Nuclear dependence of sulfur mustard-mediated cell death

Although sulfur mustard (SM) has been reported to be a DNA alkylating agent, it is not clear how much of the cytotoxicity of this agent is secondary to DNA damage. To test the hypothesis that the presence of a nucleus is required for the toxicity of sulfur mustard, enucleated endothelial cytoplasts were treated with SM. Using a combination of biochemical and microscopic assays, we demonstrate that some aspects of SM-induced cell death may be dependent on the presence of a nucleus, while others may not be. For example, it was found that cytoskeletal changes, such as loss of stress fibers and rounding, proceed in response to sulfur mustard treatment even in the absence of a nucleus. However, significant further increases in caspase activity and the associated phosphatidylserine translocation were not observed in cytoplasts treated with 500 μM SM for 6 h (following a 20-h recovery at the end of cytoplast preparation). In contrast, cytoplasts treated with chelerythrine, an agent previously reported to induce rapid apoptosis, demonstrated increases in caspase activity in cytoplasts comparable to that observed in the nucleated cells. This indicates that sulfur mustard-induced alkylation of nuclear DNA may be an important stimulus for activation of caspases in nucleated cells. Interestingly, the baseline caspase activity in cytoplasts was greater than in nucleated cells. Analysis of the time course of caspase activation in untreated adherent cytoplasts indicated that the activity increases initially and then stabilizes by 8 h to a low level that was comparable to the level observed at 26 h in untreated cytoplasts. This indicates that cytoplasts are able to tolerate stable low levels of caspase activity and not proceed immediately into the execution phase of apoptosis. The cytoplast model may be quite useful in the toxicological assessment of agents that are thought to exert their toxicity through DNA damage.