Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures

• Published in: Toxicology letters Vol. 191; no. 2-3; pp. 260 - 267
• Main Authors: Heinrich, A., Balszuweit, F., Thiermann, H., Kehe, K.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ireland Ltd 15-12-2009
• Subjects: Adenylate Kinase - metabolism; Apoptosis; Apoptosis - drug effects; Assay; Biological Assay; Cell Count; Cell death; Cell Death - drug effects; Cell Line; Cell Survival - drug effects; Chemical warfare agents; Chemical Warfare Agents - toxicity; DNA Fragmentation - drug effects; Enzyme-Linked Immunosorbent Assay; Humans; Indicators and Reagents; Keratinocytes - drug effects; Luminescence; Mustard Gas - toxicity; Necrosis; Sulfur mustard; Assay; Chemical warfare agents; Cell death; Sulfur mustard; Apoptosis; Necrosis
• ISSN: 0378-4274; 1879-3169
• DOI: 10.1016/j.toxlet.2009.09.008

Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes (endonucleases, DNase I, NUC 18), which finally cut the chromatin into specific formations of 180–200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISAplus® (CDDE). In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant.
To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight® Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISAplus® and the ToxiLight® Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300μM for 30min, Lewisite (L) 60μM for 5min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent.
This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients.