Convenient Fluorometric Method to Study Sulfur Mustard-Induced Apoptosis in Human Epidermal Keratinocytes Monolayer Microplate Culture

摘要

Sulfur mustard, a vesicant, is a chemical warfare as well as a potentialterrorism 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: (a) cell death due to SM begins via apoptosis that proceeds to necrosis via an apoptotic-necrotic continuum and (b) inhibiting apoptosis decreases SMinduced microvesication in vivo. To study the mechanisms of SM-induced apoptosisand its prevention in vitro, we have established a convenient fluorometric apoptosis assay using monolayer human epidermal keratinocytes (HEK) adaptable for multi-well 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 ofwhich 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.