Use of Fluorescent Dyes and Spectrofluorometry to Observe Evidence of Vesicant Toxicity in Human Epidermal Cells
Abstract
Normal human epidermal keratinocyes (NHEK) show multiple dose-related biochemical ranges at 3 hours after in vitro exposure to a vesicant compound, 2- chloroethyl ethyl sulfide (CEES) CEES in ethanol was diluted to 0.8, 8.0 and 80 mM concentrations in cell culture medium over confluent NHEK on gel-coated membranes of Millipore Millicells or in NHEK suspensions. A site-specific fluorescent dye was incubated with each NHEK layer for 1 hr prior to comparisons of normal and challenged NHEK within a Cytofluor 2300 spectrofluorometer. Reduced fluorescence from loss of all dye probes indicated severe membrane damage with 80 mM CEES in medium. Intracellular increases in Ca++, evidence of altered mitochondrial activity, and decreases in pH, glutathione levels and lysosomal integrity, were observed with 0.8 and 8.0 mM CEES in the culture medium. Control studies performed with Testskin and another human epidermal model suggest that dermal substitutes and transportation stresses can influence results with the dye probe/Cytofluor 2300 method. However, the feasibility of using the described methods to observe vesicant biochemical effects, screen antivesicants and perform other toxicological studies with NHEK models is supported by the results of the preliminary studies.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 13, 1993
- Accession Number
- ADP008760
Entities
People
- Laura S. Rhoads
- Millard M. Mershon
- Robert G. Van Buskirk
Organizations
- United States Army Medical Research Institute of Chemical Defense