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Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver

Open AccessPublished:December 09, 2010DOI:https://doi.org/10.1016/j.jhep.2010.11.006
      Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis).

      Keywords

      Abbreviations:

      ACC (acetyl-CoA carboxylase), APAP (acetaminophen), AZT (zidovudine), CAR (constitutive androstane receptor), ChREBP (carbohydrate responsive element-binding protein), CoA (coenzyme A), CPT (carnitine palmitoyltransferase), CYP (cytochrome P450), ddI (didanosine), d4T (stavudine), DILI (drug-induced liver injury), FAO (fatty acid oxidation), GSH (reduced glutathione), GST (glutathione S-transferase), JNK (c-Jun-N-terminal kinase), LCFA (long-chain fatty acid), MPTP (mitochondrial permeability transition pore), MTP (microsomal triglyceride transfer protein), MRC (mitochondrial respiratory chain), mtDNA (mitochondrial DNA), NAFLD (nonalcoholic fatty liver disease), NAPQI (N-acetyl-p-benzoquinone imine), NASH (nonalcoholic steatohepatitis), NRTI (nucleoside reverse transcriptase inhibitor), OXPHOS (oxidative phosphorylation), PPAR (peroxisome proliferator-activated receptor), PXR (pregnane X receptor), ROS (reactive oxygen species), SREBP-1c (sterol regulatory element-binding protein-1c), TNFα (tumor necrosis factor-α), TCA (tricarboxylic acid cycle), TZD (thiazolidinedione), VPA (valproic acid), VLDL (very-low density lipoprotein), WAT (white adipose tissue)

      Introduction

      More than a 1000 drugs of the modern pharmacopoeia can induce liver injury with different clinical presentations [
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      ]. In addition, DILI can lead to the withdrawal of drugs from the market or earlier during clinical trials, thus causing huge financial losses. A recent retrospective study indicates that the risk of DILI is enhanced when the administered daily dosage is higher than 50 mg or when the drug undergoes significant liver metabolism [
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      The mechanisms of DILI are not always known, but when they are investigated mitochondrial dysfunction is often present [
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      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
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      • Labbe G.
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      Mitochondrial involvement in drug-induced liver injury.
      ]. Importantly, drug-induced mitochondrial dysfunction can be due to the drug itself and/or to reactive metabolites generated through cytochrome P450-mediated metabolism [
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      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
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      • Labbe G.
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      ,
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      Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice.
      ]. Mitochondrial dysfunction is a generic term, which includes alteration of different metabolic pathways and damage to mitochondrial components. In addition, these mitochondrial disturbances can have a variety of deleterious consequences, such as oxidative stress, energy shortage, accumulation of triglycerides (steatosis), and cell death. Regarding steatosis, recent investigations suggest that besides mitochondrial dysfunction several other mechanisms could be involved. Before discussing the main mechanisms involved in drug-induced mitochondrial dysfunction and lipid dysmetabolism, we shall recall some important features pertaining to the central role of mitochondria in cell death and energy homeostasis. We will also bring to mind some aspects of lipid metabolism not directly related to mitochondria and the most relevant effects of the adipose hormones adiponectin and leptin on liver function. Finally, this review will also evoke the main factors that could predispose some patients to DILI, in particular when hepatotoxicity is due to mitochondrial dysfunction or due to impaired lipid homeostasis.

      Mitochondrial structure and functions

      Mitochondrial membrane permeabilization and cell death

      Mitochondria are organelles with two membranes surrounding a space (matrix) containing various enzymes and the mitochondrial genome (mtDNA) (Fig. 1). The inner membrane, which also harbors many enzymes, behaves as a barrier that is poorly permeable to various molecules [
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      The ninth sir hans Krebs lecture. Compartmentation and communication in living systems. Ligand conduction: a general catalytic principle in chemical, osmotic and chemiosmotic reaction systems.
      ]. Thus, this membrane contains transporters allowing the entry of endogenous compounds (ADP, fatty acids, glutathione, pyruvic acid) and possibly xenobiotics as well.
      Figure thumbnail gr1
      Fig. 1Schematic representation of mitochondrial fatty acid β-oxidation and oxidative phosphorylation in liver mitochondria. In contrast to short-chain and medium-chain fatty acids (not shown), the entry of long-chain (C14–C18) fatty acid (LCFA) within mitochondria requires a specific shuttle system involving four steps. (A) LCFAs are activated into LCFA-coenzyme A (acyl-CoA) thioesters by long-chain acyl-CoA synthetases (ACS) located in the outer mitochondrial membrane. (B) The long-chain acyl-CoA is converted into an acyl-carnitine derivative by carnitine palmitoyltransferase-1 (CPT 1) in the outer mitochondrial membrane. (C) This acyl-carnitine derivative is then translocated across the inner mitochondrial membrane into the mitochondrial matrix by carnitine-acylcarnitine translocase. (C) Finally, carnitine palmitoyltransferase-2 (CPT 2), located on the matrix side of the inner mitochondrial membrane, transfers the acyl moiety from carnitine back to coenzyme A. LCFA-CoA thioesters are then oxidized into acetyl-CoA moieties via the β-oxidation process. Acetyl-CoA moieties directly generate ketone bodies (mainly acetoacetate and β-hydroxybutyrate) which are liberated into the plasma to be used by extra-hepatic tissues for energy production. Mitochondrial fatty acid oxidation (FAO) generates NADH and FADH2, which transfer their electrons (e) to the mitochondrial respiratory chain (MRC), thus regenerating NAD+ and FAD used for other β-oxidation cycles. Within the MRC, electrons are sequentially transferred to different polypeptide complexes (numbered from I to IV) embedded within the inner membrane. The final transfer of the electrons to oxygen takes place at the level of complex IV which oxidizes cytochrome c (c). The flow of electrons within the MRC is coupled with the extrusion of protons (H+) from the mitochondrial matrix to the intermembrane space, which creates the mitochondrial transmembrane potential, Δψm. When energy is needed (i.e. when ATP levels are low), these protons re-enter the matrix through the F0 portion of the ATP synthase (also referred to as complex V), thus liberating energy that is used to phosphorylate ADP into ATP. The whole metabolic process which couples substrate oxidation to ATP synthesis is referred to as oxidative phosphorylation (OXPHOS). It is noteworthy that OXPHOS requires the mitochondrial DNA (mtDNA) since it encodes 13 MRC polypeptides, which are embedded within complexes I, III, IV, and V.
      In some pathophysiological circumstances, the mitochondrial membranes can lose their structural and functional integrity, in particular after the opening of the mitochondrial permeability transition pores (MPTP) [
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blachly-Dyson E.
      • Di Lisa F.
      • et al.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ]. These pores involve at least 4 candidate proteins, namely the peripheral benzodiazepine receptor (PBR), the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and cyclophilin D [
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blachly-Dyson E.
      • Di Lisa F.
      • et al.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ]. The later protein (a modulator of the pore rather than a MPTP component per se [
      • Di Lisa F.
      • Bernardi P.
      A CaPful of mechanisms regulating the mitochondrial permeability transition.
      ]) is able to bind the immunosuppressive drug cyclosporin A that therefore reduces the opening probability of the MPTP. In contrast, several drugs and toxic compounds, but also high levels of some endogenous derivatives (e.g. calcium, fatty acids, and bile salts) can induce MPTP opening. As the latter event strongly alters mitochondrial function and structure, it can endanger cell life. However, the exact pathway whereby the cell will die (namely apoptosis or necrosis) depends on the number of mitochondria harboring opened MPTP [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Pessayre D.
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      Mitochondrial involvement in drug-induced liver injury.
      ,
      • Malhi H.
      • Gores G.J.
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      Apoptosis and necrosis in the liver: a tale of two deaths?.
      ].
      Indeed, MPTP opening can profoundly disturb ATP synthesis, through the loss of inner mitochondrial membrane integrity. If numerous mitochondria present opened MPTP, ATP stores will slump rapidly and necrosis will occur through a sudden rise in intracellular calcium levels because ATP is mandatory for the activity of the plasma membrane calcium ATPase (PMCA), an enzyme responsible for calcium extrusion out of the cell. In contrast, if MPTP opening takes place only in some mitochondria, ATP levels will be maintained thanks to undamaged organelles. However, the rare mitochondria involved in MPTP opening will swell allowing the release of different pro-apoptotic proteins including the apoptosis inducing factor (AIF), several caspases, and cytochrome c [
      • Fulda S.
      • Galluzzi L.
      • Kroemer G.
      Targeting mitochondria for cancer therapy.
      ]. This key protein of the respiratory chain (Fig. 1), when released in the cytoplasm, can bind to the Apaf-1 protein and ATP thus initiating the apoptotic pathway through the activation of caspases 9 and 3. Consequently, MPTP opening in a few mitochondria can also have deleterious consequences [
      • Malhi H.
      • Gores G.J.
      • Lemasters J.J.
      Apoptosis and necrosis in the liver: a tale of two deaths?.
      ,
      • Pessayre D.
      • Haouzi D.
      • Fau D.
      • Robin M.A.
      • Mansouri A.
      • Berson A.
      Withdrawal of life support, altruistic suicide, fratricidal killing and euthanasia by lymphocytes: different forms of drug-induced hepatic apoptosis.
      ].
      Several important points must be discussed regarding mitochondrial membrane permeabilization. Firstly, MPTP opening initially permeabilizes the mitochondrial inner membrane without alteration of the outer membrane. However, MPTP opening causes an equilibration of solutes with molecular masses up to 1500 Da and the massive entry of water into the matrix, which causes unfolding of the inner membrane and mitochondrial swelling. The latter event thus induces outer membrane rupture and the release of several mitochondrial proteins located in the intermembrane space (e.g. cytochrome c and AIF), which trigger apoptotis [
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blachly-Dyson E.
      • Di Lisa F.
      • et al.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ,
      • Fulda S.
      • Galluzzi L.
      • Kroemer G.
      Targeting mitochondria for cancer therapy.
      ,
      • Kroemer G.
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      Mitochondrial control of cell death.
      ]. Secondly, mitochondrial membrane permeabilization can induce the release of cytochrome c and other cytotoxic proteins without any rupture of the mitochondrial outer membrane [
      • Fulda S.
      • Galluzzi L.
      • Kroemer G.
      Targeting mitochondria for cancer therapy.
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      • Racoeur C.
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      • et al.
      ]. This scenario requires the formation of pores within this membrane thanks to the association of two pro-apoptotic proteins belonging to the Bcl-2 family, namely Bak (already located in the outer membrane) and Bax (which is recruited from the cytosol) [
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blachly-Dyson E.
      • Di Lisa F.
      • et al.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ,
      • Fulda S.
      • Galluzzi L.
      • Kroemer G.
      Targeting mitochondria for cancer therapy.
      ]. Importantly, mitochondrial outer membrane permeabilization through the formation of Bax/Bak pores is not sensitive to cyclosporin A [
      • Balakirev M.Y.
      • Zimmer G.
      Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.
      ,
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      • Trudovishnikov A.S.
      • Lukyanova L.D.
      • Mironova G.D.
      Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca2+-induced pore: tissue specificity, age profile and dependence on the animal’s adaptation to hypoxia.
      ]. Thus, whatever the mechanism involved in membrane permeabilization, this event can strongly alter mitochondrial function and structure, and thus lead to cell death. Finally, it is noteworthy that the MPTP structure seems to be different from one tissue to another. This may explain why some organs could be more or less vulnerable to certain permeability transition inducers [
      • Eliseev R.A.
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      • Goldman A.
      • Rosier R.N.
      • et al.
      Role of cyclophilin D in the resistance of brain mitochondria to the permeability transition.
      ,
      • Mirandola S.R.
      • Melo D.R.
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      • Castilho R.F.
      3-Nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions.
      ].

      Liver mitochondria and energy homeostasis

      In most mammalian cells, mitochondria provide the most part of the energy necessary for cell homeostasis, especially during fasting periods [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Maianski N.A.
      • Geissler J.
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      • Roos D.
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      Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis.
      ,
      • Wallace D.C.
      • Fan W.
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      Mitochondrial energetics and therapeutics.
      ]. Mitochondrial ATP synthesis is possible thanks to the oxidative degradation of endogenous substrates, such as pyruvate (generated from glycolysis), fatty acids, and amino acids. Pyruvate oxidation takes place in the tricarboxylic acid cycle (TCA, also called Krebs cycle), whereas fatty acid degradation within mitochondria is mediated by β-oxidation (Fig. 1).
      In order to undergo the β-oxidation pathway fatty acids must cross the mitochondrial membranes. Whereas short-chain and medium-chain fatty acids freely enter the mitochondria, long-chain fatty acids (LCFAs) can cross the mitochondrial membranes only by means of a multienzymatic system requiring coenzyme A and l-carnitine as cofactors (Fig. 1). In this system, carnitine palmitoyltransferase 1 (CPT1) catalyses the rate limiting step of LCFA oxidation as this enzyme can be strongly inhibited by malonyl-CoA, an endogenous derivative synthesized during de novo lipogenesis [
      • Faye A.
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      • Esnous C.
      • Price N.T.
      • Gobin S.
      • Jackson V.N.
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      Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.
      ,
      • McGarry J.D.
      • Leatherman G.F.
      • Carnitine Foster.D.W.
      Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA.
      ].
      Inside the mitochondria, short-chain and medium-chain fatty acids are activated in acyl-CoA molecules by specific acyl-CoA synthases, whereas long-chain fatty acyl-carnitine intermediates are transformed back to their corresponding acyl-CoA thioesters thanks to CPT2 (Fig. 1). Whatever the length of their carbon chain, acyl-CoA derivatives are then cut down sequentially thanks to the β-oxidation process that generates acetyl-CoA moieties and shorter fatty acids that enter new β-oxidation cycles (Fig. 1). These acetyl-CoA moieties are immediately used for the synthesis of ketone bodies (mainly acetoacetate and β-hydroxybutyrate) released in the blood and oxidized in extra-hepatic tissues, such as kidney, muscle, and brain (Fig. 1). Because mitochondrial β-oxidation and ketogenesis play a fundamental role in energy homeostasis [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Hashimoto T.
      • Cook W.S.
      • Qi C.
      • Yeldandi A.V.
      • Reddy J.K.
      • Rao M.S.
      Defect in peroxisome proliferator-activated receptor alpha-inducible fatty acid oxidation determines the severity of hepatic steatosis in response to fasting.
      ], a severe deficiency in fatty acid oxidation (FAO) can lead to multiple organ failure and death of the patient [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Shekhawat P.S.
      • Matern D.
      • Strauss A.W.
      Fetal fatty acid oxidation disorders, their effect on maternal health and neonatal outcome: impact of expanded newborn screening on their diagnosis and management.
      ].
      FAO deficiency can be associated with reduced plasma ketone bodies, accumulation of acyl-carnitine derivatives and dicarboxylic acids in plasma (or urine), and severe hypoglycemia [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Shekhawat P.S.
      • Matern D.
      • Strauss A.W.
      Fetal fatty acid oxidation disorders, their effect on maternal health and neonatal outcome: impact of expanded newborn screening on their diagnosis and management.
      ]. Low blood glucose could be due to reduced hepatic gluconeogenesis and increased extra-hepatic utilization [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Derks T.G.
      • van Dijk T.H.
      • Grefhorst A.
      • Rake J.P.
      • Smit G.P.
      • Kuipers F.
      • et al.
      Inhibition of mitochondrial fatty acid oxidation in vivo only slightly suppresses gluconeogenesis but enhances clearance of glucose in mice.
      ]. Although hypoketonemia is usually observed in genetic disorders of mitochondrial FAO, hyperketonemia can be observed during drug-induced alteration of mitochondrial β-oxidation [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ]. A probable mechanism is the occurrence of drug-induced impairment of the TCA cycle in extra-hepatic tissues consuming high amounts of ketone bodies [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Senior A.E.
      • Sherratt H.S.
      A comparison of the effects on blood glucose and ketone-body levels, and of the toxicities, of pent-4-enoic acid and four simple fatty acids.
      ].
      Oxidative degradation of pyruvate and fatty acids produces acetyl-CoA molecules and also reduced cofactors [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Mitchell P.
      The ninth sir hans Krebs lecture. Compartmentation and communication in living systems. Ligand conduction: a general catalytic principle in chemical, osmotic and chemiosmotic reaction systems.
      ]. Indeed, several dehydrogenases involved in the TCA cycle and β-oxidation are using NAD+ and FAD to generate NADH and FADH2, which give their electrons and protons to the mitochondrial respiratory chain (MRC) (Fig. 1). Electrons are sequentially transferred to different multi-protein complexes of the MRC and finally to cytochrome c oxidase (complex IV), which safely reduces oxygen into water in the presence of protons (Fig. 1). Importantly, electron transfer within MRC is associated with the ejection of protons from the matrix to the intermembrane space of the mitochondria, thus generating a large membrane potential Δψm [
      • Mitchell P.
      The ninth sir hans Krebs lecture. Compartmentation and communication in living systems. Ligand conduction: a general catalytic principle in chemical, osmotic and chemiosmotic reaction systems.
      ,
      • Wallace D.C.
      A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine.
      ]. When cells need energy, protons are reentering the matrix thanks to the F0 portion of the ATP synthase (complex V) thus releasing part of the potential energy of Δψm. This energy is then used by the F1 portion of the ATP synthase for the phosphorylation of ADP into ATP (Fig. 1). Some drugs able to abolish ADP phosphorylation (and thus ATP synthesis) without inhibiting substrate oxidation are referred to as oxidative phosphorylation (OXPHOS) uncouplers [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Fromenty B.
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      • Larrey D.
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      Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and II.
      ].

      Mitochondrial production of reactive oxygen species

      A major feature of the mitochondria is the production of reactive oxygen species (ROS) through the activity of the MRC [
      • Wallace D.C.
      • Fan W.
      • Procaccio V.
      Mitochondrial energetics and therapeutics.
      ,
      • Seifert E.L.
      • Estey C.
      • Xuan J.Y.
      • Harper M.E.
      Electron transport chain-dependent and -independent mechanisms of mitochondrial H2O2 emission during long-chain fatty acid oxidation.
      ]. Indeed, a small fraction of electrons entering the MRC can prematurely escape from complexes I and III and directly react with oxygen to generate the superoxide anion radical. This radical is then dismutated by the mitochondrial manganese superoxide dismutase (MnSOD) into hydrogen peroxide (H2O2), which is detoxified into water by the mitochondrial glutathione peroxidase (GPx) that uses reduced glutathione (GSH) as a cofactor. Hence, in the normal (non-diseased) state, most of the ROS generated by the MRC are detoxified by the mitochondrial anti-oxidant defenses. The remaining (i.e. non-detoxified) ROS diffuse out of mitochondria and serve as second messengers to trigger cellular processes such as mitogenesis [
      • Wallace D.C.
      • Fan W.
      • Procaccio V.
      Mitochondrial energetics and therapeutics.
      ].
      However, this detoxification process can be overwhelmed in different pathophysiological circumstances. This occurs in particular in case of GSH depletion within liver mitochondria, which reduces greatly their capability to detoxify H2O2 since they do not have catalase [
      • Mari M.
      • Morales A.
      • Colell A.
      • Garcia-Ruiz C.
      • Fernandez-Checa J.C.
      Mitochondrial glutathione, a key survival antioxidant.
      ]. Depletion of mitochondrial GSH below a critical threshold thus favors H2O2 accumulation by impairing its detoxification. This in turn triggers mitochondrial dysfunction, MPTP opening, activation of c-Jun-N-terminal kinase (JNK), and cell death [
      • Fernandez-Checa J.C.
      • Kaplowitz N.
      Hepatic mitochondrial glutathione: transport and role in disease and toxicity.
      ,
      • Jones D.P.
      • Lemasters J.J.
      • Han D.
      • Boelsterli U.A.
      • Kaplowitz N.
      Mechanisms of pathogenesis in drug hepatotoxicity.
      ]. Chronic ethanol intoxication, fasting, and malnutrition are diseased states favoring GSH depletion, in particular within mitochondria.
      Mitochondrial anti-oxidant enzymes can also be overwhelmed when MRC is chronically impaired. Indeed, a partial block in the flow of electrons greatly increases the probability of monoelectronic reduction of oxygen and superoxide anion production within the complexes I and III [
      • Brookes P.S.
      Mitochondrial H(+) leak and ROS generation: an odd couple.
      ,
      • Tahara E.B.
      • Navarete F.D.
      • Kowaltowski A.J.
      Tissue-, substrate- and site-specific characteristics of mitochondrial reactive oxygen species generation.
      ]. High steady state levels of ROS then damage OXPHOS proteins, cardiolipin, and mtDNA [
      • Demeilliers C.
      • Maisonneuve C.
      • Grodet A.
      • Mansouri A.
      • Nguyen R.
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      Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice.
      ,
      • Sanz A.
      • Caro P.
      • Ayala V.
      • Portero-Otin M.
      • Pamplona R.
      • Barja G.
      Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins.
      ,
      • Velsor L.W.
      • Kovacevic M.
      • Goldstein M.
      • Leitner H.M.
      • Lewis W.
      • Day B.J.
      Mitochondrial oxidative stress in human hepatoma cells exposed to stavudine.
      ]. This oxidative damage aggravates mitochondrial dysfunction to further augment electron leakage and ROS formation, thus leading to a vicious circle [
      • Fromenty B.
      • Robin M.A.
      • Igoudjil A.
      • Mansouri A.
      • Pessayre D.
      The ins and outs of mitochondrial dysfunction in NASH.
      ].

      The mitochondrial genome

      A unique feature of mitochondria is the dual genetic origin of the OXPHOS proteins (ca. 100) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Wallace D.C.
      • Fan W.
      • Procaccio V.
      Mitochondrial energetics and therapeutics.
      ]. Whereas the most part of these polypeptides are encoded by the nuclear genome and subsequently imported within the mitochondria, 13 MRC polypeptides are instead encoded by the mitochondrial genome, a small piece of circular doubled-stranded DNA located within the mitochondrial matrix (Fig. 1). In a single cell there are several hundred (or thousand) copies of mtDNA whose replication occurs continuously, even in cells that do not divide [
      • Igoudjil A.
      • Begriche K.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors.
      ,
      • Wallace D.C.
      • Fan W.
      Energetics, epigenetics, mitochondrial genetics.
      ]. Permanent mtDNA replication by the DNA polymerase γ thus allows the maintenance of constant mtDNA levels in cells despite continuous removal of the most dysfunctional and/or damaged mitochondria [
      • Kim I.
      • Rodriguez-Enriquez S.
      • Lemasters J.J.
      Selective degradation of mitochondria by autophagia.
      ].
      Most cells (including hepatocytes) have a surplus of mtDNA copies, and can, therefore, tolerate a substantial depletion of mtDNA. Classically, it is considered that the number of normal mtDNA copies must fall below 20–40% of basal levels to induce mitochondrial dysfunction and severe adverse events [
      • Igoudjil A.
      • Begriche K.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors.
      ,
      • Ducluzeau P.H.
      • Lachaux A.
      • Bouvier R.
      • Duborjal H.
      • Stepien G.
      • Bozon D.
      • et al.
      Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion.
      ,
      • Rossignol R.
      • Faustin B.
      • Rocher C.
      • Malgat M.
      • Mazat J.P.
      • Letellier T.
      Mitochondrial threshold effects.
      ]. The few mtDNA copies remaining within each mitochondrion are not able to provide enough MRC polypeptides, thus leading to OXPHOS impairment and secondary inhibition of mitochondrial FAO and TCA cycle. Another key feature of mtDNA is its high sensitivity to ROS-induced oxidative damage and mutations due to its proximity to the inner membrane (a major source of ROS), the absence of protective histone, and an incomplete repertoire of mitochondrial DNA repair enzymes [
      • Demeilliers C.
      • Maisonneuve C.
      • Grodet A.
      • Mansouri A.
      • Nguyen R.
      • Tinel M.
      • et al.
      Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice.
      ,
      • Igoudjil A.
      • Begriche K.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors.
      ,
      • Croteau D.L.
      • Stierum R.H.
      • Bohr V.A.
      Mitochondrial DNA repair pathways.
      ,
      • Dianov G.L.
      • Souza-Pinto N.
      • Nyaga S.G.
      • Thybo T.
      • Stevnsner T.
      • Bohr V.A.
      Base excision repair in nuclear and mitochondrial DNA.
      ].

      Lipid and carbohydrate metabolism in extramitochondrial compartments

      Besides mitochondria, other organelles (or extra-mitochondrial enzyme systems) can be involved in FAO. For instance, peroxisomes degrade long-chain and very long-chain fatty acids but not medium-chain and short-chain fatty acids. The first step of peroxisomal FAO continuously generates H2O2 through acyl-CoA oxidase (ACO) activity [
      • Schönfeld P.
      • Dymkowska D.
      • Wojtczal L.
      Acyl-CoA-induced generation of reactive oxygen species in mitochondrial preparations is due to the presence of peroxisomes.
      ,
      • Van den Branden C.
      • Kerckaert I.
      • Roels F.
      Peroxisomal β-oxidation from endogenous substrates. Demonstration through H2 O2 production in the unanaesthetized mouse.
      ], and thus oxidative stress can occur during fatty acid overload and/or peroxisomal proliferation due to an imbalance between intraperoxisomal H2O2 production and its removal by catalase [
      • Arnaiz S.L.
      • Travacio M.
      • Llesuy S.
      • Boveris A.
      Hydrogen peroxide metabolism during peroxisome proliferation by fenofibrate.
      ]. Several cytochromes P450 (CYPs) such as CYP4A and CYP2E1 also oxidize fatty acids although the CYP-mediated oxidation involves only the terminal ω (or the ω-1) carbon of the aliphatic chain [
      • Hardwick J.P.
      Cytochrome P450 omega hydroxylase (CYP4) function in fatty acid metabolism and metabolic diseases.
      ,
      • Laethem R.M.
      • Balazy M.
      • Falck J.R.
      • Laethem C.L.
      • Koop D.R.
      Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1.
      ]. Interestingly, ω-hydroxylated fatty acids are further converted into dicarboxylic acids that can induce mitochondrial dysfunction [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Passi S.
      • Picardo M.
      • Nazzaro-Porro M.
      • Breathnach A.
      • Confaloni A.M.
      • Serlupi-Crescenzi G.
      Antimitochondrial effect of saturated medium chain length (C8–C13) dicarboxylic acids.
      ]. Although most of the CYPs are found within the endoplasmic reticulum, some of them such as CYP2E1 can have a mitochondrial localization [
      • Bai J.
      • Cederbaum A.I.
      Overexpression of CYP2E1 in mitochondria sensitizes HepG2 cells to the toxicity caused by depletion of glutathione.
      ,
      • Bansal S.
      • Liu C.P.
      • Sepuri N.B.
      • Anandatheerthavarada H.K.
      • Selvaraj V.
      • Hoek J.
      • et al.
      Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress.
      ,
      • Robin M.A.
      • Anandatheerthavarada H.K.
      • Biswas G.
      • Sepuri N.B.
      • Gordon D.M.
      • Pain D.
      • et al.
      Bimodal targeting of microsomal CYP2E1 to mitochondria through activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation.
      ].
      Mitochondrial, peroxisomal, and microsomal FAO is strongly regulated by peroxisome proliferator-activated receptor α (PPARα), a nuclear receptor and transcription factor, which can be stimulated by endogenous fatty acids or synthetic drugs (fibrates) [
      • Alaynick W.A.
      Nuclear receptors, mitochondria and lipid metabolism.
      ]. PPARα stimulation increases the expression of the mitochondrial enzymes CPT1, medium-chain acyl-CoA dehydrogenase (MCAD) and HMG-CoA synthase (involved in ketone body synthesis), the peroxisomal ACO, and the microsomal CYP4A [
      • Hsu M.H.
      • Savas U.
      • Griffin K.J.
      • Johnson E.F.
      Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells.
      ,
      • Schoonjans K.
      • Staels B.
      • Auwerx J.
      The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation.
      ]. Besides PPARα, other transcription factors regulating hepatic FAO include forkhead box A2 (FoxA2) and cAMP-response element-binding protein (CREB) that are activated during fasting periods by low insulinemia and high glucagonemia, respectively [
      • Begriche K.
      • Knockaert L.
      • Massart J.
      • Robin M.A.
      • Fromenty B.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH): are there drugs able to improve it?.
      ].
      On the contrary, the metabolic and hormonal context after a meal favors lipid synthesis with a concomitant reduction of the FAO pathway. Indeed, high plasma levels of insulin and glucose, respectively, activate the sterol regulatory element-binding protein-1c (SREBP-1c) and carbohydrate responsive element-binding protein (ChREBP) that both increase the hepatic expression of key enzymes involved in glycolysis (e.g. glucokinase and l-pyruvate kinase) and de novo lipogenesis (e.g. acetyl-CoA carboxylase and fatty acid synthase). Lipogenesis is associated with the accumulation of the CPT1 inhibitor malonyl-CoA, thus reducing the flux of mitochondrial LCFA oxidation [
      • Faye A.
      • Borthwick K.
      • Esnous C.
      • Price N.T.
      • Gobin S.
      • Jackson V.N.
      • et al.
      Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.
      ,
      • McGarry J.D.
      • Leatherman G.F.
      • Carnitine Foster.D.W.
      Carnitine palmitoyltransferase I. The site of inhibition of hepatic fatty acid oxidation by malonyl-CoA.
      ].
      It is worthy to mention herein that hepatic SREBP-1c and ChREBP can be abnormally activated in obese and diabetic individuals thus favoring fatty liver. Another mechanism that could contribute to fatty liver in these patients is the permanent and unrepressed triglycerides lipolysis taking place in the expanded adipose tissue (due to insulin resistance), which leads to a massive influx of free fatty acids in the hepatocytes [
      • Begriche K.
      • Knockaert L.
      • Massart J.
      • Robin M.A.
      • Fromenty B.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH): are there drugs able to improve it?.
      ]. Besides SREBP-1c and ChREBP, other transcription factors could play a significant role in de novo lipogenesis (at least in some metabolic contexts) such as PPARγ and pregnane X receptor (PXR). Both transcription factors are nuclear receptors that can be activated by different endogenous and exogenous ligands [
      • Desvergne B.
      • Feige J.N.
      • Casals-Casas C.
      PPAR-mediated activity of phthalates: a link to the obesity epidemic?.
      ,
      • Moreau A.
      • Téruel C.
      • Beylot M.
      • Albalea V.
      • Tamasi V.
      • Umbdenstock T.
      • et al.
      A novel pregnane X receptor and S14-mediated lipogenic pathway in human hepatocytes.
      ].
      Once synthesized, fatty acids combine with glycerol to generate triglycerides. These lipids are subsequently incorporated into VLDL particles, which are normally secreted into the plasma unless this route of lipid secretion is impaired. VLDL synthesis requires not only triglycerides but also apolipoproteins B and CIII. Furthermore, VLDL assembly within the endoplasmic reticulum requires the microsomal triglyceride transfer protein (MTP) whose expression is reduced by insulin [
      • Kamagate A.
      • Qu S.
      • Perdomo G.
      • Su D.
      • Kim D.H.
      • Slusher S.
      • et al.
      FoxO1 mediates insulin-dependent regulation of hepatic VLDL production in mice.
      ]. In the plasma, VLDL particles are hydrolyzed by lipoprotein lipase (LPL), thus allowing the release of free fatty acids that will be either oxidized in different extra-hepatic tissues (e.g. heart, skeletal muscles) or re-esterified into triglycerides in the adipose tissue. LPL is usually not expressed in the adult liver except in some pathophysiological situations such as obesity [
      • Pardina E.
      • Baena-Fustegueras J.A.
      • Llamas R.
      • Catalan R.
      • Galard R.
      • Lecube A.
      • et al.
      Lipoprotein lipase expression in livers of morbidly obese patients could be responsible for liver steatosis.
      ].

      Impact of leptin and adiponectin on lipid and carbohydrate metabolism

      Besides insulin and glucagon, hormones secreted by the adipose tissue (referred to as adipokines) can also play a salient role in lipid homeostasis. Among these adipokines, leptin, and adiponectin present an “anti-steatotic” action by decreasing de novo lipogenesis and activating mitochondrial FAO, in particular by reducing the intracellular levels of malonyl-CoA [
      • Ahima R.S.
      Adipose tissue as an endocrine organ.
      ,
      • Begriche K.
      • Massart J.
      • Fromenty B.
      Effects of β-aminoisobutyric acid on leptin production and lipid homeostasis: mechanisms and possible relevance for the prevention of obesity.
      ]. Indeed, leptin and adiponectin can induce the phosphorylation of the lipogenic enzyme acetyl-CoA carboxylase (ACC), thus leading to its inactivation and the subsequent reduction of malonyl-CoA synthesis [
      • Begriche K.
      • Massart J.
      • Fromenty B.
      Effects of β-aminoisobutyric acid on leptin production and lipid homeostasis: mechanisms and possible relevance for the prevention of obesity.
      ,
      • Yamauchi T.
      • Kamon J.
      • Minokoshi Y.
      • Ito Y.
      • Waki H.
      • Uchida S.
      • et al.
      Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.
      ]. Both adipokines also control carbohydrate homeostasis in several tissues including the liver [
      • Yamauchi T.
      • Kamon J.
      • Minokoshi Y.
      • Ito Y.
      • Waki H.
      • Uchida S.
      • et al.
      Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.
      ,
      • Kalra S.P.
      Central leptin gene therapy ameliorates diabetes type 1 and 2 through two independent hypothalamic relays; a benefit beyond weight and appetite regulation.
      ].
      Leptin also strongly regulates food intake. Consequently, low leptinaemia can induce obesity and associated metabolic disorders, such as dyslipidemia, type 2 diabetes, and fatty liver [
      • Begriche K.
      • Massart J.
      • Fromenty B.
      Effects of β-aminoisobutyric acid on leptin production and lipid homeostasis: mechanisms and possible relevance for the prevention of obesity.
      ,
      • Begriche K.
      • Lettéron P.
      • Abbey-Toby A.
      • Vadrot N.
      • Robin M.A.
      • Bado A.
      • et al.
      Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice.
      ,
      • Farooqi I.S.
      • O’Rahilly S.
      Leptin: a pivotal regulator of human energy homeostasis.
      ]. However, total leptin deficiency is particularly rare in humans. In contrast, common obesity is associated with high leptinemia (a consequence of leptin resistance) and low adiponectinemia, which plays a major role in the pathophysiology of type 2 diabetes and fatty liver [
      • Marra F.
      • Bertolani C.
      Adipokines in liver diseases.
      ,
      • Zhou M.
      • Xu A.
      • Tam P.K.
      • Lam K.S.
      • Chan L.
      • Hoo R.L.
      • et al.
      Mitochondrial dysfunction contributes to the increased vulnerabilities of adiponectin knockout mice to liver injury.
      ]. Finally, while leptin favors inflammation, fibrogenesis, and angiogenesis, adiponectin prevents these different events [
      • Marra F.
      • Bertolani C.
      Adipokines in liver diseases.
      ].

      Drug-induced mitochondrial dysfunction and liver injury

      Drug-induced adverse events and mitochondrial toxicity

      The view that drugs could disturb mitochondrial function emerged several decades ago when clinical studies reported in some medicated individuals the occurrence of symptoms usually observed in patients presenting a mitochondrial disease of genetic origin or a Reye’s syndrome (whose physiopathology involves severe mitochondrial dysfunction) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ]. For instance, several studies reported in the late 70’s and early 80’s the occurrence of a Reye-like syndrome in epileptic patients treated with valproic acid (VPA) [
      • Gerber N.
      • Dickinson R.G.
      • Harland R.C.
      • Lynn R.K.
      • Houghton L.D.
      • Antonias J.I.
      • et al.
      Reye-like syndrome associated with valproic acid therapy.
      ,
      • Zimmerman H.J.
      • Ishak K.G.
      Valproate-induced hepatic injury: analyses of 23 fatal cases.
      ]. Likewise, myopathy, lactic acidosis, and hepatic steatosis have been reported in the late 80’s and early 90’s in patients treated with the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) zidovudine (AZT), zalcitabine (ddC), didanosine (ddI) and stavudine (d4T) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Arnaudo E.
      • Dalakas M.
      • Shanske S.
      • Moraes C.T.
      • DiMauro S.
      • Schon E.A.
      Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy.
      ,
      • Lai K.K.
      • Gang D.L.
      • Zawacki J.K.
      • Cooley T.P.
      Fulminant hepatic failure associated with 2′,3′-dideoxyinosine (ddI).
      ,
      • Le Bras P.
      • D’Oiron R.
      • Quertainmont Y.
      • Halfon P.
      • Caquet R.
      Metabolic, hepatic and muscular changes during zidovudine therapy: a drug-induced mitochondrial disease?.
      ]. Since then, the list of drugs inducing adverse events due to mitochondrial dysfunction has not ceased to grow year after year.
      Regarding drug-induced liver diseases, different mechanisms of mitochondrial dysfunction have been described thus far, including membrane permeabilization, OXPHOS impairment, FAO inhibition, and mtDNA depletion (Table 1) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Pessayre D.
      • Mansouri A.
      • Berson A.
      • Fromenty B.
      Mitochondrial involvement in drug-induced liver injury.
      ]. Importantly, DILI due to mitochondrial toxicity has led to the interruption of clinical trials, or drug withdrawal after marketing, in particular when the benefit/risk ratio was deemed to be too low for the patient’s healthiness (Table 2). Moreover, some marketed drugs have received Black Box warnings from drug agencies due to mitochondrial dysfunction and related hepatotoxicity (Table 3) [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Nadanaciva S.
      • Will Y.
      The role of mitochondrial dysfunction and drug safety.
      ].
      Table 1Hepatotoxic drugs and their corresponding deleterious effects on mitochondrial function and genome. Note that the absence of cross indicates that the toxic effect has not been reported to date for the corresponding drug and that for different compounds listed below some of the mitochondrial effects have been observed only in vitro.
      aAbbreviations: FAO, fatty acid oxidation; MPTP, mitochondrial permeability transition pores; MRC, mitochondrial respiratory chain; mtDNA, mitochondrial DNA; OXPHOS, oxidative phosphorylation.
      bInhibition of mitochondrial FAO through impairment of FAO enzyme(s) and/or depletion in L-carnitine and coenzyme A.
      cInhibition of the MRC through impairment of enzyme(s) involved in electron transfer or ADP phosphorylation.
      dMitochondrial effects of APAP via its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI).
      Table 2Examples of drugs, the potential of which to cause mitochondrial dysfunction and DILI has led to the interruption of clinical trials, or their withdrawal after marketing.
      aAbbreviation: NSAID, nonsteroidal anti-inflammatory drug.
      Table 3Examples of marketed drugs able to induce hepatotoxicity due to mitochondrial dysfunction, which have received Black Box warnings from drug agencies.
      aAbbreviation: nucleoside reverse transcriptase inhibitors.

      Drug-induced mitochondrial alterations and cytolytic hepatitis

      Cytolytic hepatitis encompasses a wide spectrum of liver injury of different severity since the destruction of hepatocytes (i.e. cytolysis) can involve a variable amount of the hepatic mass. Consequently, the mildest forms are characterized by an isolated increase in plasma alanine aminotransferase (ALT) and asparate aminotransferase (AST), whereas in the most severe cases fulminant hepatitis can occur thus requiring liver transplantation [
      • Björnsson E.
      The natural history of drug-induced liver injury.
      ]. As already mentioned, hepatocyte cytolysis occurring in vivo can be the consequence of necrosis or apoptosis. While necrosis leads to the destruction of the plasma membrane and the release in the extracellular milieu of different cell components such as transaminases and lactate dehydrogenase (LDH), apoptosis is generally associated with a discreet removal of the dying cells by neighboring macrophages [
      • Pessayre D.
      • Haouzi D.
      • Fau D.
      • Robin M.A.
      • Mansouri A.
      • Berson A.
      Withdrawal of life support, altruistic suicide, fratricidal killing and euthanasia by lymphocytes: different forms of drug-induced hepatic apoptosis.
      ,
      • Jaeschke H.
      • Gores G.J.
      • Cederbaum A.I.
      • Hinson J.A.
      • Pessayre D.
      • Lemasters J.J.
      Mechanisms of hepatotoxicity.
      ]. However, the removal of a large number of apoptotic cells can induce the recruitment of inflammatory cells and the subsequent overproduction of ROS and cytokines that promote cell necrosis [
      • Ramaiah S.K.
      • Jaeschke H.
      Role of neutrophils in the pathogenesis of acute inflammatory liver injury.
      ]. Thus, apoptosis in liver can also be associated in vivo with secondary necrosis and elevated plasma transaminases [
      • Lawson J.A.
      • Fisher M.A.
      • Simmons C.A.
      • Farhood A.
      • Jaeschke H.
      Inhibition of Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice.
      ,
      • Bajt M.L.
      • Ho Y.S.
      • Vonderfecht S.L.
      • Jaeschke H.
      Reactive oxygen as modulator of TNF and fas receptor-mediated apoptosis in vivo: studies with glutathione peroxidase-deficient mice.
      ].

      Drug-induced MPTP opening

      MPTP opening is one mechanism whereby drugs can induce cytolytic hepatitis (Table 1) [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Balakirev M.Y.
      • Zimmer G.
      Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.
      ,
      • Berson A.
      • Cazanave S.
      • Descatoire V.
      • Tinel M.
      • Grodet A.
      • Wolf C.
      • et al.
      The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: protection by albumin.
      ,
      • Kaufmann P.
      • Török M.
      • Hänni A.
      • Roberts P.
      • Gasser R.
      • Krähenbühl S.
      Mechanisms of benzarone and benzbromarone-induced hepatic toxicity.
      ,
      • Trost L.C.
      • Lemasters J.J.
      The mitochondrial permeability transition: a new pathophysiological mechanism for Reye’s syndrome and toxic liver injury.
      ,
      • Berson A.
      • Descatoire V.
      • Sutton A.
      • Fau D.
      • Maulny B.
      • Vadrot N.
      • et al.
      Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation.
      ,
      • Masubuchi Y.
      • Kano S.
      • Horie T.
      Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones.
      ]. Among these drugs, disulfiram can also induce mitochondrial membrane permeabilization through a MPTP-independent mechanism [
      • Balakirev M.Y.
      • Zimmer G.
      Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.
      ]. Studies pertaining to drug-induced MPTP are sometimes performed in mitochondria de-energized with oligomycin and in the presence of high concentrations of calcium (e.g. from 10 to 50 μM). Since these conditions have a profound impact on MPTP opening [
      • Bernardi P.
      • Krauskopf A.
      • Basso E.
      • Petronilli V.
      • Blachly-Dyson E.
      • Di Lisa F.
      • et al.
      The mitochondrial permeability transition from in vitro artifact to disease target.
      ], it is difficult to extrapolate some data to the in vivo situation.
      The precise mechanisms whereby drugs can induce MPTP opening are not known although recent investigations suggest at least three hypotheses, which are not mutually exclusive.
      Firstly, drugs can interact with some MPTP components. For instance, alpidem could trigger mitochondrial membrane permeabilization and cell death through its binding to PBR which is located on the outer membrane [
      • Berson A.
      • Descatoire V.
      • Sutton A.
      • Fau D.
      • Maulny B.
      • Vadrot N.
      • et al.
      Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation.
      ].
      Secondly, drug-induced oxidative stress can favor the oxidation of regulatory thiol groups located within some MPTP components [
      • Masubuchi Y.
      • Suda C.
      • Horie T.
      Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice.
      ,
      • Balakirev M.Y.
      • Zimmer G.
      Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.
      ,
      • Kowaltowski A.J.
      • Castilho R.F.
      • Vercesi A.E.
      Mitochondrial permeability transition and oxidative stress.
      ]. This mechanism could occur with disulfiram and acetaminophen (APAP) that both induce major oxidative stress [
      • Masubuchi Y.
      • Suda C.
      • Horie T.
      Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice.
      ,
      • Balakirev M.Y.
      • Zimmer G.
      Mitochondrial injury by disulfiram: two different mechanisms of the mitochondrial permeability transition.
      ,
      • Hinson J.A.
      • Roberts D.W.
      • James L.P.
      Mechanisms of acetaminophen-induced liver necrosis.
      ]. As regards APAP, it is, however, unclear whether this drug induces MPTP opening via GSH depletion, or through the direct interaction of its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) with some (still uncharacterized) MPTP components. Indeed, NAPQI is able to bind covalently to mitochondrial proteins and this could have deleterious effect not only on MPTP but also on mitochondrial respiration and FAO [
      • Burcham P.C.
      • Harman A.W.
      Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes.
      ,
      • Chen C.
      • Krausz K.W.
      • Shah Y.M.
      • Idle J.R.
      • Gonzalez F.J.
      Serum metabolomics reveals irreversible inhibition of fatty acid β-oxidation through the suppression of PPARα activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.
      ,
      • Ruepp S.U.
      • Tonge R.P.
      • Shaw J.
      • Wallis N.
      • Pognan F.
      Genomics and proteomics analysis of acetaminophen toxicity in mouse liver.
      ].
      Thirdly, drugs such as APAP and cisplatin could cause mitochondrial permeability transition through an activation of JNK or other endogenous MPTP inducers [
      • Hinson J.A.
      • Roberts D.W.
      • James L.P.
      Mechanisms of acetaminophen-induced liver necrosis.
      ,
      • Kaplowitz N.
      • Shinohara M.
      • Liu Z.X.
      • Han D.
      How to protect against acetaminophen: don’t ask for JUNK.
      ,
      • Sohn J.H.
      • Han K.L.
      • Kim J.H.
      • Rukayadi Y.
      • Hwang J.K.
      Protective effects of macelignan on cisplatin-induced hepatotoxicity is associated with JNK activation.
      ]. Regarding APAP, several studies suggest that JNK activation is related to ROS generation and, therefore, APAP-induced oxidative stress could promote MPTP opening through direct and indirect pathways [
      • Jones D.P.
      • Lemasters J.J.
      • Han D.
      • Boelsterli U.A.
      • Kaplowitz N.
      Mechanisms of pathogenesis in drug hepatotoxicity.
      ,
      • Kaplowitz N.
      • Shinohara M.
      • Liu Z.X.
      • Han D.
      How to protect against acetaminophen: don’t ask for JUNK.
      ].

      Drug-induced OXPHOS impairment

      Drugs can also induce cell death through a direct impairment of OXPHOS (Table 1), which reduces ATP synthesis. As already mentioned, severe ATP depletion inhibits calcium extrusion from the cell thus leading to its intracellular accumulation. This in turn activates proteases, endonucleases, and phospholipases that participate in the destruction (or the disorganization) of cell constituents including the plasma membrane and cytoskeleton, thus leading to necrosis [
      • Pessayre D.
      • Haouzi D.
      • Fau D.
      • Robin M.A.
      • Mansouri A.
      • Berson A.
      Withdrawal of life support, altruistic suicide, fratricidal killing and euthanasia by lymphocytes: different forms of drug-induced hepatic apoptosis.
      ,
      • Dong Z.
      • Saikumar P.
      • Weinberg J.M.
      • Venkatachalam M.A.
      Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement of serine but not cysteine proteases.
      ]. In fact, drug-induced OXPHOS impairment can occur through different mechanisms.
      The first mechanism is OXPHOS uncoupling without subsequent inhibition of the MRC. In this case, substrate oxidation is maintained (since electron transfer within the MRC is not altered) although ATP synthesis is strongly hindered. Indeed, OXPHOS uncouplers are usually protonophores, namely molecules that are protonated in the mitochondrial intermembrane space thus generating cationic compounds that take advantage of the membrane potential Δψm to cross the inner membrane. Consequently, protons are entering the matrix independently of ATP synthase thus causing a drop of ATP synthesis. Drugs that induce OXPHOS uncoupling without subsequent inhibition of the MRC are for instance the nonsteroidal anti-inflammatory drug (NSAID) nimesulide and the anti-Alzheimer drug tacrine [
      • Berson A.
      • Cazanave S.
      • Descatoire V.
      • Tinel M.
      • Grodet A.
      • Wolf C.
      • et al.
      The anti-inflammatory drug, nimesulide (4-nitro-2-phenoxymethane-sulfoanilide), uncouples mitochondria and induces mitochondrial permeability transition in human hepatoma cells: protection by albumin.
      ,
      • Berson A.
      • Renault S.
      • Lettéron P.
      • Robin M.A.
      • Fromenty B.
      • Fau D.
      • et al.
      Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction.
      ]. Other NSAIDs such as salicylic acid and ibuprofen are also OXPHOS uncouplers but their uncoupling effect is so mild that it may not induce deleterious consequences in vivo [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Tokumitsu Y.
      • Lee S.
      • Ui M.
      In vitro effects of nonsteroidal anti-inflammatory drugs on oxidative phosphorylation in rats liver mitochondria.
      ]. Finally, OXPHOS uncoupling can be associated with other mitochondrial effects that present a more harmful impact on cell viability. For instance, although diclofenac both uncouples OXPHOS and favors MPTP opening only the latter effect could be responsible for cell injury [
      • Lim M.S.
      • Lim P.L.
      • Gupta R.
      • Boelsterli U.A.
      Critical role of free cytosolic calcium, but not uncoupling, in mitochondrial permeability transition and cell death induced by diclofenac oxidative metabolites in immortalized human hepatocytes.
      ].
      The second mechanism is OXPHOS uncoupling with subsequent inhibition of the MRC activity, thus leading to a secondary impairment of substrate oxidation such as FAO. Unfortunately, the precise mechanism whereby these drugs alter electron transfer within the MRC is unknown. Actually, the dual effect of some drugs on OXPHOS (i.e. uncoupling followed by inhibition) seems to be concentration-dependent and “isolated” uncoupling nevertheless can be observed for low concentrations of these drugs. Drug-induced dual effect on OXPHOS has been described with amiodarone, perhexiline, alpidem, tamoxifen, and buprenorphine [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Berson A.
      • Descatoire V.
      • Sutton A.
      • Fau D.
      • Maulny B.
      • Vadrot N.
      • et al.
      Toxicity of alpidem, a peripheral benzodiazepine receptor ligand, but not zolpidem, in rat hepatocytes: role of mitochondrial permeability transition and metabolic activation.
      ,
      • Berson A.
      • Fau D.
      • Fornacciari R.
      • Degove-Goddard P.
      • Sutton A.
      • Descatoire V.
      • et al.
      Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.
      ,
      • Card J.W.
      • Lalonde B.R.
      • Rafeiro E.
      • Tam A.S.
      • Racz W.J.
      • Brien J.F.
      • et al.
      Amiodarone-induced disruption of hamster lung and liver mitochondrial function: lack of association with thiobarbituric acid-reactive substance production.
      ,
      • Deschamps D.
      • DeBeco V.
      • Fisch C.
      • Fromenty B.
      • Guillouzo A.
      • Pessayre D.
      Inhibition by perhexiline of oxidative phosphorylation and the β-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Waldhauser K.M.
      • Török M.
      • Ha H.R.
      • Thomet U.
      • Konrad D.
      • Brecht K.
      • et al.
      Hepatocellular toxicity and pharmacological effect of amiodarone and amiodarone derivatives.
      ]. A dual effect has also been described for salicylic acid but strong MRC inhibition induced by this drug occurs for concentrations in the millimolar range [
      • Deschamps D.
      • Fisch C.
      • Fromenty B.
      • Berson A.
      • Degott C.
      • Pessayre D.
      Inhibition by salicylic acid of the activation and thus oxidation of long chain fatty acids. Possible role in the development of Reye’s syndrome.
      ,
      • Doi H.
      • Horie T.
      Salicylic acid-induced hepatotoxicity triggered by oxidative stress.
      ]. Finally, while drug-induced MRC blockage can participate in the inhibition of mitochondrial FAO, some drugs, such as amiodarone, perhexiline, and tamoxifen can also directly inhibit FAO enzymes such as CPT1, as discussed below [
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Fromenty B.
      • Fisch C.
      • Labbe G.
      • Degott C.
      • Deschamps D.
      • Berson A.
      • et al.
      Amiodarone inhibits the mitochondrial β-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice.
      ,
      • Kennedy J.A.
      • Unger S.A.
      • Horowitz J.D.
      Inhibition of carnitine palmitoyltransferase-1 in rat heart and liver by perhexiline and amiodarone.
      ].
      A third mechanism is an inhibition of the MRC activity without any prior OXPHOS uncoupling. This situation has been described for instance with the anti-androgen drug nilutamide [
      • Berson A.
      • Schmets L.
      • Fisch C.
      • Fau D.
      • Wolf C.
      • Fromenty B.
      • et al.
      Inhibition by nilutamide of the mitochondrial respiratory chain and ATP formation. Possible contribution to the adverse effects of this antiandrogen.
      ].

      Drug-induced severe inhibition of mitochondrial β-oxidation and microvesicular steatosis

      Some drugs can induce microvesicular steatosis (Table 4) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Hautekeete M.L.
      • Degott C.
      • Benhamou J.P.
      Microvesicular steatosis of the liver.
      ,
      • Coghlan M.E.
      • Sommadossi J.P.
      • Jhala N.C.
      • Many W.J.
      • Saag M.S.
      • Johnson V.A.
      Symptomatic lactic acidosis in hospitalized antiretroviral-treated patients with human immunodeficiency virus infection: a report of 12 cases.
      ,
      • Danan G.
      • Trunet P.
      • Bernuau J.
      • Degott C.
      • Babany G.
      • Pessayre D.
      • et al.
      Pirprofen-induced fulminant hepatitis.
      ,
      • Kleiner D.E.
      • Gaffey M.J.
      • Sallie R.
      • Tsokos M.
      • Nichols L.
      • McKenzie R.
      • et al.
      Histopathologic changes associated with fialuridine hepatotoxicity.
      ,
      • Le Bricquir Y.
      • Larrey D.
      • Blanc P.
      • Pageaux G.P.
      • Michel H.
      Tianeptine: an instance of drug-induced hepatotoxicity predicted by prospective experimental studies.
      ], which is sometimes referred to as microsteatosis. Microvesicular steatosis is a potentially severe liver lesion that can be associated with liver failure, encephalopathy, and profound hypoglycemia thus leading to the death of some patients. Liver pathology shows the presence of numerous cytoplasmic lipid droplets, which can be stained with oil red O [
      • Hautekeete M.L.
      • Degott C.
      • Benhamou J.P.
      Microvesicular steatosis of the liver.
      ,
      • Genève J.
      • Hayat-Bonan B.
      • Labbe G.
      • Degott C.
      • Lettéron P.
      • Fréneaux E.
      • et al.
      Inhibition of mitochondrial beta-oxidation of fatty acids by pirprofen. Role in microvesicular steatosis due to this nonsteroidal anti-inflammatory drug.
      ]. Hepatic cytolysis and increased plasma transaminases can also be observed to a variable degree. Amiodarone, although being able to induce “pure” microvesicular steatosis in a few patients [
      • Jones D.B.
      • Mullick F.G.
      • Hoofnagle J.H.
      • Baranski B.
      Reye’s syndrome-like illness in a patient receiving amiodarone.
      ,
      • Lewis J.H.
      • Mullick F.
      • Ishak K.G.
      • Ranard R.C.
      • Ragsdale B.
      • Perse R.M.
      • et al.
      Histopathologic analysis of suspected amiodarone hepatotoxicity.
      ], most often provokes macrovacuolar steatosis (occasionally associated with microvesicular steatosis) and steatohepatitis. Microvesicular steatosis or mixed steatosis has seldom been reported with troglitazone in addition to other lesions, such as necroinflammation, fibrosis, and cholestasis [
      • Caldwell S.H.
      • Hespenheide E.E.
      • von Borstel R.W.
      Myositis, microvesicular hepatitis, and progression to cirrhosis from troglitazone added to simvastatin.
      ,
      • Fukano M.
      • Amano S.
      • Sato J.
      • Yamamoto K.
      • Adachi H.
      • Okabe H.
      • et al.
      Subacute hepatic failure associated with a new antidiabetic agent, troglitazone: a case report with autopsy examination.
      ,
      • Kohlroser J.
      • Mathai J.
      • Reichheld J.
      • Banner B.F.
      • Bonkovsky H.L.
      Hepatotoxicity due to troglitazone: report of two cases and review of adverse events reported to the United States Food and Drug Administration.
      ]. Microvesicular steatosis can be also observed during ethanol intoxication, Reye’s syndrome, acute fatty liver of pregnancy, and several inborn errors of mitochondrial FAO and OXPHOS [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Hautekeete M.L.
      • Degott C.
      • Benhamou J.P.
      Microvesicular steatosis of the liver.
      ,
      • Fromenty B.
      • Grimbert S.
      • Mansouri A.
      • Beaugrand M.
      • Erlinger S.
      • Rötig A.
      • et al.
      Hepatic mitochondrial DNA deletion in alcoholics: association with microvesicular steatosis.
      ,
      • Rinaldo P.
      • Yoon H.R.
      • Yu C.
      • Raymond K.
      • Tiozzo C.
      • Giordano G.
      Sudden and unexpected neonatal death: a protocol for the postmortem diagnosis of fatty acid oxidation disorders.
      ].
      Table 4Examples of drugs inducing microvesicular steatosis.
      aAbbreviations: NRTIs, nucleoside reverse transcriptase inhibitors; NSAID, nonsteroidal anti-inflammatory drug.
      Whatever its etiology, microvesicular steatosis results primarily from a severe inhibition of the mitochondrial FAO (Fig. 2) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Fréneaux E.
      • Labbe G.
      • Lettéron P.
      • Le Dinh T.
      • Degott C.
      • Genève J.
      • et al.
      Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic.
      ,
      • Renaud D.L.
      • Edwards V.
      • Wilson G.J.
      • Tein I.
      Glucose-free medium exacerbates microvesicular steatosis in cultured skin fibroblasts of genetic defects of fatty acid oxidation. A novel screening test.
      ]. Although other metabolic pathways could also be impaired [
      • Lettéron P.
      • Sutton A.
      • Mansouri A.
      • Fromenty B.
      • Pessayre D.
      Inhibition of microsomal triglyceride transfer protein: another mechanism for drug-induced steatosis in mice.
      ], these additional mechanisms most probably play a secondary role in the pathophysiology and severity of microvesicular steatosis.
      Figure thumbnail gr2
      Fig. 2Metabolic consequences of severe inhibition of mitochondrial fatty acid β-oxidation. A severe impairment of mitochondrial fatty acid oxidation (FAO) can induce accumulation of free fatty acids and triglycerides (thus explaining microvesicular steatosis), reduced ATP synthesis and lower production of ketone bodies. Inhibition of FAO also decreases gluconeogenesis through different mechanisms including lower ATP production and reduced pyruvate carboxylase (PC) activity. Low plasma levels of ketone bodies (or reduced ketone bodies utilization) and hypoglycemia are thus responsible for a profound energy deficiency in extra-hepatic tissues. The accumulation of free fatty acids (and some of their metabolites such as dicarboxylic acids) could play a major role in the pathophysiology of microvesicular steatosis. Indeed, these lipid derivatives can impair mitochondrial function through different mechanisms, thus reinforcing drug-induced inhibition of FAO.
      A primary consequence of severe inhibition of mitochondrial FAO is an accumulation of fatty acids that are either esterified into triglycerides or that remain as a free form, which can reinforce mitochondrial dysfunction (Fig. 2) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Belosludtsev K.N.
      • Saris N.E.
      • Belosludtseva N.V.
      • Trudovishnikov A.S.
      • Lukyanova L.D.
      • Mironova G.D.
      Physiological aspects of the mitochondrial cyclosporin A-insensitive palmitate/Ca2+-induced pore: tissue specificity, age profile and dependence on the animal’s adaptation to hypoxia.
      ,
      • Wu X.
      • Zhang L.
      • Gurley E.
      • Studer E.
      • Shang J.
      • Wang T.
      • et al.
      Prevention of free fatty acid-induced hepatic lipotoxicity by 18-β-glycyrrhetinic acid through lysosomal and mitochondrial pathways.
      ]. Another major consequence is an impairment of energy output in the liver but also in extra-hepatic tissues attributable to lower ketone body production (or utilization). Importantly, reduced mitochondrial FAO hampers hepatic gluconeogenesis as a consequence of ATP shortage and pyruvate carboxylase inhibition, which can lead to severe hypoglycemia in some individuals (Fig. 2) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ]. Finally, severe impairment of mitochondrial FAO is associated with an accumulation in plasma and urines of fatty acid derivatives, such as acyl-carnitine and acyl-glycine esters and dicarboxylic acids [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Matsumoto J.
      • Ogawa H.
      • Maeyama R.
      • Okudaira K.
      • Shinka T.
      • Kuhara T.
      • et al.
      Successful treatment by direct hemoperfusion of coma possibly resulting from mitochondrial dysfunction in acute valproate intoxication.
      ].
      Drug-induced severe inhibition of mitochondrial FAO can result from several mechanisms and some drugs impair this metabolic pathway by interacting with different mitochondrial enzymes [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ]. These mechanisms can be classified into four different categories.
      Firstly, drugs, such as ibuprofen, tianeptine, amiodarone, tamoxifen, and VPA can directly inhibit one or several mitochondrial FAO enzymes (Table 1) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Baldwin G.S.
      • Murphy V.J.
      • Yang Z.
      • Hashimoto T.
      Binding of nonsteroidal antiinflammatory drugs to the α-subunit of the trifunctional protein of long chain fatty acid oxidation.
      ,
      • Fromenty B.
      • Fréneaux E.
      • Labbe G.
      • Deschamps D.
      • Larrey D.
      • Lettéron P.
      • et al.
      Tianeptine, a new tricyclic antidepressant metabolized by beta-oxidation of its heptanoic side chain, inhibits the mitochondrial oxidation of medium and short chain fatty acids in mice.
      ]. VPA-induced severe FAO inhibition is probably due to Δ2,4-VPA-CoA and other reactive metabolites which irreversibly inactivate FAO enzyme(s) (Fig. 3) [
      • Rettenmeier A.W.
      • Prickett K.S.
      • Gordon W.P.
      • Bjorge S.M.
      • Chang S.L.
      • Levy R.H.
      • et al.
      Studies on the biotransformation in the perfused rat liver of 2-n-propyl-4-pentenoic acid, a metabolite of the antiepileptic drug valproic acid. Evidence for the formation of chemically reactive intermediates.
      ,
      • Silva M.F.
      • Ruiter J.P.
      • Ijlst L.
      • Jakobs C.
      • Duran M.
      • de Almeida I.T.
      • et al.
      Differential effect of valproate and its Delta2- and Delta4-unsaturated metabolites, on the beta-oxidation rate of long-chain and medium-chain fatty acids.
      ]. Likewise, APAP may inhibit FAO enzymes through the generation of its reactive metabolite NAPQI [
      • Chen C.
      • Krausz K.W.
      • Shah Y.M.
      • Idle J.R.
      • Gonzalez F.J.
      Serum metabolomics reveals irreversible inhibition of fatty acid β-oxidation through the suppression of PPARα activation as a contributing mechanism of acetaminophen-induced hepatotoxicity.
      ]. This may explain why this analgesic drug induces steatosis in some individuals [
      • Biour M.
      • Ben Salem C.
      • Chazouillères O.
      • Grangé J.D.
      • Serfaty L.
      • Poupon R.
      Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs.
      ,
      • Ramachandran R.
      • Kakar S.
      Histological patterns in drug-induced liver disease.
      ]. Unfortunately, the FAO enzymes inhibited by these drugs have not always been identified, although CPT1 (Fig. 1) could be a key target. Indeed, this enzyme can be inhibited by VPA (Fig. 3), amiodarone, and tamoxifen [
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Kennedy J.A.
      • Unger S.A.
      • Horowitz J.D.
      Inhibition of carnitine palmitoyltransferase-1 in rat heart and liver by perhexiline and amiodarone.
      ,
      • Aires C.C.
      • Ijlst L.
      • Stet F.
      • Prip-Buus C.
      • de Almeida I.T.
      • Duran M.
      • et al.
      Inhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis.
      ]. Interestingly, troglitazone is able to inhibit long-chain acyl-CoA synthase (ACS) (Fig. 1), thus impairing the mitochondrial entry of LCFAs [
      • Fulgencio J.P.
      • Kohl C.
      • Girard J.
      • Pégorier J.P.
      Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats.
      ].
      Figure thumbnail gr3
      Fig. 3Mechanisms of valproic acid-induced inhibition of mitochondrial fatty acid β-oxidation. Valproic acid (VPA, or dipropylacetic acid) is an analogue of medium-chain fatty acid which freely enters the mitochondrion and generates a coenzyme A ester (VPA-CoA) within the mitochondrial matrix. This VPA-CoA derivative can inhibit carnitine palmitoyltransferase-1 (CPT 1), an enzyme catalyzing the rate limiting step of the mitochondrial entry and β-oxidation of long-chain fatty acids. Furthermore, the generation of the VPA-CoA ester reduces mitochondrial levels of CoA, which is a cofactor mandatory for fatty acid oxidation (FAO). A second mechanism which could play a major role in VPA-induced inhibition of FAO is the cytochrome P450 (CYP)-mediated generation of Δ4-VPA (a VPA metabolite which presents a double bond between carbons 4 and 5, respectively). Indeed this metabolite also enters the mitochondrion to generate Δ2,4-VPA-CoA, a reactive metabolite able to covalently bind to (and thus inactivate) FAO enzymes. The generation of Δ4-VPA can be enhanced by a co-treatment with phenytoin and phenobarbital which are CYP inducers.
      Secondly, drugs can impair mitochondrial FAO through the generation of coenzyme A and/or l-carnitine esters, thus decreasing the levels of these major FAO cofactors (Fig. 1). This mechanism has been shown for VPA (Fig. 3), salicylic acid, and ibuprofen [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Deschamps D.
      • Fisch C.
      • Fromenty B.
      • Berson A.
      • Degott C.
      • Pessayre D.
      Inhibition by salicylic acid of the activation and thus oxidation of long chain fatty acids. Possible role in the development of Reye’s syndrome.
      ,
      • Fréneaux E.
      • Fromenty B.
      • Berson A.
      • Labbe G.
      • Degott C.
      • Lettéron P.
      • et al.
      Stereoselective and nonstereoselective effects of ibuprofen enantiomers on mitochondrial beta-oxidation of fatty acids.
      ,
      • Silva M.F.
      • Ruiter J.P.
      • Ijlst L.
      • Allers P.
      • ten Brink H.J.
      • Jakobs C.
      • et al.
      Synthesis and intramitochondrial levels of valproyl-coenzyme A metabolites.
      ].
      Thirdly, mitochondrial FAO can be secondarily impaired as a result of severe inhibition of the MRC [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ]. Indeed, the MRC allows the constant regeneration of FAD and NAD+ required for the enzymatic reactions catalyzed, respectively, by the FAO enzymes acyl-CoA dehydrogenases and 3-hydroxyacyl-CoA dehydrogenases (Fig. 1). Inhibition of FAO secondarily to MRC impairment could occur with amiodarone (Fig. 4), perhexiline, tamoxifen, and buprenorphine [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Fromenty B.
      • Fisch C.
      • Berson A.
      • Lettéron P.
      • Larrey D.
      • Pessayre D.
      Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and II.
      ,
      • Berson A.
      • Fau D.
      • Fornacciari R.
      • Degove-Goddard P.
      • Sutton A.
      • Descatoire V.
      • et al.
      Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.
      ,
      • Deschamps D.
      • DeBeco V.
      • Fisch C.
      • Fromenty B.
      • Guillouzo A.
      • Pessayre D.
      Inhibition by perhexiline of oxidative phosphorylation and the β-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ]. Interestingly, these amphiphilic drugs can be protonated within the intermembrane space of the mitochondria thus generating cationic compounds entering the matrix thanks to the membrane potential Δψm (Fig. 4) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Pessayre D.
      • Mansouri A.
      • Berson A.
      • Fromenty B.
      Mitochondrial involvement in drug-induced liver injury.
      ,
      • Fromenty B.
      • Fisch C.
      • Berson A.
      • Lettéron P.
      • Larrey D.
      • Pessayre D.
      Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and II.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ]. Besides OXPHOS uncoupling, this allows their mitochondrial accumulation and the subsequent inhibition of both FAO and MRC enzymes. Whereas relatively low concentrations of these amphiphilic drugs can inhibit directly FAO enzyme(s), higher concentrations are required in order to impair the MRC [
      • Fromenty B.
      • Fisch C.
      • Berson A.
      • Lettéron P.
      • Larrey D.
      • Pessayre D.
      Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and II.
      ,
      • Berson A.
      • Fau D.
      • Fornacciari R.
      • Degove-Goddard P.
      • Sutton A.
      • Descatoire V.
      • et al.
      Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation.
      ,
      • Deschamps D.
      • DeBeco V.
      • Fisch C.
      • Fromenty B.
      • Guillouzo A.
      • Pessayre D.
      Inhibition by perhexiline of oxidative phosphorylation and the β-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Fromenty B.
      • Fisch C.
      • Labbe G.
      • Degott C.
      • Deschamps D.
      • Berson A.
      • et al.
      Amiodarone inhibits the mitochondrial β-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice.
      ]. Thus, accumulation of these amphiphilic drugs within the mitochondria eventually inhibits FAO through a dual mechanism. Finally, although tetracycline derivatives can also reduce the MRC activity [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Yu H.Y.
      • Wang B.L.
      • Zhao J.
      • Yao X.M.
      • Gu Y.
      • Li Y.
      Protective effect of bicyclol on tetracycline-induced fatty liver in mice.
      ], it is still unclear whether these drugs inhibit mitochondrial FAO through MRC impairment or by a direct mechanism.
      Figure thumbnail gr4
      Fig. 4Mechanisms of amiodarone-induced impairment of oxidative phosphorylation and mitochondrial fatty acid β-oxidation. Amiodarone (Am) is an amphiphilic compound which harbors a protonable nitrogen within its diethyl-aminoethoxy moiety. In the intermembrane space of mitochondria (which is an acidic milieu) Am undergoes a protonation to generate Am+. This cationic derivative thus freely enters the mitochondrion thanks to the mitochondrial transmembrane potential Δψm. The entry of the protonated molecule Am+ has two major consequences regarding oxidative phosphorylation (OXPHOS) and mitochondrial fatty acid oxidation (FAO): (1) a rapid and transient uncoupling of OXPHOS since protons are not entering the matrix through ATP synthase; (2) a progressive accumulation of Am+ within the mitochondrial matrix which induces the subsequent inhibition of different enzymes involved in the mitochondrial respiratory chain (MRC) and FAO. Hence, amiodarone-induced inhibition of FAO could result from the direct inhibition of FAO enzymes (such as CPT 1) and to an impairment of the MRC activity at the level of complexes I and II.
      Fourthly, drugs can impair mitochondrial FAO and induce microvesicular steatosis by reducing mtDNA levels (Table 1). Indeed, profound mtDNA depletion induces MRC impairment and secondary inhibition of FAO. This has been shown for the antiviral fialuridine (FIAU), AZT, d4T, and ddI, which all inhibit the mtDNA polymerase γ [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Igoudjil A.
      • Begriche K.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors.
      ,
      • Lewis W.
      • Levine E.S.
      • Griniuviene B.
      • Tankersley K.O.
      • Colacino J.M.
      • Sommadossi J.P.
      • et al.
      Fialuridine and its metabolites inhibit DNA polymerase γ at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.
      ,
      • Walker U.A.
      • Venhoff N.
      Uridine in the prevention and treatment of NRTI-related mitochondrial toxicity.
      ]. Low mtDNA levels can also be associated with lactic acidosis resulting from the inhibition of the TCA cycle [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Cornejo-Juarez P.
      • Sierra-Madero J.
      • Volkow-Fernandez P.
      Metabolic acidosis and hepatic steatosis in two HIV-infected patients on stavudine (d4T) treatment.
      ,
      • Walker U.A.
      • Bäuerle J.
      • Laguno M.
      • Murillas J.
      • Mauss S.
      • Schmutz G.
      • et al.
      Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine.
      ]. Tamoxifen and tacrine can also induce hepatic mtDNA depletion although it is still unclear whether this mechanism plays a major pathophysiological role [
      • Pessayre D.
      • Mansouri A.
      • Berson A.
      • Fromenty B.
      Mitochondrial involvement in drug-induced liver injury.
      ,
      • Berson A.
      • Renault S.
      • Lettéron P.
      • Robin M.A.
      • Fromenty B.
      • Fau D.
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      Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ]. Both tamoxifen and tacrine reduce mtDNA synthesis by interacting with the mitochondrial topoisomerases [
      • Berson A.
      • Renault S.
      • Lettéron P.
      • Robin M.A.
      • Fromenty B.
      • Fau D.
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      Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ].
      Figure thumbnail fx7

      Drug-induced alterations of hepatic lipid metabolism inducing macrovacuolar steatosis

      With some drugs (Table 5) [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Castéra L.
      • Kalinsky E.
      • Bedossa P.
      • Tertian G.
      • Buffet C.
      Macrovesicular steatosis induced by interferon alfa therapy for chronic myelogenous leukaemia.
      ,
      • Farrell G.C.
      Drugs and steatohepatitis.
      ,
      • Grismer L.E.
      • Gill S.A.
      • Harris M.D.
      Liver biopsy in psoriatic arthritis to detect methotrexate hepatotoxicity.
      ,
      • Zorzi D.
      • Laurent A.
      • Pawlik T.M.
      • Lauwers G.Y.
      • Vauthey J.N.
      • Abdalla E.K.
      Chemotherapy-associated hepatotoxicity and surgery for colorectal liver metastases.
      ], liver triglycerides accumulate as a large (often single) lipid vacuole displacing the nucleus at the periphery of the hepatocyte. This liver lesion is commonly referred to as macrovacuolar steatosis [
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Delzenne N.M.
      • Hernaux N.A.
      • Taper H.S.
      A new model of acute liver steatosis induced in rats by fasting followed by refeeding a high carbohydrate-fat free diet. Biochemical and morphological analysis.
      ]. Several drugs responsible for this hepatic lesion can also induce a mixed form of fat accumulation with macrovacuolar steatosis in some hepatocytes and microvesicular steatosis in others. It is possible that the size of the fat droplets could depend on the nature of some proteins wrapping the lipids (e.g. perilipin and adipophilin) and/or their content in free fatty acids [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Bickel P.E.
      • Tansey J.T.
      • Welte M.A.
      PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.
      ]. Alternatively, the coexistence of both types of steatosis could result from the occurrence of different mechanisms of toxicity in distinct hepatocytes.
      Table 5Examples of drugs inducing macrovacuolar steatosis and steatohepatitis.
      aAbbreviation: NRTIs, nucleoside reverse transcriptase inhibitors.
      Macrovacuolar steatosis is also observed in a large number of obese and diabetic patients, even in those that do no drink alcohol. That is why it is often referred to as nonalcoholic fatty liver in the context of obesity and related metabolic disorders [
      • Begriche K.
      • Knockaert L.
      • Massart J.
      • Robin M.A.
      • Fromenty B.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH): are there drugs able to improve it?.
      ,
      • Begriche K.
      • Lettéron P.
      • Abbey-Toby A.
      • Vadrot N.
      • Robin M.A.
      • Bado A.
      • et al.
      Partial leptin deficiency favors diet-induced obesity and related metabolic disorders in mice.
      ,
      • Brunt E.M.
      Pathology of nonalcoholic fatty liver disease.
      ]. In these disorders, hepatic steatosis primarily results from two mechanisms: 1) an increased delivery of free fatty acids to the liver which is the consequence of insulin resistance in adipose tissue (that favors triglycerides hydrolysis); and, 2) a stimulation of de novo hepatic lipogenesis, which is mainly due to hyperinsulinemia and hyperglycemia that activate the transcription factors SREBP-1c and ChREBP, respectively [
      • Begriche K.
      • Knockaert L.
      • Massart J.
      • Robin M.A.
      • Fromenty B.
      Mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH): are there drugs able to improve it?.
      ,
      • Begriche K.
      • Igoudjil A.
      • Pessayre D.
      • Fromenty B.
      Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.
      ,
      • Fabbrini E.
      • Sullivan S.
      • Klein S.
      Obesity and nonalcoholic fatty liver disease: biochemical, metabolic, and clinical implications.
      ].
      Ethanol intoxication frequently induces macrovacuolar steatosis although microvesicular steatosis can be also observed [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Breitkopf K.
      • Nagy L.E.
      • Beier J.I.
      • Mueller S.
      • Weng H.
      • Dooley S.
      Current experimental perspectives on the clinical progression of alcoholic liver disease.
      ]. Ethanol-induced fatty liver results from different mechanisms including increased hepatic uptake of fatty acids and de novo lipogenesis, impaired PPARα signaling, mitochondrial dysfunction and reduced secretion of triglycerides [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Crabb D.W.
      • Galli A.
      • Fischer M.
      • You M.
      Molecular mechanisms of alcoholic fatty liver: role of peroxisome proliferator-activated receptor alpha.
      ,
      • Day C.P.
      • Yeaman S.J.
      The biochemistry of alcohol-induced fatty liver.
      ,
      • Donohue T.M.
      Alcohol-induced steatosis in liver cells.
      ,
      • Ji C.
      • Chan C.
      • Kaplowitz N.
      Predominant role of sterol response element binding proteins (SREBP) lipogenic pathways in hepatic steatosis in the murine intragastric ethanol feeding model.
      ]. Some of these effects could be due to reduced adiponectin secretion by the adipose tissue and elevated expression of tumor necrosis factor-α (TNFα, which both favor lipid synthesis and reduced mitochondrial FAO [
      • Chen X.
      • Sebastian B.M.
      • Tang H.
      • McMullen M.M.
      • Axhemi A.
      • Jacobsen D.W.
      • et al.
      Taurine supplementation prevents ethanol-induced decrease in serum adiponectin and reduces hepatic steatosis in rats.
      ,
      • Song Z.
      • Zhou Z.
      • Deaciuc I.
      • Chen T.
      • McClain C.J.
      Inhibition of adiponectin production by homocysteine: a potential mechanism for alcoholic liver disease.
      ,
      • Zeng T.
      • Xie K.Q.
      Ethanol and liver: recent advances in the mechanisms of ethanol-induced hepatosteatosis.
      ].
      Regarding drug-induced macrovacuolar steatosis, different mechanisms seem involved (Fig. 5), and a single molecule can alter several metabolic pathways.
      Figure thumbnail gr5
      Fig. 5Mechanisms of drug-induced macrovacuolar steatosis and steatohepatitis. Drugs can induce macrovacuolar steatosis through at least four different mechanisms: (1) by inducing a moderate impairment of mitochondrial fatty acid oxidation (FAO); (2) by decreasing the secretion of very-low density lipoprotein (VLDL); (3) by directly activating transcription factors involved in hepatic lipogenesis, such as SREBP-1c, PPARγ, and PXR, and; (4) by favoring the occurrence of insulin resistance and hyperinsulinemia, which can be the consequence of obesity or lipoatrophy (i.e. a reduction of body fatness). It is noteworthy that the progression of steatosis into steatohepatitis in some patients involves the production of reactive oxygen species (ROS), which is responsible for oxidative stress and lipid peroxidation. These deleterious events subsequently trigger the production of different cytokines such as TNFα and TGFβ that favor necroinflammation and fibrosis. Although the mitochondria produce the majority of ROS through the alteration of the mitochondrial respiratory chain (MRC), other sources could involve peroxisomal FAO and microsomal cytochromes P450 (CYPs).
      Firstly, a moderate inhibition of mitochondrial FAO could play a role with amiodarone, perhexiline, tamoxifen, NRTIs, and glucocorticoids [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Larosche I.
      • Lettéron P.
      • Fromenty B.
      • Vadrot N.
      • Abbey-Toby A.
      • Feldmann G.
      • et al.
      Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver.
      ,
      • Jia Y.
      • Viswakarma N.
      • Fu T.
      • Yu S.
      • Rao M.S.
      • Borensztajn J.
      • et al.
      Conditional ablation of mediator subunit MED1 (MED1/PPARBP) gene in mouse liver attenuates glucocorticoid receptor agonist dexamethasone-induced hepatic steatosis.
      ,
      • Lettéron P.
      • Brahimi-Bourouina N.
      • Robin M.A.
      • Moreau A.
      • Feldmann G.
      • Pessayre D.
      Glucocorticoids inhibit mitochondrial matrix acyl-CoA dehydrogenases and fatty acid beta-oxidation.
      ]. However, some of these drugs could induce stronger inhibition of mitochondrial FAO in a few patients thus leading to the occurrence of microvesicular steatosis, as previously mentioned.
      Secondly, a reduction of hepatic VLDL secretion has been described with amiodarone, perhexiline, and tetracycline which all inhibit MTP activity [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Lettéron P.
      • Sutton A.
      • Mansouri A.
      • Fromenty B.
      • Pessayre D.
      Inhibition of microsomal triglyceride transfer protein: another mechanism for drug-induced steatosis in mice.
      ]. D4T was shown to reduce MTP mRNA expression in cultured rat hepatocytes but MTP activity was not assessed [
      • Igoudjil A.
      • Massart J.
      • Begriche K.
      • Descatoire V.
      • Robin M.A.
      • Fromenty B.
      High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes.
      ]. Interestingly, small molecules inhibiting MTP have been tested in order to lower blood lipids, but the clinical usefulness of this therapeutic strategy has been hampered by their potential to induce hepatic steatosis [
      • Chandler C.E.
      • Wilder D.E.
      • Pettini J.L.
      • Savoy Y.E.
      • Petras S.F.
      • Chang G.
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      CP-346086: an MTP inhibitor that lowers plasma cholesterol and triglycerides in experimental animals and in humans.
      ,
      • Wierzbicki A.S.
      • Hardman T.
      • Prince W.T.
      Future challenges for microsomal transport protein inhibitors.
      ].
      Thirdly, increased cellular uptake of fatty acids could play a significant role with some compounds. This mechanism has been proposed for efavirenz which activates AMP-activated protein kinase (AMPK) most probably as a consequence of mitochondrial complex I inhibition and reduced ATP synthesis [
      • Blas-García A.
      • Apostolova N.
      • Ballesteros D.
      • Monleón D.
      • Morales J.M.
      • Rocha M.
      • et al.
      Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells.
      ]. Indeed, AMPK activation promotes fatty acid uptake into the cell through the fatty acid transporter FAT/CD36 in addition to its stimulating role on mitochondrial FAO [
      • Thomson D.M.
      • Winder W.W.
      AMP-activated protein kinase control of fat metabolism in skeletal muscle.
      ]. Thus, efavirenz-induced lipid accumulation in hepatocytes is likely favored by the concomitant increased uptake of extracellular fatty acids and impaired mitochondrial FAO [
      • Blas-García A.
      • Apostolova N.
      • Ballesteros D.
      • Monleón D.
      • Morales J.M.
      • Rocha M.
      • et al.
      Inhibition of mitochondrial function by efavirenz increases lipid content in hepatic cells.
      ].
      Figure thumbnail fx8

      Mechanisms involved in the progression of steatosis into steatohepatitis

      Several drugs can induce steatohepatitis (Table 5) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Lewis J.H.
      • Mullick F.
      • Ishak K.G.
      • Ranard R.C.
      • Ragsdale B.
      • Perse R.M.
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      Histopathologic analysis of suspected amiodarone hepatotoxicity.
      ,
      • Farrell G.C.
      Drugs and steatohepatitis.
      ,
      • Hu B.
      • French S.W.
      2′,3′-Dideoxyinosine-induced Mallory bodies in patients with HIV.
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      • Ozon A.
      • Cetinkaya S.
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      • Sen Y.
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      Inappropriate use of potent topical glucocorticoids in infants.
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      • Pinto H.C.
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      • Valente A.
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      Tamoxifen-associated steatohepatitis – report of three cases.
      ,
      • Simon J.B.
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      Amiodarone hepatotoxicity simulating alcoholic liver disease.
      ], a potentially severe liver lesion characterized by the presence of necroinflammation, fibrosis, and Mallory bodies. In the context of drug-induced steatohepatitis, fat accumulates usually as large vacuoles, although microvesicular steatosis can also be present in some hepatocytes. Inflammation and fibrosis can be of variable severity and occasionally cirrhosis occurs with drugs, such as amiodarone, perhexiline, and didanosine [
      • Lewis J.H.
      • Mullick F.
      • Ishak K.G.
      • Ranard R.C.
      • Ragsdale B.
      • Perse R.M.
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      Histopathologic analysis of suspected amiodarone hepatotoxicity.
      ,
      • Farrell G.C.
      Drugs and steatohepatitis.
      ,
      • Maida I.
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      ,
      • Pessayre D.
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      • Degott C.
      • Potet F.
      • Benhamou J.P.
      • Feldmann G.
      Perhexiline maleate-induced cirrhosis.
      ,
      • Puli S.R.
      • Fraley M.A.
      • Puli V.
      • Kuperman A.B.
      • Alpert M.A.
      Hepatic cirrhosis caused by low-dose oral amiodarone therapy.
      ]. Importantly, drug-induced steatohepatitis shares many pathological and clinical features with alcoholic steatohepatitis and nonalcoholic steatohepatitis (NASH).
      Although there are still some unsolved issues about the mechanisms involved in the progression of steatosis into steatohepatitis, there is evidence for a key role of mitochondrial dysfunction (Fig. 5). Indeed, several drugs causing steatohepatitis are able to impair the mitochondrial OXPHOS process and inhibit the MRC (Fig. 5) [
      • Fromenty B.
      • Pessayre D.
      Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity.
      ,
      • Labbe G.
      • Pessayre D.
      • Fromenty B.
      Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies.
      ,
      • Pessayre D.
      • Mansouri A.
      • Berson A.
      • Fromenty B.
      Mitochondrial involvement in drug-induced liver injury.
      ,
      • Yamamoto N.
      • Oliveira M.B.
      • Campello Ade P.
      • Lopes L.C.
      • Klüppel M.L.
      Methotrexate: studies on the cellular metabolism. I. Effect on mitochondrial oxygen uptake and oxidative phosphorylation.
      ]. Actually, inhibition of the MRC could not only participate to fat deposition but also to ROS overproduction. However, other (i.e. nonmitochondrial) sources of ROS are probably involved, such as peroxisomal FAO, or microsomal CYPs [

      Hardwick JP, Osei-Hyiaman D, Wiland H, Abdelmegeed MA, Song BJ. PPAR/RXR regulation of fatty acid metabolism and fatty acid ω-hydroxylase (CYP4) isozymes: implications for prevention of lipotoxicity in fatty liver disease. PPAR Res 2009;2009:952734, doi:10.1155/2009/952734.

      ,
      • Leclercq I.A.
      • Farrel G.C.
      • Field J.
      • Bell D.R.
      • Gonzalez F.J.
      • Robertson G.R.
      CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.
      ].
      ROS, whatever their sources, can then trigger peroxidation of polyunsaturated fatty acids, a degradative process generating reactive aldehydic derivatives, such as malondialdehyde and 4-hydroxynonenal [
      • Leclercq I.A.
      • Farrel G.C.
      • Field J.
      • Bell D.R.
      • Gonzalez F.J.
      • Robertson G.R.
      CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.
      ,
      • Berson A.
      • De Beco V.
      • Lettéron P.
      • Robin M.A.
      • Moreau C.
      • El Kahwaji J.
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      Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.
      ,
      • Demori I.
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      • Fugassa E.
      • Burlando B.
      Combined effects of high-fat diet and ethanol induce oxidative stress in rat liver.
      ]. Importantly, ROS and lipid peroxidation products activate Kupffer and stellate cells that play a role in inflammation and fibrogenesis, respectively (Fig. 5) [
      • Begriche K.
      • Igoudjil A.
      • Pessayre D.
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      Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it.