Copper mediated Oxidative Stress in murine embryonary fibroblasts and lipid peroxidation in rat liver isolated mitochondria.

SAPORITO MAGRIÑÁ, C.; MUSACCO SEBIO, R; CORANALÓ, L; BORNER, C; REPETTO, M

Buenos Aires

Congreso; II International Congress in Translational Medicine. Maestría Internacional en Ciencias Biomédicas (IMBS). Universidad de Freiburg (Alemania) y Universidad de Buenos Aires.; 2015

Maestría en Ciencias Biomédicas. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires.

Excess copper (Cu) systemically, as observed in Wilson´s disease, generates damage and cell death in various organs, mainly liver and brain. The interaction between free Cu and H2O2 produced by the cells create a situation of oxidative stress that triggers cell death. Objectives: To determine the production of pro-oxidant species after overload Cu and study whether cell death occurs through an apoptotic, necrotic or mixed mechanism. Results: in murine embryonic fibroblasts (MEFs), 400 uM Cu are sufficient to produce a decrease in viability of 40% in 24 hours while hepatocytes exposed to 300 uM Cu show substantial morphological changes and cell death. This concentration of Cu leads to increased oxidation of dichlorofluorescein (DCFH) 500% at 8 hours. The oxidation of DCFH is completely prevented in the presence of protein synthesis inhibitors such as cycloheximide and actinomycin D. The N-acetylcysteine, it is not able to prevent cell death, nor diminish the oxidation of dichlorofluorescein. Deferoxamine, a chelating Fe3 + was the only molecule capable of reducing the oxidation of dichlorofluorescein and partially prevent cell death. Caspase activity was not found increased or hepatocytes, or MEFs, nor the cleavage of caspase 3 which does not involve cell death apoptotic pathway. Microscopic analysis of hepatocytes showed typical morphology of necrotic hepatocytes and absence of apoptotic bodies. Conclusion: Cu induced cell death is necrotic purely event and generation of prooxidant species dependent cell exposed to the metal. The oxidation of DCFH mediated protein synthesis involves a different way to the interaction between the H2O2 and the free Cu in the production of oxidative stress at least in MEFs.