Overall, the experiments detailed in this work show that CEES treatment in LPS-stimulated RAW264.7 murine macrophages transiently inhibits intracellular NO generation by interfering with iNOS expression rather than by direct inhibition of iNOS enzymatic activity. CEES (as well as HD) undergo rapid hydrolysis in aqueous solutions and this may account, in part, for the transitory nature of its inhibiting effect on iNOS induction . LPS is a major component of the cell wall of gram-negative bacteria and is known to trigger a variety of inflammatory reactions in macrophages and other cells expressing CD14 receptors [23, 24]. LPS is ubiquitous and is present in serum, tap water, and dust. Military and civilian personnel would, indeed, always have some degree of exposure to environmental LPS.
LPS stimulation of macrophages is known to involve the activation of protein phosphorylation by kinases as well as the activation of nuclear transcription factors such as NF-κB [25–28]. An important consequence of NF-κB activation in macrophages is the induction of iNOS expression followed with highly elevated NO production . Nitric oxide has been demonstrated to have an important role in promoting cell death; however, the precise nature of this role varies with cell type and the dose. Low levels of nitric oxide protect RAW 264.7 macrophages from hydrogen peroxide induced apoptosis , however, nitric oxide has also been reported to induce apoptosis in J774 macrophages . Nitric oxide can induce cell death through energy depletion-induced necrosis and oxidant-induced apoptosis.
We are currently exploring the potential molecular mechanism(s) whereby CEES interferes with iNOS expression in LPS stimulated macrophages. It is possible that GSH depletion caused by CEES determines iNOS expression. There are strong evidences suggesting that thiol depletion and iNOS expression are interrelated [30–32]. For example, LPS stimulated macrophages depleted of GSH exhibit a decreased level of iNOS protein and nitrite production . Similarly, both in vitro  and in vivo  studies show that hepatocytes depleted of GSH have a diminished production of nitric oxide which is primarily due to a decreased level of iNOS mRNA. Vos et al.  have also presented evidence showing that GSH modulation of iNOS expression in hepatocytes is correlated with NF-kB activation, i.e., GSH depletion is associated with a lack of NF-kB activation. The influence of GSH depletion is not, however, consistent in all cell types. Glucose induced reduction of GSH in intestinal epithelial cells is associated with NF-kB activation and upregulation of iNOS gene expression .
It is also possible that CEES decreases iNOS expression by interfering with the LPS-induced activation of transcription factor NF-κB and/or signal transducer and activator of transcription-1α (STAT-1α). It is interesting, therefore, that Gray  has found that both CEES and HD inhibit the in vitro binding of transcription factor activating protein-2 (AP-2) via alkylating the AP-2 DNA consensus binding sequence rather than by direct damage to the AP-2 protein. Furthermore, it is significant that neither CESS nor its hydrolysis products were found to damage the AP-2 transcription factoring in a manner that prevented its DNA binding . Similar experiments have yet to be done with NF-κB. Chen et al.  have also found that nitrogen mustard (bis(2-chloroethul) methylamine) similarly inhibits the binding of AP-2 to its consensus sequence. Nitrogen mustard also was shown to inhibit the binding of NF-κB to the GC-rich consensus sequence due to the interactions with DNA . It is possible, therefore, that CEES also alkylates the NF-κB consensus sequence thereby preventing the binding of the NF-κB to the iNOS promoter. LPS and/or cytokine-inducible NF-κB binding elements of the murine iNOS promoter have been identified , and they are rich of guanine, which is the major alkylation site for HD or CEES. The possible effect of CEES on iNOS promoter regulation is currently being explored.
Although the activation of NF-κB due to mustard or CEES exposure have been shown in various cell lines [7, 37, 39], the detailed mechanism of this event is still unclear. Recent report  showed that NF-κB-driven gene expression has maximum at 9 hours in HD treated keratinocytes. In contrast, in a guinea pig model, Chatterjee et al.  have shown that NF-κB activation in lung tissues occurs shortly after CEES expose (1 hour), then disappears within 2 hours completely. However, in our experiments we did not observe any short term stimulating effect of CEES on NO production or iNOS expression (data not shown). Notably, the electrophoretic mobility shift assays used by Chatterjee et al. to measure NF-κB activation show only the state of NF-κB protein complex and provide no information regarding its binding to the DNA consensus sequences.
The physiological significance of potentially decreased iNOS expression by exposure to CEES or HD is not known. Considerable evidence, however, supports the view that nitric oxide production via iNOS plays a key role in wound healing [41–43]. Animal studies  have shown that the iNOS knockout mice have impaired wound healing that is reversed by iNOS gene transfer. Soneja et al.  have suggested that wound healing could be accelerated under circumstances where oxidative stress is minimized and nitric oxide production enhanced. We have initiated work to explore the role of antioxidants in preventing HD induced pathology in skin.