Involvement of reactive oxygen species on the apoptotic mechanism induced by IFN-alpha2b in rat preneoplastic liver

QUIROGA A; ALVAREZ ML; PARODY JP; RONCO MT; FRANCÉS D; PISANI GB,; CARNOVALE CE; CARRILLO MC

Montevideo, Uruguay

Congreso; Free Radicals in Montevideo 2007. V Meeting of SFRBM-South American Group. V International Conference on Peroxinitrite and Reactive Nitrogen Species; 2007

In a previous work, in a rat liver preneoplastic model, we have demonstrated that IFN-á2b reduces the number and volume of altered hepatic foci (AHF) inducing apoptosis through a mechanism mediated by TGF-â1. In this study, it was analyzed the involvement of reactive oxygen species (ROS) on the apoptotic mechanism induced by IFN-á2b in rat preneoplastic liver. For in vitro studies, male Wistar rats were subjected to a 2-phase model of hepatic preneoplasia. Hepatocytes were obtained and cultured at different times: a) without any treatment, b) with IFN-á2b, c) with IFN-á2b plus anti-TGF-â1, d) with IFN-á2b plus ascorbic acid (ASC). IFN-á2b induced increase of TGF-â1 mRNA levels at 4 h of culture and a two-fold increase of TGF-b1 secretion at 7h of culture. ASC totally abolished the increase of TGF-â1 mRNA levels, and TGF-â1 levels in the culture media remained unchanged. IFN-á2b-treatment increased hepatocytes apoptosis since 20h of culture onwards. ASC or anti-TGF-b1 completely abolished it. After IFN-á2b treatment there were 2 peaks of ROS at 1h and 9h of culture. Anti-TGF-â1 did not block the production of the first peak of ROS whereas totally blocked the appearance of the second one. ASC abolished the production of both peaks. IFN-á2b induced NADPH oxidase activity increase at 1h of culture, reaching a maximum at 9h. Anti-TGF-â1 did not block activation at 1h, but did at 9h. Always, ASC totally blocked NADPH oxidase activation. Then, we determined the relevance of ROS on the onset of the apoptotic process in vivo in the whole preneoplastic liver. Treatment of preneoplastic rats with IFN-á2b + ASC abolished the IFN-á2b apoptotic effects observed in IFN-á2b-treated rats. Results indicate that IFN-á2b induces hepatocytic TGF-â1 production and secretion  by induction of ROS formation through the activation of a NADPH oxidase complex. TGF-â1, induces programmed cell death. It was also demonstrated that treatment of rats with IFN-á2b plus ascorbic acid, abolishes the apoptotic effect of IFN-á2b in rat preneoplastic livers, increasing the foci volume. In conclusion, these findings strongly suggest that ROS have a fundamental role as signaling molecules in the IFN-á2b-induced apoptosis in hepatic preneoplastic cells.