Metabolic capacities for debrisoquin, sparteine, mephenytoin, nifedipine, and midazolam, which are substrates of polymorphic CYP2D6, CYP2C19, and CYP3A, have been reported to exhibit, in many cases, remarkable interindividual and ethnic differences. These ethnic differences are partly associated with genetic differences. In the case of the drug transporter ABCB1/MDR1, interindividual differences in its transporter activities toward various clinical drugs are also attributed to several ABCB1/MDR1 genetic polymorphisms. In this review, the existence and frequency of various low-activity alleles of drug metabolizing enzymes as well as populational drug metabolic capacities are compared among several different races or ethnicities. Distribution of nonsynonymous ABCB1/MDR1 SNPs and haplotype frequency in various races are summarized, with the association of nonsynonymous SNPs with large functional alterations as a rare event.

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...

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12-and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the ␣6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices ␣8 and ␣9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.

Three non-synonymous single nucleotide polymorphisms (SNPs) in the CYP3A4 gene were found in 34 cell lines derived from Japanese individuals. These three SNPs (T185S, L293P, and T363M)(1) have been previously reported, but little is known about the effect that these polymorphisms, especially T185S, have on catalytic activity. We measured testosterone hydroxylation in wild-type CYP3A4 and these three variants using a mammalian expression system. Testosterone 6beta-, 2beta-, and 15beta-hydroxylations by the variant CYP3A4 forms T363M (<40%) and T185S (<60%) were reduced as compared with the wild-type in transient expression assays. L293P was similar to the wild-type in testosterone 6beta- and 2beta-hydroxylase activities. Western blot analysis confirmed lower amounts of CYP3A4 protein in the T363M and T185S variants than in the wild-type. Interestingly, Northern blot analysis showed no significant difference among mRNA levels between the wild-type and variants. These results suggest that the T363M and T185S substitutions in CYP3A4 affect either protein expression or stability. These established cell lines provided useful CYP3A4 SNP information in the Japanese.

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SummaryWe previously found that thiamine mitigates metabolic disorders in spontaneously hypertensive rats, harboring defects in glucose and fatty acid metabolism. Mutation of thiamine transporter gene SLC19A2 is linked to type 2 diabetes mellitus. The current study extends our hypothesis that thiamine intervention may impact metabolic abnormalities in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, exhibiting obesity and metabolic disorders similar to human metabolic syndrome. Male OLETF rats (4 wk old) were given free access to water containing either 0.2% or 0% of thiamine for 21 and 51 wk. At the end of treatment, blood parameters and cardiac functions were analyzed. After sacrifice, organs weights, histological findings, and hepatic pyruvate dehydrogenase (PDH) activity in the liver were evaluated. Thiamine intervention averted obesity and prevented metabolic disorders in OLETF rats which accompanied mitigation of reduced lipid oxidation and increased hepatic PDH activity. Histological evaluation revealed that thiamine alleviated adipocyte hypertrophy, steatosis in the liver, heart, and skeletal muscle, sinusoidal fibrosis with formation of basement membranes (called pseudocapillarization) which accompanied significantly reduced expression of laminin ␤ 1 and nidogen-1 mRNA, interstitial fibrosis in the heart and kidney, fatty degeneration in the pancreas, thickening of the basement membrane of the vasculature, and glomerulopathy and mononuclear cell infiltration in the kidney. Cardiac and renal functions were preserved in thiamine treatment. Thiamine has a potential to prevent obesity and metabolic disorders in OLETF rats.

RA175, a new member of the immunoglobulin superfamily, is highly expressed during neuronal differentiation of P19 embryonal carcinoma cells. In situ hybridization showed that RA175 mRNA was detected in the developing nervous system, as well as the epithelium of the various non-neuronal tissues of mouse embryo. In contrast with the epithelia of the non-neuronal tissues, RA175 mRNA was not co-expressed with Sonic hedgehog mRNA and Patched mRNA during brain morphogenesis. RA175 mRNA was highly expressed in the anterior horn cells and the peripheral nervous system at embryonic day (E) 11.5 and in the central nervous system at E14.5-E18.5, but its expression decreased after birth and was undetectable in the adult mouse brain.

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.