Biomedical subjects

Masuhiro Nishimura

Publications and source records attributed to Masuhiro Nishimura.

At least 19 recordsLinked to original sources

PubMed2006

Identification of the positive regulatory and distinct core regions of promoters, and transcriptional regulation in three types of mouse phospholipid hydroperoxide glutathione peroxidase.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is transcribed into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar types, from one gene by alternative transcription using different first exons, Ia and Ib. We investigated the regulatory mechanisms of the expressions of the three types of PHGPx using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis. Here we report a draft of the positive regulatory region and the core promoter regions of PHGPx in several cell lines. From promoter deletion analysis we identified the three distinct core regions of mitochondrial PHGPx, non-mitochondrial PHGPx and nucleolar PHGPx. The core promoter activity of non-mitochondrial PHGPx was high in L929 cells, but relatively low for mitochondrial and nucleolar PHGPx. We also identified the positive regulatory region of mitochondrial PHGPx by deletion and mutation analysis of 5'-flanking regions of mitochondrial PHGPx. This region could regulate the promoter activity of non-mitochondrial PHGPx; however, up-regulation by this region was normally suppressed by the upstream region in somatic cells. Electrophoretical mobility shift analysis demonstrated that a specific transcription factor complex bound to this region in adult testis, but not in young testis and different sizes of complexes bound to this region between testis and brain.

Age Factors↗

PubMed2006

Effects of prototypical drug-metabolizing enzyme inducers on mRNA expression of housekeeping genes in primary cultures of human and rat hepatocytes.

Quantitative real-time RT-PCR was used to investigate the effects of prototypical drug-metabolizing enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) on mRNA expression levels of the housekeeping genes beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-glucuronidase (GUSB), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolylisomerase A (PPIA), TATA box binding protein (TBP), and transferrin receptor (TFRC) in primary cultures of cryopreserved human and rat hepatocytes. The mRNA levels of ACTB, GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in human hepatocytes were constant at all concentrations of inducers. However, the mRNA level of GAPDH relative to HPRT1 in rat hepatocytes was markedly increased by Rif. The mRNA levels of GAPDH, GUSB, PPIA, TBP, and TFRC relative to HPRT1 in rat hepatocytes were significantly increased by Dex. ACTB and HPRT1 are suitable internal controls for evaluating mRNA expression levels in primary cultures of human and rat hepatocytes after Rif, Dex, or Ome exposure.

Adult↗

PubMed2006

Inducibility of UDP-glucuronosyltransferase 1As by beta-naphthoflavone in HepG2 cells.

UDP-glucuronosyltransferases (UGTs) are conjugation enzymes, which are regulated in a tissue-specific manner by endogenous and environmental factors. In this study, we focused on UGT1A isoforms (UGT1A1, UGT1A6 and UGT1A9), mainly expressed in the human liver, and examined the inducibility of UGT1As by beta-naphthoflavone (BNF) in human hepatoma HepG2 cells. The cells were pretreated for 72 h with BNF at concentrations of 25, 50 and 100 microM. 7-Ethyl-10-hydroxycamptothecin (SN-38) glucuronidation, used as a probe for UGT1A1, showed sigmoidal kinetics with a Hill coefficient (n) of 1.2-1.3 in control and BNF-pretreated HepG2 cells. The Vmax values were significantly increased 3.6- to 4.3-fold by BNF, whereas there was no significant change in the S50 values by BNF at any concentration examined. On the other hand, 4-methylumbelliferone (4-MU) glucuronidation as a probe for UGT1A6 and UGT1A9 in the control and BNF-pretreated HepG2 cells exhibited a biphasic kinetic pattern. Although Km1 values for the low-Km phase were similar between the control and BNF-pretreated HepG2 cells, Km2 values for the high-Km phase of BNF-pretreated HepG2 cells were reduced to 54-69% of control HepG2 cells. The values of Vmax1 and Vmax2 for the low- and high-Km phases, respectively, were significantly increased 1.9- to 2.6-fold by BNF at 25 and/or 50 microM but not 100 microM. With respect to Vmax (Vmax1 and Vmax2) and Vmax/Km (Vmax1/Km1 and Vmax2/Km2), the values were significantly increased 2.0- to 3.2-fold by BNF at all concentrations examined. Furthermore, real-time reverse transcription polymerase chain reaction using TaqMan probes demonstrated that BNF concentration-dependently induced mRNA levels of UGT1A1 but not UGT1A6 or UGT1A9 in HepG2 cells (1.3- to 6.0-fold). These results suggest that the inducibility of UGT1A isoforms in HepG2 cells by BNF is different from other aryl hydrocarbon receptor agonists previously reported, and should provide useful information for the prediction of drug-drug interactions and toxicological assessment of environmental chemicals.

Cell Line, Tumor↗

PubMed2006

Various irrigation fluids affect postoperative brain edema and cellular damage during experimental neurosurgery in rats.

BACKGROUND: This study was conducted to investigate how various irrigation fluids used during neurosurgical procedures affect the degree of postoperative brain edema and cellular damage during experimental neurosurgery in rats. METHODS: The cerebral cortex was exposed and incised crosswise with a surgical knife under irrigation with an artificial CSF, lactated Ringer's solution, or normal saline. Four hours after injury, irrigation was stopped and brain tissue samples were obtained from injured and uninjured sites. Specific gravity, cerebrovascular permeability, and TTC staining of the samples were evaluated. Incision and irrigation of the brain were not performed on the control group. RESULTS: At the injured site, specific gravities of the samples in the normal saline group and the lactated Ringer's solution group were significantly lower than the specific gravity in the artificial CSF group. The EB concentration was significantly higher in the lactated Ringer's solution group and relatively high in the normal saline group as compared with the artificial CSF group. TTC staining did not differ significantly between the artificial CSF group and the control group. It was significantly lower in the lactated Ringer's solution group and the normal saline group than in the control group and the artificial CSF group. CONCLUSIONS: As compared with normal saline and lactated Ringer's solution, artificial CSF reduced postoperative brain edema, cerebrovascular permeability, and cellular damage in sites injured by experimental neurosurgery in rats.

Animals↗

PubMed2006

Soybean oil fat emulsion to prevent TPN-induced liver damage: possible molecular mechanisms and clinical implications.

Long-term total parenteral nutrition (TPN) is known to be associated with cholestasis and hepatic steatosis, which can be lethal in infants who cannot be fed orally. The present review focuses on the metabolic complications in the liver that may occur due to the excessive administration of fat-free TPN. We have recently developed an infant rat model of hepatic dysfunction and steatosis induced by overdose of fat-free TPN. In this model, plasma levels of liver enzymes in the fat-free TPN group were found to be significantly higher than in the other groups (i.e., the oral diet and fat-containing TPN groups). Pathological examination showed hepatomegaly and severe fatty changes without cholestasis in the liver of infant rats that received fat-free TPN. We clearly demonstrated that the addition of soybean oil emulsion to the TPN regimen prevented hepatic dysfunction and fatty changes. In the present review, we discuss the molecular mechanism of the hepatic dysfunction induced by fat-free TPN and the role of soybean oil fat emulsion in the TPN regimen. We also discuss the clinical implications of soybean oil-containing TPN solutions and point out the importance of including fat in the TPN regimen in order to prevent the hepatic abnormalities associated with the excessive administration of fat-free TPN.

Animals↗

PubMed2006

Assessment of induction of cytochrome P450 by NO-1886 (ibrolipim), a lipoprotein lipase-promoting agent, in primary cultures of human hepatocytes and in female rat liver.

The mRNA levels of human cytochrome P450 (CYP)2Cs and CYP3As in primary cultures of freshly isolated human hepatocytes were assessed after exposure to NO-1886 and rifampicin, a typical inducer of CYP3As. mRNA levels were analyzed by real-time RT-PCR using an ABI PRISM 7700 Sequence Detector system. Exposure to NO-1886 for 24 hr at a concentration of less than 10 microM showed only a tendency to reduce or increase the expression levels of CYP2C8, CYP2C9, CYP2C19, CYP3A4, or CYP3A5 mRNA. A higher concentration (50 microM) of NO-1886 induced an increase in CYP2C8 mRNA and a decrease in CYP2C19 mRNA, and these changes continued after additional culture for 24 hr in fresh medium without NO-1886. The expression level of CYP3A4 mRNA after exposure to NO-1886 for 24 hr at 50 microM was about twice that in controls. Following additional culture for 24 hr in fresh medium without NO-1886, the expression of CYP3A4 mRNA was comparable to that in controls. On the other hand, the expression levels of CYP2C9 and CYP3A5 mRNA showed small and variable changes in each donor even at a high concentration (50 microM) of NO-1886. Furthermore, the pharmacokinetics of NO-1886 during repeated oral administration for 14 days was studied in female rats. The pharmacokinetic parameters of NO-1886 were nearly the same on days 1, 7, and 14 of repeated administration. The hepatic microsomal content of CYP isoforms was not affected by repeated administration for 7 days at a dose of 1 to 30 mg/kg in female rats, although the total CYP content was increased at a dose of 30 mg/kg. The expression levels of CYP1A2, CYP2B2, CYP2C12, and CYP2E1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2, 10, or 50 microM. The expression levels of CYP3A1 mRNA in primary cultures of rat hepatocytes were not affected by exposure to NO-1886 at 2 or 10 microM, but were increased, with large individual variation, by exposure at 50 microM. The mRNA expression levels in rat hepatocytes exposed to concentrations comparable to free plasma levels did not change significantly, which was consistent with the equivalence in the in vivo plasma concentrations observed on days 1 and 14 of repeated administration. These results suggest that repeated administration of NO-1886 at clinical doses does not significantly affect the expression levels of CYP isoforms in human liver, although the mRNA levels of the CYP isoforms involved in the metabolism of NO-1886 were increased by exposure to higher concentrations of NO-1886 in human hepatocytes in vitro.

Animals↗

PubMed2006

Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes.

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Tissue-specific mRNA expression profiles of human phase I metabolizing enzymes except for cytochrome P450 and phase II metabolizing enzymes.

Pairs of forward and reverse primers and TaqMan probes specific to each of 52 human phase I metabolizing enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, dihydropyrimidine dehydrogenase, epoxide hydrolase, esterase, flavin-containing monooxygenase, monoamine oxidase, prostaglandin endoperoxide synthase, quinone oxidoreductase, and xanthene dehydrogenase) and 48 human phase II metabolizing enzymes (acetyltransferase, acyl-CoA:amino acid N-acyltransferase, UDP-glucuronosyltransferase, glutathione S-transferase, methyltransferase, and sulfotransferase) were prepared. The mRNA expression level of each target enzyme 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 Detection System. Further, individual differences in the mRNA expression of representative human phase I and II metabolizing enzymes in the liver were also evaluated. The mRNA expression profiles of the above phase I and phase II metabolizing enzymes in 23 different human tissues were used to identify the tissues exhibiting high transcriptional activity for these enzymes. These results are expected to be valuable in establishing drug metabolism-mediated screening systems for new chemical entities in new drug development and in research concerning the clinical diagnosis of disease.

Cytochrome P-450 Enzyme System↗

PubMed2006

Evaluation of human CYP1A2 and CYP3A4 mRNA expression in hepatocytes from chimeric mice with humanized liver.

We investigated and compared the expression of human CYPs mRNA in primary cultures of cryopreserved human hepatocytes and in chimeric mice constructed by transplanting hepatocytes from the same human donors. Analysis was performed by real-time reverse-transcription polymerase chain reaction. Initial expression levels for the 12 human CYPs mRNA in chimeric mouse hepatocytes were higher than those in human hepatocytes, but a low correlation coefficient was observed (r=0.690). After 24 h of culture, the correlation remained low (r=0.699). The medium was replaced with fresh medium without human epidermal growth factor, and after 48 h of culture, expression of the 12 human CYPs mRNA were very similar in human hepatocytes and chimeric mouse hepatocytes, and a higher correlation coefficient was observed (r=0.809). After 72 h of culture, the correlation remained high (r=0.873). The ratio of human CYP1A2 mRNA to beta-actin mRNA in chimeric mouse hepatocytes decreased quickly during the first 24 h of culture, and then remained constant. Expression profiles of human CYP1A2 mRNA in chimeric mouse hepatocytes were similar to those in human hepatocytes after exposure of beta-naphthoflavone. CYP3A4 mRNA expression was increased significantly by rifampicin (Rif) exposure in human hepatocytes, whereas Rif-induced increases in CYP3A4 mRNA expression in chimeric mouse hepatocytes was seen for two of the three donors. In conclusion, we demonstrated that expression and induction of human CYPs in human hepatocytes can be reproduced in chimeric mouse hepatocytes.

Animals↗

PubMed2005

Expression of human phase II enzymes in chimeric mice with humanized liver.

We clarified that major human cytochrome P450 (P450) enzymes were expressed in a chimeric mouse line established recently in Japan, in which the liver could be replaced by more than 80% with human hepatocytes. In this study, we investigated major human phase II enzymes such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione S-transferase (GST) in the livers of chimeric mice by mRNA, protein, and enzyme activity using reverse transcription-polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Human UGT, SULT, NAT, and GST mRNA were expressed in the liver of the chimeric mice, and UGT2B7, SULT1E1, SULT2A1, and GSTA1 proteins could be detected. The expression of mRNA and protein was correlated with the human albumin (hAlb) concentration in mouse blood, the replacement of which by human hepatocytes could be estimated by the hAlb concentration in the blood of the chimeric mice, because the chimeric mice produce human albumin. The enzyme activities, such as morphine 6-glucuronosyltransferase activity and estrone 3-sulfotransferase activity, activities that are specific to humans but not to mice, were increased in a hAlb concentration-dependent manner. The chimeric mice with humanized liver with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human phase II enzymes and enzyme activities as those of the donor. In conclusion, the chimeric mice exhibited an efficient capacity of drug conjugation similar to that in humans. These chimeric mice expressed human phase II enzymes as well as P450s, suggesting that they could be a useful animal model in drug development.

Animals↗

PubMed2005

Involvement of SULT1A3 in elevated sulfation of 4-hydroxypropranolol in Hep G2 cells pretreated with beta-naphthoflavone.

Pretreatment of Hep G2 cells with beta-naphthoflavone (BNF 1-25microM) significantly increased cytosolic sulfation activities of 4-hydroxypropranolol (4-OH-PL) racemate. The profile was similar to those of sulfations towards dopamine and triiodothyronine in the same cytosolic fractions. Kinetic studies of 4-OH-PL sulfation in Hep G2 cytosolic fractions revealed that V(max) values increased but apparent K(m) values remained unchanged following the BNF pretreatment. Among five recombinant human SULT isoforms (SULT1A1, -1A3, -1B1, -1E1 and -2A1) examined, only SULT2A1 did not show 4-OH-PL sulfation activities under the conditions used. SULT1A3 and -1E1 exhibited an enantioselectivity of 4-OH-R-PL sulfation>4-OH-S-PL sulfation, which agreed with that of BNF-pretreated Hep G2 cells as well as of nontreated cells, whereas SULT1A1 and -1B1 showed a reversed enantioselectivity (R<S). In kinetic studies of 4-OH-PL sulfations by four kinds of human SULT isoforms, apparent K(m) values for SULT1A3 were the lowest, and the parameters were close to those of Hep G2 cytosolic fractions. Real time RT-PCR using TaqMan probes demonstrated that the mRNA levels of SULT1A3 increased following BNF pretreatment, which paralleled the results from Western blotting showing the elevated levels of SULT1A3 proteins. These results suggest that the induction of SULT1A3 is mainly responsible for the elevated 4-OH-PL sulfation activities following the pretreatment of Hep G2 cells with BNF.

Arylsulfotransferase↗

PubMed2005

CYP2C19 genotype affects diazepam pharmacokinetics and emergence from general anesthesia.

OBJECTIVES: Diazepam is widely used to relieve preoperative anxiety in patients. The objective of this study was to investigate the effects of polymorphism in CYP2C19 and the effects of CYP3A4 messenger ribonucleic acid (mRNA) content in blood on recovery from general anesthesia and on diazepam pharmacokinetics. METHODS: Sixty-three Japanese patients were classified into the following 3 genotype (phenotype) groups on the basis of polymerase chain reaction-restriction fragment length polymorphism analysis of CYP2C19 polymorphism: no variants, *1/*1 (extensive metabolizer [EM]); 1 variant, *1/*2 or *1/*3 (intermediate metabolizer [IM]); and 2 variants, *2/*2, *2/*3, or *3/*3 (poor metabolizer [PM]). We assessed the effects of these polymorphisms and of CYP3A4 mRNA content in the lymphocytes on the patients' recovery from general anesthesia. RESULTS: CYP2C19 genotyping analysis in the 63 subjects showed that 32%, 46%, and 22% of subjects were classified into the EM, IM, and PM groups, respectively. The PM subjects showed a larger area under the curve representing the concentration of diazepam over a 24-hour period (AUC(0-24)) (2088 +/- 378 ng/mL.h(-1), P = .0259), lower clearance of diazepam (0.049 +/- 0.009 L.h(-1).kg(-1), P = .0287), and longer emergence time (median, 18 minutes; 25th-75th percentile range, 13-21 minutes; P < .001) in comparison with subjects in the EM group (AUC(0-24), 1412 +/- 312 ng/mL; clearance, 0.074 +/- 0.018 L.h(-1).kg(-1); and emergence time, 10 minutes, 8-12 minutes [median and 25th-75th percentile range]). The IM group also showed a longer emergence time (median, 13 minutes; 25th-75th percentile range, 9-20 minutes; P < .001) and a larger variation in this parameter in comparison with the EM group. The distributions of the CYP2C19 genotype were significantly different between the 2 groups (rapid emergence <20 minutes, slow emergence >20 minutes) (P = .0148). The mean value of the CYP3A4 mRNA level in the slow-emergence group (mean +/- SD, 4.80 +/- 3.99 x10(-10)) was significantly lower than that of the rapid-emergence group (mean +/- SD, 12.50 +/- 11.90 x10(-10)) (P = .0315). However, there was no significant correlation between emergence time and CYP3A4 mRNA levels (r = 0.239, P = .0601). CONCLUSION: We found that the CYP2C19 genotype affects diazepam pharmacokinetics and emergence from general anesthesia and that the slow-emergence group possesses lower levels of CYP3A4 mRNA than are found in the rapid-emergence group.

Actins↗

PubMed2005

Soybean oil in total parenteral nutrition maintains albumin and antioxidant enzyme mRNA levels.

Changes in albumin and antioxidant enzyme mRNA expression in infant rat liver following administration of total parenteral nutrition (TPN) with/without soybean oil emulsion were studied. Infant rats were divided into three groups: group 1=oral diet, group 2=TPN without fat, and group 3=TPN with 20% of calories from soybean oil emulsion. The period of TPN administration was 4 d. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in group 2 than in the other groups, with similar levels seen in the other groups. Albumin, Cu, Zn-superoxide dismutase, and glutaredoxin 1 mRNA expression levels were lower in group 2 than in the other groups, with similar levels seen in the other groups. Catalase mRNA expression was higher in group 1 than in the other groups, with the lowest level seen in group 2. Soybean oil emulsion should be included in TPN regimens to prevent down-regulation of albumin and antioxidant enzyme mRNA expression.

Albumins↗

PubMed2005

Soybean oil fat emulsion prevents cytochrome P450 mRNA down-regulation induced by fat-free overdose total parenteral nutrition in infant rats.

Changes in the mRNA expression of hepatic cytochrome P450 (CYP) isoenzymes associated with overdose of fat-free or fat-containing total parenteral nutrition (TPN) were investigated 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 calories from fat (soybean oil emulsion). After TPN administration for 4 d, serum aspartate aminotransferase (AST) levels in group 2 were significantly increased (p<0.01) compared with the other groups. The mRNA expression of hepatic CYP isoenzymes in group 2 decreased to 0.76 to 31% of that in group 1 (p<0.01), but that in group 3 was maintained at 32 to 84% of that in group 1. These results indicate the importance of including fat in TPN regimens to prevent not only hepatic dysfunction but also mRNA down-regulation of liver CYP isoenzymes.

Animals↗

PubMed2005

Major role of soybean oil in preventing decrease in CYP4A1 mRNA content relative to PPARalpha mRNA down-regulation in infant rats.

Changes in the levels of CYP4A1, PPARalpha, and RXRalpha mRNA expression in the liver following overdose of fat-free or fat-containing total parenteral nutrition (TPN) were studied in 3-week-old male Sprague-Dawley rats. The rats were divided into three groups: group 1, oral diet; group 2, fat-free TPN; and group 3, TPN with 20% of calories from soybean oil emulsion. Levels of CYP4A1, PPARalpha, and RXRalpha mRNA in the fat-free TPN group were significantly lower than those in the other groups. Levels of CYP4A1 and PPARalpha mRNA were strongly correlated (r=0.849), and levels of CYP4A1 and RXRalpha mRNA were weakly correlated (r=0.618). This is the first report of a strong correlation between the levels of CYP4A1 and PPARalpha mRNA following overdose of fat-free or fat-containing TPN in infant rats. Our results also indicate that it is important to include fat in TPN regimens in order to prevent CYP4A1 mRNA down-regulation, which may be related to changes in PPARalpha mRNA levels in the liver.

Animals↗

PubMed2005

Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells.

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.

Antineoplastic Agents↗

PubMed2005

Tissue-specific mRNA expression profiles of human toll-like receptors and related genes.

The mRNA expression levels of 10 toll-like receptors (TLRs) and 21 related genes in total RNA from pooled specimens of fetal human tissues (brain and liver), from single and pooled specimens of various adult human tissues (adrenal gland, brain, heart, kidney, liver, lung, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, testis, thymus, thyroid gland, trachea, and uterus), and from two cell lines (Hep G2 and HeLa) were analyzed by real-time reverse transcription PCR. The mRNA expression of the 10 human TLRs was successfully detected in all of the tissues and in HeLa cells. TLR2, TLR3, TLR6, and TLR9 were consistently expressed in Hep G2 cells, but TLR1, TLR4, TLR5, TLR7, TLR8, and TLR10 showed no or very weak expression in these cells. The mRNA expression of many TLR-related genes (ICAM1, CD14, MyD88, LY96, TRIF, TICAM2, TIRAP, CD83, SOCS1, TNFAIP3, TOLLIP, IRAK1, IRAK2, IRAK4, and TRAF6) was successfully detected in all of the tissues and cell lines. The mRNA expression of CD80, CD86, IRAK3, and CCL2 was successfully detected in all of the tissues and cell lines except for Hep G2 cells. The mRNA expression of CCL5 was successfully detected in all of the tissues and cell lines except for fetal brain. The mRNA expression of CXCL10 was successfully detected in all of the tissues except for fetal and adult brain. These results provide valuable information for studies concerning the regulation of TLR-related genes.

Adolescent↗

PubMed2005

Induction of human CYP1A2 and CYP3A4 in primary culture of hepatocytes from chimeric mice with humanized liver.

Chimeric mice with near-completely humanized liver were constructed by transplantating hepatocytes from a Japanese and Caucasian donor. In the present study, we investigated the induction of human CYP1A2 and CYP3A4 mRNA in a primary culture of the cryopreserved chimeric mouse hepatocytes. beta-naphthoflavone (beta-NF) and rifampicin (Rif) were used as typical cytochrome P450 (CYP) inducers for CYP1A2 and CYP3A4, respectively. Analysis was performed by the real-time reverse-transcription polymerase chain reaction method. CYP1A2 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 3.8-, 6.3-, and 3.3-fold by 5 microM beta-NF exposure, respectively, compared with the 0.1% DMSO treated control (p<0.01). CYP3A4 mRNA in the primary culture of chimeric mouse hepatocytes in mice No. 1, 2, and 3 was significantly increased 8.4-fold (p<0.001), 2.2-fold (p<0.01), and 2.3-fold (p<0.05) by 50 microM Rif exposure, respectively, compared with the 0.1% DMSO treated control. The present study demonstrated that a primary culture of cryopreserved hepatocytes from chimeric mice with humanized liver could be used for evaluating the induction of drug metabolizing enzymes in human. This in vitro method may be a useful method for screening the induction potency of new drug candidates on drug metabolizing enzymes.

Adolescent↗