Mechanism-based integrated assay systems for the prediction of drug-induced liver injury.

Drug-induced liver injury (DILI) can cause hepatic failure and result in drug withdrawal from the market. It has host-related and compound-dependent mechanisms. Preclinical prediction of DILI risk is very challenging and safety assessments based on animals inadequately forecast human DILI risk. In contrast, human-derived in vitro cell culture-based models could improve DILI risk prediction accuracy. Here, we developed and validated an innovative method to assess DILI risk associated with various compounds. Fifty-four marketed and withdrawn drugs classified as DILI risks of "most concern", "less concern", and "no concern" were tested using a combination of four assays addressing mitochondrial injury, intrahepatic lipid accumulation, inhibition of bile canalicular network formation, and bile acid accumulation. Using the inhibitory potencies of the drugs evaluated in these in vitro tests, an algorithm with the highest available DILI risk prediction power was built by artificial neural network (ANN) analysis. It had an overall forecasting accuracy of 73%. We excluded the intrahepatic lipid accumulation assay to avoid overfitting. The accuracy of the algorithm in terms of predicting DILI risks was 62% when it was constructed by ANN but only 49% when it was built by the point-added scoring method. The final algorithm based on three assays made no DILI risk prediction errors such as "most concern " instead of "no concern" and vice-versa. Our mechanistic approach may accurately predict DILI risks associated with numerous candidate drugs.

Development of a next generation risk assessment framework for the evaluation of skin sensitisation of cosmetic ingredients.

All cosmetic products placed onto the market must undergo a risk assessment for human health to ensure they are safe for consumers, including an assessment of skin sensitisation risk. Historically, in vivo animal test methods were used to identify and characterise skin sensitisation hazard, however non-animal and other new approach methodologies (NAMs) are now the preferred and mandated choice for use in risk assessment for cosmetic ingredients. The experience gained over the last three decades on how to conduct risk assessments based upon NAMs has allowed us to develop a non-animal, next generation risk assessment (NGRA) framework for the assessment of skin sensitisers. The framework presented here is based upon the principles published by the International Cooperation on Cosmetic Regulation (ICCR) and is human relevant, exposure led, hypothesis driven and designed to prevent harm. It is structured in three tiers and integrates all relevant information using a weight of evidence (WoE) approach that can be iterated when new information becomes available. The initial tier (TIER 0) involves a thorough review of the existing information including; identification of the use scenario/consumer exposure; characterisation of the chemical purity and structure; in silico predictions; existing data pertaining to skin sensitisation hazard (historical or non-animal); the identification of suitable read-across candidates with supporting hazard identification/characterisation information and application of exposure-based waiving. Considering all information identified in TIER 0, the next step is the generation of a hypothesis (TIER 1). All data are considered in an exposure-led WoE approach, taking into account an initial view on whether a chemical is likely to be a skin sensitiser or not, choice of defined approach (DA) and availability of read-across candidates. If existing information is insufficient for concluding the risk assessment, the generation of additional information may be required to proceed (TIER 2). Such targeted testing could involve refinement of the exposure estimation or generation of data from in vitro or in chemico NAMs. Once sufficient information is available, the final stage of the NGRA framework is the determination of a point of departure (POD), characterising uncertainty and comparing to the consumer exposure in a WoE. Thorough evaluation of the sources of uncertainty is essential to ensure transparency and build trust in new risk assessment approaches. Although significant progress has been made, industry must continue to share its experience in skin sensitisation NGRA via case studies to demonstrate that this new risk assessment approach is protective for consumers. Dialogue and collaboration between key stakeholders, i.e. risk assessors, clinicians and regulators are important to gain mutual understanding and grow confidence in new approaches.

Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation.

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.

Evaluation of immune-mediated idiosyncratic drug toxicity using chimeric HLA transgenic mice.

Immune-mediated idiosyncratic drug toxicity (IDT) is a rare adverse drug reaction, potentially resulting in death. Although genome-wide association studies suggest that the occurrence of immune-mediated IDT is strongly associated with specific human leukocyte antigen (HLA) allotypes, these associations have not yet been prospectively demonstrated. In this study, we focused on HLA-B*57:01 and abacavir (ABC)-induced immune-mediated IDT, and constructed transgenic mice carrying chimeric HLA-B*57:01 (B*57:01-Tg) to determine if this in vivo model may be useful for evaluating immune-mediated IDT. Local lymph node assay (LLNA) results demonstrated that percentages of BrdU+, IL-2+, and IFN-γ+ in CD8+ T cells of ABC (50 mg/kg/day)-applied B*57:01-Tg mice were significantly higher than those in littermates (LMs), resulting in the infiltration of inflammatory cells into the ear. These immune responses were not observed in B*57:03-Tg mice (negative control). Furthermore, oral administration of 1% (v/v) ABC significantly increased the percentage of CD44highCD62Llow CD8+ memory T cells in lymph nodes and spleen derived from B*57:01-Tg mice, but not in those from B*57:03-Tg mice and LMs. These results suggest that B*57:01-Tg mice potentially enable the reproduction and evaluation of HLA-B*57:01 and ABC-induced immune-mediated IDT.

Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes.

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.

Prediction of the Clinical Risk of Drug-Induced Cholestatic Liver Injury Using an In Vitro Sandwich Cultured Hepatocyte Assay.

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.

Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.

Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein.

Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ((59)Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca(2+) uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca(2+) uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein.

Key determinants of the circulatory exposure of organic anions: differences in hepatic uptake between multidrug resistance-associated protein 2 (Mrp2)-deficient rats and wild-type rats.

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-17ß-glucuronide (E217ßG). 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.

Sustained intrahepatic glutathione depletion causes proteasomal degradation of multidrug resistance-associated protein 2 in rat liver.

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.

Ezrin regulates the expression of Mrp2/Abcc2 and Mdr1/Abcb1 along the rat small intestinal tract.

Multidrug resistance-associated protein 2 (MRP2)/ATP-binding cassette protein C2 (ABCC2) and multidrug resistance protein 1 (MDR1)/ABCB1 are well-known efflux transporters located on the brush border membrane of the small intestinal epithelia, where they limit the absorption of a broad range of substrates. The expression patterns of MRP2/ABCC2 and MDR1/ABCB1 along the small intestinal tract are tightly regulated. Several reports have demonstrated the participation of ERM (ezrin/radixin/moesin) proteins in the posttranslational modulation of MRP2/ABCC2 and MDR1/ABCB1, especially with regard to their membrane localization. The present study focused on the in vivo expression profiles of MRP2/ABCC2, MDR1/ABCB1, ezrin, and phosphorylated ezrin to further elucidate the relationship between the efflux transporters and the ERM proteins. The current results showed good correlation between the phosphorylation status of ezrin and Mrp2/Abcc2 expression along the gastrointestinal tract of rats and between the expression profiles of both ezrin and Mdr1/Abcb1 in the small intestine. We also demonstrated the involvement of conventional protein kinase C isoforms in the regulation of ezrin phosphorylation. Furthermore, experiments conducted with wild-type (WT) ezrin and a T567A (Ala substituted Thr) dephosphorylated mutant showed a decrease in membrane surface-localized and total expressed MRP2/ABCC2 in T567A-expressing vs. WT ezrin-expressing Caco-2 cells. In contrast, T567A- and WT-expressing cells both showed an increase in membrane surface-localized and total expressed MDR1/ABCB1. These findings suggest that the phosphorylation status and the expression profile of ezrin differentially direct MRP2/ABCC2 and MDR1/ABCB1 expression, respectively, along the small intestinal tract.

Interaction of Mrp2 with radixin causes reversible canalicular Mrp2 localization induced by intracellular redox status.

Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress.

Mechanistic differences in permeation behavior of supersaturated and solubilized solutions of carbamazepine revealed by nuclear magnetic resonance measurements.

A solid dispersion (SPD) of carbamazepine (CBZ) with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was prepared by the spray drying method. The apparent solubility (37 °C, pH 7.4) of CBZ observed with the SPD was over 3 times higher than the solubility of unprocessed CBZ. The supersaturated solution was stable for 7 days. A higher concentration of CBZ in aqueous medium was also achieved by mixing with Poloxamer 407 (P407), a solubilizing agent. From permeation studies of CBZ using Caco-2 monolayers and dialysis membranes, we observed improved CBZ permeation across the membrane in the supersaturated solution of CBZ/HPMC-AS SPD. On the contrary, the CBZ-solubilized P407 solution exhibited poor permeation by CBZ. The chemical shifts of CBZ on the (1)H NMR spectrum from CBZ/HPMC-AS SPD solution were not altered significantly by coexistence with HPMC-AS. In contrast, an upfield shift of CBZ was observed in the CBZ/P407 solution. The spin-lattice relaxation time (T(1)) over spin-spin relaxation time (T(2)) indicated that the mobility of CBZ in the HPMC-AS solution was much lower than that in water. Meanwhile, the mobility of CBZ in P407 solution was significantly higher than that in water. NMR data indicate that CBZ does not strongly interact with HPMC-AS. CBZ mobility was suppressed due to self-association and microviscosity around CBZ, which do not affect permeation behavior. Most of the CBZ molecules in the CBZ/P407 solution were solubilized in the hydrophobic core of P407, and a few were free to permeate the membrane. The molecular state of CBZ, as evaluated by NMR measurements, directly correlated with permeation behavior.

StemPanTox: A fast and wide-target drug assessment system for tailor-made safety evaluations using personalized iPS cells.

An alternative model that reliably predicts human-specific toxicity is necessary because the translatability of effects on animal models for human disease is limited to context. Previously, we developed a method that accurately predicts developmental toxicity based on the gene networks of undifferentiated human embryonic stem (ES) cells. Here, we advanced this method to predict adult toxicities of 24 chemicals in six categories (neurotoxins, cardiotoxins, hepatotoxins, two types of nephrotoxins, and non-genotoxic carcinogens) and achieved high predictability (AUC = 0.90-1.00) in all categories. Moreover, we screened for an induced pluripotent stem (iPS) cell line to predict the toxicities based on the gene networks of iPS cells using transfer learning of the gene networks of ES cells, and predicted toxicities in four categories (neurotoxins, hepatotoxins, glomerular nephrotoxins, and non-genotoxic carcinogens) with high performance (AUC = 0.82-0.99). This method holds promise for tailor-made safety evaluations using personalized iPS cells.

Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes.

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury.

Evaluation of Parent- and Metabolite-Induced Mitochondrial Toxicities Using CYP-Introduced HepG2 cells.

Mitochondrial toxicity is an important factor to predict drug-induced liver injury (DILI). Previous studies have focused predominantly on mitochondrial toxicities due to parent forms, and no study has adequately evaluated metabolite-induced mitochondrial toxicity. Moreover, previous studies have used HepG2 cells, which lack many cytochrome P450 (CYP) genes. To overcome this problem, CYP-introduced HepG2 cells were constructed using several gene transfer technologies, including adenoviruses and plasmids. However, these methods only led to a transient expression of CYP genes. In the present study, usefulness of four CYPs introduced-HepG2 (TC-Hep) cells previously constructed through mammalian artificial chromosome technology were examined, especially from the perspective of mitochondrial toxicity. First, we evaluated the effects of known compounds, such as rotenone and flutamide, on mitochondrial toxicity and cell death in TC-Hep cells cultured in galactose conditions. Expectedly, rotenone-induced cell death ameliorated because rotenone was metabolized by CYPs into inactive form(s) and flutamide-induced cell death increased in TC-Hep cells. Second, we evaluated five compounds that caused liver injury in clinical phase and were discontinued during pharmaceutical development. The present in vitro tool suggested that three of the five compounds caused metabolite-induced mitochondrial toxicities. In conclusion, the present in vitro tool could easily and inexpensively detect metabolite-induced mitochondrial toxicity; hence, it can be useful for predicting DILI in preclinical phase.

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices.

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.

Successful energy shift from glycolysis to mitochondrial oxidative phosphorylation in freshly isolated hepatocytes from humanized mice liver.

Mitochondrial toxicity is a factor of drug-induced liver injury. Previously, we reported an in vitro rat hepatocyte assay where mitochondrial toxicity was more sensitively evaluated, using sugar resource substitution and increased oxygen supply. Although this method could be applicable to human cell-based assay, cryopreserved human hepatocyte (CHH) has some disadvantages/uncertainty, including unstable same donor supply and potential organelle damage due to cryopreservation. Herein, we compared the mitochondrial functions of freshly-isolated hepatocytes from humanized chimeric mice liver (PXB-cells) and three CHH lots to determine the better cell source for mitochondrial toxicity assay. Two CHH lots declined after replacing glucose with galactose. To confirm the shift in energy production from glycolysis to oxidative phosphorylation, lactate and oxygen consumption rate (indicators of glycolytic activity and mitochondrial oxidative phosphorylation, respectively) were measured. In PXB-cells, lactate amount decreased, while oxygen consumption in 100 min increased. These effects were less evident in CHH. The cytotoxicity of the select respiratory chain inhibitors was enhanced in PXB-cells upon sugar replacement, but no change occurred with negative control drugs (bicalutamide and metformin). Altogether, PXB-cells was less vulnerable to sugar resource substitution than CHH. The substitution activated mitochondrial function and enhanced cytotoxicity of respiratory chain inhibitors in PXB-cells.

Hypoxia/reoxygenation exacerbates drug-induced cytotoxicity by opening mitochondrial permeability transition pore: Possible application for toxicity screening.

Recently, mitochondrial dysfunction is thought of as an important factor leading to a drug-induced liver injury. Our previous reports show that mitochondria-related toxicity, including respiratory chain inhibition (RCI) and reactive oxygen species (ROS) induction, can be detected by the modification of sugar resource substitution and high oxygen condition. However, this in vitro model does not detect mitochondrial permeability transition (MPT)-induced toxicity. Another study with a lipopolysaccharide-pre-administered rodent model showed that ischemia/reperfusion induced ROS, sensitized the susceptibility of MPT pore opening and, finally developed drug-induced liver toxicity. Based on this result, the present study investigated the effect of hypoxia/reoxygenation (H/R) treatment mimicking the ischemia/reperfusion on MPT-dependent toxicity, aiming to construct a system that can evaluate MPT by drugs in hepatocytes. Mitochondrial ROS were enhanced by H/R treatment only in the galactose culture condition. Amiodarone, benzbromarone, flutamide and troglitazone which induced MPT pore opening led to hepatocyte death only in combination with H/R and galactose. Moreover, this alteration was significantly suppressed in hepatocytes lacking cyclophilin D. In conclusion, MPT-induced cytotoxicity can be detected by activating mitochondrial function and H/R. This cell-based assay system could evaluate MPT induced-cytotoxicity by drugs, besides RCI and ROS induction.

Correlation between apical localization of Abcc2/Mrp2 and phosphorylation status of ezrin in rat intestine.

The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 +/- 2.1 nmol/cm to 18.1 +/- 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.