Mechanisms of hepatocellular toxicity associated with the components of St. John's Wort extract hypericin and hyperforin in HepG2 and HepaRG cells.

St. John's Wort preparations are used for the treatment of mild to moderate depression. They are usually well tolerated but can cause adverse reactions including liver toxicity in rare cases. To date, the mechanism(s) underlying the hepatotoxicity of St. John's Wort extracts are poorly investigated. We studied the hepatocellular toxicity of hypericin and hyperforin as the two main ingredients of St. John's Wort extracts in HepG2 and HepaRG cells and compared the effects to citalopram (a synthetic serotonin uptake inhibitor) with a special focus on mitochondrial toxicity and oxidative stress. In HepG2 cells, hypericin was membrane-toxic at 100 µM and depleted ATP at 20 µM. In HepaRG cells, ATP depletion started at 5 µM. In comparison, hyperforin and citalopram were not toxic up to 100 µM. In HepG2 cells, hypericin decreased maximal respiration starting at 2 µM and mitochondrial ATP formation starting at 10 µM but did not affect glycolytic ATP production. Hypericin inhibited the activity of complex I, II and IV of the electron transfer system and caused mitochondrial superoxide accumulation in cells. The protein expression of mitochondrial superoxide dismutase 2 (SOD2) and thioredoxin 2 (TRX2) and total and reduced glutathione decreased in cells exposed to hypericin. Finally, hypericin diminished the mitochondrial DNA copy number and caused cell necrosis but not apoptosis. In conclusion, hypericin, but not hyperforin or citalopram, is a mitochondrial toxicant at low micromolar concentrations. This mechanism may contribute to the hepatotoxicity occasionally observed in susceptible patients treated with St. John's Wort preparations.

Imatinib and Dasatinib Provoke Mitochondrial Dysfunction Leading to Oxidative Stress in C2C12 Myotubes and Human RD Cells.

Tyrosine kinase inhibitors (TKIs) can cause skeletal muscle toxicity in patients, but the underlying mechanisms are mostly unclear. The goal of the current study was to better characterize the role of mitochondria in TKI-associated myotoxicity. We exposed C2C12 murine myoblasts and myotubes as well as human rhabdomyosarcoma cells (RD cells) for 24 h to imatinib (1-100 µM), erlotinib (1-20 µM), and dasatinib (0.001-100 µM). In C2C12 myoblasts, imatinib was membrane toxic at 50 µM and depleted the cellular ATP pool at 20 µM. In C2C12 myotubes exposed to imatinib, ATP depletion started at 50 µM whereas membrane toxicity was not detectable. In myoblasts and myotubes exposed to dasatinib, membrane toxicity started at 0.5 µM and 2 µM, respectively, and the ATP drop was visible at 0.1 µM and 0.2 µM, respectively. When RD cells were exposed to imatinib, ATP depletion started at 20 µM whereas membrane toxicity was not detectable. Dasatinib was membrane toxic at 20 µM and depleted the cellular ATP pool already at 0.5 µM. Erlotinib was not toxic in both cell models. Imatinib (20 µM) and dasatinib (1 µM) reduced complex I activity in both cell models. Moreover, the mitochondrial membrane potential (Δψm) was dissipated for both TKIs in myotubes. In RD cells, the Δψm was reduced only by dasatinib. Both TKIs increased mitochondrial superoxide accumulation and decreased the mitochondrial copy number in both cell lines. In consequence, they increased protein expression of superoxide dismutase (SOD) 2 and thioredoxin 2 and cleavage of caspase 3, indicating apoptosis in C2C12 myotubes. Moreover, in both cell models, the mRNA expression of Sod1 and Sod2 increased when RD cells were exposed to dasatinib. Furthermore, dasatinib increased the mRNA expression of atrogin-1 and murf-1, which are important transcription factors involved in muscle atrophy. The mRNA expression of atrogin-1 increased also in RD cells exposed to imatinib. In conclusion, imatinib and dasatinib are mitochondrial toxicants in mouse C2C12 myotubes and human RD cells. Mitochondrial superoxide accumulation induced by these two TKIs is due to the inhibition of complex I and is probably related to impaired mitochondrial and myocyte proliferation.

Lapatinib Activates the Kelch-Like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in HepG2 Cells.

The receptor tyrosine kinase inhibitor lapatinib, indicated to treat patients with HER2-positive breast cancer in combination with capecitabine, can cause severe hepatotoxicity. Lapatinib is further associated with mitochondrial toxicity and accumulation of reactive oxygen species. The effect of lapatinib on the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, the major cellular defense pathway against oxidative stress, has so far not been studied in detail. In the present study, we show that lapatinib (2-20 µM) activates the Keap1-Nrf2 pathway in HepG2 cells, a hepatocellular carcinoma-derived cell line, in a concentration-dependent manner upon 24 h of treatment. Lapatinib stabilized the transcription factor Nrf2 at concentrations ≥5 µM and caused its nuclear translocation. Well-established Nrf2 regulated genes (Nqo1, Gsta1, Gclc, and Gclm) were upregulated at lapatinib concentrations ≥10 µM. Furthermore, cellular and mitochondrial glutathione (GSH) levels increased starting at 10 µM lapatinib. As a marker of oxidative stress, cellular GSSG significantly increased at 10 and 20 µM lapatinib. Furthermore, the gene expression of mitochondrial Glrx2 and SOD2 were increased upon lapatinib treatment, which was also observed for the mitochondrial SOD2 protein content. In conclusion, lapatinib treatment for 24 h activated the Keap1-Nrf2 pathway in HepG2 cells starting at 10 µM, which is a clinically relevant concentration. As a consequence, treatment with lapatinib increased the mRNA and protein expression of antioxidative and other cytoprotective genes and induced GSH synthesis, but these measures could not completely block the oxidative stress associated with lapatinib.

Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called "legal highs". The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

The uricosuric benzbromarone disturbs the mitochondrial redox homeostasis and activates the NRF2 signaling pathway in HepG2 cells.

The uricosuric benzbromarone is a mitochondrial toxicant associated with severe liver injury in patients treated with this drug. Since dysfunctional mitochondria can increase mitochondrial superoxide (O2•-) production, we investigated the consequences of benzbromarone-induced mitochondrial oxidative stress on the hepatic antioxidative defense system. We exposed HepG2 cells (a human hepatocellular carcinoma cell line) to increasing concentrations of benzbromarone (1-100 µM) for different durations (2-24 h), and investigated markers of antioxidative defense and oxidative damage. At high concentrations (≥50 µM), benzbromarone caused accumulation of mitochondrial superoxide (O2•-) and cellular reactive oxygen species (ROS). At concentrations >50 µM, benzbromarone increased the mitochondrial and cellular GSSG/GSH ratio and increased the oxidized portion of the mitochondrial thioredoxin 2. Benzbromarone stabilized the transcription factor NRF2 and caused its translocation into the nucleus. Consequently, the expression of the NRF2-regulated antioxidative proteins superoxide dismutase 1 (SOD1) and 2 (SOD2), glutathione peroxidase 1 (GPX1) and 4 (GPX4), as well as thioredoxin 1 (TRX1) and 2 (TRX2) increased. Finally, upregulation of NRF2 by siRNA-mediated knock-down of KEAP1 partially protected HepG2 cells from benzbromarone-induced membrane damage and ATP depletion. In conclusion, benzbromarone increased mitochondrial O2•- accumulation and activates the NRF2 signaling pathway in HepG2 cells, thereby strengthening the cytosolic and mitochondrial antioxidative defense. Impaired antioxidative defense may represent a risk factor for benzbromarone-induced hepatotoxicity.

PGC-1α plays a pivotal role in simvastatin-induced exercise impairment in mice.

AIM: Statins decrease cardiovascular complications, but can induce myopathy. Here, we explored the implication of PGC-1α in statin-associated myotoxicity. METHODS: We treated PGC-1α knockout (KO), PGC-1α overexpression (OE) and wild-type (WT) mice orally with 5 mg simvastatin kg-1  day-1 for 3 weeks and assessed muscle function and metabolism. RESULTS: In WT and KO mice, but not in OE mice, simvastatin decreased grip strength, maximal running distance and vertical power assessed by ergometry. Post-exercise plasma lactate concentrations were higher in WT and KO compared to OE mice. In glycolytic gastrocnemius, simvastatin decreased mitochondrial respiration, increased mitochondrial ROS production and free radical leak in WT and KO, but not in OE mice. Simvastatin increased mRNA expression of Sod1 and Sod2 in glycolytic and oxidative gastrocnemius of WT, but decreased it in KO mice. OE mice had a higher mitochondrial DNA content in both gastrocnemius than WT or KO mice and simvastatin exhibited a trend to decrease the citrate synthase activity in white and red gastrocnemius in all treatment groups. Simvastatin showed a trend to decrease the mitochondrial volume fraction in both muscle types of all treatment groups. Mitochondria were smaller in WT and KO compared to OE mice and simvastatin further reduced the mitochondrial size in WT and KO mice, but not in OE mice. CONCLUSIONS: Simvastatin impairs skeletal muscle function, muscle oxidative metabolism and mitochondrial morphology preferentially in WT and KO mice, whereas OE mice appear to be protected, suggesting a role of PGC-1α in preventing simvastatin-associated myotoxicity.

Exogenous Iron Increases Fasciocidal Activity and Hepatocellular Toxicity of the Synthetic Endoperoxides OZ78 and MT04.

The synthetic peroxides OZ78 and MT04 recently emerged as fasciocidal drug candidates. However, the effect of iron on fasciocidal activity and hepatocellular toxicity of these compounds is unknown. We investigated the in vitro fasciocidal activity and hepatocellular toxicity of OZ78 and MT04 in absence and presence of Fe(II)chloride and hemin, and conducted a toxicological study in mice. Studies were performed in comparison with the antimalarial artesunate (AS), a semisynthetic peroxide. Fasciocidal effects of OZ78 and MT04 were confirmed and enhanced by Fe2+ or hemin. In HepG2 cells, AS reduced cellular ATP and impaired membrane integrity concentration-dependently. In comparison, OZ78 or MT04 were not toxic at 100 µM and reduced the cellular ATP by 13% and 19%, respectively, but were not membrane-toxic at 500 µM. The addition of Fe2+ or hemin increased the toxicity of OZ78 and MT04 significantly. AS inhibited complex I, II, and IV of the mitochondrial electron transport chain, and MT04 impaired complex I and II, whereas OZ78 was not toxic. All three compounds increased cellular reactive oxygen species (ROS) concentration-dependently, with a further increase by Fe2+ or hemin. Mice treated orally with up to 800 mg OZ78, or MT04 showed no relevant hepatotoxicity. In conclusion, we confirmed fasciocidal activity of OZ78 and MT04, which was increased by Fe2+ or hemin. OZ78 and MT04 were toxic to HepG2 cells, which was explained by mitochondrial damage associated with ROS generation in the presence of iron. No relevant hepatotoxicity was observed in mice in vivo, possibly due to limited exposure and/or high antioxidative hepatic capacity.

Mitochondrial oxidative stress plays a critical role in the cardiotoxicity of sunitinib: Running title: Sunitinib and oxidative stress in hearts.

Sunitinib is cardiotoxic, but the mechanisms are not entirely clear. We aimed to enlarge our knowledge about the role of mitochondria in cardiac toxicity of sunitinib in vitro and in vivo. For this reason, we studied the toxicity of sunitinib on cardiac H9c2 cells exposed for 24 h, permeabilized rat cardiac fibers exposed for 15 min and in mice treated orally with sunitinib for 2 weeks (7.5 mg/kg/day). In H9c2 cells exposed for 24 h, sunitinib was more cytotoxic under galactose (favoring mitochondrial metabolism) compared to glucose conditions (favoring glycolysis). Sunitinib dissipated the mitochondrial membrane potential starting at 10 µM under glucose and at 5 µM under galactose conditions. Sunitinib reduced activities of mitochondrial enzyme complexes of the electron transport chain (ETC), increased mitochondrial ROS accumulation and decreased the cellular GSH pool. Electron microscopy revealed swollen mitochondria with loss of cristae. Accordingly, sunitinib caused caspase 3 activation and DNA fragmentation in H9c2 cells. Co-exposure with mito-TEMPO (mitochondrial-specific ROS scavenger) for 24 h prevented ATP and GSH depletion, as well as the increases in H2O2 and caspase 3/7 activity observed with sunitinib. In mice, treatment with sunitinib for two weeks increased plasma concentrations of troponin I and creatine kinase MB, indicating cardiomyocyte damage. The activity of enzyme complexes of the ETCwas decreased, mitochondrial ROS were increased and cleavage of caspase 3 was increased, suggesting cardiomyocyte apoptosis. In conclusion, mitochondrial damage with ROS accumulation appears to be an important mechanism of cardiotoxicity associated with sunitinib, eventually leading to apoptotic cell death.

Hepatotoxicity Due to Azole Antimycotic Agents in a HLA B*35:02-Positive Patient.

We will present a 42-year-old woman with acute myeloid leukemia and pulmonary aspergillosis. She was treated with several antifungal agents, including three triazoles. Voriconazole, posaconazole, and isavuconazole all led to hepatocellular liver injury. Voriconazole administration led to a peak alanine aminotransferase (ALT) value of 1,793 U/L (normal range, 9-59 U/L). After posaconazole and isavuconazole treatment, ALT rose over 500 U/L. The typical course of events, exclusion of differential diagnoses, and normalization of the liver function tests (LFTs) after stopping the triazoles were highly suspicious for a drug-induced liver injury (DILI). Interestingly, our patient carries a rare HLA B allele (HLA B*35:02), which occurs in less than 1% of the population and is known to be associated with minocycline-induced liver injury. Over the course of 4 months, the patient received two induction chemotherapies and afterward underwent a successful allogenic hematopoietic stem cell transplantation. Her liver function recovered rapidly and favorable clinical findings concerning the aspergillosis led to a de-escalation of the antifungal treatment to prophylactic dose fluconazole. Delayed hepatotoxicity suggested a dose dependency and a cumulative effect. The question of a common pathophysiology and a cross-toxicity was raised. At the present time, only a few case reports describe cross-toxicity or its absence after rechallenge with different azoles. The pathophysiology is not well understood. Ketoconazole was found to impair rat mitochondrial function in vitro. Further investigations showed cell membrane toxicity and ATP depletion in isolated human liver cancer cells. Our case report suggests a cross-toxicity, dose-dependency, and a possible genetic predisposition of triazole-induced liver injury.

Molecular Toxicological Mechanisms of Synthetic Cathinones on C2C12 Myoblasts.

Synthetic cathinones are popular psychoactive substances that may cause skeletal muscle damage. In addition to indirect sympathomimetic myotoxicity, these substances could be directly myotoxic. Since studies in myocytes are currently lacking, the aim of the present study was to investigate potential toxicological effects by synthetic cathinones on C2C12 myoblasts (mouse skeletal muscle cell line). We exposed C2C12 myoblasts to 3-methylmethcathinone, 4-methylmethcathinone (mephedrone), 3,4-methylenedioxymethcathinone (methylone), 3,4-methylenedioxypyrovalerone (MDPV), alpha-pyrrolidinovalerophenone (α-PVP), and naphthylpyrovalerone (naphyrone) for 1 or 24 h before cell membrane integrity, ATP content, mitochondrial oxygen consumption, and mitochondrial superoxide production was measured. 3,4-Methylenedioxymethamphetamine (MDMA) was included as a reference compound. All investigated synthetic cathinones, as well as MDMA, impaired cell membrane integrity, depleted ATP levels, and increased mitochondrial superoxide concentrations in a concentration-dependent manner in the range of 50⁻2000 µM. The two pyrovalerone derivatives α-PVP and naphyrone, and MDMA, additionally impaired basal and maximal cellular respiration, suggesting mitochondrial dysfunction. Alpha-PVP inhibited complex I, naphyrone complex II, and MDMA complex I and III, whereas complex IV was not affected. We conclude that, in addition to sympathetic nervous system effects and strenuous muscle exercise, direct effects of some cathinones on skeletal muscle mitochondria may contribute to myotoxicity in susceptible synthetic cathinone drugs users.

Sunitinib induces hepatocyte mitochondrial damage and apoptosis in mice.

Reports concerning hepatic mitochondrial toxicity of sunitinib are conflicting. We therefore decided to conduct a toxicological study in mice. After having determined the highest dose that did not affect nutrient ingestion and body weight, we treated mice orally with sunitinib (7.5 mg/kg/day) for 2 weeks. At the end of treatment, peak sunitinib plasma concentrations were comparable to those achieved in humans and liver concentrations were approximately 25-fold higher than in plasma. Sunitinib did not affect body weight, but increased plasma ALT activity 6-fold. The activity of enzyme complexes of the electron transport chain (ETC) was decreased numerically in freshly isolated and complex III activity significantly in previously frozen liver mitochondria. In previously frozen mitochondria, sunitinib decreased NADH oxidase activity concentration-dependently in both treatment groups. The hepatic mitochondrial reactive oxygen species (ROS) content and superoxide dismutase 2 expression were increased in sunitinib-treated mice. Protein and mRNA expression of several subunits of mitochondrial enzyme complexes were decreased in mitochondria from sunitinib-treated mice. Protein expression of PGC-1α, citrate synthase activity and mtDNA copy number were all decreased in livers of sunitinib-treated mice, indicating impaired mitochondrial proliferation. Caspase 3 activation and TUNEL-positive hepatocytes were increased in livers of sunitinib-treated mice, indicating hepatocyte apoptosis. In conclusion, sunitinib caused concentration-dependent toxicity in isolated mitochondria at concentrations reached in livers in vivo and inhibited hepatic mitochondrial proliferation. Daily mitochondrial insults and impaired mitochondrial proliferation most likely explain hepatocellular injury observed in mice treated with sunitinib.

Effect of the Catechol-O-Methyltransferase Inhibitors Tolcapone and Entacapone on Fatty Acid Metabolism in HepaRG Cells.

Tolcapone and entacapone are catechol-O-methyltransferase inhibitors used in patients with Parkinson's disease. For tolcapone, patients with liver failure have been reported with microvesicular steatosis observed in the liver biopsy of 1 patient. We therefore investigated the impact of tolcapone and entacapone on fatty acid metabolism in HepaRG cells exposed for 24 h and on acutely exposed mouse liver mitochondria. In HepaRG cells, tolcapone induced lipid accumulation starting at 100 µM, whereas entacapone was ineffective up to 200 µM. In HepaRG cells, tolcapone-inhibited palmitate metabolism and activation starting at 100 µM, whereas entacapone did not affect palmitate metabolism. In isolated mouse liver mitochondria, tolcapone inhibited palmitate metabolism starting at 5 µM and entacapone at 50 µM. Inhibition of palmitate activation could be confirmed by the acylcarnitine pattern in the supernatant of HepaRG cell cultures. Tolcapone-reduced mRNA and protein expression of long-chain acyl-CoA synthetase 1 (ACSL1) and protein expression of ACSL5, whereas entacapone did not affect ACSL expression. Tolcapone increased mRNA expression of the fatty acid transporter CD36/FAT, impaired the secretion of ApoB100 by HepaRG cells and reduced the mRNA expression of ApoB100, but did not relevantly affect markers of fatty acid binding, lipid droplet formation and microsomal lipid transfer. In conclusion, tolcapone impaired hepatocellular fatty acid metabolism at lower concentrations than entacapone. Tolcapone increased mRNA expression of fatty acid transporters, inhibited activation of long-chain fatty acids and impaired very low-density lipoprotein secretion, causing hepatocellular triglyceride accumulation. The findings may be relevant in patients with a high tolcapone exposure and preexisting mitochondrial dysfunction.

Mechanisms of mitochondrial toxicity of the kinase inhibitors ponatinib, regorafenib and sorafenib in human hepatic HepG2 cells.

Previous studies have shown that certain kinase inhibitors are mitochondrial toxicants. In the current investigation, we determined the mechanisms of mitochondrial impairment by the kinase inhibitors ponatinib, regorafenib, and sorafenib in more detail. In HepG2 cells cultured in galactose and exposed for 24 h, all three kinase inhibitors investigated depleted the cellular ATP pools at lower concentrations than cytotoxicity occurred, compatible with mitochondrial toxicity. The kinase inhibitors impaired the activity of different complexes of the respiratory chain in HepG2 cells exposed to the toxicants for 24 h and in isolated mouse liver mitochondria exposed acutely. As a consequence, they increased mitochondrial production of ROS in HepG2 cells in a time- and concentration-dependent fashion and decreased the mitochondrial membrane potential concentration-dependently. In HepG2 cells exposed for 24 h, they induced mitochondrial fragmentation, lysosome content and mitophagy as well as mitochondrial release of cytochrome c, leading to apoptosis and/or necrosis. In conclusion, the kinase inhibitors ponatinib, regorafenib, and sorafenib impaired the function of the respiratory chain, which was associated with increased ROS production and a drop in the mitochondrial membrane potential. Despite activation of defense measures such as mitochondrial fission and mitophagy, some cells were liquidated concentration-dependently by apoptosis or necrosis. Mitochondrial dysfunction may represent a toxicological mechanism of hepatotoxicity associated with certain kinase inhibitors.

Mechanisms of toxicity associated with six tyrosine kinase inhibitors in human hepatocyte cell lines.

Tyrosine kinase inhibitors have revolutionized the treatment of certain cancers. They are usually well tolerated, but can cause adverse reactions including liver injury. Currently, mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors are only partially clarified. We therefore aimed at investigating the toxicity of regorafenib, sorafenib, ponatinib, crizotinib, dasatinib and pazopanib on HepG2 and partially on HepaRG cells. Regorafenib and sorafenib strongly inhibited oxidative metabolism (measured by the Seahorse-XF24 analyzer) and glycolysis, decreased the mitochondrial membrane potential and induced apoptosis and/or necrosis of HepG2 cells at concentrations similar to steady-state plasma concentrations in humans. In HepaRG cells, pretreatment with rifampicin decreased membrane toxicity (measured as adenylate kinase release) and dissipation of adenosine triphosphate stores, indicating that toxicity was associated mainly with the parent drugs. Ponatinib strongly impaired oxidative metabolism but only weakly glycolysis, and induced apoptosis of HepG2 cells at concentrations higher than steady-state plasma concentrations in humans. Crizotinib and dasatinib did not significantly affect mitochondrial functions and inhibited glycolysis only weakly, but induced apoptosis of HepG2 cells. Pazopanib was associated with a weak increase in mitochondrial reactive oxygen species accumulation and inhibition of glycolysis without being cytotoxic. In conclusion, regorafenib and sorafenib are strong mitochondrial toxicants and inhibitors of glycolysis at clinically relevant concentrations. Ponatinib affects mitochondria and glycolysis at higher concentrations than reached in plasma (but possibly in liver), whereas crizotinib, dasatinib and pazopanib showed no relevant toxicity. Mitochondrial toxicity and inhibition of glycolysis most likely explain hepatotoxicity associated with regorafenib, sorafenib and possibly pazopanib, but not for the other compounds investigated.

Hepatocellular Toxicity Associated with Tyrosine Kinase Inhibitors: Mitochondrial Damage and Inhibition of Glycolysis.

Tyrosine kinase inhibitors (TKIs) are anticancer drugs with a lesser toxicity than classical chemotherapeutic agents but still with a narrow therapeutic window. While hepatotoxicity is known for most TKIs, underlying mechanisms remain mostly unclear. We therefore aimed at investigating mechanisms of hepatotoxicity for imatinib, sunitinib, lapatinib and erlotinib in vitro. We treated HepG2 cells, HepaRG cells and mouse liver mitochondria with TKIs (concentrations 1-100 µM) for different periods of time and assessed toxicity. In HepG2 cells maintained with glucose (favoring glycolysis), all TKIs showed a time- and concentration-dependent cytotoxicity and, except erlotinib, a drop in intracellular ATP. In the presence of galactose (favoring mitochondrial metabolism), imatinib, sunitinib and erlotinib showed a similar toxicity profile as for glucose whereas lapatinib was less toxic. For imatinib, lapatinib and sunitinib, cytotoxicity increased in HepaRG cells induced with rifampicin, suggesting formation of toxic metabolites. In contrast, erlotinib was more toxic in HepaRG cells under basal than CYP-induced conditions. Imatinib, sunitinib and lapatinib reduced the mitochondrial membrane potential in HepG2 cells and in mouse liver mitochondria. In HepG2 cells, these compounds increased reactive oxygen species production, impaired glycolysis, and induced apoptosis. In addition, imatinib and sunitinib impaired oxygen consumption and activities of complex I and III (only imatinib), and reduced the cellular GSH pool. In conclusion, imatinib and sunitinib are mitochondrial toxicants after acute and long-term exposure and inhibit glycolysis. Lapatinib affected mitochondria only weakly and inhibited glycolysis, whereas the cytotoxicity of erlotinib could not be explained by a mitochondrial mechanism.

The catechol-O-methyltransferase inhibitors tolcapone and entacapone uncouple and inhibit the mitochondrial respiratory chain in HepaRG cells.

The catechol-O-methyltransferase inhibitor tolcapone causes hepatotoxicity and mitochondrial damage in animal models. We studied the interaction of tolcapone with mitochondrial respiration in comparison to entacapone in different experimental models. In HepaRG cells (human cell-line), tolcapone decreased the ATP content (estimated IC50 100±15µM) and was cytotoxic (estimated IC50 333±45µM), whereas entacapone caused no cytotoxicity and no ATP depletion up to 200µM. Cytochrome P450 induction did not increase the toxicity of the compounds. In HepaRG cells, tolcapone (not entacapone) inhibited maximal complex I- and complex II-linked oxygen consumption. In intact mouse liver mitochondria, tolcapone stimulated state 2 complex II-linked respiration and both compounds inhibited state 3 respiration of complex IV. Mitochondrial uncoupling was confirmed for both compounds by stimulation of complex I-linked respiration in the presence of oligomycin. Inhibition of complex I, II and IV for tolcapone and of complex I and IV for entacapone was directly demonstrated in disrupted mouse liver mitochondria. In HepaRG cells, tolcapone-induced inhibition of mitochondrial respiration was associated with increased lactate and ROS production and hepatocyte necrosis. In conclusion, both compounds uncouple oxidative phosphorylation and inhibit mitochondrial enzyme complexes. Tolcapone is a more potent mitochondrial toxicant than entacapone. Mitochondrial toxicity is a possible mechanism for tolcapone-associated hepatotoxicity.

Hepatocellular Toxicity of Imidazole and Triazole Antimycotic Agents.

Hepatotoxicity has been described for all antimycotic azoles currently marketed. A possible mechanism involving mitochondrial dysfunction has been postulated for ketoconazole, but not for the other azoles. The aim of the current investigations was to study the toxicity of different azoles in human cell models and to find out mechanisms of their toxicity. In HepG2 cells, posaconazole and ketoconazole were cytotoxic starting at 20 and 50 µM and decreased the cellular ATP content starting at 5 and 10 µM, respectively. In HepaRG cells, cytotoxicity started at 20 and 100 µM for posaconazole and ketoconazole, respectively, and was slightly accentuated by cytochrome P450 3A4 induction with rifampicin and 1A2 with 3-methylcholantrene. Voriconazole and fluconazole were not cytotoxic. In isolated mouse liver mitochondria, ketoconazole impaired membrane potential and complex I activity, whereas the other azoles were not toxic. In HepG2 cells exposed for 24 h, both posaconazole and ketoconazole (but not fluconazole or voriconazole) decreased the mitochondrial membrane potential, impaired the function of enzyme complexes of the electron transport chain, were associated with mitochondrial superoxide accumulation, decreased mitochondrial DNA and induced apoptosis. In HepG2 cells with mitochondrial dysfunction induced by the vitamin B12 antagonist hydroxy-cobalamin[c-lactam], cytotoxicity and/or ATP depletion was more accentuated than in untreated cells. We conclude that ketoconazole and posaconazole are mitochondrial toxicants starting at concentrations, which can be reached in vivo. Cytotoxicity and ATP depletion are more accentuated in cells with mitochondrial damage, suggesting that preexisting mitochondrial dysfunction is a susceptibility factor for hepatotoxicity associated with these drugs.

IGF-1 prevents simvastatin-induced myotoxicity in C2C12 myotubes.

Statins are generally well tolerated, but treatment with these drugs may be associated with myopathy. The mechanisms of statin-associated myopathy are not completely understood. Statins inhibit AKT phosphorylation by an unclear mechanism, whereas insulin-like growth factor (IGF-1) activates the IGF-1/AKT signaling pathway and promotes muscle growth. The aims of the study were to investigate mechanisms of impaired AKT phosphorylation by simvastatin and to assess effects of IGF-1 on simvastatin-induced myotoxicity in C2C12 myotubes. C2C12 mouse myotubes were exposed to 10 µM simvastatin and/or 10 ng/mL IGF-1 for 18 h. Simvastatin inhibited the IGF-1/AKT signaling pathway, resulting in increased breakdown of myofibrillar proteins, impaired protein synthesis and increased apoptosis. Simvastatin inhibited AKT S473 phosphorylation, indicating reduced activity of mTORC2. In addition, simvastatin impaired stimulation of AKT T308 phosphorylation by IGF-1, indicating reduced activation of the IGF-1R/PI3K pathway by IGF-1. Nevertheless, simvastatin-induced myotoxicity could be at least partially prevented by IGF-1. The protective effects of IGF-1 were mediated by activation of the IGF-1R/AKT signaling cascade. Treatment with IGF-1 also suppressed muscle atrophy markers, restored protein synthesis and inhibited apoptosis. These results were confirmed by normalization of myotube morphology and protein content of C2C12 cells exposed to simvastatin and treated with IGF-1. In conclusion, impaired activity of AKT can be explained by reduced function of mTORC2 and of the IGF-1R/PI3K pathway. IGF-1 can prevent simvastatin-associated cytotoxicity and metabolic effects on C2C12 cells. The study gives insight into mechanisms of simvastatin-associated myotoxicity and provides potential targets for therapeutic intervention.

Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 µmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

Impaired mitochondrial function in HepG2 cells treated with hydroxy-cobalamin[c-lactam]: A cell model for idiosyncratic toxicity.

The vitamin B12 analog hydroxy-cobalamin[c-lactam] (HCCL) impairs mitochondrial protein synthesis and the function of the electron transport chain. Our goal was to establish an in vitro model for mitochondrial dysfunction in human hepatoma cells (HepG2), which can be used to investigate hepatotoxicity of idiosyncratic mitochondrial toxicants. For that, HepG2 cells were treated with HCCL, which inhibits the function of methylmalonyl-CoA mutase and impairs mitochondrial protein synthesis. Secondary, cells were incubated with propionate that served as source of propionyl-CoA, a percursor of methylmalonyl-CoA. Dose-finding experiments were conducted to evaluate the optimal dose and treatment time of HCCL and propionate for experiments on mitochondrial function. 50 µM HCCL was cytotoxic after exposure of HepG2 cells for 2d and 10 and 50 µM HCCL enhanced the cytotoxicity of 100 or 1000 µM propionate. Co-treatment with HCCL (10 µM) and propionate (1000 µM) dissipated the mitochondrial membrane potential and impaired the activity of enzyme complex IV of the electron transport chain. Treatment with HCCL decreased the mRNA content of mitochondrially encoded proteins, whereas the mtDNA content remained unchanged. We observed mitochondrial ROS accumulation and decreased mitochondrial SOD2 expression. Moreover, electron microscopy showed mitochondrial swelling. Finally, HepG2 cells pretreated with a non-cytotoxic combination of HCCL (10 µM) and propionate (100 µM) were more sensitive to the mitochondrial toxicants dronedarone, benzbromarone, and ketoconazole than untreated cells. In conclusion, we established and characterized a cell model, which could be used for testing drugs with idiosyncratic mitochondrial toxicity.