Flow cytometric analysis of toxicity by vesicating agents in human cells in vitro

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Abstract
Some alkylating agents are capable of causing severe blisters (vesication) when they contact human skin. The biochemical basis of vesication is undefined. In this study, we have attempted to establish in vitro models for studying vesication. We have utilized human peripheral blood lymphocytes (PBL) and commercially obtained human epidermal keratinocytes (HEK) with flow cytometry to assess cytotoxicity, DNA changes, and biochemical alterations induced by sulfur mustard (HD) and its monofunc-tional analogue, chloroethyl ethyl sulfide (CEES). Cytotoxic dose-response curves, in the dose range 10−6-10−3 M, have been generated for both alkylating agents using propidium iodide uptake. HD showed a 2–20-fold higher cytotoxic potency than did CEES. HEKs were 10 times more resistant to the cytotoxic effects of both compounds than were human lymphocytes. Cells in the S phase of the cell cycle were most sensitive to the cytotoxic effects of HD. Flow cytometric DNA histogram analysis demonstrated a blockage by both alkylating agents in the late Gl/early S phase of the cell cycle. The peak fluorescent channel positions of the diploid DNA at 24 hr exhibited a slight increase in dye uptake that might be associated with alkylation damage or DNA repair. These results showed that human cells in culture provide models for evaluating the biochemical consequences of alkylation and also may be useful in testing prophylactic and therapeutic compounds against vesication. Flow cytometry provided a rapid and sensitive technique to complement the use of in vitro cellular models in toxicologic studies.

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