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Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation
Published on: June 17, 2015
Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic
P T Theunissen1, J F Robinson, J L A Pennings
1Laboratory for Health Protection Research, National Institute for Public Health and Environment (RIVM), Bilthoven, The Netherlands. Peter.Theunissen@rivm.nl
Abstract:
Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTn morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO-BP) were identified after 24h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO-BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO-BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO-BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.
Insights
This study shows that transcriptomics can identify toxic concentrations in developmental toxicity testing using the neural stem cell test (ESTn). Gene expression changes reveal compound-specific effects and concentration-dependent toxicity, aiding alternative testing strategies.
Area of Science:
- Toxicology
- Developmental Biology
- Genomics
Background:
- Reducing animal use in regulatory toxicology necessitates alternative testing methods.
- The in vitro murine neural embryonic stem cell test (ESTn) is a promising alternative for neurodevelopmental toxicity assessment.
- Integrating toxicogenomics can enhance predictivity and elucidate mechanisms of developmental toxicity.
Purpose of the Study:
- To investigate the concentration-dependent effects of six diverse compounds on the ESTn transcriptome and neural differentiation.
- To identify gene expression changes and associated biological processes affected by these compounds.
- To assess the potential of transcriptomics for distinguishing toxic from non-toxic concentrations.
Main Methods:
- Exposure of the ESTn to six compounds (acetaldehyde, carbamazepine, flusilazole, MEHP, penicillin G, phenytoin) at various concentrations.
- Assessment of changes in cell morphology, neural differentiation, and cytotoxicity.
- Transcriptomic analysis (gene expression profiling) after 24h exposure at equipotent differentiation-inhibiting concentrations.
- Gene Ontology Biological Process (GO-BP) enrichment analysis.
Main Results:
- Five out of six compounds altered ESTn morphology and neural differentiation in a concentration-dependent manner (penicillin G showed no effect).
- Transcriptomic analysis revealed both compound-specific and common gene expression changes, with acetaldehyde, carbamazepine, and flusilazole inducing more robust changes than MEHP, phenytoin, and penicillin G.
- Genes involved in embryonic morphogenesis, neuron differentiation, and Wnt signaling were differentially regulated by acetaldehyde, carbamazepine, and flusilazole.
- Gene expression and GO-BP enrichment demonstrated concentration dependence, enabling discrimination between toxic and non-toxic concentrations.
Conclusions:
- Transcriptomic profiling in the ESTn effectively captures concentration-dependent effects of developmental toxicants.
- The study provides insights into the molecular mechanisms underlying neurodevelopmental toxicity for diverse compounds.
- This approach supports the development of transcriptomics-based assays for regulatory developmental toxicity testing, reducing animal use.
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