![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResults and DiscussionReferences |
|---|
).
CYP2C8 plays important roles in metabolizing therapeutic drugs such as the
anticancer drug paclitaxel (Rahman et al.,
1994), the antidiabetes drug troglitazone
(Yamazaki et al., 1999), the
antiarrhythmic drug amiodarone (Ohyama et
al., 2000), and the HMG-CoA reductase inhibitor cerivastatin
(Muck, 2000). In addition,
CYP2C8 also metabolizes retinoids and arachidonic acid
(Zeldin et al., 1996;
Nadin and Murray, 1999).
Several polymorphisms have been described for the CYP2C8 gene
(Fig. 1). CYP2C8*1A is a wild type
(Klose et al., 1999).
CYP2C8*1B (C-271A) and CYP2C8*1C
(T-370G) have been reported to possess a single nucleotide polymorphism
(SNP1) in the
5'-flanking region (Bahadur et al.,
2002). However, no significant differences in protein levels were
reported between samples with a homozygous CYP2C8*1A and
those possessing either the CYP2C8*1B or
CYP2C8*1C alleles
(Bahadur et al., 2002). Dai et
al. (2001) reported that
CYP2C8*2 (I269F) and CYP2C8*3 (R139K,
K399R) showed decreased paclitaxel 6
-hydroxylase activity.
CYP2C8*4 possesses a SNP causing I264M
(Bahadur et al., 2002). It has
also been reported that median paclitaxel 6-hydroxylase activity in
liver microsomes from heterozygotes of CYP2C8*4 was lower
than that in the wild type, although the difference was not significant
(Bahadur et al., 2002). Soyama
et al. (2002) reported that
CYP2C8*5 (deletion of adenine 475) would be expected to
cause amino acid alterations from codon 159 and an early stop codon at residue
177. Soyama et al. (2001) also
found a SNP leading P404A in a Japanese subject. It has been reported that the
clearance of paclitaxel 6-hydroxylation of the variant P404A was
reduced in comparison with that of the wild type because of the labile protein
(Soyama et al., 2001). Bahadur
et al. (2002) found a SNP
leading L390S in a white subject, although its effects on the function have
never been investigated. In addition to these alleles, several variants of
T130N, E154D, N193K, K249R, and H411L have been found in comparison with six
separate full-length or partial CYP2C8 cDNA sequences (SWISSPROT)
(Kimura et al., 1987;
Okino et al., 1987;
Ged et al., 1988;
Shephard et al., 1989;
Kolyada, 1990;
Zeldin et al., 1995). It is
not clear whether these alleles exist as polymorphisms or represent sequence
errors or cloning artifacts. There is little information concerning CYP2C8
polymorphisms in the Japanese population. In the present study, we screened
for previously described polymorphisms in the coding region of this gene in
Japanese using PCR-restriction fragment length polymorphism (RFLP) or allele
specific (AS)-PCR analyses.
|
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
Human Genomic DNA Samples. Written informed consent was obtained from 360 healthy Japanese subjects. This study was approved by the Ethics Committees of Kanazawa University and Medca Japan. No subjects were taking any medications.
Genotyping of CYP2C8 Alleles. Genomic DNA was extracted from peripheral lymphocytes using the Puregene DNA isolation kit. DNA sequences of primers used in this study were as follows:
5S 5'-AAA GTA AAA GAA CAC CAA GC-3'
5AS 5'-AAA CAT CCT TAG TAA ATT ACA-3'
3S 5'-AGG CAA TTC CCC AAT ATC TC-3'
3AS 5'-CAG GAT GCG CAA TGA AGA C-3'
A475del-wt 5'-TCA CCC ACC CTT GGT TTT T-3'
A475del-mt 5'-TCA CCC ACC CTT GGT TTT C-3'
T130N-wt 5'-ATC CCA AAA TTC CGC AAG G-3'
T130N-mt 5'-ATC CCA AAA TTC CGC AAG T-3'
N193K-S 5'-AGA CAC TTG GGG TTA AA-3'
N193K-AS-wt 5'-TCA TCA GGG TGA GAA AA-3'
N193K-AS-mt 5'-TCA TCA GGG TGA GAA AT-3'
K249R-S 5'-CCA AAC ACT GCA TAT TCT CAT-3'
K249R-AS-wt 5'-CAG TGA TGC TTG GTG TTC TT-3'
K249R-AS-mt 5'-CAG TGA TGC TTG GTG TTC TC-3'
L390S-wt 5'-CAC AAC CAT AAT GGC ATT-3'
L390S-mt 5'-CAC AAC CAT AAT GGC ATC-3'
P404A-wt 5'-TGA CAA AGA ATT TCC TAA TC-3'
P404A-mt 5'-TGA CAA AGA ATT TCC TAA TG-3'
8S 5'-TTC CAT TTT AAA CCA TAA TCT-3'
8AS 5'-ATC ATG GAC AAA TAG CAA TT-3'
Polymorphisms of the CYP2C8 gene were analyzed by PCR-RFLP or AS-PCR methods (Table 1). Genomic DNA samples (0.1 µg) were added to the PCR mixtures (25 µl) consisting of 1 X PCR buffer [67 mM Tris-HCl buffer (pH 8.8), 16.6 mM (NH4)2SO4, 0.45% Triton X-100, 0.02% gelatin], 1.5 or 2.0 mM MgCl2, 0.4 µM primers, 250 µM dNTPs, and 1 U of Taq DNA polymerase. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 30 s for 30 cycles. The final extension step was performed at 72°C for 5 min. Primer sets, restriction enzymes for PCR-RFLP analyses, annealing temperatures, and fragment length are summarized in Table 1.
|
|
Results and Discussion |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
;
2002). Soyama et al.
(2002) reported the existence
of one heterozygote of CYP2C8*5 out of 54 Japanese
subjects, resulting in an allele frequency of 0.009. Soyama et al.
(2001) also detected
polymorphisms of CYP2C8*3 and P404A using 73 established
cell lines derived from different Japanese individuals with apparent allele
frequencies of <0.007. There is no information concerning other
polymorphisms of the CYP2C8 gene in the Japanese population. Since no
apparent effects of the polymorphisms in the 5'-flanking region
(CYP2C8*1B and CYP2C8*1C) on the
levels of expression were proven, the SNPs in the coding region were
investigated in the present study. We developed PCR-RFLP or AS-PCR methods for
genotyping of nonsynonymous SNPs of the CYP2C8 gene to determine
their allele frequencies in the Japanese population. In all reactions, the
expected PCR product at the correct length was obtained. In the PCR-RFLP
analyses, restriction enzymes used in the present study digest PCR product of
wild type at lease one site. In the mutated PCR product, a recognition site of
the restriction enzyme is expected to disappear owing to the mutation. As
shown in Figs. 2, A and B, we
confirmed that the restriction enzymes used for five PCR-RFLP analyses exactly
worked. For AS-PCR analyses, positive controls of wild type and mutant type
were made by PCR using wild-specific primer and mutant-specific primer,
respectively, with loose PCR condition (low anneal temperature). With these
controls, we confirmed that the wild-specific primer and mutant-specific
primer can specifically anneal to the wild type and mutant type, respectively,
with the PCR conditions in Table
1. Typical data are shown in
Fig. 2C. Using these PCR-RFLP
and AS-PCR analyses, we screened for the various polymorphisms in at least 200
Japanese DNA samples. The results are summarized in
Table 2. A single subject was
genotyped as heterozygote of CYP2C8*5
(Fig. 2D), and the allele
frequency was 0.0025. The DNA sequences around the polymorphic site of the
CYP2C8 gene in the heterozygote were confirmed with direct sequencing
analysis of the PCR product (data not shown). The other SNPs were not found in
the Japanese subjects in the present study. The inconsistency of the allele
frequencies obtained in the present study and the studies by Soyama et al.
(2001,
2002) might be due to the
difference in the sample sizes.
|
|
The allele frequencies of CYP2C8*2 in white subjects
and African Americans have been reported to be 0.004
(Bahadur et al., 2002) and 0.18
(Dai et al., 2001),
respectively. Dai et al. (2001)
also reported that the allele frequencies of CYP2C8*3 in
white subjects and African Americans were 0.13 and 0.02, respectively. It has
been reported that the allele frequencies of CYP2C8*3 and
CYP2C8*4 in white subjects were 0.15 and 0.075,
respectively (Bahadur et al.,
2002). The SNP of T1196C leading L390S has been found in one
heterozygous subject of 116 white subjects, resulting in an allele frequency
of 0.004 (Bahadur et al.,
2002). Thus, it is suggested that the allele frequencies of mutant
alleles in Japanese were relatively lower than those in white subjects and
African Americans.
In the present study, the alleles of T130N, E154D, N193K, K249R, and H411L
were not detected in our Japanese subjects. Bahadur et al.
(2002) also could not detect
the alleles of T130N, N193K, and H411L in 100 white subjects. Therefore, it
remains clear that these alleles exist as polymorphisms or represent sequence
errors or cloning artifacts.
In conclusion, we screened for 11 previously described polymorphisms in the coding region of CYP2C8 gene in Japanese using PCR-RFLP or AS-PCR methods and found only CYP2C8*5 allele with a frequency of 0.0025. Although several SNPs such as CYP2C8*2, CYP2C8*3, CYP2C8*4, and P404A have been reported to reduce the enzymatic activity, pharmacokinetic abnormalities of drugs metabolized by polymorphic CYP2C8 might be rare in Japanese.
Miki Nakajima
Yuto Fujiki
Kumiko Noda
Hiroki Ohtsuka
Hisashi Ohkuni
Satoru Kyo
Masaki Inoue
Yukio Kuroiwa
Tsuyoshi Yokoi
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences (M.N., Y.F., K.N., T.Y.), Department of Obstetrics and Gynecology, School of Medicine (S.K., M.I.), Kanazawa University, Kanazawa; Medca Japan Co. Ltd. (H.Oht., H.Ohk), Saitama; and Fuji Biomedix Co. Ltd. (Y.K.), Saitama, Japan
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Address correspondence to: Dr. Tsuyoshi Yokoi, Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Takara-machi 13–1, Kanazawa 920-0934, Japan. E-mail: tyokoi{at}kenroku.kanazawa-u.ac.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
-hydroxylase activity in human liver microsomes.
Biochem Pharmacol 64:
1579-1589.[CrossRef][Medline]
-hydroxytaxol by human cytochrome P450 2C8. Cancer
Res 54:
5543-5546.This article has been cited by other articles:
|
|
 |
|
  D. R. Gandara, T. Kawaguchi, J. Crowley, J. Moon, K. Furuse, M. Kawahara, S. Teramukai, Y. Ohe, K. Kubota, S. K. Williamson, et al. Japanese-US Common-Arm Analysis of Paclitaxel Plus Carboplatin in Advanced Non-Small-Cell Lung Cancer: A Model for Assessing Population-Related Pharmacogenomics J. Clin. Oncol., July 20, 2009; 27(21): 3540 - 3546. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Gardiner and E. J. Begg Pharmacogenetics, Drug-Metabolizing Enzymes, and Clinical Practice Pharmacol. Rev., September 1, 2006; 58(3): 521 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Henningsson, S. Marsh, W. J. Loos, M. O. Karlsson, A. Garsa, K. Mross, S. Mielke, L. Vigano, A. Locatelli, J. Verweij, et al. Association of CYP2C8, CYP3A4, CYP3A5, and ABCB1 Polymorphisms with the Pharmacokinetics of Paclitaxel Clin. Cancer Res., November 15, 2005; 11(22): 8097 - 8104. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Nakajima, Y. Fujiki, S. Kyo, T. Kanaya, M. Nakamura, Y. Maida, M. Tanaka, M. Inoue, and T. Yokoi Pharmacokinetics of Paclitaxel in Ovarian Cancer Patients and Genetic Polymorphisms of CYP2C8, CYP3A4, and MDR1 J. Clin. Pharmacol., June 1, 2005; 45(6): 674 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Hichiya, T. Tanaka-Kagawa, A. Soyama, H. Jinno, S. Koyano, N. Katori, E. Matsushima, S. Uchiyama, H. Tokunaga, H. Kimura, et al. FUNCTIONAL CHARACTERIZATION OF FIVE NOVEL CYP2C8 VARIANTS, G171S, R186X, R186G, K247R, AND K383N, FOUND IN A JAPANESE POPULATION Drug Metab. Dispos., May 1, 2005; 33(5): 630 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Vaclavikova, P. Soucek, L. Svobodova, P. Anzenbacher, P. Simek, F. P. Guengerich, and I. Gut DIFFERENT IN VITRO METABOLISM OF PACLITAXEL AND DOCETAXEL IN HUMANS, RATS, PIGS, AND MINIPIGS Drug Metab. Dispos., June 1, 2004; 32(6): 666 - 674. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
