ABSTRACT
Sulfur mustard (HD) is a chemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister and co-workers1’2 hypothesized that HD alkylates DNA, predisposing to DNA strand breakage and leading to subsequent biochemical and metabolic alterations that culminate in enhancement of proteolytic activity. This increased proteolysis has been proposed as the penultimate event in the dermal-epidermal separation and vesication that follows cutaneous HD exposure. 1,2 In vitro cell and tissue culture models such as human peripheral blood lymphocyte (PBL) cytotoxicity3 and morphological changes in the human skin equivalent TESTSKIN4 have been used for the investigation of HD toxicity. Enhanced proteolysis has been reported following in vitro exposure to HD in skin cultures from rabbits, 5 in PBL , 6 in epidermal keratinocytes, and in the human skin equivalent TESTSKIN . 7 ,8 In vitro exposure of human PBL to HD demonstrated that the increase in proteolytic activity is both time and temperature dependent. 9 ,1 0 Assay of PBL HD-increased proteolysis with a panel of 10 protease substrates produced a characteristic pattern or “fingerprint” that can be compared to the cleavage patterns of known proteases such as plasminogen activator. 9 ,1 0 HD-increased proteolysis in cell cultures of PBL and keratinocytes should facilitate characterization of the proteolytic activity and may be useful as a biomarker of HD-induced vesication. 11
MATERIALS AND METHODS
Peptide Substrates and Reagents
The following Boehringer Mannheim Biochemicals Chromozym substrates (enzyme specificity) were used: t-PA (plasminogen activator), PL (plasmin), TRY (trypsin), TH (thrombin), PK (plasma kallikrein), X (Factor Xa), U (urokinase), GK (glandular kallikreins), PCa (Protein C), and Xlla (Factor XII). RPMI 1640 tissue culture medium (GIBCO, Grand Island, NY) was used in assays. Histopaque (d = 1.077), trypan blue, and tissue plasminogen activator were purchased from Sigma, St. Louis, MO. Sulfur mustard (HD, 2,2'-dichlorodiethyl sulfide) with a purity of >98% was obtained from the Edgewood Research, Development and Engineering Center, Aber deen Proving Ground, MD. The protease inhibitors antipain, APMSF, aprotinin, bestatin, chymostatin, leupeptin, pepstatin, E-64 (1 (Jig/ml), ethylenediamine tetraacetic acid (EDTA, 1 ¡ig/ ml), and phosphoramidon (4 jig/ml) were obtained from Boehringer Mannheim Biochemicals, Indianapolis, IN, and the concentration of protease inhibitor incubated with assay samples was 400 |xg/ml unless otherwise stated. Human keratinocyte cultures and KGM were provided by Clonetics, San Diego, CA.
