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 (LD(50) = about 8 mu M). LO OH induced nuclear condensation and DNA fragmentation, which was preve nted by cycloheximide (a protein synthesis inhibitor) and NGF, indicat ing that LOOH triggered apoptosis in PC12h cells. LOOH produced reacti ve oxygen species (ROS) in PC12h cells in a time- and dose-dependent m anner: as measured by flow cytometry using the ROS-specific fluorescen t indicator, 6-carboxy-2,7-dichorodihydrofluorescein diacetate, di(ace toxymethyl ester) (C-DCDHF-DA), Antioxidants such as N,N'-diphenyl-p-p henylenediamine (DPPD), vitamin E and N-acetylcysteine, and a ferric i ron 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 a poptosis and increased the generation of ROS. These results indicate t hat LOOH triggers the apoptosis of PC12h cells by increasing the produ ction of ROS. A confocal analysis with the Ca2+-specific fluorescent i ndicator, fluo-3, demonstrated that LOOH at concentrations up to 200 m u M, did not increase the intracellular Ca2+ concentration. These data indicate that LOOH induces apoptosis of PC12h cells through the enhan ced production of ROS, not through increasing the permeability of Ca2.