Alamar Blue Discloses Latent Toxicity of Vesicant in Human Epidermal Model and Cells

Abstract

A new metabolic activity indicator, alamar blue (AB), was used 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-chloroethylethyl 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 or 1% (v/v) Triton X-100 exposures. Histologic 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 suggest that separation of epidermis from EpiDerm substrate is time-dependent but not necessarily proportional to in vitro CEES dosages. AB permits repeated counts and toxicologic 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.