Rhoads1, L.S., Mershon1, M.M., Van Buskirk2, R.G., 1United States Army Medical Research Institute of Chemical Defense, Drug Assessment Division, Aberdeen Proving Ground, MD; 2Dept. of Biological Sciences, State University of New York, Binghamton, NY.

A new metabolic activity indicator, alamar blue (AB), was used by researchers at the United States Army Medical Research Institute of Chemical Defense and the State University of New York at Binghamton to estimate cell counts and to observe toxicity in the same human cells at several time points. AB permitted accurate estimates of cell numbers as low as 500 cells per well when AB was incubated with normal human epidermal keratinocytes (NHEK) for 30 min. AB was used as a noninvasive fluorescent probe to analyze toxic effects of CEES (2-chloroethylenthyl sulfide), an alkylating agent and vesicant. Dose-dependent effects of 2 hr CEES or Triton X-100 exposures on confluent NHEK monolayers were revealed 4 hr postexposure, after 2 hr incubations with AB. Effects of CEES or Triton X-100 on a synthetic human epidermal model (EpiDerm) were demonstrated at 4, 24 and 48 hr postexposure. EpiDerm samples were nonviable at 4 hr after initiation of 80 mM CEES exposures, but a similar effect was delayed until 48 hr after 8.0 and 0.8 mM CEES of 1% (v/v) Triton X-100 exposures. Histological examination of the latter EpiDerm specimens revealed complete separations of epidermis and underlying dermal substitute. However, EpiDerm controls and 80 mM CEES specimens did not show similar separation. Taken together, these data suggests that separation of epidermis from EpiDerm substrate is time-dependent, but not necessarily proportional to in vitro CEES dosages. AB permits repeated counts and toxicological observations of the same cells. Therefore AB provides a uniquely valuable addition to the battery of multiple endpoint probes that are available for measurements of toxicologic responses or studies of mechanisms in cultured human cells and cellular models.


2-Chloroethylenthyl sulfide (CEES), a mustard agent, Alamar Blue, Endpoints, Alamar Blue, EpiDerm, Surfactants, Triton X-100, Triton X-100, Vesicant

Request a copy of this paper, click here.