Oxidative stress is involved in the impairment of Mrp2 activity induced by estradiol 17ß-D-glucuronide in rat hepatocytes

SALAS, GIMENA; MEDEOT, ANABELA CAROLINA; SCHUCK, VIRGINIA SOLEDAD; MISZCZUK, GISEL SABRINA; ANDERMATTEN, ROMINA B.; CIRIACI, NADIA; RAZORI, MARÍA VALERIA; SÁNCHEZ POZZI, ENRIQUE J.; ROMA, MARCELO G.; BASIGLIO, CECILIA L.; CROCENZI, FERNANDO A.

Reunión virtual

Congreso; Reunión de la Sociedad de Biociencias (SAIC-SAI-SAFIS) 2020; 2020

SAIC-SAI-SAFIS

Estradiol 17β-D-glucuronide (E17G) is an endogenous metaboliteof estradiol which mediates the intrahepatic cholestasis of pregnancy. E17G impairs canalicular secretion via several kinase-mediated signaling pathways which leads to endocytosis and intracellularretention of canalicular transporters, contributing the MEK-ERK1/2pathway to the second process. Oxidative stress has also beenshown to trigger these effects on canalicular transporters. We studied the possible role of oxidative stress in E17G-induced impairmentof Mrp2 function by assessing the canalicular vacuolar accumulation(cVA) of glutathione-S-methylfluorescein (GS-MF) in rat hepatocytecouplets (RHC). The possible E17G-induced increase in intracellular reactive oxygen species (ROS) was assessed fluorometricallyin primary cultured rat hepatocytes (PCH) by the 2,7-dichlorofluorescin-diacetate (DCFH-DA) assay. A probable role of ROS inE17G-induced Mrp2 function impairment was evaluated by preincubating RHC for 15 min with the antioxidants vitamin C (VitC), mannitol (Man), N,N?-diphenyl-p-phenylenediamine (DPPD), and also withapocynin (Apo), a specific inhibitor of the ROS-producing enzymeNADPH oxidase (NOX), prior to a 20-min exposure to E17G; a potential role of MEK-ERK1/2 pathway was assessed by its inhibitionwith the MEK inhibitor PD98059 (PD). E17G increased intracellularROS by 43±9 %[p<0.05 vs control (C)] after 15 min in PCH andApo completely prevented this effect. E17G reduced the cVA of GSMF with respect to C by 45±2%(p<0.05). This was prevented bythe antioxidants (VitC: 61±5%, Man: 71±7%, DPPD: 69±6%, p<0.05vs E17G and C) and by Apo (72±5%, p<0.05 vs E17G and C); PDprevented E17G-induced effect on cVA of GS-MF similarly to Apoand showed no additive effects when added together, suggestingthat they share the same pathway. These results propose a role ofNOX-generated ROS in the E17G-induced impairment of Mrp2 activity in rat hepatocytes via the MEK-ERK1/2 pathway.