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Title: Pretreatment of isolated human peripheral blood lymphocytes with l-oxothiazolidine 4-carboxylate reduces sulfur mustard cytotoxicity

Abstract

Despite 70 years of research, there appears to be no satisfactory prophylaxis or treatment for the vesicant chemical warfare agent sulfur mustard (HD). Attempts to modify cytotoxicity of HD are now focusing on the use of intracellular 'scavengers' to interact with sulfur mustard before it can react with critical targets within the cell. Glutathione (GSH) is known to react readily with HD and is involved in the major metabolic pathway to HD detoxification. Glutathione level within the cell was raised 40-60% over control values by pretreatment of quiescent human peripheral blood lymphocytes (PBL) with 10 mM L-oxothiazolidine-4-carboxylate (OTC), a masked cysteine precursor. This increase in glutathione level was not toxic to the cells as judged by trypan blue dye exclusion and reached a maximum level in 48 hrs. PBL pretreated with 10 mM OTC for 48 hrs were harvested, washed, and exposed to 10, 50, or 100 uM HD. After an additional 48 hrs of incubation at 37 deg C, cytotoxicity was measured by propidium iodide dye uptake using flow cytometry. Pretreatment with OTC led to a 20% decrease in cytotoxicity with 10 uM HD, an 11% decrease in cytotoxicity with 50 uM HD, and an 8% decrease in cytotoxicitymore » with 100 uM HD. Cytotoxicity of HD was not influenced by addition of 10 mM OTC 2 hrs after HD exposure. These results suggest that biochemical manipulation of intracellular GSH level may provide an important pretreatment regimen to reduce the cytotoxicity of HD.« less

Authors:
;
Publication Date:
Research Org.:
Army Medical Research Inst. of Chemical Defense, Aberdeen Proving Ground, MD (United States)
OSTI Identifier:
7239939
Alternate Identifier(s):
OSTI ID: 7239939
Report Number(s):
AD-P-008767/6/XAB
Resource Type:
Technical Report
Resource Relation:
Other Information: This article is from 'Proceedings of the Medical Defense Bioscience Review (1993) Held in Baltimore, Maryland on 10-13 May 1993. Volume 1', AD-A275 667, p141-147
Country of Publication:
United States
Language:
English
Subject:
45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEFENSE; 63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.; CHEMICAL WARFARE AGENTS; TOXICITY; WEAPONS 450600* -- Military Technology, Weaponry, & National Defense-- Chemical & Biological-- (1990); 560300 -- Chemicals Metabolism & Toxicology

Citation Formats

Gross, C.L., and Smith, W.J. Pretreatment of isolated human peripheral blood lymphocytes with l-oxothiazolidine 4-carboxylate reduces sulfur mustard cytotoxicity. United States: N. p., 1993. Web.
Gross, C.L., & Smith, W.J. Pretreatment of isolated human peripheral blood lymphocytes with l-oxothiazolidine 4-carboxylate reduces sulfur mustard cytotoxicity. United States.
Gross, C.L., and Smith, W.J. Thu . "Pretreatment of isolated human peripheral blood lymphocytes with l-oxothiazolidine 4-carboxylate reduces sulfur mustard cytotoxicity". United States. doi:.
@article{osti_7239939,
title = {Pretreatment of isolated human peripheral blood lymphocytes with l-oxothiazolidine 4-carboxylate reduces sulfur mustard cytotoxicity},
author = {Gross, C.L. and Smith, W.J.},
abstractNote = {Despite 70 years of research, there appears to be no satisfactory prophylaxis or treatment for the vesicant chemical warfare agent sulfur mustard (HD). Attempts to modify cytotoxicity of HD are now focusing on the use of intracellular 'scavengers' to interact with sulfur mustard before it can react with critical targets within the cell. Glutathione (GSH) is known to react readily with HD and is involved in the major metabolic pathway to HD detoxification. Glutathione level within the cell was raised 40-60% over control values by pretreatment of quiescent human peripheral blood lymphocytes (PBL) with 10 mM L-oxothiazolidine-4-carboxylate (OTC), a masked cysteine precursor. This increase in glutathione level was not toxic to the cells as judged by trypan blue dye exclusion and reached a maximum level in 48 hrs. PBL pretreated with 10 mM OTC for 48 hrs were harvested, washed, and exposed to 10, 50, or 100 uM HD. After an additional 48 hrs of incubation at 37 deg C, cytotoxicity was measured by propidium iodide dye uptake using flow cytometry. Pretreatment with OTC led to a 20% decrease in cytotoxicity with 10 uM HD, an 11% decrease in cytotoxicity with 50 uM HD, and an 8% decrease in cytotoxicity with 100 uM HD. Cytotoxicity of HD was not influenced by addition of 10 mM OTC 2 hrs after HD exposure. These results suggest that biochemical manipulation of intracellular GSH level may provide an important pretreatment regimen to reduce the cytotoxicity of HD.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Thu May 13 00:00:00 EDT 1993},
month = {Thu May 13 00:00:00 EDT 1993}
}

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  • Glutathione (GSH) is the major nonprotein thiol that can protect cells from damage due to electrophilic alkylating agents by forming conjugates with the agent. Sulfur mustard (HD) is an electrophilic alkylating agent that has potent mutagenic, carcinogenic, cytotoxic, and vesicant properties. Compounds that elevate or reduce intracellular levels of GSH may produce changes in cytotoxicity induced by sulfur mustard. Pretreatment of human peripheral blood lymphocytes (PBL) for 72 hr with 1 mM buthionine sulfoximine (BSO), which reduces intracellular GSH content to approximately 26% of control, appears to sensitize these in vitro cells to the cytotoxic effects of 10 AM HDmore » but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NA C), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 LAIHD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.« less
  • Sulfur mustard is a chemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increasemore » in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.« less
  • It has been proposed that sulfur mustard (HD) may indirectly activate poly(ADP-ribose) polymerase (PADPRP) by alkylating cellular DNA (Papirmeister et al., 1985). Activation of PADPRP results in the depletion of cellular NAD+ which initiates a series of biochemical processes that have been proposed to culminate in blister formation. Preventing PADPRP activation and NAD+ depletion should inhibit blister formation. Niacinamide is both an inhibitor of PADPRP and a precursor for NAD+ synthesis. The present study was undertaken to determine whether niacinamide can protect against HD-induced microvesication in cutaneously exposed hairless guinea pigs. Each site was exposed to HD for 8 minmore » by means of a vapor cup. Niacinamide (750 mg/kg, ip) given as a 30-min pretreatment inhibited microvesicle formation by 50% after HD application. However, niacinamide given 2 hr after HD application did not reduce microvesicle formation. There was no benefit when niacinamide was given as both a pretreatment and treatment when compared to niacinamide given only as a pretreatment. The reduction in microvesication 24 hr after HD did not correlate with skin NAD+ content. Niacinamide did not reduce the degree of erythema or edema. Ballooning degeneration of basal epidermal cells was present in some niacinamide pretreated HD exposure sites.« less
  • Lymphocyte were utilized as a model for investigating HD effects on resting cells. Lymphocytes exposed to HD demonstrated a concentration dependent decrease in ATP, NAD, and viability. The decrease began in 15 minutes for ATP, 2 hours for NAD, and 6 hours for viability. All three of these HD initiated biochemical changes can be blocked by poly (ADP-ribose) polymerase inhibitors (PADPRPI). To completely inhibit HD initiated ATP, NAD, and viability decreases the PADPRPI had to be present at time 0, 1, and 4 hours respectfully. The amount of protection conferred by the PADPRPI in the viability assay decreased in amore » linear manner with the delay of the addition and the concentration of the inhibitor from 6-12 hours post HD exposure. There was a good correlation between IC50 to inhibit poly (ADP-ribose) polymerase and EC50 prevention of HD initiated cell death (r=O.94). Thus, three in vitro assays which can measure biochemical and pathologic changes induced by HD in G sub 0 lymphocytes have been developed. These assays have been employed to study the ability of candidate antidotes to prevent HD initiated changes. Benzamidine analogs, including the F.D.A. approved vitamin niacinamide, have been shown to be effective at inhibiting all of these changes.« less
  • 2,2`-Dichlorodiethyl sulfide (sulfur mustard), HD, 1,1`thiobis(2-chloroethane) is a potent vesicant which can cause severe lesions to skin, lung, and eyes. There is no convenient in vitro or in vivo method(s) to objectively measure the damage induced by HD; therefore, a simple in vitro method was developed using human peripheral lymphocytes to study HD-induced cytotoxicity. The cytotoxicity of HD was measured using dye exclusion as an indicator of human lymphocyte viability. Exposure to HD resulted in both a time- and a concentration-dependent cytotoxic effect on human lymphocytes. Using this in vitro assay, the effectiveness of various therapeutics (niacin, niacinamide, and 3-aminobenzamide)more » in preventing HD-induced cytotoxicity was studied. Niacinamide and 3-aminobenzamide prevented the cytotoxic effects of HD for up to 2 days.« less