N-acetylcysteine and endothelial cell injury by sulfur mustard

Understanding the underlying mechanisms of cell injury and death induced by the chemical warfare vesicant sulfur mustard (HD) will be extremely helpfu l in the development of effective countermeasures to this weapon of terror. We have found recently that HD induces both apoptosis and necrosis in endo thelial cells (Toxicol, Appl, Pharmacol. 1996; 141: 568-583). Pretreatment of the endothelial cells for 20 h with the redox-active agent N-acetyl-L-cy steine (NAC) selectively prevented apoptotic death induced by HD, In this s tudy, we tested the hypotheses that pretreatment with NAC acts through two different pathways to minimize endothelial injury by HD: NAC pretreatment a cts via a glutathione (GSH)-dependent pathway; and NAC pretreatment acts to suppress HD-induced activation of the nuclear transcription factor NF kapp aB. We used a fluorescence microscopic assay of apoptotic nuclear features to assess viability and electrophoretic mobility shift assays (EMSAs) to as sess the activity of NF kappaB following exposure to HD, The cells were tre ated with 0-10 mM GSH for 1 h prior to and during exposure to 0 or 500 muM HD for 5-6 h, Cells were also treated with 50 mM NAC or 200 muM buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, alone or in combination o vernight prior to exposure to 0 Or 500 muM HD for 5-6 h, Externally applied GSH up to a concentration of 5 mM had no toxic effect on the cells. Mild t oxicity was associated with 10 mM GSH alone. There was a dose-related enhan cement of viability when 2.5 and 5 mM GSH were present during the HD exposu re. Pretreatment with BSO alone had no discernible toxicity. However, pretr eatment with this inhibitor of GSH synthesis potentiated the toxicity of HD , Pretreatment with 50mM NAG, as previously reported, provided substantial protection. Combining pretreatment with both BSO and NAC eliminated the pro tective effect of NAC pretreatment alone on HD injury. These observations a re highly suggestive that NAC enhances endothelial survival via GSH-depende nt effects and confirms and extends the work of others with different model s that externally supplied GSH alone may be a fairly effective countermeasu re against HD injury of endothelium. We next examined the hypothesis that H D may activate the nuclear transcription factor NF kappaB by performing EMS As with nuclear extracts of endothelial cells following exposure to 0, 250 or 500 muM HD. This demonstrated an up to 2.5-fold increase (scanning densi tometry) in activation of NF kappaB binding to its consensus sequence induc ed by 500 muM HD after 5 h of HD exposure. Paradoxically, treatment of the endothelial cells alone with 50mM NAC activated NF kappaB, although HD-indu ced activation of NF kappaB was partially suppressed by NAC at 5 h, Factor NF kappaB is an important transcription factor for a number of cytokine gen es (e,g, tumor necrosis factor, TNF), which can be activated following stre ss in endothelial cells. Taken together, these observations suggest that th e 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 NF kappaB. Under some conditions, NAC may act as an oxidizing agent and thus increase NF kappaB activity. The NF kappaB-dependent gene expression m ay be important in inducing endothelial cell death as well as in generating a local inflammatory reaction associated with the release of endothelial-d erived cytokines. Copyright (C) 2000 John Wiley & Sons, Ltd.