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.

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.

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.

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.

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.

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.

Functional modulation of liver mitochondria in lipopolysaccharide/drug co-treated rat liver injury model.

Drug-induced liver injury is not readily detectable using conventional animal studies during pre-clinical drug development. To address this problem, other researchers have proposed the use of co-administration of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, and a drug. Using this approach, liver injury that is otherwise not detected following drug administration alone can be successfully identified. Previous studies have demonstrated that such injury is suppressed by heparin; therefore, the mechanism may involve coagulation-dependent ischemia. However, it has not been established how LPS-induced ischemia might sensitize hepatocytes to a potentially hepatotoxic drug. In the present study, we aimed to determine the effect of LPS-induced ischemia on liver mitochondrial function and downstream toxicologic responses. Consistent with previous findings, plasma alanine transaminase (ALT) activity was higher in rats co-administered with LPS (1 mg/kg) and diclofenac (100 mg/kg), but reduced by heparin. Liver mRNA expression of Hmox1, encoding heme oxygenase-1, an oxidative stress indicator, was three times higher at 2 hr after LPS administration. Furthermore, respiratory activity via mitochondrial complex II, lipid peroxidation in mitochondria, and the susceptibility to mitochondrial permeability transition pore opening in response to diclofenac administration were significantly increased by LPS administration. The increase in plasma ALT activity and the sensitization to mitochondrial permeability transition pore opening were reduced by the co-administration of heparin. In conclusion, LPS-induced transient ischemia disrupts respiratory chain complex activities, enhances reactive oxygen species production, especially in mitochondria, and sensitizes mitochondria to permeability transition pore opening when testing a potentially hepatotoxic drug in vivo.

Mild depolarization is involved in troglitazone-induced liver mitochondrial membrane permeability transition via mitochondrial iPLA2 activation.

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.

In vitro bile acid-dependent hepatocyte toxicity assay system using human induced pluripotent stem cell-derived hepatocytes: Current status and disadvantages to overcome.

Cholestatic drug-induced liver injury (DILI) is a type of hepatotoxicity. Its underlying mechanisms are dysfunction of bile salt export pump (BSEP) and multidrug resistance-associated protein 2/3/4 (MRP2/3/4), which play major roles in bile acid (BA) excretion into the bile canaliculi and blood, resulting in accumulation of BAs in hepatocytes. The sandwich-cultured hepatocyte (SCH) model can simultaneously analyze hepatic uptake and biliary excretion. Therefore, we investigated whether sandwich-cultured human induced pluripotent stem cell (iPS cell)-derived hepatocytes (SCHiHs) are suitable for evaluating cholestatic DILI. Fluorescent N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5ß-cholestan-26-oyl)-2'-aminoethanesulfonate (tauro-nor-THCA-24-DBD, a BSEP substrate) was accumulated in bile canaliculi, which supports the presence of a functional bile canaliculi lumen. MRP2 was highly expressed in the Western blot analysis, whereas the mRNA expression of BSEP was hardly detectable. MRP3/4 mRNA levels were maintained. Of the 22 compounds known to cause DILI with BAs, 7 showed significant cytotoxicity. Most high-risk drugs were detected using the developed SCHiH system. However, a shortcoming was the considerably low expression level of BSEP, which prevented the detection of some relevant drugs whose risks should be detected in primary human hepatocytes.

Inhibition of biliary network reconstruction by benzbromarone delays recovery from pre-existing liver injury.

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.

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.

Increased susceptibility to troglitazone-induced mitochondrial permeability transition in type 2 diabetes mellitus model rat.

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.

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.

Identification of Bile Acids Responsible for Inhibiting the Bile Salt Export Pump, Leading to Bile Acid Accumulation and Cell Toxicity in Rat Hepatocytes.

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 Inhibits Bile Acid Amidation: A Possible Risk Factor for Liver Injury.

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.

Use of Primary Rat Hepatocytes for Prediction of Drug-Induced Mitochondrial Dysfunction.

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.

Metabolic Activation of Cholestatic Drug-Induced Bile Acid-Dependent Toxicity in Human Sandwich-Cultured Hepatocytes.

We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BAtox, i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BAtox. There was a tendency that BAtox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BAtox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BAtox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BAtox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BAtox.

Inhibition of bile canalicular network formation in rat sandwich cultured hepatocytes by drugs associated with risk of severe liver injury.

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 (<60% of control). Effects on cell viability were negligible or minimal as confirmed by lactate dehydrogenase leakage and cellular ATP content assays. For the potent 15 drugs, IC50 values were determined. Finally, maximum daily dose divided by the inhibition constant gave good separation of the highest risk of severe liver toxicity drugs such as troglitazone, benzbromarone, flutamide, and amiodarone from lower risk drugs. In conclusion, inhibitory effect on the bile canalicular network formation observed in in vitro sandwich cultured hepatocytes evaluates a new aspect of drug toxicity, particularly associated with aggravation of liver injury.

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.

Establishment of a Drug-Induced, Bile Acid-Dependent Hepatotoxicity Model Using HepaRG Cells.

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.