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