The cytotoxic effect of HD, and its analogue CEES, is believed to involve an increased generation of damaging free radicals and ROS [8, 11–13, 32]. The data presented here show that LPS in combination with CEES induces intracellular GSH and thiol depletion as well as increased levels of protein carbonyls. In experiments with various human cell lines we have found that GSH depletion is relatively rapid as it occurs within first hour of incubation (data not shown). Thus, it is likely that this depletion is due, in large part, by a direct reaction of CEES with GSH. The measurement of protein carbonyls is one of the best indices for oxidative stress due to the stability of protein carbonyls and sensitivity of the measurement . Cellular thiols are important markers of the redox state of the cell. In particular, GSH is one of the major components of the intracellular redox system and a key intracellular antioxidant that functions as a substrate for glutathione peroxidase which detoxifies both hydrogen peroxide and lipid hydroperoxides [34, 35]. Depletion of intracellular stores of GSH plays an important role in the development of oxidative stress [12, 13, 36]. Recent work also suggests that the anti-apoptotic protein Bcl-2 directly interacts with GSH to regulate an important mitochondrial GSH pool that influences mitochondrial oxidative stress and subsequent apoptosis . It is highly possible that both sulphur and nitrogen mustards possess a similar ability to deplete cellular thiols and induce protein oxidation.
Taken together, our data strongly suggest that CEES induces oxidative stress in stimulated macrophages. Moreover, the pattern of oxidative stress parallels the pattern observed for CEES cytotoxicity, i.e., cytotoxicity and oxidative stress are amplified in cells treated with both CEES and LPS. The addition of 5–10 mM NAC, a well characterized water-soluble antioxidant, was found to be very effective in minimizing CEES toxicity in stimulated macrophages and in preventing GSH depletion. Our data suggests that NAC can be added five hours before or even five hours after CEES and still exert a cytoprotective effect. Das et al.  recently found that NAC in drinking water was effective in reducing CEES-induced lung toxicity to Guinea pigs. Fan et al.  have shown that liposomal encapsulated NAC delivered intratracheally was more effective than free NAC against acute respiratory distress syndrome in a rat model. It is interesting, therefore, that McClintock et al.  have shown that reducing agents (NAC or GSH), as well as some anti-oxidant enzymes, delivered via liposomes, can substantially diminish CEES-induced injury in rat lungs. We are currently formulating an optimal antioxidant liposome preparation for treating either lung or skin induced CEES/HD injury.
We previously reported that CEES induces a transient loss of iNOS protein expression in LPS stimulated RAW 264.7 macrophages but does not inhibit the enzymatic activity of iNOS. Based of the work of others [22–25], we hypothesized that NAC treatment would not only be protective against CEES toxicity but would also restore NO production in LPS stimulated macrophages treated with CEES. Our results indicate, however, that this was not the case. Our data did, however, show that NAC effectively increases cell viability, increases GSH levels and reduces oxidative stress in LPS stimulated macrophages treated with CEES.
CEES could inhibit iNOS protein synthesis by a number of possible molecular mechanisms which we are currently exploring . It is generally accepted that both the transcription factor NF-κB and STAT-1 play central roles in the LPS induction of iNOS [17, 40]. It is possible that CEES/HD could inhibit the NF-κB and/or the STAT-1 pathways in RAW 264.7 macrophages and consequently block iNOS gene expression. For instance, CEES could alkylate the NF-κB consensus nucleotide binding sequences thereby preventing the binding of activated NF-κB to the iNOS promoter and block the subsequent production of iNOS mRNA and protein expression. Previous studies in vitro have shown that DNA alkylation by CEES [41, 42] or by nitrogen mustard  can inhibit the DNA binding of transcription factor AP2 or NF-κB.
Alternatively, the DNA binding ability of the NF-κB and/or STAT-1 transcription factors could be reduced by direct covalent modification by CEES or as an indirect result of GSH depletion, i.e., redox regulation. Nishi et al.  have found, for example, that the cysteine-62 (Cys-62) residue of the p50 NF-κB protein subunit is oxidized in the cytoplasm but reduced in the nucleus, and that the reduced form is essential for NF-κB DNA binding. It is possible that CEES could rapidly react with Cys-62 of the p50 NF-κB subunit and prevent its DNA binding. However, since NAC was found to restore GSH levels without restoring iNOS activity (see Figures 1 and 7), it is unlikely that a GSH redox modulation of the p50 Cys-62 is the molecular mechanism for CEES induced loss of iNOS protein in LPS-stimulated macrophages. This cannot, however, be completely ruled out based on our current data.
Moreover, there is evidence suggesting that alkylating agents do not inhibit but rather promote NF-κB activation. It is known that CEES or HD treated cells release elevated levels of TNF-α and also show NF-κB activation both in vitro and in vivo as measured by electrophoretic mobility shift assays (EMSAs)[7, 45, 46]. Minsavage and Dillman recently demonstrated that NF-κB is activated by HD treatment in human cell lines via nonclassical p53-dependent pathway . Collectively, these data suggest that the inhibition of iNOS expression by CEES or HD could be due to downregulation of the STAT-1 and/or classical NF-κB pathway. We are currently exploring these various molecular mechanisms.
In the work presented here, we also tested the ability of polymyxin B to block the effect of LPS. Polymyxin B binds to the lipid A domain of LPS and neutralizes its activity. Our data show that polymyxin B effectively inhibits CEES toxicity in LPS stimulated cells. In vivo, LPS could directly enhance CEES/HD toxicity in cells with functional CD14 receptors or by triggering the release of pro-inflammatory cytokines, such as TNF-α and IL-1β, by immune cells. We have previously demonstrated that inflammatory cytokines also enhance CEES cytotoxicity .