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


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 



basement membrane component


Dulbecco's modified Eagle's medium


extracellular matrix


Englebreth-Holm-Swarm sarcoma


sulfur mustard


heparan sulfate proteoglycan


keratinocyte growth medium


normal human keratinocytes


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Burgeson RE. In: Rohrbach DH, Timpl R, eds. Molecular and cellular aspects of the basement membrane. San Diego: Academic Press; 1993:49–66.Google Scholar
  2. Carter WG, Wayner EA, Bouchard TS, Kaur P. The role of integrins α2β1 and α3β1 in cell-cell and cell-substrate adhesion of human epidermal cells. J Cell Biol. 1990;110:1387–407.Google Scholar
  3. Engel J, Hunter I, Schulthess T, Beck K, Dixon T, Parry D. Assembly of laminin isoforms by triple-and double-stranded coiled-coil structure. Biochem Soc Trans 1991;19:839–43.Google Scholar
  4. Fine J-D. 19-DEJ-1, a monoclonal antibody to the hemidesmo-some-anchoring filament complex, is the only reliable immunohistochemical probefor all major forms of junctional epidermolysis bullosa. Arch Dermatol. 1990;126:1187–90.Google Scholar
  5. Fine J-D. Structure and antigenicity of the skin basement membrane zone. J Cutan Pathol. 1991;18:401–9.Google Scholar
  6. Fine J-D. Redmar DA, Goodman AL. Sequence of reconstitution of seven basement membrane components following split-thickness wound induction in primate skin. Arch Dermatol. 1987;123:1174–8.Google Scholar
  7. Fine J-D, Johnson LB, Cronce D et al. Intracytoplasmic retention of type VII collagen and dominant dystrophic epidermolysis bullosa: reversal of defect following cessation of or marked improvement in disease activity. J Invest Dermatol. 1993:101:232–6.Google Scholar
  8. Furie MB, Frey AB, Rifkin DB. Location of a gelatin-binding region of human plasma fibronectin. J Biol Chem. 1980;255:4391–4.Google Scholar
  9. Fyrand O. Studies on fibronectin in the skin. Br J Dermatol. 1979;101:263–70.Google Scholar
  10. Goodlad GAJ. Cross-linking of collagen by S-and N-mustard. Porton, Wilts, UK: Defence Experimental Establishment, 1956; CDE Technical Paper no. 573. DITC, AD/119 934.Google Scholar
  11. Goodman SL, Gundula R, Von der Mark K. The E8 subfragment of laminin promotes locomotion of myoblasts over extracellular matrix. J Cell Biol. 1989;109:709–809.Google Scholar
  12. Hartl L, Oberbaumer I, Deutzmann R. The N terminus of laminin A chain is homologous to the B chains. Eur J Biochem. 1988;173:629–35.Google Scholar
  13. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992;69:11–25Google Scholar
  14. King JR, Monteiro-Riviere NA. Cutaneous toxicity of 2-chloroethyl methyl sulfide in isolated perfused porcine skin. Toxicol Appl Pharmacol. 1990;104:167–79.Google Scholar
  15. King JR, Peters BP, Monteiro-Riviere NA. Laminin in the cutaneous basement membrane as a potential target in lewisite vesication. Toxicol Appl Pharmacol. 1994;126:164–73.Google Scholar
  16. Kleinman HK, Cannon FB, Laurie GW et al. Biological activities of laminin. J Cell Biochem. 1985:27:317–25.Google Scholar
  17. Laemmli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. 1970;277:680–5.Google Scholar
  18. Marinkovich MP, Lunstrum GP, Keene DR, Burgeson RE. The dermal-epidermal junction of human skin contains a novel laminin variant. J Cell Biol. 1992;119:695–702.Google Scholar
  19. Monteiro-Riviere NA, Imman AO. Histochemical localization of three basement membrane epitopes with sulfur mustard induced toxicity in porcine skin. Toxicologist. 1993;13:58.Google Scholar
  20. Monteiro-Riviere NA, King JR, Riviere JE. Mustard induced vesication in isolated perfused skin: biochemical, physiological, and morphological studies. In: Proceedings of the Medical Defense Bioscience Review. Maryland: US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground; 1991:159–62.Google Scholar
  21. Monteiro-Riviere NA, Inman AO, Spoo JW, Rogers RA, Riviere JE. Studies on the pathogenesis of bis (2-chloroethyl) sulfide (HD) induced vesication in porcine skin. In: Proceedings of the Medical Defense Bioscience Review. Maryland: US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground; 1993:31–40.Google Scholar
  22. Monteiro-Riviere NA, Zhang JZ, Inman AO, Brooks JD, Riviere JE. Mechanisms of cutaneous vesication. DAMD 17-92-C-2071, NTIS, ADA283085;1994:42–55.Google Scholar
  23. Papirmeister B. Excitement in vesicant research — yesterday, today and tomorrow. In: Proceedings of the Medical Defense Bioscience Review. Maryland: US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground; 1993:1–14.Google Scholar
  24. Papirmeister B, Feister AJ, Robinson SI, Ford RD. Medical defense against mustard gas: toxic mechanisms and pharmacological implications. Boca Raton: CRC Press: 1991.Google Scholar
  25. Paulsson M. Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Mol Biol. 1992;27:93–127.Google Scholar
  26. Peters BP, Hartle RJ, Krzesicki RF et al. The biosynthesis, processing, and secretion of laminin by human choriaocarcinoma cells. J Biol Chem. 1985;27:14732–42.Google Scholar
  27. Riviere JE, Brooks JD, Williams PL, Monteiro-Riviere NA. Toxicokinetics of topical sulfur mustard penetration, disposition, and vascular toxicity in isolated perfused porcine skin. Toxicol Appl Pharmacol. (in press).Google Scholar
  28. Sakashiti S, Engvall E, Ruoslahti E. Basement membrane glycoprotein laminin binds to heparin. FEBS Lett. 1980;116:243.Google Scholar
  29. Sasaki M, Yamada Y. The laminin B2 chain has a multidomain structure homologous to the B1 chain. J Biol Chem. 1987;262:17111–7.Google Scholar
  30. Sasaki M, Kato S, Kohno K, Martin GR, Yamada Y. Sequence of the cDNA encoding the laminin B1 chain reveals a multidomain protein containing cysteine-rich repeats. Proc Natl Acad Sci USA. 1987;84:935–9.Google Scholar
  31. Sasaki M, Kleinman HK, Huber H, Deutzmann R, Yamada Y. Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains. J Biol Chem. 1988;263:16536–44.Google Scholar
  32. Shimizu Y, Van Seventer GA, Horgan KJ, Shaw S. Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin. J Immunol. 1990;145:59–67.Google Scholar
  33. Timpl R. Structure and biological activity of basement membrane proteins. Eur J Biochem. 1989;180:487–502.Google Scholar
  34. Vojvodic V, Moilosavljevic Z, Boskovic B, Bojanic N. The protective effect of different drugs in rats poisoned by sulfur andnnitrogen mustard. Fundam Appl Toxicol. 1985;5:S160–8.Google Scholar
  35. Zhang Z, Riviere JE, Monteiro-Riviere NA. Evaluation of protective effects of sodium thiosulfate, cysteine, niacinamide, and indomethacin on sulfur mustard-treated isolated perfused porcine skin. Chem Biol Interact. 1995; (in press).Google Scholar

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

Personalised recommendations