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 ELISA(plus) (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 ELISA(plus) 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 300 microM for 30 min,
Lewisite (L) 60 microM for 5 min 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.