Cutaneous Toxicity of Surfactants in Normal Human Keratinocytes Assessed by Cytotoxicity, Arachidonic Acid Release, and Regulation of Interleukin-lα mRNA

Abstract

Development and validation of in vitro methods for cutaneous irritation testing is aimed at establishing simple and reliable assays that eliminate the need for animals in toxicity testing. It is therefore important to identify in vitro end points that are predictive of in vivo toxicity. Identification of proinflamma-tory mediators and cytokines in keratinocytes may represent an early event in cutaneous inflammation. Three end points were evaluated: cytotoxicity [(crystal violet staining (CVS)], the release of [3H]arachidonic acid (AAR), and the regulation of the proinflammatory cytokine interleukin-1α (IL-lα) message in keratinocytes as a potential in vitro assay for surfactants. Cultured normal human epidermal keratinocytes were treated with various concentrations of three surfactants (SDS, Triton X-100, and Tween 20) of different in vivo potencies. The CVS50 1 h (surfactant concentrations yielding 50% viability) and AAR50 (surfactant concentrations causing 50% release of labelled arachidonic acid), both revealed the same rank order of irritancy, with SDS, Triton X-100, and Tween 20 in decreasing order of irritancy. Regulation of cytokine IL-lα mRNA was measured by semi-quantitative reverse transcription-polymerase chain reaction analysis. All three surfactants upregulated the expression of IL-lα mRNA when compared with the control at various concentrations. The CVS (1 h) and AAR assays demonstrated utility in rank ordering of chemicals from the same class as per in vivo results. At the 24-h time point IL-1 α message was upregulated and no definite stratification of surfactant-induced IL-1α mRNA was seen. These data suggest that the AAR and upregulation of IL-1α message are contributing factors in the surfactant-induced irritant/toxic effect. Key Words: Surfactants—Keratinocytes—Cytotoxicity—Arachidonic acid release—Cytokine—In vitro.