Pierre-Jean Ferron

Thomas Gicquel

Bruno Mégarbane

Bruno Clément

Bernard Fromenty
email: bernard.fromenty@inserm.fr

Treatments in Covid-19 patients with pre-existing metabolic dysfunctionassociated fatty liver disease: a potential threat for drug-induced liver injury?

Keywords:

Obese patients who often present metabolic dysfunction-associated fatty liver disease (MAFLD) are at risk of severe presentation of coronavirus disease 2019 . These patients are more likely to be hospitalized and receive antiviral agents and other drugs required to treat acute respiratory distress syndrome and systemic inflammation, combat bacterial and fungal superinfections and reverse multiorgan failure. Among these pharmaceuticals, antiretrovirals such as lopinavir/ritonavir and remdesivir, antibiotics and antifungal agents can induce drug-induced liver injury (DILI), whose mechanisms are not always understood. In the present article, we hypothesize that obese COVID-19 patients with MAFLD might be at higher risk for DILI than non-infected healthy individuals or MAFLD patients.

These patients present several concomitant factors, which individually can favour DILI: polypharmacy, systemic inflammation at risk of cytokine storm, fatty liver and sometimes nonalcoholic steatohepatitis (NASH) as well as insulin resistance and other diseases linked to obesity.

Hence, in obese COVID-19 patients, some drugs might cause more severe (and/or more frequent) DILI, while others might trigger the transition of fatty liver to NASH, or worsen pre-existing steatosis, necroinflammation and fibrosis. We also present the main mechanisms whereby drugs can be more hepatotoxic in MAFLD including impaired activity of xenobiotic-metabolizing enzymes, mitochondrial dysfunction, altered lipid homeostasis and oxidative stress. Although comprehensive investigations are needed to confirm our hypothesis, we believe that the current epidemic of obesity and related metabolic diseases has extensively contributed to increase the number of cases of DILI in COVID-19 patients, which may have participated in presentation severity and death.

COVID-19, obesity and related metabolic diseases

Since late 2019, the emergence of a new coronavirus called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) has become a worldwide threat for human health. This coronavirus is responsible for a self-limiting disease of the upper airways, called coronavirus disease 2019 (COVID- [START_REF] Boeckmans | COVID-19 and drug-induced liver injury: a problem of plenty or a petty point?[END_REF], which may result in intensive care unit (ICU) admission and fatalities [START_REF] Jothimani | COVID-19 and Liver[END_REF]. Several studies suggested that obesity, hypertension and diabetes greatly increase the risk of severe and prolonged COVID-19 presentation [START_REF] Argenziano | Characterization and clinical course of 1000 patients with coronavirus disease 2019 in New York: retrospective case series[END_REF][START_REF] Hajifathalian | Obesity is associated with worse outcomes in COVID-19: Analysis of Early Data From New York City[END_REF][START_REF] Simonnet | High Prevalence of Obesity in Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Requiring Invasive Mechanical Ventilation[END_REF]. However, it is still unclear why obesity and related metabolic disorders enhance COVID-19 severity. It has been postulated that the chronic pro-inflammatory state associated with these metabolic diseases may play a role, at least in part due to an activation of the reninangiotensin system [START_REF] Bornstein | Endocrine and metabolic link to coronavirus infection[END_REF][START_REF] Engin | Two important controversial risk factors in SARS-CoV-2 infection: Obesity and smoking[END_REF][START_REF] Hussain | COVID-19 and diabetes: Knowledge in progress[END_REF]. Hence, this pre-existing pro-inflammatory background seems to favour the cytokine storm, which may result in the multi-organ failure observed in severe COVID-19 [START_REF] Muniyappa | COVID-19 pandemic, coronaviruses, and diabetes mellitus[END_REF][START_REF] Ryan | Is Adipose Tissue a Reservoir for Viral Spread, Immune Activation, and Cytokine Amplification in Coronavirus Disease 2019?[END_REF][START_REF] Omarjee | Targeting T-cell senescence and cytokine storm with rapamycin to prevent severe progression in COVID-19[END_REF].

Importantly, metabolic dysfunction-associated fatty liver disease (MAFLD, the term now replacing nonalcoholic fatty liver disease or NAFLD), which is present in the majority of obese people, was also found to be associated with severe COVID-19 [START_REF] Ji | Non-alcoholic fatty liver diseases in patients with COVID-19: A retrospective study[END_REF][START_REF] Targher | Risk of severe illness from COVID-19 in patients with metabolic dysfunction-associated fatty liver disease and increased fibrosis scores[END_REF].

COVID-19, liver function tests and histology

Liver function tests (LFTs) classically include the measurement in plasma (or serum) of alanine and aspartate aminotransferases (ALT and AST, reflecting hepatocyte cytolysis), alkaline phosphatase and γ-glutamyltransferase (ALP and GGT, reflecting cholestasis) as well as bilirubin and albumin (reflecting alterations of specific hepatic biological functions). A prolonged prothrombin time (PT) can also reflect an alteration of the hepatic synthesis of different coagulation factors. Abnormal LFTs is observed in up to 54% of COVID-19 patients, although severe liver dysfunction seems rare and mostly associated with poor disease outcome [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF]. The most frequent abnormal LFT in COVID-19 patients is an elevation in ALT and AST, which is commonly mild (i.e. <5 times the upper reference limit) [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF]. Patients admitted to the ICU are more likely to present high levels of ALT, AST and bilirubin as well as prolonged PT [START_REF] Jothimani | COVID-19 and Liver[END_REF][START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF]. Different factors seem to cause abnormal LFTs and liver dysfunction in COVID-19 patients. SARS CoV-2 by itself may play a role because its receptor ACE2 (angiotensin-converting enzyme 2) was detected in cholangiocytes and to a much lesser degree in hepatocytes, whereas SARS CoV-2 RNA was found in liver samples [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF]. Hence, elevation in ALP and GGT in some COVID-19 patients J o u r n a l P r e -p r o o f may reflect viral-induced cholangiocyte injury [START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF]. However, the direct cytopathic effects of the virus have been questioned by some authors [START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF][START_REF] Philips | SARS-CoV-2 related liver impairment -perception may not be the reality[END_REF]. The cytokine storm may also be involved since different pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) can be harmful to the liver [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Fan | Clinical Features of COVID-19-Related Liver Functional Abnormality[END_REF][START_REF] Zhang | Liver injury in COVID-19: management and challenges[END_REF]. Prolonged hypoxia due to acute respiratory distress syndrome (ARDS), shock and blood hyperviscosity may play a role in critically ill patients [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Zhang | Liver injury in COVID-19: management and challenges[END_REF]. The role of pre-existing liver disease such as MAFLD and cirrhosis should be considered in some patients [START_REF] Jothimani | COVID-19 and Liver[END_REF][START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF]. Finally, druginduced liver injury may be involved [START_REF] Jothimani | COVID-19 and Liver[END_REF][START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Boeckmans | COVID-19 and drug-induced liver injury: a problem of plenty or a petty point?[END_REF], as discussed below in Section 4.

Post-mortem liver histology performed in some COVID-19 patients showed different liver lesions including lobular focal necrosis, apoptosis, microvesicular and macrovacuolar steatosis, inflammatory infiltrates in the portal areas and sinusoidal dilatation [START_REF] Jothimani | COVID-19 and Liver[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF][START_REF] Philips | SARS-CoV-2 related liver impairment -perception may not be the reality[END_REF]. However, these lesions are not specific and may be observed in different settings such as MAFLD, sepsis or DILI [START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Philips | SARS-CoV-2 related liver impairment -perception may not be the reality[END_REF].

Nonetheless, the presence of microvesicular steatosis in some patients suggests severe mitochondrial dysfunction, which can be induced (or favoured) by numerous factors including congenital enzymatic deficiencies and drugs [START_REF] Fromenty | Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity[END_REF].

Treatments in COVID-19 patients

COVID-19 patients can be treated by two types of drugs. On the first hand, various drugs with anti-SARS CoV-2 properties are currently being tested although their clinical benefit is still uncertain, and sometimes controversial. The main drugs currently under investigation include lopinavir/ritonavir combination, remdesivir and hydroxychloroquine, which can be associated with azithromycin (Table 1). Regarding the association of the protease inhibitors (PIs) lopinavir/ritonavir, it should be mentioned that the second PI is used to enhance lopinavir pharmacological effect by inhibiting cytochrome P450 3A4 (CYP3A4) activity [START_REF] Li | CPY3A4-Mediated Lopinavir Bioactivation and Its Inhibition by Ritonavir[END_REF]. Readers are invited to peruse recent reviews regarding the pharmacological properties of these different drugs [START_REF] Barlow | Review of Emerging Pharmacotherapy for the Treatment of Coronavirus Disease[END_REF][START_REF] Martinez | Compounds with Therapeutic Potential against Novel Respiratory 2019 Coronavirus[END_REF][START_REF] Mégarbane | Hydroxychloroquine and Azithromycin to Treat Patients With COVID-19: Both Friends and Foes?[END_REF][START_REF] Sanders | Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review[END_REF]. These reviews also discuss the possible effectiveness of other drugs with anti-SARS CoV-2 effects.

On the other hand, COVID-19 patients may receive drugs routinely used during the course of viral illnesses with possible onset of bacterial and fungal superinfections, ARDS, systemic inflammation and even multi-organ failure. These drugs include antibiotics such as macrolides (other than azithromycin) and beta-lactams, antifungal agents such as azole derivatives, immunomodulatory agents such as corticosteroids (e.g. dexamethasone, methylprednisolone) and monoclonal antibodies such as the anti-interleukin-6 (IL-6) receptor tocilizumab or the anti-IL-1 receptor anakinra [START_REF] Barlow | Review of Emerging Pharmacotherapy for the Treatment of Coronavirus Disease[END_REF][START_REF] Martinez | Compounds with Therapeutic Potential against Novel Respiratory 2019 Coronavirus[END_REF][START_REF] Sanders | Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review[END_REF][START_REF] Filocamo | Use of anakinra in severe COVID-19: A case report[END_REF]. Anticoagulant treatments, mostly heparin and low-molecular-weight heparin, the J o u r n a l P r e -p r o o f sympathomimetic amine norepinephrine and different sedative agents (e.g. midazolam, propofol, fentanyl) can also be required in critically ill COVID-19 patients. Notably, although acetaminophen (APAP, or paracetamol) is rarely prescribed as antipyretic drug in ICU patients, this drug is frequently used prior to hospitalization [START_REF] Rodríguez-Morales | Gastroenterologists, Hepatologists, COVID-19 and the Use of Acetaminophen[END_REF]. Hence, clinicians should not overlook the possibility of major acetaminophen self-administration in COVID-19 patients. This analgesic and antipyretic drug will be discussed below.

Drug-induced hepatotoxicity in COVID-19

Although liver injury is not the primary cause of death in COVID-19, elevated serum ALT and AST levels are often observed in hospitalized patients [START_REF] Jothimani | COVID-19 and Liver[END_REF][START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF][START_REF] Fan | Clinical Features of COVID-19-Related Liver Functional Abnormality[END_REF][START_REF] Zhang | Liver injury in COVID-19: management and challenges[END_REF][START_REF] Garrido | Review article: COVID-19 and liver disease-what we know on 1st May 2020[END_REF]. Hepatotoxicity may represent the first cause of liver injury [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF][START_REF] Fan | Clinical Features of COVID-19-Related Liver Functional Abnormality[END_REF][START_REF] Zhang | Liver injury in COVID-19: management and challenges[END_REF][START_REF] Garrido | Review article: COVID-19 and liver disease-what we know on 1st May 2020[END_REF], although it should be underlined that assessing DILI in a clinical setting where abnormal LFTs can be induced by pre-existing liver diseases and COVID-19 is a particularly difficult issue. Many drugs prescribed in these patients are potentially hepatotoxic. For instance, lopinavir, ritonavir, remdesivir, hydroxychloroquine, azithromycin and tocilizumab can induce liver injury, sometimes even severe (Table 1). Other drugs such as carbapenems (e.g. imipenem, meropenem), azole derivatives (e.g. ketoconazole, fluconazole), corticosteroids and propofol can also be hepatotoxic [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF]LiverTox: Clinical and Research Information on Drug-Induced Liver Injury[END_REF]. DILI and other adverse effects in COVID-19 patients may be precipitated by acute cardiovascular failure and kidney injury, resulting in drug overdose by reducing drug metabolism and elimination. Notably, acetaminophen-induced liver injury can be favoured by increased hepatic CYP2E1 activity [START_REF]LiverTox: Clinical and Research Information on Drug-Induced Liver Injury[END_REF], which is often observed in individuals with heavy alcohol consumption or in patients suffering from MAFLD, as discussed in section 7.1.

Clearly, future investigations will be required to determine which of these drugs could be involved in the elevated serum aminotransferases commonly observed in COVID-19 patients. It should be pointed out that accountability assessment in cases with suspected DILI is mainly based on chronological and clinical criteria as well as the elimination of other causes of liver injury [START_REF] Meunier | Drug-Induced Liver Injury: Biomarkers, Requirements, Candidates, and Validation[END_REF]. Drug accountability can be evaluated by different methods such as the Roussel Uclaf Causality Assessment Method (RUCAM) [START_REF] Meunier | Drug-Induced Liver Injury: Biomarkers, Requirements, Candidates, and Validation[END_REF][START_REF] Danan | RUCAM in Drug and Herb Induced Liver Injury: The Update[END_REF]. Of note, other methodological approaches are required for causality assessment of suspected DILI in patients with pre-existing nonalcoholic steatohepatitis (NASH) [START_REF] Regev | Consensus: guidelines: best practices for detection, assessment and management of suspected acute druginduced liver injury during clinical trials in patients with nonalcoholic steatohepatitis[END_REF].

Hypothesis: are Covid-19 patients with pre-existing MAFLD at higher risk for DILI?

J o u r n a l P r e -p r o o f Herein, we hypothesize that obese COVID-19 patients with MAFLD may be at higher risk for DILI than non-infected healthy individuals or MAFLD patients. This assumption is based on the fact that these patients present several concomitant factors, which individually can favour DILI (Fig. 1).

First, as previously mentioned, many drugs prescribed in COVID-19 patients are potentially harmful for the liver, and thus polypharmacy is expected to enhance the risk of hepatotoxicity. Noteworthy, numerous drug-drug interactions potentially exist with the pharmaceuticals prescribed in COVID-19 patients, thus enhancing the risk of toxicity [START_REF] Hodge | Drug interactions: a review of the unseen danger of experimental COVID-19 therapies[END_REF]. Second, the cytokine storm potentially associated with severe COVID-19 can favour DILI. It has been shown that different pro-inflammatory cytokines such as TNF-α, IL1-β and IL-6 can significantly enhance the hepatotoxicity potential of numerous drugs including antibiotics [START_REF] Cosgrove | Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity[END_REF][START_REF] Sharanek | Pro-inflammatory cytokines enhance dilatation of bile canaliculi caused by cholestatic antibiotics[END_REF]. Third, MAFLD might even further enhance the risk of DILI.

There are increasing clinical reports (sometimes confirmed by experimental studies) suggesting that some drugs can be considered as more hepatotoxic in obese patients with MAFLD, when compared with lean individuals [START_REF] Tarantino | A prospective study of acute drug-induced liver injury in patients suffering from non-alcoholic fatty liver disease[END_REF][START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Lammert | Patients With Chronic Liver Disease Suggestive of Nonalcoholic Fatty Liver Disease May Be at Higher Risk for Drug-Induced Liver Injury[END_REF][START_REF] Li | Metabolic Comorbidities and Risk of Development and Severity of Drug-Induced Liver Injury[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF].

DILI in MAFLD seems to occur as two distinct clinical situations [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF]. First, some drugs such as acetaminophen and some antibiotics may cause more severe and/or more frequent acute liver injury (Table 2) [START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] García-Román | Acetaminophen-Induced Liver Damage in Hepatic Steatosis[END_REF]. Regarding, acetaminophen, it should be pointed out that its hepatotoxicity is mainly observed after overdose but some cases of liver injury (sometimes severe) may also occur with recommended therapeutic dosing [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Claridge | Acute liver failure after administration of paracetamol at the maximum recommended daily dose in adults[END_REF][START_REF] Bouvet | Fatal acetaminophen poisoning with hepatic microvesicular steatosis in a child after repeated administration of therapeutic doses[END_REF]. Other pharmaceuticals including corticoids, antiretroviral agents and methotrexate seem to trigger the transition of simple fatty liver to NASH or worsen pre-existing steatosis, necroinflammation and fibrosis [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Meunier | Chemotherapy-associated steatohepatitis[END_REF][START_REF] Mori | Incidence, predictive factors, and severity of methotrexate-related liver injury in rheumatoid arthritis: A longitudinal cohort study[END_REF]. The mechanisms whereby some pharmaceuticals can be more hepatotoxic in MAFLD are presented in section 7. Lastly, it should be underlined that not all drugs may be at risk in MAFLD. For instance, statins and amiodaroneinduced hepatotoxicity does not seem to be more frequent in MAFLD patients [START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF].

How to test this hypothesis?

Because our hypothesis is predicting a non-established fact, cohort or registry studies are required in order to determine whether obese COVID-19 patients present increased risk of DILI. However, it should be kept in mind that hepatotoxicity is a rare event since DILI occurs at the most in about 1 per 10,000 treated patients [START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF]. This clinical approach can be complemented by experimental investigations in appropriate experimental models of MAFLD. Recently, we set up a cellular model of MAFLD by using metabolically competent HepaRG cells incubated with stearic acid (100 µM) for 7 days [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF] or with a mixture of stearic and oleic acids (150 µM each) for 14 days [START_REF] Bucher | Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure[END_REF][START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF][START_REF] Bucher | Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo[END_REF]. These in J o u r n a l P r e -p r o o f vitro models of MAFLD were characterized by reduced CY3A4 activity and increased CYP2E1 activity, thus reproducing what has been reported in clinical studies [START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Brill | Impact of Obesity on Drug Metabolism and Elimination in Adults and Children[END_REF][START_REF] Cobbina | Non-alcoholic fatty liver disease (NAFLD) -pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[END_REF], as discussed below.

In addition, these models unveiled increased cytotoxicity of acetaminophen [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF], troglitazone [START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF] and a mixture of benzo[a]pyrene and ethanol [START_REF] Bucher | Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure[END_REF][START_REF] Bucher | Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo[END_REF] in MAFLD cells compared with non-steatotic cells.

Regarding acetaminophen [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF], these in vitro investigations confirmed previous studies carried out in obese mice with MAFLD [START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] García-Román | Acetaminophen-Induced Liver Damage in Hepatic Steatosis[END_REF][START_REF] Aubert | Differences in Early Acetaminophen Hepatotoxicity between Obese ob/ob and db/db Mice[END_REF]. In contrast, we found that ritonavir was less cytotoxic in MAFLD cells compared with the non-steatotic cells [START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF]. Nonetheless, this cellular model of MAFLD is useful to determine whether drugs such as lopinavir, remdesivir, hydroxychloroquine and azithromycin are more toxic in this dysmetabolic state. The HepaRG cell model will also be useful to perform mechanistic studies in particular by investigating mitochondrial dysfunction, de novo lipogenesis, reactive oxygen species (ROS) overproduction and endoplasmic reticulum (ER) stress [START_REF] Bucher | Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure[END_REF][START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF][START_REF] Bucher | Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo[END_REF][START_REF] Allard | Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanismbased toxicity[END_REF], as discussed below.

Importantly, the above-described in vitro models of DILI using cells incubated with fatty acids should be refined in order to tally with the COVID-19 context. To this end, steatotic HepaRG cells could be cultured in the presence of SARS CoV-2-like particles. However, it should be mentioned that these cells express low levels of ACE2 and transmembrane serine protease 2 (TMPRSS2), which is also involved in the viral cellular entry [START_REF] Bornstein | Endocrine and metabolic link to coronavirus infection[END_REF][START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF]. Hence, HepaRG cells will need to be engineered to stably express these proteins. It will also be important to perform HepaRG culture in the presence of different pro-inflammatory cytokines and low oxygen levels to mimic the cytokine storm and hypoxia, respectively. Although these in vitro models are difficult to set up, they may provide invaluable information regarding drug-induced hepatotoxicity in the setting of COVID-19.

How can drugs be more hepatotoxic in MAFLD?

The mechanisms whereby drugs and xenobiotics are more hepatotoxic in MAFLD are not fully understood and are currently the matter of extensive in vitro and in vivo investigations due to the worldwide epidemic of obesity [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Bucher | Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure[END_REF][START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF][START_REF] Bucher | Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo[END_REF][START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF]. These mechanisms are currently known only for a few compounds [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF]. Hence, the mechanisms of DILI in MAFLD proposed herein represent the state of the art.

Acute liver injury in MAFLD

J o u r n a l P r e -p r o o f Some drugs may induce more severe acute liver injury in MAFLD because this disease is associated with altered activity of different xenobiotic-metabolizing enzymes (XMEs) including cytochromes P450 (CYPs) [START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Brill | Impact of Obesity on Drug Metabolism and Elimination in Adults and Children[END_REF][START_REF] Cobbina | Non-alcoholic fatty liver disease (NAFLD) -pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[END_REF]. This can increase the generation of toxic metabolites, or conversely impair different detoxification pathways (Fig. 2) [START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Brill | Impact of Obesity on Drug Metabolism and Elimination in Adults and Children[END_REF][START_REF] Cobbina | Non-alcoholic fatty liver disease (NAFLD) -pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[END_REF][START_REF] Zuckerman | A Review of the Toxicologic Implications of Obesity[END_REF]]. For instance, human MALFD is often associated with increased CYP2E1 activity and reduced CYP3A4 activity and also with higher glucuronide formation, at least with some drugs such as lorazepam and acetaminophen [START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Brill | Impact of Obesity on Drug Metabolism and Elimination in Adults and Children[END_REF][START_REF] Cobbina | Non-alcoholic fatty liver disease (NAFLD) -pathogenesis, classification, and effect on drug metabolizing enzymes and transporters[END_REF]. Although acetaminophen undergoes higher glucuronide formation in MAFLD, its increased hepatotoxicity in this metabolic disease seems primarily linked to enhanced CYP2E1 activity, which generates greater hepatic amounts of the highly toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI) [START_REF] Michaut | Acetaminophen-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF][START_REF] Aubert | Differences in Early Acetaminophen Hepatotoxicity between Obese ob/ob and db/db Mice[END_REF].

Increased generation of toxic metabolites or higher concentrations of the parent drugs can trigger cell death by necrosis and apoptosis. Regardless of the involved drugs, cell death commonly involves mitochondrial dysfunction that causes severe impairment of oxidative phosphorylation (OXPHOS), ATP shortage and necrosis. Alternatively, drug-induced mitochondrial dysfunction induces the release of pro-apoptotic factors such as cytochrome c and apoptosis inducing factor (AIF), thus leading to caspase activation and apoptosis. These mitochondrial events in necrosis and apoptosis are associated with ROS overproduction and oxidative stress as well as ER stress (Fig. 2), which can already be present in MAFLD [START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF][START_REF] Chen | Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease, Free Radic[END_REF]. Hence, drugs can worsen pre-existing impairment of mitochondrial respiratory chain (MRC) and OXPHOS, ROS overproduction and different cellular stresses. Readers are invited to peruse previous reviews to get further information on drug-induced mitochondrial dysfunction and cell death [START_REF] Pessayre | Central role of mitochondria in drug-induced liver injury[END_REF][START_REF] Pessayre | Mitochondrial Involvement in Drug-Induced Liver Injury[END_REF][START_REF] Ramachandran | Mitochondrial dysfunction as a mechanism of drug-induced hepatotoxicity: current understanding and future perspectives[END_REF][START_REF] Andrade | Drug-induced liver injury[END_REF].

The mechanisms whereby MAFLD is frequently associated with CYP2E1 induction are still unclear. Some investigations suggest the role of insulin resistance and fatty acids such as stearic acid (Fig. 2) [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF][START_REF] Massart | Cytochrome P450 2E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis[END_REF]. In keeping with the role of specific lipid derivatives and possibly free fatty acids, previous investigations reported that accumulation of hepatic triglycerides is not sufficient by itself to enhance CYP2E1 activity [START_REF] Aubert | Differences in Early Acetaminophen Hepatotoxicity between Obese ob/ob and db/db Mice[END_REF]. Interestingly, ketone bodies may also play a significant role in hepatic CYP2E1 induction [START_REF] Massart | Cytochrome P450 2E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis[END_REF]. Acetone and acetoacetate have been shown to increase CYP2E1 protein expression in rodent liver by reducing CYP2E1 protein degradation [START_REF] Massart | Cytochrome P450 2E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis[END_REF][START_REF] Abdelmegeed | Acetoacetate Induces CYP2E1 Protein and Suppresses CYP2E1 mRNA in Primary Cultured Rat Hepatocytes[END_REF]. In addition, acetoacetate enhances translation of Cyp2e1 mRNA in the rat [START_REF] Abdelmegeed | Acetoacetate Induces CYP2E1 Protein and Suppresses CYP2E1 mRNA in Primary Cultured Rat Hepatocytes[END_REF]. However, although both mitochondrial fatty acid oxidation (mtFAO) and ketogenesis are deemed to be enhanced in early MAFLD [START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF][START_REF] Sunny | Mitochondrial Adaptation in Nonalcoholic Fatty Liver Disease: Novel Mechanisms and Treatment Strategies[END_REF], ketogenesis per se seems to be progressively impaired during the evolution of the disease towards NASH [START_REF] Sunny | Mitochondrial Adaptation in Nonalcoholic Fatty Liver Disease: Novel Mechanisms and Treatment Strategies[END_REF][START_REF] Fletcher | Impaired ketogenesis and increased acetyl-CoA oxidation promote hyperglycemia in human fatty liver[END_REF]. Hence, the role of ketone bodies in CYP2E1 induction may only be transient in MAFLD. Finally, high blood glucose concentration may increase CYP2E1 expression in liver, even though conflicting results have been reported [START_REF] Massart | Cytochrome P450 2E1 should not be neglected for acetaminophen-induced liver injury in metabolic diseases with altered insulin levels or glucose homeostasis[END_REF].

J o u r n a l P r e -p r o o f

Much less is known regarding the mechanisms whereby MAFLD is commonly associated with lower expression and activity of CYP3A4. Previous investigations in the HepaRG cell line also suggested the role of stearic acid [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF], but it is still unknown why this long-chain saturated fatty acid can concomitantly decrease and increase the expression and activity of CYP3A4 and CYP2E1, respectively. Another study pointed to a role of the fibroblast growth factor 21-pregnane X receptor (PXR) pathway [START_REF] Woolsey | A Fibroblast Growth Factor 21-Pregnane X Receptor Pathway Downregulates Hepatic CYP3A4 in Nonalcoholic Fatty Liver Disease[END_REF], but recent investigations showed that the nuclear receptor PXR is not involved in MAFLD-associated CYP3A4 downregulation [START_REF] Zeng | CYP3A suppression during diet-induced nonalcoholic fatty liver disease is independent of PXR regulation[END_REF].

In contrast to CYP2E1 which metabolizes only a few drugs in addition to acetaminophen [START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF],

CYP3A4 plays a major role in the metabolism of numerous pharmaceuticals including ritonavir and other antiretroviral PIs, remdesivir, hydroxychloroquine, midazolam and other benzodiazepines, fentanyl and different antibiotics [START_REF] Li | CPY3A4-Mediated Lopinavir Bioactivation and Its Inhibition by Ritonavir[END_REF][START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF][START_REF] Guengerich | Cytochrome P-450 3A4: Regulation and Role in Drug Metabolism[END_REF][START_REF] Josephson | Drug-drug interactions in the treatment of HIV infection: focus on pharmacokinetic enhancement through CYP3A inhibition: Drug-drug interactions in the treatment of HIV infection[END_REF][START_REF] Jallouli | Plaquenil Lupus Systemic Study Group, Determinants of hydroxychloroquine blood concentration variations in systemic lupus erythematosus[END_REF][START_REF] Yang | What do we know about remdesivir drug interactions?[END_REF]. Hence, MAFLD-associated reduction in CYP3A4 activity impairs the biotransformation of several drugs prescribed in COVID-19 patients. The risk of drug-induced hepatotoxicity will be enhanced or reduced depending on whether CYP3A4 is involved in drug detoxication or in the generation of toxic reactive metabolites (Fig. 2). For instance, troglitazone is suspected to have induced severe hepatotoxicity in MAFLD due to lower CYP3A4mediated biotransformation of this antidiabetic drug [START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF]. Finally, it should be pointed that numerous drugs such as ritonavir, different antibiotics such as macrolides and fluoroquinolones and several antifungal azole derivatives such as ketoconazole and itraconazole are potent CYP3A4 inhibitors [START_REF] Josephson | Drug-drug interactions in the treatment of HIV infection: focus on pharmacokinetic enhancement through CYP3A inhibition: Drug-drug interactions in the treatment of HIV infection[END_REF][START_REF] Pea | Pharmacokinetic Aspects of Treating Infections in the Intensive Care Unit: Focus on Drug Interactions[END_REF]. Hence, these drugs could significantly reinforce MAFLD-associated impairment of CYP3A4 activity.

Drug-induced aggravation of MAFLD

As previously mentioned, different drugs can aggravate pre-existing liver lesions classically observed in MAFLD including fatty liver, necroinflammation and fibrosis, or trigger the transition of simple fatty liver to NASH (Table 3) [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Meunier | Chemotherapy-associated steatohepatitis[END_REF][START_REF] Mori | Incidence, predictive factors, and severity of methotrexate-related liver injury in rheumatoid arthritis: A longitudinal cohort study[END_REF]. Regardless of the pathologic situation, previous investigations with several drugs and other xenobiotics strongly suggest a significant role of mitochondrial dysfunction, ROS overproduction and ER stress (Fig. 3) [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Bucher | Possible Involvement of Mitochondrial Dysfunction and Oxidative Stress in a Cellular Model of NAFLD Progression Induced by Benzo[a]pyrene/Ethanol CoExposure[END_REF][START_REF] Bucher | Co-exposure to benzo[a]pyrene and ethanol induces a pathological progression of liver steatosis in vitro and in vivo[END_REF].

Exacerbation of pre-existing fatty liver could involve mtFAO inhibition but also impairment of very low-density lipoprotein (VLDL) secretion and increased de novo lipogenesis (DNL), namely enhanced synthesis of fatty acids from carbohydrates (Fig. 3) [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF].

Many drugs can impair mtFAO by different mechanisms including direct inhibition of mtFAO

enzymes such as carnitine palmitoyltransferase 1 (CPT1) and different acyl-CoA dehydrogenases as J o u r n a l P r e -p r o o f well as direct impairment of MRC and OXPHOS [START_REF] Fromenty | Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity[END_REF][START_REF] Pessayre | Mitochondrial Involvement in Drug-Induced Liver Injury[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF]. OXPHOS can also be impaired indirectly by alteration of mitochondrial DNA (mtDNA) homeostasis such as inhibition of mtDNA replication or translation [START_REF] Fromenty | Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Fromenty | Alteration of mitochondrial DNA homeostasis in drug-induced liver injury[END_REF]. All these adverse effects are not mutually exclusive. For instance, some drugs such as acetaminophen, amiodarone and tamoxifen can directly inhibit both mtFAO and MRC [START_REF] Fromenty | Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity[END_REF][START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF]. In addition, acetaminophen and troglitazone can impair MRC activity directly or indirectly through mtDNA depletion [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Fromenty | Alteration of mitochondrial DNA homeostasis in drug-induced liver injury[END_REF]. Finally, it should be mentioned that drug-induced inhibition of mtFAO could not only worsen pre-existing steatosis but also necrosis since this metabolic pathway is a major source of energy in hepatocytes [START_REF] Fromenty | Inhibition of mitochondrial fatty acid oxidation in drug-induced hepatic steatosis[END_REF][START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver[END_REF].

Impairment of VLDL secretion can be caused by direct inhibition of the activity of microsomal triglyceride transfer protein (MTTP, also referred to as MTP), an ER enzyme playing a key role in apolipoprotein B (apoB) lipidation [START_REF] Lettéron | Inhibition of microsomal triglyceride transfer protein: Another mechanism for drug-induced steatosis in mice[END_REF]. Alternatively, drugs can impair VLDL secretion by inducing ER stress and subsequent degradation of mRNAs of key proteins involved in VLDL assembly including MTTP, apoB and apoC3 [START_REF] Allard | Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanismbased toxicity[END_REF]. Of note, ER stress can be induced by numerous pharmaceuticals such as PIs (e.g. ritonavir, lopinavir, indinavir) and other antiretroviral agents, acetaminophen and several nonsteroidal anti-inflammatory drugs (NSAIDs) including diclofenac and indomethacin [START_REF] Allard | Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanismbased toxicity[END_REF][START_REF] Foufelle | Role of endoplasmic reticulum stress in drug-induced toxicity[END_REF].

Drug-induced enhanced hepatic DNL has been reported (or greatly suspected) with different drugs such as antiretroviral agents, amiodarone, benzbromarone, corticosteroids, indomethacin, rosiglitazone, sulindac and tetracyclines [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Allard | Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanismbased toxicity[END_REF]. For some of these drugs (e.g. amiodarone, rosiglitazone), mechanistic studies suggested an activation of lipogenic transcription factors such as peroxisome proliferator activated receptor gamma (PPARγ) and sterol regulatory element binding protein-1c (SREBP1c) [START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Allard | Drug-induced hepatic steatosis in absence of severe mitochondrial dysfunction in HepaRG cells: proof of multiple mechanismbased toxicity[END_REF]. With other drugs (e.g. antiretroviral agents, corticosteroids) activation of DNL could be secondary to insulin resistance and concomitant hyperinsulinemia, which in turn activates SREBP1c in the liver [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF][START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver[END_REF].

Much less is known regarding the mechanisms whereby drugs can exacerbate pre-existing inflammation and fibrosis. Previous data with some drugs and other xenobiotics suggested an important role of ROS overproduction and subsequent oxidative stress, which in turn can favour the production of pro-inflammatory cytokines (e.g. TNF-α, IL1-β, IL-6) and transforming growth factor-β (TGF-β), a key pro-fibrotic cytokine (Fig. 3) [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Mitchell | Protection against Hepatocyte Mitochondrial Dysfunction Delays Fibrosis Progression in Mice[END_REF]. In the setting of drug-induced toxicity and MAFLD, ROS overproduction can be secondary to the dysfunctional mitochondria, which can produce large amounts of superoxide anion at the level of MRC complexes I and III [START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF][START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver[END_REF].

Alternatively, ROS overproduction can be caused by CYP2E1 induction because this enzyme can produce superoxide anion and hydrogen peroxide during its catalytic cycle [START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Knockaert | Mechanisms of mitochondrial targeting of cytochrome P450 2E1: physiopathological role in liver injury and obesity: Mitochondrial CYP2E1[END_REF]. Finally, because J o u r n a l P r e -p r o o f CYP2E1 is located in the ER but also in mitochondria, ROS overproduction in these organelles can be secondary to MRC impairment and increased mitochondrial CYP2E1 expression [START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Knockaert | Mechanisms of mitochondrial targeting of cytochrome P450 2E1: physiopathological role in liver injury and obesity: Mitochondrial CYP2E1[END_REF][START_REF] Guengerich | Cytochrome P450 2E1 and its roles in disease[END_REF].

Concluding remarks

Hospitalized COVID-19 patients are often polymedicated, which can greatly increase the risk of drugdrug interactions and drug-induced adverse events. Comorbidities such as obesity, MAFLD and diabetes may additionally enhance the risk of hepatotoxicity. Even though liver injury is not the primary cause of death in COVID-19 patients, hepatic dysfunction could undoubtedly worsen the patient's condition, for instance by reducing the synthesis of coagulation proteins including factors II, V, VII, X, and fibrinogen [START_REF] Lescot | Acquired Liver Injury in the Intensive Care Unit[END_REF]. However, it should be emphasized that severe liver disease with impaired coagulation factor synthesis is rare in the setting of COVID-19, which actually is mostly associated with mild-to-moderate elevation of ALT and AST [START_REF] Bertolini | Abnormal liver function tests J o u r n a l P r e -p r o o f in COVID-19 patients: relevance and potential pathogenesis[END_REF][START_REF] Alqahtani | Liver injury in COVID-19: The current evidence[END_REF][START_REF] Li | Characteristics and Mechanism of Liver Injury in 2019 Coronavirus Disease[END_REF].

Because a vaccine against SARS CoV-2 has not been marketed yet, antiviral drugs will still be administered to COVID-19 patients in the next few months. Thus, it would be important to identify drugs responsible for a specific increased risk of liver injury in COVID-19 patients, especially in those with obesity and related metabolic diseases. To this end, well-designed prospective clinical investigations should show that LFTs are normal in COVID-19 patients before their hospitalization in order to demonstrate that hepatic dysfunction occurring after admission is induced by the virus or triggered by drugs and not related to any significant pre-existing liver disease. Experimental investigations will also be required to confirm that MAFLD can significantly enhance the risk of druginduced hepatotoxicity and investigate the involved mechanisms. Although rodent models of obesity and MAFLD can be used to reach these aims [START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF], lipid-laden hepatocytes can be more useful for preliminary drug screening [START_REF] Guillou | Drug-Induced Alterations of Mitochondrial DNA Homeostasis in Steatotic and Nonsteatotic HepaRG Cells[END_REF][START_REF] Luo | Palmitate Increases the Susceptibility of Cells to Drug-Induced Toxicity: An In Vitro Method to Identify Drugs With Potential Contraindications in Patients With Metabolic Disease[END_REF]. However, these in vitro models should evolve towards more complex experimental systems with steatotic cells cultured not only with drugs but also in the presence of SARS CoV-2-like particles, pro-inflammatory cytokines and hypoxia. All these comprehensive scientific investigations should allow to refute or confirm the hypothesis that obese COVID-19 patients with pre-existing MAFLD might be at higher risk for DILI than non-infected healthy individuals or MAFLD patients. Nevertheless, regardless of the results, data from such clinical and experimental studies will undoubtedly benefit to COVID-19 patients but also to the scientific community. Finally, beyond the COVID-19 crisis, the worldwide epidemic of obesity is expected to increase the number of cases of DILI and possibly other drug-related adverse events.

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Table 1 Liver toxicity of the main drugs with anti-SARS-CoV-2 properties and/or anti-inflammatory effects currently tested in COVID-19 patients

Drugs Pharmacological class

Hepatotoxicity

Mechanisms of hepatotoxicity

Lopinavir / ritonavir Antiretroviral protease inhibitors (PIs)

Ritonavir can induce mild to moderate (<6N) elevation in aminotransferases in ~44% of patients. Mostly presence of cytolysis but cholestasis, steatosis and fibrosis can also be present.

Lopinavir seems less hepatotoxic than ritonavir.

References: [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF]LiverTox: Clinical and Research Information on Drug-Induced Liver Injury[END_REF][START_REF] Wang | A Unifying Ontology to Integrate Histological and Clinical Observations for Drug-Induced Liver Injury[END_REF][START_REF] Olry | Drug-Induced Liver Injury and COVID-19 Infection: The Rules Remain the Same[END_REF] Mechanisms of PI-induced hepatic toxicity seem to be multiple with involvement of mitochondrial dysfunction, oxidative stress and endoplasmic reticulum stress.

PIs could also be hepatotoxic via indirect mechanisms, in particular by inducing lipodystrophy and insulin resistance.

References: [START_REF] Pessayre | Central role of mitochondria in drug-induced liver injury[END_REF][START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: Mechanistic diversity and deleterious consequences for the liver[END_REF][START_REF] Foufelle | Role of endoplasmic reticulum stress in drug-induced toxicity[END_REF][START_REF] Hoss | Lopimune-induced mitochondrial toxicity is attenuated by increased uncoupling protein-2 level in treated mouse hepatocytes[END_REF]

Remdesivir

Antiviral nucleoside analogue

Mild to moderate (<6N) elevation in aminotransferases in ~22% of patients.

Reference: [START_REF] Garrido | Review article: COVID-19 and liver disease-what we know on 1st May 2020[END_REF][START_REF] Leegwater | Drug-induced liver injury in a COVID-19 patient: potential interaction of remdesivir with Pglycoprotein inhibitors[END_REF] Currently unknown. References: [START_REF] Biour | Drug-induced liver injury; fourteenth updated edition of the bibliographic database of liver injuries and related drugs[END_REF][START_REF]LiverTox: Clinical and Research Information on Drug-Induced Liver Injury[END_REF][START_REF] Martinez | Clinical and Histologic Features of Azithromycin-Induced Liver Injury[END_REF][START_REF] Olry | Drug-Induced Liver Injury and COVID-19 Infection: The Rules Remain the Same[END_REF] Possible role of mitochondrial dysfunction and oxidative stress. Cholestasis is not related to the inhibition of liver canalicular bile salt export pump (BSEP).

Hydroxychloroquine

References: [START_REF] Yang | An updated review on druginduced cholestasis: Mechanisms and investigation of physicochemical properties and pharmacokinetic parameters[END_REF][START_REF] Li | Antibiotic-induced Elevations of Plasma Bile Acids in Rats Independent of Bsep Inhibition[END_REF][START_REF] Woodhead | Analyzing the Mechanisms Behind Macrolide Antibiotic-Induced Liver Injury Using Quantitative Systems Toxicology Modeling[END_REF][START_REF] Jiang | Mitochondrial Toxicity of Azithromycin Results in Aerobic Glycolysis and DNA Damage of Human Mammary Epithelia and Fibroblasts[END_REF] Tocilizumab Interleukin-6 (IL-6) receptor antagonist (monoclonal antibody)

Elevation in aminotransferases and acute liver injury, sometimes severe.

Reference: [START_REF] Muhović | First case of drug-induced liver injury associated with the use of tocilizumab in a patient with COVID-19[END_REF] Currently unknown.

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Table 2 Examples of drugs shown (or suspected) to induce more frequent and/or more severe acute hepatitis in the context of obesity and metabolic dysfunction-associated fatty liver disease (MAFLD). Further information is provided in previous articles [START_REF] Tarantino | A prospective study of acute drug-induced liver injury in patients suffering from non-alcoholic fatty liver disease[END_REF][START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Lammert | Patients With Chronic Liver Disease Suggestive of Nonalcoholic Fatty Liver Disease May Be at Higher Risk for Drug-Induced Liver Injury[END_REF][START_REF] Li | Metabolic Comorbidities and Risk of Development and Severity of Drug-Induced Liver Injury[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF].

Drug Therapeutic class

Acetaminophen (APAP) Example of drugs shown (or suspected) to aggravate pre-existing metabolic dysfunction-associated fatty liver disease (MAFLD), or to favor the transition of pre-existing fatty liver to nonalcoholic steatohepatitis (NASH). Further information is provided in previous articles [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF][START_REF] Bessone | Review article: druginduced liver injury in the context of nonalcoholic fatty liver disease -a physiopathological and clinical integrated view[END_REF][START_REF] Lammert | Patients With Chronic Liver Disease Suggestive of Nonalcoholic Fatty Liver Disease May Be at Higher Risk for Drug-Induced Liver Injury[END_REF][START_REF] Li | Metabolic Comorbidities and Risk of Development and Severity of Drug-Induced Liver Injury[END_REF][START_REF] Allard | Drug-induced liver injury in obesity and nonalcoholic fatty liver disease[END_REF]. According to our hypothesis, obese COVID-19 patients with metabolic dysfunction-associated fatty liver disease (MAFLD) may be at higher risk for drug-induced liver injury (DILI) than non-infected healthy individuals or MAFLD patients. This higher risk may be secondary to different factors including multiple drug administration (i.e. polypharmacy) to treat COVID-19, pre-existing fatty liver and possibly non-alcoholic steatohepatitis (NASH), altered activity of cytochromes P450 and other xenobiotic-metabolizing enzymes secondary to MAFLD and insulin resistance (as illustrated in Fig. 2)

Drug

and the cytokine storm reflecting severe systemic inflammation.

  PPARγ, peroxisome proliferator activated receptor gamma J o u r n a l P r e -p r o o fTable 3

  Figure 1.

Figure 2 .

 2 Figure 2.Some drugs can cause more severe (and/or more frequent) acute liver injury in individuals with metabolic dysfunction-associated fatty liver disease (MAFLD), which is often associated with obesity and insulin resistance. Such enhanced risk in MAFLD is in relation to altered activity of cytochromes P450 (CYPs) and other xenobiotic-metabolizing enzymes (XMEs), which can increase the generation of toxic metabolites, or conversely impair detoxification pathways. In turn, higher hepatic concentrations of toxic metabolites or of the parent drugs can induce more severe acute liver injury through mitochondrial dysfunction, oxidative stress and endoplasmic reticulum (ER) stress, which can already be present in MAFLD. All these deleterious events may lead to hepatocyte necrosis or apoptosis (not shown). It is still unclear why MAFLD is associated with impaired activity of CYPs and other XMEs. Insulin resistance may play a role directly, or indirectly via hyperinsulinemia, increased free fatty acids and hyperketonemia. These events may directly affect XMEs or impair the expression and activity of nuclear receptors regulating XME transcription.

Figure 3 .Figure 3

 33 Figure 3.In patients with metabolic dysfunction-associated fatty liver disease (MAFLD), some drugs can worsen pre-existing fatty liver, necroinflammation and fibrosis (situation ), or induce a faster progression of simple fatty liver to nonalcoholic steatohepatitis (NASH) (situation ). Aggravation of pre-existing fatty liver can be secondary to drug-induced inhibition of mitochondrial fatty acid oxidation (mtFAO) and to impairment of very low-density lipoprotein (VLDL) secretion. Drugs can also enhance de novo lipogenesis either directly by activating different lipogenic nuclear receptors

Acknowledgements

We are grateful to the Agence Nationale de Sécurité du Médicament (ANSM) for the financial support granted for the PREVITOX network. We are also grateful to INSERM (Institut National de la Santé et de la Recherche Médicale) for its constant financial support.

Author contributions

Bernard Fromenty drafted the original manuscript. Pierre-Jean Ferron, Thomas Gicquel, Bruno Mégarbane and Bruno Clément critically amended the manuscript. All the authors have read and approved the final version of the manuscript.

Declaration of competing interest

The authors have no conflict of interest in relation to this work.

J o u r n a l P r e -p r o o f

Author contributions

Bernard Fromenty drafted the original manuscript. Pierre-Jean Ferron, Thomas Gicquel, Bruno Mégarbane and Bruno Clément critically amended the manuscript. All the authors have read and approved the final version of the manuscript.

J o u r n a l P r e -p r o o f

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:


Frédéric Delarue
email: frederic.delarue@upmc.fr

S Bernard

K Sugitani

F Robert

R Tartèse

S.-V Albers

Rémi Duhamel

Sylvain Pont

Sylvie Derenne

Evidence for motility in 3.4 Gyr-old organic-walled microfossils ?

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Archean carbonaceous microfossils illustrate the widespread presence of life on Earth as early as ca. 3.4 billion years ago (Westall et al., 2006;[START_REF] Sugitani | Biogenicity of Morphologically Diverse Carbonaceous Microstructures from the ca. 3400 Ma Strelley Pool Formation, in the Pilbara Craton, Western Australia[END_REF][START_REF] Wacey | Microfossils of sulphurmetabolizing cells in 3.4-billion-year-old rocks of Western Australia[END_REF][START_REF] Alleon | Chemical nature of the 3.4 Ga Strelley Pool microfossils[END_REF][START_REF] Delarue | Out of rock: A new look at the morphological and geochemical preservation of microfossils from the 3.46 Gyr-old Strelley Pool Formation[END_REF]. However, the interpretation of the Archean palaeobiological record is fraught with difficulties pertaining to fossilization and burial-induced degradation processes, as illustrated by intense debates over the past couple of decades [START_REF] Schopf | Laser-Raman imagery of Earth's earliest fossils[END_REF][START_REF] Brasier | Questioning the evidence for Earth's oldest fossils[END_REF][START_REF] Wacey | 3.46 Ga Apex chert 'microfossils' reinterpreted as mineral artefacts produced during phyllosilicate exfoliation[END_REF]).

Remnants of early life forms all have experienced burial and thermal alteration for billions of years, which led to the degradation of many pristine biological traits [START_REF] Javaux | Challenges in evidencing the earliest traces of life[END_REF]. Therefore, Archean putative microfossils tend to exhibit simple morphological shapes (e.g., spheroidal, filamentous, film, and lenticular forms) that can also be abiotically produced [START_REF] Garcia-Ruiz | Self-Assembled Silica-Carbonate Structures and Detection of Ancient Microfossils[END_REF][START_REF] Cosmidis | Self-assembly of biomorphic carbon/sulfur microstructures in sulfidic environments[END_REF], precluding, in turn, any simple morphological distinction between genuine biological remnants and mineral/organic biomorphs. Because of the lack of taxonomically informative features [START_REF] Javaux | Challenges in evidencing the earliest traces of life[END_REF], morphological criteria alone are generally considered as insufficient to assess the biological nature of ancient traces of life in the Archean geological record [START_REF] Brasier | A fresh look at the fossil evidence for early Archaean cellular life[END_REF]. As a result, the ancient fossil record has not yet conveyed a complete picture of ancient biodiversity. Here, we report the discovery of 3.4 billion years old organic microfossils from the Strelley Pool Formation (SPF) from Western Australia exhibiting exceptionally preserved morphological traits indicative of active motility.

Results and Discussion

Observations of thin sections of SPF reveal the existence of tailed organic-walled microfossils (Fig. 1a,b). These tailed organic-walled microfossils are exclusively observed within the main siliceous sedimentary matrix precluding their introduction during hydrothermal fluid circulation post 3.4 Ga.

Raman spectra of tailed specimens chemically isolated from the mineral matrix are typical of those of disordered carbonaceous materials having undergone a low-grade metamorphism (Fig. 2a; [START_REF] Pasteris | Necessary, but Not Sufficient: Raman Identification of Disordered Carbon as a Signature of Ancient Life[END_REF][START_REF] Delarue | The Raman-Derived Carbonization Continuum: A Tool to Select the Best Preserved Molecular Structures in Archean Kerogens[END_REF]. Their Raman line shapes suggest that these microfossils experienced peak temperatures of approximately 250-300 °C [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland): RSCM in low-grade metasediments[END_REF]. Raman first-order spectra of studied SPF tailed microfossils are similar to those previously determined on syngenetic microfossils from the same geological formation observed in thin sections [START_REF] Lepot | Texture-specific isotopic compositions in 3.4Gyr old organic matter support selective preservation in cell-like structures[END_REF][START_REF] Sugitani | Microfossil assemblage from the 3400Ma Strelley Pool Formation in the Pilbara Craton, Western Australia: Results form a new locality[END_REF], on freshly fractured faces [START_REF] Alleon | Chemical nature of the 3.4 Ga Strelley Pool microfossils[END_REF], and in acid maceration residue [START_REF] Delarue | Out of rock: A new look at the morphological and geochemical preservation of microfossils from the 3.46 Gyr-old Strelley Pool Formation[END_REF]. Therefore, these tailed organic-walled microfossils should be regarded as syngenetic as they were subjected to the maximum metamorphic temperature registered by their host rock.

If Raman spectroscopy is a useful tool to assess syngeneity, it is not sufficient to determine the biogenicity of putative remains of ancient life [START_REF] Pasteris | Necessary, but Not Sufficient: Raman Identification of Disordered Carbon as a Signature of Ancient Life[END_REF]. Energy-dispersive X-ray spectroscopy data show that the studied specimens essentially contain C and O (Fig. 2b), confirming their organic nature, while nanoscale secondary ion mass spectrometry reveals significant levels of nitrogen and, in one specimen, phosphorus (Figs. 2d, f-g). The presence of these key elements of cell walls, proteins, and nucleic acids are consistent with a biological origin. Spatially resolved chemical investigations exploiting X-ray absorption confirm the heterogeneous chemical nature of the investigated organic-walled microfossils as at least three different chemical structures could be distinguished in a given specimen (Fig. 3). Specimens contain some highly graphitic organic materials with almost no nitrogen and a X-ray absorption spectrum exhibiting a broad peak of conjugated aromatic groups (285.5 eV) and the excitonic absorption feature of planar domains of highly conjugated π systems (291.7 eV; [START_REF] Bernard | XANES, Raman and XRD study of anthracene-based cokes and saccharose-based chars submitted to hightemperature pyrolysis[END_REF]. Closely associated are N-poor materials with XANES spectra similar to those of thermally-altered kerogen with an intense absorption peak at 285 eV (aromatic or olefinic groups), a relatively broad absorption feature at 287.5 eV (aliphatic carbons), and an absorption feature at 286.6 eV (imine, nitrile, carbonyl and/or phenol groups; [START_REF] Bernard | XANES, Raman and XRD study of anthracene-based cokes and saccharose-based chars submitted to hightemperature pyrolysis[END_REF][START_REF] Guillou | XANES-Based Quantification of Carbon Functional Group Concentrations[END_REF].

Specimens also contain N-rich compounds (N/C ~ 0.22) with XANES spectra that exhibit clear contributions of quinones or cyclic amides (284.5 eV), aromatic or olefinic carbons (285.1 eV), imine, nitrile, carbonyl and/or phenol groups (286.6 eV), aliphatics (287.7 eV) and amides (288.2 eV).

Altogether, the chemical structure of the SPF specimen investigated here is consistent with the preservation of partially degraded biomolecules.

From a morphological point of view, the organic-walled microfossils are leaf-shaped vesicles ranging from 30 to 84 µm in length and from 16 to 37 µm in width (Fig. 1). They exhibit classic taphonomical degradation features, including folds and tears (Figs. 1c-g). The preparation of ultrathin foils using focused ion beam illustrates their relative limited thickness, ranging from 200 to 500 nm (Fig. 3). Some specimens also exhibit a specific morphological feature, a lash-like appendage protruding from the leafshaped cell (Figs. 1c,d).

Based on analogous morphological traits observed in past and in modern microorganisms, several origins/functions may be hypothesised to explain the occurrence of this lash-like appendage. A number of extant microorganisms exhibit a micrometric tube-like appendage called prostheca, which can be involved in anchoring cells to organic and mineral surfaces, in nutrient uptake or in asexual reproduction by budding at its tip [START_REF] Curtis | Stalk formation of Brevundimonas and how it compares to Caulobacter crescentus[END_REF]. To assess whether the observed lash-like appendage is a remnant of an ancient prosthecum, we propose an Appendage Shape Index (ASI) based on the ratio between the width of the appendage and of the parent cell (Fig. 4). Compilation of morphometric data on extant microorganisms suggests that ASI ranges between 15 and 45% in prostheca, while the lash-like appendages observed in SPF microfossils are characterized by ASI ranging between 2.2 and 5.8 %, consistently with those observed on modern archaella, flagella and cilia (Fig. 4). In addition, a prosthecum consists in an extension of the cellular membrane, implying a structural continuity between the microorganism body and the base of the prosthecum [START_REF] Javaux | Recognizing and interpreting the fossils of early eukaryotes[END_REF]. On the studied SPF specimens, we observed an anchoring attachment point and a filament-like appendage, indicative of two distinct structural subunits (Fig. 1). Based on these morphometric and structural features, the lash-like appendages observed in SPF microfossils cannot be considered as remnants of a prosthecum. As far as we are aware, and in accordance with their ASI (Fig. 4), such distinct external and functional subunits can only be assigned to locomotory organelles. However, the lash-like appendages observed in SPF microfossils are between 0.7 and 1.2 µm in diameter, which is much larger than those reported for archaella, flagella, and cilia reaching ca. 10, 20, and 200 nm, respectively (Jarell and McBride, 2008;[START_REF] Beeby | Propulsive nanomachines: the convergent evolution of archaella, flagella, and cilia[END_REF]. Large cell dimensions (Ø > 10 µm) is a morphological feature commonly observed in Precambrian organic-walled microfossils [START_REF] Javaux | Organic-walled microfossils in 3.2-billion-year-old shallow-marine siliciclastic deposits[END_REF][START_REF] Sugitani | Biogenicity of Morphologically Diverse Carbonaceous Microstructures from the ca. 3400 Ma Strelley Pool Formation, in the Pilbara Craton, Western Australia[END_REF]2015;Balidukay et al., 2016;[START_REF] Loron | Organic-walled microfossils from the late Mesoproterozoic to early Neoproterozoic lower Shaler Supergroup (Arctic Canada): Diversity and biostratigraphic significance[END_REF]. Overall, the consistency of ASI values for SPF microfossils compared to those for archaella, flagella and cilia suggests that proportions between cell size and functional morphological traits/organelles may have persisted over the Earth's history. However, the SPF microfossils' lash-like appendages do not meet standard structural features (for instance, a curved hook connecting the filament to the basal body in flagella) observed on locomotory organelles from any organism of the three extant domains of life (see [START_REF] Khan | Assembly, Functions and Evolution of Archaella, Flagella and Cilia[END_REF]. Therefore, we propose that the lash-like appendage observed in some SPF microfossils may be a remain of a proto-locomotory organelle from a common ancestor or, alternatively, of an unknown and extinct domain of life.

Previous observations of 3.4-3.0 billion years-old flanged microfossils implied passive motility, microbial planktons drifting depending on its surrounding environment to engender movement [START_REF] House | Carbon isotopic analyses of ca. 3.0 Ga microstructures imply planktonic autotrophs inhabited Earth's early oceans[END_REF][START_REF] Sugitani | Early evolution of large micro-organisms with cytological complexity revealed by microanalyses of 3.4 Ga organic-walled microfossils[END_REF][START_REF] Oehler | Large and robust lenticular microorganisms on the young Earth[END_REF][START_REF] Kozawa | Early Archean planktonic mode of life: Implications from fluid dynamics of lenticular microfossils[END_REF]. To date, the oldest evidence for active motility was recorded as tubular sedimentary structures in 2.1 billion years old Francevillian sedimentary series (Gabon, El Albani et al., 2019). The preservation of lash-like appendages in some SPF microfossils suggests that some microorganisms were capable of active motility -a mechanism whereby microorganisms can direct where they go (Pollitt and Diggle, 2017) -as early as 3.4 Gyr ago.

Since it likely provided them with the ability to move in the water column or at the surface of organic and/or mineral surfaces, this evolutionary morphological innovation marks a major step in the history of life on Earth, and provide a more complex picture of the Archean biodiversity. It suggests that microorganisms were already able to escape harsh environments, adapt their feeding strategies moving towards more favorable nutrient sources, and colonize new ecological niches less than a billion years after the Earth became habitable [START_REF] Javaux | Challenges in evidencing the earliest traces of life[END_REF].

Methods

Chemical isolation of microfossils

Organic-walled microfossils were isolated from the SPF carbonaceous black chert sample using a modified version of the classical acid maceration procedure [START_REF] Delarue | Out of rock: A new look at the morphological and geochemical preservation of microfossils from the 3.46 Gyr-old Strelley Pool Formation[END_REF]. A 'soft' acid maceration procedure was applied in order to minimize both potential physical and chemical degradations of organic microstructures. Prior to acid maceration, about 30 g of rock samples were fragmented into ~3 g rock chips rather than crushed into finer grains. Rock chips were cleaned using ultrapure water and a mixture of dichloromethane/methanol (v/v: 2/1). Rock chips were then directly placed in a Teflon vessel filled with a mixture of HF (40%, reagent grade) /HCl (37%; reagent grade; v/v: 9/1) at room temperature. After 48 hours, successive centrifugation and rinsing steps using ultrapure water were performed until reaching neutrality. The residual material was suspended in ethanol and filtered on polycarbonate filters (pore Ø = 10 µm). After ethanol evaporation, polycarbonate filters were fixed on carbon tape and coated with 20 nm of gold to prevent further contamination by atmospheric deposits and further analyses with SEM-EDXS and NanoSIMS

Scanning electron microscopy and Energy Dispersive X-Ray Spectroscopy (SEM-EDXS)

SEM-EDXS imaging and analysis were performed on gold-coated filters using a TESCAN VEGA II at the French National Museum of Natural History (MNHN) operated with an accelerating voltage of 15 kV.

Raman spectroscopy

Raman microspectroscopy was carried out using a Renishaw InVIA microspectrometer equipped with a 532 nm green laser. The laser was focused on the sample by using a DMLM Leica microscope with a 50× objective. The spectrometer was first calibrated with a silicon standard before the analytical session.

For each target, we determined the Raman shift intensity in the 1000 to 2000 cm -1 spectral window that includes the first-order defect (D) and graphite (G) peaks. A laser power below 1 mW was used to prevent any thermal alteration during spectrum acquisition. Spectra acquisition was achieved after three iterations using a time exposure of 10 seconds. Raman microspectroscopy was performed on gold-coated organic surfaces implying a slight lowering of the D bands in comparison to the G one (see [START_REF] Delarue | Out of rock: A new look at the morphological and geochemical preservation of microfossils from the 3.46 Gyr-old Strelley Pool Formation[END_REF] for details)

Nanoscale secondary ion mass spectrometry

Isolated microfossils were analyzed using a CAMECA NanoSIMS 50 ion probe using a Cs+ primary ion beam. Before measurements, pre-sputtering was performed over 30 × 30 µm2 areas for ca. 8 minutes using a 500 pA primary current (750 µm aperture diaphragm) to avoid surficial contamination, and achieve Cs+ saturation fluence and constant secondary ion count rates. Analyses were then carried out using a 10 pA primary current (200 µm aperture diaphragm) on smaller areas to avoid pre-sputtering edge artifacts. The secondary molecular species 12 C 14 N -and 31 P -were collected simultaneously in electron multipliers. The NanoSIMS raw data were corrected for a 44 ns dead time on each electron multiplier and processed using the Limage software.

Focused ion beam (FIB)

FIB ultrathin sections were extracted from the organic microfossils using an FEI Strata DB 235 (IEMN, Lille, France). Milling at low gallium ion currents minimizes common artefacts including: local gallium implantation, mixing of components, creation of vacancies or interstitials, creation of amorphous layers, redeposition of the sputtered material on the sample surface and significant changes in the speciation of carbon-based polymers.

Scanning transmission X-ray microscopy

XANES investigations were conducted using the HERMES STXM beamline at the synchrotron SOLEIL (Gif-sur-Yvette, France). Carbon contamination on beamline optics was constantly removed thanks to a continuous flow of pure O2. The well-resolved 3p Rydberg peak of gaseous CO2 at 294.96 eV was used for energy calibration. Collecting image stacks at energy increments of 0.1 eV with a dwell time of ≤ 1 ms per pixel prevented irradiation damage. The estimations of N/C values and the normalization of the C-XANES spectra shown here were done using QUANTORXS [START_REF] Bernard | XANES, Raman and XRD study of anthracene-based cokes and saccharose-based chars submitted to hightemperature pyrolysis[END_REF]. was computed according to the ratio between the width of appendage (archaellum, flagellum, cilium and prosthecum) and of its parent cell (×100). Each width of appendage and of its parent cell was determined graphically following micrographs and images previously published in [START_REF] Southam | Isolation, characterization, and cellular insertion of the flagella from two strains of the archaebacterium Methanospirillum hungatei[END_REF], [START_REF] Poindexter | Caulobacter and Asticcacaulis stalk bands as indicators of stalk age[END_REF], [START_REF] Furuno | Characterization of polar-flagellar-length mutants in Vibrio alginolyticus[END_REF], [START_REF] Wustman | Extracellular Matrix Assembly in Diatoms (Bacillariophyceae) (I. A Model of Adhesives Based on Chemical Characterization and Localization of Polysaccharides from the Marine Diatom Achnanthes longipes and Other Diatoms)[END_REF]), Qintero et al. (1998), Wang et al. (2001[START_REF] Miller | Chemotaxis of Silicibacter sp. Strain TM1040 toward Dinoflagellate Products[END_REF], [START_REF] Bergholtz | On the identity of karlodinium veneficum and description of karlodinium armiger sp. nov. (dinophyceae), based on light and electron microscopy, nuclear-encoded lsu rdna, and pigment composition[END_REF][START_REF] Vasilyeva | Asticcacaulis benevestitus sp. nov., a psychrotolerant, dimorphic, prosthecate bacterium from tundra wetland soil[END_REF], [START_REF] Wagner | A nutrient uptake role for bacterial cell envelope extensions[END_REF], [START_REF] Kanbe | Characterization of Two Sets of Subpolar Flagella in Bradyrhizobium japonicum[END_REF], [START_REF] Abraham | Phylogeny by a polyphasic approach of the order Caulobacterales, proposal of Caulobacter mirabilis sp. nov., Phenylobacterium haematophilum sp. nov. and Phenylobacterium conjunctum sp. nov., and emendation of the genus Phenylobacterium[END_REF]), Nge et al. (2008), [START_REF] Pyatibratov | Alternative flagellar filament types in the haloarchaeon Haloarcula marismortui[END_REF], [START_REF] Siano | Unarmoured and thin-walled dinoflagellates from the Gulf of Naples, with the description of Woloszynskia cincta sp. nov. (Dinophyceae, Suessiales)[END_REF], [START_REF] Craveiro | Ultrastructure and Large Subunit rDNA-Based Phylogeny of Sphaerodinium cracoviense, an Unusual Freshwater Dinoflagellate with a Novel Type of Eyespot: Sphaerodinium ultrastructure and phylogeny[END_REF], [START_REF] Wang | First observation of Karlodinium veneficum from the East China Sea and the coastal waters of Germany[END_REF], [START_REF] Abraham | The Family Hyphomonadaceae[END_REF], Chang [START_REF] Lim | A bloom of Karlodinium australe (Gymnodiniales, Dinophyceae) associated with mass mortality of cage-cultured fishes in West Johor Strait, Malaysia[END_REF], [START_REF] Albers | The archaellum: how archaea swim[END_REF], [START_REF] Deng | The RNA Chaperone Hfq Is Involved in Colony Morphology, Nutrient Utilization and Oxidative and Envelope Stress Response in Vibrio alginolyticus[END_REF], [START_REF] Kinosita | Direct observation of rotation and steps of the archaellum in the swimming halophilic archaeon Halobacterium salinarum[END_REF]Nishizaka (2016), Sugitomo et al. (2016), [START_REF] Curtis | Stalk formation of Brevundimonas and how it compares to Caulobacter crescentus[END_REF], [START_REF] Leander | Euglenida[END_REF] . ASI determined on archaella, flagella and cilia are indicated in green while those determined on prostheca are indicated in pink. The area delimited by dotted lines indicate ASI determined on four lash-like appendages observed on tailed SPF organic-walled microfossils. ASI ranges from 4.8 to 5.8 % and from 2.2 to 3.3 % in organic-walled microfossil observed in thin sections (n = 2) and in the acid maceration residue (n = 2), respectively. ASI is likely overestimated in thin sections as a consequence of shadows occurring at the edge of organic-walled microfossils.

Figure 1 :

 1 Figure 1: Thin section micrographs and scanning electron microscopy images of tailed organic microfossils. (a-b) Micrographs presenting two specimens embedded in the main mineral matrix of the studied SPF chert. (c-f) SEM images of tailed organic microfossils isolated by acid maceration. (c,d) Exceptionally-well preserved leaf-shaped vesicles presenting a locomotory organelle composed of an attachment point and of a lash-like appendage (e-g) Corresponding degraded organic-walled microfossils. A taphonomic degradation gradient is observed from the left to the right. Classic taphonomical degradation features, including folds and tears, are observed.

Figure 2 :

 2 Figure 2: Raman spectra, energy-dispersive X-ray spectra and nanoscale secondary ion mass spectrometry images. (a) First-order Raman spectra determined on isolated tailed-organic walled microfossils and (b) corresponding energy-dispersive X-ray spectra. Green and purple lines indicate that spectra were acquired on specimens shown in panels c and e, respectively. (c, e) SEM images of organicwalled microfossils investigated by EDX, Raman spectroscopy and NanoSIMS. Green and purple squares indicate areas probed by NanoSIMS. (d, f) the 12 C 14 N -ion images illustrate the presence of nitrogen. (g) the 31 P -image illustrates significant levels of phosphorus. No significant level of 31 P -was recorded on the second specimen shown in panel c.

Figure 3 :

 3 Figure 3: Scanning transmission X-ray microscopy -based X-ray absorption near edge structure characterization. (a) SEM image of the specimen from which a focused ion beam foil has been extracted (green line). (b) SEM image of the focused ion beam foil evidencing the low thickness of the specimen. The green square indicates the area investigated using STXM. c, Carbon -X-ray absorption near edge structure spectra of the organic materials composing the investigated specimen.

Figure 4 :

 4 Figure 4: Compilation of Appendage Shape Indices determined on extant microorganisms. ASI

Acknowledgments

We thank V. Rouchon and O. Belhadj (CRCC) for Raman spectroscopy and D. Troadec (IEMN) for FIB extraction.. We also acknowledge The National NanoSIMS Facility at the MNHN, supported by MNHN, CNRS, Région Ile de France, and Ministère de l'Enseignement Supérieur et de la Recherche. Special thanks go to Stefan Stanescu and Sufal Swaraj for their expert support with the HERMES STXM beamline at SOLEIL. The HERMES beamline (SOLEIL) is supported by the CNRS, the CEA, the Region Ile de France, the Departmental Council of Essonne and the Region Centre. This work was supported by the Programme National de Planétologie (PNP) of CNRS/INSU, co-funded by CNES R.T. also acknowledges the UK Science and Technology Facilities Council (grant ST/P005225/1) for financial support.

Authors contributions

F.D. designed the study and supervised most of the analyses. K.S. provided the samples investigated.