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Design principles of concentration-dependent transcriptome deviations in drug-exposed differentiating stem cells.

Waldmann T, Rempel E, Balmer NV, König A, Kolde R, Gaspar JA, Henry M, Hescheler J, Sachinidis A, Rahnenführer J, Hengstler JG, Leist M - Chem. Res. Toxicol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Doerenkamp-Zbinden Chair for in Vitro Toxicology and Biomedicine, University of Konstanz , 78457 Konstanz, Germany.

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.

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Overlap of GOs affectedby low, medium and high VPA concentrations.(A) Up-regulated and (B) down-regulated PS were analyzed for oGO,and then the identified oGO were compared between drug concentrations.The overlaps of oGO at VPA concentrations of 350 μM (low), 550μM (medium), and 1000 μM are displayed as Venn diagrams.Right: The oGOs from specified areas of the diagrams (see legend;550 ∧ 1000 means overlap area of 550 μM and 1000 μMcircles) were grouped according to superordinate cell biological processes,and the absolute numbers within these groups are displayed.
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fig4: Overlap of GOs affectedby low, medium and high VPA concentrations.(A) Up-regulated and (B) down-regulated PS were analyzed for oGO,and then the identified oGO were compared between drug concentrations.The overlaps of oGO at VPA concentrations of 350 μM (low), 550μM (medium), and 1000 μM are displayed as Venn diagrams.Right: The oGOs from specified areas of the diagrams (see legend;550 ∧ 1000 means overlap area of 550 μM and 1000 μMcircles) were grouped according to superordinate cell biological processes,and the absolute numbers within these groups are displayed.

Mentions: Also, oGO between different VPA concentrations were comparedina Venn diagram to visualize the degree of overlap. This analysis showedthat oGO groups behave more similarly to TFBS than to PS (Figure 4 and Table S5, Supporting Information): For up- and down-regulated genes, most oGO groups started to beoverrepresented at 350 μM, and relatively few oGO groups areadded at higher concentrations. These findings are illustrated athigher detail in dendrograms (Figure S1, SupportingInformation). A relatively large fraction of oGO groups overrepresentedat 350 μM overlaps with oGO groups overrepresented at 550 and1000 μM (n = 55). For down-regulated genes,oGO groups began to emerge at slightly higher concentrations, withonly 6 oGO groups being overrepresented at 350 μM. The overallfeatures remained the same: 22 of the 25 GO overrepresented at 1000μM were already overrepresented at 550 μM (Figure 4B). Again, we also classified the oGOs in superordinatecell biological processes. Also, on these higher levels, the resultswere confirmed. Taken together, these analyses illustrate differentprogression models for genes on the one side and oGO groups and TFBSon the other. Increasing VPA concentrations lead to substantial numbersof additionally up- or down-regulated genes. However, most oGO groupsand TFBS were already overrepresented at 350 or 450 μM withrelatively little added at higher concentrations.


Design principles of concentration-dependent transcriptome deviations in drug-exposed differentiating stem cells.

Waldmann T, Rempel E, Balmer NV, König A, Kolde R, Gaspar JA, Henry M, Hescheler J, Sachinidis A, Rahnenführer J, Hengstler JG, Leist M - Chem. Res. Toxicol. (2014)

Overlap of GOs affectedby low, medium and high VPA concentrations.(A) Up-regulated and (B) down-regulated PS were analyzed for oGO,and then the identified oGO were compared between drug concentrations.The overlaps of oGO at VPA concentrations of 350 μM (low), 550μM (medium), and 1000 μM are displayed as Venn diagrams.Right: The oGOs from specified areas of the diagrams (see legend;550 ∧ 1000 means overlap area of 550 μM and 1000 μMcircles) were grouped according to superordinate cell biological processes,and the absolute numbers within these groups are displayed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958134&req=5

fig4: Overlap of GOs affectedby low, medium and high VPA concentrations.(A) Up-regulated and (B) down-regulated PS were analyzed for oGO,and then the identified oGO were compared between drug concentrations.The overlaps of oGO at VPA concentrations of 350 μM (low), 550μM (medium), and 1000 μM are displayed as Venn diagrams.Right: The oGOs from specified areas of the diagrams (see legend;550 ∧ 1000 means overlap area of 550 μM and 1000 μMcircles) were grouped according to superordinate cell biological processes,and the absolute numbers within these groups are displayed.
Mentions: Also, oGO between different VPA concentrations were comparedina Venn diagram to visualize the degree of overlap. This analysis showedthat oGO groups behave more similarly to TFBS than to PS (Figure 4 and Table S5, Supporting Information): For up- and down-regulated genes, most oGO groups started to beoverrepresented at 350 μM, and relatively few oGO groups areadded at higher concentrations. These findings are illustrated athigher detail in dendrograms (Figure S1, SupportingInformation). A relatively large fraction of oGO groups overrepresentedat 350 μM overlaps with oGO groups overrepresented at 550 and1000 μM (n = 55). For down-regulated genes,oGO groups began to emerge at slightly higher concentrations, withonly 6 oGO groups being overrepresented at 350 μM. The overallfeatures remained the same: 22 of the 25 GO overrepresented at 1000μM were already overrepresented at 550 μM (Figure 4B). Again, we also classified the oGOs in superordinatecell biological processes. Also, on these higher levels, the resultswere confirmed. Taken together, these analyses illustrate differentprogression models for genes on the one side and oGO groups and TFBSon the other. Increasing VPA concentrations lead to substantial numbersof additionally up- or down-regulated genes. However, most oGO groupsand TFBS were already overrepresented at 350 or 450 μM withrelatively little added at higher concentrations.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Doerenkamp-Zbinden Chair for in Vitro Toxicology and Biomedicine, University of Konstanz , 78457 Konstanz, Germany.

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.

Show MeSH
Related in: MedlinePlus