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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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Statins lower the serum low-density lipoprotein cholesterol and prevent cardiovascular events by inhibiting 3-hydroxy-3-methyl-glutaryl-CoA reductase. Although the safety of statins is documented, many patients ingesting statins may suffer from skeletal muscle-associated symptoms (SAMS). Importantly, SAMS are a common reason for stopping the treatment with statins. Statin-associated muscular symptoms include fatigue, weakness and pain, possibly accompanied by elevated serum creatine kinase activity. The most severe muscular adverse reaction is the potentially fatal rhabdomyolysis. The frequency of SAMS is variable but in up to 30% of the patients ingesting statins, depending on the population treated and the statin used. The mechanisms leading to SAMS are currently not completely clarified. Over the last 15 years, several research articles focused on statin-induced mitochondrial dysfunction as a reason for SAMS. Statins can impair the function of the mitochondrial respiratory chain, thereby reducing ATP and increasing ROS production. This can induce mitochondrial membrane permeability transition, release of cytochrome c into the cytosol and induce apoptosis. In parallel, statins inhibit activation of Akt, mainly due to reduced function of mTORC2, which may be related to mitochondrial dysfunction. Mitochondrial dysfunction by statins is also responsible for activation of AMPK, which is associated with impaired activation of mTORC1. Reduced activation of mTORC1 leads to increased skeletal muscle protein degradation, impaired protein synthesis and stimulation of apoptosis. In this paper, we discuss some of the different hypotheses how statins affect skeletal muscle in more detail, focusing particularly on those related to mitochondrial dysfunction and the impairment of the Akt/mTOR pathway., (Copyright © 2019. Published by Elsevier Ltd.)

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 H 2 O 2 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: Lower limb ischemia-reperfusion results in skeletal muscle mitochondrial alterations, production of reactive oxygen species (ROS), and remote organ impairments that are largely involved in patient prognosis. However, whether ischemia without reperfusion increases ROS production and precedes mitochondrial alteration and whether mitochondrial dysfunction occurs early in remote organs is unknown. This study determined muscle mitochondrial function and ROS production after ischemia alone, or followed by two periods of reperfusion, and investigated heart, lung, liver, kidney, and brain mitochondrial functions after lower limb ischemia-reperfusion., Methods: Wistar rats were randomized into four groups: sham (aortic exposure but no ischemia, n = 9), I3 (ischemia alone induced by aortic cross-clamping for 3 hours, n = 9), I3R10' and I3R2 (aortic cross-clamping, followed by reperfusion for 10 minutes [n = 8] or 2 hours [n = 9]). Blood lactate, alanine aminotransferase, aspartate aminotransferase, and creatinine were measured. Mitochondrial respiratory chain complexes I, II, III, and IV activities and mitochondrial coupling (acceptor control ratio) were analyzed using a Clark oxygen electrode in skeletal muscle, lung, heart, brain, liver, and kidney. ROS production was determined using dihydroethidium staining in muscle, heart, liver, and kidney. Inflammation was also investigated in remote organs (heart, liver, and kidney) using monocyte-macrophage-2 antibody staining., Results: Lactate level increased after ischemia in all groups. In muscle, ROS increased significantly after ischemia alone (+324% ± 66%; P = .038), normalized after 10 minutes of reperfusion, and increased again at 2 hours of reperfusion (+349.2 ± 67%; P = .024). Interestingly, mitochondrial function was unaffected by ischemia alone or followed by 10 minutes of reperfusion, but maximal mitochondrial oxidative capacity (6.10 ± 0.51 vs. 4.24 ± 0.36 μmol/min/g, -30%; P < .05) and mitochondrial coupling decreased after 2 hours of reperfusion (1.93 ± 0.17 vs. 1.33 ± 0.07, -45%; P < .01), in sham and I3R2 rats, respectively. Despite increased serum aspartate aminotransferase (×13; P < .0001), alanine aminotransferase (×6; P = .0019), and creatinine (×3; P = .0004), remote organs did not show mitochondrial alteration, inflammation, or ROS production enhancement after 2 hours of reperfusion., Conclusions: Oxidative stress precedes skeletal muscle mitochondrial dysfunction during lower limb ischemia. Such a kinetic explains the efficacy of ischemic preconditioning and supports that therapy should be conducted even during ongoing ischemia, suggesting that ischemic preconditioning might be a successful approach., (Copyright © 2014 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

Dronedarone is an amiodarone-like antiarrhythmic drug associated with severe liver injury. Since dronedarone inhibits mitochondrial respiration and β-oxidation in vitro, mitochondrial toxicity may also explain dronedarone-associated hepatotoxicity in vivo. We therefore studied hepatotoxicity of dronedarone (200mg/kg/day for 2 weeks or 400mg/kg/day for 1 week by intragastric gavage) in heterozygous juvenile visceral steatosis (jvs(+/-)) and wild-type mice. Jvs(+/-) mice have reduced carnitine stores and are sensitive for mitochondrial β-oxidation inhibitors. Treatment with dronedarone 200mg/kg/day had no effect on body weight, serum transaminases and bilirubin, and hepatic mitochondrial function in both wild-type and jvs(+/-) mice. In contrast, dronedarone 400mg/kg/day was associated with a 10-15% drop in body weight, and a 3-5-fold increase in transaminases and bilirubin in wild-type mice and, more accentuated, in jvs(+/-) mice. In vivo metabolism of intraperitoneal (14)C-palmitate was impaired in wild-type, and, more accentuated, in jvs(+/-) mice treated with 400mg/kg/day dronedarone compared to vehicle-treated mice. Impaired β-oxidation was also found in isolated mitochondria ex vivo. A likely explanation for these findings was a reduced activity of carnitine palmitoyltransferase 1a in liver mitochondria from dronedarone-treated mice. In contrast, dronedarone did not affect the activity of the respiratory chain ex vivo. We conclude that dronedarone inhibits mitochondrial β-oxidation in and ex vivo, but not the respiratory chain. Jvs(+/-) mice are slightly more sensitive for the effect of dronedarone on mitochondrial β-oxidation than wild-type mice. The results suggest that inhibition of mitochondrial β-oxidation is an important mechanism of hepatotoxicity associated with dronedarone., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Irrespective of the organ involved, restoration of blood flow to ischemic tissue is vital, although reperfusion per se is deleterious. In the setting of vascular surgery, even subtle skeletal muscle ischemia contributes to remote organ injuries and perioperative and long-term morbidities. Reperfusion-induced injury is thought to participate in up to 40% of muscle damage. Recently, the pathophysiology of lower limb ischemia-reperfusion (IR) has been largely improved, acknowledging a key role for mitochondrial dysfunction mainly characterized by impaired mitochondrial oxidative capacity and premature mitochondrial permeability transition pore opening. Increased oxidative stress triggered by an imbalance between reactive oxygen species (ROS) production and clearance, and facilitated by enhanced inflammation, appears to be both followed and instigated by mitochondrial dysfunction. Mitochondria are both actors and target of IR and therapeutic strategies modulating degree of ROS production could enhance protective signals and allow for mitochondrial protection through a mitohormesis mechanism., (Copyright © 2014. Published by Elsevier Ltd.)

Objective: Muscular injuries contribute to perioperative and long-term morbidity after vascular surgery in humans. We determined whether local and remote ischemic postconditioning might similarly decrease muscle mitochondrial dysfunction through reduced oxidative stress., Methods: Eighteen male Black-6 mice were divided in three groups: (1) sham mice had no ischemia (sham), (2) ischemia-reperfusion (IR) mice underwent 2-hour tourniquet-induced ischemia on both hind limbs, followed by 2-hour reperfusion, and (3) postconditioning (PoC) mice underwent four bouts of 30-second reperfusion and 30-second ischemia at the onset of reperfusion on the right limb; thus, the right limb underwent local PoC and left limb underwent remote PoC (rPoC). Maximal oxidative capacity (V(max)) of the gastrocnemius muscle mitochondrial respiratory chain was measured. Oxidative stress was evaluated by dihydroethidium staining. Expressions of genes involved in antioxidant defense (superoxide dismutase [SOD1], SOD2, glutathione peroxidase [GPx]), apoptosis (Bax, BclII), and inflammation (interleukin-6) were determined by quantitative real-time polymerase chain reaction. Muscle inflammation was determined using immunohistochemistry., Results: IR reduced V(max) (8.5 ± 2.2 vs 10.2 ± 1.8 μmol O(2)/min/g dry weight; P = .034), and increased dihydroethidium staining (134.8%; P = .039). IR decreased GPx expression (-47.9%; P = .048) and increased the proapoptotic marker Bax (255.5%; P = .020). Local PoC and rPoC further increased these deleterious effects. PoC decreased V(max) to 4.4 ± 1.4 μmol O(2)/min/g dry weight (sham vs PoC, -56.9% [P < .001]; IR vs PoC, -48.2% [P < .001]). rPoC similarly reduced V(max) to 5.1 ± 1.9 μmol O(2)/min/g dry weight (sham vs PoC, -50.0% [P < .001]; IR vs PoC, -40.0% [P = .001]). Dihydroethidium staining was further increased by PoC (207.2%; P = .002) and rPoC (305.4%; P < .001) compared with sham and was associated with macrophage infiltration. Local PoC increased SOD1, SOD2, and the antiapoptotic Bcl-2, and rPoC increased Bax (391.6%; P < .001) and the Bax/BclII ratio (621.7%; P < .001)., Conclusions: Local and remote ischemic postconditioning further increased injury by enhancing mitochondrial dysfunction, oxidative stress production, and inflammation. Caution should be applied when considering ischemic postconditioning in vascular surgery., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Objective: This study investigated whether remote (rIPC) and local ischemic preconditioning (IPC) similarly limit skeletal muscle dysfunction induced by aortic cross-clamping., Methods: Rats were divided in three groups: the sham-operated control group (C) underwent surgery without clamping. The ischemia-reperfusion group (IR) had 3 hours of ischemia induced by aortic clamping and collateral vessels ligation, followed by 2 hours of reperfusion. The IPC group had, before prolonged ischemia, three bouts of 10 minutes of ischemia and 10 minutes of reperfusion on the right hind limb. Thus, right hind limbs had local IPC and left hind limbs had rIPC. Complexes I, II, III, and IV activities of the mitochondrial respiratory chain of the gastrocnemius muscle were measured using glutamate-malate (V(max)), succinate (V(succ)), and N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (V(TMPD)). Expressions of genes involved in apoptosis (Bax, Bcl-2) and antioxidant defense (superoxide dismutase 1 [SOD 1], SOD2, glutathione peroxidase [GPx]) were determined by quantitative real-time polymerase chain reaction. Glutathione was also measured., Results: Right and left hind limb mitochondrial functions were similar in controls and after IR. IR reduced V(max) (-21.2%, 6.6 ± 1 vs 5.2 ± 1 μmol O(2)/min/g dry weight, P = .001), V(succ) (-22.2%, P = .032), and V(TMPD) (-22.4%, P = .033), and increased Bax (63.4%, P = .020) and Bax/Bcl-2 ratio (+84.6%, P = .029). SODs and GPx messenger RNA were not modified, but glutathione tended to be decreased after IR. Local IPC and rIPC counteracted similarly these deleterious effects, restoring mitochondrial maximal oxidative capacities and normalizing Bax, the Bax/Bcl-2 ratio, and glutathione., Conclusions: Remote ischemic preconditioning protection against IR injury is equivalent to that achieved by local IPC. It might deserve a broader use in clinical practice., Clinical Relevance: Acute and chronic ischemia induce mitochondrial dysfunction in human skeletal muscles, and improving muscle mitochondrial function improves subjects’ status. Compared with local ischemic preconditioning (IPC), remote IPC (rIPC) appears easier to perform and is safer for the vessel and territory involved in ischemic injury. This study demonstrates that the muscle protection afforded by rIPC is equivalent to that achieved by IPC. Acknowledging that IPC procedures should be specifically adapted to patient characteristics to be successful, our results support a broader use of rIPC in the setting of vascular surgery., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)