Generation of free radicals during lipid hydroperoxide-triggered apoptosis in PC12h cells

The compound 13-L-hydroperoxylinoleic acid (LOOH) triggered the death of clonal rat pheochromocytoma PC12h cells (LD50 = about 8 microM). LOOH induced nuclear condensation and DNA fragmentation, which was prevented by cycloheximide (a protein synthesis inhibitor) and NGF, indicating that LOOH triggered apoptosis in PC12h cells. LOOH produced reactive oxygen species (ROS) in PC12h cells in a time- and dose-dependent manner, as measured by flow cytometry using the ROS-specific fluorescent indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Antioxidants such as N,N'-diphenyl-p-phenylenediamine (DPPD), vitamin E and N-acetylcysteine, and a ferric iron chelator, deferoxamine, inhibited the LOOH-triggered apoptosis and simultaneously decreased the generation of ROS, whereas an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO), enhanced the apoptosis and increased the generation of ROS. These results indicate that LOOH triggers the apoptosis of PC12h cells by increasing the production of ROS. A confocal analysis with the Ca(2+)-specific fluorescent indicator, fluo-3, demonstrated that LOOH at concentrations up to 200 microM, did not increase the intracellular Ca2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhanced production of ROS, not through increasing the permeability of Ca2+.