Bernard Fromenty
email: bernard.fromenty@inserm.fr

Alteration of mitochondrial DNA homeostasis in drug-induced liver injury

Keywords:

Mitochondrial DNA (mtDNA) encodes for 13 proteins involved in the oxidative phosphorylation (OXPHOS) process. In liver, genetic or acquired impairment of mtDNA homeostasis can reduce ATP output but also decrease fatty acid oxidation, thus leading to different hepatic lesions including massive necrosis and microvesicular steatosis. Hence, a severe impairment of mtDNA homeostasis can lead to liver failure and death. An increasing number of investigations report that some drugs can induce mitochondrial dysfunction and drug-induced liver injury (DILI) by altering mtDNA homeostasis. Some drugs such as ciprofloxacin, antiretroviral nucleoside reverse-transcriptase inhibitors and tacrine can inhibit hepatic mtDNA replication, thus inducing mtDNA depletion.

Drug-induced reduced mtDNA levels can also be the consequence of reactive oxygen speciesmediated oxidative damage to mtDNA, which triggers its degradation by mitochondrial nucleases.

Such mechanism is suspected for acetaminophen and troglitazone. Other pharmaceuticals such as linezolid and tetracyclines can impair mtDNA translation, thus selectively reducing the synthesis of the 13 mtDNA-encoded proteins. Lastly, some drugs might alter the mtDNA methylation status but the pathophysiological consequences of such alteration are still unclear. Drug-induced impairment of mtDNA homeostasis is probably under-recognized since preclinical and post-marketing safety studies do not classically investigate mtDNA levels, mitochondrial protein synthesis and mtDNA oxidative damage.

Introduction

More than 350 drugs have been reported to induce liver injury such as hepatic cytolysis, cholestasis, steatosis (i.e. fat accretion) and steatohepatitis, or less frequently cirrhosis and liver cancer [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF] Björnsson | Categorization of drugs implicated in causing liver injury: Critical assessment based on published case reports[END_REF]. In the most severe cases, drug-induced liver injury (DILI) can require liver transplantation, or lead to the death of the patient (Fromenty and Pessayre, 1995;[START_REF] Gulmez | Risk of hospital admission for liver injury in users of NSAIDs and nonoverdose paracetamol: Preliminary results from the EPIHAM study[END_REF]. It is now acknowledged that mitochondrial dysfunction is a major mechanism whereby drugs can induce DILI [START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF][START_REF] Porceddu | Prediction of liver injury induced by chemicals in human with a multiparametric assay on isolated mouse liver mitochondria[END_REF][START_REF] Ramachandran | Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives[END_REF], although other mechanisms can be involved such as endoplasmic reticulum (ER) stress [START_REF] Foufelle | Role of endoplasmic reticulum stress in drug-induced toxicity[END_REF] and lysosomal alterations [START_REF] Woolbright | Lysosomal instability and cathepsin B release during acetaminophen hepatotoxicity[END_REF].

Most of the time, investigations on drug-induced mitochondrial dysfunction report the acute effects of pharmaceuticals on different mitochondrial parameters such as membrane potential and permeability, oxygen consumption, activity of different complexes of the respiratory chain and flux of fatty acid oxidation [START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF][START_REF] Nadanaciva | Investigating mitochondrial dysfunction to increase drug safety in the pharmaceutical industry[END_REF][START_REF] Porceddu | In vitro assessment of mitochondrial toxicity to predict drug-induced liver injury[END_REF].

However, there is an increasing number of investigations reporting that drugs can induce mitochondrial dysfunction and DILI by impairing mitochondrial DNA (mtDNA) homeostasis, for instance at the level of mtDNA replication and translation [START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF][START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Will | Mitochondrial toxicity assessment in industry -a decade of technology development and insight[END_REF]. Such mechanism of mitochondriotoxicity is most probably underrecognized because preclinical and post-marketing safety studies do not classically investigate druginduced effects on mtDNA integrity and maintenance.

In this review, I first recall the main features of mitochondria, their role in energy production and the mitochondrial genome, in particular regarding its replication, transcription, translation and repair. Next, drug-induced impairment of mtDNA homeostasis is discussed in separate sections depending on the involved mechanism including inhibition of mtDNA replication, impairment of mtDNA translation and oxidative damage to mtDNA.

A c c e p t e d M a n u s c r i p t

Main features of the mitochondria

Structure and main components

Mitochondria, whose size is ~1 µm, are intracellular organelles with two membranes (namely the outer and the inner membrane) that surround the matrix. This compartment contains numerous enzymes involved in different key oxidative pathways such as mitochondrial fatty acid oxidation (mtFAO) and pyruvate oxidation via the tricarboxylic acid (TCA) cycle. The matrix also contains the mitochondrial DNA (mtDNA) and all the components (e.g. enzymes and transcription factors) mandatory for its replication, transcription, translation and repair. However, this small genome allows only the synthesis of 13 polypeptides of the mitochondrial respiratory chain (MRC), whereas all other mitochondrial proteins (~1700) are encoded by the nuclear DNA and targeted to mitochondria (Area-Gomez and [START_REF] Area-Gomez | Mitochondrial genetics and disease[END_REF][START_REF] Borst | Small is beautiful -portrait of a mitochondrial genome[END_REF]. Notably, mitochondria share different major features with bacteria including size, double-membrane structure, mode of mtDNA transcription and some components of the mtDNA translation machinery [START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. These features are due to the fact that the precursors of mitochondria were bacteria that became endosymbiotic residents of "proto-eukaryotic" cells early in our evolutionary history [START_REF] Pessayre | Mitochondrial involvement in drug-induced liver injury[END_REF].

Production of energy

One major role of mitochondria is the production of energy so that these organelles are often referred to as the powerhouses of the cells. To this end, mitochondria oxidize many substrates including amino acids, pyruvate and fatty acids. Pyruvate, which is provided by glycolysis, is oxidized by the TCA cycle after its transformation into acetyl-CoA by the mitochondrial enzyme A c c e p t e d M a n u s c r i p t pyruvate dehydrogenase. TCA cycle includes 8 enzymes including the FAD-dependent succinate dehydrogenase (SDH, also referred to as complex II of the MRC) and 3 different NAD + -dependent dehydrogenases. Fatty acids are oxidized by the β-oxidation process, a key metabolic pathway mandatory for the preservation of normal energy output, especially during fasting. Two reactions of mtFAO are catalyzed by different FAD-dependent and NAD + -dependent dehydrogenases that have specific activities for short-chain, medium-chain, long-chain and very long-chain fatty acids (Fromenty and Pessayre, 1995;[START_REF] Houten | The biochemistry and physiology of mitochondrial fatty acid β-oxidation and its genetic disorders[END_REF].

Substrate oxidation within mitochondria is coupled to the generation of ATP in most normal physiological conditions. Indeed, oxidation of substrates such as pyruvate and fatty acids constantly generate FADH 2 and NADH. These reduced cofactors then transfer their electrons to the MRC, thus regenerating the NAD + and FAD necessary for other cycles of fuel oxidation. The electrons provided by NADH or FADH 2 migrate all the way along the respiratory chain, up to cytochrome c oxidase (COX), where they safely react with oxygen and protons to form water. Notably, electron transfer across MRC complexes I, III and IV is coupled with the extrusion of protons from the mitochondrial matrix into the intermembrane space of mitochondria, thus creating a large electrochemical potential (Δψ) across the inner membrane. When ADP is high, protons reenter the matrix through the F 0 portion of ATP synthase, causing the conversion of ADP into ATP by the F 1 portion of this enzyme [START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF][START_REF] Wallace | Mitochondrial energetics and therapeutics[END_REF]. The biochemical process linking substrate oxidation to ATP generation is referred to as oxidative phosphorylation (OXPHOS).

Production of reactive oxygen species and their deleterious effects

Mitochondria are considered as the main source of reactive oxygen species (ROS) in most cells.

Although the MRC complexes I and III are two major sites of ROS generation in mitochondria [START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF][START_REF] Wallace | Mitochondrial energetics and therapeutics[END_REF], some enzymes of the mtFAO pathway could also produce significant amount of ROS [START_REF] Kakimoto | H 2 O 2 release from the very long chain acyl-CoA dehydrogenase[END_REF][START_REF] Seifert | Electron transport chain-dependent and -independent mechanisms of mitochondrial H 2 O 2 emission during long-chain fatty acid oxidation[END_REF]. Physiological A c c e p t e d M a n u s c r i p t production of mitochondrial ROS could serves as key signaling molecules [START_REF] Diebold | Mitochondrial ROS regulation of proliferating cells[END_REF]. Fortunately, different mitochondrial enzymes can eliminate ROS such as manganese superoxide dismutase (MnSOD), peroxiredoxins and glutathione peroxidases, which necessitate reduced glutathione (GSH) as cofactor [START_REF] Pessayre | Mitochondrial involvement in drug-induced liver injury[END_REF][START_REF] Wallace | Mitochondrial energetics and therapeutics[END_REF]. Hence, mitochondrial GSH plays a key role in order to avoid the excessive accumulation of ROS and subsequent oxidative stress [START_REF] Ribas | Glutathione and mitochondria[END_REF].

In contrast, ROS overproduction can induce oxidative damage to different mitochondrial components, including lipids such as cardiolipin, proteins involved in MRC activity or other metabolic processes and mtDNA [START_REF] Musatov | Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF].

Regarding the mitochondrial genome, ROS can induce directly or indirectly the occurrence of oxidized bases such as 8-hydroxydeoxyguanosine (8-OH-dG), abasic sites (i.e. DNA sites lacking a purine or pyrimidine base), point mutations, deletions, strand breaks and also reduced mtDNA levels, as discussed later on [START_REF] Anson | Homogenous repair of singlet oxygen-induced DNA damage in differentially transcribed regions and strands of human mitochondrial DNA[END_REF][START_REF] Berneburg | Singlet oxygen mediates the UVA-induced generation of the photoaging-associated mitochondrial common deletion[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF].

Notably, the accumulation of oxidative damage in cardiolipin, MRC proteins and mtDNA can impair the activity of different MRC complexes including complexes I and III [START_REF] Musatov | Susceptibility of mitochondrial electron-transport complexes to oxidative damage. Focus on cytochrome c oxidase[END_REF], thus reinforcing ROS overproduction via a vicious circle [START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF].

Mitochondrial biogenesis

Mitochondrial biogenesis is a general program aimed at producing new functional mitochondria with intact mtDNA copies in response to different types of stress. For instance, reduced ATP production and oxidative stress are able to trigger mitochondrial biogenesis [START_REF] Gureev | Regulation of mitochondrial biogenesis as a way for active longevity: interaction between the Nrf2 and PGC-1α signaling pathways[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF]. The regulation of mitochondrial biogenesis is complex and orchestrated by different key transcription factors and coactivators including nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and peroxisome proliferator-activated receptor-γ coactivator 1α and 1β (PGC-1α and PGC-1β) [START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF][START_REF] Villena | New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond[END_REF]. The nuclear factor erythroid 2-like 2 (NFE2L2, also referred A c c e p t e d M a n u s c r i p t to as Nrf2) also plays a major role in mitochondrial biogenesis in response to oxidative stress [START_REF] Gureev | Regulation of mitochondrial biogenesis as a way for active longevity: interaction between the Nrf2 and PGC-1α signaling pathways[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF]. These transcription factors and coactivators enhance the expression of numerous nuclear DNA-encoded mitochondrial proteins as well as the mitochondrial transcription factor A (TFAM), which in turn activates mtDNA transcription [START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF][START_REF] Scarpulla | Transcriptional integration of mitochondrial biogenesis[END_REF]. Finally, it is noteworthy that AMP-activated protein kinase (AMPK) plays also a key role in mitochondrial biogenesis, in particular by activating PGC1α [START_REF] Vega | Maintaining ancient organelles: mitochondrial biogenesis and maturation[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF].

The mitochondrial genome

General features of mtDNA

Mammalian mtDNA is a 16.6 kb double-stranded circular genome present within each mitochondrion in several copies. The double-stranded structure of mtDNA consists of a guanine-rich heavy (H) strand and a light (L) strand. Notably, mtDNA encodes 13 polypeptides of the mitochondrial respiratory chain (MRC) [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF][START_REF] Wallace | Mitochondrial energetics and therapeutics[END_REF]. These polypeptides are then inserted in the inner membrane within the MRC complexes I, III, IV (cytochrome c oxidase) and V (ATP synthase), along with dozens of nuclear DNA-encoded proteins. mtDNA also encodes 2 ribosomal RNAs (rRNAs) and 22 transfer RNAs (tRNAs) that are required for protein synthesis in mitochondria [START_REF] Borst | Small is beautiful -portrait of a mitochondrial genome[END_REF]. Hence, mtDNA is mandatory for correct MRC activity, OXPHOS and ATP synthesis (Fig. 1).

Although mtDNA is not protected by histones, this genome is entirely coated by numerous molecules of TFAM [START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF][START_REF] Farge | Organization of DNA in mammalian mitochondria[END_REF]. Hence, in addition to its role in mtDNA transcription (section 2.4), TFAM might also protect the mitochondrial genome against oxidative stress [START_REF] Kang | Mitochondrial transcription factor A (TFAM): roles in maintenance of mtDNA and cellular functions[END_REF]. mtDNA, TFAM and other proteins such as mtSSB (mitochondrial single-stranded DNA-binding protein) and DNA polymerase gamma (γ) are organized A c c e p t e d M a n u s c r i p t into nucleoprotein complexes known as nucleoids, which appear to be associated with the inner mitochondrial membrane [START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF][START_REF] Farge | Organization of DNA in mammalian mitochondria[END_REF].

In the normal situation, all the mtDNA copies in a tissue are identical, a situation known as homoplasmy. However, in some pathophysiological conditions (e.g. mitochondrial diseases, aging, alcoholic liver diseases), both normal and mutated mtDNA copies may coexist within the same tissue, a situation known as heteroplasmy. Notably, most mtDNA-based diseases are functionally "recessive", so that the mutant load should be very high (e.g. ≥80%) before clinical pathology ensues [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. It is also important to bear in mind that there is an excess of mtDNA molecules in most tissues including liver. Hence, a small or moderate loss of mtDNA copies has no deleterious impact on mitochondrial function. It is deemed that the number of mtDNA copies must fall below 20 to 40% of basal levels to induce severe mitochondrial dysfunction and adverse events [START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF][START_REF] Ducluzeau | Progressive reversion of clinical and molecular phenotype in a child with liver mitochondrial DNA depletion[END_REF][START_REF] Rossignol | Mitochondrial threshold effects[END_REF]. In this context, the few mtDNA copies remaining within each mitochondrion are not able to provide enough MRC polypeptides, thus leading to OXPHOS impairment (Fig. 1). Reduced MRC activity can also lead to secondary inhibition of mtFAO and TCA cycle (Fig. 1), due to a lower mitochondrial content of NAD + and FAD generated respectively from NADH or FADH 2 [START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF]. Finally, it is noteworthy that mtDNA can be methylated although the mechanisms of methylation and demethylation in mitochondria are still poorly understood [START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF]Sharma et al., 2019). Similar to nuclear DNA, mtDNA methylation strongly regulates gene expression [START_REF] Pirola | Epigenetic modification of liver mitochondrial DNA is associated with histological severity of nonalcoholic fatty liver disease[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF].

mtDNA replication, transcription and translation

Replication of the mitochondrial genome occurs not only in dividing cells but also in post-mitotic tissues. Permanent mtDNA replication in non-dividing cells allows to keep constant cellular mtDNA levels despite continuous degradation of the most damaged and/or dysfunctional mitochondria. The Twinkle, a mitochondrial DNA helicase [START_REF] Farge | Organization of DNA in mammalian mitochondria[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF].

Two mitochondrial topoisomerases, namely Top1mt and Top2β, also regulate mtDNA replication [START_REF] Goffart | Twist and turn-topoisomerase functions in mitochondrial DNA maintenance[END_REF]. Several models of mtDNA replication have been proposed, although the "asynchronous, strand displacement" model is the most accepted so far [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF].

Transcription of human mtDNA is "prokaryotic-like". Indeed, all 37 genes encoded by human mtDNA are synthesized initially as two large polycistronic precursor transcripts, respectively encoded by the H-strand and the L-strand. The tRNA genes are then cleaved at their 5' and 3' ends by two ribonucleases, thereby releasing the tRNAs as well as the flanking rRNAs and mRNAs [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF][START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF]. Following cleavage of the precursor polycistronic RNAs, the 3' termini of the mRNAs are polyadenylated. Translation of the mature mRNAs takes place on mitochondrial ribosomes, which consist of mtDNA-encoded 12S and 16S rRNAs together with different imported ribosomal proteins [START_REF] Christian | Mechanism of protein biosynthesis in mammalian mitochondria[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF].

mtDNA repair

Similarly to nuclear DNA, the mitochondrial genome can undergo different types of damage, which require specific pathways of DNA repair in order to preserve mitochondrial function. Indeed, contrary to what has been proposed several decades ago, mitochondria do possess several of the DNA repair pathways described for the nuclear genome including a base excision repair (BER) system, a mismatch repair (MMR)-like activity, non-homologous end joining (NHEJ) and possibly homologous recombination [START_REF] Sharma | Mitochondrial DNA integrity: role in health and disease[END_REF]Sharma et al., 2019;Zinovkina, 2018). Furthermore, some components of the nucleotide excision repair (NER) pathway are present in mitochondria (Sharma et al., 2019;Zinovkina, 2018). Hence, these organelles are expected to repair most types of DNA damage A c c e p t e d M a n u s c r i p t including oxidized DNA bases, abasic sites, double-strand breaks and bulky DNA adducts [START_REF] Muftuoglu | Formation and repair of oxidative damage in the mitochondrial DNA[END_REF]Sharma et al., 2019;[START_REF] Valente | Mitochondrial DNA exhibits resistance to induced point and deletion mutations[END_REF]Zinovkina, 2018).

Besides the above-mentioned DNA repair pathways, it should be underlined that mitochondria are able to completely degrade any mtDNA molecules that present too many DNA lesions to be efficiently repaired, in particular double-strand breaks and possibly bulky DNA adducts [START_REF] Moretton | Selective mitochondrial DNA degradation following double-strand breaks[END_REF][START_REF] Valente | Mitochondrial DNA exhibits resistance to induced point and deletion mutations[END_REF]. This degradation seems to be dependent on several enzymes including MGME1 (mitochondrial genome maintenance exonuclease 1) and DNA polymerase γ, which possesses an exonuclease activity [START_REF] Nissanka | The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions[END_REF][START_REF] Peeva | Linear mitochondrial DNA is rapidly degraded by components of the replication machinery[END_REF]. Notably, the nuclease-mediated degradation of heavily damaged mtDNA molecules can cause rapid mtDNA depletion [START_REF] Nissanka | The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions[END_REF][START_REF] Peeva | Linear mitochondrial DNA is rapidly degraded by components of the replication machinery[END_REF]. This mechanism of mtDNA depletion has been proposed to explain the severe loss of mtDNA copies observed in liver after an alcoholic binge [START_REF] Mansouri | An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice[END_REF][START_REF] Mansouri | Acute ethanol administration oxidatively damages and depletes mitochondrial DNA in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants[END_REF], or carbon tetrachloride (CCl 4 ) intoxication [START_REF] Knockaert | Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver[END_REF].

If mtDNA degradation is not complete, partially degraded mtDNA molecules can generate mtDNA deletions after NHEJ-mediated DNA ligation [START_REF] Nissanka | Mechanisms of mitochondrial DNA deletion formation[END_REF]. However, it should be underscored that mtDNA deletions can also arise from errors in mtDNA replication such as slipped mispairing [START_REF] Chen | The generation of mitochondrial DNA large-scale deletions in human cells[END_REF][START_REF] Nissanka | Mechanisms of mitochondrial DNA deletion formation[END_REF]. Slipped-mispairing replication generates large mtDNA deletions that are usually flanked by small direct DNA repeats with identical sequences [START_REF] Chen | The generation of mitochondrial DNA large-scale deletions in human cells[END_REF][START_REF] Nissanka | Mechanisms of mitochondrial DNA deletion formation[END_REF]. Interestingly, oxidative stress greatly favors the occurrence of such large mtDNA deletions in human liver, for instance in the context of normal aging, Wilson's disease and alcoholic intoxication (Fromenty et al., 1995;[START_REF] Krishnan | What causes mitochondrial DNA deletions in human cells?[END_REF][START_REF] Mansouri | Premature oxidative aging of hepatic mitochondrial DNA in Wilson's disease[END_REF][START_REF] Simonetti | Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR[END_REF]. However, in these pathophysiological conditions, mtDNA deletions are present in liver only at low heteroplasmy levels (<0.1%) (Fromenty et al., 1995;[START_REF] Mansouri | Premature oxidative aging of hepatic mitochondrial DNA in Wilson's disease[END_REF][START_REF] Simonetti | Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR[END_REF]. Hence, these mtDNA deletions are most likely not pathogenic as such but may rather reflect oxidative damage of the mitochondrial genome [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF].

Finally, it is noteworthy that mtDNA repair requires DNA polymerases other than DNA polymerase gamma. For instance, it has been shown that DNA polymerase beta and PrimPol, a DNA A c c e p t e d M a n u s c r i p t primase-polymerase, are able to participate to mtDNA repair in addition to their usual role in nuclear DNA repair [START_REF] Kaufman | POLB: A new role of DNA polymerase beta in mitochondrial base excision repair[END_REF][START_REF] Torregrosa-Muñumer | PrimPol is required for replication reinitiation after mtDNA damage[END_REF]. DNA polymerases theta and zeta could also play a significant role in mtDNA maintenance (Sharma et al., 2019).

Hepatotoxic drugs impairing mtDNA homeostasis

General overview

The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) zidovudine (AZT) was the first pharmaceutical found to induce hepatotoxicity in treated patients through an impairment of mtDNA replication, resulting in severe mtDNA depletion in liver [START_REF] Chariot | Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion[END_REF][START_REF] Bras | Metabolic, hepatic and muscular changes during zidovudine therapy: a drug-induced mitochondrial disease?[END_REF]. Since then, other hepatotoxic drugs have been reported to alter mtDNA homeostasis not only by inhibiting mtDNA replication but also by impairing mtDNA translation (Fig. 2) [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Whatever its mechanism, drug-induced impairment of mtDNA replication and translation in liver can lead to hepatic cytolysis with increased plasma transaminases, steatosis, steatohepatitis and cirrhosis (Fig. 1) [START_REF] Chariot | Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Notably, steatosis can occur as microvesicular fat, thus reflecting profound inhibition of mtFAO [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Hyperlactatemia or lactic acidosis (sometimes fatal), can also occur in some patients, thus reflecting an impairment of the TCA cycle [START_REF] Margolis | A review of the toxicity of HIV medications[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Hence, severe impairment of mtDNA replication or translation can reduce MRC activity and secondarily curb the mitochondrial oxidation of different endogenous molecules such as fatty acids and pyruvate, which respectively accumulate as triglycerides and lactate [START_REF] Margolis | A review of the toxicity of HIV medications[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. The progression of steatosis towards steatohepatitis in some treated patients could be explained by oxidative stress and lipid peroxidation, which favor the occurrence of inflammation and fibrogenesis [START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Satapathy | Drug-induced fatty liver disease: An overview of pathogenesis and management[END_REF]. Finally, it is noteworthy that impairment of MRC activity and subsequent ATP shortage can not A c c e p t e d M a n u s c r i p t only explain hepatocyte necrosis (and thus hepatic cytolysis) but also cholestasis (Fig. 1), because the activity of several bile transporters is ATP dependent [START_REF] Aleo | Human drug-induced liver injury severity is highly associated with dual inhibition of liver mitochondrial function and bile salt export pump[END_REF][START_REF] Borgne-Sanchez | Mitochondrial dysfunction in drug-induced liver injury[END_REF].

For some drugs such as acetaminophen and troglitazone, mtDNA depletion is not linked to an impairment of mtDNA replication but rather to mtDNA oxidative damage (Fig. 2) [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Indeed, ROS can induce different types of DNA lesions such as single and double-strand breaks that can trigger complete mtDNA degradation by different mitochondrial enzymes including MGME1 and DNA polymerase γ, as previously mentioned [START_REF] Nissanka | The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions[END_REF][START_REF] Peeva | Linear mitochondrial DNA is rapidly degraded by components of the replication machinery[END_REF]. Although the resulting mtDNA depletion could have a pathogenic role, the main mechanism whereby these drugs induce mitochondrial dysfunction is rather a direct impairment of mitochondrial function including MRC activity and OXPHOS, as discussed below. Hence, similar to alcohol intoxication [START_REF] Cahill | Chronic ethanol consumption causes alterations in the structural integrity of mitochondrial DNA in aged rats[END_REF][START_REF] Mansouri | An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice[END_REF][START_REF] Mansouri | Acute ethanol administration oxidatively damages and depletes mitochondrial DNA in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants[END_REF], APAP and troglitazone-induced mtDNA depletion reflects the occurrence of massive oxidative stress in liver mitochondria.

Although drug-induced impairment of mitochondrial function and mtDNA homeostasis can eventually lead to DILI, propagation (and thus aggravation) of the initial liver injury can occur through different adaptive or innate immune responses. Readers are invited to read different excellent reviews regarding the role of immunity in idiosyncratic DILI (Roth et al., 2017: Woolbright andJaeschke, 2018;[START_REF] Ye | Dissecting the molecular pathophysiology of drug-induced liver injury[END_REF].

In the following sections, I present the main hepatotoxic drugs that have been shown to alter mtDNA homeostasis by inhibiting mtDNA replication or mtDNA translation as well as by inducing oxidative mtDNA damage. In the last section, other potential mechanisms of drug-induced impairment of mtDNA homeostasis are finally discussed.

Drugs altering mtDNA replication

A c c e p t e d M a n u s c r i p t

Ciprofloxacin

Ciprofloxacin is a 4-quinolone antibiotic inhibiting the bacterial DNA gyrases, which belongs to the type II topoisomerase family. Ciprofloxacin can induce extensive hepatocellular cytolysis (with possible liver failure), steatosis and cholestasis [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF] Orman | Clinical and histopathologic features of fluoroquinolone-induced liver injury[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. In cultured cells, ciprofloxacin progressively decreases mtDNA levels and impair mitochondrial respiration [START_REF] Lawrence | 4-Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells[END_REF]. In addition, further in vitro investigations suggested that ciprofloxacin could impair mtDNA replication by inhibiting the type II topoisomerase present in mitochondria [START_REF] Lawrence | Delayed cytotoxicity and cleavage of mitochondrial DNA in ciprofloxacin-treated mammalian cells[END_REF]. Ciprofloxacin might also induce hepatotoxicity via the production of free radicals and lipid peroxidation, independently of mtDNA depletion [START_REF] Gürbay | Microsomal metabolism of ciprofloxacin generates free radicals[END_REF][START_REF] Weyers | Ciprofloxacin increases hepatic and renal lipid hydroperoxides levels in mice[END_REF]. A recent study also reported that ciprofloxacin (30 to 210 µM) was able to induce the opening of the mitochondrial membrane permeability transition (MPT) pore and mitochondrial swelling in isolated rat liver mitochondria [START_REF] Oyebode | Toxicity of some broad-spectrum antibacterials in normal rat liver: the role of mitochondrial membrane permeability transition pore[END_REF]. However, such concentrations of ciprofloxacin failed to induce mitochondrial swelling in isolated mouse liver mitochondria [START_REF] Porceddu | Prediction of liver injury induced by chemicals in human with a multiparametric assay on isolated mouse liver mitochondria[END_REF].

Fialuridine

The nucleoside analogue 1-(2-deoxy-2-fluoro-β-d-arabinofuranosyl)-5-iodouracil (FIAU), or fialuridine, has been developed in the early 90s for the treatment of chronic hepatitis B. However, while preclinical safety studies and a short pilot study in patients did not reveal any signs of hepatotoxicity, phase II clinical trials conducted by the National Institute of Health were prematurely interrupted during the 13 th week due to serious adverse effects [START_REF] Attarwala | TGN1412: From discovery to disaster[END_REF][START_REF] Mak | DNA polymerase inhibitors for treating hepatitis B: a safety evaluation[END_REF]. Indeed, FIAU induced in several patients unmanageable lactic acidosis, microvesicular steatosis and liver failure requiring liver transplantation, or leading to death (Fromenty and Pessayre, 1995;[START_REF] Honkoop | Mitochondrial injury. Lessons from the fialuridine trial[END_REF][START_REF] Mckenzie | Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B[END_REF]. New in vivo and in vitro investigations after this A c c e p t e d M a n u s c r i p t unexpected tragedy showed that FIAU strongly inhibits DNA polymerase γ and triggers mtDNA depletion by an unusual mechanism [START_REF] Lewis | 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[END_REF][START_REF] Lewis | Depletion of mitochondrial DNA, destruction of mitochondria, and accumulation of lipid droplets result from fialuridine treatment in woodchucks (Marmota monax)[END_REF][START_REF] Semino-Mora | Mitochondrial and cellular toxicity induced by fialuridine in human muscle in vitro[END_REF].

Indeed, unlike NRTIs whose incorporation into a growing chain of mtDNA interrupts mtDNA replication (section 4.2.4), FIAU can be incorporated into mtDNA without immediately blocking mtDNA replication. This is because FIAU carries a 3'-hydroxyl group on the sugar moiety, thus allowing the subsequent incorporation of other nucleotides by the mitochondrial DNA polymerase γ.

However, when several adjacent molecules of FIAU are successively incorporated into a growing chain of mtDNA, DNA polymerase γ is strongly inhibited. This subsequently blocks further mtDNA replication and leads to severe mtDNA depletion in liver [START_REF] Lewis | 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[END_REF].

The fact that FIAU hepatotoxicity was not detected in preclinical safety studies is particularly puzzling. A likely explanation is that FIAU was unable to inhibit mtDNA replication in the common animal species used in these studies, in particular rats and dogs [START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF]. In contrast, FIAU was able to induce hepatic mtDNA depletion in woodchucks (Marmota monax) due to the high rate of incorporation of this drug within the woodchuck mitochondrial genome [START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF][START_REF] Lewis | Depletion of mitochondrial DNA, destruction of mitochondria, and accumulation of lipid droplets result from fialuridine treatment in woodchucks (Marmota monax)[END_REF]. Interestingly, the woodchuck model was chosen afterwards in order to evaluate the mitochondrial toxicity of any novel anti-HBV nucleoside analogues [START_REF] Attarwala | TGN1412: From discovery to disaster[END_REF]. Previous investigations suggested that interspecies differences in the susceptibility toward FIAU-induced hepatotoxicity could be related to the subcellular expression of the equilibrative nucleoside transporter 1 (ENT1) [START_REF] Lee | Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine[END_REF], but other hypotheses were recently put forward [START_REF] Hendriks | Mechanisms of chronic fialuridine hepatotoxicity as revealed in primary human hepatocyte spheroids[END_REF].

Finally, the FIAU tragedy ghoulishly illustrates the fact that mitochondrial toxicity linked to mtDNA replication impairment can occur long after treatment initiation when the mtDNA reserve becomes too low to allow proper mitochondrial function (section 3.1).

Ganciclovir

A c c e p t e d M a n u s c r i p t

The nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl) guanine, or ganciclovir, is an antiviral drug effective on different viruses including herpes simplex viruses, varicella-zoster virus, cytomegaloviruses and Epstein-Barr virus. Ganciclovir is actually a prodrug that is transformed in the infected cells into ganciclovir-triphosphate, the active form of ganciclovir. This phosphorylation is preferentially carried out in the infected cells because non-infected cells are less able to perform the first phosphorylation step. Ganciclovir triphosphate exerts its antiviral activity by impairing the synthesis of viral DNA by two mechanisms: competitive inhibition of the viral DNA polymerases and direct incorporation into the viral DNA, which slows down its elongation. Indeed, ganciclovir has two hydroxyl groups so that its incorporation into DNA does not terminate DNA replication. Nonetheless, the incorporated ganciclovir molecules are expected to distort the DNA helix and impede the next round of DNA replication [START_REF] Foti | Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement[END_REF][START_REF] Thust | Comparison of the genotoxic and apoptosis-inducing properties of ganciclovir and penciclovir in Chinese hamster ovary cells transfected with the thymidine kinase gene of herpes simplex virus-1: implications for gene therapeutic approaches[END_REF]. Hepatic cytolysis, cholestasis and diffuse fibrosis have been reported in some patients treated by this drug [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF] Shea | Ganciclovir hepatotoxicity[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF].

Ganciclovir can also be administered for the treatment of different cancers in association with an adenoviral vector harboring the gene encoding the herpes simplex virus thymidine kinase (HSVtk), a so-called "suicide gene" [START_REF] Düzgüneş | Origins of suicide gene therapy[END_REF]. The principle of this combined therapy is the HSVtkmediated conversion of ganciclovir into ganciclovir-monophosphate, which is further activated by cellular kinases into ganciclovir-triphosphate, thus impairing DNA synthesis and leading to apoptosis of the dividing cancer cells [START_REF] Düzgüneş | Origins of suicide gene therapy[END_REF]. However, this therapy can lead in treated patients to hepatotoxicity and other side effects such as skin rash, thrombocytopenia and anemia [START_REF] Immonen | AdvHSV-tk gene therapy with intravenous ganciclovir improves survival in human malignant glioma: a randomised, controlled study[END_REF][START_REF] Shalev | Suicide gene therapy toxicity after multiple and repeat injections in patients with localized prostate cancer[END_REF][START_REF] Sterman | Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma[END_REF]. Interestingly, HSVtk/ganciclovir was found to induce serious hepatic dysfunction in rats with swollen hepatocytes with enlarged nuclei, scant necrosis and apoptosis, microvesicular and macrovacuolar steatosis and infiltration of the liver with hematopoietic and immune-response cells such as macrophages [START_REF] Herraiz | Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion[END_REF][START_REF] Van Der Eb | Severe hepatic dysfunction after adenovirusmediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration[END_REF].

Different in vitro and in vivo studies showed that HSVtk/ganciclovir-induced hepatotoxicity was associated with mtDNA depletion and mitochondrial dysfunction characterized by a profound decrease A c c e p t e d M a n u s c r i p t in COX activity and hyperlactatemia [START_REF] Herraiz | Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion[END_REF][START_REF] Van Der Eb | Ganciclovir nucleotides accumulate in mitochondria of rat liver cells expressing the herpes simplex virus thymidine kinase gene[END_REF]. Interestingly, some of these investigations showed that ganciclovir is incorporated into mtDNA in addition to nuclear DNA [START_REF] Herraiz | Liver failure caused by herpes simplex virus thymidine kinase plus ganciclovir therapy is associated with mitochondrial dysfunction and mitochondrial DNA depletion[END_REF]. Thus, it is tempting to speculate that the incorporated ganciclovir molecules may distort the mitochondrial genome and impede its replication, similar to what has been proposed for nuclear DNA [START_REF] Foti | Solution structure of a DNA decamer containing the antiviral drug ganciclovir: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement[END_REF][START_REF] Thust | Comparison of the genotoxic and apoptosis-inducing properties of ganciclovir and penciclovir in Chinese hamster ovary cells transfected with the thymidine kinase gene of herpes simplex virus-1: implications for gene therapeutic approaches[END_REF].

NRTIs

NRTIs were the first family of drugs developed for the treatment of acquired immune deficiency syndrome (AIDS) after it was discovered that these compounds could strongly inhibit the human immunodeficiency virus (HIV) reverse transcriptase [START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF]. The main NRTIs include stavudine (d4T), zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), lamivudine (3TC) and newer molecules such as abacavir (ABC), emtricitabine (EMT) and tenofovir (TEN) (Fromenty and Pessayre, 1995;[START_REF] Margolis | A review of the toxicity of HIV medications[END_REF]. These drugs are 2',3'-dideoxynucleoside analogues in which the hydroxyl group (-OH) in the 3' position on the sugar ring is replaced by either an hydrogen atom or another group unable to form a phosphodiester bond.

The remarkable benefit of NRTIs is unfortunately overshadowed by the occurrence in a significant number of HIV infected patients of severe adverse effects such as pancreatitis, lactic acidosis, myopathy and liver failure (Fromenty and Pessayre, 1995;[START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF]. NRTI-induced hepatotoxicity is particularly observed with d4T, ddI and AZT. Indeed, these drugs can induce different types of liver lesion including cholestasis, microvesicular and macrovacuolar steatosis, steatohepatitis and cirrhosis [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF]Fromenty and Pessayre, 1995;[START_REF] Margolis | A review of the toxicity of HIV medications[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. The newer NRTIs seem to be less hepatotoxic [START_REF] Soriano | Antiretroviral drugs and liver injury[END_REF].

Notably, the spectrum of NRTI-induced adverse effects is similar to the clinical manifestations observed in patients with genetic OXPHOS disorders (Fromenty and Pessayre, 1995;[START_REF] Chariot | Zidovudine-induced mitochondrial disorder with massive liver steatosis, myopathy, lactic acidosis, and mitochondrial DNA depletion[END_REF].

A c c e p t e d M a n u s c r i p t

It is now acknowledged that most of the side effects induced by NRTIs are the consequence of their deleterious effects on the mitochondrial genome. Indeed, these drugs are able to inhibit mtDNA replication, thus inducing profound mtDNA depletion and OXPHOS impairment (Fromenty and Pessayre, 1995;[START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF]. NRTIs act as chain terminators after their incorporation into the growing chain of mtDNA by the DNA polymerase γ. If the NRTI molecule is not removed by the proofreading activity of this DNA polymerase, no other nucleotides can be incorporated because the DNA chain now lacks a 3'-OH end [START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF].

The ability of NRTIs to inhibit DNA polymerase γ greatly varies among the analogues. Indeed, in vitro studies on purified DNA polymerase γ indicated that the potency of the inhibition is as follows: ddC > ddI >d4T >> 3TC > AZT > ABC [START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Soriano | Antiretroviral drugs and liver injury[END_REF]. Notably, this order of potency is also observed in human liver as the so-called "D-drugs" (ddC, ddI, d4T) appear to inhibit DNA polymerase γ and induce mtDNA depletion more strongly than other NRTIs [START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF]. Interestingly, similar to fialuridine (section 4.2.2), NRTI-induced mtDNA depletion can explain why these drugs can induce liver injury in some patients several weeks or months after treatment initiation [START_REF] Soriano | Antiretroviral drugs and liver injury[END_REF][START_REF] Margolis | A review of the toxicity of HIV medications[END_REF].

In addition to mtDNA depletion, NRTIs are able to induce the occurrence of mtDNA deletions and point mutations, possibly as a consequence of mitochondrial oxidative stress [START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Liang | Nucleoside reverse transcriptase inhibitors induced hepatocellular mitochondrial DNA lesions and compensatory enhancement of mitochondrial function and DNA repair[END_REF][START_REF] Payne | Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations[END_REF]. However, it is still unclear whether these mtDNA mutations play a significant role in NRTI-induced mitochondrial dysfunction. Finally, it is noteworthy that experimental studies showed that some NRTIs such as AZT and d4T can induce mitochondrial and metabolic toxicity through mechanisms unrelated to the inhibition of DNA polymerase γ and mtDNA depletion [START_REF] Igoudjil | Mitochondrial, metabolic and genotoxic effects of antiretroviral nucleoside reverse-transcriptase inhibitors[END_REF][START_REF] Igoudjil | High doses of stavudine induce fat wasting and mild liver damage without impairing mitochondrial respiration in mice[END_REF][START_REF] Igoudjil | High concentrations of stavudine impair fatty acid oxidation without depleting mitochondrial DNA in cultured rat hepatocytes[END_REF][START_REF] Lund | Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs[END_REF].

Tacrine

A c c e p t e d M a n u s c r i p t

Tacrine, or tetrahydroaminoacridine, is a reversible cholinesterase inhibitor prescribed for Alzheimer's disease. However, this drug increases plasma ALT activity in 50% of recipients and can induce severe liver injury in some individuals [START_REF] Berson | Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. Hepatic steatosis has also been reported [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF]. Tacrine is able to inhibit mtDNA synthesis in vitro and in vivo and to extensively deplete hepatic mtDNA after chronic treatment in mice [START_REF] Mansouri | Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver[END_REF][START_REF] Robertson | Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells[END_REF]. Some investigations suggest that tacrine could impede mtDNA replication through topoisomerase inhibition [START_REF] Mansouri | Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver[END_REF]. However, it is noteworthy that another mechanism of mitochondrial toxicity seem to be involved in tacrine-induced liver injury. Indeed, tacrine is able to uncouple OXPHOS, decrease membrane potential and reduce ATP synthesis via a protonophoric mechanism similar to amiodarone and perhexiline [START_REF] Berson | Uncoupling of rat and human mitochondria: a possible explanation for tacrine-induced liver dysfunction[END_REF][START_REF] Park | Tacrine, an oral acetylcholinesterase inhibitor, induced hepatic oxidative damage, which was blocked by liquiritigenin through GSK3-beta inhibition[END_REF][START_REF] Robertson | Elucidation of mitochondrial effects by tetrahydroaminoacridine (tacrine) in rat, dog, monkey and human hepatic parenchymal cells[END_REF].

Tamoxifen

Tamoxifen is an antiestrogenic drug used in the treatment of advanced breast cancer. This anticancer drug can, however, induce acute and chronic liver injury, in particular steatosis, steatohepatitis and cirrhosis [START_REF] Bruno | Incidence and risk factors for non-alcoholic steatohepatitis: prospective study of 5408 women enrolled in Italian tamoxifen chemoprevention trial[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. Tamoxifen was found to induce a progressive reduction in hepatic mtDNA levels after chronic treatment in mice, with a strong mtDNA depletion after 28 days [START_REF] Larosche | Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver[END_REF]. Further in vitro investigations suggested that impairment of mtDNA replication could be due to tamoxifen intercalation between mtDNA bases and subsequent topoisomerase inhibition [START_REF] Larosche | Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver[END_REF]. In contrast to these investigations, a 14-day treatment with tamoxifen failed to reduce mtDNA levels in human differentiated hepatoma HepaRG cells [START_REF] Begriche | Mitochondrial dysfunction induced by xenobiotics: involvement in steatosis and steatohepatitis[END_REF], whereas the same treatment protocol with the NRTI zalcitabine (ddC) induced a severe mtDNA depletion (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. It is possible that a longer treatment with tamoxifen might have been needed in order to induce significant mtDNA depletion in HepaRG cells. Finally, it should be underlined that tamoxifen presents other A c c e p t e d M a n u s c r i p t mitochondriotoxic effects in particular OXPHOS uncoupling via a protonophoric mechanism and mtFAO inhibition [START_REF] Cardoso | Mechanisms of the deleterious effects of tamoxifen on mitochondrial respiration rate and phosphorylation efficiency[END_REF][START_REF] Larosche | Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver[END_REF].

Drugs impairing mtDNA translation

Linezolid

The oxazolidinone linezolid is an antibiotic particularly efficient against drug-resistant pathogens including the Gram-positive bacteria Enterococcus faecium and Staphylococcus aureus as well as Mycobacterium tuberculosis [START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF]. Like many other antibiotics, linezolid impairs bacterial growth by inhibiting protein synthesis via its binding to bacterial ribosomes [START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF]. Moderate to serious liver injury can occur in some patients after several weeks of treatment, with the occurrence of increased plasma transaminases and several types of hepatic lesions such as macrovacuolar and microvesicular steatosis as well as bile duct damages [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] De Bus | Severe drug-induced liver injury associated with prolonged use of linezolid[END_REF][START_REF] De Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Vinh | Linezolid: a review of safety and tolerability[END_REF]. Prolonged administration of linezolid can also induce lactic acidosis, myopathy, pancreatitis, stroke-like episodes, neuropathy and renal failure [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF][START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF]. Similar to NRTIs (section 4.2.4), the spectrum of linezolid-induced adverse effects is similar to the clinical manifestations observed in patients with genetic defects of OXPHOS (Fromenty and Pessayre, 1995;[START_REF] Vinh | Linezolid: a review of safety and tolerability[END_REF].

Several investigations support the view that linezolid-induced side effects are secondary to a strong inhibition of mammalian mitochondrial protein synthesis via its binding to mitochondrial ribosomes [START_REF] Garrabou | Influence of mitochondrial genetics on the mitochondrial toxicity of linezolid in blood cells and skin nerve fibers[END_REF][START_REF] Mckee | Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones[END_REF][START_REF] Nagiec | Oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis[END_REF][START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF]. Indeed, mammalian mitochondrial ribosomes have some similarities with bacterial ribosomes, in particular at the level of the linezolid-binding site [START_REF] Leach | The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria[END_REF][START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF].

Linezolid interaction with the mitochondrial ribosomes is expected to impair the ribosomal binding A c c e p t e d M a n u s c r i p t of aminoacyl tRNAs [START_REF] Sun | Incorporation of a chiral gem-disubstituted nitrogen heterocycle yields an oxazolidinone antibiotic with reduced mitochondrial toxicity[END_REF]. The subsequent inhibition of mtDNA translation thus reduces the activity of MRC complexes that contain mtDNA-encoded proteins [START_REF] De Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Garrabou | Reversible inhibition of mitochondrial protein synthesis during linezolid-related hyperlactatemia[END_REF][START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF][START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF]. Interestingly, it has been reported that the risk of linezolid-induced mitochondrial dysfunction and secondary adverse effects such as lactic acidosis could be increased by several genetic predispositions such as mutations or polymorphisms in the 12S and 16S rRNA genes of the mitochondrial genome, or the mtDNA haplogroups U and J1 [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Garrabou | Influence of mitochondrial genetics on the mitochondrial toxicity of linezolid in blood cells and skin nerve fibers[END_REF][START_REF] Santini | Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes[END_REF]. However, further investigations will be needed in order to determine whether this mtDNA polymorphism also increases the risk of linezolid-induced liver injury.

Tetracyclines

Tetracyclines are broad-spectrum antibiotics which prevent bacterial growth by inhibiting bacterial protein synthesis. The tetracycline family includes many compounds including tetracycline, doxycycline, minocycline, methacycline and lymecycline. These drugs can induce different types of liver lesion including cholestasis, necrosis and steatosis, classically as microvesicular fat [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. Microvesicular steatosis was particularly observed several decades ago when tetracyclines were administered at very high intravenous doses [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF]Fromenty and Pessayre, 1995). Synthetic tetracyclines such as doxycycline and minocycline are now preferred to tetracycline because of easier dose schedules and faster gastrointestinal absorption when taken with food.

Tetracycline derivatives have been shown to inhibit mtDNA translation by a mechanism similar to linezolid, namely via their binding to the mitochondrial ribosomes [START_REF] Chukwudi | rRNA binding sites and the molecular mechanism of action of the tetracyclines[END_REF][START_REF] Mckee | Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones[END_REF][START_REF] Moullan | Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research[END_REF][START_REF] Protasoni | Mitochondria as oncotarget: a comparison between the tetracycline analogs doxycycline and COL-3[END_REF]. The glycylcycline antibiotic tigecycline, a tetracycline analogue, is also able to impair mitochondrial protein synthesis [START_REF] Skrtić | Inhibition of mitochondrial translation as a therapeutic strategy for human acute myeloid leukemia[END_REF], an effect which could have led to hepatotoxicity, acidosis, progressive kidney failure and death in a 20 A c c e p t e d M a n u s c r i p t patient treated with this drug for several weeks [START_REF] Vandecasteele | Tigecycline-induced inhibition of mitochondrial DNA translation may cause lethal mitochondrial dysfunction in humans[END_REF]. However, it is noteworthy that tetracyclines can also inhibit mtFAO through a mechanism apparently independent on mtDNA translation impairment [START_REF] Fréneaux | Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic[END_REF][START_REF] Labbe | Effects of various tetracycline derivatives on in vitro and in vivo beta-oxidation of fatty acids, egress of triglycerides from the liver, accumulation of hepatic triglycerides, and mortality in mice[END_REF]. However, the exact mechanism whereby tetracyclines inhibit mtFAO is still unknown.

Other hepatotoxic drugs

Recently, in vitro investigations carried out in the human differentiated hepatoma HepaRG cells revealed that the antidepressant imipramine and the antiretroviral protease inhibitor ritonavir reduced the levels of the mtDNA-encoded ND1 and COX2 proteins, whereas the amount of COX4 and other nuclear DNA-encoded mitochondrial proteins was unaffected (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. These mitochondrial effects were associated with a significant decrease in complex I levels and activity (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. These results strongly suggest that both imipramine and ritonavir are able to impair mtDNA translation, although further investigations would be required to decipher the exact mechanism of this deleterious effect. Actually, these results were totally unexpected because mitochondrial protein synthesis is thought to be impaired only by antibiotics such as linezolid (section 4.3.1) and tetracyclines (section 4.3.2). Finally, in addition to mechanistic studies, supplementary investigations should be carried out to determine whether impaired mtDNA translation could be involved in imipramine and ritonavir-induced liver injury [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF].

Drugs inducing oxidative mtDNA damage

Acetaminophen

Acetaminophen (N-acetyl-p-aminophenol, APAP or paracetamol) is one of the most popular drugs used for the management of pain and hyperthermia. Although APAP is usually considered as safe, the A c c e p t e d M a n u s c r i p t inadvertent or intentional ingestion of an excessive dose of this drug can cause massive hepatocellular necrosis and acute liver failure, which can be fatal [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Ramachandran | Acetaminophen hepatotoxicity[END_REF]. Steatosis, in particular microvesicular fat, can be observed in residual viable hepatocytes [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF]. Recent articles thoroughly reviewed the mechanisms whereby this drug can induce severe liver injury [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Moles | Mitochondrial-lysosomal axis in acetaminophen hepatotoxicity[END_REF][START_REF] Ramachandran | Acetaminophen hepatotoxicity[END_REF]. Briefly, APAP-induced hepatotoxicity is caused by cytochrome P450-mediated generation of the highly reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). Indeed, this electrophilic metabolite depletes hepatic glutathione (GSH), increases cell calcium, alters mitochondrial function, enhances ROS and peroxynitrite generation and activates c-Jun N-terminal kinase, to finally trigger liver cell necrosis [START_REF] Banerjee | The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes[END_REF][START_REF] Moles | Mitochondrial-lysosomal axis in acetaminophen hepatotoxicity[END_REF][START_REF] Ramachandran | Acetaminophen hepatotoxicity[END_REF].

APAP induces mitochondrial dysfunction by different mechanisms including NAPQI-mediated inhibition of different MRC complexes and possibly of some components of the mtFAO pathway [START_REF] Barbier-Torres | The mitochondrial negative regulator MCJ is a therapeutic target for acetaminophen-induced liver injury[END_REF][START_REF] Chrøis | Acetaminophen toxicity induces mitochondrial complex I inhibition in human liver tissue[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Lee | Targeting mitochondria with methylene blue protects mice against acetaminophen-induced liver injury[END_REF]. Another mechanism whereby APAP might alter MRC activity is mtDNA depletion. Indeed, investigations in mice showed that hepatic mtDNA levels were rapidly depleted after an APAP overdose, possibly due to the mtDNA damage caused by peroxynitrite and ROS [START_REF] Aubert | Differences in early acetaminophen hepatotoxicity between obese ob/ob and db/db mice[END_REF][START_REF] Cover | Peroxynitrite-induced mitochondrial and endonuclease-mediated nuclear DNA damage in acetaminophen hepatotoxicity[END_REF][START_REF] Du | Induction of mitochondrial biogenesis protects against acetaminophen hepatotoxicity[END_REF][START_REF] Ge | Inhibition of mitochondrial complex I by rotenone protects against acetaminophen-induced liver injury[END_REF]. Interestingly, one of these studies showed that hepatic levels of 11 of the 13 mtDNA-encoded mRNAs were significantly decreased 24 hours after 300 mg/kg APAP [START_REF] Ge | Inhibition of mitochondrial complex I by rotenone protects against acetaminophen-induced liver injury[END_REF], probably as a consequence of mtDNA depletion. However, it cannot be excluded that oxidatively damaged mRNAs might have been specifically degraded by mitochondrial RNases [START_REF] Barchiesi | Transcription, processing, and decay of mitochondrial RNA in health and disease[END_REF]. Another mechanism of reduced mtDNA-encoded mRNAs might be an impaired transcription of the remaining mtDNA molecules. Indeed, previous investigations reported that powerful free radical initiators such as 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH) were able to strongly inhibit the de novo synthesis of mitochondrial RNAs (Fromenty and Pessayre, 1995;[START_REF] Kristal | Antioxidants reduce peroxyl-mediated inhibition of mitochondrial transcription[END_REF]. It is noteworthy that fragments of mtDNA and nuclear DNA can be 22 A c c e p t e d M a n u s c r i p t detected in the circulation of patients with APAP-induced liver injury, thus reflecting massive DNA degradation during APAP hepatotoxicity [START_REF] Mcgill | The mechanism underlying acetaminophen-induced hepatotoxicity in humans and mice involves mitochondrial damage and nuclear DNA fragmentation[END_REF][START_REF] Mcgill | Serum mitochondrial biomarkers and damage-associated molecular patterns are higher in acetaminophen overdose patients with poor outcome[END_REF]. Lastly, it should be mentioned that induction of mitochondrial biogenesis with the sirtuin 1 (SIRT1) activator SRT1720 significantly protected mice against late liver injury after APAP overdose [START_REF] Du | Induction of mitochondrial biogenesis protects against acetaminophen hepatotoxicity[END_REF].

Troglitazone

The thiazolidinedione troglitazone was the first peroxisome proliferator-activated receptor-γ (PPARγ) agonist used for the treatment of type 2 diabetes. However, this drug has been withdrawn from the market in 2000 after the occurrence of numerous cases of serious and sometimes fatal liver injury [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Yokoi | Troglitazone[END_REF]. In most patients, the histopathologic changes included massive necrosis but other lesions such as cholestasis, microvesicular and macrovacuolar steatosis, fibrosis and cirrhosis have also been reported [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Wang | A unifying ontology to integrate histological and clinical observations for drug-induced liver injury[END_REF]. Previous articles extensively reviewed the mechanisms whereby troglitazone can induce liver injury [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Masubuchi | Metabolic and non-metabolic factors determining troglitazone hepatotoxicity: a review[END_REF][START_REF] Yokoi | Troglitazone[END_REF]. Briefly, troglitazone is able to impair mitochondrial function by different mechanisms including opening of the MPT pore and inhibition of the different MRC complexes [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Nadanaciva | Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration[END_REF][START_REF] Okuda | Multiple mechanisms underlying troglitazone-induced mitochondrial permeability transition[END_REF]. Interestingly, troglitazone hepatotoxicity in mice is significantly favored in the context of partial deficiency in MnSOD (encoded by the sod2 gene) [START_REF] Lee | Integrative toxicoproteomics implicates impaired mitochondrial glutathione import as an off-target effect of troglitazone[END_REF][START_REF] Ong | Troglitazone-induced hepatic necrosis in an animal model of silent genetic mitochondrial abnormalities[END_REF], a major mitochondrial antioxidant enzyme [START_REF] Azadmanesh | A review of the catalytic mechanism of human manganese superoxide dismutase[END_REF][START_REF] Degoul | Homozygosity for alanine in the mitochondrial targeting sequence of superoxide dismutase and risk for severe alcoholic liver disease[END_REF]. In addition, troglitazone could also indirectly impair MRC activity by inducing mtDNA depletion, possibly as a consequence of oxidative mtDNA damage such as strand breaks [START_REF] Hu | Characterizing the mechanism of thiazolidinedione-induced hepatotoxicity: An in vitro model in mitochondria[END_REF][START_REF] Rachek | Troglitazone, but not rosiglitazone, damages mitochondrial DNA and induces mitochondrial dysfunction and cell death in human hepatocytes[END_REF]. Hence, this mechanism of troglitazone-induced mtDNA depletion seems similar to what has been proposed for APAP (section 4.4.1), ethanol [START_REF] Mansouri | An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice[END_REF][START_REF] Mansouri | Acute ethanol administration oxidatively damages and depletes mitochondrial DNA in mouse liver, brain, heart, and skeletal muscles: protective effects of antioxidants[END_REF] and CCl 4 [START_REF] Knockaert | Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver[END_REF]. Recently, two studies reported that the antiepileptic drug valproic acid (VPA) disturbed the methylation status of the mitochondrial genome [START_REF] Wolters | Nuclear and mitochondrial DNA methylation patterns induced by valproic acid in human hepatocytes[END_REF][START_REF] Wolters | Integrated 'omics analysis reveals new drug-induced mitochondrial perturbations in human hepatocytes[END_REF]. More specifically, a 2-day treatment of human hepatocytes with VPA (15 mM) increased the methylation of mtDNA [START_REF] Wolters | Integrated 'omics analysis reveals new drug-induced mitochondrial perturbations in human hepatocytes[END_REF], whereas a longer treatment (e.g. 5 days) in the same cellular model was associated with a hypomethylation of the mitochondrial genome [START_REF] Wolters | Nuclear and mitochondrial DNA methylation patterns induced by valproic acid in human hepatocytes[END_REF].

However, further investigations will be required in order to determine whether disturbance of the methylation status of the mitochondrial genome can alter mitochondrial function and play a role in VPA-induced liver injury. Although this hypothesis is attractive, it should be reminded that numerous studies showed that VPA-induced hepatotoxicity is mainly the consequence of an impairment of mtFAO per se and possibly of MRC activity [START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF]Fromenty and Pessayre, 1995;[START_REF] Massart | Drug-induced mitochondrial toxicity[END_REF]. Finally, it is noteworthy that VPA treatment can trigger liver failure in individuals with preexisting mtDNA depletion due to mutations in the human DNA polymerase gamma (POLG) gene [START_REF] Schon | Alterations of mitochondrial DNA in liver diseases[END_REF][START_REF] Li | Valproic acidinduced hepatotoxicity in Alpers syndrome is associated with mitochondrial permeability transition pore opening-dependent apoptotic sensitivity in an induced pluripotent stem cell model[END_REF]. This susceptibility is probably secondary to an aggravation of preexisting MRC dysfunction but not of mtDNA depletion by itself. Indeed, in vitro investigations reported that VPA was not able to reduce mtDNA levels, although only myoblasts were investigated in this study [START_REF] Stewart | Polymerase γ gene POLG determines the risk of sodium valproate-induced liver toxicity[END_REF]. It was also reported that long-term therapy with different anti-HBV drugs increased mtDNA methylation in peripheral blood mononuclear cells in treated patients [START_REF] Madeddu | DNMT1 modulation in chronic hepatitis B patients and hypothetic influence on mitochondrial DNA methylation status during long-term nucleo(t)side analogs therapy[END_REF]. However, no investigation was carried out in liver. Thus, further studies will be necessary in order to determine whether higher mtDNA methylation could play a role in liver injury induced by these drugs.

Lastly, it is noteworthy that drugs may alter hepatic mtDNA levels via a general effect on mitochondrial biogenesis. However, only a few investigations have dealt with this issue. For instance, the antitubercular drug isoniazid was found to impair mitochondrial biogenesis in HepG2 cells via a PGC1α-dependent pathway but mtDNA levels were not measured in this study (Zhang et , 2017). Conversely, other drugs were found to stimulate mitochondrial biogenesis and increase mtDNA levels in cultured hepatocytes. Indeed, non-cytotoxic concentrations of APAP (0.5 and 1 mM) increased the mtDNA copy number in HepG2 cells, an effect that might have been triggered by NFE2L2 (Nrf2) activation (Zhang et al., 2016). We also found that mtDNA levels were enhanced in differentiated HepaRG cells treated with imipramine, amiodarone, carbamazepine, terbinafine and troglitazone, which were used at concentrations inducing slight or no cytotoxicity (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. For amiodarone and imipramine, the robust increase in phospho-AMPKα levels and PGC1α mRNA expression could suggest a bona fide activation of mitochondrial biogenesis (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. However, we did not identify in this study the cue(s) responsible for AMPK activation such as increased AMP/ATP ratio, ROS and reactive nitrogen species [START_REF] Cardaci | Redox implications of AMPK-mediated signal transduction beyond energetic clues[END_REF][START_REF] Sid | Role of AMPK activation in oxidative cell damage: Implications for alcohol-induced liver disease[END_REF]. For amiodarone, carbamazepine and troglitazone, we also found that the increase in mtDNA levels observed in treated HepaRG cells was significantly blunted when cells were concomitantly incubated with fatty acids (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. Hence, these results suggest that adaptive mitochondrial biogenesis triggered by some drugs might be impaired in the context of nonalcoholic liver disease (NAFLD) (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF]. Interestingly, basal hepatic mitochondrial biogenesis seems to be impaired in NAFLD [START_REF] Aharoni-Simon | Fatty liver is associated with impaired activity of PPARγ-coactivator 1α (PGC1α) and mitochondrial biogenesis in mice[END_REF][START_REF] Handa | Reduced adiponectin signaling due to weight gain results in nonalcoholic steatohepatitis through impaired mitochondrial biogenesis[END_REF].

Conclusions

Drug-induced impairment of hepatic mtDNA homeostasis and secondary mitochondrial dysfunction can have tragic consequences for patients with the occurrence of liver failure and severe lactic acidosis, which can eventually lead to death. Although this mechanism of mitochondrial toxicity might be rarer than direct inhibition of key mitochondrial enzymes involved in OXPHOS, TCA or mtFAO, it might actually be underestimated. Indeed, preclinical and postmarketing safety studies do not classically include specific investigations in order to detect drug-A c c e p t e d M a n u s c r i p t induced effects on mtDNA integrity and maintenance. Different reasons could explain this fact. For instance, investigations looking for impairment of mtDNA translation require particular assays or tools such as an in vitro mitochondrial protein synthesis assay [START_REF] Mckee | Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones[END_REF][START_REF] Nagiec | Oxazolidinones inhibit cellular proliferation via inhibition of mitochondrial protein synthesis[END_REF], or antibodies specifically targeting mtDNA-encoded polypeptides (Le [START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF][START_REF] Will | Mitochondrial toxicity assessment in industry -a decade of technology development and insight[END_REF]. Moreover, in vitro or in vivo detection of drug-induced alteration of mtDNA homeostasis and its functional consequences can require several days (or weeks) of treatment [START_REF] Larosche | Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver[END_REF][START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF][START_REF] Mansouri | Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver[END_REF][START_REF] Will | Mitochondrial toxicity assessment in industry -a decade of technology development and insight[END_REF]. Hence, rapid and high-throughput assays cannot detect such mechanism of mitochondrial toxicity. Thus, efforts should be made in the future in order to look more often for mtDNA homeostasis impairment, in particular during preclinical safety studies. Hopefully, these endeavors might improve the safety profile of drugs that will enter clinical trials in the future. Finally, it should be underlined that investigations on mtDNA homeostasis cannot be restricted to antiviral drugs and antibiotics. Indeed, other pharmacological classes could be involved, as illustrated for example by studies on tacrine, tamoxifen and imipramine [START_REF] Larosche | Tamoxifen inhibits topoisomerases, depletes mitochondrial DNA, and triggers steatosis in mouse liver[END_REF][START_REF] Guillou | Drug-induced alterations of mitochondrial DNA homeostasis in steatotic and nonsteatotic HepaRG cells[END_REF][START_REF] Mansouri | Tacrine inhibits topoisomerases and DNA synthesis to cause mitochondrial DNA depletion and apoptosis in mouse liver[END_REF]. Note and Simon Bucher. I am also grateful to Eric Schon who welcome me in his laboratory at Columbia University for my post-doctoral training. This allowed me to discover many aspects of the mitochondrial genome, which is small and beautiful [START_REF] Borst | Small is beautiful -portrait of a mitochondrial genome[END_REF]. Zhang, T., Zhang, Q., Guo, J., Yuan, H., Peng, H., Cui, L., Yin, J., Zhang, L., Zhao, J., Li, J., White, A., Carmichael, P.L., Westmoreland, C., Peng, S., 2016. Non-cytotoxic concentrations of acetaminophen induced mitochondrial biogenesis and antioxidant response in HepG2 cells. Environ. Toxicol. Pharmacol. 46, 71-79. Zhang, T., Ikejima, T., Li, L., Wu, R., Yuan, X., Zhao, J., Wang, Y., Peng, S., 2017

  s c r i p t mtDNA replication machinery involves different proteins including DNA polymerase γ, mtSSB and

  Other mechanisms of drug-induced impairment of mtDNA homeostasis

Figure 1 .

 1 Figure 1. Deleterious consequences of impaired mitochondrial DNA homeostasis. Normal

Figure 2 .

 2 Figure 2. Drugs inducing impairment of mitochondrial DNA homeostasis and involved

  Figure1

Acknowledgments

I am grateful to INSERM (Institut National de la Santé et de Recherche Médicale) for its constant financial support. I would also like to thank different current and previous collaborators for their significant contribution to investigations carried out in the field of mitochondrial DNA and hepatotoxicity. In particular, I have a special thought for my mentor Dominique Pessayre, but also for Abdellah Mansouri, Alain Berson, Anissa Igoudjil, Caroline Maisonneuve, Christine Demeilliers, Dounia Le Guillou, Isabelle Gaou, Isabelle Larosche, Karima Begriche, Marie-Anne Robin, Reine

Conflict of interest disclosure

The author declares that there is no conflict of interest regarding this work.