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