OXIDATIVE STRESS IN A CLONAL CELL-LINE OF NEURONAL ORIGIN - EFFECTS OF ANTIOXIDANT ENZYME MODULATION

The effects of intracellularly generated H2O2 on cell viability, morph ology, and biochemical markers of injury have been investigated in a c lonal cell line of neuronal origin (140-3, mouse neuroblastoma x rat g lioma) as a cell culture model for the role of oxidative stress in the long-term loss of neurons in the brain. The H2O2 was generated from t he redox cycling of menadione, or by the oxidation of serotonin cataly zed by monoamine oxidase, to simulate the effect of amine neurotransmi tter turnover. Incubation with menadione at concentrations as low as 1 0 muM for several hours resulted in significant losses of cell viabili ty and altered morphology. Similar effects were evident in the presenc e of serotonin only after incubation overnight with concentrations > 1 mM. The cytotoxicity of either agent was potentiated by preincubation with specific inhibitors of two enzymes important to cellular antioxi dant defenses, 3-amino-1,2,4-trazole for catalase and 1,3-bis(chlorome thyl)-1-nitrosourea for glutathione reductase. Activity of another ant ioxidant enzyme of particular importance to antioxidant defenses in br ain, the selenoprotein glutathione peroxidase, was stimulated fourfold by growth of cultures in the presence of sodium selenite as a source of active-site Se for the enzyme. The only effect of the selenite on o ther functionally coupled antioxidant enzymes was a decrease in activi ty of superoxide dismutase at concentrations >200 nM. The selenite sub stantially protected cells against oxidative stress induced by combina tions of menadione, 3-amino-1,2,4-trazole, and 1, 3-bis(chloromethyl)- 1-nitrosourea, but was only marginally effective with serotonin as a s ource of oxidative stress. The monoamine oxidase inhibitor pargyline i ncreased cell survival in the presence of serotonin, demonstrating the role of this enzyme in its cytotoxicity. DNA damage (single strand br eaks), but not lipid peroxidation, correlated with the cytotoxic effec ts of menadione.