IDENTIFICATION OF POTENTIAL IN VITRO ENDPOINTS FOR SKIN IRRITATION USING PROTEOMIC AND MICROARRAY ANALYSIS.
Understanding the mechanistic basis of the human skin irritation response is key to the development of relevant in vitro test systems for the predictive identification of skin irritation hazards. Recent progress in the development of proteomic and microarray technologies means that tools for the identification and investigation of important biochemical events in the processes of skin irritation are now available. This study was designed to identify proteins (and genes encoding proteins) which may be involved in the skin irritation response, following exposure of a reconstructed human skin model (EpiDermTM) to the skin irritant sodium lauryl sulphate (SLS). EpiDerm cultures were treated in triplicate with a non-cytotoxic dose of SLS (0.1mg/ml, as determined by the MTT assay and histological examination) for 15min, 1h, 2h, 4h and 24h. Proteomics was performed using 2D-gel electrophoresis (Multiphor II and DALT system, (APB)) in combination with mass spectroscopy to investigate the protein expression profile and identify proteins of interest. In addition microarray analysis was performed using DermArray (Research Genetics) cDNA arrays covering 5000 genes of relevance to skin biology. 67 proteins of potential interest were selected and identified from a range of 2D-gels (1st dimension : pH 4-7, 4-5, 5-6, 6-9 and 6-11). Of the proteins selected, 35 were upregulated, 19 downregulated and the expression of 4 remained unchanged following exposure to SLS. Data indicated that post-translational modification occurred at an early time point (15 min) for calmodulin-like skin protein and involucrin following exposure of EpiDerm to SLS. Epithelial cell marker protein demonstrated post-translational modification after 1h exposure to SLS. These results demonstrate the differential regulation of a number of proteins in response to a known irritant, which could represent potential new in vitro markers of skin irritation.
Colipa, Cutaneous irritancy, Cutaneous irritation, Cutaneous toxicity, Cytokeratin 1, DNA Repair, Dermal irritancy, Dermal irritation, EpiDerm, Gene expression, ICAM-1, IFN-gamma, IFW, IL-1, IL-1a, Involucrin, Matrix metalloproteinase 2, Microarray, PCR, Proteins, Proteomics, RNA, SLS, Skin irritancy, Skin irritation, Sodium lauryl sulfate, Transcription factors, Transforming growth factor-beta, Tumor supressors, UV excision repair gene
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