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  • br Introduction Although not fully understood the

    2020-05-15


    Introduction Although not fully understood, the molecular toxicology of sulfur mustard (bis(2-chloroethyl)sulfide, SM; CAS–Nr. 505-60-2), an alkylating warfare agent, has been attributed to DNA alkylation [1], [2], [3]. The same principle holds true for other alkylating compounds such as nitrogen mustards or monofunctional agents such as the SM analog CEES (2-chloroethyl ethyl sulfide) [4], [5], [6]. The resulting DNA adducts can be used for the analytical verification of SM exposure [7]. The procedures required are complex, however, when they are based on sensitive mass spectrometry methods. In general, it is highly unlikely that such techniques are available in conflict zones. Attempts have thus been made to develop convenient detection methods that can be used in the field. In most cases, patients will not present immediately after SM exposure because SM-induced clinical symptoms typically occur after a latency Rufinamide of several hours [8], [9], [10]. When they do present, the detection of free, unbound alkylating agent is highly unlikely. For this reason, available rapid detections systems such as the Securetec Sulfur Mustard Detector©, which specifically detects free SM by antibody labeling, cannot be used in this context [11], [12]. The detection of SM DNA adducts extracted from exposed tissue (e.g. blister roofs) may be helpful in such a scenario. A slot blot-based method has been suggested for this purpose [13], [14].
    Materials and methods
    Results
    Discussion A significant decrease in fluorescence was observed after the treatment of pure DNA with alkylating compounds. This decrease was characterized by a dose-response relationship. Mass spectrometry and additional fluorescence measurements ruled out the covalent modification of EthBr by SM. The alkylation of DNA by SM results in around 14–20% of DNA crosslinks [3], [19], [20]; up to 50% of these crosslinks are assumed to represent interstrand crosslinks [21]. We thus assumed that DNA crosslinks may have caused DNA condensation and impaired the access of the fluorescent dyes to the DNA. Further experiments using bifunctional N-alkylating agents (i.e. HN-3) revealed an even more pronounced decrease in fluorescence, which underlined our hypothesis. However, some bifunctional and crosslinking agents (i.e. HN-1, HN-2) had only a minor effect on fluorescence. These agents were shown to exhibit slower kinetics with regard to DNA alkylation [14], [22], [23], [24]. This may also be a reason for the lower toxicity of these compounds [25]. We thus assumed that, with regard to HN-1 and HN-2, lower reactivity was the reason for the only minor effects that we observed in our experiments. In order to counteract DNA condensation, we used restriction enzymes [26] to cleave alkylated DNA into small fragments. DNA fragments were thought to be more easily accessible for DNA dyes. Our results indeed demonstrated that the use of digestion enzymes restored the fluorescence signal of stained, alkylated DNA. The decrease in fluorescence intensity may, however, also be due to quenching effects. It is reported that especially chloride ions (Cl−) may impact fluorescence [27]. A single SM molecule is able to release two Cl− ions either due to SM hydrolysis or during DNA alkylation [2], [28]. In addition to the release of Cl− from the SM molecule, two protons per SM molecule are also produced, which results in the formation of hydrochloric acid [2], [29], [30], [31]. To mimic this fact, we performed additional experiments using NaCl or HCl instead of alkylating compounds. While NaCl had no effect on fluorescence, the use of HCl decreased fluorescence in a manner comparable to the alkylating compounds. Based on the results, we concluded that neither crosslinks nor Cl− but instead H+ were responsible for decreasing the fluorescence of SM-exposed DNA in aqueous, unbuffered solutions. This hypothesis was confirmed by repeating the experiments and using defined buffer systems (e.g. PBS, Tris-HCl) instead of water. In this case, the decrease in fluorescence was clearly prevented, especially when we used PBS or Tris-HCl. Other buffer systems that we tested were also suitable but were less effective.