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.

Non-viral gene delivery of the oncotoxic protein NS1 for treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

Retrospective analysis of adverse drug reactions leading to short-term emergency hospital readmission.

AIMS OF THE STUDY: Adverse drug reactions (ADRs) are an important cause of hospital admissions. Insufficient data are available about the frequency and characteristics of ADR-related emergency readmissions in Switzerland. The aim of this retrospective study was to characterise ADRs related to short-term emergency readmissions in a large Swiss University Hospital and to assess their reporting frequency. METHODS: Electronic records of all patients discharged from the University Hospital Bern within a 12-month period (1 January to 31 December 2012) and emergency readmission within 30 calendar days were reviewed. Case inclusion required a known ADR. Cases with intentional overdosing, lack of compliance or insufficient documentation were excluded. Identified ADR-related readmission cases were searched in the Swiss ADR reporting system to assess reporting rate. RESULTS: There were 1294 emergency readmissions among the 4792 readmissions (14% of all admissions) within 30 days after discharge. We identified 270 cases of ADR-related readmissions, corresponding to 21% of emergency readmissions and 6% of all readmissions within 30 days. The most frequent ADRs were gastrointestinal disorders (26%), infections and infestations (19%), and nervous system disorders (10%). The most frequent drug classes leading to ADRs were antineoplastic/immunomodulating (35%) and antithrombotic agents (25%). Only 8 (3%) of the 270 cases were reported to the Swiss ADR reporting system. CONCLUSION: ADR-related readmissions constituted a considerable part of short-term emergency readmissions. Despite being a relevant cause for rehospitalisation, only a minority of the ADRs were reported to the regulatory authorities. Strategies to prevent ADR-related readmissions and to improve reporting rates are needed.

Antiseizure drugs and risk of developing smoking-related chronic obstructive pulmonary disease or lung cancer: A population-based case-control study.

AIMS: To determine whether enzyme-inducing antiseizure drugs (ASDs) affect the risk of developing chronic obstructive pulmonary disease (COPD) or lung cancer in smokers. METHODS: Cases of COPD and lung cancer and matched controls without these conditions were identified from a population of smokers with ≥1 prescription for any type of ASD in the Clinical Practice Research Datalink UK database of patients managed in primary care (1995-2016). A matched case-control study was performed utilising multivariate logistic regression analyses of exposure to enzyme-inducing ASDs compared to non-enzyme-inducing ASDs. The duration of ASD exposure and level of tobacco exposure were also assessed. RESULTS: We identified 5952 incident COPD and 1373 incident lung cancer cases, and 59 328 and 13 681 matched controls, respectively. Compared with never use, ever use of enzyme-inducing ASDs was associated with slightly decreased risk estimates of COPD (adjusted odds ratio: 0.85, 95% confidence interval: 0.81-0.89) and lung cancer (adjusted odds ratio: 0.82, 95% confidence interval: 0.73-0.92). These risk estimates were attenuated in heavy smokers. CONCLUSION: We found slightly decreased risk estimates of COPD and lung cancer among smokers taking enzyme-inducing ASDs and hypothesise that this may be related to induction of detoxification of tobacco-specific lung toxins.

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.

Valproic acid affects fatty acid and triglyceride metabolism in HepaRG cells exposed to fatty acids by different mechanisms.

Treatment with valproate is associated with hepatic steatosis, but the mechanisms are not fully elucidated in human cell systems. We therefore investigated the effects of valproate on fatty acid and triglyceride metabolism in HepaRG cells, a human hepatoma cell line. In previously fatty acid loaded HepaRG cells, valproate impaired lipid droplet disposal starting at 1 mM after incubation for 3 or 7 days. Valproate increased the expression of genes associated with fatty acid import and triglyceride synthesis, but did not relevantly affect expression of genes engaged in fatty acid activation. Valproate impaired mitochondrial fatty acid metabolism by inhibiting ß-ketothiolase and the function of the electron transport chain, which was associated with increased mitochondrial reactive oxygen species production. Valproate increased the mitochondrial DNA copy number per HepaRG cell, possibly as a consequence of impaired mitochondrial function. Valproate decreased the hepatocellular mRNA and protein expression of the fatty acid binding protein 1 (FABP1) and of the microsomal triglyceride transfer protein (MTTP) at 1 mM and increased the hepatocellular concentration of free fatty acids. Furthermore, valproate decreased protein expression and excretion of ApoB100 in HepaRG cells at 1 mM, reflecting impaired formation and excretion of very low-density lipoprotein (VLDL). In conclusion, valproate increased the hepatocellular triglyceride content by multiple mechanisms, whereby impaired expression of FABP1 and MTTP as well as impaired VLDL formation and excretion appeared to be dominant. Valproate caused cell death mainly by apoptosis, which may be a consequence of mitochondrial oxidative stress and increased hepatocellular concentration of free fatty acids.

Anti-αLß2 antibodies reveal novel endocytotic cross-modulatory functionality.

BACKGROUND AND PURPOSE: Antibodies targeting cell surface receptors are considered to enable highly selective therapeutic interventions for immune disorders and cancer. Their biological profiles are found, generally, to represent the net effects of antibody-target interactions. The former therapeutic anti-integrin αLß2 antibody efalizumab seems to defeat this paradigm by eliciting, via mechanisms currently unknown, much broader effects than would be predicted based on its target specificity. EXPERIMENTAL APPROACH: To elucidate the mechanisms behind these broad effects, we investigated in primary human lymphocytes in vitro the effects of anti-αLß2 antibodies on the expression of αLß2 as well as unrelated α4 integrins, in comparison to Fab fragments and small-molecule inhibitors. KEY RESULTS: We demonstrate that anti-αLß2 mAbs directly induce the internalization of α4 integrins. The endocytotic phenomenon is a direct consequence of their antibody nature. It is inhibited when monovalent Fab fragments or small-molecule inhibitors are used. It is independent of crosslinking via anti-Fc mAbs and of αLß2 activation. The cross-modulatory effect is unidirectional and not observed in a similar fashion with the α4 integrin antibody natalizumab. CONCLUSION AND IMPLICATIONS: The present study identifies endocytotic cross-modulation as a hitherto unknown non-canonical functionality of anti-αLß2 antibodies. This cross-modulation has the potential to fundamentally alter an antibody's benefit risk profile, as evident with efalizumab. The newly described phenomenon may be of relevance to other therapeutic antibodies targeting cluster-forming receptors. Thus, pharmacologists should be cognizant of this action when investigating such antibodies.

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.

Predictive impact of antibiotics in patients with advanced non small-cell lung cancer receiving immune checkpoint inhibitors : Antibiotics immune checkpoint inhibitors in advanced NSCLC.

PURPOSE: In this study, we test the hypothesis that the use of ATB reduces the efficacy of PD(L)1-targeting mAb. METHODS: We included patients with locally advanced, inoperable or metastatic, EGFR wildtype and ALK-negative non-small-cell lung cancer (NSCLC) who received a PD(L)1 targeting mAb (immune checkpoint inhibitor, ICI) between January 2013 and December 2017. The primary study objective was to assess the predictive impact of ATB use within 2 months prior to starting ICI treatment on overall survival from the time of starting ICI treatment (OS-ICI). RESULTS: 33 out of 218 evaluable patients (15.1%) received ATB within 2 months prior to starting ICI treatment. The use of ATB prior to starting ICI was associated with a lower rate of radiological response (18.2 vs. 28.3%, respectively, P = 0.02). PFS was significantly shorter in patients receiving ATB within 2 months prior to ICI compared to those not receiving ATB (median PFS 1.4 vs. 5.5 months, HR = 2.22, P < 0.01). OS-ICI was significantly shorter in NSCLC patients receiving ATB within 2 months prior to ICI compared to those not receiving ATB (median OS-ICI 1.8 vs. 15.4 months, HR = 2.61, P < 0.01; adjusted HR = 3.73, P < 0.01). CONCLUSION: The results of this study suggest that ATB may have a deleterious effect in patients with advanced NSCLC receiving ICI treatment, and more research seems to be justified to explore potential mechanisms.

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.

Acute Liver Failure in a Patient Treated With Metamizole.

We report on a patient who developed acute liver failure while being treated with metamizole. After liver transplantation, the patient recovered rapidly. Liver biopsy showed massive necrosis and lobular infiltration of lymphocytes. A lymphocyte transformation test performed 20 months after transplantation was positive for metamizole. In vitro investigations with N-methyl-4-aminoantipyrine (MAA) and 4-aminoantipyrine (AA), the two active metabolites of metamizole, did not reveal relevant toxicity in HepG2 and HepaRG cells. The demonstration of activated lymphocytes by the lymphocyte transformation test and the absence of relevant cytotoxicity by MAA and AA in hepatocyte cell lines suggest an immunological mechanism of metamizole-associated hepatotoxicity.

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.

Toxicity of metamizole on differentiating HL60 cells and human neutrophil granulocytes.

Metamizole is an analgesic and antipyretic with a superior analgesic efficacy than paracetamol. Since metamizole can cause neutropenia and agranulocytosis, it is currently used in only few countries. In a previous study, we have shown that N-methyl-4-aminoantipyrine (MAA), the active metamizole metabolite, reacts with hemin and forms an electrophilic metabolite that is toxic for HL60 cells, but not for mature neutrophil granulocytes. In the current study, we investigated the toxicity of hemin (12.5 µM) and MAA (100 µM) on differentiating HL60 cells. In undifferentiated HL60 cells, hemin decreased the viability and this effect was significantly increased by MAA. Similarly, hemin/MAA was more toxic than hemin alone on human cord blood cells. At 3 days (metamyelocyte stage) and 5 days of differentiation (mature neutrophils), hemin/MAA was not toxic on HL60 cells, whereas hemin alone was still toxic. No toxicity was observed on freshly isolated human neutrophils. The protein expression of enzymes responsible for hemin metabolism increased with HL60 cell differentiation. Inhibition of heme oxygenase-1 or cytochrome P450 reductase increased the toxicity of hemin and hemin/MAA in undifferentiated, but only for hemin in differentiated HL60 cells. Similar to the enzymes involved in hemin metabolism, the protein expression of enzymes involved in antioxidative defense and the cellular glutathione pool increased with HL60 cell differentiation. In conclusion, HL60 cells become resistant to the toxicity of hemin/MAA and partly also of hemin during their differentiation. This resistance is associated with the development of heme metabolism and of the antioxidative defense system including the cellular glutathione pool.

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.

Para-Halogenation Affects Monoamine Transporter Inhibition Properties and Hepatocellular Toxicity of Amphetamines and Methcathinones.

Halogenated derivatives of amphetamine-type stimulants are appearing on the drug market, often with altered pharmacological profile and sometimes different legal status compared to the non-halogenated substances. The aim of the present study was to investigate the pharmacological profile and hepatocellular toxicity of para-halogenated amphetamines and cathinones. The potential of amphetamine, 4-fluoroamphetamine, 4-chloroamphetamine, methcathinone, 4-fluoromethcathinone, and 4-chloromethcathinone to inhibit the monoamine transporters for norepinephrine, dopamine, and serotonin was determined in transporter-transfected human embryonic kidney 293 cells. Cell membrane integrity, ATP content, oxygen consumption rate, and superoxide levels were measured in human hepatoma HepG2 cells after exposure to the substances for 24 h. All compounds inhibited the norepinephrine transporter at submicromolar concentrations and the dopamine transporter at low micromolar concentrations. The selectivity of the compounds to inhibit the dopamine versus serotonin transporter decreased with increasing size of the para-substituent, resulting in potent serotonin uptake inhibition for the halogenated derivatives. All substances depleted the cellular ATP content at lower concentrations (0.25-2 mM) than cell membrane integrity loss occurred (≥0.5 mM), suggesting mitochondrial toxicity. The amphetamines and 4-chloromethcathinone additionally impaired the mitochondrial respiratory chain, confirming mitochondrial toxicity. The following toxicity rank order for the para-substituents was observed: chloride > fluoride > hydrogen. In conclusion, para-halogenation of stimulants increases the risk for serotonergic neurotoxicity. Furthermore, para-halogenation may increase hepatic toxicity mediated by mitochondrial impairment in susceptible users.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

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.