TRANSIENT ROS PRODUCTION INVOLVED IN TAUROLITHOCHOLATE-INDUCED IMPAIRMENT OF MRP2 FUNCTION IS MAINLY DERIVED FROM HEPATOCYTE NOX ACTIVATION

ANDERMATTEN, ROMINA B.; CIRIACI, NADIA; SCHUCK, VIRGINIA S.; FAZZI, AGUSTINA; BAROSSO, IR; SANCHEZ POZZI, EJ

Mar del Plata

Congreso; Reunión Sociedades de Biociencias 2022; 2022

Sociedad Argentina de Investigación Clínica

Cholestatic agents, such as the bile salt taurolithocholate (TLC), affect bile flow, in part, by impairing the activity of the canalicular transporters, among them Mrp2 (Multidrug Resistance-associated Protein 2). We have recently demonstrated that TLC induces a rapid and transient increase in intracellular reactive oxygen species (ROS) levels, which mediate the alteration of Mrp2 function. In vitro assays with antioxidants prevented this effect, suggesting a key role of these species in TLC-induced cholestasis. Our aim was to characterize the source of ROS production triggered by TLC. Hence, we decided to study the involvement of NADPH oxidase (NOX) and to elucidate its relationship with other proteins implicated in TLC-activated signaling pathways. Methodology: Isolated rat hepatocytes (IRH) were pretreated with NOX inhibitor apocynin (Apo 300 µM, 30 min) followed by treatment with TLC (2.5 µM) for 5 min. After washing, 2-7-dichlorofluorescein diacetate (5 µM, 30 min) was added and the fluorescence signal produced by 2-7-dichlorofluorescein (DCF) was determined. In another set of experiments, treatments were done in presence of inhibitors for proteins implicated in TLC cholestasis model, such as S1PR2 antagonist (JTE-013, 10 µM) and p38 MAPK and PKC inhibitors (SB203580, SB 1 μM) and bisindolylmaleimide, Bis 100 nM, respectively). Results: (% of Control±SEM; n=3) As previously reported, TLC treatment increased intracellular ROS production at 5 min in IRH (130±3a ). Pretreatment with Apo completely prevented this effect (105±5b ). On the other hand, the rise of ROS levels induced by TLC was also prevented in cells treated with SB (98±2b ) or Bis (94±8b ), whereas in presence of JTE-013 no change was observed (127±15a ). a p