N -acetylcysteine and endothelial cell injury by sulfur mustard

Abstract

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpful in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endothelial cells ( Toxicol. Appl. Pharmacol . 1996; 141: 568–583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N -acetyl-L-cysteine (NAC) selectively prevented apoptotic death induced by HD. In this study, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment acts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NFΚB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to assess the activity of NFΚB following exposure to HD. The cells were treated with 0–10 mM GSH for 1 h prior to and during exposure to 0 or 500 ΜM HD for 5–6 h. Cells were also treated with 50 mM NAC or 200 ΜM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination overnight prior to exposure to 0 or 500 ΜM HD for 5–6 h. Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild toxicity was associated with 10 mM GSH alone. There was a dose-related enhancement of viability when 2.5 and 5 mM GSH were present during the HD exposure. Pretreatment with BSO alone had no discernible toxicity. However, pretreatment with this inhibitor of GSH synthesis potentiated the toxicity of HD. Pretreatment with 50 mM NAC, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the protective effect of NAC pretreatment alone on HD injury. These observations are highly suggestive that NAC enhances endothelial survival via GSH-dependent effects and confirms and extends the work of others with different models that externally supplied GSH alone may be a fairly effective countermeasure against HD injury of endothelium. We next examined the hypothesis that HD may activate the nuclear transcription factor NFΚB by performing EMSAs with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 ΜM HD. This demonstrated an up to 2.5-fold increase (scanning densitometry) in activation of NFΚB binding to its consensus sequence induced by 500 ΜM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50 mM NAC activated NFΚB, although HD-induced activation of NFkB was partially suppressed by NAC at 5 h. Factor NFΚB is an important transcription factor for a number of cytokine genes (e.g. tumor necrosis factor, TNF), which can be activated following stress in endothelial cells. Taken together, these observations suggest that the protective effects of NAC may be mediated by enhanced GSH synthesis. The increased GSH may act to scavenge HD and also prevent oxidative activation of NFΚB. Under some conditions, NAC may act as an oxidizing agent and thus increase NFΚB activity. The NFΚB-dependent gene expression may be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-derived cytokines. Copyright © 2000 John Wiley & Sons, Ltd.