Sensitive Bioanalytical Methods for Mustard Gas Exposure Diagnosis

Sulfur mustard (SM, 2, 2'-dichlorodiethyl sulfide) is an alkylating vesicating agent. The injuries resulting from SM exposure are mainly characterized by epithelial damage of the tissues through which it is absorbed, i.e., skin, eye, and respiratory tract. The skin blistering action of SM is not seen until about 12–24 hr after exposure. This time lag provides a window of opportunity for an early diagnosis of SM exposure and medical intervention. Laminin-5, a 440-kDa heterotrimer consisting of α3, β3 and γ2 subunits, and integrin α6β4 are responsible for maintaining a stable attachment of the epidermis to the dermis. Recent studies have shown that in skin, SM exposure significantly reduces the expression of both laminin-5 and α6β4 integrin and destabilizes epidermaldermal attachment, leading to vesication. Nitrogen mustard (NM) is also a vesicant that works by a similar mechanism as SM. In the present study, we studied the effects of SM on laminin-5 subunits by western blotting and immunofluorescence analyses utilizing antibodies against the respective subunits. SM degraded both laminin-5 β3 and γ2 chains in a concentration (50–300 μM) and time (1–16 hr) -dependent manner. The minimum mustard concentration and time for laminin-5 degradation were approximately 50 μM and 1–3 hr, respectively. The laminin-5 degradation effect was specific for SM and NM and was not seen for the other alkylating agents tested. We also found that 200 μM SM or NM degraded the integrin β4 unit of α6β4. An immunochromatographic assay, which is also called a strip assay, was developed and used to detect laminin-γ2 degradation using polyclonal and monoclonal antibodies against laminin-5 γ2. The results of these studies provide important information regarding the mechanism of mustard-induced laminin-5 and integrin degradation that can be used to develop strategies to prevent skin damage, thereby promoting the recovery of mustard-damaged skin.