Influence of acetaminophen vehicle on regulation of transporter gene expression during hepatotoxicity

Researchers who study acetaminophen (APAP) hepatotoxicity use either a 50% propylene glycol solution or saline as a diluent. Previous studies demonstrated differential expression of hepatobiliary transporter mRNA in mice treated with a toxic dose of APAP dissolved in 50% propylene glycol. The purpose of this study was to determine whether using saline as a diluent for APAP alters regulation of transporter gene expression during hepatotoxicity. Male C57BL/6J mice received acetaminophen (APAP 400 mg/kg, i.p. in saline) or saline (20 ml/kg). Plasma and liver samples were collected at 24 and 48 h for assessment of alanine aminotransferase (ALT) activity and gene expression. It was determined that plasma ALT activity was elevated at 24 and 48 h after APAP administration. Using the branched DNA signal amplification assay, reductions in organic anion-transporting polypeptides Oatp1a1, Oatp1b2, sodium/taurocholate-cotransporting polypeptide (Ntcp), and bile salt export pump (Bsep) mRNA were observed in APAP-treated mice. In contrast, multidrug resistance-associated proteins Mrp1, Mrp2, Mrp3, and Mrp4, as well as multidrug resistance proteins Mdr1a and Mdr1b genes, were increased following APAP. No changes in Oatp1a4, Mdr2, or breast cancer resistance protein (Bcrp) mRNA were observed. Alterations in transporter gene expression in this study were similar to those reported previously using propylene glycol as diluent. With the exceptions of Oatp1a1, Ntcp, and Mrp1, these data mirror previous results suggesting that the solution used to dissolve APAP may alter the susceptibility of mice to hepatotoxicity, but only minimally change the regulation of transporter gene expression.     

Constitutive expression of various xenobiotic and endobiotic transporter mRNAs in the choroid plexus of rats.

The aim of this study was to quantitatively determine the constitutive expression levels of various transporter mRNAs in rat choroid plexus. To provide a reference for the relative expression levels, the expression of various transporter mRNAs in choroid plexus were compared with that in liver, kidney, and ileum. The mRNA levels of multidrug resistance protein (Mrp)1, 2, 3, 4, 5, and 6; multidrug resistance (Mdr)1a, 1b, and 2; organic anion transporting polypeptide (Oatp)1, 2, 3, 4, 5, 9, 12, and Oat-K (1/2); organic anion transporter (Oat)1, 2, and 3; organic cation transporter (Oct)1, 2, 3, N1, and N2; bile acid transporters sodium taurocholate cotransporting polypeptide (Ntcp), bile salt excretory protein (Bsep), and ileal bile acid transporter (Ibat); divalent metal transporter 1 (DMT1), Menke's and Wilson's metal transporters; equilibrative nucleotide transporters (Ent) 1 and 2, and constitutive nucleotide transporters (Cnt)1 and 2; peptide transporters (Pept)1 and 2; as well as ATP-binding cassette (Abc)G5 and 8 were measured in choroid plexus by the branched DNA signal amplification method. Mrp1, 4, and 5, Oatp3, Menke's transporter, DMT1, Ent1, and Pept2 mRNAs were expressed in choroid plexus at higher levels than in liver, kidney, or ileum. OctN1 and N2, Oatp2, Oat2 and 3, and Cnt1 and 2 mRNAs expressions were detectable in choroid plexus, but the levels were lower compared with that in liver, kidney, or ileum. The remaining transporters [Mrp2, Mrp3, Oct1, Oct2, Oatp1, Oatp4, Oatp5, Oatp12, Oat-K (1/2), Ntcp, Bsep, Ibat, Mdr1a, Mdr1b, Mdr2, Oat1, Ent2, Pept1, AbcG5, AbcG8] were expressed at very low levels in choroid plexus. The constitutive expression levels of different transporters in choroid plexus may provide an insight into the range of xenobiotics that can potentially be transported by the choroid plexus, thereby providing a means of xenobiotic detoxification in the brain.     

Organic anion transporting polypeptide 1a1 null mice are sensitive to cholestatic liver injury

Organic anion transporting polypeptide 1a1 (Oatp1a1) is predominantly expressed in livers of mice and is thought to transport bile acids (BAs) from blood into liver. Because Oatp1a1 expression is markedly decreased in mice after bile duct ligation (BDL). We hypothesized that Oatp1a1-null mice would be protected against liver injury during BDL-induced cholestasis due largely to reduced hepatic uptake of BAs. To evaluate this hypothesis, BDL surgeries were performed in both male wild-type (WT) and Oatp1a1-null mice. At 24 h after BDL, Oatp1a1-null mice showed higher serum alanine aminotransferase levels and more severe liver injury than WT mice, and all Oatp1a1-null mice died within 4 days after BDL, whereas all WT mice survived. At 24 h after BDL, surprisingly Oatp1a1-null mice had higher total BA concentrations in livers than WT mice, suggesting that loss of Oatp1a1 did not prevent BA accumulation in the liver. In addition, secondary BAs dramatically increased in serum of Oatp1a1-null BDL mice but not in WT BDL mice. Oatp1a1-null BDL mice had similar basolateral BA uptake (Na(+)-taurocholate cotransporting polypeptide and Oatp1b2) and BA-efflux (multidrug resistance-associated protein [Mrp]-3, Mrp4, and organic solute transporter α/β) transporters, as well as BA-synthetic enzyme (Cyp7a1) in livers as WT BDL mice. Hepatic expression of small heterodimer partner Cyp3a11, Cyp4a14, and Nqo1, which are target genes of farnesoid X receptor, pregnane X receptor, peroxisome proliferator-activated receptor alpha, and NF-E2-related factor 2, respectively, were increased in WT BDL mice but not in Oatp1a1-null BDL mice. These results demonstrate that loss of Oatp1a1 function exacerbates cholestatic liver injury in mice and suggest that Oatp1a1 plays a unique role in liver adaptive responses to obstructive cholestasis.     

Biliary excretion of pravastatin and taurocholate in rats with bile salt export pump (Bsep) impairment

The bile salt export pump (BSEP) is expressed on the canalicular membrane of hepatocytes regulating liver bile salt excretion, and impairment of BSEP function may lead to cholestasis in humans. This study explored drug biliary excretion, as well as serum chemistry, individual bile acid concentrations and liver transporter expressions, in the SAGE Bsep knockout (KO) rat model. It was observed that the Bsep protein in KO rats was decreased to 15% of that in the wild type (WT), as quantified using LC–MS/MS. While the levels of Ntcp and Mrp2 were not significantly altered, Mrp3 expression increased and Oatp1a1 decreased in KO animals. Compared with the WT rats, the KO rats had similar serum chemistry and showed normal liver transaminases. Although the total plasma bile salts and bile flow were not significantly changed in Bsep KO rats, individual bile acids in plasma and liver demonstrated variable changes, indicating the impact of Bsep KO. Following an intravenous dose of deuterium labeled taurocholic acid (D4‐TCA, 2 mg/kg), the D4‐TCA plasma exposure was higher and bile excretion was delayed by approximately 0.5 h in the KO rats. No differences were observed for the pravastatin plasma concentration–time profile or the biliary excretion after intravenous administration (1 mg/kg). Collectively, the results revealed that these rats have significantly lower Bsep expression, therefore affecting the biliary excretion of endogenous bile acids and Bsep substrates. However, these rats are able to maintain a relatively normal liver function through the remaining Bsep protein and via the regulation of other transporters. Copyright © 2016 John Wiley & Sons, Ltd.     

Effect of various antibiotics on modulation of intestinal microbiota and bile acid profile in mice

Antibiotic treatments have been used to modulate intestinal bacteria and investigate the role of intestinal bacteria on bile acid (BA) homeostasis. However, knowledge on which intestinal bacteria and bile acids are modified by antibiotics is limited. In the present study, mice were administered various antibiotics, 47 of the most abundant bacterial species in intestine, as well as individual BAs in plasma, liver, and intestine were quantified. Compared to the two antibiotic combinations (vancomycin + imipenem and cephalothin + neomycin), the three single antibiotics (metronidazole, ciprofloxacin and aztreonam) have less effect on intestinal bacterial profiles, and thus on host BA profiles and mRNA expression of genes that are important for BA homeostasis. The two antibiotic combinations decreased the ratio of Firmicutes to Bacteroidetes in intestine, as well as most secondary BAs in serum, liver and intestine. Additionally, the two antibiotic combinations significantly increased mRNA of the hepatic BA uptake transporters (Ntcp and Oatp1b2) and canalicular BA efflux transporters (Bsep and Mrp2), but decreased mRNA of the hepatic BA synthetic enzyme Cyp8b1, suggesting an elevated enterohepatic circulation of BAs. Interestingly, the two antibiotic combinations tended to have opposite effect on the mRNAs of most intestinal genes, which tended to be inhibited by vancomycin + imipenem but stimulated by cephalothin + neomycin. To conclude, the present study clearly shows that various antibiotics have distinct effects on modulating intestinal bacteria and host BA metabolism.     

Characterization of hepatobiliary organic anion transporters in Long-Evans Cinnamon rats

The liver plays important roles in the detoxification of xenobiotics. Hepatobiliary transporters contribute to hepatic uptake and efflux processes of xenobiotecs. Expressions of these transporters may be modulated under the condition of hepatic failure. Long-Evans Cinnamon (LEC) rats provide a pertinent model for basic and clinical studies on hepatitis. However, only a few reports describing the properties of hepatobiliary transporters in LEC rats have appeared in the literature. We investigated the expression levels of hepatobiliary transporters in LEC rats by real-time RT-PCR. We found that hepatic expressions of three sinusoidal organic anion transporters, Ntcp, Oatp1a1 and Oatp1a4, were decreased in LEC rats. However, no significant difference of the expressions of Mrp2 and Bsep, organic anion transporters located on canalicular membrane, were found between Wistar rats and LEC rats.     

Down-regulation of hepatic transporters for BSP in rats with indomethacin-induced intestinal injury

Previous reports have demonstrated that an intestinal injury causes hypofunctions of the liver associated with down-regulations of cytochrome P450, but an influence on hepatic transporters remains unclear. Here, we tested hepatic transporter functions in a rat model of bowel injury using indomethacin (IDM). After administration of IDM (8.5 mg/kg, i.p., 3 d), the rats suffered the intestinal impairment indicated by a reduction of alkaline phosphatase activity in mucosa. In vivo pharmacokinetic experiments of bromosulfophthalein (BSP) showed that there was a reduction in its plasma elimination rate and cumulative biliary excretion in IDM-treated rats and systemic and biliary clearances reduced to nearly 50% of the control group. Protein expressions in plasma membrane and mRNA levels of organic anion transporting polypeptide 1b2 (Oatp1b2) and multidrug resistance-associated protein 2 (Mrp2), which play hepatic BSP uptake and biliary excretion, respectively, in the liver were significantly reduced following the IDM treatment. In portal plasma, the levels of proinflammatory cytokines were unchanged, while the level of nitric oxide metabolites (NO2- + NO3-) increased to 6.5-fold that of the control. The time-course on IDM treatment indicated that, firstly, intestinal injury was induced, the NO level increased, and the hepatic Oatp1b2 and Mrp2 expression began to fall followed by an increase in plasma ALT. In conclusion, IDM-induced injury to the small intestine causes the hypofunction of hepatic Oatp1b2 and Mrp2 independently on the hepatic impairment, and NO arising from bowel injury may be one of key factors for it through the remote effect.     

雷公藤甲素致小鼠肝损伤对转运体Mrp2和Oatp2的影响

目的：通过多药耐药相关蛋白2（multidrug resistance protein 2,Mrp2）和有机阴离子转运多肽2（organic anion transporting polypeptides 2,Oatp2）初步探究雷公藤甲素诱导小鼠肝损伤的机制。方法：雄性ICR小鼠单次灌胃给予雷公藤甲素（1.0 mg·kg^-1）24 h后称重,摘眼球采血后分离血清,测定血清生化指标;取肝组织做病理切片;采用免疫印迹法检测肝脏组织中Mrp2和Oatp2的蛋白表达量。结果：与对照组相比,雷公藤甲素组肝重指数显著增加（P<0.05）,部分血清生化指标显著上升（P<0.05）,且肝细胞发生核碎裂和脂肪变性。雷公藤甲素组Oatp2的表达较对照组显著升高了32.79%（P<0.05）,而Mrp2的表达较对照组显著下降了45.47%（P<0.01）。结论：雷公藤甲素可能通过上调肝细胞膜转运体Oatp2和下调Mrp2,扰乱肝内胆红素和胆汁酸代谢排泄平衡,可能是雷公藤甲素诱导肝损伤的机制之一。     

Lipopolysaccharide-mediated regulation of hepatic transporter mRNA levels in rats.

The function of hepatic transporters is to move organic substances across sinusoidal and canalicular membranes. During extrahepatic cholestasis, transporters involved in the movement of substances from blood to bile, such as sodium/taurocholate-cotransporting polypeptide (Ntcp) and multidrug resistance protein 2 (Mrp2), are down-regulated, whereas others that transport chemicals from liver to blood, such as Mrp3, are up-regulated. Unlike extrahepatic cholestasis, where transporter expression responds to the stress of accumulating bile constituents, lipopolysaccharide (LPS)-induced intrahepatic cholestasis may be directly caused by alterations in transporter expression. The aim of this study was to quantitatively determine the effect of LPS on transporter expression and study the mechanism(s) by which LPS alters mRNA levels of major hepatic transporters in Sprague-Dawley rats. Hepatic mRNA levels of Mrp2, Mrp6, multiple drug resistance protein 1a (Mdr1a), organic anion-transporting polypeptide 1 (Oatp1), Oatp2, Oatp4, Ntcp, bile salt export pump, organic cation transporter 1 (Oct1), and organic anion transporter 3 (Oat3) were dramatically decreased, beginning approximately 6 h after LPS administration, whereas Mrp5 and Oat2 levels were unchanged. In contrast, LPS increased mRNA levels of Mrp1, Mrp3, and Mdr1b concurrently with the down-regulated transporters. Pretreatment with dexamethasone, which decreases the release of cytokines, reversed the reduction of Mdr1a, Oatp1, Oatp2, Oct1, and Ntcp mRNA following LPS administration. Furthermore, dexamethasone pretreatment also prevented the LPS-mediated increase in Mrp1, Mrp3, and Mdr1b, whereas pretreatment with aminoguanidine or gadolinium chloride, an inhibitor of inducible nitric oxide synthetase and a Kupffer cell toxicant, respectively, had no effect on the LPS-induced changes. The concurrent repression and induction of various transporters, as well as dexamethasone abatement of both LPS-mediated repression and induction, indicates that these responses may be mediated through similar pathways.     

Minimal role of hepatic transporters in the hepatoprotection against LCA-induced intrahepatic cholestasis.

The multidrug resistance-associated proteins (Mrps) are a family of adenosine triphosphate-dependent transporters that facilitate the movement of various compounds, including bile acids, out of hepatocytes. The current study was conducted to determine whether induction of these transporters alters bile acid disposition as a means of hepatoprotection during bile acid-induced cholestasis. Lithocholic acid (LCA) was used to induce intrahepatic cholestasis. C57BL/6 mice were pretreated with corn oil (CO) or known transporter inducers, phenobarbital (PB), oltipraz (OPZ), or TCPOBOP (TC) for 3 days prior to cotreatment with LCA and inducer for 4 days. Histopathology revealed that PB and TC pretreatments provide a protective effect from LCA-induced toxicity, whereas OPZ pretreatment did not. Both PB/LCA and TC/LCA cotreatment groups also had significantly lower alanine aminotransferase values than the LCA-only group. In TC/LCA cotreated mice compared with LCA only, messenger RNA (mRNA) expression of uptake transporters Ntcp and Oatp4 was significantly increased, as were sinusoidal efflux transporters Mrp3 and Mrp4. Although in PB/LCA cotreated mice, the only significant change compared with LCA-only treatment was an increase in uptake transporter Oatp4. Oatp1 was reduced in all groups compared with CO controls. No significant changes in mRNA expression were observed in Oatp2, Bsep, Mrp2, Bcrp, Mrp1, Mrp5, or Mrp6. Mrp4 protein expression was induced in the OPZ/LCA and TC/LCA cotreated groups, whereas Mrp3 protein levels remained unchanged between groups. Protein expression of Mrp1 and Mrp5 was increased in the unprotected LCA-only and OPZ/LCA mice. Thus, transporter expression did not correlate with histologic hepatoprotection, however, there was a correlation between hepatoprotection and significantly reduced total liver bile acids in the PB/LCA and TC/LCA cotreated mice compared with LCA only. In conclusion, changes in transporter expression did not correlate with hepatoprotection, and therefore, transport may not play a critical role in the observed hepatoprotection from LCA-induced cholestasis in the C57BL/6 mouse.     

Effect of acetaminophen on expression and activity of rat liver multidrug resistance-associated protein 2 and P-glycoprotein

We evaluated the effect of acetaminophen (APAP), given as a single, 1g/kg body weight dose, on expression and activity of rat liver multidrug resistance-associated protein 2 (Mrp2) and P-glycoprotein (P-gp), two major canalicular drug transporters. The studies were performed 24h after administration of the drug. APAP induced an increase in plasma membrane content of Mrp2 detected by western blotting, consistent with increased detection of the protein at the canalicular level by immunoflourescence microscopy. In vivo biliary excretion of dinitrophenyl-S-glutathione, a well known Mrp2 substrate, was slightly but significantly increased by APAP, agreeing well with upregulation of the transporter. Basal biliary excretion of oxidized glutathione, an endogenous Mrp2 substrate, was also increased by APAP, likely indicating increased hepatic synthesis as a result of APAP-induced oxidative stress followed by accelerated canalicular secretion mediated by Mrp2. APAP also increased the expression of P-gp detected by western blotting and immunofluorescence microscopy as well as the in vivo biliary secretory rate of digoxin, a model P-gp substrate. Because specific APAP-conjugated metabolites are Mrp2 substrates, we postulate that induction of Mrp2 by APAP may represent an adaptive mechanism to accelerate liver disposition of the drug. In addition, increased Mrp2-mediated elimination of oxidized glutathione may be essential in maintaining the redox equilibrium in the hepatocyte under conditions of APAP-induced oxidative stress.     

利福平对小鼠肝细胞胆汁酸转运体Bsep和Mrp2表达与定位的影响

目的利福平(Rifampicin,RIF)具有肝毒性,但其机制尚不清楚。本研究在RIF诱导的肝内胆汁淤积小鼠中,探讨RIF对肝细胞胆汁酸转运体胆汁酸输出泵(bile salt exportpump,Bsep)和多药抵抗相关蛋白-2(multidrug resistance-associated protein-2,Mrp2)表达和定位影响。方法 48只♀ICR小鼠随机分为4组,RIF1wk组:经灌胃给予RIF(200mg.kg-1.d-1),连续1周,于末次给药后6h取材;RIF6h组:单次灌胃给予RIF(200mg.kg-1)后6h取材;RIF1周对照组(CON1wk)与RIF6h对照组(CON6h):经灌胃给予等容积生理盐水。所有小鼠均收集血液和肝组织,常规生化检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、总胆红素(TB)和结合胆红素(DB),并检测小鼠血清和肝组织总胆汁酸(TBA)水平。HE染色分析肝组织病理改变。RT-PCR测定肝脏肝细胞胆汁酸转运体Bsep和Mrp2mRNA表达。免疫荧光法分析Bsep和Mrp2在肝细胞的位置。结果给予RIF1周后,小鼠血清TB由(1.25±0.69)μmol.L-1上升至(65.73±12.08)μmol.L-1,上升近70倍,DB由(0.77±0.40)μmol.L-1上升至(53.33±12.43)μmol.L-1,上升约80倍,ALP由(110.2±13.8)U.L-1上升至(279.5±80.4)U.L-1,上升约1.5倍,TBA由(3.15±0.89)μmol.L-1上升至(13.54±6.51)μmol.L-1,上升约5倍并伴有血清ALT和AST轻度升高;肝脏组织TBA由(0.15±0.04)μmol.g-1liver上升至(0.30±0.19)μmol.g-1liver,上升约2倍;肝脏组织HE染色显示肝细胞出现脂肪变性、轻度坏死和炎症。单次给予RIF6h后血清TB、DB、ALP、ALT、AST和TBA明显上升,但未观察到小鼠肝脏组织病理发生改变。免疫荧光分析显示,给予小鼠RIF1wk与单次给予RIF6h后肝细胞中Bsep和Mrp2的定位发生了改变。而无论单次给予RIF还是连续给药1周,肝细胞Bsep和Mrp2mRNA表达水平均未发生变化。结论肝细胞胆汁酸转运体Bsep和Mrp2定位改变可能是RIF诱发肝内胆汁淤积的重要机制。     

Efflux transporter expression and acetaminophen metabolite excretion are altered in rodent models of nonalcoholic fatty liver disease.

Efflux transporters are responsible for the excretion of numerous xenobiotics and endobiotics and thus play an essential role in proper liver and kidney function. Nonalcoholic fatty liver diseases (NAFLDs) comprise a spectrum of disorders that range from simple fatty liver (SFL) to nonalcoholic steatohepatitis (NASH). Although the precise events leading to NAFLD are unclear, even less is known about the effects on efflux transporter expression and drug disposition. The purpose of this study was to determine the effect of NAFLD on efflux transporter expression in rat liver as well as on acetaminophen (APAP) metabolite excretion. To simulate SFL and NASH, rats were fed either a high-fat (HF) or a methionine- and choline-deficient (MCD) diet for 8 weeks. In the livers of MCD rats, there were striking increases in both mRNA and protein levels of multidrug resistance-associated protein (Mrp) 3, Mrp4, and breast cancer resistance protein, as well as increased Mrp2 protein. After administration of a nontoxic dose of APAP, biliary concentrations of APAP-sulfate, APAP-glucuronide (APAP-GLUC), and APAP-glutathione were reduced in MCD rats. The effects of the HF diet on both transporter expression and APAP disposition were by comparison far less dramatic than the MCD diet-induced alterations. Whereas APAP-sulfate levels were also decreased in MCD rat plasma, the levels of the Mrp3 substrate APAP-GLUC were elevated. Urinary elimination of APAP metabolites was identical between groups, except for APAP-GLUC, the concentration of which was 80% higher in MCD rats. These studies correlate increased hepatic Mrp3 protein in the MCD model of NASH with increased urinary elimination of APAP-GLUC. Furthermore, the proportional shift in elimination of APAP metabolites from bile to urine indicates that MCD-induced alterations in efflux transporter expression can affect the route of drug elimination.     

Coordinated expression of multidrug resistance-associated proteins (Mrps) in mouse liver during toxicant-induced injury.

Following acute chemical injury, hepatocytes are generally more resistant to toxicant re-exposure. Alterations in expression of hepatobiliary transport systems may contribute to this resistance by preventing accumulation of potentially toxic chemicals. Previous data demonstrate the concomitant reduction of uptake transporter and induction of efflux transporter mRNA during chemical liver injury. The present study further characterizes the expression of multidrug resistance-associated proteins 1-4 (Mrp1-4), breast cancer resistance protein (Bcrp) and sodium-taurocholate co-transporting polypeptide (Ntcp) in mouse liver following administration of the hepatotoxicants acetaminophen (APAP) and carbon tetrachloride (CCl4). Mice received hepatotoxic doses of APAP (400 mg/kg), CCl4 (10 or 25 microl/kg), or vehicle, ip. Livers were collected at 6, 24, and 48 h for Western blot quantification and immunofluorescence analysis. Protein expression of Bcrp was unchanged with treatment. Ntcp levels were preserved in APAP-exposed livers and reduced to 30-50% of control after CCl4. Conversely, Mrp1-4 expression was differentially up-regulated. CCl4 increased Mrp1 (3.5-fold), Mrp2 (1.4-fold), and Mrp4 (26-fold) while reducing Mrp3 levels to 20% of control. Administration of APAP enhanced expression of Mrp2 (1.6-fold), Mrp3 (3.5-fold), and Mrp4 (16-fold). Immunostaining of liver sections obtained 48 h after hepatotoxicant treatment confirmed expression patterns of a subset of transporters (Bcrp, Ntcp, Mrp3, and Mrp4). Double immunofluorescence imaging demonstrated the simultaneous down-regulation of Ntcp and up-regulation of Mrp4 in hepatocytes adjacent to the central vein after CCl4. Altered expression of transporters may reduce the overall chemical burden of an injured liver during recovery and contribute to the resistance of hepatocytes to subsequent toxicant exposure.     

Change of drug excretory pathway by CCl4-induced liver dysfunction in rat

Liver dysfunction affects the pharmacokinetics of drugs. The liver plays an important role in drug excretion as well as drug metabolism and pharmacokinetics. In the present study, the relationship between changes in the cefmetazole (CMZ) excretory pathway and the degree of liver dysfunction induced by CCl(4) treatment was investigated. CMZ is mainly excreted as an unchanged form in feces in control rats. Depending on the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), urinary CMZ excretion was increased, whereas fecal CMZ excretion was decreased in rat with liver dysfunction. The AUC of CMZ in rats with severe liver dysfunction was approximately 2-fold higher than that in control rats. Since drug transporters could be involved in drug excretion, changes in the expression of representative hepatic drug transporters in liver dysfunction were investigated by rat DNA microarray. Basolateral solute carrier transporters such as Ntcp, Oct1, and Oatp2 were decreased and basolateral ATP-binding cassette transporters such as Mrp3 and Mrp4 were increased by the CCl(4) treatment. On the other hand, canalicular Mrp2 and Bsep were decreased, but Mdr1 was increased. However, the transporter system for CMZ has not been identified yet. In conclusion, we clarified that the fecal and urinary excretory profiles of CMZ were changed clearly depending on the serum AST and ALT levels in liver dysfunction. The changes in the CMZ excretory pathway might be responsible for the changes in the expression of drug transporters.     

Biliary efflux transporters involved in the clearance of rosuvastatin in sandwich culture of primary rat hepatocytes

Rosuvastatin (a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor) has been shown to be excreted mostly unchanged into the bile; interactions on the level of hepatic apical efflux transporters may represent a risk of liver toxicity. So far, controversial and insufficient data are available concerning transporters involved in the elimination process. This study was designed to elucidate, which transporters take part in the biliary clearance of rosuvastatin using sandwich-cultured primary rat hepatocytes. The canalicular efflux of rosuvastatin was measured in the presence of inhibitors: Ko 134, mitoxanthrone, novobiocin for breast cancer resistance protein (Bcrp); verapamil for multidrug resistance protein (Mdr1); benzbromarone, sulfasalazine, probenecid for multidrug resistance associated protein (Mrp 2); and cyclosporine A, glibenclamide, troglitazone for bile salt export pump (Bsep). Mrp2 inhibitors decreased the biliary efflux of rosuvastatin most potently by 78.9%, 35%, 54.1%; benzbromarone, probenecid, sulfasalazine, respectively, while Bcrp and Bsep inhibitors showed much less effect (29.1%, 23.0% ,30.0%; Ko 134, mitoxanthrone, novobiocin, respectively, and 32.6%, 29.3%, 20.6%, glibenclamide, cyclosporine A, troglitazone, respectively). The marked decline of canalicular transport by Mrp2 inhibitors suggests major role of Mrp2 in this process; however, Bcrp and Bsep might also contribute to the biliary elimination of rosuvatatin in sandwich-cultured rat hepatocytes.