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 50 mM NAG, as previously reported, provided substantial
protection. Combining pretreatment with both BSO and NAC eliminated the pr
otective effect of NAC pretreatment alone on HD injury. These observations
are highly suggestive that NAC enhances endothelial survival via GSH-depend
ent effects and confirms and extends the work of others with different mode
ls that externally supplied GSH alone may be a fairly effective countermeas
ure against HD injury of endothelium, We next examined the hypothesis that
HD may activate the nuclear transcription factor NF kappaB by performing EM
SAs 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 dens
itometry) in activation of NF kappaB binding to its consensus sequence indu
ced by 500 muM HD after 5 h of HD exposure. Paradoxically, treatment of the
endothelial cells alone with 50 mM NAC activated NF kappaB, although HD-in
duced activation of NF kappaB was partially suppressed by NAC at 5 h, Facto
r NF kappaB is an important transcription factor for a number of cytokine g
enes (e.g. tumor necrosis factor, TNF), which can be activated following st
ress in endothelial cells. Taken together, these observations suggest that
the protective effects of NAC may be mediated by enhanced GSH synthesis. Th
e increased GSH may act to scavenge HD and also prevent oxidative activatio
n of NF kappaB, Under some conditions, NAC may act as an oxidizing agent an
d thus increase NF kappaB activity. The NF kappaB-dependent gene expression
may be important in inducing endothelial cell death as well as in generati
ng a local inflammatory reaction associated with the release of endothelial
-derived cytokines, Copyright (C) 2000 John Wiley & Sons, Ltd.