1. The human liver UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), have been studied using microsomes from human liver and insect cells expressing human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B15). 2. The glucuronidation of SN-38 was catalysed by UGT1A1, UGT1A3, UGT1A6 and UGT1A9 as well as by liver microsomes. Among these UGT isoforms, UGT1A1 showed the highest activity of SN-38 glucuronidation at both low (1 microM) and high (200 microM) substrate concentrations. The ranking in order of activity at low and high substrate concentrations was UGT1A1 > UGT1A9 > UGT1A6 > UGT1A3 and UGT1A1 > UGT1A3 > UGT1A6 > or = UGT1A9, respectively. 3. The enzyme kinetics of SN-38 glucuronidation were examined by means of Lineweaver-Burk analysis. The activity of the glucuronidation in liver microsomes exhibits a monophasic kinetic pattern, with an apparent Km and Vmax of 35.9 microM and 134 pmol min(-1) mg(-1) protein, respectively. The UGT isoforms involved in SN-38 glucuronidation could be classified into two types: low-Km types such as UGT1A1 and UGT1A9, and high-Km types such as UGT1A3 and UGT1A6, in terms of affinity toward substrate. UGT1A1 had the highest Vmax followed by UGT1A3. Vmax of UGT1A6 and UGT1A9 were approximately 1/9 to 1/12 of that of UGT1A1. 4. The activity of SN-38 glucuronidation by liver microsomes and UGT1A1 was effectively inhibited by bilirubin. Planar and bulky phenols substantially inhibited the SN-38 glucuronidation activity of liver microsomes and UMT1A9, and/or UGT1A6. Although cholic acid derivatives strongly inhibited the activity of SN-38 glucuronidation by UGT1A3, the inhibition profile did not parallel that in liver microsomes. 5. These results demonstrate that at least four UGT1A isoforms are responsible for SN-38 glucuronidation in human livers, and suggest that the role and contribution of each differ substantially.

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ABSTRACT:Cytochrome P450 (P450) 2B6 is a hepatic enzyme of potential importance for the metabolism of clinically used drugs and environmental or abused toxicants. Genetic polymorphisms of CYP2B6 (CYP2B6*2, CYP2B6*3, CYP2B6*4, CYP2B6*5, CYP2B6*6 and CYP2B6*7; wild-type, CYP2B6*1) were found previously in white and Japanese populations. In the present study, the goal was to investigate the effects of amino acid substitutions on CYP2B6 function. Wild-type (CYP2B6.1) and all of the known variants of CYP2B6 (CYP2B6.2, CYP2B6.3, CYP2B6.4, CYP2B6.5, CYP2B6.6, and CYP2B6.7) were transiently expressed in COS-1 cells, and their 7-ethoxy-4-trifluoromethylcoumarin O-deethylation activities were determined. The levels of the variant CYP2B6 proteins were relatively low compared with that of CYP2B6.1, although the differences were not significant. The activities of 7-ethoxy-4-trifluoromethylcoumarin O-deethylation on the basis of the CYP2B6 protein level at low (0.5 M) and high (50 M) substrate concentrations varied among wild-type and variant CYP2B6 proteins. All CYP2B6 enzymes showed typical Michaelis-Menten kinetics. The K m value of CYP2B6.6 was significantly higher than that of CYP2B6.1. Those CYP2B6 variants having a Lys262Arg substitution (CYP2B6.4, CYP2B6.6, and CYP2B6.7) showed increased values for V max and V max /K m , whereas the kinetic parameters of CYP2B6.2 and CYP2B6.3 were not affected by the corresponding amino acid substitution. These results may mean that Lys262 in combination with other amino acid residues such as Gln172 and Arg487 is associated with the CYP2B6 function and that the genetic polymorphism of CYP2B6 leads to interindividual differences in xenobiotic metabolism.

The cytochrome P450 gene superfamily consists of a group of hemoproteins that catalyze the oxidative metabolism of a wide variety of exogenous chemicals such as drugs, steroids, carcinogens, toxins and endogenous compounds like steroids and fatty acids. The CYP2C8 gene is assigned to chromosome 10q24.1 1) and composed of 9 exons.2) This form of P450 is expressed in liver and many extrahepatic tissues including kidney, adrenal gland, brain, uterus and so on.2) CYP2C8 plays important roles in metabolizing therapeutic drugs: an anticancer drug paclitaxel, 3,4) an HMG-CoA reductase inhibitor cerivastatin, 5) an antiepileptic drug carbamazepine, 6) an antidiabetes drug troglitazone, 7) and an antiarrhythmic drug amiodarone. 8) CYP2C8 is also implicated in the oxidation of retinoids and fatty acids including all-transretinoic acid and arachidonic acid. 9,10) Information on single nucleotide polymorphisms (SNPs), gene duplications and splice variants of P450 genes has recently accumulated, and some SNPs have been associated with reduced or augmented metabolic activity. 11,12) As for CYP2C8, the rates of CYP2C8-catalyzed paclitaxel 6a-hydroxylation, and rosiglitazone N-demethylation and p-hydroxylation have been reported to differ up to 38-fold in microsomes from panels of human livers. 4,13) However, little information on CYP2C8 polymorphisms is available, especially for the Japanese population. In the present study, we detected 9 SNPs in the CYP2C8 gene using 73 established cell lines derived from different Japanese individuals. For 3 coding SNPs which gave amino acid alterations, we assessed their metabolic activities with paclitaxel as a substrate using microsomal fractions from Hep G2 cells transfected with wild-type or variant CYP2C8 cDNAs. MATERIALS AND METHODSReagents Paclitaxel, 6a-hydroxy paclitaxel, and human CYP2C8-expressed insect microsomes were obtained from DNA Extraction Established cell lines derived from the 73 Japanese individuals were obtained from the Health Science Research Resources Bank (Osaka, Japan) or directly from the Japanese Collection of Research Bioresources, National Institute of Health Sciences (Tokyo, Japan). The cells were cultured according to the banks' instructions. DNA extraction was carried out from approximately 5ϫ10 7 cells using a Blood and Cell Culture DNA Kit (Qiagen, Hilden, Germany). The concentrations of the obtained DNA were determined by UV absorbance, and the DNA solutions were stored in 4°C until sequence analysis.Sequencing of CYP2C8 Exons DNA fragments were amplified from genomic DNA with the appropriate set of CYP2C8-specific primers shown in Table 1 designed in intronic regions based on a genomic contig sequence (NT 008878). Then the PCR products purified using a PCR Product Pre-Sequencing Kit (USB Co., Cleveland, OH, U.S.A.) were directly sequenced on both strands using an ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, U.S.A.). The excess dye was removed by a DyeEx96 plate (Qiagen). The elutants were analyzed on an ABI Prism 370...