New Methods to Detect Sulfur Mustard (SM) and SM-Induced Skin Damage

Abstract

Sulfur mustard (SM) is a strong vesicant and a bifunctional alkylating agent targeting mainly nuclear DNA. By introducing several modifications, mostly in guanine and adenine, it severely damages the DNA, and consequently the cell. Because of its bifunctionality, it also creates crosslinks between guanine bases of the same or a different strand (17% of total alkylation). The high capability of SM to alkylate DNA was on one hand utilized to develop a rapid handheld diagnostic kit to identify SM on exposed skin and on the other hand to biomonitor SM DNA damaging effects on skin cells. A sensitive small size immunochromatographic test-strip system (SM-Detector) was developed, together with Securetec AG, Munich, Germany. For detection, a capture oligonucleotide was fixed on the test strip, which reacts with free SM on skin surfaces. An anti-SM-DNA-adduct antibody (TNO, The Hague, Netherlands) was used for visualisation of the SM adduct. The SM-Detector was evaluated by the Bundeswehr Medical Service in laboratory experiments and under field conditions. The latter was conducted in military training scenarios during a NATO exercise in Canada. In order to test the SM-Detector for direct measurement SM (2–200 μmol/L, 50 μL) was spread on pig skin. Another scenario included a soldier exploring a cave exposed with SM vapour. The SM-Detector was fixed to a side pocket of the individual protective suit and showed a positive reaction due to SM vapour in the air. The Comet assay is a rapid in vitro test system to detect DNA damage on a single cell level. However, SM-induced DNA damage cannot be determined using the standard Comet assay, since the alkylating agent forms crosslinks between the DNA strands, preventing migration in the electrical field. In order to improve the detection of SM-induced DNA damage, we utilized repair enzymes, for example formamido-pyrimidine-gly-cosylase (FPG), endoglycosylase III (ENDO III) and 3-methyladenine-glycosylase (AAG). These enzymes specifically recognize modifications of the DNA induced by SM and create additional strand breaks, which in turn can be detected by the Comet assay. The lowest detectable SM concentration using the modified Comet assay is 30 nmol/L when using FPG, 10 nmol/L in case of ENDO III and 30 nmol/L after AAG treatment. In summary, the handheld SM detector (SMD) is able to detect the agent, both as a vapour and on skin surfaces. The Comet assay modified by a precursory incubation with repair enzymes significantly raises the sensitivity of the detection of SM induced DNA damage in skin cells, thus allowing a biomonitoring of SM effects in skin cells below vesicating SM concentrations.