Low-dose acetaminophen induces early disruption of cell-cell tight junctions in human hepatic cells and mouse liver

Dysfunction of cell-cell tight junction (TJ) adhesions is a major feature in the pathogenesis of various diseases. Liver TJs preserve cellular polarity by delimiting functional bile-canalicular structures, forming the blood-biliary barrier. In acetaminophen-hepatotoxicity, the mechanism by which tissue cohesion and polarity are affected remains unclear. Here, we demonstrate that acetaminophen, even at low-dose, disrupts the integrity of TJ and cell-matrix adhesions, with indicators of cellular stress with liver injury in the human hepatic HepaRG cell line, and primary hepatocytes. In mouse liver, at human-equivalence (therapeutic) doses, dose-dependent loss of intercellular hepatic TJ-associated ZO-1 protein expression was evident with progressive clinical signs of liver injury. Temporal, dose-dependent and specific disruption of the TJ-associated ZO-1 and cytoskeletal-F-actin proteins, correlated with modulation of hepatic ultrastructure. Real-time impedance biosensing verified in vitro early, dose-dependent quantitative decreases in TJ and cell-substrate adhesions. Whereas treatment with NAPQI, the reactive metabolite of acetaminophen, or the PKCα-activator and TJ-disruptor phorbol-12-myristate-13-acetate, similarly reduced TJ integrity, which may implicate oxidative stress and the PKC pathway in TJ destabilization. These findings are relevant to the clinical presentation of acetaminophen-hepatotoxicity and may inform future mechanistic studies to identify specific molecular targets and pathways that may be altered in acetaminophen-induced hepatic depolarization

Endoplasmic reticulum stress precedes oxidative stress in antibiotic-induced cholestasis and cytotoxicity in human hepatocytes

International audienceEndoplasmic reticulum (ER) stress has been associated with various drug–induced liver lesions but its participation in drug-induced cholestasis remains unclear. We first aimed at analyzing liver damage caused by various hepatotoxic antibiotics, including three penicillinase-resistant antibiotics (PRAs), i.e. flucloxacillin, cloxacillin and nafcillin, as well as trovafloxacin, levofloxacin and erythromycin, using human differentiated HepaRG cells and primary hepatocytes. All these antibiotics caused early cholestatic effects typified by bile canaliculi dilatation and reduced bile acid efflux within 2 h and dose-dependent enhanced caspase-3 activity within 24 h. PRAs induced the highest cholestatic effects at non cytotoxic concentrations. Then, molecular events involved in these lesions were analyzed. Early accumulation of misfolded proteins revealed by thioflavin-T fluorescence and associated with phosphorylation of the unfolded protein response sensors, eIF2α and/or IRE1α, was evidenced with all tested hepatotoxic antibiotics. Inhibition of ER stress markedly restored bile acid efflux and prevented bile canaliculi dilatation. Downstream of ER stress, ROS were also generated with high antibiotic concentrations. The protective HSP27-PI3K-AKT signaling pathway was activated only in PRA-treated cells and its inhibition increased ROS production and aggravated caspase-3 activity. Overall, our results demonstrate that (i) various antibiotics reported to cause cholestasis and hepatocellular injury in the clinic can also induce such effects in in vitro human hepatocytes; (ii) PRAs cause the strongest cholestatic effects in the absence of cytotoxicity; (iii) cholestatic features occur early through ER stress; (iv) cytotoxic lesions are observed later through ER stress-mediated ROS generation; and (v) activation of the HSP27-PI3K-AKT pathway protects from cytotoxic damage induced by PRAs only. © 2017 Elsevier Inc

A dynamic mathematical model of bile acid clearance in HepaRG cells

International audienceA dynamic model based on ordinary differential equations that describes uptake, basolateral and canalicular export of taurocholic acid (TCA) in human HepaRG cells is presented. The highly reproducible inter-assay experimental data were used to reliably estimate model parameters. Primary human hepatocytes were similarly evaluated to establish a mathematical model, but with notably higher inter-assay differences in TCA clearance and bile canaliculi dynamics. By use of the HepaRG cell line, the simultaneous TCA clearance associated to basolateral uptake, canalicular and sinusoidal efflux, was predicted. The mathematical model accurately reproduced the dose-dependent inhibition of TCA clearance in the presence and absence of the prototypical cholestatic drugs cyclosporine A (CsA) and chlorpromazine. Rapid inhibition of TCA clearance and recovery were found to be major characteristics of CsA. Conversely, the action of chlorpromazine was described by slow onset of inhibition relative to inhibition of TCA clearance by CsA. The established mathematical model, validated by the use of these 2 prototypical cholestatic drugs and the integration of bile canalicular dynamics, provides an important development for the further study of human hepatobiliary function, through simultaneous temporal and vectorial membrane transport of bile acids in drug-induced cholestasis

Differential impact of inflammation on diclofenac-induced cytotoxicity in differentiated and undifferentiated HepaRG cells

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Selective bile canalicular changes induced by cholestatic drugs

International audienceMany drugs have been reported to induce cholestasis in a dose-dependent or independent (idiosyncratic) manner. Intrahepatic cholestasis represents around 40% of drug-induced injuries and up to 40% of all cases remain unpredictable. Our aim is to investigate the mechanisms underlying drug-induced cholestasis and to improve its prediction in humans using the metabolic competent human HepaRG cell line within the framework of the European project MIP-DILI. Previous data from our laboratory have shown that cholestatic drugs can cause either constriction or dilatation of bile canaliculi associated with deregulation of the Rho-kinase pathway in HepaRG cells (Sharanek et al. submitted). In the present work we have analyzed the effects of 8 cholestatic and 8 hepatotoxic (non-cholestatic) or non-toxic drugs on the dynamics of bile canaliculi using phase-contrast imaging, the activity of the Rho-kinase pathway by western blotting and the expression/activity of canalicular and basolateral transporters. The 8 cholestatic drugs were correctly identified as causing either constriction or dilatation of bile canaliculi while 7/8 non-cholestatic drugs were ineffective. Only one behaved as a false positive. Moreover, 7/7 cholestatic drugs modulated the phosphorylation level of the myosin light chain, a downstream effector of the Rho-kinase pathway. 6/6 of the tested non-cholestatic drugs did not alter the phosphorylation level of the myosin light chain. Transcripts levels of six major basolateral and canalicular transporters involved in uptake or efflux of bile acids (BSEP, NTCP, OATP-B, MRP2, MRP3, MRP4) were also determined after a 24 h treatment. A decrease in NTCP and BSEP expression was found for 6/6 cholestatic compounds. Moreover, 2/2 non-cholestatic drugs were ineffective. Furthermore, BSEP (efflux) and NTCP (influx) activities were measured after 2 and 24 h treatments: they showed a dose-dependent decrease for 6/6 and 5/5 cholestatic drugs respectively. By contrast, 2/2 non-cholestatic drugs had no effect on these activities. In summary, our preliminary results suggest that analysis of alterations of bile canaliculus structures associated with modulation of the Rho-kinase pathway are promising predictive markers of drug-induced cholestasis

Changes in bile acid profiles induced by cholestatic drugs in HepaRG hepatocytes cultured in bile acid-enriched medium

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Dose-dependent intracellular accumulation of endogenous bile acids in cyclosporine A-treated HepaRG hepatocytes

International audienceCholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Its accurate prediction represents a major challenge since up to now it is unpredictable in 40% of all cases. Various in vitro cell models are used to investigate uptake and biliary excretion of bile acids (BAs) aiming to predict cholestatic injury. However, previous studies showed that no human liver cell line did exhibit normal production of BAs. Furthermore, no study has reported yet intracellular accumulation of endogenous BAs in hepatocyte cultures following treatment with a cholestatic drug. We have evaluated the capacity of the human HepaRG cell line to produce BAs and analyzed intracellular changes in BA content and profiles after treatment by the cholestatic drug cyclosporine A (CsA). Our data show that HepaRG cells synthesized, conjugated and excreted normal primary BAs in a serum-free medium at daily levels comparable to those measured in conventional primary human hepatocytes. A 4-h treatment with CsA in serum-free medium resulted in a concentration-dependent intracellular accumulation and changes in the profiles of endogenous BAs associated with occurrence of cholestatic features while after 24 h BAs were decreased in the cellular layer and increased in the supernatant. The latter effects resulted from inhibition of BSEP and NTCP, the main BA efflux and uptake transporters, inhibition of CYP7A1, CYP8B1 and CYP27A1 which are involved in the initial steps of BA synthesis and enhanced expression of the basolateral transporters MRP3 and MRP4 which is recognized as a compensatory mechanism of BA excretion. Importantly, when CsA-treated HepaRG cells were incubated in a medium containing 2% bovine serum they did not accumulate endogenous BAs; however, after 24-h they showed a dose-dependent accumulation of bovine serum lithocholic acid in a non-sulfoconjugated form resulting from an inhibition of SULT2A1. In summary, our work bring the first demonstration that an in vitro human liver cell model, the HepaRG cell line, is able to produce, conjugate and secrete BAs and that a transient accumulation of endogenous BAs concomitantly to occurrence of various other cholestatic features can be observed following treatment with a cholestatic drug. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side-effects

Alteration of the Rho-kinase pathway activity disorganizes bile canaliculi dynamics and perturbs bile acid clearance in drug-induced cholestasis

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Evaluation of cholestatic potential of endothelin receptor antagonists

International audience52nd Congress of the European-Societies-of-Toxicology (EUROTOX), Seville, Spain, Sep 201