The hepatocellular bile acid transporter Ntcp facilitates uptake of the lethal mushroom toxin α-amanitin

Abstract

Hepatotoxicity caused by the mushroom poison α-amanitin is an unusual but serious cause of death and liver transplantation. Understanding the mechanisms of α-amanitin uptake may lead to rational therapeutic approaches. Because older data suggested that a sodium-dependent bile acid transporter is responsible for α-amanitin uptake, we tested the hypothesis that Na⁺-taurocholate cotransporter polypeptide (Ntcp) facilitates hepatocellular α-amanitin uptake. Human hepatoblastoma cells (HepG2), cells that have lost native Ntcp expression, were stably transfected with the rat Ntcp gene. Taurocholate uptake by the transfected cells exhibited a physiologically normal K _m and V _max. A toxicologically relevant functional assay for α-amanitin uptake was developed by measuring its ability to block cytokine-induced synthesis of interleukin-1 receptor antagonist (IL-1Ra) mRNA. Treatment with interleukin-1β (10 ng/ml) and interleukin-6 (100 ng/ml) increased IL-1Ra mRNA abundance 8.6-fold and 15.6-fold in HepG2 cells and Ntcp-transfected cells, respectively. Pretreatment of transfected cells with 1 µM α-amanitin for 6–10 h almost completely blocked induction of IL-1Ra mRNA (1.9-fold induction) whereas pretreatment of non-transfected cells did not block induction of IL-1Ra mRNA (21.6-fold induction, P<0.02 compared with stimulated transfected cells without α-amanitin). These findings demonstrate that Ntcp may be an important mediator of α-amanitin uptake by the liver.