THE GENERATION OF A HUMAN DERMAL EQUIVALENT TO ASSESS THE POTENTIAL CONTRIBUTION OF HUMAN DERMAL FIBROBLASTS TO THE SULFUR MUSTARD-INDUCED VESICATION RESPONSE

1 A human dermal equivalent (HDE) gel was constructed from rat tail te ndon collagen (type 1) and human dermal fibroblasts (HFs). Histologica l studies revealed that the HFs within the HDE gel matrix assumed the shape of differentiated dermal fibroblasts and were metabolically viab le as determined by the MTT assay. 2 The HDE system was developed to d etermine if viable, differentiated HFs have the potential to contribut e to tissue damage by releasing the proteolytic enzyme elastase follow ing exposure to sulphur mustard (HD). Elastase was measured, using the substrate suc-ala-ala-val-p-nitroanilide (SAAVNA), because of its ass ociation with various human pathological bullous skin diseases. An add itional elastase substrate (suc-ala-ala-ala-p-nitroanilide; SAAANA) wa s also used. A miniaturised assay was employed to measure lactate dehy drogenase (LDH), a cytosolic enzyme released following damage to the c ell membrane. 3 Elastase levels (measured with SAAVNA) increased to ov er 740% of those in control culture medium at 24 h after exposure of t he HDE to HD (2 mM) and may therefore be part of the mechanism associa ted with dermoepidermal separation and blistering in humans following exposure of skin to HD. LDH was released from the HDE after exposure t o HD in a time dependent fashion, suggesting a steady leakage of cytos olic constituents after the initial exposure. 4 The results suggest th at differentiated human dermal fibroblasts have the potential to contr ibute to the development of the vesication response by releasing prote ases such as elastase extracellularly after HD exposure. These types o f studies cannot be conducted in humans on ethical grounds because of the mutagenic properties of HD. The HDE model therefore has an importa nt advantage in studies on the mechanism of action of HD.