Sensitive Bioanalytical Methods for Mustard Gas Exposure Diagnosis

Abstract : 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 3, 3 and 2 subunits, and integrin 6 4 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 6 4 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 3 and 2 chains in a concentration (50 300 M) and time (1 16 hr) -dependent manner. The minimum mustard concentration and time for laminin-5 degradation were approximately 50 M 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 M SM or NM degraded the integrin 4 unit of 6 4. An immunochromatographic assay, which is also called a strip assay, was developed and used to detect laminin- 2 degradation using polyclonal and monoclonal antibodies against laminin-5 2. 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.