ORIGIN OF REACTIVE OXYGEN SPECIES INVOLVED IN THE DECREASE IN MRP2 FUNCTION PRODUCED BY IL-1ß

SCHUCK, VIRGINIA S.; ANDERMATTEN, ROMINA B.; CIRIACI, NADIA; FAZZI, AGUSTINA; SALAS, GIMENA; MEDEOT, ANABELA C.; BAROSSO, ISMAEL R.; SANCHEZ POZZI, EJ

Mar del Plata

Congreso; Reunión Sociedades de Biociencias 2022; 2022

Sociedad Argentina de Investigación Clínica

Inflammatory cytokines produce alterations in the location and function of canalicular transporters and could mediate the biliary secretory failure observed in inflammatory-associated pathologies such as sepsis. This action of cytokines is mediated by activation of signaling proteins. We have previously demonstrated that IL-1ss impairment of hepatocanalicular transporters activity, such as that of multidrug resistance-associated protein 2 (Mrp2) is mediated by reactive oxygen species (ROS). We proposed to evaluate the involvement of NADPH oxidase (NOX) in the production of ROS by IL-1 ss. Methodology: Isolated rat hepatocytes (HAR) were treated with IL-1ss (10ng/ml) for 5, 10, 15 and 20. Then, cells were washed, 2-7-dichlorofluorescein diacetate (DCFH, 5 µM, 30 min) was added and finally the fluorescence signal produced by its oxidized product 2-7-dichlorofluorescein (DCF) was determined at 498 nm excitation and 522 nm emission. The fluorescence intensity of DCF is proportional to the intracellular ROS production. To evaluate the origin of IL-1ss-dependent ROS production, cytokine treatment was repeated for 20 min in the presence of the NOX inhibitor apocynin (Apo 300 µM) and the antioxidant vitamin C (Vit C 100 µM) Result: (% of Control ± SEM; n=3-4) IL -1ss increased intracellular ROS production in hepatocytes, at least up to 20 min (148.7±14a). At this time (20 min of IL-1ss treatment), the co-incubation with APO (APO+IL: 89.5±5b) and VITC (Vit C+IL: 91.2±5b) prevented this increment of ROS. a different from control, b different from IL-1ss Conclusion: IL-1ss-induced ROS production is mediated by NOX. This oxidase may play a key role in the cellular signaling responsible for IL-1ss-induced cholestasis. 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 was 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±7 a ) 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±6 a ), TLC+VitC (92±3 b ), TLC+Man (94±7 b), pointing out ROS as possible modulators of Mrp2 internalization. a p