-
[show abstract]
[hide abstract]
ABSTRACT: Measurements of protein-conjugated acrolein (PC-Acro), IL-6 and CRP in plasma were useful for identifying silent brain infarction with high sensitivity and specificity. The aim of this study was to determine whether acrolein causes increased production of IL-6 and CRP in thrombosis model mice and cultured cells. In mice with photochemically induced thrombosis, acrolein produced at the locus of infarction increased the level of IL-6 and then CRP in plasma. This was confirmed in cell culture systems - acrolein stimulated the production of IL-6 in mouse neuroblastoma Neuro-2a cells, mouse macrophage-like J774.1 cells and human umbilical vein endothelial cells (HUVEC), and IL-6 in turn stimulated the production of CRP in human hepatocarcinoma cells. The level of IL-6 mRNA was increased by acrolein through an increase in phosphorylation of the transcription factors, c-Jun and NF-κB p65. Furthermore, CRP stimulated IL-6 production in mouse macrophage-like J774.1 cells and HUVEC. IL-6 functioned as a protective factor against acrolein toxicity in Neuro-2a cells and HUVEC. These results show that acrolein stimulates the synthesis of IL-6 and CRP, which function as protecting factors against acrolein toxicity, and that the combined measurement of PC-Acro, IL-6 and CRP is effective for identification of silent brain infarction. This article is protected by copyright. All rights reserved.
Journal of Neurochemistry 06/2013; · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Treatment with benzbromarone (BBR), a potent uricosuric drug, can be associated with liver injury. Recently, we reported that culture of human hepatocellular carcinoma FLC-4 cells on micro-space cell culture plates could increase the functional expression of drug-metabolizing enzymes including CYP3A4 and CYP2C9, which are involved in 1'-hydroxylation and 6-hydroxylation of BBR, respectively. Therefore, we examined whether BBR and its two metabolites (1'-hydroxy BBR and 6-hydroxy BBR) have cytotoxic effects in FLC4 cells cultured on micro-space cell culture plates. The present study showed that BBR and 1'-hydroxy BBR, but not 6-hydroxy BBR, have cytotoxic effects in cells cultured on micro-space cell culture plates. BBR-induced cytotoxicity was decreased by CYP3A inhibitors (itraconazole and ketoconazole), an Nrf2 activator (tert-butylhydroquinone) and a GSH precursor (N-acetyl-L-cystein). In contrast, BBR-induced cytotoxicity was increased by a GSH biosynthesis inhibitor (buthionine sulfoximine) and an inhibitor of NAD(P)H quinone oxidoreductase 1 (dicoumarol). These results suggested that metabolic activation of 1'-hydroxy BBR via CYP3A, formation of quinone metabolites and the decrease in GSH levels were involved in the BBR-induced cytotoxicity observed in FLC4 cells cultured on micro-space cell culture plates.
Drug Metabolism and Pharmacokinetics 12/2012; · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6β-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes. Copyright © 2012 John Wiley & Sons, Ltd.
Biopharmaceutics & Drug Disposition 08/2012; 33(8):466-73. · 2.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human hepatocellular carcinoma cell lines cultured in a monolayer show negligible activities of drug-metabolizing enzymes such as cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs). Here, we show that culture of human hepatocellular carcinoma FLC-4 cells on 24-well plates arrayed with uniform micro-sized compartments on the bottom of the plates (micro-space cell culture plates) resulted in increased expression of drug-metabolizing enzymes (CYP1A2, CYP2C9, CYP3A4, UGT1A1, etc.) and nuclear receptors (pregnane X receptor, constitutive androstane receptor, etc.). When cells were treated with a typical CYP3A substrate (triazolam), CYP2C9 substrate (diclofenac) or UGT1A1 substrate (SN-38), large amounts of their metabolites were detected in the medium of cells cultured on micro-space cell culture plates. The formation of metabolites from triazolam, diclofenac and SN-38 was strongly inhibited by co-treatment with a CYP3A inhibitor (ketoconazole), CYP2C9 inhibitor (sulfaphenazole) and UGT1A1 inhibitor (ketoconazole), respectively. On the other hand, formation of metabolites was not observed in the medium of cells cultured in a monolayer. Finally, the cytotoxic effect of aflatoxin B1 was more potent in cells cultured on micro-space cell culture plates than in cells cultured in a monolayer. The results suggest that FLC-4 cells cultured on micro-space cell culture plates are useful for studying drug metabolism and drug-induced hepatotoxicity.
Drug Metabolism and Pharmacokinetics 03/2012; 27(5):478-85. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytochrome P450 (CYP) is deeply involved in the metabolism of chemicals including pharmaceuticals. Therefore, polymorphisms of this enzyme have been widely studied to avoid unfavorable side effects of drugs in chemotherapy. In this work, we performed computational analysis of the mechanism of the decrease in enzymatic activity for three typical polymorphisms in CYP 2C9 species: *2, *3, and *5. Based on the equilibrated structure obtained by molecular dynamics simulation, the volume of the binding pocket and the fluctuation of amino residues responsible for substrate holding were compared between the wild type and the three variants. Further docking simulation was carried out to evaluate the appropriateness of the binding pocket to accommodate substrate chemicals. Every polymorphic variant was suggested to be inferior to the wild type in enzymatic ability from the structural viewpoint. F-G helices were obviously displaced outward in CYP2C9*2. Expansion of the binding pocket, especially the space near F' helix, was remarkable in CYP2C9*3. Disappearance of the hydrogen bond between K helix and β4 loop was observed in CYP2C9*5. The reduction of catalytic activity of those variants can be explained from the deformation of the binding pocket and the consequent change in binding mode of substrate chemicals. The computational approach is effective for predicting the enzymatic activity of polymorphic variants of CYP. This prediction will be helpful for advanced drug design because calculations forecast unexpected change in drug efficacy for individuals.
Journal of Computational Chemistry 11/2010; 31(15):2746-58. · 4.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Pregnane X receptor (PXR), constitutive androstane receptor (CAR) and liver X receptor (LXR) are intracellular sensors for foreign chemicals and/or endogenous compounds. Docking of a ligand into the ligand-binding domain (LBD) of a nuclear receptor induces conformational changes and switches the nuclear receptor into an active conformation. In this study, we examined whether assembly assays to exploit the ligand-dependent interaction of N- and C-terminal regions of the LBD could be used for detection of ligands for PXR, CAR and LXR. Rifampicin, CITCO and T1317 significantly enhanced interactions for human PXR, human CAR and human LXR, respectively. The effects of ligands on the interaction of LBDs in PXR and CAR reflected the species differences in ligand response of PXR and CAR. In conclusion, it appears that the present assay, which exploits the interaction between N- and C-terminal regions of LBDs, is applicable to identify ligands.
Drug metabolism letters. 04/2010; 4(2):88-94.
-
[show abstract]
[hide abstract]
ABSTRACT: Nuclear receptor constitutive androstane/active receptor (CAR) is well known as a transcription factor regulating many genes that encode drug-metabolizing enzymes and factors modulating hepatic gluconeogenesis. However, there have been few studies on regulation of the CAR gene itself. In this study, we examined the involvement of retinoic acid receptor alpha (RAR alpha) in transcriptional regulation of the CAR gene in the liver. The expression levels of CAR mRNA in human primary hepatocytes and HepG2 cells were increased by all-trans retinoic acid. Activities of the human CAR promoter containing a region (termed cRARE) located at +1453/+1469 within intron 1 were increased by co-expression of RAR alpha in HepG2 cells. In addition, introduction of mutation into cRARE abolished transcriptional activation of the promoter by RAR alpha. The results of gel mobility shift assay and chromatin immunoprecipitation assay showed that RAR alpha was bound to cRARE. These results suggest that RAR alpha transactivated the human CAR gene by binding to cRARE located at +1453/+1469 within intron 1 of the gene. In contrast, the rat CAR gene was not activated by exposure to all-trans retinoic acid, probably due to the lack of a region corresponding to cRARE in the human CAR gene. Although the physiological significance of RAR alpha-dependent up-regulation of CAR in the human liver remains to be clarified, retinoid metabolism may be regulated by the up-regulation of CAR.
Biochemical pharmacology 03/2010; 80(1):129-35. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) often causes serious drug-drug interactions. To examine species differences in MBI of CYP3A between humans and rodents, we compared MBI potencies of five representative CYP3A inhibitors in human, rat and mouse liver microsomes. Among the inhibitors studied, erythromycin and clarithromycin exhibited markedly weaker MBI effects on CYP3A activity in rat and mouse liver microsomes compared to human liver microsomes. Results of spectroscopic experiments showed that erythromycin and clarithromycin form a metabolic intermediate complex with human liver microsomes but not with rat or mouse liver microsomes. In contrast, troleandomycin, diltiazem and nicardipine form a metabolic intermediate complex with rat and mouse liver microsomes, although some differences in MBI potency among species were observed. Parameters for MBI potency (k(inact)/K(I) ratio) and reversible inhibition (IC(50)) were negatively correlated (r=-0.820, p=0.0003), suggesting that the different affinities of CYP3A inhibitor for CYP3A may partly contribute to the different MBI potencies of inhibitor among species. Taken together, the results suggest that there are species differences in MBI of CYP3A in humans, rats and mice, which should be considered when rodents are used as in vivo models for MBI-mediated drug-drug interaction study.
Drug Metabolism and Pharmacokinetics 01/2010; 25(1):93-100. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The constitutive androstane receptor (CAR; NR1I3) is a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters. However, studies on regulation of CAR target genes via up- or down-regulation of CAR are limited. In this study, we examined the effects of PPARalpha agonists (ciprofibrate, bezafibrate, fenofibrate and WY14643) on the expression of CAR and its target gene CYP2B1/2 in rat primary hepatocytes. Results from real-time PCR analysis showed that CAR and CYP2B1/2 mRNAs exhibit increases in response to all PPARalpha agonists studied (5 to 10-folds of control). Pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, completely suppressed increase in CYP2B1/2 mRNA in response to ciprofibrate, suggesting that protein synthesis is required in this process. In addition, the induction of CAR by ciprofibrate on the protein level was observed with nuclear extracts as well as total cell lysates. These results indicate that CYP2B1/2 mRNAs are induced by PPARalpha agonists and that this effect is accompanied by increase in the expression of CAR gene at both mRNA and nuclear protein levels. Activated PPARalpha may increase functional CAR protein, which can induce the expression of CAR target genes such as CYP2B.
Drug Metabolism and Pharmacokinetics 01/2010; 25(1):108-11. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the present study, we analyzed the function of a novel mutation (c.1628T>G, p.Leu543Trp) in the solute carrier organic anion transporter (SLCO) 1B1 gene, encoding organic anion transporting polypeptide (OATP) 1B1, which was identified in a patient with pravastatin-induced myopathy. OATP1B1 variants carrying the mutation (OATP1B1*1a+c.1628T>G or *1b+c.1628T>G) showed a reduced transporting activity toward typical substrates and pravastatin compared with the activity of the references (OATP1B1*1a or *1b). This was due to reduction in V(max) values of the variants, not due to change in their K(m) values. OATP1B1*1b+c.1628T>G was normally expressed on the plasma membrane of HEK293 cells at the same level as that of OATP1B1*1b. Taken together, our results suggest that the mutation c.1628T>G (p.Leu543Trp) reduced the function of OATP1B1 probably due to decrease in turnover rate of one OATP1B1 molecule rather than impairment of protein sorting to the plasma membrane.
The Pharmacogenomics Journal 03/2009; 9(3):185-93. · 4.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Mammalian carboxylesterases comprise a multigene family, the gene products of which are localized in the endoplasmic reticulum. The carboxylesterases catalyze the hydrolysis of various xenobiotics and endogenous substrates such as ester, amide and thioester bonds and are thought to function mainly in drug metabolism. We have suggested the possibility that individual variation of human liver carboxylesterase activity causes the difference in expression levels of CES1A isozymes. However, little is known about the transcriptional regulation of human carboxylesterase genes. In the present study, we isolated two CES genes encoding human carboxylesterase CES1A, which were designated as CES1A1 (AB119997) and CES1A2 (AB119998). These genes were identical except for exon 1 and the 5' regulatory element. We investigated the transcriptional regulation of these two CES genes. A reporter gene assay and electrophoretic mobility shift assay demonstrated that Sp1 and C/EBPalpha could bind to each responsive element of the CES1A1 promoter but that the Sp1 and C/EBP could not bind to the responsive element of the CES1A2 promoter. Thus, CES1A1 mRNA expression level is much higher than the expression level of CES1A2 mRNA in the liver and lung. It is thought that these results provide information on individual variation of human carboxylesterase isozymes.
Drug Metabolism and Pharmacokinetics 02/2008; 23(1):73-84. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Pregnane X receptor (PXR; NR1I2), a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters, is abundantly expressed in the human liver. However, studies on the molecular mechanism of human PXR gene regulation are limited. In this study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) in the transcriptional regulation of the human PXR gene in the human liver. The activities of the human PXR promoter containing the direct repeat 1 (DR1) element located at -88/-76 of the promoter were significantly increased by co-expression of HNF4alpha in the human hepatocellular carcinoma cell line. In addition, introduction of mutation into the DR1 element abolished the transcriptional activation of the human PXR promoter by exogenous HNF4alpha. The results of gel mobility shift assays and chromatin immunoprecipitation assays showed that HNF4alpha was bound to the promoter region containing the DR1 element. A knock-down of HNF4alpha by siRNA significantly decreased expression levels of endogenous PXR mRNA in HepG2 cells. Furthermore, expression levels of PXR mRNA positively correlated with those of HNF4alpha mRNA in 18 human liver samples. These results suggested that HNF4alpha transactivated the human PXR gene by binding to the DR1 element located at -88/-76 of the promoter and was involved in the expression of PXR in the human liver.
Drug Metabolism and Pharmacokinetics 02/2008; 23(1):59-66. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The aim of the present study was to clarify the factors responsible for interindividual variability of organic anion transporting polypeptide (OATP, gene symbol SLCO) 1B1 mRNA expression level in the human liver.
OATP1B1 mRNA expression levels were determined by real-time PCR in 31 human liver samples. The results were analyzed in relation to a single nucleotide polymorphism (-11187G>A) located in the promoter region and levels of hepatocyte nuclear factor (HNF) 1alpha mRNA.
There was a 4.9-fold interindividual variability of OATP1B1 mRNA expression level in the livers analyzed, which was not associated with -11187G>A polymorphism. Accordingly, the -11187G>A polymorphism did not alter the SLCO1B1 gene promoter activity in luciferase assays. On the other hand, OATP1B1 mRNA levels showed a significant correlation with HNF1alpha mRNA levels (r=0.83, P<0.0001). This correlation was consistent with the results of luciferase assays and chromatin immunoprecipitation assays showing functional interaction between HNF1alpha and SLCO1B1 gene promoter.
Our results suggest that HNF1alpha is an essential regulator of OATP1B1 mRNA expression and thus the level of HNF1alpha expression is one of the major determinants of interindividual variability in OATP1B1 mRNA expression.
Pharmaceutical Research 12/2007; 24(12):2327-32. · 4.09 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: UDP-glucuronosyltransferases (UGTs) catalyze phase-II biotransformation reaction of a variety of substances. Among the UGT1A isoforms, UGT1A1, UGT1A3, UGT1A4, UGT1A6 and UGT1A9 are predominantly expressed in the liver. Interindividual variability in expression of these isoforms would cause interindividual differences in drug response, toxicity and cancer susceptibility. In the present study, we investigated the interindividual variability in UGT1A mRNA expression and whether hepatocyte nuclear factor 1alpha (HNF1alpha) and HNF4alpha were factors responsible for their variability in human livers. The amounts of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, HNF1alpha and HNF4alpha mRNA in 18 human livers were measured by quantitative real-time polymerase chain reaction. The largest and smallest interindividual differences in expression levels were observed in UGT1A1 (8.6-fold) and UGT1A4 (2.5-fold) mRNA, respectively. The amounts of HNF1alpha and HNF4alpha mRNA were strongly correlated with the amount of UGT1A9 mRNA and moderately correlated with that of UGT1A6 mRNA, whereas no significant correlation was found with the amounts of UGT1A1, UGT1A3 and UGT1A4 mRNA. Our results suggest that HNF1alpha and HNF4alpha are the factors involved in the interindividual variability of UGT1A6 and UGT1A9 mRNA expression. Further studies of other transcription factors are needed to clarify the factor(s) determining the interindividual variations in UGT1A1, UGT1A3 and UGT1A4 mRNA expression.
Drug Metabolism and Pharmacokinetics 11/2007; 22(5):391-8. · 2.32 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Diphenhydramine is widely used as an over-the-counter antihistamine. However, the specific human cytochrome P450 (P450) isozymes that mediate the metabolism of diphenhydramine in the range of clinically relevant concentrations (0.14-0.77 microM) remain unclear. Therefore, P450 isozymes involved in N-demethylation, a main metabolic pathway of diphenhydramine, were identified by a liquid chromatography-mass spectrometry method developed in our laboratory. Among 14 recombinant P450 isozymes, CYP2D6 showed the highest activity of diphenhydramine N-demethylation (0.69 pmol/min/pmol P450) at 0.5 microM. CYP2D6 catalyzed diphenhydramine N-demethylation as a high-affinity P450 isozyme, the K(m) value of which was 1.12 +/- 0.21 microM. In addition, CYP1A2, CYP2C9, and CYP2C19 were identified as low-affinity components. In human liver microsomes, involvement of CYP2D6, CYP1A2, CYP2C9, and CYP2C19 in diphenhydramine N-demethylation was confirmed by using P450 isozyme-specific inhibitors. In addition, contributions of these P450 isozymes estimated by the relative activity factor were in good agreement with the results of inhibition studies. Although an inhibitory effect of diphenhydramine on the metabolic activity of CYP2D6 has been reported previously, the results of the present study suggest that it is not only a potent inhibitor but also a high-affinity substrate of CYP2D6. Therefore, it is worth mentioning that the sedative effect of diphenhydramine might be caused by coadministration of CYP2D6 substrate(s)/inhibitor(s). In addition, large differences in the metabolic activities of CYP2D6 and those of CYP1A2, CYP2C9, and CYP2C19 could cause the individual differences in anti-allergic efficacy and the sedative effect of diphenhydramine.
Drug Metabolism and Disposition 02/2007; 35(1):72-8. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: MKC-963, (R)-1-(1-cyclohexylethylamino)-4-phenylphthalazine, a potent inhibitor of platelet aggregation, was synthesized and used in clinical trials in the 1990s. In the process of clinical study, it was found that urinary excretion ratios for 6beta-hydroxycortisol and free cortisol increased significantly in parallel with decreases in the plasma concentrations of MKC-963 after repeated oral administration of the compound to healthy volunteers. These findings suggested that MKC-963 caused autoinduction (defined as the ability of a drug to induce enzymes that enhance its own metabolism, resulting in dispositional tolerance) in humans, and clinical studies using the compound were stopped. This experience prompted us to reevaluate the effects of this compound on CYP3A4 using primary human hepatocytes and cDNA-expressed human cytochrome P450 (P450) enzymes to determine whether the autoinduction of MKC-963 metabolism in humans could have been predicted if these in vitro systems had been used for the evaluation of MKC-963 in the preclinical study. The results of in vitro study showed that MKC-963 increased CYP3A4 mRNA expression level and activity of testosterone 6beta-hydroxylation to extents similar to those observed with rifampicin in primary human hepatocytes. In addition, approximately 90% of the MKC-963 metabolism in human liver microsomes was estimated to be attributable to CYP3A4. These in vitro findings are in good agreement with the results of clinical study, suggesting that studies using human hepatocytes and cDNA-expressed human P450s are useful for assessing the autoinductive nature of compounds under development before starting clinical studies.
Drug Metabolism and Disposition 07/2006; 34(6):950-4. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: CYP2C9 and CYP2C19 are clinically important drug-metabolizing enzymes. The expression level of CYP2C9 is much higher than that of CYP2C19, although the factor(s) responsible for the difference between the expression levels of these genes is still unclear. It has been reported that hepatocyte nuclear factor 4alpha (HNF4alpha) plays an important role in regulation of the expression of liver-enriched genes, including P450 genes. Thus, we hypothesized that HNF4alpha contributes to the difference between the expression levels of these genes. Two direct repeat 1 (DR1) elements were located in both the CYP2C9 and CYP2C19 promoters. The upstream and downstream elements in these promoters had the same sequences, and HNF4alpha could bind to both elements in vitro. The transactivation levels of constructs containing two DR1 elements of the CYP2C9 promoter were increased by HNF4alpha, whereas those of the CYP2C19 promoter were not increased. The introduction of mutations into either the upstream or downstream element in the CYP2C9 gene abolished the responsiveness to HNF4alpha. We also examined whether HNF4alpha could bind to the promoter regions of the CYP2C9 and the CYP2C19 genes in vivo. The results of chromatin immunoprecipitation assays showed that HNF4alpha could bind to the promoter region of the CYP2C9 gene but not to that of the CYP2C19 promoter in the human liver. Taken together, our results suggest that HNF4alpha is a factor responsible for the difference between the expression levels of CYP2C9 and CYP2C19 in the human liver.
Drug Metabolism and Disposition 07/2006; 34(6):1012-8. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: SLCO1B1*5 and SLCO1B1*15 have been reported to reduce the clearance of pravastatin in healthy volunteers. However, there remains controversy in the effects of SLCO1B1*5 on the activity of OATP1B1 in vitro. In addition, the effect of SLCO1B1*15 on the function of OATP1B1 has not been studied using cDNA-expression systems. Object of the present study was to study the influence of SLCO1B1*5, *15 and *15+C1007G, a novel haplotype found in a patient with pravastatin-induced myopathy, on the functional properties of OATP1B1 by transient expression systems of HEK293 and HeLa cells using endogenous conjugates and statins as substrates.
Transporting assays for endogenous substrates were performed using tritium labeled estradiol-17beta-D-glucuronide and estrone-3-sulfate. Quantitation of pravastatin, atorvastatin, cerivastatin and simvastatin were carried out using HPLC tandem mass spectrometry.
The transporting activities of cells expressing SLCO1B1*5, *15 and *15+C1007G decreased significantly but those of SLCO1B1*1b, *1a+C1007G and *1b+C1007G were not altered for all of the substrates tested except for simvastatin. Kinetic analysis of pravastatin and atorvastatin showed that Km values were not altered but Vmax values decreased significantly in cells expressing SLCO1B1*5, *15 and *15+C1007G. Immunocytochemical study showed that SLCO1B1*5, *15 and *15+C1007G proteins are localized not only at the plasma membrane but also in the intracellular space.
These findings suggest that 521T>C, existing commonly in SLCO1B1*5, *15 and *15+C1007G, is the key single nucleotide polymorphism (SNP) that determines the functional properties of SLCO1B1*5, *15 and *15+C1007G allelic proteins and that decreased activities of these variant proteins are mainly caused by a sorting error produced by this SNP.
Pharmacogenetics and Genomics 08/2005; 15(7):513-22. · 3.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Constitutive active (or androstane) receptor (CAR, NR1I3), a member of the nuclear receptor family, is a major regulator for induction of cytochrome P450 2B (CYP2B) genes by phenobarbital. Phenobarbital-like inducer, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is a potent mouse CAR ligand that has been used to study CAR target genes in mice but does not activate human CAR (hCAR) or rat CAR (rCAR). Although 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) was reported to be an hCAR agonistic ligand, activation of hCAR by CITCO in cell-based reporter assay was weak. Therefore, we performed a screening of 50 drugs and chemicals using cell-based reporter assays to identify activators of hCAR. Among them, HMG-CoA reductase inhibitors (cerivastatin, simvastatin, fluvastatin, and atorvastatin) enhanced the hCAR-mediated transcriptional activation of phenobarbital-responsive enhancer module reporter gene by up to 3-fold. Similar activation by HMG-CoA reductase inhibitors was also observed with mouse and rat CARs. On the other hand, pravastatin did not activate hCAR at the concentrations tested (up to 30 microM). The extent of activation by the HMG-CoA reductase inhibitors was stronger than that by CITCO. Cerivastatin, simvastatin, fluvastatin, and atorvastatin induced CYP2B6 mRNA in stable hCAR-expressed FLC7 cells but not in original FLC7 cells. Therefore, we concluded that CAR mediates the effects of HMG-CoA reductase inhibitors on the induction of CYP2B genes, although HMG-CoA reductase inhibitors also activate pregnane X receptor. HMG-CoA reductase inhibitors such as cerivastatin would be useful to study for elucidating molecular and cellular mechanisms of hCAR.
Drug Metabolism and Disposition 08/2005; 33(7):924-9. · 3.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at -1732/-1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (-256/-233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription.
Journal of Biological Chemistry 03/2005; 280(5):3458-66. · 4.77 Impact Factor
