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T Shiraga

Publications and source records attributed to T Shiraga.

35 records · Page 2Linked to original sources
PubMed1994

Isolation and characterization of an alicyclic amine N-sulfotransferase from female rat liver.

An alicyclic amine N-sulfotransferase sulfonating 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP) was purified from female rat liver cytosol and showed a homogenous band with a molecular weight of 30500 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme, designated NST-1, catalyzed sulfonation not only of the alicyclic amine but also dehydroepiandrosterone, a typical substrate of hydroxysteroid sulfotransferase (STs), but had little sulfonating activity towards 2-naphthol, a typical substrate of aryl STs. The N-terminal amino acid sequence for the first 24 residues had a high homology with those of rat liver hydroxysteroid STs. Therefore, it is suggested that NST-1 is classified as a member of the hydroxysteroid ST. Immunoblot analysis of male and female rat liver cytosol, carried out by using rabbit antisera raised against NST-1, indicated that the female cytosol contained a higher level of the enzyme than that of male. The marked sex difference in the expression level of NST-1 was in good accordance with the previous demonstration that female rat liver cytosol catalyzed sulfonation of PTHP to a greater extent than that of male.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1994

Isolation and characterization of four cytochrome P450 isozymes from untreated and phenobarbital-treated beagle dogs.

Four different forms of cytochrome P450 (P450) were purified from liver microsomes of untreated or phenobarbital (PB)-treated male beagle dogs using HPLC techniques, and designated as DUT-1, DPB-1, DPB-2 and DPB-3, respectively. Specific contents of the purified DUT-1, DPB-1, DPB-2 and DPB-3 were 13.3, 9.6, 15.6 and 12.2 nmol/mg protein, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the monomeric molecular weights of DUT-1, DPB-1, DPB-2 and DPB-3 were estimated to be 57.5, 50.0, 47.0 and 50.0 kDa, respectively. The absolute spectra of the oxidized forms indicated that they exist in the low-spin state of heme in their oxidized forms. The NH2-terminal amino acid sequence of DUT-1 was unique and different from that of any other P450 so far reported. DUT-1 was active in the omega-hydroxylation of lauric acid. The amino-terminal sequences of DPB-1, DPB-2 and DPB-3 suggested that they belong to the P450 3A, 2C and 2B gene families, respectively. DPB-3 was a major form of P450 in PB-treated dog liver microsomes. Purified DPB-1 catalyzed nifedipine and (+)- and (-)-nilvadipine oxidations, as well as testosterone 6 beta-hydroxylation in the reconstituted system. These activities were enhanced 3- to 5-fold by the addition of cytochrome b5. DPB-2 and DPB-3 catalyzed aminopyrine N-demethylation, 7-ethoxycoumarin O-deethylation, biphenyl 4-hydroxylation and testosterone 16 alpha-hydroxylation. We believe that DUT-1 is a new form not purified previously.

Amino Acid Sequence↗
PubMed1994

Effect of repeated injection of somatomedin C on drug metabolizing enzyme activities in rats.

The effect of somatomedin C on the hepatic drug-metabolizing enzyme system in rat liver after sc injection, a clinically applicable dosing route, was studied. Although liver weight was slightly increased after somatomedin C (1 mg/kg) was injected for 7 days, no significant changes were observed in other dosing group (0.1 and 10 mg/kg). There were no significant effects of somatomedin C on cytochrome P-450 and b5 contents, and NADPH-cytochrome c reductase, aminopyrine demethylase, aniline hydroxylase, and ethoxyresorufin deethylase. Additionally, somatomedin C treatment did not affect the activities of 2 alpha-, 2 beta-, 6 beta- and 16 alpha-hydroxytestosterone and androstenedione formation from testosterone. Changes relation to dosing period were also examined with 1 mg/kg of somatomedin C. In this case, hepatic levels of cytochrome P-450 and b5, NADPH-cytochrome c reductase and drug oxidations did not differ significantly among groups treated for 3, 7, or 14 days. These results suggest that somatomedin C has no effect on the hepatic mixed-function oxidase system.

Animals↗
PubMed1993

Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the activation of 6-aminochrysene in liver microsomes from phenobarbital-treated rats and from human samples HL-4, HL-13 and HL-18 but not HL-16. In contrast, 1,1,1-trichloropropane-2,3-oxide, an inhibitor of epoxide hydrolase activity, enhanced the activation of 6-aminochrysene catalyzed by liver microsomes from beta-naphthoflavone-treated rats and from human samples HL-16 but not HL-4, HL-13 and HL-18. Inclusion of purified rat epoxide hydrolase to the reconstituted system containing rat and human P4501A enzymes caused a decrease in the rates of 6-aminochrysene activation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Effects of twenty-three drugs on the metabolism of FK506 by human liver microsomes.

We investigated the effects of 23 drugs on the metabolism of FK506 by human liver microsomes. Acyclovir, amphotericin B, cefixime, cefotaxime, ciprofloxacin, cyclosporin A, diltiazem, enoxacin, erythromycin, ethinyl estradiol, fluconazole, fosfomycin, kanamycin, lincomycin, loxoprofen, minocyclin, nifedipine, nilvadipine, norethindrone, ofloxacin, phenobarbital, prednisolone, or rifampicin was added to the reaction media at equimolar or at ten times an excess molar ratio of the substrate concentration; their effects on FK506 metabolism were examined. Drugs known to be the substrate of cytochrome P-450 3A inhibited the metabolism of FK506, and among the drugs tested, the inhibition by cyclosporin A and nifedipine was the strongest.

Anti-Infective Agents↗
PubMed1993

Perinatal development of amine, alcohol and phenol sulfoconjugations in the rat.

Development of the sulfoconjugating activities for amine, alcoholic and phenolic compounds was studied in hepatic 105,000 g supernatants of fetal and newborn rats. All activities in the fetus at the late stage of pregnancy were negligible or very low when compared with those of the adult female level. Amine and alcohol sulfoconjugating activities were low 2 days after birth, increased with age, and attained the adult female level 17 days after birth. In contrast, phenol sulfoconjugating activity was nearly half the level of adult female rats in the neonates 2 days after birth and was relatively constant before maturation. There were no sex-related differences in any of the activities in the immature rats, but in adult animals the activities for amine and alcohol were higher in the females than in the males; the opposite was observed for phenol sulfoconjugation.

Age Factors↗
PubMed1992

Effect of repeated oral doses of a novel immunosuppressive macrolide lactone on hepatic mixed-function oxidase system in the rat. Comparative study with ciclosporin.

Effect of pretreatment of rats with FK506((-)-(1R,9S,12S,13R,14S,17R,18E,21S,23S,24R,25S,27R)-17-allyl-1,14- dihydroxy-12-[(E)-2-[(1R,3R,4R)-4-hydroxy-3methoxycyclohexyl]-1- methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4- azatricyclo-[22.3.1.0(4.9)]octacos-18-ene-2,3,10,16-tetrone hydrate, CAS 104987-11-3) on microsomal cytochrome P-450 system and oxidations of the administered drug and other model substrates were studied and compared with those of a pharmacologically related drug, ciclosporin (cyclosporin A). Oral treatment of male Sprague-Dawley rats with FK506 (0.4, 2 or 10 mg/kg/d) for 7 days did not decrease microsomal content of total cytochrome P-450 in livers, but rather increased the content in groups with the dose of 0.4 or 10 mg/kg to the levels of 126-130% of the control. Microsomal NADPH-cytochrome c reductase activities were decreased up to 67% of the control with the increasing dose of FK506 and to 62% in a group treated orally with cyclosporin A (25 mg/kg/d for 7 days), although another microsomal electron-transport component, cytochrome b5, was rather increased in all the treated groups. Treatment with FK506 or cyclosporin A did not reduce but slightly increased microsomal activities of aniline hydroxylation, p-nitroanisole O-demethylation and O-ethoxyresorufin O-deethylation. Microsomal depropylation of 7-propoxycoumarin, a typical P-450IIIA-substrate, was also not reduced in all dose groups of FK506, while it was decreased by the treatment with 25 mg/kg cyclosporin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

N-sulphoconjugation of alicyclic, alkyl- and aryl-amines in vivo and in vitro.

Radioactive 35S in 3'-phosphoadenosine 5'-phosphosulphate was incorporated into alicyclic, alkyl- and aryl- amines in the presence of hepatic 105 000 g supernatants of female rats. 4-Phenylpiperazine, 4-phenyl-1,2,3,6-tetrahydropyridine (PTHP), 1,2,3,4-tetrahydroisoquinoline, N-methylbenzylamine, desmethylzotepine and desmethylzimeldine showed the highest conjugation with 35SO3 among the amines tested. Incorporation of 35SO3 into alicyclic and alkyl-amines was higher at pH 10.0 than at pH 7.4 but the incorporation into arylamines was the opposite. A greater amount of 35SO3 was incorporated into the secondary alkylamines than the corresponding primary amines. Radioactive reaction products were identified as N-sulphoconjugates of amines by comparison on t.l.c. with synthetic authentic compounds. Reaction products of desmethylimipramine (DMI) and PTHP in vitro were isolated as their sulphoconjugates, identified by comparison of field desorption mass spectra, u.v. spectra, retention time on h.p.l.c. and RF values on t.l.c. with synthetic standards. DMI N-sulphonate and PTHP N-sulphonate were detected in the body of female rats treated orally with DMI and PTHP, respectively. These results indicate that N-sulphoconjugation is a common metabolic pathway of alicyclic, alkyl- and aryl-amines in vivo and in vitro.

Adenine Nucleotides↗
PubMed1986

Age- and sex-related changes in amine sulphoconjugations in Sprague-Dawley strain rats. Comparison with phenol and alcohol sulphoconjugations.

Age- and sex-related changes in sulphoconjugation were investigated in hepatic 105 000 g supernatants of three-week-old (young), seven-week-old (young adult), one-year-old (middle-aged) and two-year-old (old) Sprague-Dawley rats in the presence of biologically active sulphate. The supernatants of seven-week-old, one-year-old and two-year-old female rats catalysed the sulphoconjugations of alcohol (tiaramide as substrate), and alicyclic amine, alkylamine and arylamine more rapidly than those of the respective males, but the supernatants of three-week-old rats did not exhibit significant sex-related differences. The sulphoconjugations by the supernatants of female rats were substantially constant during ageing. On the other hand, the supernatants of seven-week-old male rats catalysed phenol (beta-naphthol as substrate) sulphoconjugation more rapidly than those of seven-week-old females, but the supernatants of two-year-old rats did not exhibit this sex-related difference. These results indicate that the substrates for sulphoconjugation could be divided into at least two groups: an alcohol and amine type, and a phenol type.

Age Factors↗
PubMed1983

Terminal N-sulphoconjugation of an alicyclic amine (piperazine), identified as a new metabolite of tiaramide in mouse and rat.

A new metabolite of tiaramide, 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazineethanol, found in mouse urine was identified as potassium 4-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-1-piperazinesulphonate (M-I). Sex differences in the excretion of M-I were noted in the mouse following oral administration of tiaramide and female mice excreted about 4.6-fold more M-I in urine than males. M-I could not be detected in the urine of male or female rats treated orally with tiaramide. After oral dosing with 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine (DETR), M-I was detected in the urine, and the urinary excretion by male and female rats was similar to that in mice.

Analgesics↗
PubMed1983

Metabolism of tiaramide in vitro. III. Sulphoconjugation of alicyclic amine and alcoholic hydroxyl group by hepatic 105 000 g supernatants.

Sulphoconjugation of the alicyclic secondary amine, DETR (the N-dealkylated metabolite of tiaramide) and of the alcoholic hydroxyl group of tiaramide were observed in liver 105 000 g supernatants of rats and mice but hardly detected in hepatic microsomes. The activities depended on active sulphate, 3'-phosphoadenosine 5'-phosphosulphate, or its generating system. Sulphoconjugation of DETR and tiaramide by the supernatants of female rats and mice proceeded more rapidly than those of males, and sex differences were observed irrespective of sulphate donors, the active sulphate or its generating system. Sulphoconjugation of the alicyclic amine and of the alcohol exhibited different pH optima and different susceptibilities to salts.

Animals↗
PubMedDate not supplied

Isolation, identification, and biological activities of oxidative metabolites of FK506, a potent immunosuppressive macrolide lactone.

To characterize structures and biological activities of FK506 metabolites, FK506 was incubated with liver microsomes prepared from phenobarbital-treated rats in the presence of NADPH generating system under aerobic condition. Oxidative metabolites formed in the reaction medium were isolated and identified. Purified samples were analyzed by HPLC, mass spectrometry, and NMR spectroscopy. M-I, M-II, and M-III were the O-demethylated metabolites at the 13-, 31-, and 15-positions of FK506, respectively, and M-IV was the monohydroxylated metabolite at the 12-position. M-I was the dominant metabolite in this reaction system. M-II and M-III retained the tetrahydropyrane ring in their structures like FK506, but M-I and M-IV had rearranged structures in which the tetrahydropyrane ring was changed to a tetrahydrofuran ring. Measuring the immunosuppressive activity in the mouse mixed lymphocyte reaction system, IC50 values for M-I, M-II, M-III, M-IV, and FK506 were 1.65, 0.23, > 127, 5.52, and 0.15 nM, respectively. Reactivity of the metabolites with mouse anti-FK506 monoclonal antibody was studied and immunocross-reactivity of M-I, M-II, M-III, and M-IV with the antibody were nil, 109.0, 90.5, and 8.8% of FK506, respectively. These results indicate that rat hepatic microsomes oxidatively metabolize FK506 to four metabolites, and some of them exhibit pharmacological activity.

Animals↗