Cytotoxic effects of an oxidative stress on neuronal-like pheochromocytoma cells (PC12)

Although the generation of oxygen derivatives during ischemia and reperfusion is generally held as a major event in the process leading to neuronal death, the biochemical mechanisms responsible for cell degeneration remain poorly understood. To better understand the toxicity induced by oxidative stress in neural tissue, we have tested the effect of an exogenous hydroperoxide, cumene hydroperoxide (CHP), on the metabolism and viability of PC12 cells. Addition of CHP in the culture medium leads to significant cell death that becomes perceptible at concentrations above 1 mu M and reaches a maximum (80-90% toxicity) at 100 mu M. A time-course study shows that Trypan blue uptake is preceded by a rapid phase of cell rounding and detachment from the substratum (within 30 min) followed by a progressive uptake of the dye (60-120 min). During this 2-hr period, we failed to observe any major signs of membrane lipoperoxidation (such as MDA production or fatty acid release). On the other hand, we observed that cell death is preceded by a striking decrease in cellular ATP content and in the retention of rhodamine 123 (within 15-30 min of treatment); thus, suggesting that the mitochondria may be the primary target of hydroperoxide action.