Sulfur mustard (2, 2′-chloroethyl sulfide [CAS 505-60-2], mustard gas, NATO Standard Agreement designation; HD) and related chloroethyl compounds () are alkylating agents and potent vesicants. It was last employed in combat in the conflict between Iraq and Iran in the 1980s. Erythema, blistering, and necrosis follow dermal contact. Conjunctivitis and/or corneal opacity result from exposure of the eyes. Inhalation can lead to pneumonia, chronic bronchitis and asthma, while systemic symptoms have radiomimetic characteristics. Injury is dose dependent and initially painless (Papirmeister et al., 1991
; Somani, 1992
). It is interesting that the dermal reaction to sulfur mustard exposure in humans is different from that in most other species; no animal blisters like man (Marshall et al., 1919
). Recent in vitro
work (Sabourin et al., 2000
; Smith et al., 2001
) has revealed the initiation of proinflammatory cytokine production at the cellular level starting several hours after exposure. However, the initial biochemical lesion, the event that ultimately sets off cytokine production and characteristic symptoms, remains unidentified.
Compounds spontaneously forming “onium” ions
Several lines of inquiry have indicated that this initial lesion might involve free radicals. For example, reports in the literature show a strong parallel between the cellular and biochemical effects of sulfur mustard exposure and the effects of free radical damage (Papirmeister et al., 1991
; Somani, 1992
; Halliwell and Gutteridge, 1989
). Additionally, when an electron rich center such as a sulfur, nitrogen or selenium is in a vicinal relationship to a halide such as one finds with these chloroethyl compounds (), it favors intramolecular cyclization to form an energetically strained, reactive three-membered cyclic onium ion () (Yang et al., 1988
). Onium ions, cyclic and acyclic, form spontaneously in solutions of mustards (Kang and Spears, 1987
; Ross, 1962
; Bartlett et al., 1949
). Electrochemical studies have shown that the one electron reduction of onium ions results in free radical production (Saeva and Morgan, 1984
). Reports of the direct electrochemical reduction of sulfonium ions in aqueous systems (Chambers, 1978
) indicated the mechanistic feasibility of this process in vivo
and raised the possibility of enzymatic reduction as the source of mustard free radicals.
The rapid Sn1 mediated internal cyclization of sulfur mustard with loss of chlorine to form the cyclic sulfonium ion
Additional evidence of free radical participation in mustard toxicity arose as part of a study designed to evaluate the potential for interaction among military deployment-related chemicals at the level of metabolism. We investigated the effect of sulfur mustard on the microsomal cytochrome P450 drug metabolizing system using NADP, an NADPH generating system and mouse liver microsomes induced with phenobarbital. The results indicated that mustard inhibited the O-demethylation of p
-nitroanisole by cytochrome P450 (Brimfield and Hodgson, 2005
). To clarify where mustard might be exerting its effect, we simplified the mixture from the previous work by using purified NADPH-cytochrome P450 reductase (CYPOR) in place of microsomes, and cytochrome c as a terminal electron acceptor in place of the microsomal cytochrome P450. Employing this system we were able to follow electron transport spectrophotometrically by measuring cytochrome c reduction (Brimfield and Hodgson, 2005
). The rate of cytochrome c reduction was slowed in the presence of mustard just as the rate of p
-nitroanisole O-demethylation had been. That effect provided an indication that the flavoprotein reductase itself (the only remaining enzyme in the test system) was the probable site of mustard interaction. This phenomenon has also been reported with hydrazines which are known to impair the oxidative metabolism of co-administered substrates in the microsomal drug metabolizing system and also to be enzymatically activated to carbon free radicals (Muakkassah et al., 1981
; Reed, 1976
; Lee and Lucier, 1976
Taken together those results suggested that mustard was inhibiting all cytochrome P450 activity by uncoupling electron transport at the flavoenzyme reductase and diverting electrons directly to xenobiotic reduction. This mode of toxicant transformation can lead to free radical formation (Testa, 1995
) and lends weight to our earlier speculation about enzymatic onium ion reduction. Ubiquitous pyridine nucleotide-driven flavoenzyme reductases such as NADPH-cytochrome P450 reductase [EC188.8.131.52] (Tew, 1993
), the reductase domain of neuronal nitric oxide synthase [EC 184.108.40.206] (Steuhr et al., 1991
), the cytosolic thioredoxin-disulfide reductase [EC 220.127.116.11] (Gray et al., 2007
) and others (Halliwell and Gutteridge, 1999
) appear to act as good electron sources for this one-electron reduction process.
It has been known for some time that onium compounds such as diphenyliodonium ion and the dipyridylium ions of the herbicides paraquat and diquat are capable of uncoupling extramitochondrial electron transport and being reduced in place of cytochrome P450 or other terminal electron acceptors (Testa, 1995
; Halliwell and Gutteridge, 1999
). The diphenyliodonium cation offers an especially interesting example. When it undergoes single electron enzymatic reduction by either cytochrome P450 reductase or the reductase domain of nitric oxide synthase it rapidly fragments to yield iodobenzene and a phenyl free radical (Tew, 1993
; Steuhr et al., 1991
) capable of forming radical adducts (O’Donnell et al., 1994
). This provided additional evidence that free radical production might be an early step in the mechanism of mustard toxicity. As a result, we hypothesized that a process mechanistically analogous to that seen with diphenyliodonium and paraquat led to the cytochrome P450 reductase (CYPOR) inhibition that we had observed and explained the parallel biochemical effects of free radical and mustard exposure. Spontaneously formed onium ions could be enzymatically reduced to yield carbon-centered free radicals which reacted with and damaged cellular macromolecules leading to toxicity.
The detection of free radical formation from a mustard in the presence of NADPH and a flavoenzyme reductase in vitro would prove the feasibility of this mechanism. Since an electron paramagnetic resonance (EPR) signal is the preferred method for free radical detection, we proceeded with the development of a spin trapping technique able to detect carbon-centered free radicals emanating from the enzymatic one electron reduction of onium ions originating with the mustards.