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Tsuyoshi Yokoi

Publications and source records attributed to Tsuyoshi Yokoi.

71 records · Page 4Linked to original sources
PubMed2002

Identification of catalase in human livers as a factor that enhances phenytoin dihydroxy metabolite formation by human liver microsomes.

We have reported previously that the formation of a 3',4'-dihydroxylated metabolite of phenytoin (3',4'-diHPPH) by human liver microsomal cytochrome P450 (P450) is enhanced by the addition of human liver cytosol [Komatsu et al., Drug Metab Dispos 2000;28:1361-8]. The enhancing factor was determined in this study. The addition of cytosolic proteins precipitated by 50% ammonium sulfate to incubation mixtures increased the rate of microsomal 3',4'-diHPPH formation. This fraction was separated further by diethylaminoethyl-, carboxymethyl-, and hydroxyapatite-column chromatography. The amino acid sequence of the purified protein of approximately 55kDa by electrophoresis revealed this protein to be a catalase. The addition of purified or authentic catalase to the incubation mixtures increased the rates of microsomal 3',4'-diHPPH formation from 3'- and 4'-hydroxylated metabolites and from phenytoin in a concentration-dependent manner. In reconstituted systems containing CYP2C9, CYP2C19, and CYP3A4, the formation of 3',4'-diHPPH was also enhanced by catalase to different extents. This is the first report that catalase in livers enhances drug oxidation activities catalyzed by P450 in human liver microsomes.

Catalase↗
PubMed2002

CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability.

Comparing bufuralol 1'-hydroxylase activity among liver microsomes prepared from individuals whose CYP2D6 genotypes had been determined, we found that the activity tended to decrease depending on the number of the CYP2D6*10 allele. Pre-incubation of liver microsomes from individuals homozygous for the CYP2D6*10 allele resulted in a decrease in the enzyme activity more rapidly than those from individuals homozygous for the CYP2D6*1, suggesting that not only the catalytic activity but also the thermal stability of the enzyme appeared to be affected by the genetic polymorphism. To confirm this hypothesis, the kinetic parameters of CYP2D6.1 and CYP2D6.10 were compared for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation using microsomes prepared from yeast transformed with plasmids carrying CYP2D6 cDNAs (*1A and *10B). Kinetic studies of these CYP2D6 forms indicated clear differences in the metabolic activities between the wild (CYP2D6.1) and the mutant enzymes (CYP2D6.10). Bufuralol 1(')-hydroxylase activity in microsomes of yeast expressing CYP2D6.10 was rapidly decreased by heat treatment, supporting the idea that the thermal stability of the enzyme was reduced by amino acid replacement from Pro (CYP2D6.1) to Ser (CYP2D6.10). These data strongly suggest that the thermal instability together with the reduced intrinsic clearance of CYP2D6.10 is one of the causes responsible for the known fact that Orientals show lower metabolic activities than Caucasians for drugs metabolized mainly by CYP2D6, because of a high frequency of CYP2D6*10 in Orientals.

Alleles↗
PubMed2002

Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences.

Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[ a]pyrene and 7-nitrobenz[ a]anthracene were weaker than those of their parents benzo[ a]pyrene and benz[ a]anthracene, respectively. This study demonstrated that NPAHs as well as PAHs induced human CYP1A1, CYP1A2, and CYP1B1 in a chemical-, CYP isoform-, and cell-specific manner. Furthermore, the cell-specific induction of the CYP1 family was not related to the expression levels of aryl hydrocarbon receptor, aryl hydrocarbon nuclear translocator, or estrogen receptors alpha and beta.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes.

A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.

Chromatography, High Pressure Liquid↗
PubMed2002

Evaluation of approach to predict the contribution of multiple cytochrome P450s in drug metabolism using relative activity factor: effects of the differences in expression levels of NADPH-cytochrome P450 reductase and cytochrome b(5) in the expression system and the differences in the marker activities.

The concept of relative activity factor (RAF) to extrapolate data obtained with recombinant cytochrome P450(CYP)s to human liver microsomes has been proposed. To evaluate the approach to predict the contribution of multiple CYPs using RAF, we investigated the effects of the differences in the expression levels of NADPH-cytochrome P450 reductase (OR) and cytochrome b(5) (b(5)) in recombinant CYPs from baculovirus-infected insect cells and the differences in the marker activities. Because we previously clarified that azelastine, an antiallergy and antiasthmatic drug, is N-demethylated by CYP1A2, CYP2D6, and CYP3A4 in humans, the reaction was used as a model. For calculation of RAF, three lots of recombinant CYP1A2, CYP2D6, and CYP3A4 from baculovirus-infected insect cells with different expression levels of OR and b(5) were used. The OR/CYP ratios for recombinant CYP1A2, CYP2D6, and CYP3A4 were 3.9-4.8, 5.1-8.7, and 8.0-11.3, respectively. The b(5)/CYP ratio for recombinant CYP3A4 was 2.1-18.7. As marker activities, ethoxyresorufin O-deethylation and phenacetin O-deethylation for CYP1A2, bufuralol 1'-hydroxylation and debrisoquin 4-hydroxylation for CYP2D6, testosterone 6beta-hydroxylation and midazolam 1'-hydroxylation for CYP3A4 were compared. Our results indicated that the differences in the expression levels of OR and b(5) coexpressed in baculovirus-infected insect cells would not be a critical factor for the quantitative prediction using RAF. In addition, we confirmed that differences in the marker activities did not significantly affect the calculation of RAF values, when the marker activities are specific for a certain CYP isoform. It was suggested that the RAF approach using recombinant CYPs from baculovirus-infected insect cells coexpressing OR (and b(5) if required) could be valuable for the prediction of the contribution of each CYP in drug metabolism.

Animals↗
PubMed2002

Roles of NADPH-P450 reductase and apo- and holo-cytochrome b5 on xenobiotic oxidations catalyzed by 12 recombinant human cytochrome P450s expressed in membranes of Escherichia coli.

Drug oxidation activities of 12 recombinant human cytochrome P450s (P450) coexpressed with human NADPH-P450 reductase (NPR) in bacterial membranes (P450/NPR membranes) were determined and compared with those of other recombinant systems and those of human liver microsomes. Addition of exogenous membrane-bound NPR to the P450/NPR membranes enhanced the catalytic activities of CYP2C8, CYP2C9, CYP2C19, CYP3A4, and CYP3A5. Enhancement of activities of CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, and CYP2E1 in membranes was not observed after the addition of NPR (4 molar excess to each P450). Exogenous purified human cytochrome b5 (b5) further enhanced catalytic activities of CYP2A6, CYP2B6, CYP2C8, CYP2E1, CYP3A4, and CYP3A5/NPR membranes. Catalytic activities of CYP2C9 and CYP2C19 were enhanced by addition of b5 in reconstituted systems but not in the P450/NPR membranes. Apo b5 (devoid of heme) enhanced catalytic activities when added to both membrane and reconstituted systems, except for CYP2E1/NPR membranes and the reconstituted system containing purified CYP2E1 and NPR. Catalytic activities in P450/NPR membranes fortified with b5 were roughly similar to those measured with microsomes of insect cells coexpressing P450 with NPR (and b5) and/or human liver microsomes, based on equivalent P450 contents. These results suggest that interactions of P450 and NPR coexpressed in membranes or mixed in reconstituted systems appear to be different in some human CYP2 family enzymes, possibly due to a conformational role of b5. P450/NPR membrane systems containing b5 are useful models for prediction of the rates for liver microsomal P450-dependent drug oxidations.

Cell Membrane↗
PubMed2002

Genetic polymorphisms in human CYP2A6 gene causing impaired nicotine metabolism.

AIMS: Previously, we determined the phenotyping of in vivo nicotine metabolism and the genotyping of the CYP2A6 gene (CYP2A6*1 A, CYP2A6*1B, CYP2A6*2, CYP2A6*3, CYP2A6*4 and CYP2A6*5 ) in 92 Japanese and 209 Koreans. In the study, we found one Korean and four Japanese subjects genotyped as CYP2A6*1B/CYP2A6*4 who revealed impaired nicotine metabolism, although other many heterozygotes of CYP2A6*4 demonstrated normal nicotine metabolism (CYP2A6*4 is a whole deletion type). After our previous report, several CYP2A6 alleles, CYP2A6*6 (R128Q), CYP2A6*7 (I471T), and CYP2A6*8 (R485L), have been reported. The purpose of the present study was to clarify whether the impaired nicotine metabolism can be ascribed to these CYP2A6 alleles. Furthermore, we also determined whether the subjects possessing CYP2A6*1x2 (duplication) reveal higher nicotine metabolism. METHODS: Genotyping of CYP2A6 alleles, CYP2A6*6, CYP2A6*7, CYP2A6*8, and CYP2A6*1x2 was determined by PCR. RESULTS: The five poor metabolizers were re-genotyped as CYP2A6*7/CYP2A6*4, suggesting that a single nucleotide polymorphism (SNP) causing I471T decreases nicotine metabolism in vivo. Furthermore, we found that two subjects out of five with a lower potency of nicotine metabolism possessed SNPs of CYP2A6*7 and CYP2A6*8 simultaneously. The novel allele was termed CYP2A6*10. In the 92 Japanese and 209 Koreans, the CYP2A6*6 allele was not found. The allele frequencies of CYP2A6*7, CYP2A6*8, and CYP2A6*10 were 6.5%, 2.2%, and 1.1%, respectively, in Japanese, and 3.6%, 1.4%, and 0.5%, respectively, in Koreans. The CYP2A6*1x2 allele was found in only one Korean subject (0.5%) whose nicotine metabolic potency was not very high. CONCLUSIONS: It was clarified that the impaired in vivo nicotine metabolism was caused by CYP2A6*7 and CYP2A6*10 alleles.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

Interindividual differences in nicotine metabolism and genetic polymorphisms of human CYP2A6.

Nicotine is widely consumed throughout the world, and exerts a number of physiological effects. After nicotine is absorbed through the lungs by cigarette smoking, it undergoes extensive metabolism in humans. Nicotine is mainly metabolized to cotinine by cytochrome P450 (CYP) 2A6. CYP2A6 can metabolize some pharmaceutical agents such as halothane, valproic acid, and fadrozole, and activate tobacco-specific nitrosamines. There are large interindividual differences in nicotine metabolism, and it has been found that the interindividual differences are attributed to the genetic polymorphisms of CYP2A6 gene. This review describes the techniques for determination of in vivo nicotine metabolism, characteristics of each human CYP2A6 alleles, and ethnic differences. The relationship between CYP2A6 genetic polymorphism and potency of nicotine metabolism, smoking behavior, and cancer risk are extensively reviewed. Finally, the usefulness of nicotine metabolism for phenotyping of CYP2A6 in individuals and implication of the significance of CYP2A6 genetic polymorphism in a clinical perspective are discussed.

Animals↗
PubMed2002

Involvement of multiple UDP-glucuronosyltransferase 1A isoforms in glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin in human liver microsomes.

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide. Phenytoin is oxidized to 4'-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4'-HPPH is metabolized to 4'-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4'-HPPH O-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4'-HPPH, and UDP-glucuronic acid was identified as 4'-HPPH O-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4'-HPPH O-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 microM of 4'-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4'-HPPH O-glucuronide. The 4'-HPPH O-glucuronidation in pooled human liver microsomes was inhibited by beta-estradiol as a typical substrate for UGT1A1 (IC(50) = 21.1 microM) and imipramine as a typical substrate for UGT1A4 (IC(50) = 57.7 microM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC(50) = 167.1 microM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC(50) = 287.6 microM) were not prominent. The interindividual difference in the 4'-HPPH O-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4'-HPPH O-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4'-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.

Biotransformation↗
PubMed2002

Troglitazone glucuronidation in human liver and intestine microsomes: high catalytic activity of UGT1A8 and UGT1A10.

Troglitazone glucuronidation in human liver and intestine microsomes and recombinant UDP-glucuronosyltransferases (UGTs) were thoroughly characterized. All recombinant UGT isoforms in baculovirus-infected insect cells (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) exhibited troglitazone glucuronosyltransferase activity. Especially UGT1A8 and UGT1A10, which are expressed in extrahepatic tissues such as stomach, intestine, and colon, showed high catalytic activity, followed by UGT1A1 and UGT1A9. The kinetics of the troglitazone glucuronidation in the recombinant UGT1A10 and UGT1A1 exhibited an atypical pattern of substrate inhibition when the substrate concentration was over 200 micro M. With a Michaelis-Menten equation at 6 to 200 micro M troglitazone, the K(m) value was 11.1 +/- 5.8 micro M and the V(max) value was 33.6 +/- 3.7 pmol/min/mg protein in recombinant UGT1A10. In recombinant UGT1A1, the K(m) value was 58.3 +/- 29.2 micro M and the V(max) value was 12.3 +/- 2.5 pmol/min/mg protein. The kinetics of the troglitazone glucuronidation in human liver and jejunum microsomes also exhibited an atypical pattern. The K(m) value was 13.5 +/- 2.0 micro M and the V(max) value was 34.8 +/- 1.2 pmol/min/mg for troglitazone glucuronidation in human liver microsomes, and the K(m) value was 8.1 +/- 0.3 micro M and the V(max) was 700.9 +/- 4.3 pmol/min/mg protein in human jejunum microsomes. When the intrinsic clearance was estimated with the in vitro kinetic parameter, microsomal protein content, and weight of tissue, troglitazone glucuronidation in human intestine was 3-fold higher than that in human livers. Interindividual differences in the troglitazone glucuronosyltransferase activity in liver microsomes from 13 humans were at most 2.2-fold. The troglitazone glucuronosyltransferase activity was significantly (r = 0.579, p < 0.05) correlated with the beta-estradiol 3-glucuronosyltransferase activity, which is mainly catalyzed by UGT1A1. The troglitazone glucuronosyltransferase activity in pooled human liver microsomes was strongly inhibited by bilirubin (IC(50) = 1.9 micro M), a typical substrate of UGT1A1. These results suggested that the troglitazone glucuronidation in human liver would be mainly catalyzed by UGT1A1. Interindividual differences in the troglitazone glucuronosyltransferase activity in S-9 samples from five human intestines was 8.2-fold. The troglitazone glucuronosyltransferase activity in human jejunum microsomes was strongly inhibited by emodin (IC(50) = 15.6 micro M), a typical substrate of UGT1A8 and UGT1A10, rather than by bilirubin (IC(50) = 154.0 micro M). Therefore, it is suggested that the troglitazone glucuronidation in human intestine might be mainly catalyzed by UGT1A8 and UGT1A10.

Catalysis↗
PubMed2002

Characterization of nicotine and cotinine N-glucuronidations in human liver microsomes.

The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K(m) and V(max) values were 33.1 +/- 28.1 micro M and 60.0 +/- 21.0 pmol/min/mg and 284.7 +/- 122.0 micro M and 124.0 +/- 44.0 pmol/min/mg for the high- and low-affinity components, respectively, in human liver microsomes (n = 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K(m) = 1.9 +/- 0.3 mM, V(max) = 655.6 +/- 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K(i) = 3.9 and 3.3 micro M), imipramine as a substrate for UGT1A4 (K(i) = 6.1 and 2.7 micro M), and propofol as a substrate for UGT1A9 (K(i) = 6.0 and 12.0 micro M). The nicotine N-glucuronidation (50 micro M nicotine) in 14 human liver microsomes was significantly (r = 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r = 0.425) or cotinine N-glucuronidation (r = 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.

Cotinine↗
PubMed2002

Formation of a novel quinone epoxide metabolite of troglitazone with cytotoxicity to HepG2 cells.

Troglitazone, an oral antidiabetic drug, was reported to cause adverse hepatic effects in certain individuals, leading to its withdrawal from the market. After incubation of troglitazone (100 microM) with the human hepatoma cell line, HepG2 cells, and human primary hepatocytes for 48 to 72 h, an unknown peak was detected in the cell culture. The formation of this peak from troglitazone (100 microM) was also catalyzed by expressed CYP3A4, and further HPLC analysis revealed that there were three metabolites (metabolite A, B, and C) in the peak. The major metabolite, metabolite C (M-C) was identified as an epoxide of a quinone metabolite of troglitazone by comparison with a synthetic authentic standard using tandem mass spectrometry, (1)H NMR, and (13)C NMR analyses. The other two metabolites (M-A and M-B) were stereoisomers with the same molecular weight as M-C, probably produced from M-C by intramolecular rearrangement at the epoxide moiety. M-C showed a weak cytotoxicity in HepG2 cells at low concentrations, as assessed by the crystal violet-staining assay. Since epoxides are generally regarded as the chemically reactive species, M-C may play a role in idiosyncrasy of troglitazone hepatotoxicity via individual differences either in the formation or degradation of this metabolite.

Antineoplastic Agents↗
PubMed2002

Imipramine N-glucuronidation in human liver microsomes: biphasic kinetics and characterization of UDP-glucuronosyltransferase isoforms.

A method for the direct determination of imipramine N-glucuronidation in human liver microsomes by high-performance liquid chromatography with UV detection was developed. Imipramine was incubated with human liver microsomes and UDP-glucuronic acid. The Eadie-Hofstee plots of imipramine N-glucuronidation in human liver microsomes were biphasic. For the high-affinity component, the K(m) was 97.2 +/- 39.4 microM and the V(max) was 0.29 +/- 0.03 nmol/min/mg of protein. For the low-affinity component, the K(m) was 0.70 +/- 0.29 mM and the V(max) was 0.90 +/- 0.28 nmol/min/mg of protein. The imipramine N-glucuronosyltransferase activities were not detectable in two samples of human jejunum microsomes. Among recombinant UDP-glucuronosyltransferases (UGTs) in baculovirus-infected insect cells (Supersomes or Bacurosomes) or human B-lymphoblastoid cells tested in the present study (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15), only UGT1A4 showed imipramine N-glucuronosyltransferase activity. The activity in UGT1A4 Supersomes was higher than that in recombinant UGT1A4 expressed in human B-lymphoblastoid cells at all imipramine concentration tested. The kinetics of imipramine N-glucuronidation in UGT1A4 Supersomes did not fit the Michaelis-Menten plot, showing a K(m) of >1 mM. In contrast, in UGT1A4 expressed in human B-lymphoblastoid cells, K(m) was 0.71 +/- 0.36 mM and the V(max) was 0.11 +/- 0.03 nmol/min/mg of protein. Interindividual differences in the imipramine N-glucuronidation in liver microsomes from 14 humans were at most 2.5-fold. The imipramine N-glucuronosyltransferase activities in 11 human liver microsomes were significantly (r = 0.817, P < 0.005) correlated with the glucuronosyltransferase activities of trifluoperazine, a typical substrate of UGT1A4. This is the first report of the biphasic kinetics of imipramine N-glucuronide in human liver microsomes.

Animals↗
PubMed2002

Genetic polymorphisms of human organic anion transporters OATP-C (SLC21A6) and OATP-B (SLC21A9): allele frequencies in the Japanese population and functional analysis.

Genetic polymorphisms of human organic anion transporting polypeptides OATP-C (SLC21A6) and OATP-B (SLC21A9) in the Japanese population were analyzed. The allele frequencies of OATP-C*1a, OATP-C*1b (N130D), OATP-C*1c (R152K and D241N), and OATP-C*5 (V174A) were 35.2, 53.7, 0, and 0.7%, respectively, in 267 healthy Japanese subjects. In the OATP-C gene, we found a novel allele called OATP-C*15 possessing two single nucleotide polymorphisms (SNPs), N130D and V174A, simultaneously. The allele frequency of OATP-C*15 was 3.0%. The allele frequencies of OATP-B*1, OATP-B*2 (T392I), and OATP-B*3 (S486F) were 69.1, 0, and 30.9%, respectively. For functional analysis, each OATP-C and OATP-B allele was expressed in human embryonic kidney (HEK293) cells, and the kinetics of uptake of [(3)H]estrone-3-sulfate was determined. In the case of OATP-C alleles, no significant alteration in K(m) or V(max) values of [(3)H]estrone-3-sulfate uptake was observed, even when the V(max) values were corrected for the expression levels of OATP-C protein. In contrast, V(max), corrected with the expression level of OATP-B*3, was decreased to 42.5% of OATP-B*1, whereas the K(m) values were comparable. Since the frequency of the OATP-B*3 allele was high (30.9%) in our subjects, the SNP of S486F may affect the physiological function and/or pharmacological effects of OATP-B substrates in vivo.

Amino Acid Substitution↗
PubMed2002

A mutation in the flavin-containing monooxygenase 3 gene and its effects on catalytic activity for N-oxidation of trimethylamine in vitro.

To clarify the mutation of the flavin-containing monooxygenase (FMO) 3 gene causing fish-odor syndrome, we analyzed the FMO3 gene of a Thai subject who possibly suffered from fish-odor syndrome. A novel mutation, a single-base substitution from G to A at the position of 265 (G265A), was identified in exon 3. The mutation caused an amino acid substitution from valine to isoleucine at residue 58 (V58I). The mutated FMO3 protein with V58I exhibited the reduced trimethylamine N-oxidase activity when it was expressed in E. coli. The V(max)/K(m) value for the activity of the mutant-type FMO3 was about 5 times lower than that for the wild-type FMO3.

Journal Article↗
PubMed2002

Activities of cytochrome p450 enzymes in liver and kidney microsomes from systemic carnitine deficiency mice with a gene mutation of carnitine/organic cation transporter.

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.

Journal Article↗