Outlining the molecular targets in copper toxicity. The protective role of GSH.

KOOK, L; DESIMONE, M; REPETTO, M; SAPORITO MAGRIÑÁ, C.; BOERRIES, M; ANDRIEUX, G; BORNER, C; TUTTOLOMONDO, MV

Buenos Aires

Congreso; III International Congress of Translational Medicine; 2016

Maestría Internacional en Ciencias Biomédicas, Facultad de Farmacia y Bioquímica y Facultad de Medicina, UBA

An enhanced intracellular copper (Cu) content, due to either a defective extrusion from the cell or a high extracellular concentration of the metal entails potentially deleterious effects which end up in the demise of the cell. While the increased Cu is long acknowledged as the cause of Wilson Disease and is also related to several other pathologies, the actual molecular events underlying the toxicity of the metal are still obscure. Objective: To identify endogenous protective molecules in Cu overload and outline the mechanism of Cu toxicity. Methodology: Cell viability (Annexin-V/7AAD by FACS), ?SH content (spectrophotometrically by reaction with DTNB), prooxidant species production (DCFH assay by FACS), Cu content (atomic absorption spectrometry), gene expression profile (RNA expression microarrays) were assessed after Cu exposure in 5 cell lines. Results: Cells exposed to high Cu concentration (100-1400 µM) showed a linear increase in cell death after a lag phase of 10-12 h. Intracellular Cu starts to build up after 1 h of exposure. An enhancement in prooxidant species production takes place over a broader time-window 3-7 in a cell dependent manner. Interestingly, GSH does not become oxidized during the first 7 hours of exposure. In accordance, addition of N-acetylcysteine (NAC) does not prevent cell death. However, addition of GSH as well as its oxidized form GSSG to the medium fully prevents the toxicity of the metal. GSH depletion by buthionine-sulfoximine (BSO) dramatically enhances cell death upon Cu exposure which cannot be rescued by replenishment of soluble ?SH groups by means of NAC on top of GSH depleted cells indicating that GSH is likely to be acting as a protective molecule, independently of its ?SH group. Cu exposure leads to a ubiquitous phosphorylation of JNK and may lead to caspase 3 cleavage and activation in a cell dependent manner. Although, even when active, caspase 3 plays a secondary role in cell death since its inhibition by ZVAD has a minor effect on the survival of the cell. Finally, the gene expression profile shows a largely increased expression of heat shock proteins, ER stress response related genes, autophagy and genes related with proteasome degradation processes such as ubiquitin. In accordance, a large number of genes under control of transcription factors involved in protein folding show a consistent enhanced expression. Discussion: Cu toxicity is likely related to the high reactivity of the metal. However, the molecular targets once the metal enters the cell are uncertain. The capacity of the metal to undergo redox cycling while giving rise to the highly reactive hydroxyl radical by Fenton chemistry makes the redox hypothesis a tempting explanation to the toxicity of the metal. However, according to these results, the manipulation of the free ?SH pool by exogenous addition of NAC had no effect on cell death. Moreover, the fact that GSSG as well as GSH addition result in a protective effect suggests the capacity of GSH to act independently of its ?SH group, and redox active part of the molecule. Interestingly, the gene expression profile suggests that Cu ions interact with proteins, thus leading to improper folding and heat shock response followed by enhanced proteasome degradation and autophagy. Conclusion: Cu induced cell death is delayed by intracellular GSH which acts independently of the ?SH. The metal overload may trigger caspase 3 activation. However, apoptosis is only secondary to a more critical caspase independent event.