William J. Smith

Biochemical manipulation of intracellular glutathione levels influences cytotoxicity to isolated human lymphocytes by sulfur mustard

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 μM HD but not to higher HD concentrations. Pretreatment of PBL for 48 hr with 10 mM N-acetyl cysteine (NAC), which elevates intracellular glutathione levels to 122% of control, appears to partially protect these in vitro cells from the cytotoxic effects of 10 μM HD but not to higher HD concentrations. Augmentation of intracellular levels of glutathione may provide partial protection against cytotoxicity of sulfur mustard.

The use of human epidermal keratinocytes in culture as a model for studying the biochemical mechanisms of sulfur mustard toxicity.

Human epidermal keratinocytes in culture were studied to evaluate their usefulness in demonstrating toxic events following exposure to sulfur mustard. Exposure of keratinocytes to sulfur mustard over a concentration range of 1–1000 μM HD, reduced NAD+ levels from 96% to 32% of control levels. When keratinocytes were exposed to a concentration of 300 μM HD, NAD+ levels began to fall at 1 hour and reached a plateau of 47% of control levels at 4 hours. Niacinamide, an inhibitor of the enzyme poly(ADP-ribose) polymerase, partially protected mustard-exposed cells against NAD+ depletion. It also protected cellular viability as assessed by vital staining 24 hours after exposure. This protection was not seen in long-term (72 hr) cultures. These studies suggest that human epidermal keratinocytes in culture can serve as a usefulin vitro model for research into the biochemical mechanisms of sulfur mustard-induced cutaneous injury.

Sulfur mustard-increased proteolysis following in vitro and in vivo exposures

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that, the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.

Cytometric Analysis of DNA Changes Induced by Sulfur Mustard

AbstractSulfur mustard is an alkylating agent that causes severe, potentially debilitating blisters following cutaneous exposure. Its mechanism of pathogenesis is unknown and no antidote exists. The biochemical basis of sulfur mustards vesicating activity has been hypothesized to be a cascade of events beginning with alkylation of DNA.Using human cells in culture, we have assessed the effects of sulfur mustard on cell cycle activity using flow cytometry with propidium iodide. Two distinct patterns emerged: a G1/S interface block at concentrations equivalent to vesicating doses (> 50 μM) and a G2 block at 10-fold lower concentrations. In addition, noticeable increases in amount of dye uptake were observed at 4 and 24 hr after sulfur mustard exposure. These increases are believed to be related to DNA repair activities and can be prevented by treatment of the cells with niacinamide, which inhibits DNA repair. Other drugs that provide alternative alkylating sites or inhibit cell cycle progression were shown ...

Calmodulin, poly(ADP–ribose)polymerase and p53 are targets for modulating the effects of sulfur mustard

We describe two pathways by which the vesicating agent sulfur mustard (HD) may cause basal cell death and detachment: induction of terminal differentiation and apoptosis. Following treatment of normal human epidermal keratinocytes (NHEK) with 10 or 100 μmM HD, the differentiation‐specific keratin pair K1/K10 was induced and the cornified envelope precursor protein, involucrin, was cross‐linked by epidermal transglutaminase. Fibronectin levels were reduced in a time‐ and dose‐dependent manner. The rapid increase in p53 and decrease in Bcl‐2 levels was consistent not only with epidermal differentiation but with apoptosis as well. Further examination of biochemical markers of apoptosis following treatment of either NHEK or human papillomavirus (HPV)‐immortalized keratinocytes revealed a burst of poly(ADP–ribose) synthesis, specific cleavage of poly(ADP–ribose)polymerase (PARP) in vivo and in vitro into characteristic 89 and 24 kDa fragments, processing of caspase‐3 into its active form and the formation of DNA ladders. The intracellular calcium chelator BAPTA suppressed the differentiation markers, whereas antisense oligonucleotides and chemical inhibitors specific for calmodulin blocked both markers of differentiation and apoptosis. Modulation of p53 levels utilizing retroviral constructs expressing the E6, E7 or E6 + E7 genes of HPV‐16 revealed that HD‐induced apoptosis was partially p53‐dependent. Finally, immortalized fibroblasts derived from PARP −/− ‘knockout mice’ were exquisitely sensitive to HD‐induced apoptosis. These cells became HD resistant when wild‐type PARP was stably expressed in these cells. These results indicate that HD exerts its effects via calmodulin, p53 and PARP‐sensitive pathways. Copyright

Pretreatment of Human Epidermal Keratinocytes with D,L-Sulforaphane Protects Against Sulfur Mustard Cytotoxicity

ABSTRACT Sulfur mustard (SM) is a powerful cytotoxic agent as well as a potent vesicant, mutagen, and carcinogen. This compound reacts with glutathione (GSH) and forms GSH-SM conjugates that appear to be excreted through the mercapturic acid pathway in mammals. The question of whether glutathione-S-transferases (GST) are involved in enzymatic formation of these conjugates remains unresolved. In previous studies, ethacrynic acid (EAA), a putative inhibitor of this transferase, and oltipraz, a known inducer,were ineffective in modulating this enzyme in cultured normal human epidermal keratinocytes (NHEK) so this hypothesis could not be tested. Higher levels of intracellular GSH appeared to be solely responsible for resistance of EAA-pretreated cells to SM. A better inducer of GST was needed to test whether this enzyme could be used to modify cytotoxicity following SM exposure. D,L-sulforaphane (DLS), a compound from broccoli extract known to be a potent inducer of this enzyme, was tested for GST induction in cultured NHEK. The enzyme levels increased optimally (40%) in these cells within 4 hours using 0.5 μg DLS/mL over a 48 hour incubation period. When the drug was removed by washing, and pretreated cells were challenged with 0–200 μM SM, there was a 10%–15% increase in survival at 24 hours compared with non-pretreated SM controls. This protective effect due to increased levels of GST was abolished at 300 μM sulfur mustard, where there was no difference in survival between pretreated and non-pretreated controls. Glutathione levels were also assessed and showed no increase at 4 hours in cultured NHEK with DLS pretreatment and appear not to be responsible for this protection against SM.

Effect of sulfur exposure on protease activity in human peripheral blood lymphocytes

Sulfur mustard is a waemical 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 ethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase 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.

Therapeutic Options to Treat Sulfur Mustard Poisoning - The Road Ahead

For the past 15 years the international research community has conducted a basic and applied research program aimed at identifying a medical countermeasure against chemical threat vesicant, or blistering, agents. The primary emphasis of this program has been the development of therapeutic protection against sulfur mustard and its cutaneous pathology-blister formation. In addition to the work on a medical countermeasures, significant research has been conducted on the development of topical skin protectants and medical strategies for wound healing. This review will focus on the pharmacological strategies investigated, novel therapeutic targets currently under investigation and therapeutic approaches being considered for transition to advanced development. Additionally, we will review the expansion of our understanding of the pathophysiological mechanisms of mustard injury that has come from this research. While great strides have been made through these investigations, the complexity of the mustard insult demands that further studies extend the inroads made and point the way toward better understanding of cellular and tissue disruptions caused by sulfur mustard.

Sulfur mustard exposure enhances Fc receptor expression on human epidermal keratinocytes in cell culture: implications for toxicity and medical countermeasures.

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 µmol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.

Sulfur Mustard Increases Elastase-Like Activity in Homogenates of Hairless Guinea Pig Skin

AbstractThe pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Enhanced proteolysis activity has been postulated as the cause of HD-induced dermal epidermal separation. Using a chromogenic assay, we previously reported that HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolysis of the peptide substrate Tosyl-gly-pro-arg-p-nitroanilide, Chromozym TH (TH). Elastaise activity has been used as a biomarker of inflammation in skin. In this study we assayed human leukocyte elastase (HLE) and soluble extracts of biopsies from control and HD-exposed hairless guinea pig skin with both the TH and the HLE (N-methoxysuccinyl-ala-ala-pro-val-p-nitroanilide) substrates. HD-increased proteolysis of both substrates indicates that multiple enzymes may contribute to HD-induced pathology. The increased activities of an inflammation-associated enzyme such as elastase as early as 3-6 hr after HD exposure of hairless guinea pig skin indicate that inflammat...