# Mitochondrial dysfunction in drug-induced hepatic steatosis: recent findings and current concept

Annie Borgne-Sanchez, Bernard Fromenty

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Annie Borgne-Sanchez, Bernard Fromenty. Mitochondrial dysfunction in drug-induced hepatic steatosis: recent findings and current concept. Clinics and Research in Hepatology and Gastroenterology, 2025, 49 (3), pp.102529. 10.1016/j.clinre.2025.102529. hal-04926763

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Submitted on 3 Feb 2025

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Review

# Mitochondrial dysfunction in drug-induced hepatic steatosis: Recent findings and current concept

Annie Borgne-Sanchez<sup>a</sup>, Bernard Fromenty<sup>b,*</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

**Keywords:**

Liver

Steatosis

Drug

Toxicity

Mitochondria

## ABSTRACT

Mitochondrial activity is necessary for the maintenance of many liver functions. In particular, mitochondrial fatty acid oxidation (FAO) is required for energy production and lipid homeostasis. This key metabolic pathway is finely tuned by the mitochondrial respiratory chain (MRC) activity and different transcription factors such as peroxisome proliferator-activated receptor α (PPARα). Many drugs have been shown to cause mitochondrial dysfunction, which can lead to acute and chronic liver lesions. While severe inhibition of mitochondrial FAO would eventually cause microvesicular steatosis, hypoglycemia, and liver failure, moderate impairment of this metabolic pathway can induce macrovacuolar steatosis, which can progress in the long term to steatohepatitis and cirrhosis. Drugs can impair mitochondrial FAO through several mechanisms including direct inhibition of FAO enzymes, sequestration of coenzyme A and L-carnitine, impairment of the activity of one or several MRC complexes and reduced PPARα expression. In drug-induced macrovacuolar steatosis, non-mitochondrial mechanisms can also be involved in lipid accumulation including increased *de novo* lipogenesis and reduced very-low-density lipoprotein secretion. Nonetheless, mitochondrial dysfunction and subsequent oxidative stress appear to be key events in the progression of steatosis to steatohepatitis. Patients suffering from metabolic dysfunction-associated steatotic liver disease (MASLD) and treated with mitochondriotoxic drugs should be closely monitored to reduce the risk of acute liver injury or a faster transition of steatosis to steatohepatitis. Therapies based on the mitochondrial cofactor L-carnitine, the antioxidant N-acetylcysteine, or thyromimetics might be useful to prevent or treat drug-induced mitochondrial dysfunction, steatosis, and steatohepatitis.

## 1. Introduction

Drugs can induce several types of liver injury such as acute and chronic hepatitis, cholestasis, ductopenia, phospholipidosis, and steatosis. The latter lesion is considered pathologic when at least 5 % of hepatocytes have visible intracellular lipid droplets. More specifically, steatosis corresponds to the hepatic accumulation of lipids, mainly triglycerides, albeit other lipids species can also accumulate [1]. The exact prevalence of drug-induced steatosis is currently unknown, although two studies suggest that it could be relatively common. Indeed, the first study reported that 12 % of the hepatotoxic drugs could induce steatosis [2], while the second one reported that about one-fourth of the cases of suspected drug-induced liver injury (DILI) is characterized by the presence of steatosis [3].

This review seeks to present the latest insights into the role of mitochondrial dysfunction in drug-induced steatosis, which can

manifest in two forms, namely microvesicular and macrovacuolar steatosis [4-7]. Because we published a literature review on this topic in 2019 [6], this update incorporates data reported since then. For this purpose, we performed a PubMed search of literature published in the English language using several terms including "drug-induced hepatic steatosis", "drug-induced steatosis", "drugs and steatosis", "drug-induced hepatotoxicity", "drug-induced mitochondrial dysfunction", and "drugs and mitochondrial dysfunction". This data-mining analysis was also completed by using Google Scholar®. However, because readers are not necessarily familiar with mitochondrial function, a brief section will be devoted to this matter. Next, we present the main mechanisms by which drugs can induce microvesicular or macrovacuolar steatosis through mitochondrial dysfunction. Non-mitochondrial mechanisms are also briefly mentioned. We also discuss the issue of drug-induced impairment of mitochondrial function in patients with metabolic dysfunction-associated steatotic liver disease (MASLD, formerly named

<footnote><sup>a</sup> MITOLOGICS S.A.S., 172 rue de Charonne, 75011 Paris, France</footnote>

<footnote><sup>b</sup> INSERM, INRAE, Univ Rennes, Institut NUMECAN, UMR_S1317, 35000 Rennes, France</footnote>

<footnote>* Corresponding author.
E-mail address: bernard.fromenty@inserm.fr (B. Fromenty).</footnote>
nonalcoholic fatty liver disease or NAFLD) [8]. In addition, we review the present knowledge on some therapeutic options to prevent or treat drug-induced steatosis. Finally, we consider other issues regarding drug-induced mitochondrial dysfunction, steatosis, and liver injury such as adaptive mechanisms, the mode of cell death, and gender difference.

## 2. General overview of mitochondrial function

### 2.1. Mitochondrial substrate oxidation and energy synthesis

Mitochondria are intracellular organelles with two membranes that surround the matrix. The inner membrane harbors all the components of the mitochondrial respiratory chain (MRC), whereas the matrix contains enzymes involved in key oxidative pathways and the mitochondrial genome (Fig. 1). A key role of hepatic mitochondria is ATP synthesis via the oxidation of substrates such as pyruvate, amino acids, and fatty acids of different chain lengths. The metabolic process that allows the tight coupling between substrate oxidation and ATP synthesis (Fig. 1), called oxidative phosphorylation (OXPHOS), is finely controlled by numerous circulating and intrahepatocellular factors [9,10]. Any significant alteration of the OXPHOS process can lead to severe liver disorders [4, 11].

Fatty acids are oxidized within the mitochondria via the β-oxidation

pathway, which generates acetyl-CoA, NADH, and FADH₂ (Fig. 1). This pathway requires coenzyme A (CoA) and L-carnitine and involves several enzymes with specific activities according to their chain length [4,12]. During fasting periods, the acetyl-CoA molecules resulting from mitochondrial fatty acid oxidation (FAO) generate ketone bodies, mostly acetoacetate and β-hydroxybutyrate. Ketone bodies are then oxidized in extra-hepatic tissues via the tricarboxylic acid (TCA) cycle to provide energy (Fig. 1). Outside the fasting periods, acetyl-CoA is mostly oxidized by the hepatic TCA into CO₂, NADH, and FADH₂.

As previously mentioned, mitochondrial FAO can oxidize fatty acids (FAs) with different chain lengths [12,13]. These FAs include long-chain FAs (LCFAs, between 14 and 20 carbons), medium-chain FAs (MCFAs, between 8 and 12 carbons), and short-chain FAs (SCFAs, <6 carbons). Whereas SCFAs and MCFAs freely enter mitochondria, the entry of LCFAs into these organelles requires several enzymes including carnitine palmitoyltransferase 1 (CPT1), carnitine/acylcarnitine translocase (CACT), and CPT2 (Fig. 1). During mitochondrial FAO, all FAs undergo four consecutive reactions generating one acetyl-CoA molecule and a shortened FA (Fig. 1), which can be further oxidized by other cycles of β-oxidation. Hence, in physiological situations, FAs are entirely oxidized in acetyl-CoA molecules (for instance, 8 molecules for palmitic acid). Notably, two reactions of mitochondrial β-oxidation are catalyzed by different FAD- and NAD⁺-dependent dehydrogenases that have specific

<figure><img src="image_3.png" /></figure>

**Fig. 1.** Mitochondrial fatty acid oxidation and energy production: mechanism involved in drug-induced hepatic steatosis. The entry of long-chain fatty acids (LCFAs) into mitochondria requires a 4-step shuttle system. First, LCFAs are activated into LCFA-coenzyme A (Acyl-CoA) esters by an acyl-CoA synthetase (ACS) located in the outer mitochondrial membrane (OMM). Second, long-chain acyl-CoA esters are converted into acylcarnitines by carnitine palmitoyltransferase-1 (CPT1) also located in the OMM. Then, acylcarnitines are translocated across the inner mitochondrial membrane (IMM) into the mitochondrial matrix by carnitine-acylcarnitine translocase (CACT). Finally, carnitine palmitoyltransferase-2 (CPT2), located in the IMM, transfers the acyl moiety from carnitine back to CoA. In contrast, short and medium-chain fatty acids (SCFA and MCFA) freely enter mitochondria and are activated into acyl-CoA esters by another ACS. The acyl-CoA esters (Cn) of different lengths are then oxidized via the mitochondrial fatty acid oxidation (FAO) pathway into fatty acyl-CoA esters shortened by 2 carbons (Cn-2) and an acetyl-CoA molecule. Whereas the Cn-2 fatty acyl-CoA esters re-enter a new β-oxidation cycle (i.e. four steps), acetyl-CoA moieties can generate ketone bodies, which are released into the plasma and used by extra-hepatic tissues for energy production. Two types of FAO dehydrogenases use NAD⁺ and FAD, which are reduced to NADH and FADH₂, respectively. These reduced cofactors transfer their electrons (e⁻) to the mitochondrial respiratory chain (MRC), thus regenerating NAD⁺ and FAD used for other β-oxidation cycles. In the MRC, electrons are sequentially transferred to different polypeptide complexes (numbered from I to IV), which are embedded in the IMM. The final transfer of the electrons to oxygen occurs at the level of complex IV (cytochrome c oxidase). The flow of electrons within the MRC is coupled to the extrusion of FADH₂ and NADH-derived protons (H⁺) from the mitochondrial matrix to the intermembrane space, which creates the mitochondrial transmembrane potential, Δψm. When energy is needed, these protons re-enter the matrix through ATP synthase (complex V), thus liberating energy used to phosphorylate ADP into ATP. Mitochondrial DNA (mtDNA) encodes 13 polypeptides that are embedded within complexes I, III, IV and V. Prototypical drugs impairing mitochondrial FAO, MRC activity, or mtDNA homeostasis are indicated in red boxes.
activities for SCFAs, MCFAs, and LCFAs. For instance, there are 4 different FAD-dependent dehydrogenases including short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD), and very long-chain acyl-CoA dehydrogenase (VLCAD) [4,12]. Of note, LCAD and VLCAD have overlapping enzyme activities for LCFA dehydrogenation [13].

Mitochondrial FAO is regulated by different transcription factors including peroxisome proliferator-activated receptor α (PPARα), PPARβ/δ, forkhead box A1 (FoxA1) and FoxA2, cAMP-response element-binding protein (CREB), and hepatocyte nuclear factor-4α (HNF4α) [6,14]. AMP-activated protein kinase (AMPK) and several mitochondrial NAD⁺-dependent protein deacetylase sirtuins (SIRTs) also regulate mitochondrial FAO and other mitochondrial metabolic pathways [15,16]. Notably, some of these factors such as CREB, AMPK, and some SIRTs (e.g. SIRT1) are also involved in mitochondrial biogenesis, a complex program aimed at generating new and healthy mitochondria [17,18].

As previously mentioned, mitochondrial FAO and TCA cycle generate NADH and FADH₂, which are key for MRC-mediated energy production. Indeed, these reduced cofactors feed electrons and protons into the electron transport chain (ETC) to create a large electrochemical potential (Δψm) mandatory for ATP production (Fig. 1). In parallel, this process regenerates oxidized NAD⁺ and FAD which are required for other cycles of substrate oxidation. While the ETC is composed of 4 different multiprotein complexes (numbered from I to IV), ATP synthesis is catalyzed by only one complex, namely complex V (Fig. 1).

Thirteen polypeptides of the MRC are encoded by mitochondrial DNA (mtDNA) (Fig. 1), while the remaining polypeptides are encoded by nuclear DNA. mtDNA homeostasis is tightly regulated, in particular by several factors required for mtDNA replication, transcription, transcript maturation, translation, and repair [11,19]. Permanent mtDNA replication keeps a constant mtDNA copy number in hepatocytes despite continuous degradation of the most damaged and/or dysfunctional mitochondria [11]. It has been estimated that several hundreds to thousands of mtDNA copies exist in most mammalian somatic cells including hepatocytes [20]. This large reserve of mtDNA molecules makes it possible to cope with low or moderate losses of mtDNA copies without impairing MRC activity [11].

## 2.2. Mitochondrial substrate oxidation and production of reactive oxygen species

Mitochondria can generate large amounts of reactive oxygen species (ROS), which can be involved in cell signaling. For instance, ROS activate different transcription factors such as nuclear factor erythroid 2-like 2 (NFE2L2, also termed Nrf2), which contributes to the antioxidant response and mitochondrial biogenesis [11,21]. Although the ETC complexes I and III have long been thought to be the main sites of mitochondrial ROS production, recent investigations showed that large amounts of ROS can also be generated by the TCA cycle enzyme α-ketoglutarate dehydrogenase (αKGDH) [22]. Notably, this enzyme can produce ROS in mitochondria fueled with fatty acids of different chain lengths [22]. Interestingly, other investigations reported that ROS can be generated by some enzymes of the β-oxidation pathway such as VLCAD [11,23]. Hence, mitochondrial FAO appears to be a major metabolic pathway that can directly or indirectly produce ROS.

## 2.3. Other key features of mitochondria and mitochondrial function

Hepatic mitochondria have other important features which deserve to be briefly mentioned in this review.

1) There is ample evidence that the ketone bodies acetoacetate and β-hydroxybutyrate can have biological effects beyond energy production in extrahepatic tissues. Indeed, ketone bodies can exert

cellular effects through changes transduced by epigenetic, post-translational, and cell surface signaling mechanisms [24].

2) Mitochondria possess a non-selective channel, called mitochondrial permeability transition pore (mPTP), that forms in the inner mitochondrial membrane under some conditions such as elevated concentrations of calcium in the matrix and oxidative stress [25]. While transient mPTP opening can exert different physiological roles including at the level of cellular bioenergetics and ROS production, prolonged mPTP opening can lead to OXPHOS impairment, organelle swelling, mitochondrial membrane rupture, and cell death [25, 26].

3) Hepatic mitochondria contain cytochromes P450 such as CYP1A2 and CYP2E1, the latter metabolizing different types of exogenous and endogenous molecules including acetaminophen (APAP, also known as paracetamol), ethanol, and fatty acids [27].

4) In some pathophysiological situations, mitochondrial stress can increase the synthesis of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) [28], two mitokines whose serum levels can serve as biomarkers of mitochondrial dysfunction [29].

# 3. Impairment of mitochondrial function in drug-induced steatosis

Many studies have identified mitochondrial dysfunction as a primary mechanism of drug-induced steatosis (Fig. 2) [4,6,7,30,31], although other mechanisms may also be involved, as discussed later. By studying the ability of 45 pharmaceuticals to impair the FAO pathway in isolated mouse liver mitochondria, we recently showed that drug-induced inhibition of mitochondrial β-oxidation was able to predict the occurrence of steatosis in patients with a sensitivity and positive predictive value above 88% [32]. These investigations also confirmed that drugs can acutely impair mitochondrial FAO by directly inhibiting the β-oxidation pathway, or indirectly by impeding the MRC activity [32]. Of note, this study was not designed to detect drug-induced inhibition of β-oxidation and MRC via alteration of mtDNA homeostasis [32]. Prolonged exposure of hepatocytes to drugs would have been necessary for this purpose [33, 34].

In the context of drug-induced hepatotoxicity, the severity of mitochondrial dysfunction can vary depending on several factors, including those related to the drug (e.g. dose and duration of the treatment, drug-drug interactions...), or the patient itself (e.g. presence of underlying genetic mitochondrial diseases, metabolic disorders, alcohol abuse...) (Fig. 2) [6,35]. While severe mitochondrial dysfunction will eventually lead to microvesicular steatosis, slight or moderate impairment of the β-oxidation pathway can contribute to macrovacuolar steatosis (Fig. 2), as discussed in the following sections. This could explain why several mitochondriotoxic drugs can induce microvesicular steatosis in some individuals and macrovacuolar steatosis in others [6,35]. Noteworthy, recent investigations suggested that the fatty acid composition of the lipid droplets could be different between microvesicular and macrovacuolar steatosis [36,37]. Hence, mitochondrial dysfunction could have a major impact on fatty acid homeostasis beyond mitochondrial β-oxidation.

# 4. Mitochondrial dysfunction in drug-induced microvesicular steatosis

## 4.1. Microvesicular steatosis

Drug-induced microvesicular steatosis is a rare but potentially deadly liver disease that can be associated with liver failure, encephalopathy, multiorgan failure, and coma [4–6]. Liver histopathology classically shows the presence in the cytoplasm of several lipid droplets, which leave the nucleus at the center of the hepatocyte (Fig. 2) [38,39]. However, isolated microvesicular steatosis is rare and can be associated
<figure><img src="image_4.png" /><figcaption>**Fig. 2. Role of mitochondrial dysfunction in hepatic steatosis and steatohepatitis.** Drug-induced microvesicular or macrovacuolar steatosis depends on the degree of impairment of mitochondrial fatty acid oxidation (FAO). Severe and prolonged inhibition of mitochondrial FAO can induce microvesicular steatosis with several small lipid droplets (LD) leaving the nucleus (N) at the center of the hepatocyte. Moderate impairment of mitochondrial FAO can favor the occurrence of macrovacuolar steatosis, with a single large lipid vacuole (LV) displacing the nucleus at the periphery of the cell. Apart from situations of drug overdose, severe inhibition of mitochondrial FAO can be favored by genetic predispositions or non-genetic factors. In contrast to microvesicular steatosis which can be associated with hepatic failure, macrovacuolar steatosis is a benign liver lesion. However, in the long term, it can progress to steatohepatitis. Mitochondriotoxic drugs causing steatohepatitis commonly impair mitochondrial respiratory chain (MRC) activity. This causes an overproduction of mitochondrial reactive oxygen species (ROS), which favor lipid peroxidation (LPO) in a cellular environment enriched in fat. ROS and LPO are involved in the pathogenesis of cell death, inflammation, and fibrosis. Some mitochondriotoxic drugs causing obesity or lipoatrophy can also favor the occurrence of macrovacuolar steatosis via insulin resistance.</figcaption></figure>

with macrovacuolar steatosis [6,40]. Of note, the presence of small lipid droplets only reflects severe mitochondrial dysfunction in the injured hepatocytes and has no pathogenic role by itself [6]. Importantly, profound mitochondrial dysfunction in the liver is consistently associated with ultrastructural abnormalities of mitochondria [4,41]. Why lipids accumulate as small droplets in case of severe mitochondrial dysfunction is still unknown, although several hypotheses can be put forward. For instance, ATP deficiency linked to severe mitochondrial dysfunction might curb the growth of lipid droplets by reducing lipid synthesis or altering the expression of key proteins and enzymes wrapping the lipids [6]. In addition, the need for energy might promote triglyceride hydrolysis in the largest lipid droplets in an attempt to mobilize oxidable fatty acids [42,43]. A concomitant alteration of lysosomal function might also favor the accumulation of small lipid droplets [42].

In drug-induced microvesicular steatosis, biochemical abnormalities can include increased levels of serum transaminases (ALT and AST), thus reflecting hepatic cytolysis, severe hypoglycemia, hyperammonemia, hypoketonemia, and lactic acidosis [6,44]. Hypoglycemia and hypoketonemia could explain, at least in part, the occurrence of severe energy deficiency in extra-hepatic tissues, and hence multi-organ failure [4,6]. It should be noted that hypoketonemia could be deleterious beyond the energy deficit since ketone bodies can have epigenetic and post-translational effects [24], as already mentioned. Although not routinely measured, acylcarnitine derivatives and dicarboxylic acids can be detected in plasma and urine, thus reflecting severe impairment of mitochondrial function [6,45]. Importantly, microvesicular steatosis associated with liver failure can also be observed in other hepatic diseases linked to severe mitochondrial dysfunction such as acute fatty liver of pregnancy, some inborn errors of mitochondrial FAO, and several mitochondrial cytopathies (i.e. genetic disorders of the OXPHOS system) [4,6,46,47]. Because patients with inborn mitochondrial hepatopathies can present elevated serum levels of GDF15 and FGF21 [29], further investigations would be necessary to determine whether these mitokines are also elevated in drug-induced microvesicular steatosis. Interestingly,

previous investigations showed that the mRNA expression of GDF15 and FGF21 was rapidly increased in vitro (human and rat primary hepatocytes) and in vivo (rats) in the context of drug-induced hepatotoxicity [48]. Finally, microvesicular steatosis with liver failure can also be observed in Reye's syndrome, which is triggered by an acute viral illness (mainly influenza and varicella) and treatment with the nonsteroidal anti-inflammatory drug (NSAID) aspirin (or herbal tea containing salicylate) and possibly other medications such as the antiepileptic agent valproic acid [4,41].

## 4.2. Drugs inducing microvesicular steatosis

Table 1 gives examples of mitochondriotoxic drugs that can induce microvesicular steatosis in treated patients. Among these drugs, the antiviral fialuridine was stopped during a phase II trial, while the antidepressant amineptine, the antianginal perhexiline, and the antidiabetic troglitazone were withdrawn after marketing [6,35]. It should be noted that the analgesic/antipyretic APAP, the anesthetic isoflurane, the antibiotic linezolid, and the vitamin niacin induced microvesicular steatosis in only a few patients [6]. Therefore, additional studies are needed to confirm the intrinsic ability of these drugs to cause this liver lesion and to explore the potential involvement of other pathogenic factors, such as underlying mitochondrial genetic disorders. Information regarding other drugs inducing microvesicular steatosis can be found in previous reviews [4,6,47].

## 4.3. Mitochondrial mechanisms of drug-induced microvesicular steatosis

Drugs can induce microvesicular steatosis by different mitochondrial mechanisms (Fig. 1, Table 1). Notably, all these mechanisms can eventually lead to severe inhibition of the mitochondrial β-oxidation pathway (Fig. 2). From the available published data [4,6,7,30,31,49,50], the following mechanisms are involved in most cases of microvesicular steatosis:
<table><caption>**Table 1**
Examples of mitochondrialotoxic drugs inducing steatosis and steatohepatitis.</caption><thead><tr><th>Drug</th><th>Therapeutic class</th><th>Type of steatosis</th><th>Steatohepatitis</th><th>Possible mechanism(s) of mitochondrial dysfunction<sup>1</sup></th></tr></thead><tbody><tr><td>Acetaminophen (APAP)</td><td>Analgesic, antipyretic</td><td>Microvesicular</td><td>No</td><td>Inhibition of MRC<sup>2</sup> activity, oxidative degradation of mtDNA<sup>2</sup></td></tr><tr><td>Amiodarone</td><td>Antianginal, antiarrhythmic</td><td>Microvesicular, macrovacuolar</td><td>Yes</td><td>Inhibition of mitochondrial FAO<sup>2</sup> enzymes, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Aspirin and other salicylates</td><td>NSAID<sup>2</sup></td><td>Microvesicular</td><td>No</td><td>Sequestration of coenzyme A and l-carnitine, impairment of OXPHOS<sup>2</sup></td></tr><tr><td>Carbamazepine</td><td>Antiepileptic</td><td>Macrovacuolar</td><td>Yes</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Didanosine</td><td>Antiretroviral (anti-HIV<sup>2</sup>)</td><td>Microvesicular, macrovacuolar</td><td>Yes</td><td>Inhibition of mtDNA<sup>2</sup> replication</td></tr><tr><td>Estrogens</td><td>Estrogenic hormones</td><td>Microvesicular, macrovacuolar</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup></td></tr><tr><td>Ibuprofen</td><td>NSAID<sup>2</sup></td><td>Microvesicular</td><td>No</td><td>Sequestration of coenzyme A, inhibition of mitochondrial FAO<sup>2</sup> enzymes</td></tr><tr><td>Indinavir</td><td>Antiretroviral (anti-HIV<sup>2</sup>)</td><td>Microvesicular</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup></td></tr><tr><td>Indomethacin</td><td>NSAID<sup>2</sup></td><td>Microvesicular</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Linezolid</td><td>Antibiotic</td><td>Microvesicular</td><td>No</td><td>Inhibition of mtDNA<sup>2</sup> translation</td></tr><tr><td>Methotrexate</td><td>Antipsoriatic, anti-rheumatoid</td><td>Macrovacuolar</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Nifedipine</td><td>Antianginal, antihypertensive</td><td>Macrovacuolar</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Olanzapine</td><td>Antipsychotic</td><td>Macrovacuolar</td><td>Yes</td><td>Inhibition of mitochondrial FAO<sup>2</sup></td></tr><tr><td>Perhexiline</td><td>Antianginal</td><td>Microvesicular, macrovacuolar</td><td>Yes</td><td>Inhibition of mitochondrial FAO<sup>2</sup> enzymes, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Rifampicin</td><td>Antibiotic</td><td>Macrovacuolar</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity</td></tr><tr><td>Tamoxifen</td><td>SERM<sup>2</sup>, antineoplastic</td><td>Macrovacuolar</td><td>Yes</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity, inhibition of mtDNA<sup>2</sup> synthesis</td></tr><tr><td>Tetracycline</td><td>Antibiotic</td><td>Microvesicular</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of MRC<sup>2</sup> activity, inhibition of mtDNA<sup>2</sup> translation</td></tr><tr><td>Tianepine</td><td>Antidepressant</td><td>Microvesicular</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup></td></tr><tr><td>Troglitazone</td><td>Antidiabetic</td><td>Microvesicular</td><td>No</td><td>Inhibition of MRC<sup>2</sup> activity, oxidative degradation of mtDNA<sup>2</sup></td></tr><tr><td>Valproic acid</td><td>Antiepileptic</td><td>Microvesicular, macrovacuolar</td><td>Yes</td><td>Sequestration of coenzyme A and l-carnitine, inhibition of mitochondrial FAO<sup>2</sup> enzymes</td></tr><tr><td>Zidovudine</td><td>Antiretroviral (anti-HIV<sup>2</sup>)</td><td>Microvesicular</td><td>No</td><td>Inhibition of mitochondrial FAO<sup>2</sup>, inhibition of mtDNA<sup>2</sup> replication</td></tr></tbody></table>

1) Direct inhibition of one or several mitochondrial FAO enzymes including CPT1 and acyl-CoA dehydrogenases.

2) Sequestration of coenzyme A and l-carnitine, two cofactors essential for the mitochondrial β-oxidation pathway.

3) Inhibition of the activity of one or several MRC complexes, which limits the oxidation of NADH and FADH₂ into NAD⁺ and FAD, to cofactors mandatory for the proper functioning of the mitochondrial FAO dehydrogenases.

4) Impairment of mtDNA homeostasis including replication, transcription, and translation. In case of inhibition of mtDNA replication, which can lead to mtDNA depletion, the number of mtDNA copies must fall below 20–40 % of basal levels to induce impairment of MRC and mitochondrial FAO.

Noteworthy, these mechanisms are not mutually exclusive and the same drug can therefore alter the mitochondrial β-oxidation pathway via different mechanisms. This might depend on the drug concentration (or dose) and the duration of the treatment. For instance, low concentrations of the antianginal and antiarrhythmic drug amiodarone can directly inhibit mitochondrial FAO while higher concentrations impair the MRC activity, thus reinforcing the inhibition of this metabolic pathway [32,51]. The anti-HIV drugs zidovudine and stavudine can rapidly impair mitochondrial FAO in the absence of reduced mtDNA copy number [32,52] while longer exposure induces mtDNA depletion, which will eventually impair the MRC activity and further worsen the inhibition of β-oxidation [4,11].

Noteworthy, microvesicular steatosis can sometimes occur in the context of severe oxidative stress secondary to ROS overproduction. For instance, this liver lesion was reported with the potent free radical initiator 2,2'-azobis-(2-amidinopropane) dihydrochloride [4] or in situations of genetically- or drug-induced GSH depletion [53,54]. The exact mechanism(s) whereby oxidative stress can impair mitochondrial

FAO is still unclear albeit recent investigations pointed to a role of reduced PPARα and FOXA1 expression [54,55]. ROS overproduction also induces damage to mtDNA and other key mitochondrial components including proteins and lipids involved in MRC activity [4,11,56, 57]. Finally, it should be mentioned that different underlying genetic disorders of mitochondria can favor drug-induced microvesicular steatosis. For instance, polymorphisms (or mutations) in genes encoding MCAD, DNA polymerase γ (POLG), and manganese superoxide dismutase (SOD2) can promote drug-induced severe mitochondrial dysfunction, microvesicular steatosis, and other forms of severe liver injury [6, 35,58].

### 4.4. Auto-amplification of mitochondrial dysfunction in drug-induced microvesicular steatosis

Inhibition of mitochondrial β-oxidation can further worsen mitochondrial dysfunction via different mechanisms, thus inducing a vicious circle (Fig. 3). For instance, the accumulation of long-chain fatty acids and some of their metabolites (e.g. acyl-CoA esters and dicarboxylic acids) can impair the OXPHOS process [4] and trigger mPTP opening, or other forms of mitochondrial membrane permeabilization [26,59]. Accumulation of lipids with concomitant ROS overproduction can also promote lipid peroxidation, whose products (e.g. malondialdehyde and 4-hydroxynonenal) have many deleterious effects on cellular and mitochondrial function [4,26,60]. Noteworthy, fatty acids and lipid peroxidation can favor ROS overproduction by inducing generalized mitochondrial dysfunction [60–62], further amplifying the aforementioned vicious circle (Fig. 3). All these effects can induce necrosis, ferroptosis or apoptosis of the most dysfunctional hepatocytes, thus favoring hepatic cytolysis and eventually liver failure [4,26].

<footnote n="1">¹ Information in the column comes primarily from several articles cited in this review [4–7,11,30–32,49–51,90,159].</footnote>

<footnote n="2">² Abbreviations: FAO, fatty acid oxidation; HIV, human immunodeficiency virus; MRC, mitochondrial respiratory chain; mtDNA, mitochondrial DNA; NSAID, nonsteroidal anti-inflammatory drug; OXPHOS, oxidative phosphorylation; SERM, selective estrogen receptor modulator.</footnote>
<figure><img src="image_5.png" /><figcaption>Figure 3</figcaption></figure>

**Fig. 3.** Mechanisms of auto-amplification of mitochondrial dysfunction in drug-induced steatosis. Drug-induced mitochondrial dysfunction can undergo an auto-amplification loop by several mechanisms. Inhibition of mitochondrial fatty acid oxidation (FAO) eventually leads to the accumulation of free fatty acids that are toxic to mitochondria, for instance by favoring mitochondrial membrane permeabilization (MMP) and impairment of oxidative phosphorylation (OXPHOS). Drug-induced inhibition of mitochondrial respiratory chain (MRC) activity favors the overproduction of mitochondrial reactive oxygen species (ROS), which can cause MMP and oxidatively damage different mitochondrial components including the mitochondrial genome (not shown) and MRC polypeptides. ROS can also favor lipid peroxidation, whose products (e.g. malondialdehyde and 4-hydroxynonenal) can induce mitochondrial dysfunction and damage. Finally, ROS and subsequent oxidative stress can reduce the activity of the transcription factor peroxisome proliferator-activated receptor α (PPARα), which plays a key role in regulating mitochondrial FAO.

# 5. Mitochondrial dysfunction in drug-induced macrovacuolar steatosis and steatohepatitis

## 5.1. Macrovacuolar steatosis and steatohepatitis

In macrovacuolar steatosis, liver triglycerides commonly accumulate as a single large lipid vacuole relocating the nucleus at the periphery of the hepatocyte [38,39]. In addition to drug-induced cases, this lesion is commonly observed in individuals with obesity [10,39], those consuming excessive amounts of alcohol [6,39], and patients with various congenital disorders, such as Wilson's disease and familial hypobetalipoproteinemia [63]. Macrovacuolar steatosis can be asymptomatic, often presenting only with hepatomegaly. However, in some cases, it may progress over time to steatohepatitis, characterized by necroinflammation, ballooning degeneration of hepatocytes, and fibrosis [38,39]. At this stage, a mild to moderate increase in plasma transaminases (ALT and AST) can be observed, thus reflecting hepatic cytolysis [6,64]. Finally, whatever its etiology steatohepatitis can evolve into cirrhosis, which can lead to liver failure in the most severe cases [6, 64]. Noteworthy, fibrosis progression to cirrhosis can occur over weeks or months in drug-induced steatohepatitis, in contrast to obesity and other metabolic diseases where the evolution may take decades [64].

## 5.2. Drugs inducing macrovacuolar steatosis and steatohepatitis

Examples of drugs causing macrovacuolar steatosis are presented in Table 1. It is noteworthy that several drugs can induce macrovacuolar steatosis in some patients and microvesicular steatosis in others [4,6, 65]. In the latter group of patients, underlying genetic disorders of mitochondria could favor the occurrence of severe mitochondrial

dysfunction [6,35,58], as already mentioned. Progression of macro-vacuolar steatosis to steatohepatitis and eventually cirrhosis can occur with different pharmaceuticals such as amiodarone, didanosine, and perhexiline (Table 1) [6,40,64,66]. Although recent clinical studies question whether the antipsoriatic and anti-rheumatoid drug metho-trexate can induce steatohepatitis and fibrosis, this drug may worsen MASLD in some patients [67]. Information regarding other pharma-ceuticals inducing macrovacuolar steatosis and steatohepatitis can be found in previous reviews [4,6,47,64,66].

## 5.3. Mitochondrial involvement in drug-induced macrovacuolar steatosis and steatohepatitis

For some drugs such as amiodarone, perhexiline, rifampicin (anti-biotic), and stavudine, macrovacuolar steatosis might be associated with mild to moderate impairment of mitochondrial FAO (Fig. 2) [6,68,69]. Of note, recent investigations using public toxicogenomic data sug-gested that drug-induced steatosis could be more related to mitochon-drial dysfunction than activation of nuclear receptors involved in lipid homeostasis [70]. However, non-mitochondrial mechanisms are also involved in drug-induced macrovacuolar steatosis, as discussed in the next section.

Interestingly, drug-induced mitochondrial dysfunction seems to be also involved in the progression of macrovacuolar steatosis to steato-hepatitis. Indeed, drugs inducing steatohepatitis often impair MRC ac-tivity, which favors ROS overgeneration and lipid peroxidation in a cellular environment enriched in fat (Fig. 2) [6,7,71]. However, lipid peroxidation may also be triggered by mitochondrial ROS from sources other than MRC such as FAO and TCA cycle enzymes [22,23]. Mito-chondrial ROS and lipid peroxidation products then promote hepatocyte cell death, liver inflammation, and fibrosis by activating Kupffer and stellate cells, respectively (Fig. 2) [6,7,62,72,73]. Notably, oxidative stress can alter the activity of PPARα and more generally mitochondrial biogenesis [10,54,55], further impairing mitochondrial dysfunction (Fig. 3). Genetic predispositions that affect mitochondrial function may also contribute to the pathogenesis of drug-induced steatohepatitis; however, data on this important issue are currently lacking.

Recently, we measured the ability of 32 steatogenic and 13 non-steatogenic drugs to induce ROS overproduction in isolated mouse liver mitochondria energized with succinate [32]. However, no relationship was found between mitochondrial ROS production in vitro and the occurrence of steatosis in treated patients according to the literature and other data sources [32]. Still, further investigations should be performed with other energetic substrates, in particular fatty acids since the mitochondrial oxidation of these compounds can be an important source of ROS [11,22,23], as previously mentioned. In addition, it should be underlined that this study was not designed to investigate drug-induced steatohepatitis [32]. Additional drugs able to induce this type of liver injury should be investigated to determine whether mitochondrial ROS could predict its occurrence.

# 6. Non-mitochondrial mechanisms in drug-induced steatosis

## 6.1. Non-mitochondrial mechanisms in drug-induced microvesicular steatosis

Although there is consistent evidence that drug-induced micro-vesicular steatosis is secondary to severe impairment of mitochondrial β-oxidation, other mechanism(s) cannot be excluded. For instance, studies point to the role of lysosomes which are important organelles for recycling intracellular materials like lipids, proteins, or damaged organelles (autophagy). First, lysosomal dysfunction could favor the ac-cretion of small lipid droplets observed in microvesicular steatosis [42]. Secondly, an altered lysosomal function can impair the autophagic degradation of the most damaged mitochondria (i.e. mitophagy), thus further aggravating mitochondrial dysfunction and favoring hepatocyte
cell death [74]. In addition, impaired lysosomal degradation of phos-
pholipids can induce phospholipidosis, which can be observed with
several drugs inducing microvesicular steatosis such as amiodarone and
perhexiline [4,75]. Hepatocytes affected by phospholipidosis are char-
acterized by numerous and large lysosomes, containing lamellar bodies
visible by electron microscopy [4,75]. It should be noted that this lesion
itself appears to have limited clinical consequences [4,75]. Finally, as
already discussed, drug-induced severe oxidative stress can secondarily
induce mitochondrial dysfunction and microvesicular steatosis [4,54].

## 6.2. Non-mitochondrial mechanisms in drug-induced macrovacuolar steatosis

Although macrovacuolar steatosis is the most prevalent form of drug-
induced steatosis, it is only recently that the involvement of different
non-mitochondrial mechanisms has been shown. These mechanisms
include increased fatty acid transport into the hepatocytes, stimulation
of fatty acid synthesis from carbohydrates (i.e. de novo lipogenesis), and
impaired very-low-density lipoprotein (VLDL) secretion [6,68,76].
VLDL particles are made of neutral lipids, mostly triglycerides and
different apolipoproteins including apolipoproteins B100 (apoB100)
and apoE. While the first two mechanisms can lead to excessive free
LCFAs available for triglyceride synthesis, alteration of VLDL secretion
directly causes triglyceride accumulation. Notably, these mechanisms
are not mutually exclusive and can be associated with mild to moderate
impairment of mitochondrial β-oxidation. For instance, in the absence of
severe mitochondrial dysfunction, fat accumulation induced by amio-
darone could involve moderate inhibition of mitochondrial FAO but also
increased fatty acid uptake by the hepatocytes and activation of de novo
lipogenesis [68-70,77].

Drug-induced stimulation of *de novo* lipogenesis could involve different molecular mechanisms including activation of lipogenic transcription factors such as sterol regulatory element binding protein 1c (SREBP1c) and PPARγ [6,30,75]. Regarding drug-induced impairment of VLDL secretion, our recent investigations highlighted a frequent involvement of endoplasmic reticulum (ER) stress leading to a dramatically decreased expression of several structural proteins and enzymes involved in VLDL formation such as apolipoproteins B and C3, angiopoietin-like 3 (ANGPTL3), and microsomal triglyceride transfer protein (MTTP) [68]. Interestingly, previous investigations showed that MTTP activity could be inhibited by different drugs causing steatosis [78]. Lastly, some medications such as valproic acid could favor hepatic lipid accumulation via oxidative stress, which promotes the uptake of fatty acids and their esterification into triglycerides [79,80]. Oxidative stress could also promote steatosis by impairing triglyceride lipolysis [81,82].

6.3. Involvement of adipose tissue in drug-induced steatosis

Drugs can also promote steatosis by inducing obesity or lipoatrophy
(Fig. 2) [6,30], although it is not yet clear whether they do so system-
atically by altering mitochondrial function. For instance, by inducing
obesity valproic acid and several atypical antipsychotics (e.g. olanza-
pine, risperidone) favor insulin resistance and hyperinsulinemia, thus
stimulating hepatic de novo lipogenesis [30,83,84]. Drug-induced lip-
oatrophy can also cause insulin resistance and steatosis (Fig. 2) [6,7].
Recent investigations reported that amiodarone can induce triglyceride
lipolysis in white adipose tissue, thus leading to an increased influx of
free fatty acids to the liver [85]. Interestingly, adipose tissue lipolysis is
increasingly recognized as an important mechanism of alcohol-induced
fatty liver [86,87].

7. Drug-induced mitochondrial dysfunction and MASLD

MASLD is the most common cause of chronic liver disease due to the
global epidemic of obesity. MASLD is associated with mitochondrial

dysfunction, with a degree of severity depending on the stage of the
disease [10,88]. In the United States and China, it is estimated that 10
and 23 % of the patients with DILI, respectively, have underlying liver
disease including MASLD [89]. Because many drugs can impair mito-
chondrial function [32,90-92], they can potentially induce acute liver
injury more frequently in MASLD patients, or worsen the liver disease
[10,93-95]. For instance, mitochondrial dysfunction could be involved
in the aggravation of MASLD observed or suspected in patients treated
with stavudine and didanosine, methotrexate, and the antidiabetic agent
rosiglitazone [10,93,94]. In line with this assumption, MASLD aggra-
vation was reported in obese mice treated with rosiglitazone [96],
tetracycline [97], or olanzapine [98], three drugs reported to impair
mitochondrial function [4,32,99]. Hence, the risk of MASLD worsening
by drugs must be fully considered by clinicians since obesity and related
metabolic disorders increase the odds of polypharmacy [100]. In
particular, a complete liver biochemistry analysis should be performed
before the initiation of therapy with potentially hepatotoxic drugs [89].
Finally, it is noteworthy that mitochondrial dysfunction and damage
appear to play a key role in MASLD aggravation induced by ethanol
abuse [10,99,101,102].

8. Therapies for the prevention and treatment of drug-induced mitochondrial dysfunction and steatosis

Treatment of DILI is a major challenge for clinicians, especially in the context of severe liver injury [89,103]. Discontinuation of the suspected drugs is the most important measure in DILI and standard medical care is required for severe cases. Different medications with antioxidant and hepatoprotective effects have been proposed to treat drug-induced acute liver failure [89,103]. Still, only a few of them are clinically effective [89,103]. In addition, their efficiency in DILI patients with severe microvesicular steatosis has been poorly explored. Different treatments have also been investigated for the prevention of DILI [103,104], but little evidence has been reported regarding drug-induced steatosis. Overall, only L-carnitine and N-acetylcysteine have been consistently explored in liver steatosis and injury induced by drugs, or other causes.

8.1. *L*-carbamate

L-carnitine is a key cofactor mandatory for mitochondrial FAO of long-chain fatty acids, as previously mentioned (Fig. 1) [4,12]. Notably, this micronutrient has antioxidant, antiapoptotic, anti-inflammatory, and anti-oncogenic properties, which do not appear to be directly related to its role in the transport of long-chain fatty acids in mitochondria [105-107].

Several experimental and clinical investigations reported the po-
tential usefulness of L-carnitine supplementation to prevent valproic
acid-induced liver injury and metabolic disturbances such as L-carnitine
deficiency and hyperammonemia [4,108]. A retrospective study also
showed that L-carnitine therapy resulted in better survival amongst pa-
tients who experienced acute severe valproate-induced hepatotoxicity
[109]. However, prospective clinical investigations would be needed to
confirm that L-carnitine treatment can efficiently prevent fatal hepatic
failure in patients with already established valproic acid-induced
hepatotoxicity.

L-carnitine supplementation may also be beneficial in preventing
mitochondrial dysfunction and steatosis induced by drugs other than
valproic acid. For instance, investigations carried out in vitro (rat he-
patocytes) and in vivo (mice) showed that this cofactor alleviated
stavudine-induced impairment of mitochondrial FAO and lipid accu-
mulation in hepatocytes [52,110]. Interestingly, L-carnitine supple-
mentation was shown to reduce liver fat accumulation in rodents
intoxicated with ethanol, possibly through an improvement of mito-
chondrial function [111].

Beyond its effects on steatosis, experimental and clinical studies have
shown that L-carnitine can prevent hepatic cytolysis induced by several
drugs, including acetaminophen (APAP) [112], antituberculosis medications [113], and the antihistamine drugs cetirizine and chlorpheniramine [114]. A recent meta-analysis of randomized controlled trials (RCT) reported that L-carnitine supplementation can significantly reduce serum ALT, AST, and γ-glutamyl transpeptidase (GGT) levels in patients with different types of liver diseases including metabolic dysfunction-associated steatohepatitis (MASH, formerly named nonalcoholic steatohepatitis or NASH) and chronic hepatitis C [115]. Interestingly, RCT meta-analyses focused on MASLD reported that L-carnitine supplementation could improve liver function [116,117]. Because the safety profile of L-carnitine is excellent [108,117], the use of this mitochondrial cofactor is promising for the prevention of drug-induced steatosis and other liver pathologies characterized by fatty liver and hepatic cytolysis.

## 8.2. N-Acetylcysteine

N-acetylcysteine (NAC) is currently the only FDA-approved therapy for the treatment of APAP-induced acute liver failure [89]. Indeed, NAC administration after APAP overdose allows rapid restoration of intrahepatic levels of the endogenous antioxidant glutathione (GSH), thereby avoiding severe oxidative stress, mitochondrial dysfunction, and massive necrosis of the liver parenchyma [95]. Although APAP-induced microvesicular steatosis, yet rarely, has been reported [118], it is unknown whether NAC can prevent the occurrence of this liver lesion. Notably, NAC has proved clinically beneficial in managing non-APAP-induced acute liver failure [89,119]. However, there is no data regarding steatosis in this specific pathophysiological context.

Interestingly, several investigations in preclinical models of MASLD have shown that NAC supplementation can alleviate necroinflammation as well as the degree of liver fat deposition [120]. In MASLD and other causes of steatosis, NAC appears to reduce hepatic lipid accumulation by several mechanisms including favoring triglyceride lipolysis, reducing de novo lipogenesis, and alleviating mitochondrial dysfunction [81,120]. Altogether, these data call for future investigations to evaluate the efficiency of NAC in the treatment of drug-induced steatosis.

The safety profile of NAC is overall good although serious adverse events can occasionally occur such as allergic and anaphylactic reactions [54,121]. Rare cases of acute hepatic cytolysis and steatosis have also been reported in treated patients [2]. Of note, NAC overdose in normal mice was shown to induce acute hepatotoxicity with microvesicular steatosis [54]. Mechanistic investigations suggested severe hepatic oxidative stress, possibly secondary to reduced GSH synthesis and increased expression of CYP2E1 [54]. Interestingly, other experimental investigations showed that increased CYP2E1 and the resulting overproduction of ROS were key mechanisms in valproate-induced hepatic steatosis [79]. The recent data reported with NAC [54] epitomizes the fact that high doses of some antioxidants can have pro-oxidant effects inducing adverse events [122,123].

## 8.3. Emerging therapies

In addition to L-carnitine and NAC, other therapies may hold promise in alleviating mitochondrial dysfunction and lipid accumulation in the liver. However, their effectiveness in drug-induced hepatic steatosis has yet to be established. For example, it would be interesting to determine the efficacy of drugs with favorable effects in genetic diseases affecting mitochondrial function, such as quinone and ubiquinone derivatives (e. g. idebenone and β-tocopherolquinone) and the pan-PPAR agonist bezafibrate [124-126]. Another avenue could be using certain drugs developed to treat MASLD that have favorable effects on mitochondrial function and lipid homeostasis. For instance, thyromimetics such as resmetirom might be of interest in improving mitochondrial FAO [10]. Notably, this β-selective agonist of thyroid hormone receptor (THR) is the first drug approved by the Food and Drug Administration to treat MASH [127]. Activation of THR-β in the liver promotes FAO,

mitochondrial biogenesis, and mitophagy [127,128]. Analogs of FGF21 might also be interesting since this mitokine stimulates hepatic FAO by inducing peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α) and reduces lipogenesis by repressing SREBP1c [129]. Although thyromimetics and FGF21 analogs appear promising medications for MASLD, future investigations will be needed to determine whether they can also prevent or treat drug-induced mitochondrial dysfunction and hepatic steatosis.

# 9. Other important considerations for drug-induced steatosis and other liver lesions

## 9.1. Experimental versus clinical evidence for the role of mitochondrial dysfunction in drug-induced steatosis

It should be acknowledged here that most evidence for the role of mitochondrial dysfunction in the pathophysiology of drug-induced steatosis is primarily based on experimental studies in rodents, cultured hepatocytes, or isolated liver mitochondria [32,33,51,52,68, 75,76,78,110]. Moreover, because some of these studies were performed using high doses or concentrations of drugs, their translational value is limited. Nonetheless, some clinical investigations have highlighted the significant role of mitochondrial dysfunction in drug-induced steatosis and liver injury. For instance, microvesicular steatosis induced by valproic acid and aspirin has been associated with the detection in the liver, blood, or urine of several biomarkers of severe mitochondrial injury such as acylcarnitines and dicarboxylic acids [130,131]. Furthermore, serum or urine mitochondrial biomarkers can be found in cases of liver injury induced by APAP [132,133], a drug reported to induce microvesicular steatosis in some patients (Table 1) [118,134]. Despite these investigations on a few drugs, further clinical research is needed to firmly establish a causal relationship between drug-induced steatosis and mitochondrial dysfunction. In addition to the biomarkers mentioned above, other methodological approaches can be used to assess mitochondrial functionality in patients [10,135].

## 9.2. Role of non-mitochondrial organelles in drug-induced steatosis

Although drug-induced dysfunction of hepatic mitochondria can cause steatosis, alteration of other organelles such as lysosomes and the ER could also contribute to the pathophysiology of this lesion. For instance, it has been reported that an impairment of the autophagy-lysosome pathway could contribute to valproic acid-induced triglyceride accumulation in hepatocytes [136]. Interestingly, there is increasing evidence that lysosomal instability and dysfunction are involved in APAP-induced liver injury [137,138]. Hence, lysosomal alterations should not be overlooked when investigating drug-induced hepatotoxicity, especially steatosis [136], hepatic cytolysis [137], and phospholipidosis [4,75], as previously mentioned. The ER is another organelle deserving of attention when investigating steatosis because it plays a key role in lipid homeostasis. Some studies have shown that ER stress induced by xenobiotics including drugs can participate in hepatic steatosis and other types of liver injury [68,139,140]. Lastly, it would also be relevant to assess the effects of drugs on lipid droplets, which are organelles covered by various structural proteins and enzymes involved in lipid synthesis and hydrolysis [141]. It can be predicted that drug-induced alteration of some of these components could contribute to the accumulation of hepatic triglycerides [141,142].

## 9.3. Adaptive mechanisms in response to drug-induced mitochondrial dysfunction

Since mitochondrial dysfunction in hepatocytes can impair carbohydrate, lipid, and energy homeostasis, various adaptive mechanisms may arise to mitigate these effects. For instance, treatment of hepatocytes with sub-cytotoxic concentrations of amiodarone was associated
with increased mtDNA content, PGC1α mRNA expression, and phospho-AMPK protein levels, possibly reflecting enhanced mitochondrial biogenesis [34]. Interestingly, investigations in mice showed that mitochondrial biogenesis is important for recovery after APAP-induced liver injury [143,144]. Conversely, mitophagy (i.e. autophagy of the most damaged mitochondria) appears to be a key adaptive mechanism limiting hepatocellular damage in APAP hepatotoxicity [144]. Lastly, non-mitochondrial mechanisms could also participate in the metabolic adaptations during drug-induced mitochondrial dysfunction. For example, lower mitochondrial FAO and steatosis induced by 150 µM rifampicin were accompanied in treated hepatocytes by reduced *de novo* lipogenesis and increased VLDL secretion [68].

## 9.4. Cell death in drug-induced steatosis and steatohepatitis

Steatotic hepatocytes can undergo several types of damage when adaptive mechanisms are overwhelmed. More specifically, drug-induced steatosis can be associated with the death of a varying proportion of hepatocytes, which depends on the degree of mitochondrial dysfunction and other cellular alterations. Liver histology in patients with drug-induced steatosis can reveal hepatocellular necrosis [4,145,146] and in some occasions, apoptotic hepatocytes [44]. By creating a pro-oxidant state that promotes peroxidation of accumulated LCFAs, steatosis could foster ferroptosis (Fig. 2), a form of iron-dependent regulated cell death [147]. Importantly, ferroptosis was shown to play a significant role in the progression of steatosis to steatohepatitis in different murine models of diet-induced MASH [148–150]. However, to our knowledge, the role of ferroptosis in drug-induced steatohepatitis has not been studied.

## 9.5. Drug-induced mitochondrial dysfunction and lipid accumulation in non-liver tissues

Drug-induced mitochondrial dysfunction can cause lipid accumulation in the liver and other tissues, especially skeletal muscle. For instance, lipid droplets in the quadriceps muscle were reported in a patient treated with zidovudine and presenting myopathy, massive liver steatosis, and lactic acidosis [146]. Small lipid droplets and ultrastructural abnormalities of mitochondria were reported in the skeletal muscle of seven children treated with valproic acid [151]. In a patient with linezolid-induced hepatic steatosis, lactic acidosis, optic neuropathy, and myopathy, electron microscopy of a muscle biopsy sample revealed abnormal mitochondria and granules of lipofuscin, which are composed of lipids and oxidized proteins [152].

## 9.6. Gender difference in DILI and drug-induced mitochondrial dysfunction

To our knowledge, the issue of gender difference in drug-induced steatosis has not yet been addressed. Considering all DILI cases, current data suggest that women may not be more susceptible to DILI than men [153]. Nonetheless, gender susceptibility to DILI appears to depend on specific drugs. For instance, female dominance was observed among herb-related cases whereas male dominance was reported in amoxicillin/clavulanate-induced liver injury [153]. Interestingly, a data-mining analysis of drug-induced liver events revealed that mitochondrial dysfunction was a prominent feature of a group of drugs with female-biased reporting frequency [154]. Regarding the mitochondrial toxicant APAP [74,95], clinical data indicate that women may not have an increased risk of developing APAP-induced hepatotoxicity although they predominate in APAP-induced acute liver failure [153]. However, experimental investigations showed that APAP induced more severe liver injury in male mice [153,155]. These discrepancies highlight the need for further clinical investigations to determine the mechanisms of susceptibility to DILI, particularly regarding mitochondrial dysfunction.

## 9.7. High prevalence of fatty liver in the general population and diagnosis of DILI

Because of the worldwide obesity epidemic, the global prevalence of MASLD in the adult general population is around 30% [156]. Consistent with these data, a recent study from the United Kingdom reported high rates (ca. 43%) of undiagnosed fatty liver disease in a population of patients undergoing colonoscopy [157]. Hence, the high prevalence of MASLD in the general population might be a major confounding factor for the diagnosis of drug-induced macrovacuolar steatosis and steatohepatitis. In this regard, it is noteworthy that methotrexate was mistakenly thought to induce steatohepatitis and fibrosis before 2000 because MASH was unknown at that time [67]. Notably, recent expert consensus guidelines called for best practices to enable the detection of suspected DILI in patients with obesity-induced steatohepatitis [158].

# 10. Conclusion and perspectives

This article has reviewed the current knowledge on the role of mitochondrial dysfunction in drug-induced hepatic steatosis and steatohepatitis. Many experimental studies have revealed the primary role of impaired FAO, MRC activity, and mtDNA homeostasis (Table 1) [4–7, 11,30–32,49–51,90,159]. However, other mechanisms should be better studied such as mitophagy [18,74,144] and mitochondrial biogenesis [17,18,143]. We acknowledge that further investigations are needed to confirm the impairment of mitochondrial function in patients, particularly those with microvesicular steatosis, or steatohepatitis. For this purpose, hepatic mitochondrial function can be studied using the same methods as those used for MASLD [10,135].

The selection of the safest compounds during preclinical studies does not exclude the occurrence of severe drug-induced mitochondrial dysfunction and liver injury in some patients. Many reasons could explain such misadventures including drug-drug interactions, underlying genetic disorders of mitochondria, gender differences, and preexisting liver diseases [6,30,35,50,154,160]. In particular, the administration of potential mitochondriotoxic drugs should be carefully monitored in patients with chronic liver diseases such as alcoholic liver disease and MASLD. This may help reduce the risk of various hepatic adverse events, including acute liver injury, worsening of fatty liver, and the rapid progression of this lesion to steatohepatitis and cirrhosis [10, 94,161]. L-carnitine and NAC supplementation in these patients could also be envisaged to prevent or curb drug-induced mitochondrial dysfunction and liver injury.

Because drug-induced mitochondrial dysfunction can lead to potentially serious liver diseases, a major challenge for the pharmaceutical industry is to efficiently detect mitochondriotoxic compounds during preclinical studies. This can be achieved by different technological approaches using isolated liver mitochondria, cultured hepatocytes, or other experimental models [32,35,50,90,162]. However, some of these approaches can fail to detect mechanisms of drug-induced mitochondrial toxicity requiring several days, or weeks to manifest. For instance, this is the case of several types of alteration of mtDNA homeostasis such as impairment of mtDNA replication and translation [33,34,159]. Because drug-induced alteration of mtDNA homeostasis in the liver is not rare [11,50], drug developers should consider the potential risk of this mechanism of mitochondrial toxicity.

## Availability of data and material

Not applicable.

## CRediT authorship contribution statement

Annie Borgne-Sanchez: Writing – review & editing. Bernard Fromenty: Writing – original draft, Conceptualization.
Declaration of competing interest

Annie Borgne-Sanchez is a co-founder of MITOLOGICS S.A.S. Bernard Fromenty declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by a grant from the Agence Nationale de la Recherche (ANR-16-CE18-0010, projet MITOXDRUGS). We are grateful to INSERM (Institut National de la Santé et de la Recherche Médicale) and the University of Rennes for their constant financial support.

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