Erin K. Pias

The current study examines the contribution of mitochondria-derived reactive oxygen species (ROS) in tert-butyl-hydroperoxide (TBH)-induced apoptotic signaling using clones of undifferentiated pheochromocytoma (PC-12) cells that stably overexpress the human mitochondrial or cytoplasmic forms of superoxide dismutase (SOD) (viz. Mn-SOD or CuZn-SOD, respectively). Exposure of wild type cells to TBH caused an early generation of ROS (30 min) that resulted in cell apoptosis at 24 h. These responses were attenuated with N-acetylcysteine pretreatment; however, N-acetylcysteine was ineffective in cytoprotection when added after TBH-induced ROS formation. Stable overexpression of SOD isoforms caused a 2- and 3.5-fold elevation in CuZn-SOD and Mn-SOD activities in the cytoplasm and mitochondria, respectively, and 3-fold increases in cellular GSH content. Accordingly, the stable overexpression of Mn-SOD attenuated TBH-induced mitochondrial ROS generation and cell apoptosis. Whereas transient Mn-SOD expression similarly prevented PC-12 apoptosis, this was associated with increases in SOD activity but not GSH, indicating that cytoprotection by Mn-SOD overexpression is related to mitochondrial ROS elimination and not due to increases in cellular GSH content per se. Stable or transient CuZn-SOD overexpression exacerbated cell apoptosis in conjunction with accelerated caspase-3 activation, regardless of cell GSH levels. Collectively, our results support a role for mitochondrial ROS in TBH-induced PC-12 apoptosis that is attenuated by Mn-SOD overexpression and is independent of cellular GSH levels per se.

Oxidants are known to induce cell apoptosis. Because oxidants also elicit redox imbalance, it is difficult to distinguish the direct effects of cellular redox from that of oxidants. This study tests the hypothesis that induction of redox imbalance independent of reactive oxygen species (ROS), can induce cell apoptosis in a mitotic competent, undifferentiated cell line, PC-12. Cells grown in standard DMEM containing 25 mM glucose were treated with diamide, a thiol oxidant, at a concentration that did not generate ROS. Diamide caused a rapid increase in oxidized glutathione (GSSG) and a loss of mitochondrial cytochrome c in 15-30 min, caspase-3 activation in 2 h, and apoptosis in 24 h. N-Acetyl cysteine attenuated GSSG elevation and diamide-induced apoptosis. Incubation of cells in 5 mM glucose or inhibition of the pentose phosphate pathway maintained GSSG elevation and accelerated cell apoptosis. Collectively, these results show that loss of redox balance is an upstream event that kinetically preceded mitochondrial apoptotic signaling. A sustained redox change was not critical or necessary for apoptotic progression, but its prolongation exacerbated apoptotic death. The potentiation of apoptosis by sustained redox imbalance was correlated with decreases in NADPH supply for GSSG reduction.