Selective bile canalicular changes induced by cholestatic drugs
Résumé
Many 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.