Abstract
ABSTRACT: A series of experiments were designed to examine the potential cytotoxicity of nitric oxide (NO), or reactive species derived from NO, in HA1 fibroblasts and H2O2-resistant variants of this cell line, designated OC14 cells. A 1-h exposure at 37°C to a 1.7 mM bolus dose of NO, prepared in N2-gassed medium, significantly reduced clonogenic survival in the HA1 fibroblasts line to 60% of control cells treated with N2-gassed medium alone. The OC14 cells were found to be completely resistant (100% survival) to NO-mediated injury in comparable experiments. A second set of experiments was designed to determine the role of the intracellular antioxidant, glutathione, in protection against NO-mediated injury. Depletion of total glutathione resulted in a significant reduction in HA1 and OC14 clonogenic survival to 8% and 50% when compared with respective control cells. The effect of total glutathione depletion on NO-initiated toxicity in HA1 cells was dose- and cell-density dependent and was observed to occur within 5 min of exposure to NO. Further evidence of cytotoxicity was demonstrated by loss of trypan blue dye exclusion properties in glutathione-depleted HA1 cells after NO exposure. Other experiments demonstrated that nitrate and nitrite exposure produced no cytotoxicity in glutathione-depleted HA1 cells and that coincubation of NO-saturated medium with oxyhemoglobin inhibited NO-induced cytotoxicity in glutathione-depleted HA1 cells. These results demonstrate that 1) nitric oxide, or an NO-derived reactive nitrogen species other than nitrites or nitrates, is responsible for reduction in clonogenic survival and trypan blue dye exclusion capabilities in vitro; 2) biochemical pathways associated with cellular resistance to oxidative stress also confer resistance to NO-mediated injury in this cell model; and 3) total glutathione content determines a significant portion of cell sensitivity to NO-mediated cytotoxicity.
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Walker, M., Kinter, M., Roberts, R. et al. Nitric Oxide-Induced Cytotoxicity: Involvement of Cellular Resistance to Oxidative Stress and the Role of Glutathione in Protection. Pediatr Res 37, 41–49 (1995). https://doi.org/10.1203/00006450-199501000-00010
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DOI: https://doi.org/10.1203/00006450-199501000-00010
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