Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts

Abstract

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N7 guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N7 position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N7 guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0–300μM, 60min) or HN-1, HN-2, HN-3 (120min). After exposure, cells were scraped and DNA was isolated and normalized. 1μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3′-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30nM to 300μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.