EPR evidence of hydroxyl radical generation as an initiator of lipid peroxidation in amyloid β-protein-stimulated PC12 cells

Abstract

Recent data from several groups suggest that the primary mechanism of amyloid β-protein (Aβ) neurotoxicity may be mediated by free radicals. To evaluate this hypothesis, our aim is to make the mechanism of Aβ neurotoxicity clear, especially in the formation of free radicals. In this study, rat pheochromocytoma (PC12) cells were exposed to Aβ25–35 and confirmed free radical generations using two kinds of spin trap agents, 5,5-dimethyl-1-pyrroline- N-oxide; DMPO and α-(4-pyridyl-1-oxide)- N- tert-butylnitrone; POBN. DMPO spin adduct revealed that hydroxyl radical ( OH), while POBN spin adduct identified a lipid radical ( L) as electron paramagnetic resonance (EPR) evidence of lipid peroxidation in the process of cell damage by Aβ25–35 exposure. An Aβ cytotoxicity assay also was performed by using WST-8 reduction system and histochemical analysis. These analyses showed cell damage induced by Aβ. This study provides EPR evidence that Aβ neurotoxicity is derived from hydrogen abstraction from polyunsaturated lipid acid by hydroxyl radical as a cause of lipid peroxidation.