NADPH OXIDASE-GENERATED REACTIVE OXYGEN SPECIES ARE INVOLVED IN ESTRADIOL 17ß-D-GLUCURONIDE-INDUCED CHOLESTASIS

SALAS, G; LITTA, AA; MEDEOT, AC; SCHUK, VS; RAZORI, MV; ANDERMATTEN, RB; BASIGLIO CL; CROCENZI FA

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS); 2023

SAFIS

Introduction: Estradiol 17β-D-glucuronide (E17G) induces cholestasis by impairing the activity of canalicular transporters such asMrp2. We have recently presented evidence that E17G induces a rapid increase in intracellular reactive oxygen species (ROS)levels, which mediate the alteration of Mrp2 function and subcellular localization. We also reported that NADPH oxidase (NOX)seems to be the main source of these ROS, and evidenced that NOX shares the MEK-ERK1/2 and p38MAPK signaling pathwaysactivated by E17G, downstream these kinases. Aim: to corroborate the participation of ROS in a more physiological model andto gain more direct evidence of the involvement of NOX in the impairment of Mrp2 localization and activity. Methods: Inisolated and perfused rat livers (IPL) we evaluated the effects of the antioxidant compound, N-acetylcysteine (NAC, 1mM) on thealteration of bile flow and the biliary excretion of the Mrp2 substrate dinitrophenyl-glutathione induced by a single intraportalinjection of E17G (3 μmol/liver) or its solvent (DMSO/10% ASB). In sandwich-cultured rat hepatocytes (SCRH) we performed aknockdown of the regulatory cytosolic subunit of NOX, p47phox, with specific siRNA, evaluating its expression by RTqPCR. Intransfected SCRH treated with E17G, we evaluated the activity of Mrp2 by assessing the initial transport rate (ITR) ofglutathione-methylfluorescein by epifluorescence microscopy, and the subcellular distribution of Mrp2 by immunofluorescenceand confocal microscopy analysis. Results: In IPL, E17G rapidly decreased bile flow, which then started a slow recovery. NACprevented the initial drop in bile flow and accelerated its recovery. siRNA transfection significantly decreased p47phox mRNA