CHARACTERIZATION OF HYDROGEN-PEROXIDE TOXICITY IN CULTURED RAT FOREBRAIN NEURONS

We investigated the ability of hydrogen peroxide (H2O2) to cause apopt otic cell death in cultured rat forebrain neurons and the potential me chanisms by which oxidative stress triggers delayed neuronal death. H2 O2 (25 mu M for 5 min) reduced cell viability to 34.5 +/- 8.3% of untr eated controls 20 h after exposure, and resulted in a significant prop ortion of neurons which exhibited apoptotic nuclear morphology. Using single cell fluorescence assays, we measured H2O2-induced changes in D NA strand breaks, 2'7' dichlorofluorescin fluorescence, reduced glutat hione, intracellular free Ca2+, and mitochondrial membrane potential. DNA strand breaks in response to H2O2 were not evident immediately fol lowing exposure, but were increased 12h and 20h after exposure. Millim olar concentrations of H2O2 caused increases in the fluorescence of th e oxidant-sensitive fluorescent dye, 2'7'-dichlorofluorescin. H2O2 tre atment decreased reduced glutathione following 30 minutes of exposure using the fluorescent indicator, 5-chloromethylfluorescein diacetate, and increased intraneuronal free Ca2+ levels in a subpopulation of neu rons. Mitochondrial membrane potential, measured by rhodamine 123 loca lization was unaffected by 25 mu M H2O2, while higher concentrations o f H2O2 (10 or 30 mM) depolarized mitochondria. These studies demonstra te that H2O2 is a potent and effective neurotoxin that produces oxidat ive stress, as well as apoptotic neuronal death.