Cell Biology and Toxicology

, Volume 11, Issue 2, pp 89–101 | Cite as

Assessment of sulfur mustard interaction with basement membrane components

  • Z. Zhang
  • B. P. Peters
  • N. A. Monteiro-Riviere
Article

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.

Key words

adhesion alkylation basement membrane keratinocyte sulfur mustard vesication 

Abbreviations

BMC

basement membrane component

DEM

Dulbecco's modified Eagle's medium

ECM

extracellular matrix

EHS

Englebreth-Holm-Swarm sarcoma

HD

sulfur mustard

HSPG

heparan sulfate proteoglycan

KGM

keratinocyte growth medium

NHEK

normal human keratinocytes

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • Z. Zhang
    • 1
  • B. P. Peters
    • 1
  • N. A. Monteiro-Riviere
    • 1
  1. 1.Cutaneous Pharmacology and Toxicology CenterNorth Carolina State UniversityRaleighUSA

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