Characterization of disturbed neural crest migration as mechanism of developmental toxicity of prescription drugs

Abstract

In the last years, different individual human embryonic stem cell-based developmental toxicity test systems have been established and have been proven to offer new possibilities to explore toxicological hazard directly on relevant and non-transformed human cells. A further achievement has been the combination of these assays to comprehensive batteries able to predict human developmental toxicity. In the framework of the European project ESNATS (Embryonic Stem cell-based Novel Alternative Testing Strategies), we developed a test battery which allows the inclusion of any developmental toxicity assay, and that explores the responses of such test systems to a wide range of compounds. As a first step, we selected and characterized a heterogeneous group of compounds with a wide applicability domain, which ranged from environmental pollutants to several prescription drugs. To evaluate the feasibility of the suggested test framework, we performed the initial screen in a well-characterized assay that evaluates ‘migration inhibition of neural crest cells’ (MINC assay), which finally resulted in the identification of 11 hits (e.g. geldanamycin, arsenite, PBDE-99). Next, transcriptome analysis for some selected MINC hits was performed. The transcriptome changes triggered by these substances in human neural crest cells (NCC) were recorded and analyzed. Transcript profiling allowed a clear separation of different toxicants. Furthermore, a diagrammatic system was developed to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. KEGG pathways or overrepresented gene ontology terms). Finally, the transcript data were mined for potential markers of toxicity. We found that the inclusion of transcriptome data largely increased the information from the MINC phenotypic test. As a final step, one of the MINC-positive compounds, the prescription drug interferon-β (IFNβ), was chosen to be further characterized as potential developmental toxicity hazard. We could confirm the adverse effects of IFNβ on NCC migration in different functional assays. The analysis of transcriptome changes suggested a role of altered JAK-STAT signaling in toxicity, which was confirmed by detailed measurements of interferon effects on signaling in the presence of specific kinase inhibitors.