Assessment of sulfur mustard interaction with basement membrane components

Abstract

Bis-2-chloroethyl sulfide (sulfur mustard, HD) is a bifunctional alkylating agent which causes severe vesication characterized by slow wound healing. Our previous studies have shown that the vesicant HD disrupts the epidermal-dermal junction at the lamina lucida of the basement membrane. The purpose of this study was to examine whether HD directly modifies basement membrane components (BMCs), and to evaluate the effect of HD on the cell adhesive activity of BMCs. EHS laminin was incubated with [14C]HD, and extracted by gel filtration. Analysis of the [14C]HD-conjugated laminin fraction by a reduced sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) revealed the incorporation of radioactivity into both laminin subunits and a laminin trimer resistant to dissociation in reduced SDS-PAGE sample buffer, suggesting direct alkylation and cross-linking of EHS laminin by [14C]HD. Normal human foreskin epidermal keratinocytes were biosynthetically labeled with [35S]cysteine. 35S-labeled laminin isoforms, Ae.B1e.B2e. laminin and K.B1e.B2e. laminin (using the nomenclature of Engel), fibronectin, and heparan sulfate proteoglycan were isolated by immunoprecipitation from the cell culture medium, treated with HD or ethanol as control, and then analyzed by SDS-PAGE. On reduced SDS gels, these three BMCs not treated with HD showed the typical profile of dissociated subunits. However, HD treatment caused the appearance of higher molecular weight bands indicative of cross-linking of subunits within these BMCs. The HD scavengers sodium thiosulfate and cysteine prevented the cross-linking of BMC subunits by HD. Finally, tissue culture dishes coated with laminin or fibronectin were treated with HD or ethanol as a control, and human keratinocytes were plated on the BMC-coated surfaces. After 20 h of incubation, it was observed that cell adhesion was decreased significantly on the BMC-coated surfaces treated with HD. As expected, the preincubation of HD with cysteine diminished the HD inhibition of cell adhesion. Thus, HD alkylates adhesive macromolecules of the basement membrane zone and inhibits their cell adhesive activity. These findings support the hypothesis that the alkylation of basement membrane components by HD destabilizes the epidermal-dermal junction in the process of HD-induced vesication. The failure of the HD-alkylated BMCs to support the attachment of keratinocytes might also contribute to the slow reepithelialization of the wound site which is characteristic of HD-induced blistering.

Effects of sulfur mustard on the basal cell adhesion complex†

“…cell's keratin (K5/K14) filaments and the external laminin 5 anchoring sites of the basement membrane. We also know from recent studies that keratinocyte adhesion is decreased on laminin surfaces exposed to HD, 16 and that laminin 5 is a prime target of HD alkylation in the basement membrane of weanling pig skin. 18 It is of corresponding interest to blister formation that the detachment and terminal differentiation of basal cells have been associated with selective loss of ␣ 6 ␤ 4 -integrin and concomitant loss of its stabilizing attachments to the laminin 5 matrix.…”

“…13,14 Mutations affecting ␣ 6 ␤ 4 -integrin are known to be causative factors in junctional epidermolysis bullosa (JEB), another family of blistering skin diseases. Based on the collective information about attachment mechanisms of human epidermal keratinocytes (HEK), lesions affecting HEK in blistering skin diseases and alkylating effects of HD, [15][16][17][18] we have postulated that HD may induce blisters through disruptive alkylating effects on keratins K5 and K14, and other key molecules of the basal cell's adhesion complex. Our study indicates that this hypothesis may be correct and may provide a mechanistic approach to the development of more effective therapies for treatment of HD lesions.…”

Treatment of keratin intermediate filaments with sulfur mustard analogs

“…One hypothesis for vesicant action is the activation of an endogenous protease by SM [7][8][9][10] and an endogenous inhibitor of this protease has been identified [11,12]. Other investigators have analyzed the effects of SM on basal cell adhesion complex molecules [13][14][15][16][17], hypothesizing alkylation of basal cell adhesion complex molecules results in failure of the complex [16,18], leading to blister formation, similar to inherited skin blistering diseases [19].…”

A cutaneous full-thickness liquid sulfur mustard burn model in weanling swine: clinical pathology and urinary excretion of thiodiglycol†‡

“…basal cells as well as anchoring filaments. Separation appears to occur at the lamina lucida of the basement membrane 5,[7][8][9] and is characterized by fluid-filled blisters in man and microblisters in most animal models, including pigs. 10 High doses of HD can result in cytotoxic effects on the gastrointestinal and hematopoietic systems following systemic absorption.…”