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 H202 was the main 0 2 intermediate involved in the cytotoxicity resulting from the XO-hypoxanthine reaction.Increased glutathione peroxidase activity associated with the addition of 2 lo-' A4 selenomethionine to culture medium had a partial protective effect which could be related to an increased rate of H202 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. o 1985 Society for Experimental Biology and Medicine.

The time course of biochemical changes related to cell loss and damage during exposure to 95% O2 [DNA and protein content of dishes, lactate dehydrogenase (LDH) release] was studied in postconfluent endothelial cells isolated from pig aorta, cultured in standard medium and in medium supplemented with 2 X 10(-7) M selenomethionine (Se-Met). A fourfold increase in glutathione peroxidase (G-Px) was the only major enzymatic Se-related effect under both normoxic and hyperoxic conditions, the other antioxidant enzymes being little or not at all affected by this treatment. The addition of Se-Met had a clearcut protective action against the cytotoxic effect of O2 as shown by measurements of DNA and protein content of Petri dishes and of LDH release. On the other hand, the most sensitive O2-related effect, namely the decrease in [3H]thymidine incorporation into DNA, was not affected by Se-Met addition. These experiments suggest that some of the O2-related toxic effects (but not the inhibition of DNA synthesis) could be mediated by lipid peroxides, since they were, at least partly, prevented by a Se-Met-induced increase in G-Px activity.