DEVELOPMENT OF AN IN-VITRO CELL-CULTURE MODEL TO INVESTIGATE THE INDUCTION AND QUANTIFICATION OF OXIDATIVE STRESS AND ITS INHIBITION BY ALPHA-TOCOPHEROL

The ability of the natural antioxidant alpha-tocopherol to protect against oxidative stress in vitro was assessed. Primary cultures of chicken embryo fibroblasts (CEF) were oxidatively stressed by exposure to paraquat (PQ). Activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were measured as indices of oxidative stress. CEF incubated with 0.125-1.0 mM PQ for 18 hr exhibited increased SOD activity (P < 0.05). CAT activity increased with 0.25 mM PQ (P < 0.05). GSH-Px activity decreased significantly in the presence of PQ. No cytotoxicity, as indicated by lactate dehydrogenase release, was observed at PQ concentrations below 2 mM. Incorporation of added alpha-tocopherol (100 nM) into 0.25 mM PQ-treated CEF resulted in SOD activity not significantly different from that observed in control cells not treated with PQ. Lower levels of added alpha-tocopherol (16 nM) returned CAT to its control value. However, even at 1000 nM alpha-tocopherol, GSH-Px activity was not protected in PQ-treated cells. CEF represent a useful model to study both inducers and inhibitors of oxidative stress.