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Global gene expression profiling is useful for elucidating a drug's mechanism of action on the liver; however, such profiling in rats is not very sensitive for predicting human drug-induced liver injury, while dedifferentiated monolayers of primary human hepatocytes (PHHs) do not permit chronic drug treatment. In contrast, micropatterned cocultures (MPCCs) containing PHH colonies and 3T3-J2 fibroblasts maintain a stable liver phenotype for 4-6 weeks. Here, we used MPCCs to test the hypothesis that global gene expression patterns in stable PHHs can be used to distinguish clinical hepatotoxic drugs from their non-liver-toxic analogs and understand the mechanism of action prior to the onset of overt hepatotoxicity. We found that MPCCs treated with the clinical hepatotoxic/non-liver-toxic pair, troglitazone/rosiglitazone, at each drug's reported and non-toxic Cmax (maximum concentration in human plasma) for 1, 7, and 14 days displayed a total of 12, 269, and 628 differentially expressed genes, respectively, relative to the vehicle-treated control. Troglitazone modulated >75% of transcripts across pathways such as fatty acid and drug metabolism, oxidative stress, inflammatory response, and complement/coagulation cascades. Escalating rosiglitazone's dose to that of troglitazone's Cmax increased modulated transcripts relative to the lower dose; however, over half the identified transcripts were still exclusively modulated by troglitazone. Last, other hepatotoxins (nefazodone, ibufenac, and tolcapone) also induced a greater number of differentially expressed genes in MPCCs than their non-liver-toxic analogs (buspirone, ibuprofen, and entacapone) following 7 days of treatment. In conclusion, MPCCs allow evaluation of time- and dose-dependent gene expression patterns in PHHs treated chronically with analog drugs., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Drug-induced liver injury (DILI) is one of the serious and frequent drug-related adverse events. This adverse event is a main reason for regulatory action pertaining to drugs, including restrictions in clinical indications and withdrawal from clinical trials or the marketplace. Idiosyncratic DILI especially has become a major clinical concern because of its unpredictable nature, frequent hospitalization, need for liver transplantation and high mortality. The estimation of the potential for compounds to induce idiosyncratic DILI is very difficult in non-clinical studies because the precise mechanism of idiosyncratic DILI is still unknown. Recently, many in vitro assays which indicate a possibility of the prediction of the idiosyncratic DILI have been reported. Among these, some in vitro assays focus on the effects of compounds on mitochondrial function and the apoptotic effects of compounds on human hepatocytes. In this study, we measured oxygen consumption rate (OCR) and caspase-3/7 activity as an endpoint of mitochondrial dysfunction and apoptosis, respectively, with human hepatocytes after treatment with compounds causing idiosyncratic DILI (troglitazone, leflunomide, ranitidine and diclofenac). Troglitazone and leflunomide decreased the OCR but did not affect caspase-3/7 activity. Ranitidine increased caspase-3/7 activity but did not affect the OCR. Diclofenac decreased the OCR and increased caspase-3/7 activity. Acetaminophen and ethanol, which are also hepatotoxicants but do not induce idiosyncratic DILI, did not affect the OCR or caspase-3/7 activity. These results indicate that a combination assay of mitochondrial dysfunction and apoptosis is useful for the estimation of potential risk of compounds to induce idiosyncratic DILI.

DILI is a major safety issue during drug development and one of the leading causes for market withdrawal. Despite many efforts made in the past, the prediction of DILI using in vitro models remains very unreliable. In the present study, the well-established hepatocyte Collagen I-Matrigel™ sandwich culture was used, mimicking chronic drug treatment after multiple incubations for 14 days. Ten drugs associated with different types of specific preclinical and clinical liver injury were evaluated at non-cytotoxic concentrations. Mrp2-mediated transport, intracellular accumulation of neutral lipids and phospholipids were selected as functional endpoints by using Cellomics™ Arrayscan® technology and assessed at five timepoints (day 1, 3, 7, 10, 14). Liver specific functional impairments after drug treatment were enhanced over time and could be monitored by HCI already after few days and before cytotoxicity. Phospholipidosis-inducing drugs Chlorpromazine and Amiodarone displayed the same response as in vivo. Cyclosporin A, Chlorpromazine, and Troglitazone inhibited Mrp2-mediated biliary transport, correlating with in vivo findings. Steatosis remained difficult to be reproduced under the current in vitro testing conditions, resulting into false negative and positive responses. The present results suggest that the repeated long-term treatment of rat hepatocytes in the Collagen I-Matrigel™ sandwich configuration might be a suitable tool for safety profiling of the potential to induce phospholipidosis and impair Mrp2-mediated transport processes, but not to predict steatosis., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes., Methods: We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI)., Results: The mean (± SEM) urea production was significantly higher in sandwich cultures from freshly-isolated hepatocytes (27.88 (± 0.96) nmol/cm(2)), compared to cultures from cryopreserved hepatocytes (22.86 (± 1.91) nmol urea/cm(2)). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (± SEM) DICI values for CsA 10 μM and Tro 75 μM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 μM) and Tro (≥ 100 μM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history., Discussion: In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Drug-induced human hepatotoxicity is difficult to predict using the current in vitro systems. In this study, long-term 3D organotypic cultures of the human hepatoma HepaRG cell line were prepared using a high-throughput hanging drop method. The organotypic cultures were maintained for 3 weeks and assessed for (1) liver specific functions, including phase I enzyme and transporter activities, (2) expression of liver-specific proteins, and (3) responses to three drugs (acetaminophen, troglitazone, and rosiglitazone). Our results show that the organotypic cultures maintain high liver-specific functionality during 3 weeks of culture. The immunohistochemistry analyses illustrate that the organotypic cultures express liver-specific markers such as albumin, CYP3A4, CYP2E1, and MRP-2 throughout the cultivation period. Accordingly, the production rates of albumin and glucose, as well as CYP2E1 activity, were significantly higher in the 3D versus the 2D cultures. Toxicity studies show that the organotypic cultures are more sensitive to acetaminophen- and rosiglitazone-induced toxicity but less sensitive to troglitazone-induced toxicity than the 2D cultures. Furthermore, the EC50 value (2.7mM) for acetaminophen on the 3D cultures was similar to in vivo toxicity. In summary, the results from our study suggest that the 3D organotypic HepaRG culture is a promising in vitro tool for more accurate assessment of acute and also possibly for chronic drug-induced hepatotoxicity.

The aims of this study were to assess the utility of the PXB mouse model of a chimeric human/mouse liver in studying human-specific effects of an important human hepatotoxic drug, the PPARγ agonist, troglitazone. When given orally by gavage for 7 days, at dose levels of 300 and 600 ppm, troglitazone induced specific changes in the human hepatocytes of the chimeric liver without an effect on the murine hepatic portions. The human hepatocytes, in the vehicle-treated PXB mouse, showed an accumulation of electron-dense lipid droplets that appeared as clear vacuoles under the light microscope in H&E-stained sections. Following dosing with troglitazone, there was a loss of the large lipid droplets in the human hepatocytes, a decrease in the amount of lipid as observed in frozen sections of liver stained by Oil-red-O, and a decrease in the expression of two bile acid transporters, BSEP and MRP2. None of these changes were observed in the murine remnants of the chimeric liver. No changes were observed in the expression of three CYPs, CYP 3A2, CYP 1A1, and CYP 2B1, in either the human or murine hepatocytes, even though the baseline expression of the enzymes differed significantly between the two hepatocyte species with the mouse hepatocytes consistently showing increased expression of the protein of all three enzymes. This study has shown that the human hepatocytes, in the PXB chimeric mouse liver, retain an essentially normal phenotype in the mouse liver and, the albeit limited CYP enzymes studied show a more human, rather than a murine, expression pattern. In line with this conclusion, the study has shown a differential response of the human versus the mouse hepatocytes, and the effects observed are highly suggestive of a differential handling of the compound by the two hepatocyte species although the exact reasons are not as yet clear. The PXB chimeric mouse system therefore holds the clear potential to explore human hepatic-specific features, such as metabolism, prior to dosing human subjects, and as such should have considerable utility in drug discovery and development.

Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Inhibition of bile acid (BA) transport may contribute to the hepatotoxicity of troglitazone (TRO), a peroxisome proliferator-activated receptor gamma agonist. Typically, studies use taurocholic acid (TCA) as a model substrate to investigate effects of xenobiotics on BA disposition. However, TRO may differentially affect the transport of individual BAs, potentially causing hepatocyte accumulation of more cytotoxic BAs. The effects of TRO on the disposition of [(14)C]-labeled chenodeoxycholic acid ([(14)C]CDCA), an unconjugated cytotoxic BA, were determined in suspended hepatocytes and sandwich-cultured hepatocytes (SCH) from rats. (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571), a multidrug resistance-associated protein (MRP) inhibitor, was included to evaluate involvement of MRPs in CDCA disposition. Accumulation in cells + bile of total [(14)C]CDCA species in SCH was sixfold greater than [(3)H]TCA and unaffected by 1 and 10μM TRO; 100μM TRO and 50μM MK571 ablated biliary excretion and significantly increased intracellular accumulation of total [(14)C]CDCA species. Results were similar in Mrp2-deficient TR(-) rat hepatocytes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that taurine- and glycine-conjugated CDCA, in addition to unconjugated CDCA, accumulated in hepatocytes during the 10-min incubation. In suspended rat hepatocytes, initial [(14)C]CDCA uptake was primarily Na(+)-independent, whereas initial [(3)H]TCA uptake was primarily Na(+)-dependent; TRO and MK571 decreased [(14)C]CDCA uptake to a lesser extent than [(3)H]TCA. Unexpectedly, MK571 inhibited Na(+)-taurocholate cotransporting polypeptide and bile salt export pump. Differential effects on uptake and efflux of individual BAs may contribute to TRO hepatotoxicity. Although TCA is the prototypic BA used to investigate the effects of xenobiotics on BA transport, it may not be reflective of other BAs.

Troglitazone (TGZ), an orally active hypoglycemic agent, was found to be associated with severe drug-induced liver failure and was withdrawn from the market in 2000. Although the exact mechanism is not clear, it has been postulated that the formation of its major sulfo-conjugated metabolite (TGZS) plays an important role in its toxicity. TGZS inhibits bile salt export pump (BSEP) that causes accumulation of bile salts in liver. High concentration of bile salts causes cell death and mitochondrial dysfunction via detergent properties. One question arises whether TGZS has direct toxicity effect on human liver cells in addition to BSEP inhibition. In this study, both TGZ and chemically synthesized TGZS were incubated with normal human hepatocytes (THLE-2 cells) for measuring their cytotoxicity in vitro using the MTT assay. Glutathione (GSH) and protein carbonyl (PC) assays were further performed to measure the oxidative stress generated by these two compounds during incubation with THLE-2 cells. The results from this study indicated that TGZS (EC(50)=21.74+/-5.38 microM) was more toxic than TGZ (EC(50)=41.12+/-4.3 microM) in THLE-2 cells. The GSH and PC data further confirmed that TGZS produced greater oxidative stress in THLE-2 cells as compared to TGZ. In conclusion, our study demonstrated for the first time that TGZS has direct toxicity effect on human liver cells and may be partially responsible for the hepatotoxicity of TGZ., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Troglitazone (TGZ) is an orally active hypoglycemic agent which is used for the treatment of non-insulin-dependent diabetes mellitus. It had been associated with severe drug-induced liver failure which resulted in its withdrawal from the market in 2000. While the exact mechanism of its toxicity remains unknown, it has been postulated that the formation of toxic reactive metabolites (RMs) may play an important role in the hepatotoxicity of TGZ. The purpose of this study is to investigate the role of sulfur moiety of thiazolidinedione (TZD) nucleus in inducing liver toxicity via the formation of RMs. An analogue of TGZ, trosuccinimide (TSN), was synthesized chemically where the sulfur moiety of thiazolidinedione ring was replaced by a methylene group. Both compounds were incubated independently with human liver microsomes enriched with glutathione (GSH) and normal human hepatocytes (THLE-2 cell lines) to profile GSH-adducts using ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS). Four RM-GSH conjugates of TGZ were identified during the profiling experiments of which three were related to the sulfur moiety of the TZD ring, whereas no RM of TSN was detected in both microsomes and hepatocytes. MTT, GSH and protein carbonyl (PC) assays were performed using THLE-2 hepatocytes to measure the levels of toxicity of TGZ and TSN in vitro. Finally, peroxisome proliferator activated receptor gamma (PPAR(gamma)) binding activity was measured to check the binding affinities of both TGZ and TSN. The calculated binding affinities of TGZ and TSN were 332.2 and 1106.0 microM, respectively. Our results indicated collectively that TSN (EC(50)=138.5+/-7.32 microM) was less toxic than TGZ (EC(50)=27.2+/-4.8 microM) in THLE-2 hepatocytes. As both compounds were shown to bind to PPAR(gamma), the substitution of the TZD moiety may be beneficial from a drug design perspective. In conclusion, our study confirmed that the TZD ring of TGZ may be partially responsible for its liver toxicity in humans via the formation of RMs., (2009 Elsevier Ireland Ltd. All rights reserved.)

Troglitazone was withdrawn from the market shortly after approval for diabetes type II therapy because of strong hepatotoxic effects in man that could not be predicted from regulatory animal or in vitro studies. Another pharmaceutical that is regularly associated with adverse effects on the liver, sometimes leading to acute liver failure, is the widely used non-steroidal anti-inflammatory drug (NSAID) diclofenac. Since the underlying molecular mechanisms are not yet fully known, we treated primary rat and human hepatocyte monolayer cultures for 24h with different doses of troglitazone and diclofenac to analyze species differences related to toxicity in vitro. Metformin an antidiabetic drug which does not cause severe adverse reactions served as negative control. Human hepatocytes showed a higher sensitivity to troglitazone than rat hepatocytes, while diclofenac-induced cytotoxicity at fairly similar concentrations. By co-treatment with specific inhibitors for cytochrome P450 (CYP) 2C and CYP3A - the major phase I enzymes involved in liver xenobiotic metabolism - we could confirm the prominent role of CYP3A in the bioactivation of troglitazone as well as the role of CYP3A and CYP2C in the activation of diclofenac. Inhibition of these enzymes increased the viability of treated cells in both species. Furthermore, we were able to demonstrate marked species differences in gene expression patterns of troglitazone treated rat and human hepatocytes. In contrast to rat hepatocytes, human cells showed distinct upregulation of various CYPs, regulators of xenobiotic metabolism and marker genes for oxidative stress. In contrast, gene expression alterations in rat and human hepatocytes treated with Diclofenac were rather similar. Altogether our study showed that species-specific effects as well as indications for the mode of action of compounds can be addressed by the use of primary hepatocyte cultures from various species in combination with gene expression profiling.

Background and Aim: Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit cell growth and induce apoptosis in various cancer cells. Bile acids are also known to cause hepatocyte apoptosis through nuclear receptor-mediated mechanisms. The aim of this study was to examine the effect of two different bile acid species on the inhibitory action of PPARgamma in cell growth with paying attention to the role of the mitogen-activated protein kinase pathway as an underlying mechanism., Methods: Immortalized human hepatocytes (OUMS-29) and hepatoma cells (HepG2 and Huh7) were incubated with troglitazone (TGZ), a PPARgamma ligand with or without pre-incubation of either hydrophobic glycochenodeoxycholate (GCDC) or hydrophilic tauroursodeoxycholate (TUDC)., Results: TGZ induced cell apoptosis in all cell types, resulting in the reduction of cell viability. While pre-incubation with GCDC enhanced the apoptotic effects of TGZ, TUDC significantly attenuated it. Both bile acids enhanced p38 and c-Jun N-terminal kinase (JNK) phosphorylation in a similar way, whereas there was more drastic enhancement of extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in the presence of TUDC compared to GCDC. In addition, ERK inhibitors suppressed the action of TUDC against apoptotic effect of TGZ., Conclusion: This study demonstrates that TUDC exhibits anti-apoptotic and cytoprotective effects against TGZ-induced cell apoptosis, presumably through the ERK signaling pathway. We speculate that the administration of TUDC might be one of the potential strategies for the hepatotoxicity caused by TGZ.

Although the mechanisms and susceptibility factors of troglitazone-associated idiosyncratic liver injury have not been elucidated, experimental evidence has identified oxidant stress and mitochondrial injury as a potential hazard in vitro. In search of upstream mediators of toxicity, we hypothesized that troglitazone-induced increased mitochondrial generation of superoxide might activate the thioredoxin-2 (Trx2)/apoptosis signal-regulating kinase 1 (Ask1) signaling pathway, leading to cell death, and that, hence, the mitochondrially targeted radical scavenger, mito-carboxy proxyl (CP), would prevent the increase in superoxide net levels and inhibit mitochondrial signaling and cell injury. Immortalized human hepatocytes (HC-04) were exposed to troglitazone (0-100 microM), which caused concentration and time-dependent apoptosis after 12-24 h (ketoconazole-insensitive). We found that troglitazone rapidly dissipated the mitochondrial inner transmembrane potential (DeltaPsi(m)) and independently increased the net levels of mitochondrial superoxide by 5-fold. This was followed by a shift of the redox ratio of mitochondrial Trx2 toward the oxidized state and subsequent activation of Ask1. Cell injury, but not the decrease in DeltaPsi(m), was prevented by cyclosporin A (3 microM), indicating that mitochondrial permeabilization, but not membrane depolarization, was causally involved in cell death. Mito-CP not only decreased troglitazone-induced superoxide levels but also prevented Trx2 oxidation and activation of Ask1 and protected cells from toxic injury. These data indicate that troglitazone, but not its oxidative metabolite(s), produce intramitochondrial oxidant stress that activates the Trx2/Ask1 pathway, leading to mitochondrial permeabilization. Furthermore, the data support our concept that targeted delivery of an antioxidant to mitochondria can inhibit upstream signaling and protect from troglitazone-induced lethal cell injury.

With appropriate models, the metabolic profile of a biological system may be interrogated to obtain both significant discriminatory markers as well as mechanistic insight into the observed phenotype. One promising application is the analysis of drug toxicity, where a single chemical triggers multiple responses across cellular metabolism. Here, we describe a modeling framework whereby metabolite measurements are used to investigate the interactions between specialized cell functions through a metabolic reaction network. As a model system, we studied the hepatic transformation of troglitazone (TGZ), an antidiabetic drug withdrawn due to idiosyncratic hepatotoxicity. Results point to a well-defined TGZ transformation module that connects to other major pathways in the hepatocyte via amino acids and their derivatives. The quantitative significance of these connections depended on the nutritional state and the availability of the sulfur containing amino acids.

Agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) are a new class of oral drugs designed to treat insulin-resistant diabetes (i.e., type 2 diabetes). However, troglitazone, the first compound in the class approved by the US Food and Drug Administration (FDA) in 1997 was found to be hepatotoxic and was withdrawn from the market after reports of severe liver failure. The mechanism of PPAR gamma agonist-induced hepatotoxicity remains unknown. In this study, we examined the hepatotoxic effects of five PPAR gamma agonists (ciglitazone, pioglitazone, rosiglitazone, troglitazone, and JTT-501) on rat primary hepatocytes and human HepG2 cells. We also compared the gene expression profiles of rat primary hepatocytes after exposure to PPAR gamma agonists by using the Rat Genome Survey Microarray system from Applied Biosystems in order to understand the mechanisms of hepatotoxicities induced by PPARgamma agonists. Consistent with the hepatotoxicity data, our results demonstrate that the gene expression profiles affected by troglitazone and ciglitazone can be clearly distinguished from those by pioglitazone and rosiglitazone. Genes that are differentially expressed between the more toxic troglitazone/ciglitazone group and the less toxic rosiglitazone/pioglitazone group are involved in necrotic, apoptotic, and cell proliferative pathways. The five compounds were also clustered based on a set of molecular descriptors. The clustering based on chemical structural information is in good agreement with the clustering of compounds based on cytotoxicity or gene expression data, indicating a strong relationship between chemical structure and biological endpoints. Our work suggests that microarray analysis together with toxicological observations can be used to rank drugs for hepatotoxicity and to evaluate the safety of new compounds.

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione family are used for the treatment of type 2 diabetes mellitus due to their ability to reduce glucose and lipid levels in patients with this disease. Three thiazolidinediones that were approved for treatment are Rezulin (troglitazone), Avandia (rosiglitazone), and Actos (pioglitazone). Troglitazone was withdrawn from the market due to idiosyncratic drug toxicity. Rosiglitazone and pioglitazone are still on the market for the treatment of type 2 diabetes. The authors present data from a gene expression screen that compares the impact these three compounds have in rats, in rat hepatocytes, and in the clone 9 rat liver cell line. The authors monitored the changes in expression in multiple genes, including those related to xenobiotic metabolism, proliferation, DNA damage, oxidative stress, apoptosis, and inflammation. Compared to the other two compounds, troglitazone had a significant impact on many of the pathways monitored in vitro although no major perturbation was detected in vivo. The changes detected predict not only general toxicity but potential mechanisms of toxicity. Based on gene expression analysis, the authors propose there is not just one but multiple ways troglitazone could be toxic, depending on a patient's environment and genetic makeup, including immune response-related toxicity.

Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 microM) decreased the initial rate of taurocholate uptake approximately 3-fold; the initial uptake rate of estradiol-17beta-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased approximately 4-fold. Bosentan (100 microM) decreased the initial uptake rate of taurocholate and estradiol-17beta-D-glucuronide by approximately 12- and approximately 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 +/- 57 pmol/mg protein) was decreased significantly by troglitazone (157 +/- 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 +/- 8%) was decreased in the presence of 10 microM troglitazone (27 +/- 2%) and 100 microM bosentan (10 +/- 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion.

Background and Objectives: Damage to blood and other tissues during laser interventions depends mainly upon absorption of laser radiation by cells. The objective of this work was to evaluate the influence tissue-specific physiological factors on photo-damage thresholds of individual cells: Red blood cells (blood), hepatocytes (liver), and miocytes (heart)., Study Design/materials and Methods: Laser-induced damage to individual cells was detected and studied with Laser Load Test (LLT). Probability and thresholds of RBC damage after one laser pulse (532 nm, 10 nanoseconds) were obtained experimentally as functions of physiological conditions. Using in vitro models, we have studied influence of the oxygen level, pH, temperature, and cell heterogeneity on RBC, the inhibition of metabolic activity on miocytes and drug toxicity on hepatocytes., Results: Single laser pulse induced cell lyses through a vapor bubble. The decrease of the O2 level and temperature caused increase of damage thresholds at 532 nm. Deviation of the pH level from neutral to any side caused also the increase of the damage threshold. Inhibition of metabolism of miocytes and toxic damage to hepatocytes also resulted in the increase of the damage threshold., Conclusions: Resistance of various tissues at cell level against photo-damage significantly depends on physiological properties of cells. A general rule for such dependence is that the better the cell state the lower its threshold for laser-damage.

Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (DeltaPsi(m)) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the DeltaPsi(m) per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity.

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.

We report here our studies on troglitazone and rosiglitazone cytotoxicity in human hepatocytes isolated from multiple donors to investigate factors responsible for individual differences in sensitivity to the known hepatotoxicity of these antidiabetic drugs. Using cellular adenosine triphosphate (ATP) content as an endpoint, cytotoxicity of both drugs was evaluated in cryopreserved human hepatocytes from 37 donors. We confirmed reports of others that troglitazone was cytotoxic to human hepatocytes using cellular ATP content as an endpoint. In addition, we found that rosiglitazone, although less toxic in the study population, was cytotoxic to hepatocytes in some donors (EC(50)<100 microM). ATP content, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) metabolism, depletion of intracellular glutathione, Alamar Blue metabolism, and neutral red uptake were used as endpoints in a single donor study using freshly isolated human hepatocytes. Troglitazone appeared to be more toxic than rosiglitazone by all endpoints. From the demographic data provided to us for each donor, we were able to establish no direct correlation between cytotoxicity (expressed as EC(50) values) and age, sex, smoking status, or alcohol consumption. We conclude that troglitazone and rosiglitazone are differentially toxic to human hepatocytes, and that toxicity may be independent of age, sex, tobacco use, and alcohol use.

Troglitazone (TRO) was developed for the treatment of type II diabetes. It was withdrawn from use due to idiosyncratic liver damage and failure. The mechanism of toxicity is still not determined, moreover, it is still not clear whether toxicity is due to the parent compound or its metabolite(s). The cytotoxicity of TRO was evaluated in human hepatocytes using previously cryopreserved hepatocyte suspensions from 27 human donors. Cellular adenosine triphosphate content was used as a viability endpoint. To investigate the role of xenobiotic metabolism in TRO toxicity, the correlation between the drug metabolism activities of the hepatocytes from each donor to EC(50) values TRO cytotoxicity. The activities examined were cytochrome P450 (CYP) isoform activities (CYP2A6, CYP2D6, CYP2C19, CYP1A2, CYP2E1, CYP3A4 and CYP2C9) and phase 2 conjugation enzyme activities (phenol sulfotransferase (PST) and glucuronyl transferase (UGT)). Taken individually, none of the phase 1 or 2 enzyme activities correlated to the EC(50). However, when three enzyme activities ((CYP3A4 x UGT)/PST) were taken into account, a correlation was made (r(2)=0.53). Based on the correlation, we hypothesize that TRO and TRO sulfate are direct acting toxicants, whereas CYP3A4 oxidation and glucuronidation are detoxification pathways.

Troglitazone, an oral antidiabetic drug, was reported to cause adverse hepatic effects in certain individuals, leading to its withdrawal from the market. After incubation of troglitazone (100 microM) with the human hepatoma cell line, HepG2 cells, and human primary hepatocytes for 48 to 72 h, an unknown peak was detected in the cell culture. The formation of this peak from troglitazone (100 microM) was also catalyzed by expressed CYP3A4, and further HPLC analysis revealed that there were three metabolites (metabolite A, B, and C) in the peak. The major metabolite, metabolite C (M-C) was identified as an epoxide of a quinone metabolite of troglitazone by comparison with a synthetic authentic standard using tandem mass spectrometry, (1)H NMR, and (13)C NMR analyses. The other two metabolites (M-A and M-B) were stereoisomers with the same molecular weight as M-C, probably produced from M-C by intramolecular rearrangement at the epoxide moiety. M-C showed a weak cytotoxicity in HepG2 cells at low concentrations, as assessed by the crystal violet-staining assay. Since epoxides are generally regarded as the chemically reactive species, M-C may play a role in idiosyncrasy of troglitazone hepatotoxicity via individual differences either in the formation or degradation of this metabolite.

We examined the cytotoxicity of troglitazone toward cultured rat hepatocytes. The drug concentration- and time-dependently decreased cell viability and increased lactate dehydrogenase leakage from the cells. Troglitazone-induced cell death was characterized by "DNA ladders", condensation of nuclei, and a positive reaction to in situ nick-end labeling. The results indicate that troglitazone can cause apoptotic cell death in cultured rat hepatocytes.