| A convenient fluorometric method to study sulfur mustard-induced apoptosis in human epidermal keratinocytes monolayer microplate culture. | |
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MedLine Citation:
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PMID: 15720039 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Sulfur mustard [SM; bis-(2-chloroethyl) sulfide], which causes skin blistering or vesication [(1991). Histo- and cytopathology of acute epithelial lesions. In: Papirmeister, B., Feister, A. J., Robinson, S. I., Ford, R. D., eds. Medical Defense Against Mustard Gas: Toxic Mechanisms and Pharmacological Implications. Boca Raton: CRC Press, pp. 43-78.], is a chemical warfare agent as well as a potential terrorism agent. SM-induced skin blistering is believed to be due to epidermal-dermal detachment as a result of epidermal basal cell death via apoptosis and/or necrosis. Regarding the role of apoptosis in SM pathology in animal skin, the results obtained in several laboratories, including ours, suggest the following: 1) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic-necrotic continuum and 2) inhibiting apoptosis decreases SM-induced microvesication in vivo. To study the mechanisms of SM-induced apoptosis and its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multiwell plates (24-, 96-, or 384-well) and high-throughput applications. This assay allows replication and multiple types of experimental manipulation in sister cultures so that the apoptotic mechanisms and the effects of test compounds can be compared statistically. SM affects diverse cellular mechanisms, including endoplasmic reticulum (ER) Ca2+ homeostasis, mitochondrial functions, energy metabolism, and death receptors, each of which can independently trigger apoptosis. However, the biochemical pathway in any of these apoptotic mechanisms is characterized by a pathway-specific sequence of caspases, among which caspase-3 is a key member. Therefore, we exposed 80-90% confluent HEK cultures to SM and monitored apoptosis by measuring the fluorescence generated due to hydrolysis of a fluorogenic caspase-3 substrate (acetyl- or benzyl oxycarbonyl-Asp-Glu-Val-Asp-fluorochrome, also designated as AC-or Z-DEVD- fluorochrome) added to the assay medium. Fluorescence was measured using a plate reader. We used two types of substrates, one (Sigma-Aldrich, CASP-3-F) required cell disruption and the other (Beckman-Coulter CellProbe HT Caspase-3/7 Whole Cell Assay Kit) was cell permeable. The latter substrate was useful in experiments such as determining the time-course of apoptosis immediately following SM exposure without disruption (e.g., due to cell processing). In SM-exposed HEK, fluorescence generated from the fluorogenic caspase-3 substrate hydrolysis increased in a time (0-24 h) and concentration (0.05, 0.1, 0.15, 0.2, 0.3, 0.5 mM) dependent manner. SM caused maximum fluorescence at about 0.5 mM. However, at 2 mM SM, fluorescence decreased compared with 0.5 mM, which remains to be explained. Following 0.3 mM SM exposure, which is considered to be the in vitro equivalent of a vesicating dose in vivo (Smith, W. J., Sanders, K. M., Ruddle, S. E., Gross, C. L. (1993). Cytometric analysis of DNA changes induced by sulfur mustard. J. Toxicol.-Cut. Ocular Toxicol. 12(4):337-347.), a small fluorescence increase was observed at 6 to 8 h, which was markedly higher at 12 h. At 24 h, all SM concentrations increased fluorescence. Fluorescence increase due to SM was prevented 100% by a caspase-3-specific peptide inhibitor AC-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde, 0.1 mM), but less effectively by a general caspase inhibitor Z-VAD-FMK (benzyl oxycarbonyl-Val-Ala-Asp-fluoromethylketone, 0.01 mM), indicating that the fluorescence increase was due to caspase-3-mediated apoptosis. These results suggest potential applications of this method to study apoptosis mechanisms involving caspase-3 substrates and possibly those involving other caspase substrates. |
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Authors:
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Radharaman Ray; Stephanie Hauck; Rachel Kramer; Betty Benton |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Drug and chemical toxicology Volume: 28 ISSN: 0148-0545 ISO Abbreviation: Drug Chem Toxicol Publication Date: 2005 |
Date Detail:
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Created Date: 2005-02-21 Completed Date: 2005-05-19 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 7801723 Medline TA: Drug Chem Toxicol Country: United States |
Other Details:
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Languages: eng Pagination: 105-16 Citation Subset: IM |
Affiliation:
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Biochemical Pharmacology Branch, US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground, Maryland 21010-5400, USA. radharaman.ray@apg.amedd.army.mil |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Apoptosis
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drug effects* Caspase 3 Caspases / antagonists & inhibitors, metabolism Cell Culture Techniques / methods* Cells, Cultured Chemical Warfare Agents / toxicity* Dose-Response Relationship, Drug Fluorometry / methods* Humans Keratinocytes / drug effects*, pathology Mustard Gas / toxicity* |
| Chemical | |
Reg. No./Substance:
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0/Chemical Warfare Agents; 505-60-2/Mustard Gas; EC 3.4.22.-/CASP3 protein, human; EC 3.4.22.-/Caspase 3; EC 3.4.22.-/Caspases |
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