Estradiol 17beta-D-glucuronide-induced impairment of MRP2 activity in rat hepatocytes involves NADPH oxidase-mediated oxidative stress.

SALAS G.; MEDEOT A.; SCHUCK V.S.; ANDERMATTEN R.B.; MISZCZUK G.S.; CIRIACI N.; BAROSSO I.R.; SÁNCHEZ POZZI E.J.; ROMA M.G.; BASIGLIO C.L.; CROCENZI F.A.

Rosario, Santa Fe

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología (SAFIS) 2019; 2019

Sociedad Argentina de Fisiología (SAFIS) 2019

Estradiol 17ß-D-glucuronide (E17G) is an endogenous metabolite of estradiol that is considered a main responsible of intrahepatic cholestasis of pregnancy. E17G induces acute cholestasis in animal models via activation of several kinase-mediated signaling pathways leading to an increased endocytosis and intracellular retention of canalicular transporters and the consequent canalicular secretory failure. Oxidative stress has been shown to induce internalization of canalicular transporters and cholestasis. Here, we investigated the possible involvement of oxidative stress in E17G-induced impairment of canalicular Mrp2 function. Transport function of Mrp2 was analyzed by the measurement of canalicular vacuolar accumulation (cVA) of glutathione-S-methylfluorescein (GS-MF), a fluorescent glutathione-conjugated metabolite of chloromethylfluorescein diacetate (CMFDA) in rat hepatocyte couplets (RHC), and also by measuring the initial transport rate (ITR) of GS-MF in sandwich-cultured rat hepatocytes (SRH). The role of reactive oxygen species (ROS) in E17G-induced Mrp2 function impairment was evaluated by preincubating hepatocytes for 15 to 30 min with the antioxidant compounds vitamin C (VitC), mannitol (Man), N,N´-diphenyl 1,4-phenylenediamine (DPPD), or with apocynin (Apo), a specific inhibitor of the ROS-producing enzyme NADPH oxidase (NOX), prior to a 20-min exposure to E17G. cVA of GS-MF, expressed as percent of control, was significantly reduced by E17G (45±2, p<0.05 vs control, n=7); this effect was significantly prevented by preincubation with the antioxidant compounds (VitC: 61±5, Man: 71±7, DPPD: 69±6, p<0.05 vs E17G and control, n=4) and by inhibition of NOX with Apo (72±5, p<0.05 vs E17G and control, n=3). Neither the antioxidants nor Apo alone affected the cVA of GS-MF. Equivalent results were obtained when ITR of GS-MF was assessed in SRH after these treatments. These results support a role of ROS in the impairment of Mrp2 transport function induced by E17G in hepatocytes, probably by participating in the internalization of this transporter induced by this cholestatic compound. An increased activity of NOX induced by E17G seems to represent the source of ROS.