The cytoprotective roles of ascorbate and glutathione against nitrogen dioxide toxicity in human endothelial cells.

Abstract The depletion of human umbilical vein endothelial (HUVE) cell glutathione with buthionine sulfoximine or with sulfur amino acid-free medium potentiated the sub-lethal ( 3 H-deoxyglucose release) and lethal (lactate dehydrogenase release) cytotoxicity responses of the cells to direct exposure to NO 2 over the range 2–20 ppm. When control cells, or glutathione-depleted cells, were either pre-loaded with ascorbate (intracellular ascorbate), or washed with ascorbate-containing medium just before exposure (extracellular ascorbate), the cells were fully protected from NO 2 -dependent toxicity. Concomitant with these exposures, NO 2 caused dose-dependent depletions of both glutathione and ascorbate. Further, it was noted that the depletion of the intracellular ascorbate pool was accelerated in these glutathione depleted cells. Conversely, loading ascorbate into the cells significantly diminished NO 2 -dependent depletion of intracellular GSH. In contrast to affecting the acute cytotoxicity response of the HUVE cells to NO 2 , ascorbate supplementation of the medium of cells exposed to NO 2 at clonal density facilitated considerable protection to the colony-forming efficiency of the cells. We conclude that both ascorbate and glutathione play important protective roles in defending HUVE cells from the toxicity of NO 2 under direct exposure conditions. The results also strengthen the premise that ascorbate and glutathione co-operate in the antioxidative protection of cellular viability.