Effect of Variable Glutathione Peroxidase Activity on H 2 O 2 -Related Cytotoxicity in Cultured Aortic Endothelial Cells

Abstract

Abstract

Primary cultures of porcine aortic endothelial cells were used to assess the effects of O₂ intermediates produced by 10-40 mU/ml xanthine oxidase (XO; +2 mM hypoxanthine) or 25-100 mU/ml glucose oxidase (GO; +5 mM glucose). A 60-min incubation in the presence of the enzyme systems resulted in a dose-dependent toxic effect with evidence of cytolysis (increased LDH release) and cell loss (decrease in DNA and protein content), when these indexes were measured 24 hr after completion of the enzyme reaction. Decreased [³H]thymidine incorporation into DNA was the most sensitive index of cell dysfunction for both enzyme systems. The effects of various scavengers and enzymes indicated that H₂O₂ was the main O₂ intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction. Increased glutathione peroxidase activity associated with the addition of 2 10^-7 M selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H₂O₂ degradation. Evidence for increased DNA synthesis after injury was found in cells previously exposed to XO-hypoxanthine, the degree of increase in [³H]thymidine incorporation being dependent on the intensity of the initial cytotoxicity. Cultured endothelial cells provide a useful tool to evaluate the role of O₂ intermediates in endothelial cell injury, to test the effects of protective agents, and to study the repair process.