A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests

Abstract

BackgroundAllergic contact dermatitis is an inflammatory skin disease that affects a significant proportion of the population. This disease is caused by an adverse immune response towards chemical haptens, and leads to a substantial economic burden for society. Current test of sensitizing chemicals rely on animal experimentation. New legislations on the registration and use of chemicals within pharmaceutical and cosmetic industries have stimulated significant research efforts to develop alternative, human cell-based assays for the prediction of sensitization. The aim is to replace animal experiments with in vitro tests displaying a higher predictive power.ResultsWe have developed a novel cell-based assay for the prediction of sensitizing chemicals. By analyzing the transcriptome of the human cell line MUTZ-3 after 24 h stimulation, using 20 different sensitizing chemicals, 20 non-sensitizing chemicals and vehicle controls, we have identified a biomarker signature of 200 genes with potent discriminatory ability. Using a Support Vector Machine for supervised classification, the prediction performance of the assay revealed an area under the ROC curve of 0.98. In addition, categorizing the chemicals according to the LLNA assay, this gene signature could also predict sensitizing potency. The identified markers are involved in biological pathways with immunological relevant functions, which can shed light on the process of human sensitization.ConclusionsA gene signature predicting sensitization, using a human cell line in vitro, has been identified. This simple and robust cell-based assay has the potential to completely replace or drastically reduce the utilization of test systems based on experimental animals. Being based on human biology, the assay is proposed to be more accurate for predicting sensitization in humans, than the traditional animal-based tests.

The involvement of the Toll-like receptor signaling and Nrf2-Keap1 pathways in thein vitroregulation of IL-8 and HMOX1 for skin sensitization

“…Many molecular processes involving skin sensitization have been identified (Martin et al, 2011;van der Veen et al, 2011), the role of these mechanisms in driving biomarker expression is often not known. In recent gene profiling studies it was shown both in dendritic cells as well as in keratinocytes that the cytoprotective Nrf2-Keap1 and the innate immunity-related Toll-like receptor (TLR) signaling pathways are triggered upon exposure to chemical sensitizers (Ade et al, 2009;Johansson et al, 2011;van der Veen et al, 2013b). In this study, the role of these pathways in driving the biomarkers heme oxygenate1 (HMOX1) and interleukin (IL)-8 was further established.…”

The GARD platform for potency assessment of skin sensitizing chemicals_suppl

“…The myeloid cell line used in this study was derived from MUTZ-3 (DSMZ, Braunschweig, Germany) and maintained as described (Johansson et al, 2011(Johansson et al, , 2013. A phenotypic control analysis of the cells prior to each experiment was carried out by flow cytometry in order to confirm the cells' immature state.…”

“…The resulting biomarker signature, the GARD prediction signature (GPS), consists of 200 transcripts, which are used as input into a support vector machine (SVM) model trained on a set of reference chemicals (Johansson et al, 2011). The changes in transcription can be linked to the maturation and activation of dendritic cells (KE3) during sensitization.…”

In Vitro Approaches for Detection of Chemical Sensitization

“…Extensive genomic analysis of MUTZ-3 cells has identified genes describing eight dominating functions. The primary pathways of skin sensitization induction involve signalling through transcription factors Nrf2 and aryl hydrocarbon receptor (AHR), and protein ubiquitination [37]. Monocyte-derived dendritic cells (Mo-DCs) and human monocytic leukaemia cell line (THP-1) exposed to haptens induce the Nrf2 pathway when exposed to chemicals exerting cysteine and cysteine/lysine reactivity.…”

“…Functional and transcriptional analysis of various myeloid cell lines has clearly demonstrated the significance of the MUTZ-3 cell line as a model for functional studies of inflammatory responses [38]. The genomic allergen rapid detection (GARD) test is a MUTZ-3-based assay for assessing chemical sensitizers utilizing genomic biomarker prediction signatures to generate prediction calls of unknown chemicals as skin sensitizers, respiratory sensitizers or nonsensitizers, including irritants [37]. The predictive performance of the skinGARD was assessed in an in-house validation study.…”