Copper mediated oxidative stress in murine embryonary fibroblasts and lipid peroxidation in rat liver isolated mitochondria

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

Buenos Aires

Congreso; 2nd International Congress in Translational Medicine; 2015

Copper (Cu) is a biometal involved in a myriad of cell processes; however, when the metal concentration surpasses a certain threshold, it becomes highly toxic for living organisms. The mechanism by which it exerts its toxicity and eventual cell death is often attributed to the capacity of this metal to undergo redox cycling upon reaction with O2.- and H2O2 yielding OH. which induces oxidative stress and lipid peroxidation. Objectives: To determine whether Cu overload induces oxidative stress in cultured murine embryonary fibroblasts (MEFs) and lipid peroxidation in rat liver isolated mitochondria and study its effect on cell death and mitochondrial functionality. Results: Cu overload shows about a 50% decrease in cell survival on MEFs after 24 h of exposure to the metal. Cu overload in MEFs enhances the production of prooxidant species in a concentration and time dependent manner as assessed by DCFH oxidation. However, cell survival is not improved by addition of neither N-acetylcysteine (NAC), nor butylhydroxyanisole (BHA). Furthermore, both NAC and BHA do not seem to decrease DCFH oxidation. Interestingly, the enhanced DCFH oxidation is fully abrogated by addition of the protein synthesis inhibitors anisomycin, cycloheximide and actinomycin D. Isolated mitochondria are able to consume O2 using malate-glutamate as substrate and synthesize ATP in the presence of ADP. When Cu is added into the measurement buffer it causes a metal concentration-dependent drop in the state 3 respiration which becomes significant after a 75 μM Cu concentration. This drop in the state 3 respiration is fully abrogated by NAC and reduced glutathione (GSH). If mitochondria are allowed to respire for longer times and produce H2O2 and O2.- as byproducts of respiration, 25 μM Cu is required to see a significant drop in the state 3 respiration. This impaired respiration is accompanied by an increase in the TBARS. Interestingly, the exogenous addition of 4 mM GSH to the respiration buffer fully abrogates the increase in TBARS and both GSH and NAC had a significant effect on preventing the drop in the state 3 respiration. Conclusion: Cu overload in MEFs entails an enhanced production of prooxidant species and this situation is accompanied by cell death. This oxidative stress situation is neither controlled by NAC nor by BHA, whereas it seems to be fully abrogated by protein synthesis inhibition. The toxicity of Cu in respiring isolated mitochondria seems to be of a different kind of that in MEFs, since in isolated mitochondria, the addition of NAC and specially GSH had significant effects on preventing the drop on the state 3 respiration and GSH also fully prevented lipid peroxidation.