Novel Mechanisms of Valproate Hepatotoxicity: Impaired Mrp2 Trafficking and Hepatocyte Depolarization.

Drug-induced liver injury (DILI) remains a major challenge in drug development. Although numerous mechanisms for DILI have been identified, few studies have focused on loss of hepatocyte polarization as a DILI mechanism. The current study investigated the effects of valproate, an antiepileptic drug with DILI risk, on the cellular mechanisms responsible for loss of hepatocyte polarization. Fully polarized collagen sandwich-cultured rat hepatocytes were treated with valproate (1-20mM) for specified times (3-24hr). Hepatocyte viability was significantly decreased by 10mM and 20mM valproate. Valproate depolarized hepatocytes, even at non-cytotoxic concentrations (=5mM). Depolarization was associated with significantly decreased canalicular levels of multidrug resistance-associated protein 2 (Mrp2) resulting in reduced canalicular excretion of the Mrp2 substrate carboxydichlorofluorescein. The decreased canalicular Mrp2 was associated with intracellular accumulation of Mrp2 in Rab11-positive recycling endosomes and early endosomes. Mechanistic studies suggested that valproate inhibited canalicular trafficking of Mrp2. This effect of valproate on Mrp2 appeared to be selective in that valproate had less impact on canalicular levels of the bile salt export pump (Bsep) and no detectable effect on P-glycoprotein (P-gp) canalicular levels. Treatment with valproate for 24hr also significantly downregulated levels of tight junction-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of tight junction proteins claudin 1, claudin 2, occludin, ZO1 and ZO3. These findings reveal that two novel mechanisms may contribute to valproate hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.