Skin homogenates of hairless guinea pigs exposed percutaneously to sulfur mustard (SM) were investigated by measuring four serine protease activities (elastase, tryptase, chymase, and Asp-ase) using sensitive chromogenic substrates. The homogenate samples from skin area directly exposed to S M showed enhanced elastase activities. The tryptase (trypsin-like enzyme) activity also increased slightly; however, the chymase (chymotrypsin-like enzyme) and Aspase (cleave after aspartic acid) activities did not show any increase. The enhanced elastase activities after SM exposure indicate that inflammation is present in the SM lesions. The inhibitory potency of MeO-Suc-Ala-Ala-Pro-Val-CH2Cl (an elastase inhibitor) and two amidine derivatives (inhibitors of trypsin-like enzyme) was tested against the activities present in samples from both exposed and control tissues. The elastase inhibitor decreased the hydrolysis of elastase substrate in the samples from SM-exposed skin to a greater extent than the samples from control tissues. The two trypsin inhibitors decreased the activity in samples from exposed and control tissues equally well. These substrate and inhibitor studies facilitate the characterization of various proteases affected by SM and may be useful for elucidating the mechanism of SM-induced vesication.
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2.
Cook, R. R., McRae, B. J., and Powers, J. C.1984. Kinetics of hydrolysis of peptide thioester derivatives of arginine by human and bovine thrombins. Arch. Biochem. Biophys.234:82–88.
3.
Cowan, F. M., Broomfield, C. A., and Smith, W. J.1992. Inhibition of sulfur mustard-increased protease activity by niacinamide, N-acetyl-L-cysteine or dexamethasone. Cell Biol. Toxicol.8:129–138.
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Farmer, D. A., and Hageman, J. H.1975. Use of N-benzoyl-L-tyrosine thiobenzyl ester as a protease substrate. Hydrolysis by α-chymotrypsin and subtilisin BPN'. J. Biol. Chem.250:7366–7371.
9.
Harper, J. W., Ramirez, G., and Powers, J. C.1981. Reaction of peptidyl thiobenzyl esters with mammalian chymotrypsin like enzymes: A sensitive assay method. Anal. Biochem.118:382–387.
10.
Harper, J. W., Cook, R. R., Roberts, C. J., McLaughlin, B. J., and Powers, J. C.1984. Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin Aα, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates. Biochemistry23:2995–3002.
11.
Kam, C.-M., Hernandez, M. A., Patil, G. S., Ueda, T., Simmons, W. H., Braganza, V. J., and Powers, J. C.1995. Mammalian tissue trypsin-like enzymes: Substrate specificity and inhibitory potency of substituted isocoumarin mechanism-based inhibitors, benzamidine derivatives, and arginine fluoroalkylke-tone transition-state inhibitors. Arch. Biochem. Biophys.316:808–814.
12.
Meier, H. L., Gross, C. L., Graham, L. M., Lusco, C. T., and Johnson, J. B.1987. The prevention of 2,2′-dichlorodiethyl sulfide (sulfur mustard, HD) cytotoxicity in human lymphocytes by inhibitors of poly(ADP-ribose) polymerase. Proc. Sixth Medical Chemical Defense Biosci. Rev. 313–316.
13.
Mershon, M. M., Wade, J. V., Mitcheltree, L. W., Petrali, J. P., and Braue, E. H., Jr.1990. Hairless guinea pig bioassay for vesicant vapor exposures. Fundam. Appl. Toxicol.15:622–630.
14.
Miskin, R., and Reich, E.1980. Plasminogen activator: Induction of synthesis by DNA damage. Cell190:217.
15.
Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M.1979. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the α-protease inhibitor reactive site. J. Biol. Chem.254:4027–4032.
16.
Odake, S., Kam, C. M., Narasimhan, L., Poe, M., Blake, J. T., Krahenbuhl, O., Tschopp, J., and Powers, J. C.1991. Human and murine cytotoxic T lymphocyte serine proteases: Subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry30:2217–2227.
17.
Oleksyszyn, J., Boduszek, B., Kam, C. M., and Powers, J. C.1994. Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases. J. Med. Chem.37:226–231.
18.
Papirmeister, B.1994. Does apoptosis (programmed cell death) play a role in sulfur mustard injury?Med. Chem. Defense Bull. Res. Dev.7:1–12.
19.
Papirmeister, B., Gross, C. L., Meier, H. L., Petrali, J. P., and Johnson, J. B.1985. Molecular basis for mustard-induced vesication. Fundam. Appl. Toxicol.5:S134–S149.
20.
Papirmeister, B., Feister, A. J., Robinson, S. I., and Ford, R. D.1991. Medical defense against mustard gas: Toxic mechanisms and pharmacological implications, pp. 1–359. Boston: CRC Press.
21.
Powers, J. C., Gupton, B. F., Harley, A. D., Nishino, N., and Whitley, R. J.1977. Specificity for porcine pancreatic elastase, human leukocyte elastase, and cathepsin G. Inhibition with peptide chloromethyl ketones. Biochem. Biophys. Acta485:158–166.
22.
Powers, J. C., Tanaka, T., Harper, J. W., Minematsu, Y., Barker, L., Lincoln, D., Crumley, K. W., Fraki, J. E., Schechter, N. M., Lazarus, G. G., Nakajima, K., Nakashino, K., Neurath, H., and Woodbury, R. G.1985. Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with 4-nitroanilide substrates, and peptide chloromethyl ketone and sulfonyl fluoride inhibitors. Biochemistry24:2048–2058.
23.
Schecter, I., and Berger, A.1967. On the size of the active site in protease. 1. Papain. Biochem. Biophys. Res. Commun.27:157–162.
24.
Tanaka, T., McRae, B. J., Cho, K., Cook, R., Fraki, J. E., Johnson, D. A., and Powers, J. C.1983. Mammalian tissue trypsin-like enzymes. Comparative reactivities of human skin tryptase, human lung tryptase and bovine trypsin with peptide 4-nitroanilide and thioester substrates. J. Biol. Chem.258:13552–13557.
25.
Yoshida, N., Everitt, M. T., Neurath, H., Woodbury, R. G., and Powers, J. C.1980. Substrate specificity of two chymotrypsin-like proteases from rat mast cells. Studies with peptide 4-nitroanilides and comparison with cathepsin G. Biochemistry19:5799–5804.
26.
Yourick, J. J., Clark, C. R., and Mitcheltree, L. W.1991. Niacinamide pretreatment reduces microvesicle formation in hairless guinea pigs cutaneously exposed to sulfur mustard. Fundam. Appl. Toxicol.17:533–542.
