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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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Toxicological implications of transcriptomics responsesat differentdrug concentrations. (A) Transcriptome data were obtained for multipledrug (VPA) concentrations as in Figure 1. Toobtain a measure of the deviation of the transcriptome from normal(teratogenicity measure), two indices were calculated and used todefine the two dimensions of a developmental toxicity plane. The firstdimension reflects the extent of gene regulation. The index is thesum of the negative logarithms of the p-values ofthe 100 most regulated genes. The second dimension reflects the extentof coordinated changes in biological processes reflected by GOs. Theindex is proportional to the number of overrepresented GOs in thegene sets. The purple numbers indicate the concentrations of VPA associatedwith the data points. (B) Summary of overall findings on concentration-dependenttranscriptome deviations: drug concentrations were chosen in a wayto allow either normal neuroectodermal differentiation of human embryonicstem cells (hESC) or disturbed differentiation (teratogenic concentrationrange) or cytotoxicity. Over this large concentration range (25–1000μM), the number of deregulated genes increased continuously,once a certain threshold concentration was reached (125 μM).In contrast to this, superordinate biological regulations, as indicatedby enriched GOs (gene ontologies) or TFBS (transcription factor bindingsites), increased steeply in the teratogenic range and then more orless reached a plateau. At cytotoxic concentrations, only few additionalGOs and TFBS were overrepresented. Thus, cytotoxicity, overall generegulation, and coordinate regulation of biological processes showedlargely different concentration dependencies. There were clear lowerthresholds, and the extent of transcriptome regulation as such, withoutfurther bioinformatic analysis, did not appear to be a good measurefor teratogenicity.
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fig7: Toxicological implications of transcriptomics responsesat differentdrug concentrations. (A) Transcriptome data were obtained for multipledrug (VPA) concentrations as in Figure 1. Toobtain a measure of the deviation of the transcriptome from normal(teratogenicity measure), two indices were calculated and used todefine the two dimensions of a developmental toxicity plane. The firstdimension reflects the extent of gene regulation. The index is thesum of the negative logarithms of the p-values ofthe 100 most regulated genes. The second dimension reflects the extentof coordinated changes in biological processes reflected by GOs. Theindex is proportional to the number of overrepresented GOs in thegene sets. The purple numbers indicate the concentrations of VPA associatedwith the data points. (B) Summary of overall findings on concentration-dependenttranscriptome deviations: drug concentrations were chosen in a wayto allow either normal neuroectodermal differentiation of human embryonicstem cells (hESC) or disturbed differentiation (teratogenic concentrationrange) or cytotoxicity. Over this large concentration range (25–1000μM), the number of deregulated genes increased continuously,once a certain threshold concentration was reached (125 μM).In contrast to this, superordinate biological regulations, as indicatedby enriched GOs (gene ontologies) or TFBS (transcription factor bindingsites), increased steeply in the teratogenic range and then more orless reached a plateau. At cytotoxic concentrations, only few additionalGOs and TFBS were overrepresented. Thus, cytotoxicity, overall generegulation, and coordinate regulation of biological processes showedlargely different concentration dependencies. There were clear lowerthresholds, and the extent of transcriptome regulation as such, withoutfurther bioinformatic analysis, did not appear to be a good measurefor teratogenicity.

Mentions: The distinct concentration–responsebehavior of the regulation of genes vs GO and superordinate cell biologicalprocesses suggested that these features may be used to develop a quantitativemeasure of teratogenic activity. For this purpose, the effect of eachdrug concentration was defined by two measures, the gene regulationindex and the GO overrepresentation index. When these values wereplotted onto a coordinate system formed by the two indices, it becameevident that the distance from the origin was a useful measure ofteratogenicity (Figure 7A). This way of datapresentation also clearly revealed that VPA concentrations between125 and 450 μM resulted in a progressive deviation of normaldifferentiation (increasing numbers of oGO among deregulated PS).At higher concentrations, the extent of gene deregulation increased,but oGOs remained fairly constant. The concentrations marking thedistinct increase of teratogenicity according to this presentationcorrelate well with the VPA plasma concentrations associated withhuman or animal birth defects. The curve formed by the teratogenicityindex data (Figure 7A) differed fundamentallyfrom the cytotoxicity curve (Figure 1B). Thelatter showed strong and progressive changes at high drug concentrations,while the former did not change at high VPA levels but rather at mediumlevels. Thus, it seems feasible to define a specific teratogenicitymeasure that yields clearly different information from plain cytotoxicityin the same assay.


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)

Toxicological implications of transcriptomics responsesat differentdrug concentrations. (A) Transcriptome data were obtained for multipledrug (VPA) concentrations as in Figure 1. Toobtain a measure of the deviation of the transcriptome from normal(teratogenicity measure), two indices were calculated and used todefine the two dimensions of a developmental toxicity plane. The firstdimension reflects the extent of gene regulation. The index is thesum of the negative logarithms of the p-values ofthe 100 most regulated genes. The second dimension reflects the extentof coordinated changes in biological processes reflected by GOs. Theindex is proportional to the number of overrepresented GOs in thegene sets. The purple numbers indicate the concentrations of VPA associatedwith the data points. (B) Summary of overall findings on concentration-dependenttranscriptome deviations: drug concentrations were chosen in a wayto allow either normal neuroectodermal differentiation of human embryonicstem cells (hESC) or disturbed differentiation (teratogenic concentrationrange) or cytotoxicity. Over this large concentration range (25–1000μM), the number of deregulated genes increased continuously,once a certain threshold concentration was reached (125 μM).In contrast to this, superordinate biological regulations, as indicatedby enriched GOs (gene ontologies) or TFBS (transcription factor bindingsites), increased steeply in the teratogenic range and then more orless reached a plateau. At cytotoxic concentrations, only few additionalGOs and TFBS were overrepresented. Thus, cytotoxicity, overall generegulation, and coordinate regulation of biological processes showedlargely different concentration dependencies. There were clear lowerthresholds, and the extent of transcriptome regulation as such, withoutfurther bioinformatic analysis, did not appear to be a good measurefor teratogenicity.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Toxicological implications of transcriptomics responsesat differentdrug concentrations. (A) Transcriptome data were obtained for multipledrug (VPA) concentrations as in Figure 1. Toobtain a measure of the deviation of the transcriptome from normal(teratogenicity measure), two indices were calculated and used todefine the two dimensions of a developmental toxicity plane. The firstdimension reflects the extent of gene regulation. The index is thesum of the negative logarithms of the p-values ofthe 100 most regulated genes. The second dimension reflects the extentof coordinated changes in biological processes reflected by GOs. Theindex is proportional to the number of overrepresented GOs in thegene sets. The purple numbers indicate the concentrations of VPA associatedwith the data points. (B) Summary of overall findings on concentration-dependenttranscriptome deviations: drug concentrations were chosen in a wayto allow either normal neuroectodermal differentiation of human embryonicstem cells (hESC) or disturbed differentiation (teratogenic concentrationrange) or cytotoxicity. Over this large concentration range (25–1000μM), the number of deregulated genes increased continuously,once a certain threshold concentration was reached (125 μM).In contrast to this, superordinate biological regulations, as indicatedby enriched GOs (gene ontologies) or TFBS (transcription factor bindingsites), increased steeply in the teratogenic range and then more orless reached a plateau. At cytotoxic concentrations, only few additionalGOs and TFBS were overrepresented. Thus, cytotoxicity, overall generegulation, and coordinate regulation of biological processes showedlargely different concentration dependencies. There were clear lowerthresholds, and the extent of transcriptome regulation as such, withoutfurther bioinformatic analysis, did not appear to be a good measurefor teratogenicity.
Mentions: The distinct concentration–responsebehavior of the regulation of genes vs GO and superordinate cell biologicalprocesses suggested that these features may be used to develop a quantitativemeasure of teratogenic activity. For this purpose, the effect of eachdrug concentration was defined by two measures, the gene regulationindex and the GO overrepresentation index. When these values wereplotted onto a coordinate system formed by the two indices, it becameevident that the distance from the origin was a useful measure ofteratogenicity (Figure 7A). This way of datapresentation also clearly revealed that VPA concentrations between125 and 450 μM resulted in a progressive deviation of normaldifferentiation (increasing numbers of oGO among deregulated PS).At higher concentrations, the extent of gene deregulation increased,but oGOs remained fairly constant. The concentrations marking thedistinct increase of teratogenicity according to this presentationcorrelate well with the VPA plasma concentrations associated withhuman or animal birth defects. The curve formed by the teratogenicityindex data (Figure 7A) differed fundamentallyfrom the cytotoxicity curve (Figure 1B). Thelatter showed strong and progressive changes at high drug concentrations,while the former did not change at high VPA levels but rather at mediumlevels. Thus, it seems feasible to define a specific teratogenicitymeasure that yields clearly different information from plain cytotoxicityin the same assay.

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