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Toshiharu Horie

Publications and source records attributed to Toshiharu Horie.

11 recordsLinked to original sources
PubMed2003

Non-steroidal anti-inflammatory drugs affect the methotrexate transport in IEC-6 cells.

Methotrexate (MTX) is used not only for the cancer chemotherapy but also for the treatment of rheumatic disease, often together with non-steroidal anti-inflammatory drugs (NSAIDs). MTX is actively cotransported with H(+) in the small intestine, mediated by a reduced folate carrier (RFC). The coadministration of some NSAIDs with MTX to rats caused a decrease of MTX absorption through the small intestine. This may be due to the uncoupling effect of oxidative phosphorylation of the NSAIDs. The present study investigated whether flufenamic acid, diclofenac and indomethacin, NSAIDs, decreased ATP content of rat-derived intestinal epithelial cell line IEC-6 cells and affected the MTX transport in IEC-6 cells. The MTX uptake in IEC-6 cells was dependent on medium pH and maximum around pH 4.5-5.5. The MTX uptake was composed of a transport inhibited by 4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) and a non-saturable one. The DIDS-sensitive component in the MTX uptake showed a saturation kinetics (Michaelis-Menten constant (Km): 3.91 +/- 0.52 microM, Maximum velocity (Vmax): 94.66 +/- 6.56 pmol/mg protein/5 min). The cellular ATP content in IEC-6 cells decreased significantly at 30 min after the cells were started to incubate with the NSAIDs (250 microM flufenamic acid, 500 microM diclofenac and 500 microM indomethacin). The MTX uptake in IEC-6 cells in the presence of the NSAIDs decreased with the reduction of cellular ATP content and showed a good correlation with the ATP content (correlation coefficient: 0.982). Thus it seems likely that the ATP content in IEC-6 cells with the NSAIDs decreased due to the uncoupling effect of oxidative phosphorylation of the NSAIDs, resulting in the inhibition of the secondary active transport of MTX in IEC-6 cells. The present results also suggest that IEC-6 cells are useful to evaluate the drug interaction relating to this carrier system.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2003

Stimulation of intestinal epithelial restitution by prostaglandin E(1) analogue.

BACKGROUND: 5-Fluorouracil (5-FU) causes intestinal mucosal damage and malabsorption. We have recently reported that coadministration of 17 S,20-dimethyl- trans- lower right triangle (2)-prostaglandin E(1) (OP-1206), a stable synthetic analogue of prostaglandin E(1), with 5-FU to rats protects the small intestine from 5-FU-induced damage. Enterocyte proliferation would contribute to the restitution of the wounded intestinal mucosa. Thus, we investigated the effect of OP-1206 on the proliferation of rat jejunal crypt cells (IEC-6 cells) treated with 5-FU. METHODS: Proliferation of IEC-6 cells was evaluated in terms of [(3)H]-thymidine incorporation and using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Mucosal healing was assessed by measuring the speed of resealing across the denuded area of an IEC-6 cell monolayer. RESULTS: OP-1206 stimulated [(3)H]-thymidine incorporation into subconfluent IEC-6 cells pretreated with 5-FU and increased the number of IEC-6 cells. AH23848B, an EP4 prostaglandin receptor antagonist, blocked the OP-1206-stimulated [(3)H]-thymidine incorporation into IEC-6 cells. The speed of resealing across the denuded area of a wounded IEC-6 cell monolayer was found to increase following treatment with OP-1206. CONCLUSIONS: OP-1206 stimulated the proliferation of IEC-6 cells treated with 5-FU, indicating a possible mechanism for the protective effect of OP-1206 against 5-FU-induced damage to the small intestine. OP-1206 was shown to be active in intestinal mucosal healing.

Alprostadil↗
PubMed2002

Correlation between methotrexate-induced intestinal damage and decrease in polyamine content.

A synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] protects the small intestine from the methotrexate (MTX)-induced damage. The purpose of this study is to evaluate the protective effect of OP-1206 on the methotrexate-induced small intestinal damage in rats from the biochemical point of view. MTX (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days, and on the 6th day biochemical components in the jejunal mucosa of the treated rats were determined. The contents of DNA, RNA, proteins and polyamines (spermine and spermidine) in the jejunal mucosa of rats were markedly decreased by the MTX treatment. The coadministration of OP-1206 with MTX prevented such decreases caused by the MTX treatment. The MTX treatment decreased the incorporation of 3H-thymidine into DNA in the jejunal mucosa, while the coadministration of OP-1206 with MTX prevented it. These results indicated that OP-1206 could protect the intestinal mucosa against the biochemical effects of MTX through a trophic action on intestinal villi. Further, it should be noted that polyamines may possibly play an important role of modulation action on intestinal mucosa.

Administration, Oral↗
PubMed2002

Multidrug resistance-associated protein2 (MRP2) plays an important role in the biliary excretion of glutathione conjugates of 4-hydroxynonenal.

Glutathione (GSH) conjugates of 4-hydroxy-trans-2,3-nonenal (HNE) are the final products of lipid peroxidation. In the present study, the role of multidrug resistance-associated protein 2 (MRP2) in biliary excretion of GSH conjugates of HNE (HNE-SG) was studied in vitro by using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2 and in vivo using a mutant rat strain whose MRP2 expression is defective (Eisai-hyperbilirubinemic rats [EHBR]). A high-performance liquid chromatography method was developed to assay HNE-SG conjugates. Four diastereomeric HNE-SG conjugates could be separated with this method. Three of four HNE-SG conjugates were detectable after incubation of the cell monolayers with HNE. Expression of human MRP2 resulted in a 10-fold increase in HNE-SG conjugates excretion across the apical membrane of MDCK II cells. The four HNE-SG conjugates appeared swiftly in bile from Sprague Dawley rats after intravenous administration of HNE, whereas no detectable HNE-SG conjugates were observed in the bile of EHBR. These results demonstrate the role of MRP2 in the biliary excretion of HNE-SG conjugates.

Aldehydes↗
PubMed2002

A synthetic analog of prostaglandin E(1) prevents the production of reactive oxygen species in the intestinal mucosa of methotrexate-treated rats.

Administration of methotrexate to rats results in severe enterocolitis and death. Previous our studies showed that a synthetic analog of prostaglandin E(1), OP-1206 [17S, 20-dimethyl-trans-Delta(2)-prostaglandin E(1)] ameliorated the anticancer agent-induced enterocolitis of rats. In the current study, we have focused on the biochemical effect of OP-1206 on the methotrexate-induced intestinal inflammation implicating reactive oxygen species (ROS). Methotrexate (15 mg/kg body weight) was orally administered to rats once daily for 5 days. OP-1206 (0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days. On the 6th day, the chemiluminescence from the jejunum was measured to evaluate the generation of ROS. Spontaneous chemiluminescence from the jejunum of the methotrexate-treated rats increased significantly, compared with the control. Luminol-enhanced chemiluminescence from inflamed mucosal scrapings from the jejunum of the methotrexate-treated rats indicated more remarkable enhancement than the control rats. The treatment of OP-1206 with methotrexate showed significantly lower chemiluminescence of both the jejunum and mucosal scrapings than those of the methotrexate-treated rats. The alkaline phosphatase (ALP) activity, as a marker of small intestinal differentiation, in the intestinal mucosa of the methotrexate-treated rats decreased remarkably, but that of the methotrexate and OP-1206-treated rats was significantly higher than that of the methotrexate-treated rats. Thus, OP-1206 may possibly help the anticancer chemotherapy by protecting the small intestine from the methotrexate-induced damage.

Alprostadil↗
PubMed2002

Chemiluminescence associated with the oxidative metabolism of salicylic acid in rat liver microsomes.

Rat liver microsomal suspension (1 mg protein per ml) was incubated at 37 degrees C with 5 mM salicylic acid and 0.2 mM NADPH. The amounts of thiobarbituric acid reactive substances (TBARS) and 2,5-dihydroxybenzoic acid (2,5-DHB), an oxidative metabolite of salicylic acid increased with the incubation time. Simultaneously spontaneous chemiluminescence (CL) was found to be generated there. The addition of SKF-525A, an inhibitor of cytochrome P450 (P450), to the reaction mixture inhibited the CL generation together with the inhibition of the oxidative metabolism. The anti-oxidants and singlet oxygen scavengers like N,N-diphenylphenylenediamine (DPPD) and histidine suppressed the CL generation. The addition of 1,4-diazabicyclo [2.2.2] octane (DABCO), a singlet oxygen quencher, to the reaction mixture generating CL enhanced CL transiently and then CL decreased markedly. Thus CL observed here may possibly originate from the singlet oxygen. The CL generation was suggested to be closely related with salicylic acid-induced lipid peroxidation, and to be coupled with the oxidative metabolism mediated by P450 in rat liver microsomes.

Animals↗
PubMed2002

Multiple mechanisms in indomethacin-induced impairment of hepatic cytochrome P450 enzymes in rats.

BACKGROUND & AIMS: Indomethacin impairs liver microsomal monooxygenase activities mediated by cytochrome P450 (CYP). We investigated the inhibition mechanism and the isoform selectivity in vitro and in vivo. METHODS: In an in vitro study, liver microsomes from male Wistar rats were preincubated with indomethacin and a reduced nicotinamide adenine dinucleotide phosphate-generating system, followed by assay of monooxygenase activities indicative of several CYP isoforms. In an in vivo study, rats were intraperitoneally treated with indomethacin, followed by preparation of microsomes and the enzyme assays. RESULTS: The preincubation of microsomes with indomethacin and reduced nicotinamide adenine dinucleotide phosphate decreased CYP3A2 activity but not any other isoforms. Kinetic analysis showed the mechanism-based inactivation of CYP3A2. The metabolism of [14C]indomethacin resulted in covalent binding to microsomal protein, which was diminished by inhibiting CYP3A enzyme. Administration of indomethacin caused impairment of not only CYP3A2 but also other CYP isoforms. Rats were protected from the impairment of the CYP enzymes except CYP3A2 by depleting macrophages and inhibiting inducible nitric oxide synthase. CONCLUSIONS: Metabolism of indomethacin causes inactivation of CYP3A2, which is the result of the covalent binding of its metabolite, whereas partially selective in vivo impairment of CYP isoforms is suggested to be indirect inhibition by inflammatory mediators probably released from Kupffer cells.

Animals↗
PubMed2002

Role of mitochondrial permeability transition in diclofenac-induced hepatocyte injury in rats.

Hepatotoxicity of diclofenac has been known in experimental animals and humans but its mechanism has not been fully understood. The present study examined the role of mitochondrial permeability transition (MPT) in the pathogenesis of diclofenac-induced hepatocyte injury by using isolated mitochondria and primary culture hepatocytes from rats. Incubation of energized mitochondria with succinate in the presence of Ca(2+) and diclofenac resulted in mitochondrial swelling, leakage of accumulated Ca(2+), membrane depolarization, and oxidation of nicotinamide adenine dinucleotide phosphate and protein thiol. All of these phenomena were suppressed by coincubation of the mitochondria with cyclosporin A, a typical inhibitor of MPT, showing that diclofenac opened the MPT pore. It was also suggested that reactive oxygen species probably generated during mitochondrial respiration and/or voltage-dependent mechanism was involved in MPT, which are proposed as mechanisms of MPT by uncouplers of mitochondrial oxidative phosphorylation. Culture of hepatocytes for 24 hours with diclofenac caused a decrease in cellular ATP, leakage of lactate dehydrogenase and membrane depolarization. The hepatocyte toxicity thus observed was attenuated by coincubation of the hepatocytes with cyclosporin A and verapamil, a Ca(2+) channel blocker. In conclusion, these results showed the important role of MPT in pathogenesis of hepatocyte injury induced by diclofenac and its possible contribution to human idiosyncratic hepatotoxicity.

Animals↗
PubMed2002

Diclofenac-induced inactivation of CYP3A4 and its stimulation by quinidine.

Incubation of human liver microsomes with diclofenac in the presence of NADPH resulted in a decrease in testosterone 6 beta-hydroxylation activity. The decrease in the activity followed time- and concentration-dependent kinetics, required oxidative metabolism, and was resistant to reduced glutathione, suggesting that diclofenac causes a mechanism-based inactivation of cytochrome p450 (p450) 3A4 (CYP3A4). The inactivation was reproduced by using microsomes from B-lymphoblastoid cell lines expressing CYP3A4 instead of human liver microsomes. No other monooxygenase activities measured as indexes of p450 enzymes; CYP2C8, CYP2C9, or CYP2C19 was inactivated by the same incubation procedure. Quinidine, a stimulant of CYP3A4-mediated diclofenac 5-hydroxylation, did not affect the inactivation of CYP3A4 assessed by testosterone 6 beta-hydroxylation activity but accelerated the inactivation assessed by diazepam 3-hydroxylation activity. These results supported the idea that diclofenac 5-hydroxylation is involved in the inactivation of CYP3A4 and described for the first time a stimulation of mechanism-based inactivation attributable to CYP3A4 heterotropic cooperativity. Preincubation of human liver microsomes with 5-hydroxydiclofenac instead of diclofenac did not cause the inactivation of CYP3A4, suggesting that 5-hydroxydiclofenac is not a precursor of a postulated reactive metabolite that inactivates CYP3A4, and thus 5-hydroxylation step is critical to inactivation of CYP3A4.

Cytochrome P-450 CYP3A↗
PubMedDate not supplied

Evaluation of drug-induced hepatotoxicity by plasma retinol binding protein.

Retinol binding protein (RBP) in the plasma of rats treated with D-galactosamine was monitored to establish whether its level can be used to evaluate drug-induced hepatotoxicity. Blood was withdrawn by heart puncture at 0 hours and 12 hours after the administration of D-galactosamine (400 mg/kg body weight i.p.) to rats. Lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) in the plasma at 12 hours after the D-galactosamine administration significantly increased, while RBP in the plasma at that time significantly decreased. On the other hand, the albumin in the plasma was unaffected at 12 hours after the D-galactosamine administration. Thus RBP seems to monitor different aspects of drug-induced hepatotoxicity than LDH and ALT and to detect the drug-induced hepatotoxicity more sensitively than albumin level under the present conditions.

Alanine Transaminase↗