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Dive into the research topics where Clark L. Gross is active.

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Featured researches published by Clark L. Gross.


Cell Biology and Toxicology | 1993

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

Clark L. Gross; Joy K. Innace; Renee C. Hovatter; Henry L. Meier; William J. Smith

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.


Cell Biology and Toxicology | 1990

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

William J. Smith; Clark L. Gross; Philip Chan; Henry L. Meier

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.


Journal of Toxicology-cutaneous and Ocular Toxicology | 1993

Cytometric Analysis of DNA Changes Induced by Sulfur Mustard

William J. Smith; K. M. Sanders; S. E. Ruddle; Clark L. Gross

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 ...


Cutaneous and Ocular Toxicology | 2006

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

Clark L. Gross; Eric W. Nealley; Mary T. Nipwoda; William J. Smith

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.


Journal of Toxicology-cutaneous and Ocular Toxicology | 1992

Sulfur Mustard-Induced Biochemical Alterations in Proliferating Human Cells in Culture

William J. Smith; Kenneth M. Sanders; James E. Caulfield; Clark L. Gross

AbstractHuman epidermal keratinocytes (HEK), human epithelial tumor cells (HeLa), and actively proliferating human peripheral blood lymphocytes (PBL) were used as in vitro model systems to investigate the biochemical sequelae of sulfur mustard-induced injury. Sulfur mustard exposure of all three proliferating cell types resulted in inhibition of cell cycle progression in the early S phase. Cytotoxicity due to sulfur mustard was higher in proliferating, mitogenstimulated PBL than in resting PBL. Niacinamide, an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase, partially prevented the loss of cellular NAD + in sulfur mustard-treated cells. Niacinamide also protected against the cytotoxic effects of sulfur mustard for 24 hr. However, this protection waned when cell growth was allowed to proceed up to 72 hr.


Toxicology Mechanisms and Methods | 2003

A Rapid Colorimetric Assay for Sulfur Mustard Cytotoxicity Using Isolated Human Peripheral Blood Lymphocytes and Keratinocytes

Clark L. Gross; Eric W. Nealley; William J. Smith; Charlene M. Corun; Mary T. Nipwoda

Sulfur mustard (SM) is a potent vesicating agent that has pronounced cytotoxic effects as well as mutagenic, carcinogenic, and radiomimetic properties. Isolated human peripheral blood lymphocytes (PBLs) and human epidermal keratinocytes (HEKs) have been used as in vitro models for determining SM-induced cytotoxicity. A recently developed colorimetric assay (the CellTiter 96 AQ ueous Non-radioactive Cell Proliferation Assay) was assessed using both of the in vitro models described above. Using 24- or 96-well microplates, reproducible (± 10%) SM dose/response curves for both types of human cells were obtained using a spectrophotometric microplate reader set at 490 nm. After a 4-h incubation time, as many as 96 sample wells could be measured within 45 s using this commonly available equipment. Multiple plates of samples can be run immediately. This technique may facilitate cytotoxicity investigations of new candidate compounds for both prophylaxis of and therapy for SM intoxication.


Archive | 1999

The Use of In Vitro Systems to Define Therapeutic Approaches to Cutaneous Injury by Sulfur Mustard

William J. Smith; Margaret E. Martens; Clark L. Gross; Offie E. Clark; Fred M. Cowan; Jeffrey J. Yourick

Sulfur mustard (HD) is an alkylating agent that has been shown to have mutagenic, cytotoxic, and vesicating properties. Its use in combat situations has resulted in lethal, incapacitating, and disfiguring injuries. The principal incapacitating injuries come from the vesicating capacity of HD, i.e., production of skin blisters (1). Despite decades of medical research, the mechanism by which HD induces vesication is not known, and no effective antidotes are currently available. The studies in this chapter utilize flow cytometric, biochemical, and histopathological analyses of human and animal cells and tissues for the toxicologic assessment of HD-induced damage. These techniques allowed a unique series of experiments to be conducted that defined levels of sensitivity in human cells to both the cytotoxic and genotoxic effects of HD. These studies, in turn, have provided data that are relevant to the development of therapeutic intervention in the human pathology produced by HD.


Toxicology Mechanisms and Methods | 2004

Pretreatment of human epidermal keratinocytes in vitro with ethacrynic Acid reduces sulfur mustard cytotoxicity.

Clark L. Gross; Mary T. Nipwoda; Eric W. Nealley; William J. Smith

Sulfur mustard (SM) is a potent alkylating agent, profoundly cytotoxic, and a powerful vesicant. SM reacts quite extensively with glutathione (GSH) and forms GSH conjugates, which are presumably excreted through the mercapturic acid pathway in mammals. It is unknown whether any enzymes, such as the glutathione-S-transferases (GST), are involved in this detoxification of SM by the formation of conjugates. A prototypic inhibitor (ethacrynic acid, EAA) and a prototypic inducer (Oltipraz, OLT) of GSH-S-transferase, have been used as pretreatment compounds in human epidermal keratinocytes (HEK) to investigate the effect of enzyme levels on cytotoxicity following SM challenge from 50 μM to 300 μM. Pretreatment of HEK for 24 h with EAA doubled survival against 200 μM SM (36% viability in non-pretreated cells vs. 81% in EAA-pretreated cells) and quadrupled survival (17% viability in non-pretreated controls vs. 71% in EAA-pretreated cells), while OLT pretreatment had no effect on cytotoxicity at either SM dose. The role of GST in SM cytotoxicity could not be tested because of the lack of an effect on modulation of GST activities by these 2 drugs. Cellular levels of GSH were increased 250–300% over control values using EAA pretreatment, while OLT pretreatment did not lead to any increase in GSH. Pretreatment of HEK with buthionine sulfoximine (BSO), a known depleter of glutathione levels, reduced glutathione levels and increased cytotoxicity. This large increase in GSH appears to be solely responsible for the enhanced survivability of EAA-pretreated HEK.


Military Medicine | 2002

Sulfur mustard medical countermeasures in a nuclear environment.

William J. Smith; Clark L. Gross


Archive | 1993

Pretreatment of Isolated Human Peripheral Blood Lymphocytes with L- Oxothiazolidine 4-Carboxylate Reduces Sulfur Mustard Cytotoxicity

Clark L. Gross; William J. Smith

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William J. Smith

United States Army Medical Research Institute of Chemical Defense

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Eric W. Nealley

United States Army Medical Research Institute of Chemical Defense

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Mary T. Nipwoda

United States Army Medical Research Institute of Chemical Defense

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Henry L. Meier

Johns Hopkins University

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Jeffrey J. Yourick

Food and Drug Administration

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