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 (VPA), 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 VPA (1-20 mM) for specified times (3-24 h). Hepatocyte viability was significantly decreased by 10 and 20 mM VPA. Valproate depolarized hepatocytes, even at noncytotoxic concentrations (≤5 mM). 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 VPA inhibited canalicular trafficking of Mrp2. This effect of VPA on Mrp2 appeared to be selective in that VPA 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 VPA for 24 h also significantly downregulated levels of tight junction (TJ)-associated protein, zonula occludens 2 (ZO2), but appeared to have no effect on the levels of TJ proteins claudin 1, claudin 2, occludin, ZO1, and ZO3. These findings reveal that two novel mechanisms may contribute to VPA hepatotoxicity: impaired canalicular trafficking of Mrp2 and disruption of ZO2-associated hepatocyte polarization.