TAUROLITHOCHOLATE CONTRIBUTES TO MRP2 ACTIVITY IMPAIRMENT BY INDUCING A TRANSIENT ROS PRODUCTION

ANDERMATTEN, ROMINA B; CIRIACI, NADIA; SCHUCK, VIRGINIA S; RAZORI, M VALERIA; MEDEOT, ANABELA C; SALAS, GIMENA; BAROSSO, ISMAEL R; SANCHEZ POZZI, ENRIQUE J.

Congreso; REUNIÓN DE SOCIEDADES DE BIOCIENCIAS 2021; 2021

Previous studies in different models of cholestasis showed that reactive oxygen species (ROS)/oxidative stress mediate the internalization of the hepatocanalicular transporters, such as multidrug resistance-associated protein 2 (Mrp2). Taurolithocholate (TLC) is known to be the most pro-oxidative bile salt. However, there is no direct evidence that ROS production derived from TLC action is a mechanism involved in cholestasis pathogenesis. Herein, we evaluated a possible role of ROS in the TLC- induced impairment of Mrp2 activity.Methodology: ROS production was measured by the 2´,7´-dichlorofluorescin-diacetate (DCFH-DA) assay in primary culture rat hepatocytes. Cells were exposed to TLC (2.5 µM) at different times (5, 10, 15 and 20 min) followed by incubation with DCFH-DA (5 μM). On the other hand, isolated rat hepatocyte couplets (IRHC) were co-treated with TLC (2.5 µM) and antioxidants: vitamin C (VitC 100 µM) or mannitol (Man 60 mM) for 20 min. To analyze the TLC-induced ROS involvement in Mrp2 activity impairment, functional studies were carried out by assessing the canalicular vacuolar accumulation of its substrate glutathione methylfluorescein (GMF).Results: (% of Control±SEM; n=3-5): TLC increased intracellular ROS production in hepatocytes, reaching the maximum peak at 5 min (133±7a) and rapidly returning to control levels at 10 min (102±4). This transient production suggests the participation of ROS as signaling molecules. Pre-treatment of IRCH with both antioxidants prevented TLC-induced impairment of canalicular accumulation of GMF: TLC (67±6a), TLC+VitC (92±3b), TLC+Man (94±7b), pointing out ROS as possible modulators of Mrp2 internalization. ap