The generation of a human dermal equivalent to assess the potential contribution of human dermal fibroblasts to the sulphur mustard-induced vesication response

Abstract

<jats:p> 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. </jats:p><jats:p> 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. </jats:p><jats:p> 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. </jats:p><jats:p> 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. </jats:p>

Examination of changes in connective tissue macromolecular components of large white pig skin following application of lewisite vapour

“…Protease Table 4-Measurement of optical density (as determined by imaging densitrometry and subsequent analysis by 'Quantity One' software) of all collagen IV bands in Fig. 2A release is known to occur in vesicant-damaged skin (Lindsay and Upshall, 1995;Lindsay and Rice, 1996), potentially degrading vulnerable connective tissue components into smaller protein fragments that are extracted more efficiently, although it is possible that leakage of blood proteins into the Lewisite-exposed skin may have contributed to these raised protein levels. However, the amount of non-laminin antigen on the immuno-blot controls was relatively low and easily identified as such.…”

“…Enzymes such as elastase are known to be released by fibroblasts following exposure to sulphur mustard (Lindsay and Upshall, 1995) and human skin explants have been found to release trypsin after exposure to sulphur mustard (Lindsay and Rice, 1996). The apparently greater vulnerability of laminin to degradation may be due to its relatively 'open' asymmetric cross-shaped structure compared with the more complex structure of collagen IV, consisting of tightly packed triple-helical domains and more open non-helical domains (Tryggvason et al, 1987).…”

Xenobiotic Metabolism in Human Skin and 3D Human Skin Reconstructs: A Review

“…Furthermore glycosidase, elastase, catalase, superoxide dismutase, and peroxidase activity have been detected in a range of SE, EE and DE models [54,69,[76][77][78]. Proteomic analysis detected an increased amount of arginase protein after topical exposure of EE to sodium dodecyl sulphate [79].…”

“…Therefore, as with conventional, monolayer cultures, the test substance has to be supplemented into the culture medium. The advantage of DE above monolayer cultures is that the fibroblasts differentiate, synthesize extracellular matrix and exhibit contractile properties in a similar manner to their in vivo counterpart [54]. This model is used less than EE or SE in metabolic studies and product safety testing.…”

The use of doxycycline as a protectant against sulphur mustard in HaCaT cells

“…Elevated activities of trypsin and elastase-like activities have been found in HD exposed human skin explants . Increased elastase levels have also been reported in HD exposed cultures of a human dermal equivalent comprising normal human dermal fibroblasts in a type I collagen gel system (Lindsay and Upshall, 1995), and elevated metalloproteinase activities have been measured in explant cultures of HD exposed rabbit skin (Woessner et al, 1990).…”