Induction of mutations by CEES was first measured in a bacterial system (Gilbertet al., 1975
), and we have previously reported induced mutation rates in a human keratinocyte cell line (Powell et al., 2010
). Mutagenicity of SM in rats has been inferred from dominant lethal analysis after exposure by inhalation (Rozmiarek et al., 1973
) and gavage (Sasser et al., 1993
). The results presented above clearly show that topical application of CEES induces a significant increase in mutation frequency in the skin, using a phage-based reporter system. This is the first quantitative measurement of the in vivo
mutagenic action of a sulfur mustard in mammals, although such an effect was clearly expected based on measurements in vitro
and in lower model organisms. Mutation frequency was close to maximal in these assays at a dose of CEES that was insufficient to induce major histopathological changes in the treated skin. Furthermore, the induced mutation frequency dropped to background levels when the CEES treatment was followed by a therapeutic regimen of 13 treatments with DTP. Since we have previously shown facile adduct formation between CEES and DTP, both in a test tube (Liu et al., 2010
) and in cell culture (Powell et al., 2010
), this suggests that this scavenging action may be the primary mechanism by which DTP protects against mutagenicity in this model. For this to be the case, much of the mutagenic action of CEES must have taken place after
the one hour lag between CEES exposure and the first DTP treatment. As noted in the Introduction, it is thought that sulfur mustards remain stably in the skin for periods of time much longer than one hour, and are able to continue to produce macromolecular adducts several days following treatment (Noort et al., 2002
The lack of overt pathological changes in mouse skin treated with 200 mM CEES was somewhat unexpected, given the severe toxicity shown previously with neat CEES in C57BL/6 mice (Isidore et al., 2007
). We have found histopathological lesions similar to those described by Isidore et al. at an intermediate dose of 430 mM (unpublished data), including loss of all epidermis, extensive damage to the dermis and massive inflammation. However, Tewari-Singh and colleagues reported similar “full thickness” lesions in SKH-1/hairless mice after treatment with CEES at approximately 80 mM in acetone (Tewari-Singh et al., 2009
). This apparent difference could be a strain dependent difference in sensitivity, or a solvent effect. Indeed, when we treated C57BL/6 mice with 80 mM CEES in acetone we also observed extensive epidermal damage (unpublished results). This suggests that acetone somehow enhances the skin toxicity of CEES compared to effects with ethanol as supporting solvent.
Exposure of mouse skin to SM in the vapor phase has recently been shown to induce γ-H2AX formation, visible 1-3 days following treatment (Joseph et al., 2011
). This phosphorylation of H2AX is induced by DNA double strand breaks, mediated by the ATM/ATR/DNA-PK kinases, and plays a key role in the organization of multi-protein complexes in damaged chromatin that facilitate double strand break repair (Dickey et al., 2009
). Although classically induced by agents that directly create double strand breaks such as ionizing radiation, alkylating agents are also known to induce γ-H2AX formation (Dickey et al., 2009
). This is presumed to be due to the formation of double strand breaks during cellular attempts to repair the simple alkylation adducts in DNA. Thus, it is not surprising that the mono-functional alkylating agent CEES induces γ-H2AX foci (). The presence of multiple labeled nuclei suggests that in many keratinocytes DNA repair is still not complete three days following exposure to 400 mM CEES.
Concerns have been expressed that SM could be utilized by terrorists to harm civilian populations (Saladi et al., 2006
). In a terrorist attack on an urban population, it is likely that many people would be exposed to amounts of liquid sulfur mustard that produce obvious, albeit delayed skin toxicity. These patients would require immediate treatment, and extended hospitalization. However, it seems likely that a large number of people might receive lower doses that do not cause overt toxicity, and that this exposure could go undetected and untreated. Because CEES and SM react with nucleophiles by a common reaction mechanism, the present results suggest that some of these victims would receive mutagenic doses. It is well known that most mutagens are also carcinogens, and that chronic low-dose mustard exposure is associated with increased risk of cancer (Wada et al., 1968
; Easton et al., 1988
; Hu et al., 2002
). In experimental animals, acute treatment with SM has been shown to be carcinogenic (Heston, 1953
; Fox and Scott, 1980
). Taken together, these results suggest the possibility that civilian victims receiving a single, low dose of SM would be at increased risk for cancer development later in life. Thus, a therapeutic intervention with DTP based on our mouse model would be likely to benefit that fraction of the victims receiving a sub-toxic dose.