Bernard Fromenty
email: bernard.fromenty@inserm.fr

Although mitochondriotoxicity plays a major role in drug-induced hepatotoxicity, alteration of mitochondrial DNA (mtDNA) homeostasis has been described only with a few drugs. Because it requires long drug exposure, this mechanism of toxicity cannot be detected with investigations performed in isolated liver mitochondria or cultured cells exposed to drugs for several hours, or a few days. Thus, a first aim of this study was to determine whether a 2-week treatment with 9 hepatotoxic drugs could affect mtDNA homeostasis in HepaRG cells. Previous investigations with these drugs showed rapid toxicity on oxidative phosphorylation but did not address the possibility of delayed toxicity secondary to mtDNA homeostasis impairment. The maximal concentration used for each drug induced about 10% cytotoxicity. Two other drugs, zalcitabine and linezolid, were used as positive controls for their respective effects on mtDNA replication and translation. Another goal was to determine whether druginduced mitochondriotoxicity could be modulated by lipid overload mimicking nonalcoholic fatty liver.

Among the 9 drugs, imipramine and ritonavir induced mitochondrial effects suggesting alteration of mtDNA translation. Ritonavir toxicity was stronger in non-steatotic cells. None of the 9 drugs decreased mtDNA levels. However, increased mtDNA was observed with 5 drugs, especially in non-steatotic cells. mtDNA levels could not be correlated with the expression of key factors involved in mitochondrial biogenesis (e.g. PGC1, PGC1, AMPK). Hence, drug-induced impairment of mtDNA translation might not be rare and increased mtDNA levels could be a frequent adaptive response to slight energy shortage. Nevertheless, this adaptation could be impaired by lipid overload.

Introduction

Although mitochondrial dysfunction is deemed to be a major mechanism of drug-induced liver injury (DILI) [START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF][START_REF] Will | Mitochondrial toxicity assessment in industry--a decade of technology development and insight[END_REF], alteration of mitochondrial DNA (mtDNA) homeostasis has been described so far only with a few drugs [START_REF] Cohen | Pharmacologic effects on mitochondrial function[END_REF]Schon and Fromenty, 2016).

For instance, the antiretroviral nucleoside reverse transcriptase inhibitors (NRTIs) zalcitabine (ddC), stavudine (d4T) and zidovudine (AZT) are able to block mtDNA replication via an inhibition of the DNA polymerase , thus inducing a progressive reduction of mtDNA levels in liver [START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF]Schon and Fromenty, 2016). The antibiotics linezolid (LNZ) and tetracycline can specifically impair mtDNA translation, possibly by interacting with the mitochondrial ribosomes (De [START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Moullan | Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research[END_REF].

Because mtDNA encodes for 13 oxidative phosphorylation (OXPHOS) polypeptides, any significant alteration of mtDNA replication, transcription or translation can cause energy shortage and severe liver diseases [START_REF] Wallace | Mitochondrial Energetics and Therapeutics[END_REF]Schon and Fromenty, 2016). However, because there is an excess of mtDNA copies in most cells, OXPHOS will be impaired only after severe decrease of the mtDNA content (Schon and Fromenty, 2016). It is deemed that the number of mtDNA copies must fall below 20-40% of basal levels in order to induce a significant mitochondrial dysfunction [START_REF] Begriche | Mitochondrial dysfunction in NASH: causes, consequences and possible means to prevent it[END_REF][START_REF] Begriche | Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver[END_REF]. Importantly, other threshold effects exist in mitochondria including at the levels of mitochondrial protein synthesis and OXPHOS activity [START_REF] Rossignol | Threshold effect and tissue specificity implication for mitochondrial cytopathies[END_REF][START_REF] Rossignol | Mitochondrial threshold effects[END_REF]. These different thresholds explain why mitochondrial toxicity and liver injury induced by NRTIs and LNZ can occur in patients after several weeks or months of treatment [START_REF] Spengler | Antiretroviral drug toxicitya challenge for the hepatologist?[END_REF][START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF][START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Bus | Severe drug-induced liver injury associated with prolonged use of linezolid[END_REF].

Currently, most in vitro investigations addressing drug-induced mitochondrial toxicity are performed in isolated 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][START_REF] Marroquin | Identifying compounds that induce opening of the mitochondrial permeability transition pore in isolated rat liver mitochondria[END_REF] and in cultured cells exposed to the tested drugs for several hours, or a few days at the most [START_REF] Kamalian | The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death[END_REF][START_REF] Eakins | A combined in vitro approach to improve the prediction of mitochondrial toxicants[END_REF].

These investigations can detect direct and rapid impairment of OXPHOS and fatty acid oxidation (FAO) pathway but not mitochondrial dysfunction secondary to alterations of mtDNA homeostasis. Hence, a first aim of this study was to investigate, in the metabolically competent human hepatoma HepaRG cell line, the effects of 11 hepatotoxic drugs on mtDNA homeostasis after 2 weeks of treatment, namely LNZ, ddC, amiodarone, atorvastatin, carbamazepine, imipramine, lovastatin, perhexiline, ritonavir, terbinafine and troglitazone. The drugs ddC and LNZ were selected as model molecules to ascertain that HepaRG cells could be used as a valuable model to detect drug-induced inhibition of mtDNA replication and translation, respectively [START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF][START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF]Schon and Fromenty, 2016). The other drugs were chosen because previous studies in isolated liver mitochondria showed that they directly impaired OXPHOS at relatively low concentrations [START_REF] Fromenty | Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and complex II[END_REF][START_REF] Deschamps | Inhibition by perhexiline of oxidative phosphorylation and the beta-oxidation of fatty acids: possible role in pseudoalcoholic liver lesions[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] but these investigations did not address the possibility that their hepatotoxicity might also be mediated by a deleterious effect on mtDNA homeostasis.

The second aim of this study was to compare the long-term cytotoxicity and mitochondriotoxicity of these drugs between non-steatotic and steatotic HepaRG cells. Indeed, some drugs such as acetaminophen, halothane and methotrexate are more hepatotoxic in the context of obesity and associated nonalcoholic fatty liver disease (NAFLD) [START_REF] Fromenty | Drug-induced liver injury in obesity[END_REF][START_REF] Michaut | Acetaminophen-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]. Although the mechanisms of this increased susceptibility are not precisely known, higher formation of cytochrome P450 (CYP)-generated toxic metabolites and preexistent mitochondrial dysfunction could be involved, at least for some pharmaceuticals [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]. Because obesity and NAFLD are highly prevalent in numerous countries, it is important to determine which drugs could present a higher risk of hepatotoxicity in obese patients.

Materials and Methods

Chemicals and reagents

Amiodarone, atorvastatin, carbamazepine, imipramine, lovastatin, perhexiline, ritonavir, terbinafine, troglitazone, ddC, LNZ, testosterone, 6β-hydroxytestosterone, chlorzoxazone (CZX), dimethyl sulfoxide (DMSO), D-(+)-galactose, oleic acid, stearic acid and insulin were purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). [U-14 C]-palmitic acid was supplied by PerkinElmer (Waltham, MA). A-769662 was purchased from Santa Cruz (Santa Cruz, CA). William's E medium, glutamine, penicillin/streptomycin, Nile red and Hoechst dyes were obtained from Thermo Fischer Scientific (Waltham, MA). William's E medium without glucose was supplied by CliniSciences (Nanterre, France). Fetal Bovine Serum (FBS) was supplied by Lonza (Levallois-Perret, France). Hydrocortisone hemisuccinate was purchased from SERB Laboratories (Paris, France). Protease and phosphatase inhibitors were supplied by Roche Diagnostics (Indianapolis, IN).

Cell cultures and treatments

Native HepaRG cells were cultured as previously described [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF]. Briefly, HepaRG cells were seeded at a density of 2.6 x 10 4 cells/cm 2 and were first incubated in a William's E medium supplemented with 10% FBS, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 5 µg/ml insulin and 50 µM hydrocortisone hemisuccinate. After 2 weeks, cell differentiation was induced by culturing the HepaRG cells in the same medium supplemented with 2% DMSO for 2 additional weeks.

Cells were then maintained for 2 additional weeks in the same culture medium except for the FBS and DMSO concentrations that were set at 5 and 1%, respectively. In order to induce steatosis, HepaRG cells were treated during this 2 week-period with a mixture of stearic acid (150 M) and oleic acid (150 M).

For drug treatments, non-steatotic and steatotic HepaRG cells were incubated during the same period with each compound at different concentrations. In some experiments, cells were treated with 30 M of A-769662, a direct activator of AMP-activated protein kinase -subunit (AMPK) (Smith et al., 2016).

Stearic acid, oleic acid and the different drugs were dissolved in DMSO whose final concentration in A c c e p t e d m a n u s c r i t p t JPET #246751 7 cultures was always maintained at 1%. Whatever the treatments including A-769662, the culture medium was renewed every 2 or 3 days during the 2-week experiments. To this end, newly added medium contained each selected drug (or no drug) with or without the fatty acid mixture. For some investigations with LNZ, ddC, imipramine and ritonavir, HepaRG cells were treated for 3 days with two treatments over this time period. All investigations were performed 24 h after the last drug treatment, excepted for AMPK activation that was assessed 6 h after the last drug treatment. HepaRG cells were used at passages 11 to 15.

Primary human hepatocytes (PHH) were obtained from Biopredic International (Saint-Grégoire, France). Briefly, PHH were isolated by collagenase perfusion of peritumoral liver tissue from 4 male adult donors who underwent resection for hepatic metastases. PHH were then seeded at a density of 0.2 ×10 5 cells/cm 2 onto rat collagen I-coated plates in a medium provided by Biopredic International. This medium was discarded 12 h after cell seeding and PHH cultures were maintained for 24 h in William's E medium supplemented with 10% FBS, 2 % DMSO, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 5 µg/ml insulin and 50 µM hydrocortisone hemisuccinate. PHH were then maintained for 12 days in the same culture medium except for the FBS and DMSO concentrations that were set at 5 and 1%, respectively. PHH were treated or not for 12 days with fatty acids and drugs using the same protocol as for HepaRG cells.

Nile red staining, cellular triglycerides and cytotoxicity assays

The Nile red dye allows the staining of neutral lipids within cells. For Nile red staining, cells were washed with warm phosphate buffer saline (PBS), fixed with 4% paraformaldehyde for 20 min at room temperature and then washed three times with cold PBS. After paraformaldehyde fixation, cells were incubated with 0.05 µg/ml Nile red in PBS for 30 min at room temperature and then washed once with PBS. Nuclei were labelled with 10 µg/ml Hoechst dye and cells were observed with an ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, Berkshire, UK). Triglycerides were measured with a colorimetric kit purchased from Biovision (Milpitas, CA), using the manufacturer's recommendations.

Cytotoxicity was assessed by measuring cellular ATP levels using the CellTiter-Glo Luminescent Cell Viability assay purchased from Promega (Charbonnières, France), according to the manufacturer's instructions. Briefly, untreated and treated HepaRG cells were first incubated with the CellTiter-Glo reagent for 10 min at 37°C. Cells were then transferred in an opaque-walled multiwell plate and the luminescent signal was quantified using a POLARstar Omega microplate reader (BMG Labtech, Ortenberg, Germany).

Results were expressed in comparison to untreated cells. Fifty and 10% inhibitory concentration (IC50 and IC10) values were calculated with a 4-parameter logistic regression using GraphPad Prism software 6.0 (GraphPad, San Diego, CA).

Determination of CYP2E1 and CYP3A4 activities

Measurement of CYP2E1 and CYP3A4 activities was performed as recently described [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF]. Briefly, HepaRG cells were incubated for 6 and 2 h in phenol red-free and DMSO-free William's E medium containing 300 µM CZX or 200 µM testosterone, respectively. At the end of the incubation, cell culture media were collected and stored at -80°C until analysis. 6-Hydroxychlorzoxazone was then quantified by high-performance liquid chromatography (HPLC)-tandem mass spectrometry (Xenoblis, Rennes, France), whereas 6β-hydroxytestosterone was measured by HPLC analysis.

Assessment of mtDNA levels

Total DNA was extracted from about 3x10 5 HepaRG cells using a Blood and Cell Culture DNA Mini Kit (Qiagen, Les Ulis, France). The relative content of mtDNA was then assessed by real-time quantitative PCR analysis (RT-qPCR) using the SYBR Green PCR Master Mix (Applied Biosystem, Woolston, UK) and an Applied Biosystem 7900HT Fast Real-Time PCR System (Applied Biosystem). The primers used to amplify a portion of the mtDNA gene encoding the cytochrome c oxidase subunit 1 (MT-CO1, also referred to as COX1) were 5'-TACGTTGTAGCCCACTTCCACT-3' (forward) and 5'-AGTAACGTCGGGGCATTCCG-3' (reverse). For normalization of mtDNA content, a portion of the nDNA gene encoding the ribosomal protein S6 (RPS6) was amplified by using the following primers 5'-TGATGTCCGCCAGTATGTTG-3' (forward) and 5'-TCTTGGTACGCTGCTTCTTC-3' (reverse). The 2 -ΔΔCt method was then used to assess the relative mtDNA levels.

Isolation of RNA and measurement of gene expression

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Total RNA was extracted from about 6x10 5 HepaRG cells with the Nucleospin RNA isolation system purchased from Macherey-Nagel (Hoerdt, France), which included a DNase treatment step. RNAs were reverse-transcribed into cDNAs using the High-Capacity cDNA Reverse Transcription Kit purchased from Applied Biosystems. RT-qPCR was then performed as mentioned above. Expression of GAPDH was used as reference, and the 2 -ΔΔCt method was used to express the relative expression of each selected gene. The sequences of the primers used to measure gene expression are presented in the Table 1.

Western blot analysis

At the end of the treatments, cells were harvested, washed twice with ice-cold PBS and lysed in a RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 0.25% sodium deoxycholate, 0.1% sodium dodecylsulfate, 1 mM EDTA, 1% NP-40) supplemented with protease and phosphatase inhibitors. Twenty µg of proteins were then separated by electrophoresis on NuPAGE 4-12% gradient Bis-Tris gels (Thermo Fisher Scientific), transferred to 0.2-m nitrocellulose membranes (Bio-Rad, Hercules, CA), which were saturated with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 2 h at room temperature. Proteins were then immunoblotted with antibodies against NADH dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunits 2 and 4 (COX2 and COX4), mitochondrial ribosomal protein L11 (MRPL11), MRPL28, AMP-activated protein kinase -subunit (AMPK), phospho-AMPK and -actin, which was used as loading control. The dilution used for all antibodies was 1:1000 except for those against COX2 and -actin whose dilutions were 1:500 and 1:5000 respectively. Finally, blots were incubated with appropriate secondary antibodies, and protein bands were revealed by enhanced chemiluminescence with the Fusion-FX7 Spectra system (Vilber Lourmat, Eberhardzell, Germany). COX4 and MRPL28 antibodies were purchased from Abcam (Cambridge, UK). AMPK, phospho-AMPK (Thr172) and MRPL11 antibodies were purchased from Cell Signaling (Danvers, MA). -Actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX). ND1 and COX2 antibodies were generated by the group of Dr Anne Lombès, as previously reported [START_REF] Agier | Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations[END_REF]. Enzyme Activity Dipstick Assay kit purchased from Abcam, according to the manufacturer instructions. In the assay, complex I is immunocaptured and immuno-precipitated in active form on the dipstick that consists of a nitrocellulose membrane to which an anti-complex I monoclonal antibody is attached.

Measurement of citrate synthase activity and respiratory chain complex I levels and activity

Complex I levels and activity were measured immunochromatographically with 20 µg of proteins using a Hamamatsu MS1000 Dipstick Reader (Abcam).

Measurement of mitochondrial respiration and fatty acid oxidation

Mitochondrial respiration in the presence of L-glutamine (2 mM), glucose (10 mM) and pyruvate (1 mM) was measured in adherent HepaRG cells using an Agilent Seahorse XFe24 Analyzer and the Mito Stress Test Kit (Agilent, Santa Clara, CA), according to the manufacturer instructions. Oxygen consumption rates were normalized in each well by the number of cells estimated by the fluorescence intensity of the Hoechst dye (10 µg/ml). For some investigations, HepaRG cells were also cultured in a William's E medium without glucose but supplemented with 11.1 mM galactose. After 14 days, mitochondrial respiration was measured for each culture condition (i.e. glucose or galactose) in the presence of L-glutamine/pyruvate/glucose or Lglutamine/pyruvate/galactose with glucose and galactose at the same concentration (i.e. 10 mM). These experiments showed that HepaRG cells cultured for 14 days with glucose or galactose presented similar basal and maximal (i.e. FCCP-stimulated) mitochondrial respiration measured in the presence of glucose (Table 2). A trend towards lower maximal respiration was noticed when mitochondrial respiration was measured in the presence of galactose (Table 2). The ATP production parameter was similar between the different conditions (Table 2). Non-mitochondrial respiration and proton leak (two other parameters provided by the Mito Stress Test profile) were also unchanged between the 4 different conditions of mitochondrial assessment (data not shown). Mitochondrial fatty acid oxidation (FAO) was measured in adherent HepaRG cells using [U-14 C]palmitic acid (0.05 Ci/ml), as previously described [START_REF] Anthérieu | Induction of vesicular steatosis by amiodarone and tetracycline is associated with up-regulation of lipogenic genes in HepaRG cells[END_REF]. Results for mitochondrial FAO were normalized to the total protein content determined by the bicinchoninic acid method.

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Statistical analysis

All results are expressed as mean ± SEM (standard error of mean). Comparisons between two groups were performed with a t-test. Comparisons between multiple groups were performed with two-way analysis of variance (ANOVA). When ANOVA provided significant differences, individual means were compared with the post hoc Bonferroni test. Statistical analyses were performed using GraphPad Prism software 6.0. This article has not been copyedited and formatted. The final version may differ from this version.

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Results

Detection of drug-induced alterations of mtDNA homeostasis in HepaRG cells

ddC and LNZ are known inhibitors of mtDNA replication and translation, respectively [START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF][START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF]Schon and Fromenty, 2016). In a first series of experiments, we verified that a 2week treatment with ddC and LNZ could induce these mitochondrial effects in differentiated HepaRG cells.

A concentration of 20 M was used for each drug because our preliminary data showed no significant cytotoxicity that could interfere with the interpretation of the results. ddC strongly reduced mtDNA levels and this was associated with lower mRNA and protein expression of ND1 and COX2, two mtDNA-encoded polypeptides (Fig. 1A,B,C). However, the expression of the nDNA-encoded COX4 was not decreased (Fig. 1C). Complex I levels were significantly reduced by ddC but the activity of this complex was less affected (P=0.15) (Fig. 1D). Regarding LNZ, the 2-week treatment reduced the protein expression of ND1 and COX2, whereas COX4 protein expression, mtDNA levels and mRNA expression of MT-ND1 and MT-CO2 were not decreased (Fig. 1A,B,C). Complex I levels tended to be decreased (P=0.06), while complex I activity was unaffected (Fig. 1D). Finally, HepaRG cells were treated for 72 h with ddC (20 M) and LNZ (20 M). No mtDNA depletion was observed with ddC and the protein expression of ND1 and COX2

was not affected by LNZ (data not shown). Our 2-week treatment protocol was therefore appropriate to detect drug-induced impairment of mtDNA replication and translation as well as the functional consequences of these deleterious effects.

Validation of the HepaRG cell as a model of NAFLD

In a recent study carried out in HepaRG cells, we set up a model to study drug-induced hepatotoxicity in the context of NAFLD [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF]. One rationale of that study was to find optimal conditions of HepaRG cell culture and fatty acid incubation inducing higher CYP2E1 activity and lower CYP3A4 activity, which are both consistent alterations reported in NAFLD patients [START_REF] Aubert | Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: Mechanisms and pathophysiological role[END_REF][START_REF] Woolsey | Cytochrome P450 3A Activity and Expression in Non-Alcoholic 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]. Higher CYP2E1 activity and lower CYP3A4 activity were obtained in HepaRG cells incubated for one week with stearic acid [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF]. However, incubation of the HepaRG cells with stearic acid for 2 weeks induced significant toxicity (~15% decrease in cellular ATP levels) leading to modification of the protocol. After testing several conditions of fatty acid incubation, we found that a 2-week treatment with a mixture of stearic acid (150 M) and oleic acid (150 M) did not cause significant cytotoxicity (data not shown) and gave satisfying results regarding cellular accumulation of neutral lipids including triglycerides (Fig. 2A), mRNA expression of the lipid biomarkers apolipoprotein A4 (APOA4), perilipin 1 (PLIN1) and perilipin 2 (PLIN2, also known as ADFP) (Fig. 2B). Furthermore, CYP2E1 mRNA expression and activity were increased while CYP3A4 mRNA expression and activity were decreased (Fig. 2B,C). Thus, this protocol of fatty acid treatment was selected for the present study.

Drug-induced cytotoxicity in non-steatotic and steatotic cells

One major goal of this study was to determine whether mtDNA homeostasis could be affected in nonsteatotic and steatotic HepaRG cells by different drugs, namely amiodarone, atorvastatin, carbamazepine, imipramine, lovastatin, perhexiline, ritonavir, terbinafine and troglitazone. To this end, preliminary investigations were performed to assess the cytotoxic potential of each drug in order to select non-cytotoxic drug concentrations for further studies on mtDNA homeostasis. Hence, cytotoxicity was determined by treating HepaRG cells with increasing concentrations of each drug and IC50 and IC10 values were calculated for both non-steatotic and steatotic cells. IC50 could be calculated for all drugs except for terbinafine and carbamazepine because these drugs did not induce cytotoxicity in the range of concentrations that could be tested owing to their solubility in the culture medium (Table 3). The lack of carbamazepine and terbinafine cytotoxicity in HepaRG cells was somehow surprising because both drugs induced mitochondrial dysfunction for relatively low concentrations (<100 M) 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]. For amiodarone, atorvastatin, imipramine, lovastatin and perhexiline, IC 50 and IC 10 values were similar between non-steatotic and steatotic HepaRG cells (Table 3). For troglitazone, the IC50 and IC10 were moderately but significantly lower in steatotic cells (Table 3), thus indicating higher cytotoxicity in the context of lipid accumulation. The converse profile was observed with ritonavir with lower cytotoxicity in steatotic HepaRG cells (Table 3). For drugs with calculated IC10, the selected concentrations were close to the IC10 and IC10/5 in order to perform investigations in condition of slight cytotoxicity or no cytotoxicity, respectively. An exception was for atorvastatin in order to use the same concentration than lovastatin, which belongs to the same pharmacological class. For carbamazepine and terbinafine, for which no IC10 could be determined, arbitrary concentrations were chosen. Hence, the working concentrations of the different drugs were as follows: 4 and 20 M for amiodarone, 1 and 5 M for atorvastatin and lovastatin, 10 and 300 M for carbamazepine, 12 and 60 M for imipramine, 6 and 30 M for perhexiline, 9 and 45 M for ritonavir, 10 and 100 M for terbinafine and 10 and 50 M for troglitazone. Importantly, the highest working concentrations corresponded to about 6, 8, 24, 25, 47, 83, 100, 107 and 500 x Cmax for ritonavir, troglitazone, amiodarone, terbinafine, carbamazepine, atorvastatin, imipramine, perhexiline and lovastatin, respectively (Table 3).

Selection of drug concentrations to study mtDNA homeostasis

Thus, for all drugs except lovastatin, these concentrations were lower, equivalent or slightly above than the 100 x Cmax cutoff classically used for safety assessment in pharmaceutical industry [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] Dykens | In vitro assessment of mitochondrial dysfunction and cytotoxicity of nefazodone, Trazodone, and Buspirone[END_REF]. Lovastatin was kept in our study in order to compare its mitochondrial effects with atorvastatin, which belong to the same pharmacological class.

Effects of the 9 drugs on mtDNA levels and expression of ND1 and COX2 at the mRNA and protein levels

In a next series of investigations, we determined the effects of the 9 drugs on mtDNA homeostasis in nonsteatotic and steatotic HepaRG cells. For this purpose, HepaRG cells were treated with each drug for 2 weeks to determine their respective effects on mtDNA levels and on the mRNA and protein expression of ND1 and COX2.

None of these drugs decreased mtDNA levels (Figs. 3A, 4A and5). Instead, mtDNA levels were significantly increased with amiodarone, carbamazepine, imipramine, terbinafine and troglitazone especially for their highest concentrations (Figs. 3A and5). For amiodarone, carbamazepine and troglitazone, increased mtDNA levels were particularly observed in the absence of steatosis (Fig. 5). The mRNA expression of MT-ND1 and MT-CO2 was also enhanced by different drugs excepted for imipramine, carbamazepine and terbinafine (Figs. 3B and4B; Table 4). Overall, there was no clear 4). Six drugs also significantly increased the mRNA expression of COX4I1 (Table 4).

Next, the protein expression of ND1 and COX2 was investigated. The highest concentrations of imipramine and ritonavir reduced ND1 and COX2 protein levels (Figs. 3C and4C), whereas the other drugs did not alter the expression of these mtDNA-encoded proteins (data not shown). Ritonavir-induced reduction of ND1 and COX2 protein expression was apparently more pronounced in non-steatotic HepaRG cells as compared to steatotic cells (Fig 4C). Importantly, imipramine and ritonavir did not reduce COX4 protein levels (Figs. 3C and4C). This result, together with the above-mentioned data, strongly suggested that imipramine and ritonavir impaired mtDNA translation without affecting mtDNA replication and transcription.

Non-steatotic and steatotic HepaRG cells (2 independent cultures) were also treated for 3 days with imipramine (12 and 60 M) or ritonavir (9 and 45 M). Imipramine did not reduce ND1 and COX2 protein levels. In contrast, the highest concentration of ritonavir induced about 50% reduction of ND1 protein expression in non-steatotic HepaRG cells, whereas ND1 levels were unchanged in steatotic cells (data not shown). COX2 and COX4 protein levels were not affected by 45 M ritonavir in non-steatotic and steatotic cells. Taken together, our results suggest that ritonavir-induced impairment of mtDNA translation might occur earlier than with LNZ and imipramine.

Further investigations were also carried out in non-steatotic and steatotic PHH treated or not for 12 days with ritonavir (45 M) or imipramine (60 M). Four lots of PHH from different donors were used for these investigations. Our results indicated that ritonavir and imipramine reduced ND1 and COX2 expression in 3 different donors, although the effects were variable between these individuals (Fig. 6). However, these drugs presented no effect in another donor (data not shown). Ritonavir-induced reduction of ND1 expression was more marked in non-steatotic PHH as compared to steatotic PHH (Fig. 6). Interestingly, this effect was also observed in HepaRG cells after 3 days (data not shown) and 2 weeks of ritonavir treatment (Fig. 4). Recent data indicated that some toxicants inducing a mitochondrial stress could impair mitochondrial translation by reducing the levels of different mitochondrial ribosomal proteins including MRPL11 and MRPL28 (Quiros et al., 2017). Hence, we determined whether imipramine and ritonavir could reduce the levels of these proteins. However, MRPL11 and MRPL28 protein levels were not decreased with both drugs after 2 weeks of treatment (data not shown). No change was also observed with 20 M LNZ (data not shown).

Effects of imipramine and ritonavir on the expression of MRPL11 and MRPL28

Effects of imipramine and ritonavir on complex I levels and activity, CS activity, mitochondrial respiration and fatty acid oxidation

In order to determine whether imipramine and ritonavir-induced reduction of mtDNA-encoded protein levels could have functional consequences, complex I levels and activity were first measured at the end of the treatments. With imipramine (60 M), complex I levels and activity were particularly decreased in steatotic cells (Fig. 3D,E), whereas ritonavir (45 M) induced a stronger reduction of these parameters in non-steatotic cells (Fig. 4D,E). When data collected with both imipramine and ritonavir were taken into account, a robust correlation (R 2 =0.89; P<0.001) was observed between complex I levels and complex I activity. Furthermore, a significant correlation existed between ND1 protein expression on the one hand and complex I levels (R 2 =0.58; P=0.01), or complex I activity (R 2 =0.69; P<0.01), on the other hand. CS activity was also significantly decreased by imipramine (Fig. 3F) and ritonavir (Fig. 4F), in both nonsteatotic and steatotic cells.

Oxygen consumption was next assessed in adherent HepaRG cells. Imipramine (60 M) did not modify mitochondrial respiration (Fig. 3G) but ritonavir (45 M) significantly reduced the maximal (i.e. FCCPstimulated) respiration in non-steatotic cells (Fig. 4G). Mitochondrial -oxidation of palmitic acid was also specifically assessed in adherent HepaRG cells. Imipramine significantly reduced mitochondrial FAO, but only in steatotic HepaRG cells (Fig. 3H). Surprisingly, ritonavir induced a significant increase in mitochondrial FAO, although this effect was observed only in non-steatotic HepaRG cells (Fig. 4H). This effect in non-steatotic cells was accompanied with a significantly higher mRNA expression of peroxisome proliferator-activated receptor- (PPAR) and its target gene liver carnitine palmitoyltransferase 1 This article has not been copyedited and formatted. The final version may differ from this version.

A c c e p t e d m a n u s c r i t p t JPET #246751 17 (CPT1A), whereas the expression of medium chain acyl-CoA dehydrogenase (ACADM) and peroxisomal acyl-coenzyme A oxidase (ACOX) was unchanged (data not shown). Finally, it was noteworthy that basal mitochondrial FAO was higher in steatotic cells compared with non-steatotic cells (Figs. 3H and4H). This is consistent with several reports showing increased mitochondrial FAO in experimental and clinical NAFLD [START_REF] Begriche | Mitochondrial adaptations and dysfunctions in nonalcoholic fatty liver disease[END_REF]Sunny et al., 2016).

Effects of the 9 drugs on the mRNA expression of key factors involved in mitochondrial biogenesis

In this study, several drugs (i.e. amiodarone, carbamazepine, imipramine, terbinafine, troglitazone) significantly enhanced mtDNA levels (Figs. 3A and5). Thus, we next determined for all drugs the mRNA expression of four factors playing a key role in mitochondrial biogenesis, namely peroxisome proliferatoractivated receptor-γ coactivator 1α and 1 (PGC-1 and PGC-1), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) [START_REF] Scarpulla | Transcriptional integration of mitochondrial biogenesis[END_REF][START_REF] Villena | New insights into PGC-1 coactivators: redefining their role in the regulation of mitochondrial function and beyond[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF].

However, different profiles of expression of PGC1A, PGC1B, NRF1 and TFAM were observed among the drugs (Table 4) and there was no apparent relationship between these profiles and the ability of some of these drugs to increase mtDNA levels. An interesting observation was that basal PGC1 expression was significantly reduced by about 25% in steatotic HepaRG cells when compared to non-steatotic HepaRG cells (P<0.01) (data not shown). Finally, the expression of POLG and POLRMT was determined since these polymerases play a key role in mitochondrial biogenesis and function [START_REF] Scarpulla | Transcriptional integration of mitochondrial biogenesis[END_REF][START_REF] Vega | Maintaining ancient organelles: mitochondrial biogenesis and maturation[END_REF]. Only ritonavir, atorvastatin and lovastatin significantly increased the expression of both POLG and POLRMT, whereas carbamazepine and terbinafine reduced their expression (Table 4). No significant change was observed with the other drugs (Table 4).

Effects of the 9 drugs on AMPK activation

AMPK is another key factor in mitochondrial biogenesis and function, in particular by activating PGC1 [START_REF] Scarpulla | Transcriptional integration of mitochondrial biogenesis[END_REF][START_REF] Vega | Maintaining ancient organelles: mitochondrial biogenesis and maturation[END_REF][START_REF] Ploumi | Mitochondrial biogenesis and clearance: a balancing act[END_REF]. Thus, we determined whether the different drugs could activate AMPK by assessing the expression of phospho-AMPK. Activation of phospho-AMPK was first validated after a 14-day treatment with A-769662 (Fig. 7A,B). Five drugs, namely amiodarone, imipramine, lovastatin, terbinafine and troglitazone significantly increased phospho-AMPK levels after a 14-day treatment at their highest concentrations (Fig. 7A,B). However, for these drugs and A-769662, phospho-AMPK levels were not reduced in steatotic cells compared to the nonsteatotic cells (Fig. 7A,B). Surprisingly, carbamazepine significantly reduced phospho-AMPK levels compared with the untreated cells (Fig. 7A,B). Finally, we determined whether the expression of phospho-AMPK levels observed in all conditions of treatment correlated with the corresponding mRNA expression of PGC1. This correlation tended to be significant (P=0.09) when phospho-AMPK levels were taken into account but was highly significant (R 2 =0.52; P<0.001) when the phospho-AMPKtotal AMPK ratio was considered. In contrast, there was no correlation between phospho-AMPK levels (or the phospho-AMPKtotal AMPK ratio) with mtDNA levels.

The moderate but significant increased phospho-AMPK levels observed with 100 M terbinafine in steatotic cells was surprising since ATP levels were not reduced in this experimental condition (Table 3).

Because oxidative stress can also activate AMPK (Horie et al., 2007;[START_REF] Morales-Alamo | AMPK signaling in skeletal muscle during exercise: Role of reactive oxygen and nitrogen species[END_REF], we determined whether terbinafine increased the expression of several oxidative stress-related genes, namely nuclear factor erythroid 2-like 2 (NFE2L2, sometimes referred to as Nrf2), heme oxygenase 1 (HMOX1), NAD(P)H quinone dehydrogenase 1 (NQO1), superoxide dismutase 2 (SOD2) and tribbles pseudokinase 3 (TRIB3). However, only HMOX1 and NQO1 expression was significantly increased by 100 M terbinafine, in particular in non-steatotic cells (data not shown). Hence, energy shortage and oxidative stress were most probably not responsible for AMPK activation in steatotic HepaRG cells treated with 100 M terbinafine. This article has not been copyedited and formatted. The final version may differ from this version.

A c c e p t e d m a n u s c r i t p t JPET #246751 Discussion

Drug-induced alteration of mtDNA homeostasis has been described only with a few drugs, especially with the antiretrovirals NRTIs and some antibiotics [START_REF] Cohen | Pharmacologic effects on mitochondrial function[END_REF]Schon and Fromenty, 2016). In this study, the respective inhibitory effects of ddC (20 M) and LNZ (20 M) on mtDNA replication and translation were observed in HepaRG cells after 2 weeks (Fig. 1) but not after 3 days (data not shown). This underlines the importance of an adequate duration of cell treatment in order to detect any significant drug-induced alterations of mtDNA homeostasis and their functional consequences. Such delayed appearance of mitochondrial dysfunction is due to the existence of several threshold effects at different mitochondrial levels [START_REF] Rossignol | Threshold effect and tissue specificity implication for mitochondrial cytopathies[END_REF][START_REF] Rossignol | Mitochondrial threshold effects[END_REF]. In patients, mitochondrial toxicity and liver injury induced by NRTIs and LNZ usually occurs after several weeks or months of treatment [START_REF] Spengler | Antiretroviral drug toxicitya challenge for the hepatologist?[END_REF][START_REF] Walker | Depletion of mitochondrial DNA in liver under antiretroviral therapy with didanosine, stavudine, or zalcitabine[END_REF][START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Bus | Severe drug-induced liver injury associated with prolonged use of linezolid[END_REF]. The maximal plasma concentrations (Cmax) of ddC in patients can be up to 1 M [START_REF] Massarella | The effect of probenecid on the pharmacokinetics of zalcitabine in HIV-positive patients[END_REF]. Thus, the concentration of ddC able to inhibit mtDNA replication in our cellular model represents 20 x Cmax. Regarding LNZ, (serum) Cmax in patients can be up to 40 M (MacGowan, 2003), thus suggesting that the safety margin of this antibiotic is rather low.

Our data strongly suggest that the antidepressant drug imipramine and the antiretroviral protease inhibitor (PI) ritonavir specifically impede mitochondrial translation. Indeed, reduced ND1 and COX2 protein levels induced by imipramine (60 M) and ritonavir (45 M) were neither associated with lower expression of the corresponding mtDNA-encoded transcripts, nor with mtDNA depletion (Figs. 3 and4).

Moreover, reduced ND1 and COX2 protein levels were not accompanied by lower protein expression of COX4 (Figs. 3 and4), MRPL11 and MRPL28 (data not shown), which are nDNA-encoded mitochondrial proteins. Actually, these results were unexpected because mtDNA translation is usually impaired by antibiotics [START_REF] Cohen | Pharmacologic effects on mitochondrial function[END_REF]Schon and Fromenty, 2016). Further investigations will be required to determine whether imipramine and ritonavir could interact with the mitochondrial ribosomes, as shown for the antibiotics chloramphenicol, LNZ and tetracycline [START_REF] Vriese | Linezolid-induced inhibition of mitochondrial protein synthesis[END_REF][START_REF] Cohen | Pharmacologic effects on mitochondrial function[END_REF][START_REF] Moullan | Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research[END_REF]. Imipramine and ritonavir also reduced ND1 and COX2 protein expression in PHH from 3 different donors, although the effects were variable among these individuals (Figure 6). This suggests the existence of an inter-individual variability regarding imipramine and ritonavir-induced impairment of mitochondrial translation. Interestingly, genetic susceptibility was reported for antibiotic-induced alterations of mitochondrial protein synthesis and related adverse effects [START_REF] Hobbie | Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity[END_REF][START_REF] Garrabou | Influence of mitochondrial genetics on the mitochondrial toxicity of linezolid in blood cells and skin nerve fibers[END_REF].

Originally developed as an antiretroviral PI, ritonavir is nowadays exclusively used in HIV-positive patients as a pharmacological booster of other PIs. Indeed, by strongly inhibiting CYP3A4, ritonavir increases the bioavailability and plasma concentrations of other PIs [START_REF] Hull | Ritonavir-boosted protease inhibitors in HIV therapy[END_REF]. However, although ritonavir is a CYP3A4 inhibitor, the bioactivation of ritonavir into several metabolites is dependent of this CYP in rodent and human [START_REF] Kumar | Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes[END_REF][START_REF] Li | Metabolomic screening and identification of the bioactivation pathways of ritonavir[END_REF]. In the mouse, 26 different metabolites have been detected after ritonavir administration [START_REF] Kumar | Cytochrome P450-mediated metabolism of the HIV-1 protease inhibitor ritonavir (ABT-538) in human liver microsomes[END_REF], thus underlining the complexity of ritonavir metabolism. In this study, ritonavir-induced cytotoxicity (IC50) (Table 1) and impairment of complex I levels and activity (Fig. 4D,E) were significantly lower in steatotic HepaRG cells.

Notably, although basal CYP3A4 activity was significantly reduced in untreated steatotic HepaRG cells (Fig. 2), the remaining CYP3A4 activity in cells treated for 2 weeks with 45 M ritonavir was extremely low (maximum 8%) and equivalent between non-steatotic and steatotic cells (data not shown). Hence, CYP3A4 was probably not responsible for the lower ritonavir toxicity associated with steatosis. Further investigations would be needed to determine why ritonavir is less toxic in steatotic HepaRG cells.

Contrary to ritonavir, the antidiabetic drug troglitazone induced significantly higher cytotoxicity in steatotic HepaRG cells (Table 3). Moreover, the troglitazone-induced adaptive increase in mtDNA levels observed in non-steatotic HepaRG cells was absent in steatotic cells. Importantly, troglitazone can be toxic by itself, in particular by directly impairing mitochondrial function [START_REF] Nadanaciva | Mitochondrial impairment by PPAR agonists and statins identified via immunocaptured OXPHOS complex activities and respiration[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]. Although troglitazone can be biotransformed into several reactive metabolites, especially via CYP3A4, different investigations suggested that these metabolites are overall less toxic than troglitazone itself [START_REF] Masubuchi | Metabolic and non-metabolic factors determining troglitazone hepatotoxicity: a review[END_REF][START_REF] Yokoi | Troglitazone[END_REF][START_REF] Hosomi | Development of a highly sensitive cytotoxicity assay system for CYP3A4-mediated metabolic activation[END_REF]. Hence, our data suggest that higher troglitazone cytotoxicity in steatotic HepaRG cells could be due to lower troglitazone biotransformation, including via the CYP3A4 pathway. Troglitazone, the first marketed thiazolidinedione, was actually withdrawn from the market in 2000 because of the occurrence of numerous cases of severe liver failure in patients with type 2 [START_REF] Isley | Hepatotoxicity of thiazolidinediones[END_REF][START_REF] Labbe | Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies[END_REF][START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF]. Because type 2 diabetes most often occurs in obese individuals, troglitazone-induced severe hepatotoxicity might have been favored by preexistent fatty liver in these patients. Interestingly, several experimental investigations showed that rosiglitazone, a thiazolidinedione that is still marketed, was more hepatotoxic in obese mice (Garcia-Ruiz et al., 2007;[START_REF] Massart | Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity[END_REF].

Another important observation in our study was that several of the investigated drugs increased mtDNA levels in HepaRG cells after a treatment of 2 weeks. However, mtDNA levels in treated cells were in general higher in non-steatotic compared to steatotic cells. This pattern was particularly striking with amiodarone, carbamazepine and troglitazone (Fig. 5). Because differences in mtDNA levels could have been explained by changes in mitochondrial biogenesis, we determined the mRNA expression of several factors playing a key role in this process including PGC1A, PGC1B, NRF1, TFAM and phospho-AMPK. Moreover, POLG expression was also assessed. However, our data could not explain why some drugs increased mtDNA levels in HepaRG cells and why steatosis was associated with lesser mtDNA levels in HepaRG cells treated with amiodarone, carbamazepine and troglitazone. Further investigations will be needed in order to determine whether these effects could be secondary to changes in the activity of these factors, rather than lower mRNA expression. Alternatively, other key factors involved in mtDNA homeostasis might have played a role.

Among the different drugs, only ritonavir (45 M) significantly increased the mRNA expression of all mitochondrial-related genes investigated in this study, in both non-steatotic and steatotic HepaRG cells (Table 4). Moreover, this drug also enhanced PPAR and CPT1A mRNA expression (data not shown) and palmitate oxidation in non-steatotic cells (Fig. 4G). In contrast, mtDNA levels were unchanged and CS activity was reduced by the treatment, thus suggesting that ritonavir-induced mitochondrial adaptive response was incomplete. Whether the lack of AMPK activation (Fig. 7) might have played a role in such incomplete response will require further investigations. Notably, CS activity is frequently used as a bona fide marker of mitochondrial content in different pathophysiological situations such as inherited mitochondrial diseases [START_REF] Guillery | Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts[END_REF] and exercise training [START_REF] Bishop | Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content?[END_REF]. Hence, lower CS activity could reflect reduced mitochondrial content in ritonavir-treated cells despite higher mRNA Previous investigations in HepG2 cells reported that drug-induced cytotoxicity could be different when these cells were cultured in the presence of glucose or galactose, which was reported to favor mitochondrial metabolism [START_REF] Marroquin | Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants[END_REF][START_REF] Paech | Hepatocellular toxicity associated with tyrosine kinase inhibitors: mitochondrial damage and inhibition of glycolysis[END_REF]. In the present study, we showed that HepaRG cells cultured for 14 days with glucose or galactose presented similar mitochondrial respiration (Table 2), thus indicating that galactose was not able to augment mitochondrial metabolism in HepaRG cells. Importantly, recent investigations reported that mitochondrial function (e.g. respiration and complex II and IV activities) in differentiated HepaRG cells was close to PHH and significantly superior compared to HepG2 cells [START_REF] Peyta | Reduced cardiolipin content decreases respiratory chain capacities and increases ATP synthesis yield in the human HepaRG cells[END_REF][START_REF] Porceddu | In vitro Assessment of Mitochondrial Toxicity to Predict Drug-Induced Liver Injury[END_REF]. Taken together, these data indicate that HepaRG cells present fully functional mitochondria and that their culture with galactose is not able to further enhance mitochondrial capacity.

Because drug-induced alteration of mtDNA translation might have been overlooked in the past, especially for drugs that are not antibiotics, we would recommend to include studies on mtDNA translation to investigators wishing to better understand how some compounds can alter mitochondrial function. To do so, the HepaRG cell line is a suitable model because drug toxicity can be studied over 2 weeks (present study) or longer [START_REF] Anthérieu | Optimization of the HepaRG cell model for drug metabolism and toxicity studies[END_REF]. Moreover, this cell line could be appropriate to study drug-induced modulation of mitochondrial biogenesis. Indeed, the expression of several major mitochondrial biogenesis factors (e.g. PGC-1, PGC-1, phospho-AMPK) was significantly increased, or decreased, by several investigated drugs. Interestingly, there was a significant correlation between the PGC1 mRNA expression and the phospho-AMPKtotal AMPK ratio in treated HepaRG cells. Despite these advantages, HepaRG cells present some limitations, as previously pointed out [START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF][START_REF] Michaut | A cellular model to study drug-induced liver injury in nonalcoholic fatty liver disease: Application to acetaminophen[END_REF]. For instance, this cell line derives from one female patient and thus does not allow to disclose gender differences or genetic susceptibility. Supplementary investigations in PHH can help to tackle these issues.

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  CS) activity was measured with 20 µg of proteins using the Citrate Synthase Assay Kit from Sigma-Aldrich, according to the manufacturer recommendations. Complex I levels and activity were respectively measured with the Complex I Human Protein Quantity Dipstick Assay Kit and the Complex I

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  A c c e p t e d m a n u s c r i t p t JPET #246751 relationship between mtDNA levels and the mRNA expression of MT-ND1 and MT-CO2 (Figs. 3, 4 and 5; Table

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  nDNA and mtDNA-encoded mitochondrial proteins. Alternatively, ritonavir might directly inhibit the activity of this mitochondrial enzyme.

Fig. 1

 1 Figure Legends:

Fig. 2

 2 Fig. 2 Main features of the in vitro HepaRG cell model of steatosis induced by lipid overload

Fig. 3

 3 Fig. 3 Mitochondrial effects of imipramine

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  performed with a two-way ANOVA: T, effect of treatment, S, effect of steatosis, TxS, interaction between treatment and steatosis. Individual means were then compared with the post hoc Bonferroni test. *Significantly different from untreated NS or S cells. †Significantly different from NS cells (P<0.05).

Fig. 4

 4 Fig. 4 Mitochondrial effects of ritonavir

Fig. 5

 5 Fig. 5 Drug-induced modulation of mtDNA levels

Fig. 6 Fig. 7

 67 Fig. 6 Effect of imipramine and ritonavir on ND1, COX2 and COX4 expression in primary human

  Table 4. Effects of the 9 different drugs on the mRNA expression of different mitochondrial-related genes.

  as a fold-change of untreated non-steatotic (NS) cells, are means ± SEM for 4 independent cultures. Statistical analysis was performed with a two-way ANOVA: T, effect of treatment, S, effect of steatosis. Individual means were then compared with the post hoc Bonferroni test. *Significantly different from untreated NS or S cells. †Significantly different from NS cells (P<0.05). Data in grey boxes indicate significant differences with the two-way ANOVA. Data in bold characters indicate significant differences with the post hoc Bonferroni test.

Figure 2 :

 2 Figure 2: Main features of the in vitro HepaRG cell model of steatosis induced by lipid overload

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Table 1 .

 1 List of the primers used in this studyThis article has not been copyedited and formatted. The final version may differ from this version.

	Gene symbol	Gene name	Forward primer	Reverse primer
	APOA4	apolipoprotein A4	CAGTGTGGCAAGAAACTCCT	GTAGTCCCACATCACCGTG
	ACADM	acyl-CoA dehydrogenase, C-4 to C-12 straight	TTTGGGGAGAATGACTGAGG	TGGATCAGAACGTGCCAATA
		chain
	ACOX1	acyl-CoA oxidase 1	GATGAAGTATGCCCAGGTGAAG	CACAAGGAAGGACCTGACAAA
	COX4I1	cytochrome c oxidase subunit 4I1	CAGAATGTTGGCTACCAGGG	GGTCACGCCGATCCATATAAG
	CPT1A	carnitine palmitoyltransferase 1A	CGGGAGGAAATCAAACCAATTC	CTGGGATCCGGGAAGTATTAAA
	CYP2E1	cytochrome P450 family 2 subfamily E member 1	TTGAAGCCTCTCGTTGACCC	CGTGGTGGGATACAGCAA
	CYP3A4	cytochrome P450 family 3 subfamily A member 4	CTTCATCCAATGGACTGCATAAAT	TCCCAAGTATAACACTCTACACAGACAA
	GAPDH	glyceraldehyde-3-phosphate dehydrogenase	ATGACATCAAGAAGGTGGTG	CATACCAGGAAATGAGCTTG
	HMOX1	heme oxygenase 1	ACTTTCAGAAGGGCCAGGT	TTGTTGCGCTCAATCTCCT
	MT-CO2	mitochondrially encoded cytochrome c oxidase	CTGAACCTACGAGTACACCG	TTAATTCTAGGACGATGGGC
		subunit II
	MT-ND1	mitochondrially encoded NADH dehydrogenase	CCCTAAAACCCGCCACATCT	GAGCGATGGTGAGAGCTAAGGT
		subunit 1
	NFE2L2	nuclear factor, erythroid 2 like 2	TCAGCATGCTACGTGATGAAG	TTTGCTGCAGGGAGTATTCA
	NQO1	NAD(P)H quinone dehydrogenase 1	ACGTCCTTCAACTATGCCATG	TTACCTGTGATGTCCTTTCTGG
	NRF1	nuclear respiratory factor 1	GCCACAGCCACACATAGTATAG	CGTACCAACCTGGATAAGTGAG
	PLIN1	perilipin 1	TGGTCCTCATGATCCTCCTC	GTTGTCGATGTCCCGGAATT
	PLIN2	perilipin 2	CCATTCTACTGTTCACCTGATTGA	ACCCATGAGAGGTAGAGCTTAT
	POLG	DNA polymerase gamma, catalytic subunit	GGCTGTCCAGGAAGAGTTTATG	CCACAAGCATGAGGTGTAAGT
	POLRMT	RNA polymerase mitochondrial	GGACTCCAAGGTCAAGCAAATA	TCTTCTGCTTACGTGTGTTGG
	PPARA	peroxisome proliferator activated receptor alpha	GTTCTGGAAGCTTTGGCTTTAC	GAAAGCGTGTCCGTGATGA
	PPARGC1A (PGC1A) PPARG coactivator 1 alpha	AGTGGTGCAGTGACCAATCA	CTGCTAGCAAGTTTGCCTCA
	PPARGC1B (PGC1B) PPARG coactivator 1 beta	GTACATTCAAAATCTCTCCAGCGACATG GAGGGCTCGTTCCTCAGGGCA
	SOD2	superoxide dismutase 2	GGGTTGGCTTGGTTTCAATA	CTGATTTGGACAAGCAGCAA
	TFAM	transcription factor A, mitochondrial	GCTCAGAACCCAGATGCAAA	TGCCACTCCGCCCTATAA
	TRIB3	tribbles pseudokinase 3	CCCTGCTCACAGAGATGACA	GCAGCTGGTTTGTTTGTGAA

Table 2 .

 2 Effects of glucose and galactose on mitochondrial function in HepaRG cells HepaRG cells were cultured in a William's E medium with 11.1 mM glucose or without glucose but supplemented with 11.1 mM galactose. After 14 days, mitochondrial respiration was measured for each culture condition (i.e. glucose or galactose) in the presence of L-glutamine/pyruvate/glucose or L-glutamine/pyruvate/galactose with glucose and galactose at the same concentration (10 mM). Three mitochondrial parameters (i.e. respiration rates in nmol/min/10 6 cells) provided by the Mito Stress Test profile are shown. Results are means ± SEM for 3 independent cultures.

			Mito Stress Test profile parameters
	Substrate for	Substrate for	Basal	Maximal	ATP
	culture	respiration	respiration	respiration	production
	Glucose	Glucose	3.7 ± 0.2	17.9 ± 1.7	2.8 ± 0.3
	Galactose	Glucose	3.7 ± 0.3	17.5 ± 2.0	2.8 ± 0.3
	Glucose	Galactose	3.6 ± 0.2	16.8 ± 2.1	2.8 ± 0.2
	Galactose	Galactose	3.8 ± 0.2	15.0 ± 1.7	2.9 ± 0.1

Table 3 .

 3 Drug-induced cytotoxicity in non-steatotic and steatotic HepaRG cells Cytotoxicity was assessed by measuring cellular ATP levels and IC50 and IC10 values were subsequently calculated. Results are means ± SEM for 4 independent cultures. Statistical analysis was performed with a t-test. *Significantly different from non-steatotic HepaRG cells (P<0.05). Cmax values are from Porceddu et al. 2012. article has not been copyedited and formatted. The final version may differ from this version.

	This
	Drugs	IC50 (µM) Non-steatotic	Steatotic	IC10 (µM) Non-steatotic Steatotic	Cmax (µM)
	Amiodarone	29.8 ± 0.6	34.2 ± 0.6	24.6 ± 0.7	23.4 ± 0.7	0.81
	Atorvastatin	19.3 ± 0.5	17.0 ± 0.5	15.0 ± 0.6	11.0 ± 0.6	0.06
	Carbamazepine	> 300	> 300	> 300	> 300	6.43
	Imipramine	79.8 ± 0.5	75.6 ± 0.5	66.2 ± 0.6	63.0 ± 0.6	0.6
	Lovastatin	13.8 ± 0.6	10.8 ± 0.6	6.5 ± 0.7	4.0 ± 0.6	0.01
	Perhexiline	32.4 ± 1.8	33.0 ± 0.7	30.7 ± 0.8	30.6 ± 0.6	0.28
	Ritonavir	51.3 ± 0.5	58.8 ± 0.6*	43.8 ± 0.6	46.8 ± 0.6	7.07
	Terbinafine	> 100	> 100	> 100	> 100	4
	Troglitazone	70.4 ± 0.5	58.6 ± 0.6*	66.1 ± 0.5	51.1 ± 0.7*	6.6

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Acknowledgments:

We are grateful to the Institut National de la Santé et de la Recherche Médicale (INSERM) for its constant support.

Footnotes:

This work (including Dounia Le Guillou's PhD stipend) has been founded by Sanofi. Simon Bucher was recipient of a fellowship from the Région Bretagne and the Agence Nationale de la Recherche (STEATOX project; "ANR-13-CESA-0009").

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Conflict of interest:

Bernard Fromenty reports personal fees from Alfasigma France and Novo Nordisk, outside the submitted work.

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Pascale Blond

Alice Mattiuzzi

Hennie Valkenier

Ludovic Troian-Gautier

Jean-François Bergamini

Thomas Doneux

Erik Goormaghtigh

Vincent Raussens

Ivan Jabin

Grafting of Oligo(ethylene glycol) Functionalized Calix[4]arene-tetra-diazonium Salts for Antifouling Germanium and Gold Surfaces

Biosensors that can determine protein concentration and structure are highly desired for biomedical applications. For the development of such biosensors, the use of Fourier transformed infra-red (FTIR) spectroscopy with the attenuated internal total reflection (ATR) configuration is particularly attractive but it requires appropriate surface functionalization of the ATR optical element. Indeed, the surface has to specifically interact with a target protein in close contact with the optical element and must display antifouling properties to prevent nonspecific adsorption of other proteins. We here report robust monolayers of calix[4]arenes bearing oEGs chains, which were grafted on germanium and gold surfaces via their tetra-diazonium salts. The formation of monolayers of oEGylated calix [4]arenes was confirmed by AFM, IR and contact angle measurements. The antifouling properties of these modified surfaces were studied by ATR-FTIR spectroscopy and fluorescence microscopy and the non-specific absorption of BSA was found to be reduced by 85% compared to non-modified germanium. In other words, the organic coating by oEGylated calix[4]arenes provides remarkable antifouling properties, opening the way to the design of germanium-or gold-based biosensors.

INTRODUCTION

Proteins are involved in a very large number of biological processes. Even a small change in their concentration, post-translational modifications, or in their native structure can lead to pathologies. 1 Biosensors that enable fast and selective identification and structural characterization A c c e p t e d m a n u s c r i t p t 3 of proteins in complex mixtures represent thus an important target for research. [START_REF] Leech | Affinity Biosensors[END_REF][START_REF] Cao | Advances on Aryldiazonium Salt Chemistry Based Interfacial Fabrication for Sensing Applications[END_REF] A major challenge in the development of such biosensors is the modification of surfaces by a robust organic monolayer able to specifically interact with a protein [START_REF] Camarero | New Developments for the Site-Specific Attachment of Protein to Surfaces[END_REF] and displaying antifouling properties to prevent nonspecific adsorption phenomena.

In this context, there is an increasing interest in the use of germanium-based surfaces because this material can be readily used for Fourier transformed infra-red (FTIR) spectroscopy. 5,[START_REF] Schartner | Immobilization of Proteins in their Physiological Active State at Functionalized Thiol Monolayers on ATR-Germanium Crystals[END_REF][START_REF] Goldzstein | Ligand-Receptor Interactions in Complex Media: A New Type of Biosensors for the Detection of Coagulation Factor VIII[END_REF][START_REF] Schartner | An ATR-FTIR Sensor Unraveling the Drug Intervention of Methylene Blue, Congo Red, and Berberine on Human Tau and Aβ[END_REF] In contrast with other detection methods, FTIR spectroscopy allows to simultaneously collect a wealth of information such as secondary structure and post-translational modifications. [START_REF] Goormaghtigh | Evaluation of the Information Content in Infrared Spectra for Protein Secondary Structure Determination[END_REF][START_REF] Derenne | FTIR Spectroscopy as a Multi-Parameter Analytical Tool[END_REF] Therefore, FTIR spectroscopy with attenuated internal total reflection (ATR) configuration is currently applied to study mono-and multilayers of bioorganic samples. [START_REF] Dole | Advance Applications of Fourier Transform Infrared[END_REF] Because the evanescent wave propagates outside of the ATR element on a very short distance only, the ATR configuration allows the detection of analytes in aqueous media when they are brought in close contact with the optical element. This ATR element could for example be a Ge crystal with the appropriate geometry. Ge has the advantages over Si (a much more studied element 12 ) to have a larger refractive index (resulting in a better signal-to-noise ratio) and to be transparent in a broader spectral range, allowing the study of more important chemical groups absorbing between 1400 and 800 cm -1 . Chemisorbed thiols [START_REF] Schartner | Immobilization of Proteins in their Physiological Active State at Functionalized Thiol Monolayers on ATR-Germanium Crystals[END_REF][START_REF] Holmberg | PEGylation of Carboxylic Acid-Functionalized Germanium Nanowires[END_REF] and silane derivatives 5,[START_REF] Schartner | An ATR-FTIR Sensor Unraveling the Drug Intervention of Methylene Blue, Congo Red, and Berberine on Human Tau and Aβ[END_REF][START_REF] Nabers | An Infrared Sensor Analysing Label-Free the Secondary Structure of the Abeta Peptide in Presence of Complex Fluids[END_REF][START_REF] Voué | Biochemical Interaction Analysis on ATR Devices: a Wet Chemistry Approach for Surface Functionalization[END_REF] are generally used for the chemical modification of Ge surfaces. However, the lack of reproducible grafting protocols and of longterm stability in aqueous media of the obtained monolayers are important limitations of these methods. In contrast, the reductive grafting of aryldiazonium salts [START_REF] Cao | Advances on Aryldiazonium Salt Chemistry Based Interfacial Fabrication for Sensing Applications[END_REF][START_REF] Pinson | Attachment of Organic Layers to Conductive or Semiconductive Surfaces by Reduction of Diazonium Salts[END_REF][START_REF] Pinson | Aryl Diazonium Salts: New Coupling Agents in Polymer and Surface Science[END_REF][START_REF] Delamar | Covalent Modification of Carbon Surfaces by Grafting of Functionalized Aryl Radicals Produced from Electrochemical Reduction of Diazonium Salts[END_REF] yields stable layers on Ge. [START_REF] Collins | Organic Functionalization of Germanium Nanowires using Arenediazonium Salts[END_REF]20,[START_REF] Lefevre | Towards Organic Film Passivation of Germanium Wafers Using Diazonium Salts: Mechanism and Ambient Stability[END_REF] Nevertheless, this technique generates in situ aryl radicals that easily react with the A c c e p t e d m a n u s c r i t p t 4 already grafted aryl groups rendering the formation and organization of monolayers difficult to control. [START_REF] Combellas | Steric Effects in the Reaction of Aryl Radicals on Surfaces[END_REF][START_REF] Combellas | Sterically Hindered Diazonium Salts for the Grafting of a Monolayer on Metals[END_REF] In this regard, we have recently developed a general strategy for the formation of robust, homogeneous and well-organized organic monolayers on various surfaces thanks to the use of calix [4]arene-tetra-diazonium salts. 24,[START_REF] Troian-Gautier | Extremely Robust and Post-Functionalizable Gold Nanoparticles Coated with Calix[4]arenes via Metal-Carbon Bonds[END_REF]26 These compounds can be electrochemically or chemically reduced to produce aryl radicals that can be grafted via multiple covalent bonds. The structure and geometry of the calix [4]arenes do not allow any additional grafting, preventing the formation of multilayers. Furthermore, when grafting calix [4]arenes functionalized at the small rim with for instance appending COOH groups, the resulting calixarene layer can be further postfunctionalized under mild conditions by various chemical species. 24 An additional feature, in contrast to all the other known methods, is the preparation of binary monolayers of controlled composition in a single step from the grafting of binary mixtures of calix [4]arene-tetra-diazonium salts. 27,[START_REF] Valkenier | Controlled Functionalization of Gold Nanoparticles with Mixtures of Calix[4]arenes Revealed by Infrared Spectroscopy[END_REF] Regarding the nonspecific adsorption of proteins and biomacromolecules, this is typically prevented by surface modification with small shielding molecules or polymers. Coatings based on oligo(ethylene glycol) (oEG), polysaccharides, zwitterionic compounds or perfluoroalkyls are some examples. [START_REF] Yam | Characterization, Resistance to Protein Adsorption, and Specific Avidin-Biotin Binding of Poly(amidoamine) Dendrimers Functionalized with Oligo(ethylene glycol) on Gold[END_REF][START_REF] Banerjee | Antifouling Coatings: Recent Developments in the Design of Surfaces That Prevent Fouling by Proteins, Bacteria, and Marine Organisms[END_REF][START_REF] Ederth | Resistance of Galactoside-Terminated Alkanethiol Self-Assembled Monolayers to Marine Fouling Organisms[END_REF][START_REF] Kitano | Resistance of Zwitterionic Telomers Accumulated on Metal Surfaces Against Nonspecific Adsorption of Proteins[END_REF]33,[START_REF] Li | Protein Adsorption and Cell Adhesion Controlled by the Surface Chemistry of Binary Perfluoroalkyl/Oligo(ethylene glycol) Self-Assembled Monolayers[END_REF] As oEGs are nontoxic and nonimmunogenic, they are widely used in medical applications. [START_REF] Harris | Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications[END_REF][START_REF] Comenge | The Role of PEG Conformation in Mixed Layers: from Protein Corona Substrate to Steric Stabilization Avoiding Protein Adsorption[END_REF][START_REF] Ozcelik | Poly(ethylene glycol)-Based Coatings Combining Low-Biofouling and Quorum-Sensing Inhibiting Properties to Reduce Bacterial Colonization[END_REF][START_REF] Riedel | Complete Identification of Proteins Responsible for Human Blood Plasma Fouling on Poly(ethylene glycol)-Based Surfaces[END_REF] The dominant way through which proteins adhere to surfaces is by hydrophobic effect. [START_REF] Brash | Proteins at Interfaces II[END_REF][START_REF] Awadhiya | Agarose Bioplastic-Based Drug Delivery System for Surgical and Wound Dressings[END_REF] Uncharged oEGylated surfaces interact with water molecules, preventing this hydrophobic effect and thus effectively shielding the surface from protein fouling. It is noteworthy that the grafting of antifouling coatings on Ge has been scarcely reported. [START_REF] Nabers | An Infrared Sensor Analysing Label-Free the Secondary Structure of the Abeta Peptide in Presence of Complex Fluids[END_REF]41 A c c e p t e d m a n u s c r i t p t 5 In order to develop a new ATR device suitable for biosensing applications, we have investigated the grafting on Ge of a robust monolayer of calix [4]arenes decorated with multiple oEGs arms.

For this, calix [4]arene-tetra-diazonium salts bearing oEGs substituents of different lengths (1 and 2) and a reference compound 3 [START_REF] Fairman | Protein Resistance of Surfaces Modified with Oligo(ethylene glycol) Aryl Diazonium Derivatives[END_REF] were synthesized (Scheme 1). The feasibility of the grafting was first evaluated on standard gold surfaces through previously developed electrochemical and chemical grafting processes. 26 As Ge is a semi-conducting surface, the conventional electrochemical grafting process is not appropriate and thus only the chemical process was then used. Finally, the antifouling properties of the surfaces that were modified with oEGylated calixarenes were evaluated by FTIR spectroscopy and confocal laser scanning fluorescence microscopy upon exposure to proteins. Scheme 1. Chemical grafting of calix [4]arenes 1 and 2 or electrochemical grafting of 1a. Inset: structure of reference diazonium salt 3. Note that the representation of the grafted calixarene does not necessarily imply that its four aryl units are linked to the surface.

EXPERIMENTAL SECTION

A c c e p t e d m a n u s c r i t p t 6 Chemicals and materials. All solvents and reagents were at least of reagent grade quality and were purchased either from Alfa Aesar, Sigma-Aldrich, TCI, Roth or Acros organics. All reactions were performed under an inert atmosphere. Reactions were magnetically stirred and monitored by thin layer chromatography using Merck-Kiesegel 60F254 plates. Flash chromatography was performed with silica gel 60 (particle size 35-70 µm) supplied by Merck. Anhydrous DMF was obtained from Acros organics. Anhydrous THF was obtained from distillation on Na/benzophenone. Ultrapure water was obtained via a Millipore Milli-Q system (18.2 MΩ cm).

The fluorescent FITC-BSA (fluorescein isothiocyanate BSA conjugate) was purchased from Sigma-Aldrich as well as the BSA used for ATR-FTIR spectroscopy experiments. Gold-coated silicon wafer (1000 Å layer thickness) was purchased from Sigma-Aldrich. Both sides polished germanium squares (10 x 10 x 0.5 mm) and germanium single-crystal triangular prisms (base 6.8 mm x 45 mm length and an internal incident angle of 45°) were purchased from ACM (France).

Caution! Although we have not encountered any problem, it is noted that diazonium salts derivatives are potentially explosive and should be handled with appropriate precautions.

Both calix [4]arene-tetra-oEGs 1 and 2 were prepared following the same strategy. The detailed synthesis of compound 1 (Scheme 2) is given here, while details for the compounds 2 and 3 are reported in the SI. the organic layer was washed with H2O (3 x 150 mL) and the combined aqueous layers were extracted with CH2Cl2 (300 mL). The combined organic layers were concentrated under reduced pressure. The crude oil was purified by flash chromatography on silica gel (EtOAc/MeOH = 85:15). The oil was washed with H2O to yield the compound 6 as a yellow oil (4.2 g, 3.0 mmol, 96%). [START_REF] Zhao | Solvent-Tunable Binding of Hydrophilic and Hydrophobic Guests by Amphiphilic Molecular Baskets[END_REF] but is here prepared via another synthetic strategy. Calix [4]arene-tetra-oEG4 6 (4.2 g, 3.0 mmol) was dissolved in CH2Cl2 (100 mL). A mixture of glacial CH3COOH/fuming HNO3 (1:1) (22 mL) was added at 0°C and the reaction mixture was stirred for 5 hours at room temperature. The reaction mixture was concentrated under reduced pressure. The resulting oil was dissolved in CH2Cl2 (100 mL), the organic layer was washed with H2O (3 x 70 mL) and the combined aqueous layers were extracted with CH2Cl2 (150 mL). The combined organic layers were concentrated under reduced pressure. The crude oil was purified by flash chromatography on silica gel (DCM/Acetone = 65:35) to yield compound 8 as a yellow oil (2.5 g, 1.8 mmol, 61%).

A c c e p t e d m a n u s c r i t p t 8 1 H NMR spectrum of compound 8 is in accordance with the one reported in the literature. [START_REF] Zhao | Solvent-Tunable Binding of Hydrophilic and Hydrophobic Guests by Amphiphilic Molecular Baskets[END_REF] 1 H NMR (300 MHz, CDCl3, 298K), δ (ppm): 3.33-3.44 (m, 16H), 3.48-3.63 (m, 48H), 3.80 (t, J = 4.5 Hz, 8H), 4.24 (t, J = 4.5 Hz, 8H), 4.65 (d, J = 12 Hz, 4H), 7.58 (s, 8H). 12,32,52,72-tetrakis(2-(2-methoxyethoxy)ethoxy)-1,3,5,7(1,3)tetrabenzenacyclooctaphane-15,35,55,75-tetraamine 1a. Note that compound 1a was already described in the literature [START_REF] Zhao | Solvent-Tunable Binding of Hydrophilic and Hydrophobic Guests by Amphiphilic Molecular Baskets[END_REF] but is here prepared via another synthetic strategy. Calix [4]arene-tetra-NO2-tetra-oEG4 8 (0.901 g, 0.660 mmol) and Pd/C (53 mg, 0.498 mmol) were suspended in EtOH (18 mL). Hydrazine hydrate (3 mL, 61.8 mmol) was added dropwise and the reaction mixture was stirred for 15 hours at reflux. The reaction mixture was filtered on Celite and the Celite was washed with EtOH and CH2Cl2. The filtrate was concentrated under reduced pressure to yield the compound 1a as a yellow oil (0.798 g, 0.641 mmol, 97%). 1 H NMR spectrum of compound 1a is in accordance with the one reported in the literature. [START_REF] Zhao | Solvent-Tunable Binding of Hydrophilic and Hydrophobic Guests by Amphiphilic Molecular Baskets[END_REF] A c c e p t e d m a n u s c r i t p t 14 Hz, 4H), 3.84 (br t, 8H), 4.49 (br t, 8H), 4.72 (d, J = 14 Hz, 4H), 8.02 (br s, 8H). [START_REF] Holmberg | PEGylation of Carboxylic Acid-Functionalized Germanium Nanowires[END_REF] Gold electrodes were first polished with alumina paste (1 µm particles) and sonicated in ultrapure water for 10 minutes before being dried under a flux of nitrogen. The diazonium salt 1 was prepared in situ in 0.5 M aqueous HCl in the presence of NaNO2 (8 eq.) from the corresponding calix [4]-tetra-aniline 1a (1.5 mM). The gold substrate was functionalized through the reduction of the in situ generated diazonium cations by six voltammetric cycles, the potential being swept between +0.5 V and -0.4 V versus Ag|AgCl electrode. Once the grafting was achieved, the surfaces were thoroughly rinsed with ultrapure water and sonication was applied for 10 minutes.

Chemical grafting strategy. Caution! Piranha solution is a very strong oxidant and should be handled very carefully. Gold surfaces were first immersed in a piranha solution (H2SO4/H2O2 3:1) for 10 minutes and sonicated for 5 minutes. They were subsequently washed with concentrated sulphuric acid and with ultrapure water and then dried under argon atmosphere. Germanium surfaces were first immersed in ultrapure water and then in ethanol; sonication was applied each A c c e p t e d m a n u s c r i t p t 10 time for 5 minutes. They were then rinsed with Et2O and dried under argon atmosphere. The surfaces were dipped in a 5 mM solution of the diazonium salt (1-3) in aqueous 0.1 M sodium hydroxide for 2 hours, without stirring in order to avoid any mechanical damage of the surface by the magnetic stirrer bar (a minimal volume of solution is used). Once the grafting was achieved, all the surfaces were thoroughly washed with ultrapure water and then acetonitrile. Sonication was applied each time for 5 minutes. The surfaces were then dried under argon flow.

ATR-FTIR spectra were recorded at 22°C on a Bruker Equinox 55 FTIR spectrophotometer equipped with a liquid nitrogen-cooled mercury-cadmium-telluride (MCT) detector. The spectrophotometer was continuously purged with dried air. The target chemicals were deposited in solution on a germanium single-crystal internal reflection element (triangular prism of 6.8 x 45 mm, with an internal incidence angle of 45°) and the solvent was removed with a flow of nitrogen gas. The spectra from the grafted monolayer were obtained following the chemical grafting of the calix [4]arene target directly on the germanium internal reflection element. In each case, bare germanium was used for the background spectrum. The nonspecific adsorption experiment was measured in a flow-through cuvette with a 100 µg/mL solution of BSA in phosphate buffer media in D2O (PBS-D2O) and a flow rate of 10 µL/min. Opus software (4.2.37) was used to record 128 scans with a nominal resolution of 2 cm -1 for Figures 1 andS54 and 64 scans with a nominal resolution of 4 cm -1 for Figures 2, S55 and S56. Data were processed and analysed using the homewritten Kinetics package in Matlab R2013a by subtraction of water vapor, baseline correction, apodization at 16 cm -1 .

Confocal Laser Scanning Fluorescence Microscopy: fluorescence images were recorded with a Nikon Ti-Eclipse inverted microscope, equipped with a laser emitting at 488 nm. The emitted light was collected in the epi-fluorescence mode and a dichroic mirror was employed to select

RESULTS AND DISCUSSION

Both calix [4]arene-tetra-oEGs 1 and 2 were prepared following the strategy developed for other calix [4]arene-tetra-diazonium salts (Scheme 2). 24,26 First, a tetra O-alkylation of p-t Bucalix [4]arene constrained the macrocycle in a cone conformation and allowed the introduction of the oEGs arms on the small rim. Then, a sequence of ipso-nitration / reduction afforded the tetraanilines 1a and 2a in respectively 57 % and 52 % overall yields from p-t Bu-calix [4]arene. Finally, a diazotation reaction yielded target diazonium salts 1 and 2 whose structures were clearly confirmed by 1D and 2D NMR spectroscopy. Grafting experiments were performed on gold and germanium surfaces with both calix [4]arenes 1 and 2 as well as with the reference compound 3 for comparison (Scheme 1). Similarly to previously reported calixarenes, 26 grafting of the new oEGylated calixarenes was first evaluated on a gold substrate, using calix [4]-tetra-aniline precursor 1a, from which the corresponding tetra-diazonium salt 1 was prepared in situ by adding an excess of NaNO2 in acidic conditions. Reduction of the diazonium cations was performed by six voltammetric cycles. The density of the electrografted layer was characterized by analyzing its blocking property towards the electrochemical response of the redox probe Fe(CN)6 3-/4-. 24 Complete inhibition of the electrochemical response was found, indicating that a dense organic layer had been formed at the electrode surface (Figure S51). Having found that the electrochemical grafting methodology was applicable to the newly synthesized calix [4]arene-tetra-oEGs, we subsequently undertook the chemical grafting of the diazonium salts (1-3) on Au and Ge substrates. The previously reported protocols for the grafting of aryl diazonium salts on Ge in organic solvents requires an oxide-free Ge surface obtained by treatment with an aqueous HF solution. [START_REF] Collins | Organic Functionalization of Germanium Nanowires using Arenediazonium Salts[END_REF]20,[START_REF] Lefevre | Towards Organic Film Passivation of Germanium Wafers Using Diazonium Salts: Mechanism and Ambient Stability[END_REF] Here, the grafting procedure on Ge substrate was performed in a basic aqueous solution and involved the in situ formation of diazoates. [START_REF] Griffete | Preparation of Water-Soluble Magnetic Nanocrystals Using Aryl Diazonium Salt Chemistry[END_REF][START_REF] Doyle | Structure-Dependent Reactivity of Semiconducting Single-Walled Carbon Nanotubes with Benzenediazonium Salts[END_REF][START_REF] Podvorica | Spontaneous Grafting of Diazoates on Metals[END_REF] Under these aqueous conditions, the laborious and hazardous pre-treatment with aqueous HF is not needed, as the Ge oxide at the surface is water soluble. [START_REF] Amy | Passivation and Characterization of Germanium Surfaces[END_REF] The chemical grafting of diazonium salts 1, 2 and 3 was performed upon immersion of the Au and Ge substrates in an aqueous 0.1 M NaOH solution containing 5 mM of the desired diazonium salt for 2 hours. This procedure afforded gold surfaces Au-1, Au-2 and Au-3 and germanium surfaces Ge-1 and Ge-2 (Scheme 1). Each grafting experiment was repeated at least three times to assess the reproducibility.

ACS Paragon Plus Environment

All these modified surfaces were then characterized by contact angle measurements and the results were compared to those obtained with bare surfaces (i.e., surfaces that were treated similarly to the modified ones but in the absence of the diazonium salt). Contact angles of 62 ± 9° and 30 ± 10° were obtained respectively for bare Au and Ge substrates (Table 1). For all the modified oEGylated surfaces, the contact angle was found to be ca. 56°. These results clearly confirm the presence of the oEG layer on all the modified surfaces and highlight the reproducibility of the grafting process. Furthermore, the value of ca. 56° is in agreement with those found in literature for oEGylated surfaces. Atomic Force Microscopy (AFM) measurements were performed on the surfaces Au-1, Au-3, Ge-1 and on the bare gold and germanium substrates. All the modified surfaces exhibited a surface topography similar to that of the corresponding bare substrate, indicating a thin uniform deposit on the surface (Figure S52 and S53). Next, thicknesses of the organic layers were estimated in contact mode through scratching experiments. The AFM tip was used to scratch a square area on the modified samples by exercising a force sufficient to remove the organic part without damaging the substrate. Using the height difference between both areas of the surface profile (Figure S52 and S53), thicknesses of the organic layer of 2.4 and 3.0 nm were found for Au-1 and Ge-1 respectively (Table 1). These values correspond well with the height of the calix [4]arene-tetra-oEG4 structure (ca. 2.2 nm estimated from MM2 energy minimizations with ChemBio3D software), suggesting that a monolayer of grafted calix [4]arene-tetra-oEGs 1 was obtained on gold and germanium. In the case of the control surface Au-3, a thickness of 19.0 nm was estimated, clearly indicating the formation of multilayers. This result highlights the crucial role played by the calixarene structure in the formation of monolayers. The germanium surfaces Ge-1 and Ge-2 were then analyzed by ATR-FTIR spectroscopy. For comparison, the IR absorbance spectra of compounds 1, 2 and their corresponding anilines 1a and 2a were recorded (Figures 1 andS54). Typical bands belonging to the calix [4]arene core and oEG groups were observed both for the compounds and the corresponding monolayers. The asymmetric COC stretching from the oEG chains around 1100 cm -1 is clearly visible in all spectra. Other bands can be attributed to the calix [4]arene moieties such as the symmetric COCAr stretching around 1050 -1020 cm -1 and the aromatic ring stretching around 1460 cm -1 . The IR results thus clearly confirm the grafting of calixarenes 1 and 2 on Ge.

Figure 1. ATR-FTIR absorption spectra from 3100-2700 and 1800-800 cm -1 of modified surface Ge-1 (red), calix [4]arene-tetra-diazonium salt 1 (green) and calix [4]-tetra-aniline 1a (blue).

The nonspecific adsorption of proteins on bare Ge and on oEGylated Ge substrates was monitored by ATR-FTIR spectroscopy. Surfaces Ge-1 and Ge-2 were compared to evaluate the influence of the chain length of oEGs on the protein adsorption. A flow-through cuvette with a cm -1 ). This was not observed, indicating that the calix [4]arene monolayer is stable under aqueous conditions for at least 16 hours (Figure S55). The surfaces Ge-1 and Ge-2 were then placed under a flow (10 µL/min) of the PBS-D2O medium for 10 min in order to reach an equilibrium state under aqueous conditions. The flow was then switched to the solution of BSA in PBS-D2O for 16

hours and finally back to PBS-D2O for 5 hours. Infrared spectra were recorded every minute for the first 20 minutes and then every 10 minutes till the end of the experiment. Adsorbed BSA was characterized by two bands whose intensities were increasing over time: the amide-I' at 1640 cm - 1 , which is mainly associated with the C=O(ND) stretching vibrations and the amide-II' at 1450 cm -1 , which results mainly from in plane ND bending vibrations (Figure 2a andS56). The integral of the amide-I' band at 1640 cm -1 was calculated for each spectrum. The graph shows that the binding of BSA to surfaces Ge-1 and Ge-2 is reduced by more than 85% in comparison to bare Ge (Figure 2b). No difference was found between Ge-1 and Ge-2, showing that an oEG length of four on the calix [4]arenes is sufficient for the effective prevention of nonspecific protein adsorption on Ge. Such an efficiency in the reduction of nonspecific adsorption has been reported scarcely and, in the case of the previous systems, either a very thick coverage was necessary (e.g., polymer brushes) [START_REF] Chen | Adjustable Bioadhesive Control of PEGylated Hyperbranch Brushes on Polystyrene Microplate Interface for the Improved Sensitivity of Human Blood Typing[END_REF]49 or a limited stability was observed.

CONCLUSIONS

In conclusion, we have developed a reproducible procedure for the grafting of thin and robust organic monolayers of calix [4]arene-tetra-oEGs on Ge and Au that do not require any laborious or dangerous pre-treatment. The significant antifouling properties of these monolayers were demonstrated. The nonspecific absorption of proteins was decreased by more than 85% compared to bare Ge. Such an efficiency is unique if we consider the thickness (ca.

6 .

 6 Commercial p-t Bu-calix[4]arene (2.0 g, 3.1 mmol) was dissolved in dry DMF (50 mL). NaH (60% dispersion in oil, 0.840 g, 21.0 mmol) and 2,5,8,11tetraoxatridecan-13-yl 4-methylbenzenesulfonate 4 (7.6 g, 21.0 mmol) were added and the reaction mixture was stirred for 24 hours at 75°C under inert atmosphere. The reaction mixture was then brought to room temperature and quenched dropwise with 5 mL of HCl (0.5 M). The mixture was A c c e p t e d m a n u s c r i t p t 7 then concentrated under reduced pressure. The resulting oil was dissolved in CH2Cl2 (200 mL),

1 H 8 .

 18 NMR (300 MHz, CDCl3, 298K), δ (ppm): 1.06 (s, 36H), 3.09 (d, J = 12 Hz, 4H), 3.37 (s, 12H), 3.49-3.71 (m, 48H), 3.93 (t, J = 4.5 Hz, 8H), 4.09 (t, J = 4.5 Hz, 8H), 4.41 (d, J = 12 Hz, 4H), 6.75 (s, 8H).[START_REF] Holmberg | PEGylation of Carboxylic Acid-Functionalized Germanium Nanowires[END_REF] C NMR (100 MHz, CDCl3, 298K), δ (ppm): 31.2, 31.6, 33.9, 59.2, 70.5, 70.6, 70.6, 70.7, 70.8, 70.8, 72.1, 73.0, 125.1, 133.9, 144.7, 153.4. FTIR, ν (cm -1 ): 3412, 2952, 2921, 2899, 2867, 1644, 1480 1457, 1392, 1361, 1300, 1249, 1199, 1107, 1062, 1027, 944, 869. HRMS: calcd. for C80H128O20 (M+H) + 1409.91 found 1409.91. Note that compound 8 was already described in the literature

  1 H NMR (300 MHz, CD3OD, 298K), δ (ppm): 2.94 (d, J = 13 Hz, 4H), 3.35 (s, 12H), 3.48-3.70 (m, 48H), 3.89 (t, J = 5 Hz, 8H), 4.03 (t, J = 5 Hz, 8H), 4.40 (d, J = 13 Hz, 4H), 6.17 (s, 8H). 12,32,52,72-tetrakis(2-(2-methoxyethoxy)ethoxy)-1,3,5,7(1,3)tetrabenzenacyclooctaphane-15,35,55,75-tetrakis(diazonium) 1. Calix[4]-tetra-aniline-tetra-oEG4 1a (50 mg, 0.040 mmol) was solubilized in dry acetonitrile (0.5 mL). At -40°C, NOBF4 (46 mg, 0.400 mmol) was added and the reaction mixture was stirred for 2 hours at -40°C under inert atmosphere. The reaction mixture was concentrated under reduced pressure at room temperature to yield the compound 1 as a yellow/orange oil. Due to the well-known low stability of diazonium groups, compound 1 was used without further purification for the grafting experiments. HRMS analysis was not performed because of the low stability of compound 1 against temperature.

  C NMR (150 MHz, CD3CN, 298K), δ (ppm): 31.0, 58.9, 70.4, 70.7, 70.7, 70.9, 71.0, 71.0, 72.5, 77.5, 107.7, 135.0, 138.7, 168.7. FTIR, ν (cm -1 ): 3083, 2942, 2883, 2267, 1571, 1443, 1355, 1293, 1264, 1096, 1025, 922, 846. Electrochemical grafting strategy. Electrochemical measurements were conducted in a threeelectrode cell using a Ag|AgCl saturated KCl electrode as reference electrode, a large area platinum foil as counter electrode, and a polycrystalline gold disk (1.6 mm in diameter, from Bioanalytical Systems) as working electrode, all connected to an Autolab PGSTAT30 (Metrohm Autolab) potentiostat equipped with a ScanGen module.

A c c e p t e d m a n u s c r i t p t 11 wavelengths

 11 shorter than 540 nm. A bandpath emission filter (515-530 nm), matching well the emission spectrum of fluorescein, was placed in front of the photo-multiplier tube. A 10× magnification objective (NA = 0.30, working distance 16 mm) was employed. The images were processed with the software ImageJ.

Langmuir

A c c e p t e d m a n u s c r i t p t Scheme 2 .

 2 Scheme 2. Synthesis of calix[4]arene-tetra-oEGn-tetra-diazonium salts 1 and 2.

48

 48

Table 1 .a

 1 Contact angle measurements and thickness estimations of the grafted organic layers. Contact angle a (°) Thickness b (nm) Average values obtained by multiple analyses repeated on several surfaces. b Determined by AFM measurement (see text). c The bare surfaces were treated similarly to the modified ones but in the absence of the diazonium salt. d n.d.: not determined.

A c c e p t e d m a n u s c r i t p t 15

 15

u s c r i t p t 16 solution

 16 of bovine serum albumin (BSA, 100 µg/mL) in phosphate buffer media in D2O (PBS-D2O) at 22°C was used to analyze the nonspecific binding of BSA on the surfaces. BSA is used as model protein in many investigations because it is one of the most abundant proteins found in blood and it adheres very well to surfaces. First, the stability of the calixarene layer under the study conditions was evaluated through exposure of the surface Ge-1 to a flow (10 µL/min) of PBS-D2O buffer during 16 h. The decomposition of the organic layer should give rise to the appearance of negative absorbance bands in the regions where the calixarene signals are located (e.g., at 1100

36

 36

A c c e p t e d m a n u s c r i t p tFigure 2 .A c c e p t e d m a n u s c r i t p t 18 distinguished

 218 Figure 2. (a) ATR-FTIR absorption spectra from 1800-1350 cm -1 reported for every hour during

Figure 3 .

 3 Figure 3. Fluorescence images acquired on gold and germanium surfaces that were partially

A c c e p t e d m a n u s c r i t p t 19 toward

 19 3 nm) and the high stability of the calixarene layer. The modified Ge surfaces thus constitute promising devices for the development of ATR-FTIR-biosensors. Current developments in our laboratories are directed Page 18 of 27 ACS Paragon Plus EnvironmentLangmuir the grafting of calix[4]arene-tetra-oEGs with terminal post-functionalizable groups (e.g. COOH, azide or alkyne groups) that can serve for the controlled immobilization of proteins. s c r i t p t

H NMR (600 MHz, CD3CN,

298K), δ (ppm): 3.30 (s, 12H), 3.43-3.59 (m, 48H), 3.78 (d, J =

Devouge, S.; Conti, J.; Goldsztein, A.; Gosselin, E.; Brans, A.; Voue, M.; De Coninck, J.;

Ammar, M.; Smadja, C.; Ly, G. T.; Tandjigora, D.; Vigneron, J.; Etcheberry, A.; Taverna, M.;

Girard, A.; Geneste, F.; Coulon, N.; Cardinaud, C.; Mohammed-Brahim, T. SiGe

Santos, L.; Mattiuzzi, A.; Jabin, I.; Vandencasteele, N.; Reniers, F.; Reinaud, O.; Hapiot, P.;

Brady, R. F.; Aronson, C. L. Elastomeric Fluorinated Polyurethane Coatings for Nontoxic Fouling Control. Biofouling 2003, 19, 59-62.

Schartner, J.; Güldenhaupt, J.; Mei, B.; Rögner, M.; Muhler, M.; Gerwert, K.; Kötting, C.

ACKNOWLEDGMENT

The "Actions de Recherches Concertées" of the Fédération Wallonie-Bruxelles and the ULB (Ph.D. grant to P.B.) are acknowledged for financial support.

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:. Detailed procedure for the preparation and characterization of the synthesized compounds, NMR spectra, AFM results, and additional FTIR experiments. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*E-mail: ijabin@ulb.ac.be; vrauss@ulb.ac.be

ORCID


Diego Armando Diaz Vargas

Claude Baron

Philippe Esteban

Diego Armando

Diaz Vargas

System Engineering and Agile Management

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.

Figure 1. Agile Methods Approach

The research objective is to integrate the traditional practices of System Engineering (SE) and Project Management (PM) to coordinate stakeholders based on a collaborative decision-making using agile management (Figure 3).

  To achieve that objective we'll try: • To mapping agile concept • To define what tool can be used according to each project • To propose a methodology

Figure 3 .Figure 2 .

 32 Figure 3. Global Research Context