The role of glutathione as protective molecule in copper overload

SAPORITO, C; BORNER, C; REPETTO, M

Mar del Plata

Congreso; 61º Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2016

Wilson Disease is characterized by high copper (Cu) content in the liver and different organs due to a defective extrusion of the metal into the bile. The enhanced intracellular concentration of Cu ends up in the demise of the cell. However, the molecular events responsible for Cu toxicity are largely unknown. Objective: To outline the mechanism of toxicity of Cu and identify endogenous protective molecules in Cu overload. Methodology: Cell viability (FACS) and ?SH content (reaction with DTNB) were assessed after Cu exposure in HCT116, BEAS-2B, Hela, MEFs and Hepa1-6 cells. Results: Cells show large differences in LD50. In cells exposed to LD50, the onset of cell death takes place after 10 hours. Cu oxidized ?SH groups of reduced glutathione (GSH), N-acetylcysteine (NAC) and cysteine in vitro. However, during the first 7 hours of Cu exposure, intracellular GSH is not significantly oxidized in any cell line. In accordance, the addition of NAC (membrane permeable ?SH) has no effect on the survival of the cell. The lack of effect of NAC on cell viability implies that the oxidation of cell surface ?SH groups by Cu has no direct impact on the survival of the cell. However, the addition of GSH or GSSG to the medium largely improves cell survival upon Cu overload. This protection is likely due to the formation of complexes between Cu and GSH or GSSG, thus preventing the metal entrance into the cytosol. However, given the high GSH content, this reaction likely takes place also intracellularly. Likewise, GSH depletion by BSO drastically potentiates cell death induced by Cu while ?SH replenishment by NAC in GSH depleted cells brings no improvement to the survival. Conclusion: Cu oxidation of cell surface ?SH groups has no direct impact on cell survival. Even when Cu is present at toxic concentrations, cells are in control the intracellular ?SH state. Nevertheless, GSH performs as a major protective molecule in Cu overload although, independently of the reduced ?SH group.