A double-label technique that monitors sulfur mustard damage to nuclei and mitochondria of normal human epidermal keratinocytes in vitro

Sulfur mustard and 2-chloro ethyl ethyl sulfide (GEES, a sulfur mustard analog) is known to have immediate (minutes), long-term (hours to days), and toxic effects on human skin. Research was directed toward developing a single in vitro assay that might reflect both these short-term and long-term effects of this vesicating agent on normal human epidermal keratinocytes (NHEK) in vitro. Such an assay system would be useful in identifying and developing sulfur mustard therapeutic agents. NHEK were exposed to the monofunctional sulfur mustard analog 2-chloro ethyl ethyl sulfide for a variety of times. The effects of GEES on NHEK nuclei were assessed using the membrane-permeable SYTO nuclear stains, whereas the effects of GEES on NHEK metabolism were determined by using the nontoxic mitochondria dye Alamar blue. GEES enhanced SYTO binding in a concentration-dependent manner to the nucleus immediately subsequent to a 2-hr exposure, whereas GEES had relatively little effect on metabolic activity at this time. Fifteen to 36 hr subsequent to GEES exposure, however, Alamar blue revealed a robust, sulfur mustard-dependent effect on mitochondrial activity. To determine if both these indicator dyes could be used simultaneously, NHEK were exposed to GEES and stained with the SYTO nuclear stain 2 hr subsequent to exposure. This procedure was followed by assay of the same cell cultures with Alamar blue at 36 hr subsequent to initial GEES exposure. The data indicate that this nuclear/milochondrial double-label technique can be used to monitor the short-and long-term effects of sulfur mustard on the same culture of NHEK.