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Troglitazone, the first peroxisome proliferator-associated receptor γ agonist developed as an antidiabetic drug, was withdrawn from the market due to idiosyncratic severe liver toxicity. One proposed mechanism by which troglitazone causes liver injury is induction of mitochondrial membrane permeability transition (MPT), which occurs in a calcium-independent phospholipase A2 (iPLA2)-dependent manner at a concentration of 10 µM. MPT, induced by opening of the MPT pore, leads to the release of cytochrome c and consequent apoptosis or necrosis. In the present study, we aimed to clarify the mechanism of troglitazone-induced MPT in more detail using isolated rat liver mitochondria. We focused on extra-mitochondrial Ca2+ and membrane potential as triggers of iPLA2 activation or MPT induction. As a link between iPLA2 and MPT, we focused on cardiolipin (CL), a unique, mitochondria-specific phospholipid with four acyl chains that affects respiration, the morphology, and other mitochondrial functions. We found that (1) Ca2+ release from the mitochondrial matrix was induced prior to troglitazone-induced onset of MPT, (2) released Ca2+ was involved in troglitazone-induced MPT, (3) mild depolarization (approximately 10%) may be a trigger of troglitazone-induced MPT and (4) enhanced decomposition of CL following mitochondrial iPLA2 activation might mediate troglitazone-induced MPT.

Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments.

Inhibition of bile salt export pump (BSEP) causes hepatic accumulation of toxic bile acid (BA), leading to hepatocyte death. We reported a sandwich-cultured hepatocyte (SCH)-based model that can estimate potential cholestatic compounds by assessing their ability to induce hepatotoxicity in combination with a titrated amount of human 12 BA species. However, there is little information about the specific BAs responsible for hepatotoxicity, when BSEP is inhibited. This study measured the accumulation of each BA in rat SCHs in the presence of 10 μM cyclosporine A (CsA), which only inhibits BSEP, and 50 μM CsA, which further inhibits basolateral BA efflux transporters. The accumulation of all BAs (not significant for deoxycholic acid [DCA]) was observed in the presence of 10 μM CsA. In particular, 3 BAs (chenodeoxycholic acid [CDCA], DCA, and glyco-DCA [GDCA]) showed increased toxicity in the presence of 10 μM CsA, whereas the other BAs did not. In addition to these BAs, taurolithocholic acid, glyco-CDCA, and glycocholic acid showed increased toxicity in the presence of 50 μM CsA, but additional accumulation of these BAs could not be observed. These results indicate the inhibiting BSEP results in the accumulation of CDCA, GDCA, and partially DCA, thereby resulting in hepatotoxicity.

Troglitazone and pioglitazone were developed as thiazolidinedione-type antidiabetes drugs, but only troglitazone was withdrawn from the markets due to severe liver injury. As both troglitazone and its sulfate metabolite are strong inhibitors of the bile salt export pump (BSEP), troglitazone-induced bile acid (BA) retention is thought to be one of the underlying mechanisms of liver injury. However, pioglitazone is also a strong BSEP inhibitor, indicating other mechanisms may also be involved in troglitazone-induced BA retention. Although retention of hydrophobic BAs (eg, chenodeoxycholic acid [CDCA]: a nonamidated BA) is known to cause hepatocyte injury, little is known about the hepatic conversion of nonamidated, hydrophobic BA species into less toxic hydrophilic BAs (eg, glycochenodeoxycholic acid: amidated BA) as a mechanism of drug-induced liver injury. In this study, we, therefore, investigated the effects of troglitazone and pioglitazone on BA amidation and the role of amidated BAs in troglitazone-associated BA-mediated hepatotoxicity. We also evaluated the intracellular BA composition of human hepatocytes treated with nonamidated BA species (CDCA or deoxycholic acid [DCA]) in the presence of troglitazone or pioglitazone. Amidation of CDCA and DCA was significantly inhibited by troglitazone (IC50: 5 and 3 µmol/l, respectively), but not pioglitazone. Moreover, treatment with troglitazone led to the retention of CDCA and DCA and decrease of glycine-amidation in hepatocytes. From these results, we suggest that troglitazone-induced liver injury might be caused by the accumulation of nonamidated BAs in hepatocytes due to inhibition of BA amidation.

Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity.

Bile acid (BA) retention within hepatocytes is an underlying mechanism of cholestatic drug-induced liver injury (DILI). We previously developed an assay using sandwich-cultured human hepatocytes (SCHHs) to evaluate drug-induced hepatocyte toxicity accompanying intracellular BA accumulation. However, due to shortcomings commonly associated with the use of primary human hepatocytes (e.g., limited availability, lot-to-lot variability, and high cost), we examined if the human hepatic stem cell line, HepaRG, might also be applicable to our assay system. Consequently, mRNA expression levels of human BA efflux and uptake transporters were lower in HepaRG cells than in SCHHs but higher than in HepG2 human hepatoma cells. Nevertheless, HepaRG cells and SCHHs showed similar toxicity responses to 22 selected drugs, including cyclosporine A (CsA). CsA (10 μM) was cytotoxic toward HepaRG cells in the presence of BAs and also reduced the biliary efflux rate of [(3)H]taurocholic acid from 38.5% to 19.2%. Therefore, HepaRG cells are useful for the evaluation of BA-dependent drug toxicity caused by biliary BA efflux inhibition. Regardless, the prediction accuracy for cholestatic DILI risk was poor for HepaRG cells versus SCHHs, suggesting that our DILI model system requires further improvements to increase the utility of HepaRG cells as a preclinical screening tool.

The liver performs a variety of essential functions; hence drug-induced liver injury (DILI) is a serious concern that can ultimately lead to the withdrawal of a drug from the market or discontinuation of drug development. However, the mechanisms of drug-induced liver injury are not always clear. We hypothesized that drugs may inhibit the liver recovery process, especially bile canalicular (BC) network reformation, leading to persistent liver injury and deterioration, and tested this hypothesis in the present work. The BC structure disappeared in mice following treatment with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or thioacetamide (TAA) for 4 weeks, then reappeared after 4 weeks of receiving a normal diet. By contrast, reconstruction of the BC structure was suppressed in mice fed a diet containing 0.3% benzbromarone (BBR; which can induce fatal liver injury in clinical settings) after liver injury. Plasma ALT levels were increased significantly in mice treated with BBR after DDC or TAA treatment, compared with BBR alone. To confirm whether BBR has a direct inhibitory effect on hepatocytes, we also examined BC reformation in primary cultured mouse hepatocytes with a sandwich configuration. Under these culture conditions, the BC network rapidly reformed from days 2 and 3 after seeding. During the reformation period, BBR inhibited BC reformation significantly. These results suggest that BBR inhibits BC reconstruction and delays recovery from pre-existing liver injury.

The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs.

Mitochondrial dysfunction plays a central role in drug-induced liver injury. To evaluate drug-induced mitochondrial impairment, several isolated mitochondria- or cell line-based assays have been reported. Among them, culturing HepG2 cells in galactose provides a remarkable method to assess mitochondrial toxicity by activating mitochondrial aerobic respiration. We applied this assay to primary rat hepatocytes by culturing cells in galactose and hyperoxia to enhance the evaluation of metabolism-related drug-induced mitochondrial toxicity. Conventional culture of primary hepatocytes under high-glucose and hypoxic conditions could force cells to switch energy generation to glycolysis. By contrast, cells cultured in galactose and hyperoxia could maintain energy generation from mitochondrial aerobic respiration, which is consistent with physiological conditions, and consequently improve the susceptibility of cells to mitochondrial toxicants. Measuring the toxicities of test compounds in primary rat hepatocytes cultured in modified conditions provides a useful model to identify mitochondrial dysfunction-mediated drug-induced hepatotoxicity. © 2017 by John Wiley & Sons, Inc. Keywords: primary rat hepatocytes; mitochondrial toxicity; oxygen requirement; galactose; drug-induced liver injury

Troglitazone, a member of the thiazolidinedione class of antidiabetic drugs, was withdrawn from the market because it causes severe liver injury. One of the mechanisms for this adverse effect is thought to be mitochondrial toxicity. To investigate the characteristics of troglitazone-induced liver toxicity in more depth, the toxicological effects of troglitazone on hepatocytes and liver mitochondria were investigated using a rat model of type 2 diabetes mellitus (T2DM). Troglitazone was found to increase mitochondrial permeability transition (MPT) in the liver mitochondria of diabetic rats to a greater extent than in control rats, whereas mitochondrial membrane potential and oxidative phosphorylation were not affected. To identify the factors associated with this increase in susceptibility to MPT in diabetic rats, we assessed the oxidative status of the liver mitochondria and found a decrease in mitochondrial glutathione content and an increase in phospholipid peroxidation. Moreover, incorporation of oxidized cardiolipin, a mitochondrion-specific phospholipid, was involved in the troglitazone-induced alteration in susceptibility to MPT. In conclusion, liver mitochondria display disease-associated mitochondrial lipid peroxidation in T2DM, which facilitates the higher susceptibility to troglitazone-induced MPT. Thus, greater susceptibility of liver mitochondria may be a host factor leading to troglitazone-induced hepatotoxicity in T2DM.

Methotrexate (MTX) causes intestinal damage, resulting in diarrhea. The side effects often disturb the cancer chemotherapy. We previously reported that AGE protected the small intestine of rats from the MTX-induced damage. In the present paper, the mechanism of the protection of AGE against the MTX-induced damage of small intestine was investigated, using IEC-6 cells originating from rat jejunum crypt. The viability and apoptosis of IEC-6 cells were examined in the presence of MTX and/or AGE. The viability of IEC-6 cells exposed to MTX was decreased by the increase of MTX concentration. The MTX-induced loss of viable IEC-6 cells was almost completely prevented by the presence of more than 0.1% AGE. In IEC-6 cells exposed to MTX, the cromatin condensation, DNA fragmentation, caspase-3 activation and cytochrome c release were observed. These were preserved to the control levels by the presence of AGE. MTX markedly decreased intracellular GSH in IEC-6 cells, but the presence of AGE in IEC-6 cells with MTX preserved intracellular GSH to the control level. IEC-6 cells in G2/M stage markedly decreased 72 h after the MTX treatment, which was preserved to the control level by the presence of AGE. These results indicated that AGE protected IEC-6 cells from the MTX-induced damage. The MTX-induced apoptosis of IEC-6 cells was shown to be depressed by AGE. AGE may be useful for the cancer chemotherapy with MTX, since AGE reduces the MTX-induced intestinal damage.

Idiosyncratic drug-induced liver injury is a clinical concern with serious consequences. Although many preclinical screening methods have been proposed, it remains difficult to identify compounds associated with this rare but potentially fatal liver condition. Here, we propose a novel assay system to assess the risk of liver injury. Rat primary hepatocytes were cultured in a sandwich configuration, which enables the formation of a typical bile canalicular network. From day 2 to 3, test drugs, mostly selected from a list of cholestatic drugs, were administered, and the length of the network was semi-quantitatively measured by immunofluorescence. Liver injury risk information was collected from drug labels and was compared with in vitro measurements. Of 23 test drugs examined, 15 exhibited potent inhibition of bile canalicular network formation (

Drug-induced liver injury (DILI) is of concern to the pharmaceutical industry, and reliable preclinical screens are required. Previously, we established an in vitro bile acid-dependent hepatotoxicity assay that mimics cholestatic DILI in vivo. Here, we confirmed that this assay can predict cholestatic DILI in clinical situations by comparing in vitro cytotoxicity data with in vivo risk. For 38 drugs, the frequencies of abnormal increases in serum alkaline phosphatase (ALP), transaminases, gamma glutamyltranspeptidase (γGT), and bilirubin were collected from interview forms. Drugs with frequencies of serum marker increases higher than 1% were classified as high DILI risk compounds. In vitro cytotoxicity was assessed by monitoring lactate dehydrogenase release from rat and human sandwich-cultured hepatocytes (SCRHs and SCHHs) incubated with the test drugs (50 μM) for 24 hours in the absence or presence of a bile acids mixture. Receiver operating characteristic analyses gave optimal cutoff toxicity values of 19.5% and 9.2% for ALP and transaminases in SCRHs, respectively. Using this cutoff, high- and low-risk drugs were separated with 65.4-78.6% sensitivity and 66.7-79.2% specificity. Good separation was also achieved using SCHHs. In conclusion, cholestatic DILI risk can be successfully predicted using a sandwich-cultured hepatocyte-based assay.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

We investigated whether the species difference in the biliary excretion activity of some Mrp2 substrates was attributable to the intrinsic transport potential or the expression level of Mrp2, especially in rat and dog. Dog Mrp2 cDNA was isolated from beagle dog liver, and a vesicle transport study was performed using recombinant rat and dog Mrp2 expressed in insect Sf9 cells. The ATP-dependent transport of 17beta-estradiol 17-(beta-D-glucuronide) ([3H]E(2)17betaG) and leukotriene C4 ([3H]LTC4), normalized by the absolute protein expression level, was similar in both Mrp2s. The Mrp2 protein expression in dog liver was only 10% of that in rat liver and was comparable with the reported difference in the biliary excretion clearance of temocaprilat as Mrp2 substrate. In contrast to LTC4, unique transport kinetics for E(2)17betaG were evident in dog Mrp2. In addition to the high-affinity site with a K(m) value of 3.25 +/- 0.10 microM, which is similar to that in rat Mrp2 (4.81 +/- 1.21 microM), dog Mrp2 has an additional low-affinity site (>>75 microM), which makes a major contribution to the transport of E(2)17betaG (65% of the total transport capacity at tracer concentration). In summary, the difference in the biliary excretion activity of Mrp2 substrates between rat and dog depends on the Mrp2 protein expression level rather than the intrinsic transport activity of the transporter molecules. The unique transport properties of glucuronide conjugates by dog Mrp2 may lead to the species difference involving the drug-drug interaction or drug-induced hyperbilirubinemia on the bile canalicular membrane.

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.

Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a "bile acid-independent" mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17-beta-D-glucuronide (E(2)17 beta G; by 265%), leukotriene C4 (LTC(4); by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases.

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

Retinol-binding protein (RBP) is a specific transport protein which carries retinol in the circulation. RBP concentration in plasma and liver of rats following a large dose of acetaminophen (APAP) intraperitoneally was examined. The RBP concentration in plasma decreased significantly at 12 hr after the APAP administration, while the plasma albumin concentration was affected a little. Western blot and northern blot analyses showed marked changes in RBP but not in albumin. Thus, RBP was suggested to be more sensitive for the acute drug-induced hepatotoxicity than albumin. The decrease of RBP concentration in plasma was suggested to be caused by the dysfunction of RBP synthesis in the liver.

1. Raloxifene-6-glucuronide (R6G) is a substrate of rat multidrug resistance-associated protein 2 (Mrp2), a transporter responsible for biliary excretion of organic anions. 2. Pharmacokinetic modeling of R6G in Eisai hyperbilirubinemic rats (EHBRs), hereditary Mrp2-deficient rats, and wild-type Sprague-Dawley rats (SDRs) indicated that reduction in not only biliary excretion but also hepatic uptake of R6G influenced low clearance in EHBRs. 3. An integration plot study demonstrated that the hepatic uptake of R6G was 66% lower in EHBRs than that in SDRs. A reduction was observed for the other Mrp2 substrate Valsartan (95% lower) but not for estradiol-17β-glucuronide (E217βG). This variation may be associated with the difference in substrate specificity of transporters and/or inhibition of hepatic uptake of organic anions by endogenous substances such as bilirubin glucuronides. 4. In conclusion, incidental alteration of the hepatic uptake of organic anions should be considered as an explanation of their enhanced systemic exposure in EHBRs.

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

Multidrug resistance-associated protein 2 (MRP2) transports glutathione conjugates, glucuronide conjugates, and sulfated conjugates of bile acids. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, using the isolated membrane vesicles from Sf9 cells infected with the recombinant baculoviruses. By normalizing the transport activity for compounds by that for estradiol 17beta-D-glucuronide (E(2)17betaG), it was indicated that the site-directed mutagenesis from Lys to Met at 325 (K325M) and from Arg to Leu at 586 (R586L) results in a marked reduction in the transport for glutathione conjugates [2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene (LT) C(4)] without affecting that for 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl) benzothiazole glucuronide and taurolithocholate sulfate. In contrast to the reduced affinity for DNP-SG, the affinity for E(2)17betaG was increased severalfold in these mutant Mrp2s, suggesting the amino acids at 325 and 586 play an important role in distinguishing between glutathione and glucuronide conjugates. The comparable affinity for LTD(4), LTE(4), and LTF(4) in these mutant Mrp2s with that in wild-type Mrp2 indicates that recognition of LTC(4) metabolites by Mrp2 is different from that of LTC(4). The transport activity for glutathione conjugate was retained on R586K, whereas no such complementary cationic amino acid effect was observed in K325R. In addition, R1206M and E1208Q exhibited the loss of transport activity for the tested compounds. The results of the present study demonstrate that the charged amino acids in the transmembrane domain of rat Mrp2 may play an important role in the recognition and/or transport of its substrates.

Purpose. The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK).

The antiplatelet drug, ticlopidine (TIC), reportedly causes cholestatic liver injuries. The present study analyzed the effect of TIC on bile formation, revealing that the biliary secretion of phospholipids was significantly decreased in TIC-administered Sprague Dawley (SD) rats. However, the effect of TIC on biliary phospholipids was not observed in SD rats pretreated with diethylaminoethyl diphenylpropylacetate that inhibits cytochrome P450s (P450), or in Eisai hyperbilirubinemic rats (EHBR) lacking functional multidrug resistance-associated protein 2 (MRP2/ABCC2). These results suggest that glutathione-conjugated TIC metabolites (TIC-SGs), which were formed in the liver after P450s-mediated metabolism and were excreted extensively into bile by MRP2, mediated the observed alterations of the bile composition. Administration of TIC caused significant liver injuries in SD rats, with decreased biliary phospholipids, but not in EHBR, consistent with the in vitro observation that phospholipid-bile acid-mixed micelles moderated the cytotoxic effects of bile acids. Further analyses revealed that TIC-SGs did not directly inhibit multidrug resistance 3 P-glycoprotein (MDR3/ABCB4)-mediated phosphatidylcholine efflux in vitro. Because the diminished biliary secretion of phospholipids with TIC administration was restored by taurocholate infusion in SD rats, the decreased biliary concentration of bile acids, due to the stimulation of bile acid-independent bile flow driven by TIC-SGs, might have indirectly attenuated phospholipid secretion. In conclusion, extensive biliary excretion of TIC-SGs decreased the biliary secretion of phospholipids, which might have increased the risk of TIC-induced cholestatic liver injury.

Biliary secretion of bile acids and phospholipids, both of which are essential components of biliary micelles, are mediated by the bile salt export pump (BSEP/ABCB11) and multidrug resistance 3 P-glycoprotein (MDR3/ABCB4), respectively, and their genetic dysfunction leads to the acquisition of severe cholestatic diseases. In the present study, we found two patients with itraconazole (ITZ)-induced cholestatic liver injury with markedly high serum ITZ concentrations. To characterize the effect of ITZ on bile formation in vivo, biliary bile acids and phospholipids were analyzed in ITZ-treated rats, and it was revealed that biliary phospholipids, rather than bile acids, were drastically reduced in the presence of clinically relevant concentrations of ITZ. Moreover, by using MDR3-expressing LLC-PK1 cells, we found that MDR3-mediated efflux of [¹⁴C]phosphatidylcholine was significantly reduced by ITZ. In contrast, BSEP-mediated transport of [³H]taurocholate was not significantly affected by ITZ, which is consistent with our in vivo observations. In conclusion, this study suggests the involvement of the inhibition of MDR3-mediated biliary phospholipids secretion in ITZ-induced cholestasis. Our approach may be useful for analyzing mechanisms of drug-induced cholestasis and evaluating the cholestatic potential of clinically used drugs and drug candidates.

We previously demonstrated the increase of reactive oxygen species (ROS) production and myeloperoxidase (MPO) activity in the small intestine of methotrexate (MTX)-treated rats. In the present study, we investigated the role of ROS modulating intestinal mucosal permeability in this damage.MTX (20 mg/kg body weight) was administered to rats intravenously. N-Acetylcysteine (NAC; 80 mg/kg body wt), an antioxidant and a precursor of glutathione (GSH) was administered to rats intraperitoneally to investigate the contribution of ROS to the intestinal permeability enhancement. Intestinal permeability was evaluated by determining that of a poorly absorbable marker, fluorescein isothiocyanate-labeled dextran (FD-4; average molecular mass, 4.4 kDa) using the in vitro everted intestine technique. The occurrence of oxidative stress in the small intestine was assayed by measuring chemiluminescence and thiobarbituric acid reactive substances (TBARS) productions in mucosal homogenates of the small intestine.The mucosal permeability of FD-4 significantly (p0.01) increased in MTX-treated rats compared with control rats, as demonstrated by a twofold increase of FD-4 permeation clearance. This suggests an increase in paracellular permeability. Interestingly, the ROS production was observed preceding the increase of paracellular permeability. Treatment with NAC prevented the MTX-induced ROS production and the increase of paracellular permeability.NAC protected the small intestine of rats from MTX-induced change in paracellular permeability, suggesting that ROS played an important role in the enhanced paracellular permeability.

MRP2(/ABCC2) excretes amphiphilic organic anions into bile, and associates with detergent-resistant bile canalicular membrane domains (DRM). Here, we have evaluated sensitivities of MRP2 transport function and DRM association by titrating the cellular cholesterol content. We demonstrate that the role of cholesterol in the partitioning of MRP2 to DRM can be separated from the role of cholesterol in the function of MRP2, such that (i) cholesterol is not necessary for the polarized distribution of MRP2 at the canalicular membrane, (ii) partitioning into DRM is not required for MRP2 function, yet (iii) the presence of cholesterol is necessary for transport activity. (c) 2008 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Oxidative stress is known to be a common feature of cholestatic syndrome. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile salt-independent bile flow, under acute oxidative stress, and a series of signaling pathways finally leading to the activation of novel protein kinase C were involved in this mechanism; however, it has been unclear whether the internalized Mrp2 localization was relocalized to the canalicular membrane when the intracellular redox status was recovered from oxidative stress. In this study, we demonstrated that decreased canalicular expression of Mrp2 induced by tertiary-butyl hydroperoxide (t-BHP) was recovered to the canalicular membrane by the replenishment of GSH by GSH-ethyl ester, a cell-permeable form of GSH. Moreover, pretreatment of isolated rat hepatocytes with colchicine and PKA inhibitor did not affect the t-BHP-induced Mrp2 internalization process but did prevent the Mrp2 recycling process induced by GSH replenishment. Moreover, intracellular cAMP concentration similarly changed with the change of intracellular GSH content. Taken together, our data clearly indicate that the redox-sensitive balance of PKA/PKC activation regulates the reversible Mrp2 localization in two different pathways, the microtubule-independent internalization pathway and -dependent recycling pathway of Mrp2.

Ezetimibe (EZE) selectively blocks intestinal cholesterol absorption by interacting with Niemann-Pick C1 Like 1 (NPC1L1). After administration, EZE is extensively metabolized in liver and intestine to its phenolic glucuronide form (EZE-G) by uridine diphosphate glucuronosyltransferases (UGTs), among which UGT1A1 and 1A3 exhibit highest activity. EZE-G is excreted into bile and undergoes extensive enterohepatic recirculation. Considering the pharmacokinetic properties of EZE and an in vitro binding study showing the high affinity binding of EZE-G to NPC1L1, glucuronidation by UGTs has been believed to be essential for the pharmacological efficacy of EZE. To study the role of glucuronidation by UGTs for the cholesterol-lowering effect of EZE, in vitro and in vivo studies were performed using Gunn rats, which hereditarily lack the expression of UGT1A enzymes. The biliary excreted amount of EZE-G was reduced by 73% up to 3 h after administration of EZE (0.3 mg/kg) in Gunn rats, which is consistent with the reduction of in vitro EZE glucuronidation activity found in liver and intestinal microsome from Gunn rats. These results indicate that the formation of EZE-G in Gunn rats is much lower than that in Wistar rats. However, in vivo study showed that 0.3 mg/kg EZE, which is the clinically relevant dose, reduced cholesterol absorption in both Wistar and Gunn rats to nearly the same degree and the dose dependence was not significantly different between Wistar and Gunn rats at the range 0.001 approximately 0.3 mg/kg. These results indicate that a deficiency of UGT1A activity does not necessarily alter the cholesterol-lowering effect of EZE in rats at therapeutic doses.

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.

The absorption of nutrients is mainly mediated by specific carriers and generally retarded following gastrointestinal injury. The aim of this study was to assess the effect of repeated oral administration of 5-fluorouracil (5-FU) on the intestinal absorption of glucose by using 3-O-methyl-D-glucose (3-OMG), a glucose analogue that is not metabolized, as a probe. Repeated administration of 5-FU (60 mg/kg/day for 3 days) readily induced intestinal mucosal injury assessed by visual observation and loss of intestinal wet weight. At the same time, the carrier-dependent absorption clearance of 3-OMG was increased 1.8-fold, while the carrier-independent absorption assessed by L-glucose transport was not affected. Phloretin, a glucose transporter 2 (GLUT2) inhibitor, completely abolished the absorption of 3-OMG in both control and 5-FU-treated mice, indicating the specific effect on the carrier-dependent process. Protein and mRNA expressions of GLUT2 were significantly higher in 5-FU-treated mice compared to the control mice. Sodium (Na(+)) glucose co-transporter 1 (SGLT1) expressions were also moderately elevated in 5-FU-treated mice. Concomitantly, the uptake of D-glucose into both isolated brush border and basolateral membrane vesicles was significantly increased. These results indicate that repeated oral administration of 5-FU did not hamper, but unexpectedly induced, SGLT1 and GLUT2 expression to enhance glucose absorption.

Cholestatic and choleretic effect are well known for protein kinase C activator and inhibitor, respectively. However, post-translational regulation, especially the effect of phosphorylation status of the biliary transporters on their intrinsic transport activity has not been fully understood. In this study, effect of phosphorylation on the transport activity of Mrp2, a biliary organic anion transporter, was examined in membrane vesicles isolated from Sf9 cells co-expressing excess amount of protein kinase Calpha (PKCalpha). Mrp2-mediated transport activity was enhanced to three-fold by co-expressing PKCalpha. At the same time, phosphorylation of Mrp2 was also detected. The Km and Vmax values for the transport of [3H]estradiol-17beta-D-glucuronide exhibited a 1.5-fold decrease and a 1.9-fold increase, respectively. Probenecid (100 microM) and benzylpenicillin (1 mM), both are activator of Mrp2, did not stimulated the transport activity of phosphorylated Mrp2. On the other hand, transport activity was further stimulated by Estron-3-sulfate and taurocholic acid. Similar mechanism that occurred in the presence of probenecid and benzylpenicillin, but different from that occurred in the presence of Estron-3-sulfate and taurocholic acid seems to be involved in the stimulation. Considering the discrepancy between the previous in vivo inhibitory effect of PKC activators and our in vitro stimulatory effect of PKCalpha on Mrp2 transport activity, direct modulation of Mrp2-transport activity may be minor if any under in vivo condition.

4-Hydroxy-trans-2,3-nonenal (HNE) is a toxic end product of lipid peroxidation. This multifunctional aldehyde reacts with proteins, phospholipids, and nucleic acids, consequently activating/inactivating enzymes, affecting signal transduction and gene expression. HNE is mainly detoxified by glutathione (GSH) conjugation. In our previous report, we showed that GSH conjugates of 4-hydroxynonenal (HNE-SG) are substrates of multidrug resistance-associated protein 2 (MRP2). MRP2 has been shown to export HNE-SG conjugates into the extracellular space. In the present study, the role of MRP2 in the detoxification of HNE was studied using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2. MRP2 reduced the intracellular accumulation of HNE-SG conjugate but unexpectedly increased the susceptibility of cells to HNE. The viability of cells was reduced to approximately 70% in the presence of 62.5 microM HNE in MDCK II cells expressing MRP2, whereas MDCK II cells remained unaffected. MRP2 accelerated the elimination of intracellular GSH via a conjugation reaction with HNE (half-life of GSH was 30.1 and 12.2 min for MDCK II cells and MDCK II cells expressing MRP2, respectively). Moreover, the consumption of GSH was unlimited in MDCK II cells expressing MRP2, finally resulting in necrosis. These results indicate that MRP2 has an adverse effect during the detoxification of HNE in MDCK II cells and suggest that expression of MRP2 may enhance the damage caused by oxidative stress.

The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH(-) gradient or an inwardly directed H(+)gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.

The transport characteristics of fluorescein methotrexate (F-MTX) were studied by using the rat intestinal crypt cell line IEC-6. Enhanced accumulation of F-MTX at 4°C suggests the existence of an active efflux system. MK-571, an inhibitor of the multidrug resistance-associated protein/ATP binding cassette C (MRP/ABCC) family, also enhanced the accumulation of F-MTX. Transcellular transport of F-MTX from the apical to the basolateral compartment was 2.5 times higher than the opposite direction. This vectorial transport was also reduced by MK-571, indicating the presence of Mrp-type transporter(s) on the basolateral membrane. Mrp3 mRNA was readily detectable, and the protein was localized on the basolateral membrane. Uptake of FMTX into membrane vesicles from IEC-6 cells and Spodoptera frugiperda-9 cells expressing rat Mrp3 were both ATP dependent and saturable as a function of the F-MTX concentration. Similar Kmvalues (11.0 ± 1.8 and 4.5 ± 1.1 μM) and inhibition profiles by MK-571, estradiol-17β-d-glucuronide, and taurocholate for the ATP-dependent transport of F-MTX into these vesicles were obtained. These findings suggest that the efflux of F-MTX is mediated by Mrp3 on the basolateral membrane of IEC-6 cells.

Multidrug resistance-associated protein 2 (MRP2), along with MRPI, transports glutathione and glucuronide conjugates as well as non-conjugated organic anions. In the present study, we examined the role of charged amino acids in the transmembrane domains of rat Mrp2, conserved among MRP families, by means of sitedirected mutagenesis. A single amino acid mutation from Lys to Met at 325 (K325M), from Arg to Leu at 586 (R586L) and from Asp to Asn at 329 (D329N) resulted in a marked reduction in the transport activity of glutathione-S conjugates (2, 4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4 (LTC4)) without affecting that of the subsequent metabolites of LTC4 (LTD4, LTE4 and LTF4) or glucuronide conjugates (17β-estradiol 17-β-D-glucuronide (E217βG) and E3040-glucuronide). In contrast to the reduced affinity for DNP-SG and LTC4, the affinity for E217βG was increased several-fold in these mutants, suggesting that amino acids at 325 and 586 may play an important role in distinguishing between glutathione and glucuronide conjugates. Furthermore, R1096L acquired the ability to transport TC, which is not transported by Mrp2. Since Mrp3, which transports TC, has a neutral amino acid at 1084 corresponding to this position, the charge-dependence on the transport ability of TC was further investigated in both Mrp2 and Mrp3. In the case of Mrp2, substitution of Arg at 1096 by Lys (R1096K) and Val (RIO96V) also resulted in the acquisition of the ability to transport TC. In contrast, substitution of Leu at 1084 of rat Mrp3 by Lys (L1084KMrp3) resulted in the loss of transport activity for both E217βG and TC, although transport activity for these compounds was unaffected by substitution by Val (L1084VMrp3) or Met (L1084MMrp3). This suggests that the charged moieties at 1096 of Mrp2 and 1084 of Mrp3 do not simply govern the transport ability for TC; Arg at 1096 acts as an obstacle to the transport of TC in rat Mrp2, whereas a neutral amino acid at the corresponding position in Mrp3 (Leu at 1084) is required for transport of both TC and E217βG.

Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague–Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.