w/v) acrylamide gels. Microsomal protein (5 μg) was separated and transferred onto a nitrocellulose membrane. Immunoblot quantitation was performed using recombinant CYP3A4 (Supersomes, BD Gentest, Franklin Lakes, NJ, USA) and a mouse antibody to human CYP3A (BD Gentest) as the standard protein and primary antibody for both human CYP3A4 and CYP3A5, respectively. Horseradish peroxidase-conjugated anti-mouse immunoglobulin (BD Gentest) was used as a secondary antibody (Yamaori et al., 2004). A specific band was visualized using LAS-1000UVmini (GE Healthcare, Tokyo, Japan) and analyzed using Multi Gauge software, version 3.0 (GE Healthcare).

Statistical analyses

Statistical analysis was performed by Prism software, version 5.01 (GraphPad, San Diego, CA, USA). The differences in enzymatic activities between two genotypes were evaluated using an unpaired t-test with Welch correction. Statistical tests provided two-sided p values with a significance level of < 0.05.

Results

DNA samples from 41 Caucasian and 53 Japanese subjects were genotyped for CYP3A4 using a TaqMan assay method (Table 1). Among the Caucasian samples, 36 subjects were genotyped as CYP3A4*1/*1 but five subjects were of the CYP3A4*1/*22 genotype. The CYP3A4*22 allelic frequency was estimated to be 6.1% in this sample, similar to that reported previously in Caucasians (Elens et al., 2011b). On the other hand, there were no CYP3A4*22 alleles observed in our Japanese samples (106 alleles). The expected frequency of the CYP3A4*22 in a Japanese population was calculated to be <1%; further analysis may be important in this context.

Table 1.

CYP3A4 allele frequencies in 41 Caucasian and 53 Japanese DNA samples

3A4 allele Number (%)
Caucasian
n=82		 Japanese
n=106
*1 77 (94) 106 (100)
*22 5 (6) 0 (0)
Open in a new tab

To investigate the effects of genetic polymorphism of the CYP3A4 and CYP3A5 genes on the metabolic activities, dextromethorphan O- and N-demethylation, midazolam 1′-hydroxylation, and testosterone 6β-hydroxylation activities were determined in liver microsomes prepared from 23 Caucasian subjects genotyped for CYP3A4 (19 CYP3A4*1/*1 and four CYP3A4*1/*22) and CYP3A5 (four CYP3A5*1/*3 and 19 CYP3A5*3/*3). There were no examples of both CYP3A4*22 and CYP3A5*1 among these liver samples. CYP2D6-dependent catalytic activities (dextromethorphan O-demethylation) in liver microsomes did not differ between both the CYP3A4 and CYP3A5 genotypes (Figure 1A). On the other hand, the heterozygote group consisted of four liver microsomal preparations derived from subjects genotyped as CYP3A4*1/*22 (and also CYP3A5*3/*3) showed significantly lower CYP3A-dependent activities (N-demethylation of dextromethorphan, 1′-hydroxylation of midazolam, and 6β-hydroxylation of testosterone), compared with the wild-type (CYP3A4*1/*1) group (Figure 1B–D, *p < 0.05 and **p < 0.01). Although these typical substrates of CYP3A are also metabolized by CYP3A5, the CYP3A5 genotype did not show any significant differences under the present experimental conditions (Figure 1). CYP3A protein contents in liver microsomes from subjects genotyped as CYP3A4*1/*22 were significantly lower (28% of control) than from subjects genotyped as CYP3A4*1/*1, while there was no significant differences between CYP3A5 genotypes (Figure 2). The individuals in the CYP3A4*1/*22 heterozygote group were all genetically poor expressers of CYP3A5 in this study.

Fig. 1. Association between CYP3A4 and CYP3A5 genotypes and P450-dependent drug oxidation activities in human liver microsomes.

Fig. 1

Open in a new tab

Dextromethorphan O- (A) and N-demethylation (B), midazolam 1′-hydroxylation (C), and testosterone 6β-hydroxylation (D) activities were analyzed in liver microsomal samples from 23 Caucasians genotyped for CYP3A4 and CYP3A5. The horizontal lines indicate the mean activities, respectively. *p <0.05; **p <0.01, significantly different by unpaired t-test with Welch correction.

Fig. 2. Association between CYP3A4 and CYP3A5 genotypes and expression levels of CYP3A protein in human liver microsomes.

Fig. 2

Open in a new tab

CYP3A contents were measured in liver microsomal samples from 23 Caucasians genotyped for CYP3A4 and CYP3A5 by immunoblotting. *p <0.05, significantly different by unpaired t-test with Welch correction.

Discussion

A CYP3A4 intron 6 C>T (CYP3A4*22) allele (Elens et al., 2011a; Elens et al., 2011b; Elens et al., 2011c; Wang et al., 2011) is associated with reduced CYP3A4 activity in vivo. Wang et al. have recently reported that CYP3A4*22 is linked to reduced CYP3A4 mRNA production (without any splice variants) and limited testosterone 6β-hydroxylation activity in human livers. However, the finding regarding CYP3A4 mRNA levels has not been strongly supported by the recent study (Klein et al., 2012). Although statistically significant decreases of area-under the curve of drug metabolites per variant allele has been strongly confirmed in the in vivo cohort (Klein et al., 2012), we are not aware of clear in vitro reports of studies regarding any effects of the CYP3A4*22 allele on protein levels and multiple enzymatic function.

In the present study, decreased CYP3A protein contents (28% of control) in human liver microsomes associated with the CYP3A4*22 genotype were clearly shown for the first time, using a mouse anti-human CYP3A antibody (Figure 2). Under the present condition, CYP3A5 could be detected, along with CYP3A4 in liver microsomes; however, most Caucasians are genetically poor expressers of CYP3A5 (CYP3A5*3/*3) (Kuehl et al., 2001; van Schaik et al., 2002). In the four Caucasian liver samples that were heterozygous expressers of CYP3A5, these levels were low and < ~5 pmol CYP3A5/mg protein (Yamaori et al., 2005; Yamazaki et al., 1995). Reduced CYP3A4 protein expression levels resulted in decreased CYP3A4-mediated enzymatic activities in liver microsomal samples derived from Caucasians (24–50% of controls, an average of 37%, shown in Figure 1). Consequently, these results regarding impaired metabolic function in liver microsomes in vitro caused by the CYP3A4*22 genotype could support the recent reported findings of low clearance of clinical CYP3A-related drugs in vivo (Elens et al., 2011a; Elens et al., 2011b; Elens et al., 2011c; Elens et al., 2013; Wang et al., 2011). The reason why this CYP3A4*22 polymorphism in the intronic part of the gene can lead to lowered the mRNA and/or protein expression should be investigated in the future.

In conclusion, the CYP3A4*22 allele was associated with decreased CYP3A4 protein expression levels and resulted in decreased CYP3A4-mediated enzymatic activities. These results suggest an important role of low expression of CYP3A4 protein associated with this CYP3A4*22 allele in the individual differences in drug clearance in Caucasians. Drug therapy for the Caucasian patients harboring the CYP3A4*22 allele with CYP3A4-dependent drugs should be paid attention. Furthermore, it was also demonstrated that the frequency of the CYP3A4*22 allele shows ethnic differences. Shi et al. have recently reported that they found no CYP3A4*22 alleles in 216 Chinese subjects. Because no CYP3A4*22 allele carriers were found in a Japanese population in the present study, it may be implied that the effects of CYP3A4*22 polymorphism might be limited in Japanese subjects, which show a wide variety of inter-individual differences in inductive CYP3A4-dependent drug disposition.

Acknowledgements

This work was supported in part by the Ministry of Education, Science, Sports and Culture of Japan, the Takeda Science Foundation, and the United States Public Health Service (R37 CA090426).

References

  1. Adler G, Loniewska B, Parczewski M, Kordek A, Ciechanowicz A. Frequency of common CYP3A5 gene variants in healthy Polish newborn infants. Pharmacol Rep. 2009;61:947–951. doi: 10.1016/s1734-1140(09)70154-9. [DOI] [PubMed] [Google Scholar]
  2. Ball SE, Scatina J, Kao J, Ferron GM, Fruncillo R, Mayer P, Weinryb I, Guida M, Hopkins PJ, et al. Population distribution and effects on drug metabolism of a genetic variant in the 5' promoter region of CYP3A4. Clin Pharmacol Ther. 1999;66:288–294. doi: 10.1016/S0009-9236(99)70037-8. [DOI] [PubMed] [Google Scholar]
  3. Elens L, Becker ML, Haufroid V, Hofman A, Visser LE, Uitterlinden AG, Stricker B, van Schaik RH. Novel CYP3A4 intron 6 single nucleotide polymorphism is associated with simvastatin-mediated cholesterol reduction in the Rotterdam Study. Pharmacogenet Genomics. 2011;21:861–866. doi: 10.1097/FPC.0b013e32834c6edb. [DOI] [PubMed] [Google Scholar]
  4. Elens L, Bouamar R, Hesselink DA, Haufroid V, van der Heiden IP, van Gelder T, van Schaik RH. A new functional CYP3A4 intron 6 polymorphism significantly affects tacrolimus pharmacokinetics in kidney transplant recipients. Clin Chem. 2011;57:1574–1583. doi: 10.1373/clinchem.2011.165613. [DOI] [PubMed] [Google Scholar]
  5. Elens L, van Schaik RH, Panin N, de Meyer M, Wallemacq P, Lison D, Mourad M, Haufroid V. Effect of a new functional CYP3A4 polymorphism on calcineurin inhibitors' dose requirements and trough blood levels in stable renal transplant patients. Pharmacogenomics. 2011;12:1383–1396. doi: 10.2217/pgs.11.90. [DOI] [PubMed] [Google Scholar]
  6. Elens L, Bouamar R, Hesselink DA, Haufroid V, van Gelder T, van Schaik RH. The new CYP3A4 intron 6 C>T polymorphism (CYP3A4*22 ) is associated with an increased risk of delayed graft function and worse renal function in cyclosporine-treated kidney transplant patients. Pharmacogenet Genomics. 2012;22:373–380. doi: 10.1097/FPC.0b013e328351f3c1. [DOI] [PubMed] [Google Scholar]
  7. Elens L, Nieuweboer A, Clarke SJ, Charles KA, de Graan AJ, Haufroid V, Mathijssen RH, van Schaik RH. CYP3A4 intron 6 C>T SNP (CYP3A4*22 ) encodes lower CYP3A4 activity in cancer patients, as measured with probes midazolam and erythromycin. Pharmacogenomics. 2013;14:137–149. doi: 10.2217/pgs.12.202. [DOI] [PubMed] [Google Scholar]
  8. Garcia-Martin E, Martinez C, Pizarro RM, Garcia-Gamito FJ, Gullsten H, Raunio H, Agundez JA. CYP3A4 variant alleles in white individuals with low CYP3A4 enzyme activity. Clin Pharmacol Ther. 2002;71:196–204. doi: 10.1067/mcp.2002.121371. [DOI] [PubMed] [Google Scholar]
  9. Guengerich FP. Cytochrome P450 and chemical toxicology. Chem Res Toxicol. 2008;21:70–83. doi: 10.1021/tx700079z. [DOI] [PubMed] [Google Scholar]
  10. Inoue K, Yamazaki H, Imiya K, Akasaka S, Guengerich FP, Shimada T. Relationship between CYP2C9 and 2C19 genotypes and tolbutamide methyl hydroxylation and S-mephenytoin 4'-hydroxylation activities in livers of Japanese and Caucasian populations. Pharmacogenetics. 1997;7:103–113. doi: 10.1097/00008571-199704000-00003. [DOI] [PubMed] [Google Scholar]
  11. Klein K, Thomas M, Winter S, Nussler AK, Niemi M, Schwab M, Zanger UM. PPARA : a novel genetic determinant of CYP3A4in vitro andin vivo . Clin Pharmacol Ther. 2012;91:1044–1052. doi: 10.1038/clpt.2011.336. [DOI] [PubMed] [Google Scholar]
  12. Kronbach T, Mathys D, Umeno M, Gonzalez FJ, Meyer UA. Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Mol Pharmacol. 1989;36:89–96. [PubMed] [Google Scholar]
  13. Kuehl P, Zhang J, Lin Y, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, et al. Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet. 2001;27:383–391. doi: 10.1038/86882. [DOI] [PubMed] [Google Scholar]
  14. Lamba JK, Lin YS, Schuetz EG, Thummel KE. Genetic contribution to variable human CYP3A-mediated metabolism. Adv Drug Deliv Rev. 2002;54:1271–1294. doi: 10.1016/s0169-409x(02)00066-2. [DOI] [PubMed] [Google Scholar]
  15. Ozdemir V, Kalow W, Tang BK, Paterson AD, Walker SE, Endrenyi L, Kashuba AD. Evaluation of the genetic component of variability in CYP3A4 activity: a repeated drug administration method. Pharmacogenetics. 2000;10:373–388. doi: 10.1097/00008571-200007000-00001. [DOI] [PubMed] [Google Scholar]
  16. Rendic S. Summary of information on human CYP enzymes: human P450 metabolism data. Drug Metab Rev. 2002;34:83–448. doi: 10.1081/dmr-120001392. [DOI] [PubMed] [Google Scholar]
  17. Shi Y, Li Y, Tang J, Zhang J, Zou Y, Cai B, Wang L. Influence of CYP3A4, CYP3A5 and MDR-1 polymorphisms on tacrolimus pharmacokinetics and early renal dysfunction in liver transplant recipients. Gene. 2013;512:226–231. doi: 10.1016/j.gene.2012.10.048. [DOI] [PubMed] [Google Scholar]
  18. Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther. 1994;270:414–423. [PubMed] [Google Scholar]
  19. Shimada T, Tsumura F, Yamazaki H, Guengerich FP, Inoue K. Characterization of (+/−)-bufuralol hydroxylation activities in liver microsomes of Japanese and Caucasian subjects genotyped for CYP2D6. Pharmacogenetics. 2001;11:143–156. doi: 10.1097/00008571-200103000-00005. [DOI] [PubMed] [Google Scholar]
  20. Uno Y, Uehara S, Kohara S, Murayama N, Yamazaki H. Cynomolgus monkey CYP2D44, newly identified in liver, metabolizes bufuralol, and dextromethorphan. Drug Metab Dispos. 2010;38:1486–1492. doi: 10.1124/dmd.110.033274. [DOI] [PubMed] [Google Scholar]
  21. van Schaik RH, van der Heiden IP, van den Anker JN, Lindemans J. CYP3A5 variant allele frequencies in Dutch Caucasians. Clin Chem. 2002;48:1668–1671. [PubMed] [Google Scholar]
  22. Wang D, Guo Y, Wrighton SA, Cooke GE, Sadee W. Intronic polymorphism in CYP3A4 affects hepatic expression and response to statin drugs. Pharmacogenomics J. 2011;11:274–286. doi: 10.1038/tpj.2010.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Westlind A, Lofberg L, Tindberg N, Andersson TB, Ingelman-Sundberg M. Interindividual differences in hepatic expression of CYP3A4: relationship to genetic polymorphism in the 5'-upstream regulatory region. Biochem Biophys Res Commun. 1999;259:201–205. doi: 10.1006/bbrc.1999.0752. [DOI] [PubMed] [Google Scholar]
  24. Westlind-Johnsson A, Malmebo S, Johansson A, Otter C, Andersson TB, Johansson I, Edwards RJ, Boobis AR, Ingelman-Sundberg M. Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism. Drug Metab Dispos. 2003;31:755–761. doi: 10.1124/dmd.31.6.755. [DOI] [PubMed] [Google Scholar]
  25. Wrighton SA, Stevens JC. The human hepatic cytochromes P450 involved in drug metabolism. Crit Rev Toxicol. 1992;22:1–21. doi: 10.3109/10408449209145319. [DOI] [PubMed] [Google Scholar]
  26. Yamaori S, Yamazaki H, Iwano S, Kiyotani K, Matsumura K, Honda G, Nakagawa K, Ishizaki T, Kamataki T. CYP3A5 Contributes significantly to CYP3A-mediated drug oxidations in liver microsomes from Japanese subjects. Drug Metab Pharmacokinet. 2004;19:120–129. doi: 10.2133/dmpk.19.120. [DOI] [PubMed] [Google Scholar]
  27. Yamaori S, Yamazaki H, Iwano S, Kiyotani K, Matsumura K, Saito T, Parkinson A, Nakagawa K, Kamataki T. Ethnic differences between Japanese and Caucasians in the expression levels of mRNAs for CYP3A4, CYP3A5 and CYP3A7: lack of co-regulation of the expression of CYP3A in Japanese livers. Xenobiotica. 2005;35:69–83. doi: 10.1080/00498250400021796. [DOI] [PubMed] [Google Scholar]
  28. Yamazaki H, Inui Y, Wrighton SA, Guengerich FP, Shimada T. Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed inEscherichia coli and by human liver microsomes. Carcinogenesis. 1995;16:2167–2170. doi: 10.1093/carcin/16.9.2167. [DOI] [PubMed] [Google Scholar]
  29. Yamazaki H, Shimada T. Progesterone and testosterone hydroxylation by cytochromes P450 2C19, 2C9, and 3A4 in human liver microsomes. Arch Biochem Biophys. 1997;346:161–169. doi: 10.1006/abbi.1997.0302. [DOI] [PubMed] [Google Scholar]
  30. Yamazaki H, Nakamura M, Komatsu T, Ohyama K, Hatanaka N, Asahi S, Shimada N, Guengerich FP, Shimada T, et al. Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli. Protein Expr Purif. 2002;24:329–337. doi: 10.1006/prep.2001.1578. [DOI] [PubMed] [Google Scholar]
  31. Yamazaki H, Kiyotani K, Tsubuko S, Matsunaga M, Fujieda M, Saito T, Miura J, Kobayashi S, Kamataki T. Two novel haplotypes of CYP2D6 gene in a Japanese population. Drug Metab Pharmacokinet. 2003;18:269–271. doi: 10.2133/dmpk.18.269. [DOI] [PubMed] [Google Scholar]
  32. Yamazaki H, Okayama A, Imai N, Guengerich FP, Shimizu M. Inter-individual variation of cytochrome P4502J2 expression and catalytic activities in liver microsomes from Japanese and Caucasian populations. Xenobiotica. 2006;36:1201–1209. doi: 10.1080/00498250600944318. [DOI] [PubMed] [Google Scholar]

RESOURCES