Intervention of sulfur mustard toxicity by downregulation of cell proliferation and metabolic rates†

Abstract

Metabolically active and proliferating basal cells in the skin are most sensitive to the potent skin blistering chemical warfare compound HD (bis-(2-chloroethyl) sulfide). We previously described a Ca2+-dependent mechanism of HD (0.3-1 mM) toxicity that was inhibited by the cell-permeant Ca2+ chelator BAPTA AM (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester). We describe some cellular effects of BAPTA AM that suggest a mechanism for its protective action. Monolayer log-phase normal human epidermal keratinocytes were incubated (37 degrees C) first in keratinocyte growth medium (KGM) containing BAPTA AM (10-40 microM) for 30 min and then in KGM alone overnight prior to evaluation. The BAPTA AM inhibited cell growth in a concentration-dependent manner with some cellular degeneration above 30 microM (light microscopy). At 20-30 microM, BAPTA AM also inhibited cellular metabolic processes, as evidenced by a lower incorporation of [3H]-thymidine (DNA synthesis, 54 +/- 5%), [3H]-uridine (RNA synthesis, 29 +/- 6%) and [14C]-valine (protein synthesis, 12 +/- 2%) as well as a lower protein content per culture (30 +/- 3%) compared with corresponding untreated controls. However, 20-30 microM BAPTA AM did not cause any demonstrable cytopathology based on morphological (electron microscopy) as well as biochemical (lactate dehydrogenase release, an indicator of cell viability loss) criteria, indicating a lack of acute toxicity. These results suggest that a mechanism of protection by BAPTA AM against HD may be via decreasing some metabolic, and therefore proliferative, rates.