Neural differentiation of embryonic stem cells (ESC) is considered a promising model to perform in vitro testing for neuroactive
and neurotoxic compounds. We studied the potential of a dual reporter murine ESC line to identify bioactive and/or toxic compounds.
This line expressed firefly luciferase under the control of the neural cell-specific tubulin alpha promoter (TUBA1A), and renilla
luciferase under the control of the ubiquitous translation elongation factor 1-alpha-1 (EEF1A1) promoter. During neural differentiation,
TUBA1A activity increased, while EEF1A1 activity decreased. We first validated our test system using the known neurotoxin methyl
mercury. This compound altered expression of both reporter genes, with ESC-derived neural precursors being affected at markedly lower
concentrations than undifferentiated ESCs. Analysis of a library of 1040 bioactive compounds picked up 127 compounds with altered
EEF1A1 and/or TUBA1A promoter activity, which were classified in 4 clusters. Cluster 1 (low EEF1A1 and TUBA1A) was the largest
cluster, containing many cytostatic drugs, as well as known neurodevelopmental toxicants, psychotropic drugs and endocrine disruptors.
Cluster 2 (high EEF1A1, stable TUBA1A) was limited to three sulfonamides. Cluster 3 (high EEF1A1 and TUBA1A) was small, but
markedly enriched in neuroactive and neurotoxic compounds. Cluster 4 (stable EEF1A1, high TUBA1A) was heterogeneous, containing
endocrine disruptors, neurotoxic and cytostatic drugs. The dual reporter gene assay described here might be a useful addition to in vitro
drug testing panels. Our two-dimensional testing strategy provides information on complex response patterns, which could not be
achieved by a single marker approach.
Keywords: Developmental toxicity, embryonic stem cells, ESC, in vitro screening, neural differentiation, neurotoxicology, Cluster Analysis, Neural Cells, neurotoxic compounds, alpha promoter.
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Published on: 28 January, 2013
Page: [710 - 723]