Sulfur mustard gas (SM1
, bis-[2-chloroethyl]sulfide) and related chemical weapons have been used in warfare since World War I to maim and incapacitate enemy soldiers. Military exposures to the mustard agents have long been known to cause severe blistering of exposed skin, which is slow to heal, and damage to the cornea; the term vesicant is used to describe these actions. In addition, inhalation exposure of the aerosolized agent causes damage or destruction of the respiratory tract, including chronic bronchitis, bronchiectasis, and pulmonary fibrosis (1
). Despite extensive research efforts, little progress has been made on developing effective treatment regimens, and victims often require extended hospitalization.
There is increased concern in the Homeland Defense community (http://www.bt.cdc.gov/Agent/AgentlistChem.asp
) that these agents or related chemicals could be used by terrorists to attack the civilian population of the United States, and thus there is need to develop strategies to ameliorate the effects of such an attack. The concerns extend not only to treating the exposed human population, but also to protecting emergency personnel called upon to treat the victims and decontaminate the affected area. SM, for example, is actually a liquid under ambient conditions that can contaminate clothing and surfaces after deployment and maintain its toxic activity for some time, thus posing a threat to rescue, medical and decontamination personnel (1
). In addition, there is increasing evidence that a significant fraction of the SM dose remains biologically active within the victim's body for several days after exposure (3
The underlying cause of the various forms of toxicity is thought to be macromolecular damage. SM and the monofunctional analogs, 2-chloroethyl ethyl sulfide (CEES) and 2-chloroethyl methyl sulfide (CEMS) are direct acting, electrophilic agents that react with nucleophilic moieties in nucleic acids and proteins (5
). The reaction of SM with DNA has been well-studied (5
). The initial reaction pathway in aqueous solution is spontaneous dechlorination of one of the chloroethyl substituents, resulting in the formation of a cyclic episulfonium ion (10
), which preferentially reacts at the N7 position of deoxyguanosine to give a mono adduct. Because of the reactivity of both chloroethyl groups, further reaction with an adjacent deoxyguanosine produces cross-links in the DNA. In particular, in both yeast (12
) and mammalian cells (13
) the cytotoxicity of SM is dramatically enhanced in cells that are deficient in the nucleotide excision repair pathway; in mammalian cells, cytotoxicity due to 2-(chloroethyl) ethyl sulfide, a monofunctional analog of SM, was also found to be enhanced by NER deficiency. An elegant host cell reactivation assay based in CHO cells (13
), showed that this enhanced cytotoxicity correlated with an inability to functionally repair SM- or CEES-damaged DNA. Together, these data provide strong evidence that at the cellular level, DNA damage is the major determinant of mustard toxicity.
Numerous other chemical carcinogens also damage DNA through electrophilic intermediates that attack nucleophilic sites in DNA. Several strategies for scavenging these electrophilic carcinogens have been demonstrated previously. In particular, thiopurines have been shown to react facilely with a wide range of electrophilic toxicants (14
), and have demonstrated effective suppression of cytotoxicity, mutagenesis and carcinogenesis (14
). Several of these compounds, notably 2,6-dithiopurine (DTP), are substrates for the cellular purine transport system (19
), and can therefore be rapidly taken up by cells. These compounds, therefore, might be effective in scavenging reactive sulfur mustards.
The initial and rate-determining step in the nucleophilic substitution reactions of sulfur mustards in aqueous solution ()is dissociation of Cl-
). The episulfonium ion formed in this dissociation then reacts in a faster, bimolecular reaction with either water or an added nucleophile. An extensive series of anions was analyzed in early work for their ability to “compete” with water for reaction with the episulfonium ions derived from SM (20
). Because the reaction with water is much faster than the initial dissociation, measurement of kW
has not been reported, and the relative strengths of competing nucleophiles have been expressed as “competition factors”, which are actually the ratio k2
. Among the nucleophiles examined, one of the most reactive non-toxic compounds appears to be thiosulfate, with k2
~ 27,000 M-1
for SM and 19,000 M-1
for CEES; nucleic acids, cysteine and cysteine ethyl ester were reported to be at least an order of magnitude less reactive with SM (20
The initial reaction of the half-mustards CEES and CEMS with nucleophiles in aqueous solution follows the same mechanism as the analogous reaction of SM. Thus, many studies have been done with the half-mustards, which are less toxic and can be utilized in a standard chemical fume hood, without the need for glove boxes and special facilities. We hypothesized that DTP and other thiopurines would likely show robust nucleophilic substitution reactions with the half-mustards CEES and CEMS, and therefore may be candidates for effective prophylactic and/or therapeutic agents against SM toxicity. In the present communication, we show that three non-toxic thiopurines, 2,6-dithiopurine (DTP), 2,6-dithiouric acid (DUA) and 9-methyl-6-mercaptopurine (MMP), exhibit facile substitution reactions with these sulfur half mustards. The “competition factors” for these thiopurines, a measure of the relative reactivity of a potential scavenging agent, are found to be comparable to thiosulphate, a well-known scavenger, whereas other commonly used thiol-containing reagents, N-acetyl cysteine (NAC) and reduced glutathione (GSH) are found to be much less active at near neutral pH. More importantly, we show that at equimolar concentrations these thiopurines significantly reduce overall exposure to the reactive episulfonium ions that cause DNA damage and genotoxicity.