The generation of a human dermal equivalent to assess the potential contribution of human dermal fibroblasts to the sulphur mustard-induced vesication response.
1. A human dermal equivalent (HDE) gel was constructed from rat tail tendon collagen (type 1) and human dermal fibroblasts (HFs). Histological studies revealed that the HFs within the HDE gel matrix assumed the shape of differentiated dermal fibroblasts and were metabolically viable as determined by the MTT assay. 2. The HDE system was developed to determine if viable, differentiated HFs have the potential to contribute to tissue damage by releasing the proteolytic enzyme elastase following exposure to sulphur mustard (HD). Elastase was measured, using the substrate suc-ala-ala-val-p-nitroanilide (SAAVNA), because of its association with various human pathological bullous skin diseases. An additional elastase substrate (suc-ala-ala-ala-p-nitroanilide; SAAANA) was also used. A miniaturised assay was employed to measure lactate dehydrogenase (LDH), a cytosolic enzyme released following damage to the cell membrane. 3. Elastase levels (measured with SAAVNA) increased to over 740% of those in control culture medium at 24 h after exposure of the HDE to HD (2 mM) and may therefore be part of the mechanism associated with dermo-epidermal separation and blistering in humans following exposure of skin to HD. LDH was released from the HDE after exposure to HD in a time dependent fashion, suggesting a steady leakage of cytosolic constituents after the initial exposure. 4. The results suggest that differentiated human dermal fibroblasts have the potential to contribute to the development of the vesication response by releasing proteases such as elastase extracellularly after HD exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Lindsay
C D
CD
Chemical and Biological Defence Establishment, Porton Down, Salisbury, Wiltshire, UK.
Upshall
D G
DG
D000818:Animals; D001768:Blister / Q000139:chemically induced* / Q000201:enzymology / Q000473:pathology; D002470:Cell Survival; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D006801:Humans; D008954:Models, Biological; D009151:Mustard Gas; D051381:Rats; D012867:Skin / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology
D003470:Culture Media; D003094:Collagen; D009151:Mustard Gas