Abstract
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 tis sue 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 there fore be part of the mechanism associated with dermo- epidermal separation and blistering in humans follow ing 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 devel opment of the vesication response by releasing proteases such as elastase extracellularly after HD exposure. These types of studies cannot be conducted in humans on ethical grounds because of the mutagenic properties of HD. The HDE model therefore has an important advantage in studies on the mechanism of action of HD.
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