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European studies have revealed that the ABCB11 c.1331T>C (V444A) polymorphism (rs2287622) C-allele frequency is higher among patients with drug-induced cholestasis. Given the low incidence of this disease, however, this association has not been sufficiently elucidated. We aimed to investigate the significance of this polymorphism in Japanese patients. We determined ABCB11 V444A polymorphism frequencies and HLA genotypes in two independent drug-induced cholestasis cohorts. Expression and taurocholate transport activity of proteins from 444A variants were analyzed using Madin-Darby canine kidney II cells. In cohort 1 (n = 40), the V444A polymorphism C-allele frequency (66%) was lower than that in controls (n = 190, 78%), but this difference was not significant (P = 0.09). In cohort 2 (n = 119), comprising patients with cholestatic (n = 19), hepatocellular (n = 74), and mixed (n = 26) liver injuries, the C-allele frequency was lower among patients with cholestatic liver injury (68%) than among those with hepatocellular (75%) or mixed liver injury (83%), although this difference was not significant. In cohort 1, HLA-A*0201 was observed more frequently in patients (22%) than in controls [11%; P = 0.003; odds ratio, 2.4 (95% confidence interval, 1.4-4.0)]. Taurocholate transport activity of 444A-encoded protein was significantly lower than that of 444V-encoded protein (81% of 444V, P < 0.05) because of the reduced protein stability. In conclusion, ABCB11 444A had slightly reduced transport activity, but it did not contribute to the occurrence of drug-induced cholestasis in Japanese patients. Therefore, genetic susceptibility to acquired cholestasis may differ considerably by ethnicity.

CYP2C9 is a polymorphic enzyme that metabolizes a number of clinically important drugs. In this study, catalytic activities of seven alleles found in Japanese individuals, CYP2C9*3 (I359L), *13 (L90P), *26 (T130R), *28 (Q214L), *30 (A477T), *33 (R132Q), and *34 (R335Q), were assessed using three substrates (diclofenac, losartan, and glimepiride). When expressed in a baculovirus-insect cell system, the holo and total (apo and holo) CYP2C9 protein expression levels were similar among the wild type (CYP2C9.1) and six variants except for CYP2C9.13. A large part of CYP2C9.13 was present in the apo form P420. Compared with CYP2C9.1, all variants except for CYP2C9.34 exhibited substrate-dependent changes in K(m), V(max), and intrinsic clearance (V(max)/K(m)). For diclofenac 4'-hydroxylation, the intrinsic clearance was decreased markedly (by80%) in CYP2C9.13, CYP2C9.30, and CYP2C9.33 and variably (63-76%) in CYP2C9.3, CYP2C9.26, and CYP2C9.28 due to increased K(m) and/or decreased V(max) values. For losartan oxidation, CYP2C9.13 and CYP2C9.28 showed 2.5- and 1.8-fold higher K(m) values, respectively, and all variants except for CYP2C9.34 showed77% lower V(max) and intrinsic clearance values. For glimepiride hydroxylation, the K(m) of CYP2C9.13 was increased 7-fold, and the V(max) values of all variants significantly decreased, resulting in reductions in the intrinsic clearance by80% in CYP2C9.3, CYP2C9.13, CYP2C9.26, and CYP2C9.33 and by 56 to 75% in CYP2C9.28 and CYP2C9.30. These findings suggest the necessity for careful administration of losartan and glimepiride to patients bearing these six alleles.

Human cytochrome P450 1A2 (CYP1A2) catalyzes the metabolism of many important drugs and environmental chemicals. We previously reported three naturally occurring genetic polymorphisms (125CG, Pro42Arg, CYP1A2*15; 1130GA, Arg377Gln, *16; and 1367GA, Arg456His, *8) found in a Japanese population. In this study, these variant enzymes were expressed in Chinese hamster V79 cells, and their mRNA and protein expression levels as well as catalytic activities were determined. All three variant enzymes showed reduced protein expression levels (66% for Pro42Arg and approximately 30% for Arg377Gln and Arg456His) compared with that of the wild type (WT) without any change in mRNA expression levels. Kinetic analysis for 7-ethoxyresorufin O-deethylation revealed that V(max) and V(max)/K(m) of all three variants were less than 3 and 1% of the WT, respectively, although the K(m) value was significantly increased only in the Arg377Gln variant (approximately a 9-fold increase). Markedly reduced activities of the three variants were also observed for phenacetin O-deethylation. In the reduced CO difference spectral analysis using recombinant proteins produced in the Sf21/baculovirus system, the peak at 450 nm seen in the WT protein was hardly observed in the three variants, suggesting marked reductions in their hemoprotein formation. These results suggest that Pro42, Arg377, and Arg456 are critical amino acids for the production of catalytically active CYP1A2 holoenzyme.

Twelve single nucleotide polymorphisms (SNPs) in the human CES2 gene, which encodes a carboxylesterase, hCE-2 [human carboxylesterase 2 (EC 3.1.1.1)], have been reported in the Japanese. In this report, we have examined functional alterations of three SNPs, a nonsynonymous SNP (100C>T, R34W), an SNP at the splice acceptor site in intron 8 (IVS8-2A>G), and one newly discovered nonsynonymous SNP (424G>A, V142M). For the two nonsynonymous SNPs, the corresponding variant cDNAs were expressed in COS-1 cells. Both the R34W and V142M variants showed little esterase activities toward the anticancer agent irinotecan and two typical carboxylesterase substrates, p-nitrophenol acetate and 4-methylumbelliferyl acetate, although increased levels of cDNA-mediated protein expression were observed by Western blotting as compared with the wild type. To investigate a possible splicing aberration in IVS8-2A>G, an in vitro splicing assay was utilized and transcripts derived from CES2 gene fragments of the wild type and IVS8-2A>G were compared. Sequence analysis of the cloned transcripts revealed that IVS8-2A>G yielded mostly aberrantly spliced transcripts, including a deleted exon or a 32-bp deletion proximal to the 5' end of exon 9, which resulted in truncated hCE-2 proteins. These results suggested that 100C>T (R34W), 424G>A (V142M), and IVS8-2A>G are functionally deficient SNPs.

Ethnic differences in genetic polymorphisms in UDP-glucuronosyltransferase 1A1 (UGT1A1) were investigated among African-Americans, Caucasians, and Japanese using samples obtained from 150 individuals for each population. Genotyping of -3279T>G in the phenobarbital-responsive enhancer module, TA repeats in the TATA box, 211G>A (G71R) and 686C>A (P229Q) in exon 1, and three single nucleotide polymorphisms (SNPs) (1813C> T, 1941C>G, and 2042C>G) in the 3'-untranslated region in exon 5 was performed. Eight haplotypes of block 1 (exon 1 and its 5'-flanking region) harboring the first four variations were assigned to each individual. The dominant haplotype for African-Americans was *28b (-3279G;TA(7); 211G;686C) (0.446), whereas that for the Japanese was *1a (-3279T; TA(6);211G;686C) (0.610). Frequencies of the two haplotypes *1a and *28b were comparable in Caucasians. Haplotype *6a (-3279T;TA(6); 211A;686C) was characteristic of the Japanese, whereas haplotypes *36b and *37b (-3279T;TA(5) and TA(8);211G;686C) were found mostly in African-Americans. Although the three SNPs in block 2 (exons 2-5) were in complete linkage in the Japanese, they were not completely linked in African-Americans or Caucasians. These differences in haplotype distribution patterns among the three populations suggest the possibility of ethnic differences in toxicity profiles of drugs detoxicated by UGT1A1. A novel SNP, 686C>T (P229L), was found in an African-American. The intrinsic clearance of 7-ethyl-10-hydroxycamptothecin (SN-38) by P229L UGT1A1 expressed in COS-1 cells was about 3% of the wild type. The results of Western blotting and real-time reverse transcription-polymerase chain reaction suggest that the low glucuronidation activity of the variant was partly due to its low stability. The variation 686C>T may cause high toxicity during 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) therapy or hyperbilirubinemia in patients.

Both UDP-glucuronosyltransferase 2B4 (UGT2B4) and UGT2B7 are expressed mainly in the human liver and have several overlapping substrates; e.g., catechol estrogens, bile acids, codeine, and carvedilol. To identify novel single nucleotide polymorphisms (SNPs) and haplotypes in a Japanese population, the enhancer/promoter regions, all the exons, and the surrounding intronic regions of UGT2B4 and UGT2B7 were sequenced from 136 Japanese individuals. We found 16 and 21 polymorphisms, including 10 and 4 novel ones in UGT2B4 and UGT2B7, respectively. The novel nonsynonymous SNPs were 1364AG (K455R) and 1531TC (C511R) in UGT2B4 and 1192GA (D398N) in UGT2B7. From linkage disequilibrium analysis, several SNPs in UGT2B7 were found to be highly linked with each other. No close linkage between the SNPs in UGT2B4 and UGT2B7 was observed, indicating that each gene is located within an independent haplotype block. Thus, haplotype analysis was separately performed for the two genes. In UGT2B4, we unambiguously determined 8 haplotypes and inferred an additional 12 haplotypes using an expectation-maximization-based program. In UGT2B7, five haplotypes were unambiguously assigned and an additional eight haplotypes were inferred. The haplotype structure of UGT2B7 was more diverse than that of UGT2B4 in terms of the number of frequent SNPs. In addition, ethnic differences in the UGT2B4(*)2 and UGT2B7(*)2 haplotypes between the Japanese and the Caucasian and/or African populations were found. Our findings provide fundamental and useful information for genotyping UGT2B4 and UGT2B7 in the Japanese, and probably other populations.

Carvedilol ((+/-)-1-carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol) is metabolized primarily into glucuronide conjugates. In the present study, we identified the human UDP-glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of carvedilol by thin-layer chromatography using microsomes from human liver or insect cells expressing recombinant UGT isoforms. We observed two forms of carvedilol glucuronides, namely G1 and G2, in hepatic microsomes. The glucuronidation of carvedilol was catalyzed by at least three recombinant UGT isoforms: UGT1A1, UGT2B4, and UGT2B7. UGT2B4 formed both G1 and G2, whereas UGT1A1 and UGT2B7 were responsible for the formation of glucuronide G2 and G1, respectively. The enzyme kinetics for carvedilol glucuronidation by UGT1A1, UGT2B4, and UGT2B7 in addition to human liver microsomes were examined by Lineweaver-Burk analysis. The values of Km and Vmax for human liver microsomes were 26.6 microM and 106 pmol/min/mg protein for G1, and 46.0 microM and 44.5 pmol/min/mg protein for G2, respectively. The Km values for UGT1A1, UGT2B4, and UGT2B7 for G1 and G2 (22.1-55.1 microM) were comparable to those of the liver microsomes, whereas the Vmax values were in the range of 3.33 to 7.88 pmol/min/mg protein. The Km and Vmax/Km values for UGT2B4 and UGT2B7 for G1 were similar, whereas UGT2B4 had lower Km and higher Vmax/Km values for G2 compared with those of UGT1A1. These results suggest that G1 formation is catalyzed by UGT2B4 and UGT2B7, whereas G2 is formed by UGT2B4 and UGT1A1. These three hepatic UGT isoforms may have important roles in carvedilol metabolism.

Metabolism of administered drugs is determined by expression and activity of many drug-metabolizing enzymes, such as the cytochrome P450 (P450s) family members. Pregnane X receptor (PXR) is a master transcriptional regulator of many drug/xenobiotic-metabolizing enzymes, including P450s and drug transporters. In this study, we describe the functional analysis of four naturally occurring human PXR (hPXR) variants (R98C, R148Q, R381W, and I403V) that we have recently identified. By a reporter gene assay using the CYP3A4 promoter/enhancer reporter in COS-7 or HepG2 cells, it was found that the R98C variant failed to transactivate the CYP3A4 reporter. The R381W and I403V variants also showed varying degrees of reduction in transactivation, depending on the dose of PXR activators, rifampicin, clotrimazole, and paclitaxel. The transcriptional activities of the R148Q variant were not significantly different from that of the wild-type hPXR. The electrophoretic mobility shift assay revealed that only the R98C variant lacked DNA binding. Furthermore, the cellular localization of the hPXR proteins was analyzed. All four variants as well as the wild-type hPXR localized exclusively to the nucleus, regardless of the presence or absence of rifampicin. These data suggest that the R98C, R381W, and I403V hPXR variants, especially R98C, may influence the expression of drug-metabolizing enzymes and transporters, which are transactivated by PXR.

We found nucleotide variability in the 5'-upstream region and exonic sequences of a gene-encoding canalicular multispecific organic anion transporter/multidrug resistance-associated protein 2 (cMOAT/MRP2) by polymerase chain reaction-based sequencing using genomic DNA from 72 established cell lines derived from 72 Japanese individuals. Four single nucleotide polymorphisms (SNPs) were found in the 5'-untranslational region and 21 in the exonic regions. Of them, 14 were nonsynonymous SNPs. One deletion of seven consecutive adenines resulting in a frameshift variant was also found. Four SNPs, c-24t, g1249a (V417I), c2366t (S789F), and c3972t (I1324I), were the same as those recently reported. A strong association was found between c-24t (5'-untranslated region) and c3972t (exon 28), with the promoter activity of the former worth being compared.