In Vitro Dendritic Cell-Based Test for Skin Sensitizers Identification and Potency Estimation
Ethical and economic concerns and the recent European legislative framework triggered intensive research efforts towards the development and validation of alternative methods. We developed an in vitro predictive test to assess the skin sensitizing potential of chemicals. The test was based on the analysis and integration of intracellular signalling pathways evoked by chemicals concomitantly with gene expression modulation in skin-derived dendritic cells. In a first approach, cells were treated with four known sensitizers and two non-sensitizers, and the effects on the expression of 20 candidate genes and on the activation of several signalling pathways were analysed by real-time RT-PCR and Western blot, respectively. The genes Trxr1, Hmox1, Nqo1 and Cxcl10 and the signalling pathways p38 MAPK and JNK were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, a large number of chemicals were afterwards analysed in a blinded assay. From the total 18 compounds tested, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% and a specificity of 100%. Additionally, we also analysed the feasibility to predict the sensitizer’s potency using in vitro-generated data and several in silico-calculated descriptors. A strong correlation with LLNA EC3 values was obtained (Pearson correlation coefficient r = 0.85, p < 0.001, n = 12). Overall, our results indicate that the analysis of proposed gene and signalling pathway signatures in a mouse skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.
This work is funded by FEDER funds through the Operational Programme Competitiveness Factors—COMPETE, “ToxVitroTesting”, Quadro de Referência Estratégico Nacional (QREN)—Empresas em co-promoção project ref.: 38977 and national funds by FCT—Foundation for Science and Technology under the project grant number PTDC/SAU-OSM/099762/2008 and strategic project UID/NEU/04539/2013. João Demétrio Martins had a FCT grant number SFRH/BD/73065/2010.
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