Damien Tedoldi
email: damien.tedoldi@enpc.fr

Ghassan Chebbo
email: chebbo@enpc.fr

Arthur Gaillot
email: arthur.gaillot@supagro.fr

Daniel Pierlot

Marie-Christine Gromaire
email: gromaire@enpc.fr

Evaluation of an improved modelling approach for the retention of heavy metals in SUDS soil

Keywords: Contamination profiles, Modelling, Runoff infiltration, Soil, Sustainable Urban Drainage Systems, Validation

The ability of reactive transport models to accurately replicate the fate of contaminants in SUDS has been sparsely documented. The present work aims (i) to improve the description of the processes, including nonuniform fluxes at the surface, and (ii) to use extensive experimental data, collected in a stormwater infiltration basin, to evaluate the model's accuracy. Both the model and observations confirm that most infiltration occurs near the water inlet. Differences between the measured and predicted profiles are either attributable to the filtration of particle-bound metals, or to spatially variable sorption capacities of the soilnotably because of lower contents of organic matter.

INTRODUCTION

Source-control stormwater management appears as an efficient way to mitigate surface pollutant fluxes, but brings about some concerns regarding the fate of micropollutants in soil [START_REF] Bressy | Towards the determination of an optimal scale for stormwater quality management: Micropollutants in a small residential catchment[END_REF]. Thereupon, experimental assessments of SUDS contamination may be valuably complemented by modelling approaches, so as to gain insight into, e.g., potential downward fluxes of contaminants, or optimal designs towards pollutant retention [START_REF] Quinn | Modeling heavy metal behavior in sustainable drainage systems: a case study[END_REF]. However, the current literature comprises few attempts at validating such models with field data from SUDS. Additionally, most existing models are onedimensional, but recent investigations on surface soil contamination have evidenced that upper infiltration fluxes are highly non-uniform in such devices [START_REF] Tedoldi | Spatial distribution of heavy metals in the surface soil of source-control stormwater infiltration devices -Inter-site comparison[END_REF]. Hence, the two main objectives of the present work are (i) to develop a new modelling framework to simulate the fate of metals in SUDS soil, and (ii) to compare the predicted distributions with experimental data.

MATERIALS & METHODS

Model description. So as to take into account non-uniform infiltration, the modelled facility is divided into n homogeneous zones starting from the inflow point (Fig. 1). The fraction of the flow rate exceeding the infiltration capacity of the domain k is routed towards the domain k+1 as an upper boundary condition; the potential excess of water from the domain n causes ponding in the whole device. Within each domain, the soil is described with a classical one-dimensional model, using (i) Richards equation to replicate hydrodynamics, (ii) an advection-dispersion equation to replicate solute transport, and (iii) a Langmuir sorption isotherm to replicate physical chemistry, associated with 1 st order kinetics for a given part of the sorption sites. The corresponding formulae are presented elsewhere (e.g. [START_REF] Simunek | The HYDRUS-1D software package for simulating the one-dimensional movement of water, heat, and multiple solutes in variably-saturated media[END_REF]. Each 1D problem is solved independently with the HYDRUS-1D software package.

Validation data. A thorough characterization of a stormwater infiltration basin was undertaken. The facility collects runoff water from a small residential area, where metals (and especially Zn) are expected to be mainly in dissolved form [START_REF] Bressy | Towards the determination of an optimal scale for stormwater quality management: Micropollutants in a small residential catchment[END_REF]. The spatial distribution of Cu and Zn was determined both in the surface soil and along vertical profiles. Concurrently, hydrodynamic parameters were acquired via "BEST" infiltration tests [START_REF] Lassabatère | Beerkan estimation of soil transfer parameters through infiltration experiments[END_REF] at several locations in the basin (Fig. 2a). The soil's dispersivity was derived from inverse modelling of a conservative tracer experiment in a 50-cm-long soil column (Fig. 2b). Cu and Zn sorption characteristics were determined via batch tests (Fig. 2c-d), adjusting the initial concentrations so as to fit the isotherms in a typical range of runoff concentrations. All theoretical relationships satisfactorily fitted the data (R 2 > 0.9).

PRELIMINARY RESULTS & DISCUSSION

The last section of the inlet pipe has broken 3 years ago, so that the location of the inflow point has changed during the operation of the facility, as revealed by the spatial distribution of metals in the surface soil (Fig. 3a). The latter appears as an accurate signature of the cumulative infiltration fluxes, as replicated by the hydrologic model (Fig. 3b), which confirms that a significant part of the device is seldom reached by runoff contaminants.

The variability of runoff concentrations was not considered; their mean value was derived from the sorption isotherms, assuming that soil-water equilibrium had been reached in the upper sample from the most contaminated region of the basin (i.e. near the former water inlet, referred to as "Zone 1"): the calculated Zn concentration is 150 µg/L, which is consistent with the land use.

The model appears to be rather accurate, but does not capture the sharp gradient of concentrations in the upper centimeters of soil, and slightly underestimates the migration depth of metals (Fig. 4a). This may be attributable to the mechanical filtration of particle-bound contaminants, and/or to the fact that the soil's sorption capacities are actually variable with depth, as suggested by the vertical evolution of the volatile matter content (Fig. 4b), since organic matter is known to largely govern metal retention in soils.

CONCLUSIONS

Both the experimental results and the hydrologic model show that non-uniform processes should be taken into account, in order to improve the model's accuracy. Uncertainties remain regarding the simulated contamination profiles, then further research will be dedicated (i) to incorporate mechanical filtration in the model, and (ii) to investigate the variability of the sorption isotherm with depth, and its consequences on the predicted soil concentrations.

ACKNOWLEDGEMENTS

This research was carried out under the OPUR research program. The authors gratefully acknowledge OPUR partners for their financial support.


Matthew G Burbank

Audrey Burban

Ahmad Sharanek

Richard J Weaver

Christiane Guguen-Guillouzo

André Guillouzo
email: andre.guillouzo@univ-rennes1.fr

C G-G

Prof André Guillouzo

Early alterations of bile canaliculi dynamics and the ROCK/MLCK pathway are characteristics of drug-induced intrahepatic cholestasis

Keywords: ALB, albumin, ALDOB, aldolase B, AMI, amiodarone, APAP, acetaminophen, BA, bile acid, BC, bile canaliculi, BOS, bosentan, BSEP, bile salt export pump, BUS, buspirone, CaM, calmodulin, CIM, cimetidine, CPZ, chlorpromazine, CsA, cyclosporine A, CYP3A4, cytochrome P450 3A4, DIC, diclofenac, DILI, drug-induced liver injury, DMSO, dimethyl sulfoxide, ENT, entacapone, FIA, fialuridine, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, MDR1, multidrug resistance 1, MET, metformin, MLC, myosin-light-chain, MLC2, myosin light chain subunit-2, MLCK, myosin light chain kinase, SOD2, manganesedependent superoxide dismutase, MRP2,3,4, multidrug resistance-associated proteins 2,3,4, MTT, methylthiazoletetrazolium, NEF, nefazodone, NTCP, Na

Intrahepatic cholestasis represents 20-40% of drug-induced injuries from which a large proportion remains unpredictable. We aimed to investigate mechanisms underlying druginduced cholestasis and improve upon its early detection using human HepaRG cells and a set of 12 cholestatic and 6 non-cholestatic drugs. Bile canaliculi (BC) dynamics, Rho/myosinlight-chain (MLC) kinase pathway implication, efflux inhibition of taurocholate, a predominant BSEP substrate, and expression of the major canalicular and basolateral bile acids transporters were analyzed. We demonstrated 12 cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both BC dynamics, characterized by either dilatation or constriction, and alteration of the ROCK/MLCK signalling pathway while non cholestatic compounds, by contrast, have no effect. Co-treatment with Y-27632, a ROCK inhibitor, and calmodulin, a MLCK activator, reduced BC constriction and dilatation, respectively confirming the role of these pathways in intrahepatic drug-induced cholestasis.

By contrast, inhibition of taurocholate efflux and/or human BSEP overexpressed in membrane vesicles (published data) was not observed with all cholestatic drugs and moreover examples of non-cholestatic compounds reportedly found to inhibit BSEP.

Transcripts levels of major bile acids transporters were determined after 24h-treatment.

BSEP, NTCP and OATP-B were down-regulated with most cholestatic and some noncholestatic drugs while deregulation of MRPs was more variable, probably mainly reflecting secondary effects. Together, our results show cholestatic drugs consistently cause an early alteration of BC dynamics associated with modulation of the Rho/MLC kinases and these changes are more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis.

.

D η ϳϭϯϳϯ ϯ polypeptide; OATP-B, organic anion transporting polypeptide; PER, perhexiline; PHH, primary human hepatocytes; PIO, pioglitazone; ROCK, Rho-kinase; ROS, reactive oxygen species; RT-qPCR, real-time quantitative polymerase chain reaction; SCHH, sandwichcultured human hepatocytes; TCA, taurocholic acid; TAC, tacrolimus; TOL, tolcapone; TRO, troglitazone; [ 3 H]-TCA, [ 3 H]-taurocholic acid; XIM, ximelagatran; Y-27632, 4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride.

Introduction

Many drugs have been reported to induce liver injury in a dose-dependent or -independent manner in humans. Due to their hepatotoxicity and idiosyncrasy, a large fraction has been removed from the market or put under warning box during the last decades. A frequent manifestation of drug-induced liver injury (DILI) is represented by intra-hepatic cholestasis which is characterized by impaired hepatocellular secretion of bile, resulting in accumulation of bile acids (BA), bilirubin and cholesterol [START_REF] Padda | Drug-Induced Cholestasis[END_REF]. In several population-based studies on DILI, a cholestatic pattern has been found in 20-40% and a mixed cholestatic and hepatocellular injury pattern in 12-20% of patients [START_REF] Lee | Drug-Induced Hepatotoxicity[END_REF] with a large proportion of the drug-induced cholestatic cases reportedly unpredictable.

Early detection of DILI and more particularly cholestasis remains a real challenge for pharmaceutical industries and regulatory agencies. According to the European Medicines Agency guidelines on the investigation of drug interactions and the International Transporter Consortium (Zamek-Gliszczynski et al. 2012), inhibition of the bile salt export pump (BSEP), that plays a major role in bile acid canalicular transport (Jansen et al. 1999;[START_REF] Stieger | Role of the Bile Salt Export Pump, BSEP, in Acquired Forms of Cholestasis[END_REF], should be evaluated during drug development when evidence of cholestatic liver injury has been observed in nonclinical safety studies or in human clinical trials [START_REF] Kenna | Current Concepts in Drug-Induced Bile Salt Export Pump (BSEP) Interference[END_REF]. Indeed, many drugs reported to cause DILI have been identified as efflux transporters inhibitors [START_REF] Morgan | Interference with Bile Salt Export Pump Function Is a Susceptibility Factor for Human Liver Injury in Drug Development[END_REF][START_REF] Stieger | Role of the Bile Salt Export Pump, BSEP, in Acquired Forms of Cholestasis[END_REF][START_REF] Dawson | In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans[END_REF][START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF]), but a significant number of false positives has been found [START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF]. Moreover, compounds known to interfere with BSEP function are often not associated with significant liver cell injury in preclinical animal models although they have been related to liver damage when administered to humans [START_REF] Morgan | Interference with Bile Salt Export Pump Function Is a Susceptibility Factor for Human Liver Injury in Drug Development[END_REF][START_REF] Dawson | In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans[END_REF]) and many cholestatic drugs are not BSEP inhibitors [START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF].

Currently, various in vivo and in vitro biological approaches are used for studying BA transporters. Detection of cholestatic drugs can depend on the choice of the method and/or D η ϳϭϯϳϯ ϲ the model and its species origin. Animal models including knockout, mutant models and whole tissues can be employed but in vitro systems are more frequently used, based on membrane and cell assays [START_REF] Brouwer | In Vitro Methods to Support Transporter Evaluation in Drug Discovery and Development[END_REF]. Inverted vesicles derived from various nontransfected or transfected cell lines are widely used to analyze interactions between substrates and inhibitors with target transporters. However, a number of probe substrates and inhibitors are recommended for evaluation of human ATP-dependent transport into membrane vesicles. Polarized cells are standard models for evaluating drug transport.

Primary hepatocytes appear to be the most appropriate cell model to mimic the hepatic uptake and efflux functions. However, freshly isolated and cryopreserved hepatocytes are not polarized, and in standard culture conditions they rapidly lose their functional activities and functional bile canaliculi (BC) (Guguen-Guillouzo and Guillouzo 2010). Rat hepatocyte couplets (Graf et al. 1984;Thibault et al.1992) and primary rat and human hepatocytes in a sandwich configuration [START_REF] Swift | Sandwich-Cultured Hepatocytes: An in Vitro Model to Evaluate Hepatobiliary Transporter-Based Drug Interactions and Hepatotoxicity[END_REF]Ellis et al. 2014) have been the most widely used in vitro cell models to analyze hepatic transport processes. Sandwich-cultured human hepatocytes (SCHH) recapitulate the polarized architecture but they frequently exhibit large inter-donor and in vitro time-dependent functional variations. An alternative to SCHH is the human HepaRG cell-line which expresses features characteristic of mature hepatocytes, exhibits typical functional BC and correctly polarized distribution of transport proteins, and produces BA at levels comparable to those measured in primary human hepatocyte cultures (Guguen-Guillouzo and Guillouzo 2010;[START_REF] Azzi | Comparative Localization and Functional Activity of the Main Hepatobiliary Transporters in HepaRG Cells and Primary Human Hepatocytes[END_REF][START_REF] Sharanek | Cellular Accumulation and Toxic Effects of Bile Acids in Cyclosporine A-Treated HepaRG Hepatocytes[END_REF].

The mechanisms underlying drug-induced intrahepatic cholestasis remain poorly understood.

Besides the role of hepatobiliary transporter changes, other mechanisms, such as altered cell polarity, disruption of cell-to-cell junctions and cytoskeletal modifications, are believed to participate to the development of intrahepatic cholestasis. In normal hepatocytes BC undergo spontaneous contractions, which are essential for BA efflux [START_REF] Arias | The Biology of the Bile Canaliculus[END_REF]) and alternations in myosin light chain (MLC2) phosphorylation and dephosphorylation are required for these contractions [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]. Recently, we reported that cholestatic D η ϳϭϯϳϯ ϳ drugs can cause in vitro early constriction or dilatation of BC associated with deregulation of the Rho-kinase/myosin-light-chain-kinase (ROCK/MLCK) pathway [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]).

In the present work, we aimed to determine whether changes in BC dynamics and the ROCK/MLCK pathway are more representative targets of cholestatic drugs than the efflux transporters inhibition and whether these alterations could better discriminate cholestatic from non cholestatic compounds using human differentiated HepaRG cells. Our results showed that the 12 tested cholestatic drugs could cause BC constriction and dilatation associated with different disturbances of the ROCK/MLCK pathway. were provided from Cell Signaling Technology (Schuttersveld, The Netherlands). Secondary antibodies were obtained from Invitrogen (Saint Aubin, France). Hoechst dye was from Promega (Madison, WI). Other chemicals were of the highest reagent grade.

D η ϳϭϯϳϯ

ϴ

Materials and methods

Reagents

Cell cultures and treatments

HepaRG cells were seeded at a density of 2.6×10 4 cells/cm 2 in Williams E medium supplemented with 2mm glutamax, 100µ/ml penicillin, 100μg/ml streptomycin, 10% Hyclone bovine fetal calf serum, 5μg/ml insulin, and 50μM hydrocortisone hemisuccinate. At confluence, after a 2-week cell proliferation phase, HepaRG cells were shifted to the same medium supplemented with 1.7% dimethyl sulfoxide (DMSO) for 2 additional weeks in order to obtain confluent differentiated cultures containing equal proportions of hepatocyte-like and progenitors/primitive biliary-like cells [START_REF] Cerec | Transdifferentiation of Hepatocyte-like Cells from the Human Hepatoma HepaRG Cell Line through Bipotent Progenitor[END_REF]). These differentiated hepatic cell cultures were used for analytical assays.

D η ϳϭϯϳϯ ϵ

Primary human hepatocytes (PHH) were obtained from Biopredic International (St Gregoire, France). They were isolated by collagenase perfusion of histologically normal liver fragments from 3 adult donors undergoing resection for primary and secondary tumours (Guguen-Guillouzo et al. 1982). These three donors were caucasian males: 74-, 65-, and 71-year old for donor 1, 2 and 3, respectively. Primary cultures were obtained by seeding 1.5 x 10 5 hepatocytes per cm 2 onto plates in Williams E medium supplemented with 10% Hyclone bovine fetal calf serum, 100units/μl penicillin, 100μg/ml streptomycin, 1μg/ml insulin, 2mM

glutamine. The medium was discarded 12h after cell seeding, and cultures were thereafter maintained in serum-free medium. PHH cultures were used 3-4 days after cell seeding. For drug treatments, differentiated HepaRG cells and PHH were incubated in a medium containing 2% serum and 1% DMSO. The list and characteristics of tested drugs are displayed in Table 1. The drugs were divided into 3 groups according to their effects in clinic:

well recognized as cholestatic (group 1), rarely cholestatic (group 2) and non cholestatic (group 3). The compounds were dissolved in DMSO or water and the final concentration of the solvent did not exceed 1%.

Cell viability

Cytotoxicity of the tested drugs was evaluated using the MTT colorimetric assay. Briefly, cells were seeded in 96-or 24-well plates and exposed to various concentrations of each compound in triplicate for 24h. After medium removal, 100μl serum-and DMSO-free medium containing MTT (0.5mg/ml) was added to each well and incubated for 2h at 37°C.

The water-insoluble formazan was dissolved in 100μl of DMSO and absorbance was measured at 550nm [START_REF] Aninat | Expression of Cytochromes P450, Conjugating Enzymes and Nuclear Receptors in Human Hepatoma HepaRG Cells[END_REF]). IC20 values (the concentrations causing 20% cytotoxicity) were calculated from the concentration-responses curves.

D η ϳϭϯϳϯ ϭϬ

Time-lapse cell imaging

Phase-contrast images of HepaRG cells and PHH were captured every 10min, using timelapse phase-contrast videomicroscopy. The inverted microscope Zeiss Axiovert 200M was equipped with a thermostatic chamber (37°C and 5% CO 2 ) to maintain the cells under normal culture conditions and with an Axiocam MRm camera with a 10x objective.

Determination of canalicular lumen surfaces

Canalicular lumen surfaces appeared bright (white) and the hepatocytes/biliary cells denser (black) using a phase-contrast microscope Zeiss Axiovert 200M. Brightness parameters were adjusted to better distinguish between white and black densities and analysis was performed on at least 4 images per condition per well. White canalicular lumen was then quantified using the image J software every 10min for 24h. Data obtained during the first 4h with all tested compounds are presented and BC quantification is expressed as percent of the control (Supplement Figure 1).

MLCK and ROCK modulation

MLCK and ROCK implication in BC deformations was determined by the use of calmodulin (CaM) and Y-27632, two specific modulators. HepaRG cells were treated with CaM, a specific MLCK activator, and Y-27632, a selective ROCK inhibitor, in the presence or absence of dilatators and constrictors, respectively [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]. BC alterations after co-treatment were quantified and compared to treatment with the modulator alone as described above.

Taurocholic acid clearance

Cells were first exposed to 43.3nM [ 3 H]-TCA for 30min to induce its intracellular accumulation, then washed with standard buffer and incubated with the tested compounds for 2h in a standard buffer with Ca 2+ and Mg 2+ . After the incubation time, cells were washed Control * 100) / ([ 3 H]-TCA accumulation in cell layers Tested compound ) [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF].

Real-time quantitative polymerase chain reaction (RT-qPCR) analysis.

Total RNA was extracted from 10 6 HepaRG cells with the SV total RNA isolation system (Promega).

Western blotting analysis of p-MLC2

HepaRG cells were treated with the tested compounds for 1, 2 and 3h at a concentration causing BC alteration, then washed with cold phosphate-buffered saline and finally resuspended in cell lysis buffer supplemented with protease and phosphatase inhibitors

Statistical analysis

One-way Anova with multiple comparison test (graphpad prism 6.00) was performed to compare time-dependent MLC2 phosphorylation in different samples. Data were considered significantly different when *P < 0.05. The student t test was applied to compare values of BC quantification between treated and corresponding co-treated cultures (with CaM or Y-27632).

Data were considered as significantly different when # P < 0.001. The student t test was also applied to compare values of BA clearance and mRNA levels between treated and corresponding control cultures. Data were considered as significantly different when *P < 0.05, **P < 0.01 and ***P < 0.001. Each value corresponded to the mean ± standard error (SEM) of three independent experiments.

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Results

Cytotoxicity determination

Experiments were performed to firstly calculate IC20 values for each compound after a 24h treatment of HepaRG cells using the MTT assay (Table 2). Then, three concentrations were tested for further experiments, the highest corresponding to the IC20 value. IC10 or IC20 values are commonly selected for use in early in vitro toxicology studies [START_REF] Liguori | Trovafloxacin-Induced Gene Expression Changes in Liver-Derived in Vitro Systems: Comparison of Primary Human Hepatocytes to HepG2 Cells[END_REF][START_REF] Pomponio | In Vitro Kinetics of Amiodarone and Its Major Metabolite in Two Human Liver Cell Models after Acute and Repeated Treatments[END_REF]. When IC20 values could not be determined because of absence of toxicity, higher concentrations were tested. The IC20 values and/or final selected maximum concentrations tested are displayed in Table 2.

Bile canaliculi dynamics

BC contractions are essential for clearance of BAs; they are characterized by repeated opening and closing processes [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]. We examined BC dynamics in living cells by time-lapse imaging during at least 12h after addition of the tested compounds.

Depending on the drug, BC showed constriction, dilatation or no change. The 5 compounds from group 1 caused early alterations of BC, usually starting after 1h exposure of HepaRG cells (Figure 1). As previously reported [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF], CPZ (50µM) and CsA (50 µM) induced constriction while BOS (100 µM) caused dilatation of BC. The two other cholestatic compounds from group 1 (NEF and TRO) and 4 rarely cholestatic compounds from group 2 (DIC, ENT, PER, TAC) also caused morphological alterations of BC at the same range of concentrations, either constriction with NEF (75µM), PER (20µM) and TRO (50µM) (Supplemental video 1) or dilatation with ENT (100µM) (Supplemental video 2), DIC (200µM) and TAC (50µM) (Figure 1A and B). Two other compounds from group 2, MET and CIM, also caused alterations of BC dynamics but at much higher although non cytotoxic concentrations, i.e. constriction and dilatation at 3 and 4mM respectively (Figure 1B).

D η ϳϭϯϳϯ ϭϰ

Noticeably, differential time-or concentration-dependent BC alterations were evidenced with some cholestatic compounds. Thus, among the 5 constrictors from group 1, only CsA caused dilatation of BC at low concentrations (10µM) (Figure 1C). Interestingly, one group 2 compound, TOL (100µM), showed dilatation during the first hour followed by constriction of BC during the following hours of treatment (Figure 1D). By contrast, ENT, a dilatator from the same therapeutic family as TOL, caused strong dilatation starting within the first hour without any constriction during the following hours (Figure 1B). All other tested compounds, namely XIM, AMI, APAP, FIA, BUS and PIO (group 3), did not alter BC dynamics at non cytotoxic concentrations (Figure 2E; Supplemental video 3).

Morphological canalicular alterations induced by the tested compounds were quantified by measuring the canalicular surface using the image J software every 10min for 24h. Data obtained during the first 3h with all tested compounds are displayed in Figure 1F-I. When observed several altered BC were detected whatever the selected culture area or the tested drug. Interestingly, the results were comparable to those previously obtained using the canalicular distribution of zona occludens-1 protein to measure BC areas [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]); e.g. BOS caused 1.5-fold increase in BC surfaces using either method.

Interestingly, constrictions appeared to be irreversible while dilatations were only transient, as seen with TOL. Noticeably, AMI showed an unspecific increase in BC surface quantification starting after 1h due to appearance of intracytoplasmic vesicles corresponding to induction of phospholipidosis (Figure 1E). Drug effects on BC structures are summarized in Supplemental Table 1.

ROCK and MLCK alterations with cholestatic drugs

The myosin light chain subunit-2 (MLC2), the regulatory subunit of myosin 2, that is predominantly distributed in the pericanalicular region of hepatocytes, is known as a target of ROCK; its phosphorylation/dephosphorylation reflects the dynamic rhythm of BCs and was D η ϳϭϯϳϯ ϭϱ found to be impaired by CPZ, CsA and BOS [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]. Based on this previous study, we analyzed the 18 compounds at 3 critical time-points (i.e. 1, 2 and 3h) by western blotting. However, we first verified with 6 compounds that similar alterations of MLC2 phosphorylation occurred after 30min whatever the compound (Supplemental Figure 2).

Western blots analysis of MLC2 showed an increased phosphorylation tendency between 1 and 3 hours with constrictors (i.e. CPZ, CsA, PER, NEF, TRO and MET) and inversely dephosphorylation tendency with dilatators (i.e. TAC, BOS, CIM and DIC) (Figure 2A).

Depending on the compound, either a constrictor or a dilatator, large individual quantitative differences were observed in their effects (Figure 2B). The potent constrictors, PER, TRO and NEF, showed a much higher rate of phosphorylation than metformin after 3h treatment (Figure 2A,B). Interestingly, ENT showed a decrease of MLC2 phosphorylation during BC dilatation while TOL did not show any significant change, likely due to rapid morphological changes of BC from dilatation to constriction after one hour treatment (Figure 2B). All non cholestatic drugs did not significantly affect MLC2 phosphorylation/dephosphorylation (Figure 2C, D).

To further analyze alterations of BC dynamics by cholestatic compounds, involvement of CaM and less constriction after 1h when co-treated with Y-27632 whereas ENT showed a much lower dilatation in the presence of CaM (Figure 3E). Interestingly, a co-treatment with 3G).

Taurocholic acid clearance

The influence of all tested drugs on efflux of [ 3 H]-TCA, a predominant BSEP substrate, was assessed in HepaRG cells maintained in a standard buffer. Cells were first incubated with

Modulation of bile acids transporter expression

Genes encoding the major BA transporters can also be deregulated by cholestatic drugs and ALDOB, was also observed with most drugs, that likely reflected cell adaptation or some cytotoxicity after a 24h treatment and not a direct drug-induced cholestatic effect. Induction of CYP3A4 mRNAs with CPZ and their inhibition by CsA, TAC and BOS agreed with previous reports. SOD2 mRNA expression was increased with CsA and CPZ and was not altered with controls compounds.

Comparative analysis of BC dynamics in primary human hepatocytes

Hepatocytes from 3 different donors were analyzed by phase-contrast cell imaging.

Similar results as those described with HepaRG hepatocytes were obtained within the first hours of drug exposure, i.e. BC constriction with CPZ, CsA, NEF and TRO (Figure 5A) as well as with TOL after 2h, and BC dilatation with TOL (between 1-2h), ENT, DIC and BOS (Figure 5B); however, these effects were not observed in cells from all donors with DIC, ENT and TAC. Moreover, the rarely cholestatic drug PER that caused BC constriction in HepaRG cells, did not alter BC structures in any of the 3 PHH populations (Figure 5C). As expected, the non cholestatic drug FIA was ineffective. The time-course of appearance of BC alterations was approximately the same for all the cholestatic compounds but the extent of dilatation appeared to be reduced compared to that observed in HepaRG cells.

D η ϳϭϯϳϯ ϭϴ

Discussion

Both BC dynamics and ROCK/MLCK activities can be impaired by cholestatic drugs [START_REF] Sharanek | Rho-Kinase/myosin Light Chain Kinase Pathway Plays a Key Role in the Impairment of Bile Canaliculi Dynamics Induced by Cholestatic Drugs[END_REF]). In the current study, we demonstrated by using a set of 18 cholestatic and non cholestatic compounds that the 12 drugs classified cholestatic on the basis of reported clinical findings caused disturbances of both BC dynamics and the ROCK/MLCK pathway. By contrast, non cholestatic compounds exhibited no effect on these pathways.

Comparison of these data with TCA efflux showed some cholestatic drugs were ineffective or enhanced TCA clearance while some non cholestatic exerted an inhibitory effect.

Drug-induced impairment of BC dynamics can result in either early constriction or dilatation of the canalicular lumen. These morphological changes were observed with the two groups of cholestatic drugs, i.e either well-recognized as causing clinical cholestasis or responsible of rare cases of clinical cholestasis (Table 1). BC constriction was irreversible, representing a terminal step leading to cell death while dilatation could be reversible and did not impede, at least within 24h, cell survival. Interestingly, the two types of BC alterations could be observed with some drugs, showing either time-concentration-dependency. TOL caused first dilatation and later on constriction while CsA caused dilatation at low and constriction at high concentrations. However, other BC constrictors, i.e. CPZ, NEF, PER and TRO did not show clear evidence of dilatation prior to constriction or differences in the effects on BC dynamics at lower concentrations or shorter time. Since a cholestatic liver usually develops after chronic drug administration in patients and is characterized by dilatated BC (Imanari, et al. 1981;[START_REF] Watanabe | Motility of Bile Canaliculi in the Living Animal: Implications for Bile Flow[END_REF][START_REF] Chung | Increased Microfilaments in Hepatocytes and Biliary Ductular Cells in Cholestatic Liver Diseases[END_REF]) experiments are on-going to determine whether dilatation of BC could occur after prolonged repeated treatments with constricting drugs at lower concentrations. No BC alteration was observed with the 6 non cholestatic drugs. Noticeably, additional non cholestatic drugs have been tested, especially ambrisentan, a member of the endothelin receptor antagonists family as BOS, and ibuprofen and were found to be similarly ineffective (data not shown). 3G).

Drug inhibition studies of BSEP have mostly been performed using membrane vesicles overexpressing BSEP [START_REF] Morgan | Interference with Bile Salt Export Pump Function Is a Susceptibility Factor for Human Liver Injury in Drug Development[END_REF]. Although PHH represent a more relevant physiological in vitro model they have been only rarely used [START_REF] Kostrubsky | Inhibition of Hepatobiliary Transport as a Predictive Method for Clinical Hepatotoxicity of Nefazodone[END_REF][START_REF] Swift | Sandwich-Cultured Hepatocytes: An in Vitro Model to Evaluate Hepatobiliary Transporter-Based Drug Interactions and Hepatotoxicity[END_REF][START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF]. A large proportion of cholestatic drugs has been identified as BSEP inhibitors [START_REF] Morgan | Interference with Bile Salt Export Pump Function Is a Susceptibility Factor for Human Liver Injury in Drug Development[END_REF][START_REF] Stieger | Role of the Bile Salt Export Pump, BSEP, in Acquired Forms of Cholestasis[END_REF][START_REF] Dawson | In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans[END_REF][START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF]), but a significant number of false positives has been found, using either vesicle assays or SCHH [START_REF] Pedersen | Early Identification of Clinically Relevant Drug Interactions with the Human Bile Salt Export Pump (BSEP/ABCB11)[END_REF]. Using the TCA clearance assay we also found that not all cholestatic drugs are inhibitors of TCA clearance. Indeed, compared to deformation of BC associated with disruption of the ROCK/MLCK pathway induced by the 12 cholestatic drugs and not by the 6 non cholestatic drugs, alteration of TCA clearance was observed only with 4/7 drugs known to rarely cause cholestasis in clinic, i.e. DIC, ENT, MET and CIM.

Noticeably, if DIC and ENT inhibit BSEP using transfected vesicles, MET and CIM are not classified or only weakly shown to be inhibitors of BSEP [START_REF] Morgan | Interference with Bile Salt Export Pump Function Is a Susceptibility Factor for Human Liver Injury in Drug Development[END_REF]. Moreover, the potent cholestatic drug CPZ was not detected as a BSEP inhibitor using transfected vesicles dynamics. Together, these data clearly emphasize the importance of both cell model-based systems and assays to estimate BSEP inhibition. Obviously, in our experimental whole-cell system, TCA efflux could not be considered as reflecting only BSEP activity. Indeed, TCA clearance was reduced to around 20% of control values by CsA. This strong inhibition could be due not only to an inhibition of canalicular efflux, but also to an inhibition of basolateral efflux. By contrast, a dose-dependent increase in TCA efflux was obtained with DIC, likely reflecting an enhanced basolateral efflux and/or a reduced influx activity. Moreover, with some drugs whether cholestatic or not (for example BUS), only a slight increase in TCA efflux was observed at low concentrations while at higher concentrations efflux levels were comparable to those measured in control HepaRG cell cultures, confirming previously reported concentration-dependent effects of drugs on TCA efflux using SCHH [START_REF] Kostrubsky | Inhibition of Hepatobiliary Transport as a Predictive Method for Clinical Hepatotoxicity of Nefazodone[END_REF]. With the two compounds, MET and CIM, high non-cytotoxic concentrations were required to observe changes in BC dynamics while TCA efflux levels were comparable to those measured in controls but significantly decreased with low drug concentrations. By contrast, ENT did not inhibit TCA efflux at any of the concentrations tested. Together, these data support the conclusion that not all cholestatic drugs induce accumulation of TCA in treatment and classified them into 3 categories according to their capacity to strongly (>60%), moderately (20-60%) or not repressing the transporter. In the current study, we analysed four of these compounds. Similarly, we found a strong repression of BSEP expression with TRO and BOS that were tested at comparable concentrations but we evidenced a strong repression with NEF and CsA which were tested at higher concentrations. As shown here, the effects of cholestatic and non cholestatic drugs on BA transporters expression depended on drug concentration and the tested transporter (Supplemental Figure 3A). Noteworthy, most cholestatic drugs repressed BSEP, NTCP and OATP-B at concentrations that caused BC deformations, suggesting that in vitro experimental conditions (drug concentrations, in vitro model,…) are critical for obtaining correct results. The three BA transporters were also repressed by some non cholestatic drugs. Interestingly, it has been found that in human cholestatic liver, down-regulation of transporters also occurs at the mRNA level as observed for BSEP, NTCP, OATP (Zollner et al. 2001). More variable results were obtained with MRPs (Table 3). The most important changes were induction of MRP2-4 with CsA and TAC and of MRP2 with DIC. MRP3 upregulation has been described as a compensatory mechanism to BA accumulation in cholestatic rat liver (Donner and Keppler 2001). Since transcripts levels were measured 24h after drug addition, their changes reflected more likely secondary effects rather than direct and specific drug effects.

Interestingly, different responses were obtained with drugs of a same family as observed with TOL and ENT (as described above) as well as CsA and TAC that caused BC constriction to 100-fold lower therapeutic doses than CsA, the absence of hepatotoxic and cholestatic effects at concentrations up to 25μm fully agreed with its safety reported in clinic [START_REF] Mihatsch | The Side-Effects of Ciclosporine-A and Tacrolimus[END_REF]. Accordingly, considering the Cmax values measured in patients, i.e. 1.15 and 0.09 µM for CsA and TAC respectively, cholestatic concentrations are expected to be reached normally only in patients treated with CsA. However, considering the 12 tested cholestatic drugs, no direct correlation could be established between Cmax (Table 2) and their in vitro concentrations inducing BC deformation, confirming data reported with BSEP inhibition assays on a large set of compounds [START_REF] Dawson | In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans[END_REF][START_REF] Köck | Risk Factors for Development of Cholestatic Drug-Induced Liver Injury: Inhibition of Hepatic Basolateral Bile Acid Transporters Multidrug Resistance-Associated Proteins 3 and 4[END_REF].

BC deformation was also investigated in PHH cultures from 3 donors treated with some of the tested compounds. If both BC constriction and dilatation were induced by all cholestatic drugs except PER, donor-donor differences were observed. Only 4/8 tested cholestatic compounds were found to cause alterations or no changes of BC structures in the 3 hepatocyte populations. Among the factors that could explain donor differences were variations in drug metabolism and detoxification capacity and/or BA transporters levels.

Nevertheless, these data with PHH confirm those obtained with HepaRG cells and support the suitability of this model as a reproducible, easy to use liver cell model for screening and mechanistic studies for the identification of on drug-induced cholestasis.

In summary, early BC deformations resulting in constriction or dilatation of the canalicular lumen associated with alterations of the ROCK/MLCK pathway were observed with all tested cholestatic drugs, including those which are not BSEP inhibitors (Table 4). Together, our results favour the conclusion that alterations of BC dynamics with the involvement of the ROCK/MLCK pathway are more specific predictive markers than TCA clearance and direct BSEP inhibition to screen the cholestatic potential of new chemical entities.

  Cyclosporine A (CsA), chlorpromazine (CPZ), nefazodone (NEF), tolcapone (TOL), diclofenac (DIC), perhexiline (PER), troglitazone (TRO), tacrolimus (FK-506) (TAC), amiodarone (AMI), acetaminophen (APAP), ximelagatran (XIM), metformin (MET), entacapone (ENT), buspirone (BUS), pioglitazone (PIO), cimetidine (CIM), 4-(1-aminoethyl)-) were purchased from Sigma (St. Quentin Fallavier, France). Fialuridine (FIA) was supplied by Carbosynth (Compton, U.K). Bosentan (BOS) was obtained from Sequoia Research Products (Pangbourne, U.K). Calmodulin (CaM) was provided from Merck Chemicals (Fontenay sous Bois, France). Phalloidin fluoprobe was purchased from Interchim (Montluçon, France). [ 3 H]-taurocholic acid ([ 3 H]-TCA) was supplied by Perkin Elmer (Boston, MA). Specific antibodies against phospho-myosin light chain 2 (ser19) and HSC70

  0.5N NaOH and the remaining radiolabeled substrate was measured through scintillation counting to determine [ 3 H]-TCA clearance. [ 3 H]-TCA clearance was determined based on its accumulation in the cell layers (cells + BC) and calculated relative to the control using the following formula: [ 3 H]-TCA clearance = ([ 3 H]-TCA accumulation in (cells + BC)

  ROCK and MLCK was investigated using Y-27632, a specific inhibitor of ROCK, and CaM, a specific MLCK activator. Y-27632 (10µM) caused BC dilatation whereas CaM (5µM) alone, caused no BC alteration (Figure3A). Alteration of BC size after co-treatment with a drug and CaM or Y-27632 was quantified and compared to the drug treatment alone. As expected, a co-treatment with CaM reduced BC dilatation induced by the dilatators (i.e. BOS, DIC, CIM and TAC) (Figure3B and F). By contrast, co-treatment with Y-27632 partly counteracted the early phase of constriction induced by the constrictors (i.e. TRO, NEF, PER and MET) (Figure3C and D). TOL showed a lower dilatation during the first hour when co-treated with

  [ 3 H]-TCA for 30min and then treated for 2h with all drugs at three different concentrations, the highest causing BC alterations. [ 3 H]-TCA clearance, reflecting BAs accumulation in HepaRG hepatocytes and thus the cholestatic potential was determined by quantification of intracellular accumulation of radiolabeled TCA. The results are displayed in Figure4. Two categories of compounds could be distinguished. The first one was composed of 8 compounds that caused a dose-dependent decrease in TCA clearance after 2h treatment; it included the 5 group 1 cholestatic drugs (i.e. NEF, TRO, BOS, CPZ and CsA) as well as 3 drugs of group 2 (i.e. PER, TOL and TAC). The second category was composed of the other 10 drugs that did not inhibit TCA efflux at the concentrations used; it included 4 drugs of group 2 (i.e. DIC, ENT, MET and CIM) and the 6 drugs that are classified as non cholestatic in vivo (i.e. XIM, AMI, APAP, FIA, BUS and PIO). Noticeably, DIC caused a concentrationdependent increase of TCA clearance, reaching 4-fold at 200µM.

(

  [START_REF] Pauli-Magnus | Hepatobiliary Transporters and Drug-Induced Cholestasis[END_REF] and the deregulation of their mRNA expression is usually linked to their activity[START_REF] Bramow | Cholestasis and Regulation of Genes Related to Drug Metabolism and Biliary Transport in Rat Liver Following Treatment with Cyclosporine A and Sirolimus (Rapamycin)[END_REF]. Six major genes encoding either efflux (BSEP, MRP2, MRP3 and MRP4) or uptake (NTCP, OATP-B) transporters were analyzed by RT-qPCR after a 24h treatment with 3 concentrations of each tested drug. Different patterns depending on the compound and the tested concentration, were evidenced and are shown in

  Fig. 1. Effect of cholestatic and non cholestatic drugs on bile canaliculi dynamics inHepaRG cells

Fig

  Fig. 2. Alterations of MLC2 phosphorylation/dephosphorylation by cholestatic drugs inHepaRG cells.

Fig. 3 .

 3 Fig. 3. Alterations of the ROCK/MLCK pathway by cholestatic drugs in HepaRG cells.

Fig. 4 .Fig

 4 Fig. 4. Effects of tested drugs on bile acid clearance activity in HepaRG cells

Fig. 6 .

 6 Fig. 6. Schematic representation of the molecular targets of cholestatic drugs.

  This article has not been copyedited and formatted. The final version may differ from this version.

	DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373
	D η ϳϭϯϳϯ
	(Roche, Mannheim, Germany). Aliquots containing equivalent total protein content, as
	determined by the Bradford procedure with bovine serum albumin as the standard, were
	subjected to sodium dodecyl sulfate/12% polyacrylamide gel electrophoresis, electro-
	transferred to immobilon-p membranes, and incubated overnight with primary antibodies
	directed against p-MLC2 and HSC70. After incubation with a horseradish peroxidase-
	conjugated anti-mouse/rabbit antibody (Thermofisher Scientific, Waltham, MA), membranes
	were incubated with a chemiluminescence reagent (Millipore, Billerica, MA) and bands were
	visualized and quantified by densitometry with fusion-CAPT software (Vilber Lourmat,
	Collegien, France).
	ϭϮ

  This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ
	BOS and Y-27632 showed an increase in dilatation extent in comparison with BOS or Y-
	27632 alone (Figure
	ϭϲ

Table 3

 3 OATP-B was repressed by CsA, BOS and NEF while MRP2, MRP3 and MRP4 were overexpressed only by CsA. Most compounds of group 2, i.e. TOL, DIC, PER, MET, CIM and TAC, also repressed BSEP, NTCP and OATP-B.Moreover, MRP2, MRP3 and MRP4 were upregulated by TAC. In addition, MRP2 was upregulated by DIC and MRP3 was down-regulated by PER. Noticeably, none of the tested transporters was significantly deregulated by ENT. Some BA transporters were also deregulated by non cholestatic compounds. Thus, BUS down-regulated BSEP and NTCP while PIO and AMI inhibited BSEP and NTCP expression. In addition, transcripts of four other genes, i.e. ALB, ALDOB; CYP3A4 and SOD2, were also measured (supplemental Table 2). A slight or significant decrease in the expression of the liver-specific genes, ALB

and supplemental Figures

3-8

. Both BSEP and NTCP were strongly repressed by all D η ϳϭϯϳϯ ϭϳ group 1 cholestatic compounds. In addition,

  This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 Noteworthy, BC constriction and dilatation were associated with different alterations of the ROCK/MLCK pathway. Activation of the ROCK pathway and inhibition of the MLCK pathway associated with increased phosphorylation and dephosphorylation of MLC2 were observed

	D η ϳϭϯϳϯ
	with constrictors and dilatators respectively (Figure 6). Importantly, differential quantitative
	effects were observed, supporting the conclusion that cholestatic drugs can act by different
	mechanisms, involving different targets on the ROCK/MLCK pathway (Sharanek et al. 2016).
	The involvement of MLC2 in the occurrence of BC constriction or dilatation was also
	demonstrated by using Y-27632, a specific ROCK inhibitor, and CaM, a specific MLCK
	activator, which caused inhibition of BC constriction and dilatation respectively, giving
	support to the conclusion that disturbances of the ROCK/MLCK pathway preceded
	alterations of BC dynamics. Interestingly, BOS combined with Y-27632 had an additive effect
	on BC dilatation showing that both acted via distinct MLCK and ROCK enzymatic targets
	(Figure
	ϭϵ

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	DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373
	D η ϳϭϯϳϯ
	(Pedersen et al. 2013) whereas it appeared as a BC constrictor and an inhibitor of TCA efflux
	in the current study. This discrepancy could be attributed to CPZ-induced generation of ROS
	that cannot be observed with transfected vesicles (Anthérieu et al. 2013). Two additional
	major differences were evidenced between transfected cells and TCA clearance assays: we
	observed inhibition of TCA clearance and BC deformation with TOL and no change with PIO
	while opposite data were reported with transfected vesicles (Pedersen et al. 2013; Morgan et
	al. 2010). Noticeably, PIO has been classified as either rarely cholestatic (May et al. 2002) or
	non cholestatic (Pedersen et al. 2013) and as a potent BSEP inhibitor (Morgan et al. 2010;
	Dawson et al. 2012) or not (Kaimal et al. 2009; Pedersen et al. 2013) using transfected
	vesicles. However, in Pedersen's work PIO at 50µM did not significantly inhibit TCA efflux in
	SCHH. Our results agreed with this finding; at 100µM, PIO had no effect on TCA efflux or BC
	ϮϬ

  This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ
	HepaRG cells and that its clearance can be modulated by different mechanisms other than
	direct alteration of BSEP activity.
	Most studies have focused on interactions between drugs and BSEP without considering
	perturbation of expression of this transporter. Recently, one group (Garzel et al. 2014)
	analysed 30 drugs for their ability to perturb BSEP expression in PHH cultures after 24h
	Ϯϭ

  This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 D η ϳϭϯϳϯ ϮϮ and dilatation respectively. TAC has been reported to cause rare cases of cholestasis in infants. Accordingly, it induced dilatation at only 50μM in this study. Since TAC is used at 10

  This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ
	phosphorylation/dephosphorylation quantified using fusion-CAPT software. (C) Western blots
	of p-MLC2 obtained with group 3 molecules. AMI, amiodarone; APAP, acetaminophen; BOS,
	bosentan; BUS, buspirone; CIM, cimetidine; CPZ, chlorpromazine; CsA, cyclosporine A; DIC,
	diclofenac; ENT, entacapone; FIA, fialuridine; MET, metformin; NEF, nefazodone; PER,
	perhexiline; PIO, pioglitazone; TAC, tacrolimus; TOL, tolcapone; TRO, troglitazone; XIM,
	ximelagatran. Data are expressed in arbitrary units (A.U) and represent means ± SEM of 3
	independent experiments. One hour treatment was arbitrarily set at a fixed value of 2.5 for all
	the drugs. Data were considered significantly different when *P < 0.05.
	ϯϲ

Table 1 . Characteristics of the 18 tested drugs.

 1 This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ

Drugs Therapeutic use Clinical hepatic effects In vitro hepatic effects Main references Group 1. DRUGS involved in clinical cholestasis BOS Antihypertensive

  This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ
		Cholestasic hepatitis ;	Inhibition of mitochondrial	Spigset et al. 2003 ;
		hepatocellular necrosis	respiration ; BSEP inhibition*	Dykens et al. 2008 ;
	NEF	Antidepressant		Kostrubsky et al.
				2006 ; Morgan et al.
				2010 ; Livertox
		Serious idiosyncratic liver	Bile acid transporters inhibition ;	Watkins et al. 1998 ;
		injury ; hepatocyte necrosis ;	mitochondrial dysfunction ;	Jemnitz et al. 2010 ;
	TRO	Antidiabetic
		cholestatic hepatitis	BSEP inhibition*	Morgan et al. 2010 ;
				Livertox
		Aminotransferases elevation ;	BSEP inhibition* ; substrate for	Fattinger et al. 2001 ;
		increase in serum bile acids	OATP, NTCP and MRP2**	Mano et al. 2007 ;
				Treiber et al. 2007 ;
				Livertox
		Intrahepatic cholestasis ;	Oxidative stress ; mitochondrial	Anthérieu et al. 2013
		hepatocellular necrosis ;	membrane potential loss ;	; Padda et al. 2011 ;
	CPZ	Antipsychotic
		phospholipidosis	pericanalicular F-actin	Velayudham and
	Group 2.	Cholestasis;	disorganisation Oxidative stress ; BSEP, MRP2	Farell 2003 ; Livertox Sharanek et al. 2015
		hyperbilirubinemia ;	and MDR1 inhibition** ; bile acid	; Mihatsch et al.
		Immunosup-
	CsA	Aminotransferases elevation	transport alteration ;	1998
		pressant
			pericanalicular F-actin
			disorganisation
				ϯϵ

DRUGS involved in rare cases of clinical cholestasis

  This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ D η ϳϭϯϳϯ
					reactive metabolite	HU) unpublished
						data ; Longo et al.
						2016
	Group 3. DRUGS not involved in clinical cholestasis	CIM DIC ENT AMI APAP BUS FIA PIO	Histamine2-receptor antagonist AINS Antiparkinsonian Antiarrhythmic Analgesic and antipyretic Hypnotic Antiviral Antidiabetic (PPAR-γ agonist)	Hepatocellular-cholestatic injury Cholestasis ; aminotransferases elevation Mild jaundice and cholestatic pattern ; aminotransferases elevation Steatosis ; phopholipidosis ; aminotransferases elevation Acute liver failure ; aminotransferases elevation Rare aminotransferase elevation with no link to clinically apparent liver injury Liver failure associated with lactic acidosis ; mitochondrial toxicity No aminotransferase elevation MRP2 stimulation ; BSEP and BSEP inhibition* BSEP inhibition* ; BSEP and MRP2 inhibition** BSEP and NTCP inhibition* ; mitochondrial dysfunction Phospholipidosis ; steatosis Lactate dehydrogenass increase ; mitochondrial damage No inhibition of mitochondrial respiration Mitochondrial and cellular toxicity MRP4 inhibition* Immune toxicity: No formation of reactive	Livertox ; Mohi-ud-din et al. 2004 ; Pedersen et al. 2013 Banks et al. 1995 ; Livertox ; Pedersen et al. 2013 Fisher et al. 2002 ; Longo et al. 2016 ; Livertox ; Krajcsi P. Fromenty et al. 1995 ; Anthérieu et al. 2011 Toyoda et al. 2012 ; McGill et al. 2012 Kostrubsky et al. 2006 ; Dykens et al. 2008 ; Livertox Tujios S et al. 2011 Morgan et al. 2010 ; Livertox Keisu et al. 2010 ;
		XIM	Anticoagulant
				aminotransferase elevation	metabolites	(Solvo, HU) Kenne et al. 2008
						unpublished data
				Cholestatic hepatitis ; mixed	Bile acid accumulation ; liver	Nammour et al. 2003
				hepatocellular and cholestatic	mitochondria injury ; BSEP	; Saadi et al. 2013 ;
		MET	Antidiabetic	injury	inhibition*	Livertox ; Krajcsi P.
						(Solvo, HU)
						unpublished data
				Steatohepatitis ; rare cases of	ROS formation ; lipid	Feuer et al. 1992 ;
			Prophylactic	cholestasis	peroxidation ; BSEP and NTCP	Krajcsi P. (Solvo,
		PER
			antianginal		inhibition*	HU) unpublished
						data
				Cholestasis after	BSEP inhibition* ; BSEP and	Sharanek et al.
				transplantation	NTCP inhibition** ; BSEP	2014 ; Mihatsch et
		TAC	Immunosupressant
				(Infant) ; moderate	internalization**	al. 1998 ;
				aminotransferases elevation		Livertox
				Aminotransferases elevation ;	ROS formation ; mitochondrial	Smith et al. 2003 ;
		TOL	Antiparkinsonian	intrahepatic cholestasis	toxicity ; NTCP, BSEP, MRP2,	Morgan et al. 2013 ;
					MRP3 and MRP4 inhibition* ;	Krajcsi P. (Solvo,
						ϰϬ ϰϭ

Table 2 . Cmax, IC20 and maximum tested concentrations of the 18 tested drugs

 2 Cmax values are from the literature[START_REF] Regenthal | Drug Levels: Therapeutic and Toxic Serum/plasma Concentrations of Common Drugs[END_REF][START_REF] Chu | Transport of the Dipeptidyl Peptidase-4 Inhibitor Sitagliptin by Human Organic Anion Transporter 3, Organic Anion Transporting Polypeptide 4C1, and Multidrug Resistance P-Glycoprotein[END_REF][START_REF] Xu | Cellular Imaging Predictions of Clinical Drug-Induced Liver[END_REF] Dhillon and Keating 2009;[START_REF] Keisu | Drug-Induced Liver Injury in Humans: The Case of Ximelagatran[END_REF][START_REF] Dawson | In Vitro Inhibition of the Bile Salt Export Pump Correlates with Risk of Cholestatic Drug-Induced Liver Injury in Humans[END_REF][START_REF] Sharanek | Different Dose-Dependent Mechanisms Are Involved in Early Cyclosporine a-Induced Cholestatic Effects in hepaRG Cells[END_REF][START_REF] Schadt | Minimizing DILI Risk in Drug Discovery -A Screening Tool for Drug This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward[END_REF]. Cells were incubated for 24h with different concentrations of each drug. For cytotoxicity testing the cells were treated with varying concentrations of each drug for 24h, then assayed with the MTT colorimetric test and IC20s values were calculated.Cmax refers to the maximum serum concentration of the drug (µM). Drug abbreviations as in

Table 1 .

 1

		Cmax	IC20 (µM)	Maximun in vitro
	Drugs
		(µM)	(24h)	concentration tested (µM)
	BOS	7.4	120	100
	CPZ	0.2	50	50
	Group 1. DRUGS involved
	CsA	1.15	-	50
	clinical cholestasis
	NEF	4.26	50	75
	TRO	6.39	65	50
	Group 2.

DRUGS involved in rare cases of clinical cholestasis

  This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

	D η ϳϭϯϳϯ
	CIM	4.9	4000	4000
	DIC	8	375	200
	ENT	3.9	250	100
	MET	7.74	3000	3000
	PER	2	25	20
	TAC	0.09	60	50
	TOL	23	70	100
	AMI	0.8	20	20
	APAP	139	20000	25000
	Group 3.
	BUS	0.01	>300	100
	DRUGS not involved in
	FIA	0.64	>300	100
	clinical cholestasis
	PIO	4.2	200	100
	XIM	0.3	>300	100
				ϰϮ

Table 3 . Effects of drugs tested on transcript levels of different hepatic transporters.

 3 HepaRG cells were exposed to the 3 groups of compounds at the indicated concentration for 24h. mRNA levels were measured by RT-PCR analysis. All results are expressed relative to the levels found in control cells, arbitrarily set at a value of 1. *P<0.05, **P<0.01 and ***P<0.001 compared with untreated cells. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

		Drugs			Bile acids transporters
			BSEP	NTCP	OATB-B	MRP2	MRP3	MRP4
		Bosentan
			0.10 ± 0.01***	0.23 ± 0.06***	0.40 ± 0.02*	0.99 ± 0.16	0.62 ± 0.08	1.08 ± 0.16
		(100µM)
	Group 1.	Chlorpromazine	0,5 ± 0.2*	0.5 ± 0.1*	1.1 ± 0.1	1.2 ± 0.2	0.8 ± 0.2	1.7 ± 0.2*
		(50µM)
	DRUGS
		Cyclosporine
	involved in		0.54 ± 0.15**	0.05 ± 0.01***	0.17 ± 0.02***	1.8 ± 0.15***	1.79 ± 0.08***	2.68 ± 0.12***
		(50µM)
	clinical
		Nefazodone
	cholestasis		0.28 ± 0.08*	0.16 ± 0.06***	0.32 ± 0.07***	1.00 ± 0.1	0.90 ± 0.12	1.40 ± 0.2
		(75µM)
		Troglitazone
			0.58 ± 0.11*	0.51 ± 0.06*	0.89 ± 0.14	0.88 ± 0.08	1.11 ± 0.10	0.86 ± 0.09
		(50µM)
		Cimetidine
			0.33 ± 0.04***	0.21 ± 0.05**	0.63 ± 0.09	1.00 ± 0.31	0.89 ± 0.07	0.99 ± 0.26
		(4mM)
		Diclofenac
			0.66 ± 0.1*	0.56 ± 0.1**	0.7 ± 0.06**	1.67 ± 0.09***	0.75 ± 0.06	0.74 ± 0.02**
		(200µM)
	Group 2.
		Entacapone
	DRUGS	(100µM)	0.83 ± 0.18	1.48 ± 0.12	1.13 ± 0.18	0.93 ± 0.11	0.98 ± 0.18	1.28 ± 0.08
	involved in
		Metformin
	rare cases		0.30 ± 0.06***	0.15 ± 0.04**	0.37 ± 0.14*	1.11 ± 0.16	1,00 ± 0.15	1.05 ± 0.26
		(3mM)
	of clinical
	cholestasis	Perhexiline	0.45 ± 0.09*	0.38 ± 0.07***	0.63 ± 0.11	0.96 ± 0.13	0.52 ± 0.06**	1.43 ± 0.15
		(20µM)
		Tacrolimus
			0.44 ± 0.11**	0.23 ± 0.08***	0.43 ± 0.19*	2.04 ± 0.41*	1.64 ± 0.32*	2.60 ± 0.51*
		(50µM)
		Tolcapone	0.20 ± 0.04***	0.25 ± 0.11***	0.27 ± 0.13***	0.56 ± 0.06**	0.48 ± 0.05*	1.19 ± 0.28

Table 4 . Summary of the results on the 18 tested drugs BC

 4 deformation (i.e. constriction or dilatation), BSEP inhibition from the literature (vesicles), [ 3 H]-TCA Clearance (control=100%), BSEP mRNA expression (control=1), MLC2 phosphorylation state and MLCK/ROCK implication are summarized. Human BSEP inhibition levels in transfected vesicles are taken from the literature: a (Pedersen et al. 2013) b (Morgan et al. 2010) c (Dawson et al. 2012) d (Krajcsi P, Solvo, HU); unpublished data) and expressed as high (++), medium (+) and low or absent (-). BC, bile canaliculi; ROCK, Rho-kinase; MLCK, myosin light chain kinase; nc: no change. Drug abbreviations as in Table1.This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

				BSEP	[	3 H]-TCA	BSEP mRNA	MLC2
			BC							MLCK/ROCK
		Drugs		inhibition	Clearance	expression	phosphorylation
			deformation							implication
				(vesicles)	(ctrl = 100%)	(ctrl = 1)	state
					a,b,c,d
		BOS	Dilatation	++			64.24 ± 4.13**	0.10 ± 0.01***	Dephosphorylation	MLCK
	Group 1.				a,c,d
		CPZ	Constriction	+		49.96 ± 5.14*	0.5 ± 0.2*	Phosphorylation	ROCK
	DRUGS
	involved in	CsA	Constriction	++	a,b,c,d	13.16 ± 1.48**	0.54 ± 0.15**	Phosphorylation	ROCK
	clinical				a,b,c,d
		NEF	Constriction	++			52.46 ± 3.87***	0.28 ± 0.08*	Phosphorylation	ROCK
	cholestasis
		TRO	Constriction	++	a,b,c,d	21.97 ± 3.72***	0.58 ± 0.11*	Phosphorylation	ROCK
		CIM	Dilatation		+	a	86.25 s 12.12	0.33 ± 0.04***	Dephosphorylation	MLCK
					a,d
		DIC	Dilatation	+		384.8 ± 57.96**	0.66 ± 0.1*	Dephosphorylation	MLCK
	Group 2.
					b,d
	DRUGS	ENT	Dilatation	-		109.2 ± 16.94	0.83 ± 0.18	Dephosphorylation	MLCK
	involved in	MET	Constriction	a,d -	99.41 ± 8.45	0.30 ± 0.06***	Phosphorylation	ROCK
	rare cases of
					d
	clinical	PER	Constriction		-		41.43 ± 8.22**	0.45 ± 0.09*	Phosphorylation	ROCK
	cholestasis				b
		TAC	Dilatation		-		56.14 ± 2.71***	0.44 ± 0.11**	Dephosphorylation	MLCK
			Dilatation then		b,c,d
		TOL		+		48.36 ± 8.14**	0.20 ± 0.04***	nc	MLCK -ROCK
			constriction

Acknowledgment

We thank R. Le Guevel from the ImPACcell platform (Biosit) for imaging analysis.

Fate of Chemicals 40 (1): 130-38. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 D η ϳϭϯϳϯ Ϯϲ Dhillon, Sohita, and Gillian M. Keating. 2009. Bosentan: A Review of Its Use in the Management of Mildly Symptomatic Pulmonary Arterial Hypertension. American Journal of Cardiovascular Drugs: Drugs, Devices, and Other Interventions 9 (5): 331-50. Donner, M. G., and D. Keppler. 2001. Up-Regulation of Basolateral Multidrug Resistance Protein 3 (Mrp3) in Cholestatic Rat Liver. Hepatology (Baltimore, Md.) 34 (2): 351-59. Dykens, James A., Joseph D. Jamieson, Lisa D. Marroquin, Sashi Nadanaciva, Jinghai J. Xu, Margaret C. Dunn, Arthur R. Smith, and Yvonne Will. 2008. In Vitro Assessment of Mitochondrial Dysfunction and Cytotoxicity of Nefazodone, Trazodone, and Buspirone. Toxicological Sciences: An Official Journal of the Society of Toxicology 103 (2): 335-45. Ellis, L. C. J., M. H. Grant, G. M. Hawksworth, and R. J. Weaver. 2014. Quantification of Biliary Excretion and Sinusoidal Excretion of 5(6)-Carboxy-2',7'-Dichlorofluorescein (CDF) in Cultured Hepatocytes Isolated from Sprague Dawley, Wistar and Mrp2-Deficient Wistar (TR(-)) Rats. Toxicology in Vitro: An International Journal Published in Association with BIBRA 28 (6): 1165-75. Fattinger, K., C. Funk, M. Pantze, C. Weber, J. Reichen, B. Stieger, and P. J. Meier. 2001. The Endothelin Antagonist Bosentan Inhibits the Canalicular Bile Salt Export Pump: A Potential Mechanism for Hepatic Adverse Reactions. Clinical Pharmacology and Therapeutics 69 (4): 223-31. Feuer, G., and C. J. Di Fonzo. 1992. Intrahepatic Cholestasis: A Review of Biochemical-Pathological Mechanisms. Drug Metabolism and Drug Interactions 10 (1-2): 1-161. Fisher, Alexander, James Croft-Baker, Michael Davis, Patrick Purcell, and Allan J. McLean. 2002. Entacapone-Induced Hepatotoxicity and Hepatic Dysfunction. Movement Disorders: Official Journal of the Movement Disorder Society 17 (6): 1362-65; discussion 1397-1400. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 D η ϳϭϯϳϯ Ϯϳ Fromenty, B., and D. Pessayre. 1995. Inhibition of Mitochondrial Beta-Oxidation as a Mechanism of Hepatotoxicity. Pharmacology & Therapeutics 67 (1): 101-54. Garzel, Brandy, Hui Yang, Lei Zhang, Shiew-Mei Huang, James E. Polli, and Hongbing Wang. 2014. The Role of Bile Salt Export Pump Gene Repression in Drug-Induced Cholestatic Liver Toxicity. Drug Metabolism and Disposition: The Biological Fate of Chemicals 42 (3): 318-22. Graf, J., A. Gautam, and J. L. Boyer. 1984. Isolated Rat Hepatocyte Couplets: A Primary Secretory Unit for Electrophysiologic Studies of Bile Secretory Function. Proceedings of the National Academy of Sciences of the United States of America 81 (20): 6516-20. Guguen-Guillouzo, C., J. P. Campion, P. Brissot, D. Glaise, B. Launois, M. Bourel, and A. Guillouzo. 1982. High Yield Preparation of Isolated Human Adult Hepatocytes by Enzymatic Perfusion of the Liver. Cell Biology International Reports 6 (6): 625-28. Guguen-Guillouzo, Christiane, and Andre Guillouzo. 2010. General Review on in Vitro Hepatocyte Models and Their Applications. Methods in Molecular Biology (Clifton, N.J.) 640: 1-40. Imanari, H., H. Kuroda, and K. Tamura. 1981. Microfilaments around the Bile Canaliculi in Patients with Intrahepatic Cholestasis. Gastroenterologia Japonica 16 (2): 168-73. Jansen, P. L., S. S. Strautnieks, E. Jacquemin, M. Hadchouel, E. M. Sokal, G. J. Hooiveld, J. H. Koning, et al. 1999. Hepatocanalicular Bile Salt Export Pump Deficiency in Patients with Progressive Familial Intrahepatic Cholestasis. Gastroenterology 117 (6): 1370-79. Jemnitz, Katalin, Zsuzsa Veres, Regina Tugyi, and Laszlo Vereczkey. 2010. Biliary Efflux Transporters Involved in the Clearance of Rosuvastatin in Sandwich Culture of Primary Rat Hepatocytes. Toxicology in Vitro: An International Journal Published in Association with BIBRA 24 (2): 605-10. This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373 Financial support This work was supported by the European Community [Contract MIP-DILI-115336]. The MIP-DILI project has received support from the Innovative Medicines Initiative Joint Undertaking, resources of which are composed of financial contribution from the European Union's Seventh Framework Programme [FP7/20072013] and EFPIA companies' in kind contribution. http://www.imi.europa.eu/. Matthew G. Burbank was financially supported by a CIFRE PhD contract with Servier, and Audrey Burban and Ahmad Sharanek by the MIP-DILI project. Address reprint requests to: Prof. André Guillouzo, INSERM U991, Faculté de Pharmacie. 2 avenue L. Bernard, 35043 Rennes cedex, France. E-mail address : andre.guillouzo@univ-rennes1.fr This article has not been copyedited and formatted. The final version may differ from this version. DMD Fast Forward. Published on August 18, 2016 as DOI: 10.1124/dmd.116.071373

Authorship Contributions

Participated in research design: Matthew G. Burbank, Audrey Burban, Ahmad Sharanek, André Guillouzo.

Conducted experiments: Matthew G. Burbank

Performed data analysis: Matthew G. Burbank, André Guillouzo, Christiane Guguen-Guillouzo, Richard J. Weaver Wrote or contributed to the writing of the manuscript: Matthew G. Burbank, André Guillouzo, Richard J. Weaver (A), Cells were treated with 50µM CsA (constriction) or 100µM ENT (dilatation) for 2h. Canalicular lumen surfaces appeared bright (white) and hepatocytes/biliary cells denser (black) using a phase-contrast microscope Zeiss Axiovert 200M. Brightness parameters were adjusted to eliminate non corresponding objects and analysis was performed on at least 4 images per each condition (well). White canalicular lumen was then quantified using the image J software every 10min for 24h. The integrated density was determined and then expressed as percent of the control (T0). (B), Differences between a phase-contrast photograph and the same photograph after density adjustment. DMD #71373

CsA 50µM (CONSTRICTION) ENT 100µM (DILATATION)