Design Principles of Concentration-Dependent Transcriptome Deviations in Drug-Exposed Differentiating Stem Cells

Abstract

Information on design principles governing transcriptome changes upon transition from safe to hazardous drug concentrations or from tolerated to cytotoxic drug levels are important for the application of toxicogenomics data in developmental toxicology. Here, we tested the effect of eight concentrations of valproic acid (VPA; 25–1000 μM) in an assay that recapitulates the development of human embryonic stem cells to neuroectoderm. Cells were exposed to the drug during the entire differentiation process, and the number of differentially regulated genes increased continuously over the concentration range from zero to about 3000. We identified overrepresented transcription factor binding sites (TFBS) as well as superordinate cell biological processes, and we developed a gene ontology (GO) activation profiler, as well as a two-dimensional teratogenicity index. Analysis of the transcriptome data set by the above biostatistical and systems biology approaches yielded the following insights: (i) tolerated (≤25 μM), deregulated/teratogenic (150–550 μM), and cytotoxic (≥800 μM) concentrations could be differentiated. (ii) Biological signatures related to the mode of action of VPA, such as protein acetylation, developmental changes, and cell migration, emerged from the teratogenic concentrations range. (iii) Cytotoxicity was not accompanied by signatures of newly emerging canonical cell death/stress indicators, but by catabolism and decreased expression of cell cycle associated genes. (iv) Most, but not all of the GO groups and TFBS seen at the highest concentrations were already overrepresented at 350–450 μM. (v) The teratogenicity index reflected this behavior, and thus differed strongly from cytotoxicity. Our findings suggest the use of the highest noncytotoxic drug concentration for gene array toxicogenomics studies, as higher concentrations possibly yield wrong information on the mode of action, and lower drug levels result in decreased gene expression changes and thus a reduced power of the study.

Toxicogenomics directory of chemically exposed human hepatocytes

“…In a similar fashion, previous studies were based on the 100 genes with highest variability (Krug et al 2013;Waldmann et al 2014). We have seen that this technique offers optimal conditions to obtain an overview of compound effects.…”

“…The corresponding analysis for the downregulated genes is shown in Fig. S3 studies, we have seen that identification of 'close to cytotoxic' concentrations is challenging, especially for compounds with steep concentration effect curves (Krug et al 2013;Waldmann et al 2014). Also the method used to determine cytotoxicity is of importance.…”

In vitro systems: current limitations and future perspectives

“…Clearly, in vitro systems represent one of the most popular topics in our journal (Godoy et al 2013;Azqueta and Collins 2013;Hessel et al 2013;Jiang et al 2013;Liu et al 2013;Fraczek et al 2013;Leist and Hartung 2013). This seems to represent a general trend in toxicological sciences (Weng et al 2014;Waldmann et al 2014;Karlsson et al 2013;Onrubia et al 2013;Lake et al 2013;Houston et al 2012;Hardelauf et al 2011;Ilowski et al 2011;Gabriel et al 2012;Hammad et al 2013) but also in other disciplines of biomedical research (Zellmer et al 2010;Godoy et al 2009Godoy et al , 2010aIlowski et al 2010;Meyer et al 2011). In the Archives of Toxicology, in vitro systems are used to study mechanisms of action of toxic compounds, a traditional strategy since decades (Chen et al 2013;Qu et al 2013; One possibility would be to identify compounds for which in vivo blood concentrations (nonprotein bound) in humans are known and the risk of adverse effects in humans is clearly established.…”

Highlight report: pluripotent stem cells in translational research

“…Typically, pluripotent cells are exposed to test compounds during their differentiation processes, e.g., to the three germ layers or to neuronal cells (Shinde et al 2015;Waldmann et al 2014).…”