Elsevier

Biochemical Pharmacology

Volume 64, Issue 11, 1 December 2002, Pages 1579-1589
Biochemical Pharmacology

CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6α-hydroxylase activity in human liver microsomes

https://doi.org/10.1016/S0006-2952(02)01354-0Get rights and content

Abstract

Published cDNA sequences suggest the existence of non-synonymous single nucleotide polymorphisms in the cytochrome P450 CYP2C8. To determine whether these polymorphisms could be confirmed in a Caucasian population and to investigate whether additional polymorphisms occur in the coding and upstream regions of this gene, we screened for previously described and for novel polymorphisms using PCR-RFLP and SSCP analysis. We confirmed the existence of two of the previously detected polymorphisms which give rise to the amino acid substitutions I264M and K399R, respectively, but failed to detect three others in our population. We also confirmed that a recently identified polymorphism (R139K) is linked to K399R (CYP2C8*3) in our study population. The allele frequencies for the I264M (CYP2C8*4 allele) and the CYP2C8*3 allele were 0.075 and 0.15, respectively. Three novel polymorphisms (T-370G, C-271A and T1196C/L390S) were also detected with the upstream polymorphisms showing allele frequencies of 0.061 and 0.196, respectively, but the L390S polymorphism detected only in a single subject. An additional single subject was heterozygous for a polymorphism recently described in African–Americans (A805T; CYP2C8*2 allele). The functional significance of the two upstream polymorphisms and the CYP2C8*3 and CYP2C8*4 alleles was investigated in human liver microsomes. Samples heterozygous for CYP2C8*3 showed significantly lower paclitaxel 6α-hydroxylase activity compared with wild-type samples. Median activity associated with CYP2C8*4 also appeared lower than the wild-type but the difference was not significant. There was no evidence that either upstream polymorphism gave rise to altered CYP2C8 expression.

Introduction

The cytochrome P450 CYP2C8‡ is present at relatively high levels in most human livers [1] and plays a major role in the metabolism of several therapeutically important drugs including paclitaxel, all-trans retinoic acid, verapamil, rosiglitazone, cerivastatin, amiodarone, dapsone and amodiaquine [2], [3], [4], [5], [6], [7], [8], [9], [10]. There is some evidence for a physiological role for CYP2C8 in arachidonic acid metabolism, especially in the oxidation of arachidonic acid to the putative endothelium-derived hyperpolarising factor 11,12-epoxyeicosatrienoic acid (11,12-EET) [11], [12], though more recent reports suggest that CYP2C9 may be the major isoform involved [13]. CYP2C8 may also contribute to activation of toxicologically important compounds including benzo[a]pyrene and parathion [14], [15]. CYP2C8 shows considerable homology with other members of the CYP2C family, particularly CYP2C9, but there is little overlap in substrate specificity with, for example CYP2C8 being considerably less efficient in the oxidative metabolism of tolbutamide compared with CYP2C9 [16], whereas all-trans retinoic acid is more efficiently hydroxylated by CYP2C8 compared with CYP2C9 [3]. A recent homology model for human CYP2C isoforms suggests that there are significant differences with regard to the key amino acids within the active site of CYP2C8 compared with other CYP2Cs [17] which is also consistent with differences in xenobiotic inhibitor profiles compared with CYP2C9 as well as a variety of other P450s [18]. Many of the drug substrates metabolised by CYP2C8 are also metabolised by CYP3A4 but in a number of cases including, for example paclitaxel, the products of metabolism by CYP2C8 and CYP3A4 are different. Unlike in the case of other members of the human CYP2C family, there is currently limited information on the existence of polymorphism in CYP2C8. However, there are reports of inter-individual variation in the metabolism of several CYP2C8 substrates particularly paclitaxel and rosiglitazone [2], [6]. A number of independently determined cDNA sequences for CYP2C8 are available which suggest the existence of several genetic polymorphisms [11], [19], [20], [21], [22], [23]. We have determined whether these polymorphisms occur in vivo and have in addition screened a Caucasian population for the existence of novel polymorphisms in the exons and part of the upstream sequence. We now describe the existence of a number of SNPs in the coding and upstream sequences of CYP2C8, their population frequencies and preliminary data on their possible functional significance by studies on the relationship between genotype and enzyme activity and protein expression in a human liver bank. Some of these SNPs have recently been detected in two independent studies [24], [25].

Section snippets

Blood and liver samples

Blood samples were obtained from apparently healthy British Caucasian volunteers resident in Northeast England. These individuals have been described in detail previously [26], [27]. Sample collection was approved by the Newcastle University and Newcastle and North Tyneside joint ethical committee and all volunteers gave informed consent to their use in studies on cytochrome P450 polymorphisms. Three separate human liver banks were used. Bank 1 consisted of samples from 26 British Caucasian

Studies on known CYP2C8 cDNA sequences

At least six separate full length or partial CYP2C8 cDNA sequences have been reported as summarised in Fig. 1. Since the sequences indicated the existence of five different alleles, we developed PCR-RFLP or SSCP assays for at least one non-synonymous SNP associated with each sequence to confirm their existence and determine their population frequencies. Using these assays, we screened for the various polymorphisms in at least 100 British Caucasian DNA samples. The results obtained are

Discussion

The current investigation has shown that at least four additional CYP2C8 variant alleles including two involving non-synonymous mutations occur and has also confirmed the existence of a previously described variant [24], [25]. In addition, we have surveyed existing database sequence data on CYP2C8 and shown that while this correctly predicts the existence of two allelic variants (CYP2C8*3 and CYP2C8*4), the existence of an additional three non-synonymous polymorphisms predicted from published

Acknowledgements

We are grateful to Professor G. Hawksworth for assisting with supply of human livers, to Dr. R. Edwards for a gift of anti-CYP2C antiserum, to Helen Rees for assistance with preliminary experiments and to Lieve Van de Velde for some of the DNA extractions

References (45)

  • L.M Forrester et al.

    Relative expression of cytochrome P450 isoenzymes in human liver and association with the metabolism of drugs and xenobiotics

    Biochem. J.

    (1992)
  • A Rahman et al.

    Selective biotransformation of taxol to 6-alpha-hydroxytaxol by human cytochrome-P450 2C8

    Cancer Res.

    (1994)
  • D Busse et al.

    Cytochromes of the P450 2C subfamily are the major enzymes involved in the O-demethylation of verapamil in humans

    Naunyn Schmiedebergs Arch. Pharmacol.

    (1995)
  • H Yamazaki et al.

    Oxidation of troglitazone to a quinone-type metabolite catalyzed by cytochrome P-4502C8 and P-450 3A4 in human liver microsomes

    Drug Metab. Dispos.

    (1999)
  • S.J Baldwin et al.

    Characterization of the cytochrome P450 enzymes involved in the in vitro metabolism of rosiglitazone

    Br. J. Clin. Pharmacol.

    (1999)
  • W Muck

    Clinical pharmacokinetics of cerivastatin

    Clin. Pharmacokinet.

    (2000)
  • K Ohyama et al.

    A significant role of human cytochrome P4502C8 in amiodarone N-deethylation: an approach to predict the contribution with relative activity factor

    Drug Metab. Dispos.

    (2000)
  • H.R Winter et al.

    CYP2C8/9 mediate dapsone N-hydroxylation at clinical concentrations of dapsone

    Drug Metab. Dispos.

    (2000)
  • X.-Q Li et al.

    Amodiaquine clearance and its metabolism to N-desthylamodiaquine is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe substrate

    J. Pharmacol. Exp. Ther.

    (2002)
  • S.S Bolz et al.

    Antisense oligonucleotides against cytochrome P4502C8 attenuate EDHF-mediated Ca2+ changes and dilation in isolated resistance arteries

    FASEB J.

    (2000)
  • I Fleming et al.

    Endothelium-derived hyperpolarizing factor synthase (cytochrome P4502C9) is a functionally significant source of reactive oxygen species in coronary arteries

    Circ. Res.

    (2001)
  • C.H Yun et al.

    Roles of human liver cytochrome P4502C and cytochrome P4503A enzymes in the 3-hydroxylation of benzo(a)pyrene

    Cancer Res.

    (1992)
  • Cited by (0)

    View full text