Biomedical subjects

Masuhiro Nishimura

Publications and source records attributed to Masuhiro Nishimura.

31 records · Page 2Linked to original sources

PubMed2005

Tissue-specific mRNA expression profiles of human ATP-binding cassette and solute carrier transporter superfamilies.

Pairs of forward and reverse primers and TaqMan probes specific to each of 46 human ATP-binding cassette (ABC) transporters and 108 human solute carrier (SLC) transporters were prepared. The mRNA expression level of each target transporter was analyzed in total RNA from single and pooled specimens of various human tissues (adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, pancreas, peripheral leukocytes, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus) by real-time reverse transcription PCR using an ABI PRISM 7700 sequence detector system. In contrast to previous methods for analyzing the mRNA expression of single ABC and SLC genes such as Northern blotting, our method allowed us to perform sensitive, semiautomatic, rapid, and complete analysis of ABC and SLC transporters in total RNA samples. Our newly determined expression profiles were then used to study the gene expression in 23 different human tissues, and tissues with high transcriptional activity for human ABC and SLC transporters were identified. These results are expected to be valuable for establishing drug transport-mediated screening systems for new chemical entities in new drug development and for research concerning the clinical diagnosis of disease.

ATP-Binding Cassette Transporters↗

PubMed2005

Role of soybean oil fat emulsion in the prevention of hepatic xenobiotic transporter mRNA up- and down-regulation induced by overdose of fat-free total parenteral nutrition in infant rats.

Total parenteral nutrition (TPN) is associated with cholestasis and hepatic steatosis in human infants. The present study focused on the changes in hepatic xenobiotic transporters associated with overdose of fat-free or fat-containing TPN in infant rats. Three-week-old male Sprague-Dawley rats were divided into three groups: group 1 received an oral diet, group 2 received TPN without fat, and group 3 received TPN with 20% of its calories from fat (soybean oil emulsion). After TPN administration for 4 days, both serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which are indicators of hepatic dysfunction, in group 2 were significantly higher (p<0.001) than those in the other groups, whereas there were no differences between groups 1 and 3 in either serum AST or ALT levels. The serum bilirubin concentration in group 2 was also markedly higher than that in the other groups. Mdr2, Bsep, Mrp2, Mrp6, Oct1, and Oat2 mRNA levels were decreased in group 2 (fat-free TPN) compared with those in group 1 (oral diet), whereas Mdr1b, Mrp1, and Mrp5 mRNA levels were increased. Specifically, the level of Mdr1b mRNA in group 2 was 16 times higher (p<0.001) than that in group 1. On the other hand, the changes in these mRNA expression levels in group 3 (fat-containing TPN) were smaller than those in group 2, and specifically, the expression levels of Mdr1b, Mrp1, Mrp5, Mrp6, and Oat2 mRNA in group 3 were not significantly different from those in group 1. The results of the present study indicate that including fat in the TPN regimen is very important in preventing the mRNA up- and down-regulation of xenobiotic transporters, which is considered to be the main factor responsible for the abnormal hepatic changes such as cholestasis associated with the excessive administration of fat-free TPN.

ATP-Binding Cassette Transporters↗

PubMed2005

Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Recently, the constitutive activation of signal transducer and activator of transcription (STAT) factors has been found in a variety of human cancers. In the present study, the mRNA expression of STATs in various human NSCLC cell lines was investigated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine whether STATs can be implicated in cisplatin resistance and apoptosis inducibility. Cisplatin triggered apoptosis in Ma-46 based on biochemical and morphological findings, but not in Ma-31. The mRNA expression of STAT3 was highest in cisplatin-resistant Ma-31 and lowest in cisplatin-sensitive Ma-46. A 6-hour exposure of cancer cells to cisplatin failed to stimulate STAT3 mRNA expression. Therefore, an increased transcriptional level of constitutive STAT3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemo-resistant NSCLC cells.

Antineoplastic Agents↗

PubMed2004

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution.

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.

Amino Acid Sequence↗

PubMed2004

Cytochrome p450 gene expression levels in peripheral blood mononuclear cells in comparison with the liver.

Cytochromes p450 (CYPs) compose a superfamily of similar proteins involved in detoxification and elimination, as well as activation of a wide variety of compounds. Most CYP family members are localized in the liver. In order to assess whether peripheral blood leukocytes (PBL) are available as a surrogate for the determination of CYP gene expression levels in the liver, we compared CYP gene expression levels in PBL with those in liver tissues from patients with hepatocellular carcinoma (HCC). We measured CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 3A4, 3A5, 3A7, 4A11, 4B1 and CYP27 gene expressions in PBL and in the liver by real-time reverse-transcription (RT)-PCR. We could detect expression of CYP1A1, 1A2, P1B1, 2A6, 2B6 and 2E1 genes in PBL and all the genes except for CYP2F1 in the liver. Although gene expression levels within each subfamily were closely correlated within PBL and within the liver, a clear correlation of gene expression levels between PBL and liver tissues was found only for CYP4B1. Although inter-individual variation of the expression level of each CYP gene was wide, the induced level was proportional to the basal expression level. Therefore, monitoring of CYP gene expression levels in PBL, especially those of CYP4B1, could be available as a biomarker for monitoring of exposure to environmental pollutants and assessing the associated risk. Compared with non-tumor tissue, HCC tissues tended to show overexpression of multiple CYP genes, indicating that individualized selection and more effective administration of chemotherapeutic agents could perhaps be based on the pattern of CYP overexpression.

Adult↗

PubMed2004

Comparative analysis of ATP-binding cassette (ABC) transporter gene expression levels in peripheral blood leukocytes and in liver with hepatocellular carcinoma.

ATP-binding cassette (ABC) transporters comprise a superfamily of similar proteins involved in transmembrane transport of various substances. ABC transporter family members in the liver participate in bile formation and lipid metabolism. In order to assess whether peripheral blood leukocytes (PBL) are available as a surrogate for determination of the expression of ABC transporter genes in the liver, we compared ABC transporter gene expression levels in PBL with those in liver tissues from patients with hepatocellular carcinoma (HCC). We measured ABCA1, A2, B1-B4, C1 degrees C5, G1 and G2 gene expression levels in PBL, and cancerous and non-cancerous portions of liver from patients with hepatocellular carcinoma by means of real time reverse-transcription (RT)-PCR. We could not detect ABCC5 expression in any tissue of the liver. Close correlations between ABCA2, C1 and G1 in PBL and in non-tumor tissues of the liver were found. Compared with the non-tumor part, HCC tissue expressed lower levels of ABCA1, B4 and G2. We think monitoring of ABCA2, C1 and G1 gene expression levels in PBL will be useful for selection of anti-cancer agents and monitoring of drug resistance of HCC. Administration of chemotherapeutic agents which are substrates of ABCA1, B4 and G2 should be effective for the treatment of HCC.

ATP-Binding Cassette Transporters↗

PubMed2004

Effect of lansoprazole and rabeprazole on tacrolimus pharmacokinetics in healthy volunteers with CYP2C19 mutations.

The aim of this study was to investigate the effects of the proton pump inhibitors (PPIs), lansoprazole and rabeprazole, on tacrolimus pharmacokinetics in healthy volunteers with mutations in the cytochrome P450 (CYP) 2C19 gene (CYP2C19). An open-label crossover study was performed with 19 healthy subjects. Tacrolimus (2 mg) was administered orally with and without lansoprazole (30 mg per day for 4 days) or rabeprazole (10 mg per day for 4 days). Blood concentrations of tacrolimus were determined before and 1, 2, 4 and 8 h after dosing. Genotyping for CYP2C19 was conducted by a polymerase chain reaction-restriction fragment length polymorphism method. Coadministration of lansoprazole significantly decreased the oral tacrolimus clearance, resulting in an increase in the area under the blood concentration-time curve (AUC0-8) (control vs with lansoprazole: 29.7 +/- 3.5 vs 44.1 +/- 5.0 ng h mL(-1), P < 0.05). Large individual variation was observed in the effects of lansoprazole on tacrolimus AUC0-8 owing to CYP2C19 genotype status. The percent change for tacrolimus AUC0-8 in subjects with and without CYP2C19 mutant alleles was 81% and 29%, respectively. Coadministration of rabeprazole also increased the mean AUC0-8 of tacrolimus, but the difference was not statistically significant. These observations suggest that drug interaction between tacrolimus and lansoprazole occurs in subjects with higher lansoprazole blood concentrations corresponding to CYP2C19 genetic status. In contrast, rabeprazole has minimal effect on tacrolimus pharmacokinetics regardless of CYP2C19 genotype status.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2004

Tissue-specific mRNA expression profiles of human nuclear receptor subfamilies.

Pairs of forward and reverse primers and TaqMan probes specific to each human nuclear receptor were prepared. Analysis of the mRNA expression level of each target of 43 nuclear receptors in total RNA from single and pooled specimens of various human organs (liver, kidney, adrenal gland, lung, heart, brain, cerebellum, skeletal muscle, spleen, thymus, thyroid gland, prostate, testis, uterus, placenta, bone marrow, trachea, and salivary gland) was performed by real-time reverse transcription PCR using an ABI PRISM 7700 sequence detector system. The mRNA expression of 33 nuclear receptors (NR1A1, 1A2, 1B1, 1B2, 1B3, 1C1, 1C2, 1C3, 1D1, 1D2, 1F1, 1F2, 1F3, 1H2, 1H3, 1I1, 1I2, 2B1, 2B2, 2B3, 2C1, 2C2, 2F1, 2F2, 3A2, 3B1, 3C1, 3C2, 3C4, 4A1, 4A2, 4A3, and 6A1) was successfully detected in all of the tissues by this method. NR1H4, 2A1, and 3C3 mRNAs were not detectable in the heart, heart, and liver, respectively. NR5A2 mRNA was not detectable in either the brain or cerebellum. NR3A1 mRNA was not detectable in the small intestine, colon, brain, and cerebellum. NR5A1 mRNA was not detectable in the kidney, stomach, small intestine, and colon. NR1I3 mRNA was detected in the liver, kidney, stomach, small intestine, adrenal gland, lung, brain, skeletal muscle, thymus, thyroid gland, prostate, testis, placenta, and trachea. NR2A2 mRNA was detected in the liver, kidney, prostate, testis, uterus, and trachea. NR2E1 mRNA was detected in the adrenal gland, brain, cerebellum, testis, placenta, and bone marrow. NR2E3 mRNA was detected in the adrenal gland, thyroid gland, prostate, testis, uterus, trachea, and salivary gland. This study provides information concerning the tissue distribution of the mRNA expression of 43 human nuclear receptors. The mRNA expression profiles of CYP3A4, CYP3A5 and ABC-transporters are also shown. These results are valuable for establishing a nuclear receptor-mediated screening system for new chemical entities in new drug development.

ATP-Binding Cassette Transporters↗

PubMed2004

Effects of NO-1886 (Ibrolipim), a lipoprotein lipase-promoting agent, on gene induction of cytochrome P450s, carboxylesterases, and sulfotransferases in primary cultures of human hepatocytes.

In the present study, the effects on expression of cytochrome P450 (CYP1A1, CYP1A2, CYP3A4 and CYP3A5), carboxylesterase (CES1 and CES2) and sulfotransferase (CHST1, CHST3, CHST4, CST, SULT2A1 and TPST2) mRNA in primary cultures of cryopreserved human hepatocytes were evaluated after exposure to NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl) carbamoyl] benzylphosphonate) for 48 hr at 2, 10, and 50 microM. Analysis was performed by RT-PCR in the presence of TaqMan probe. CYP1A1 and CYP1A2 mRNA levels after exposure to 50 microM omeprazole (positive control for CYP1As) were increased by 162 (p<0.001) and 37 times (p<0.001), respectively, compared with untreated controls. However, these mRNA levels were increased by 2 times or less after exposure to NO-1886. CYP3A4 and CYP3A5 mRNA levels after exposure to 50 microM rifampicin (positive control for CYP3As) were significantly increased by 5.8 (p<0.01) and 2.0 times (p<0.01), respectively, compared with untreated controls. The CYP3A4 mRNA level after exposure to 10 microM NO-1886 was increased by 1.3 times (p<0.05). Further, the CYP3A4 mRNA level after exposure to 50 microM NO-1886 was significantly increased by 3.6 times (p<0.001). However, the CYP3A5 mRNA level after exposure to 50 microM NO-1886 was not significantly increased. CES1 and CES2 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 1.4 (p<0.05) and 2.6 times (p<0.01), respectively, compared with untreated controls. CHST1, CST and SULT2A1 mRNA levels after exposure to 50 microM NO-1886 were significantly increased by 3.8 (p<0.001), 1.8 (p<0.01) and 4.4 times (p<0.01), respectively. CHST3, CHST4 and TPST2 mRNA levels after exposure to 50 microM NO-1886 were not significantly increased. This in vitro technique using primary cultured human hepatocytes is expected to be very useful for the preclinical evaluation of the induction of drug-metabolizing enzymes in humans.

Benzamides↗

PubMed2003

Effects of dimethyl sulfoxide on the gene induction of cytochrome P450 isoforms, UGT-dependent glucuronosyl transferase isoforms, and ABCB1 in primary culture of human hepatocytes.

We investigated the gene expression of GAPDH, cytochrome P450 isoforms (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7), UGT-dependent glucuronosyl transferase isoforms (UGT1A6 and UGT1A9), and ABC transporter (ABCB1) after exposure to 0.1, 0.5, and 2.5% dimethyl sulfoxide (DMSO). GAPDH mRNA levels after exposure were less than 2 times higher or lower than control levels at all DMSO concentrations. CYP1A1, CYP1B1, CYP2A6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, and UGT1A9 mRNA levels were less than 2 times higher or lower than control levels at 0.1% and 0.5% DMSO. CYP1A2, UGT1A6, and ABCB1 mRNA levels were less than 2 times higher or lower than control levels at all DMSO concentrations. CYP2E1 and CYP3A4 mRNA levels were 2.5-3.2 times and 2.0-3.3 times higher than control levels at 2.5% DMSO, respectively. UGT1A9 mRNA levels were 0.2-0.5 times lower than control levels at 2.5% DMSO. Exposure to beta-naphthoflavone reduced CYP1A1 mRNA levels in a concentration-dependent manner for donors 1 and 2. Exposure to beta-naphthoflavone reduced CYP1A2 mRNA levels in a concentration-dependent manner for all donors. However, exposure to beta-naphthoflavone increased CYP1B1 mRNA levels for donors 2 and 3 at 2.5% DMSO. Exposure to rifampicin reduced CYP3A4 mRNA levels for all donors at 0.5% and 2.5% DMSO. Exposure to rifampicin also reduced CYP3A5 and CYP3A7 mRNA levels for donors 1 and 3 at 0.5% and 2.5% DMSO. In conclusion, a DMSO concentration of 0.1% or less may be appropriate for studying induction after drug exposure.

ATP-Binding Cassette Transporters↗

PubMed2003

Tissue distribution of mRNA expression of human cytochrome P450 isoforms assessed by high-sensitivity real-time reverse transcription PCR.

Pairs of forward and reverse primers and TaqMan probes specific to each human cytochrome P450 isoform were prepared. Analysis of the mRNA level of each CYP isoform in total RNA from pooled specimens of various human organs was performed by real-time reverse transcription PCR using an ABI PRISM 7700 sequence detector system. The expression of CYP3A4 mRNA was similar to that of CYP3A7 mRNA in the fetal liver, and CYP3A4 mRNA levels in the fetal liver were about 0.1 times lower than in the adult liver. CYP2E1 showed the highest level of mRNA expression in the liver. The mRNA expression of 30 CYP isoforms (CYP1A1, 1A2, 1B1, 2A6, 2A7, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 3A4, 3A5, 3A7, 4A11, 4F2, 4F3, 5A1, 7B1, 8A1, 8B1, 17, 26A1, 27, 27B1, 39A1, 46, and 51) in the liver was successfully detected by this method. CYP2F1, 4B1, 4F8, 11s (11A, 11B1, and 11B2), 19, and 24 mRNA levels were the highest in the lung, lung, prostate, adrenal gland, placenta, and kidney, respectively; however, the mRNA expression of these eight CYP isoforms in the liver was not detected by this method. The mRNA levels of the CYP isoforms determined in various human tissues were in good agreement with previously reported data. The method described here has the advantages of high specificity and excellent quantification over a wide range of mRNA concentrations, making it suitable for the evaluation of a large number of samples in the assessment of the expression profile of drug-metabolizing enzymes.

Cytochrome P-450 Enzyme System↗

PubMed2002

Stereoselective pharmacokinetics of BOF-4272 racemate after oral administration to rats and dogs.

The stereoselective pharmacokinetics of BOF-4272 enantiomers in rats and dogs was investigated by simultaneously measuring concentrations in arterial, portal, and venous plasma, the liver, and the kidney at 2 h after the oral administration of the racemic drug. The concentrations of BOF-4272 enantiomers were measured using high-performance liquid chromatography. The concentrations of the S(-) enantiomer in arterial, portal, and venous plasma were higher than those of the R(+) enantiomer in rats, but the opposite was found in dogs. In rats, absorption from the intestinal tract into the portal system was almost the same for the two enantiomers, whereas the hepatic uptake of the R(+) enantiomer was greater than that of the S(-) enantiomer. In dogs, absorption from the intestinal tract into the portal system was greater for the R(+) enantiomer than for the S(-) enantiomer, whereas hepatic uptake was comparable for the two enantiomers. The stereoselectivity of the renal uptake of BOF-4272 enantiomers had little effect on the stereoselectivity of enantiomers in the systemic circulation in both rats and dogs. The stereoselectivity in the systemic circulation of BOF-4272 enantiomers is therefore related to hepatic uptake in rats, and to absorption from the intestinal tract into the portal system in dogs.

Animals↗

PubMed2002

Evaluation of gene induction of drug-metabolizing enzymes and transporters in primary culture of human hepatocytes using high-sensitivity real-time reverse transcription PCR.

In the present study, the induction of drug-metabolizing enzymes and transporters was evaluated by analyzing mRNA expression in human hepatocytes after exposure to various compounds. The compounds tested included typical enzyme inducers, rifampicin and omeprazole, and controls. All experiments were performed in the presence of 0.1% DMSO. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method (RT-PCR) in the presence of TaqMan probes using an ABI PRISM 7700 Sequence Detector system. A new analytic method to quantify mRNA levels in small numbers of human hepatocytes has been developed for phase I enzymes, phase II enzymes, and transporters. The levels of CYP1A1, CYP2B6, CYP2C8, CYP3A4, CYP3A5, ADH3, and ABCG1 mRNA in human hepatocytes increased after exposure to rifampicin. The levels of CYP1A1, CYP2B6, CYP3A4, CYP3A5, and ABCG1 mRNA recovered after a change to media without rifampicin. The levels of CYP1A1, CYP1A2, CYP1B1, ALDH3, and ALDH6 mRNA increased after exposure to omeprazole, and recovered after a change to media without omeprazole. On the other hand, the levels of ADH3 and ABCB4 mRNA decreased after exposure to omeprazole, and recovered after a change to media without omeprazole. In conclusion, these results demonstrate the applicability of quantitative real-time RT-PCR to the evaluation of the gene induction and recovery of drug-metabolizing enzymes and transporters after exposure to drugs in human hepatocytes.

ATP Binding Cassette Transporter, Subfamily G, Mem↗