Transcriptomic Concentration-Response Evaluation of Valproic Acid, Cyproconazole, and Hexaconazole in the Neural Embryonic Stem Cell Test (ESTn)

Abstract

Alternative developmental toxicity assays are urgently needed to reduce animal use in regulatory developmental toxicology. We previously designed an in vitro murine neural embryonic stem cell test (ESTn) as a model for neurodevelopmental toxicity testing (Theunissen et al., 2010). Toxicogenomic approaches have been suggested for incorporation into the ESTn to further increase predictivity and to provide mechanistic insights. Therefore, in this study, using a transcriptomic approach, we investigated the concentration-dependent effects of three known (neuro) developmental toxicants, two triazoles, cyproconazole (CYP) and hexaconazole (HEX), and the anticonvulsant valproic acid (VPA). Compound effects on gene expression during neural differentiation and corresponding regulated gene ontology (GO) terms were identified after 24 h of exposure in relation to morphological changes on day 11 of culture. Concentration-dependent responses on individual gene expression and on biological processes were determined for each compound, providing information on mechanism and concentration-response characteristics. All compounds caused enrichment of the embryonic development process. CYP and VPA but not HEX significantly enriched the neuron development process. Furthermore, specific responses for triazole compounds and VPA were observed within the GO-term sterol metabolic process. The incorporation of transcriptomics in the ESTn was shown to enable detection of effects, which precede morphological changes and provide a more sensitive measure of concentration-dependent effects as compared with classical morphological assessments. Furthermore, mechanistic insight can be instrumental in the extrapolation of effects in the ESTn to human hazard assessment.

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants

“…For instance, assays have been developed that assess changes in early neural differentiation Pennings et al, 2012;Theunissen et al, 2012Theunissen et al, , 2013Krug et al, 2013b;Balmer et al, 2014;Waldmann et al, 2014;Rempel et al, 2015;Shinde et al, 2015), neurite outgrowth (Stiegler et al, 2011;Krug et al, 2013a), synaptogenesis (Harrill et al, 2011), gliogenesis (Fritsche et al, 2005) or myelination (Zurich et al, 2000(Zurich et al, , 2002.…”

Comparison of gene expression regulation in mouse- and human embryonic stem cell assays during neural differentiation and in response to valproic acid exposure

“…VPA exposure data was obtained in earlier studies of the mESTn [19] and hESTn [20]. Exposure in both assay systems was started at the onset of differentiation initiation in cell aggregates.…”

“…The VPA concentrations tested were based on human pharmacological relevant concentrations with >80% cell viability in vitro, as determined in earlier studies [18,19]. Optimal exposure concentrations were determined in earlier individual studies, performed independently, resulting in comparable concentrations of VPA exposure [18,19]. mESC had been exposed for 24 h, from day 3 in the protocol, to either 0.015 mM, 0.06 mM, 0.25 mM or 1.0 mM VPA.…”

Epigenetics and Transcriptomics to Detect Adverse Drug Effects in Model Systems of Human Development

“…Transcriptional profiling has been proposed as sensitive endpoint to distinguish neural differentiation states during normal and disturbed development [10,63,[73][74][75]. Marker genes specifying time and region of the differentiating cells can be assessed by transcriptional profiling and it has been shown that such marker gene expression during neural differentiation of embryonic stem cells occurs in a similar manner to in vivo [76].…”

“…Transcriptome analysis was found to be a suitable approach to assess alterations in the patterning of neural differentiation [63]. It allows a comprehensive description of overall changes of the cell culture during differentiation [75,[79][80][81]. Global alterations in the cell culture's transcriptomic fingerprint were found to indicate altered neural development [19,78].…”