A transcriptome-based classifier to identify developmental toxicants by stem cell testing: design, validation and optimization for histone deacetylase inhibitors.

Test systems to identify developmental toxicants are urgently needed. A combination of human stem cell technology and transcriptome analysis was to provide a proof of concept that toxicants with a related mode of action can be identified and grouped for read-across. We chose a test system of developmental toxicity, related to the generation of neuroectoderm from pluripotent stem cells (UKN1), and exposed cells for 6 days to the histone deacetylase inhibitors (HDACi) valproic acid, trichostatin A, vorinostat, belinostat, panobinostat and entinostat. To provide insight into their toxic action, we identified HDACi consensus genes, assigned them to superordinate biological processes and mapped them to a human transcription factor network constructed from hundreds of transcriptome data sets. We also tested a heterogeneous group of 'mercurials' (methylmercury, thimerosal, mercury(II)chloride, mercury(II)bromide, 4-chloromercuribenzoic acid, phenylmercuric acid). Microarray data were compared at the highest non-cytotoxic concentration for all 12 toxicants. A support vector machine (SVM)-based classifier predicted all HDACi correctly. For validation, the classifier was applied to legacy data sets of HDACi, and for each exposure situation, the SVM predictions correlated with the developmental toxicity. Finally, optimization of the classifier based on 100 probe sets showed that eight genes (F2RL2, TFAP2B, EDNRA, FOXD3, SIX3, MT1E, ETS1 and LHX2) are sufficient to separate HDACi from mercurials. Our data demonstrate how human stem cells and transcriptome analysis can be combined for mechanistic grouping and prediction of toxicants. Extension of this concept to mechanisms beyond HDACi would allow prediction of human developmental toxicity hazard of unknown compounds with the UKN1 test system.

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

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

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.

From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects.

The superordinate principles governing the transcriptome response of differentiating cells exposed to drugs are still unclear. Often, it is assumed that toxicogenomics data reflect the immediate mode of action (MoA) of drugs. Alternatively, transcriptome changes could describe altered differentiation states as indirect consequence of drug exposure. We used here the developmental toxicants valproate and trichostatin A to address this question. Neurally differentiating human embryonic stem cells were treated for 6 days. Histone acetylation (primary MoA) increased quickly and returned to baseline after 48 h. Histone H3 lysine methylation at the promoter of the neurodevelopmental regulators PAX6 or OTX2 was increasingly altered over time. Methylation changes remained persistent and correlated with neurodevelopmental defects and with effects on PAX6 gene expression, also when the drug was washed out after 3-4 days. We hypothesized that drug exposures altering only acetylation would lead to reversible transcriptome changes (indicating MoA), and challenges that altered methylation would lead to irreversible developmental disturbances. Data from pulse-chase experiments corroborated this assumption. Short drug treatment triggered reversible transcriptome changes; longer exposure disrupted neurodevelopment. The disturbed differentiation was reflected by an altered transcriptome pattern, and the observed changes were similar when the drug was washed out during the last 48 h. We conclude that transcriptome data after prolonged chemical stress of differentiating cells mainly reflect the altered developmental stage of the model system and not the drug MoA. We suggest that brief exposures, followed by immediate analysis, are more suitable for information on immediate drug responses and the toxicity MoA.

Evaluation of a human neurite growth assay as specific screen for developmental neurotoxicants.

Organ-specific in vitro toxicity assays are often highly sensitive, but they lack specificity. We evaluated here examples of assay features that can affect test specificity, and some general procedures are suggested on how positive hits in complex biological assays may be defined. Differentiating human LUHMES cells were used as potential model for developmental neurotoxicity testing. Forty candidate toxicants were screened, and several hits were obtained and confirmed. Although the cells had a definitive neuronal phenotype, the use of a general cell death endpoint in these cultures did not allow specific identification of neurotoxicants. As alternative approach, neurite growth was measured as an organ-specific functional endpoint. We found that neurite extension of developing LUHMES was specifically inhibited by diverse compounds such as colchicine, vincristine, narciclasine, rotenone, cycloheximide, or diquat. These compounds reduced neurite growth at concentrations that did not compromise cell viability, and neurite growth was affected more potently than the integrity of developed neurites of mature neurons. A ratio of the EC50 values of neurite growth inhibition and cell death of >4 provided a robust classifier for compounds associated with a developmental neurotoxic hazard. Screening of unspecific toxicants in the test system always yielded ratios <4. The assay identified also compounds that accelerated neurite growth, such as the rho kinase pathway modifiers blebbistatin or thiazovivin. The negative effects of colchicine or rotenone were completely inhibited by a rho kinase inhibitor. In summary, we suggest that assays using functional endpoints (neurite growth) can specifically identify and characterize (developmental) neurotoxicants.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

Epigenetics and transcriptomics to detect adverse drug effects in model systems of human development.

Prenatal exposure to environmental chemicals or drugs has been associated with functional or structural deficits and the development of diseases in later life. For example, developmental neurotoxicity (DNT) is triggered by lead, and this compound may predispose to neurodegenerative diseases in later life. The molecular memory for such late consequences of early exposure is not known, but epigenetic mechanisms (modification of the chromatin structure) could take this role. Examples and underlying mechanisms have been compiled here for the field of DNT. Moreover, we addressed the question as to what readout is suitable for addressing drug memory effects. We summarize how complex developmental processes can be modelled in vitro by using the differentiation of human stem cells. Although cellular models can never replicate the final human DNT phenotype, they can model the adverse effect that a chemical has on key biological processes essential for organ formation and function. Highly information-rich transcriptomics data may inform on these changes and form the bridge from in vitro models to human prediction. We compiled data showing that transcriptome analysis can indicate toxicity patterns of drugs. A crucial question to be answered in our systems is when and how transcriptome changes indicate adversity (as opposed to transient adaptive responses), and how drug-induced changes are perpetuated over time even after washout of the drug. We present evidence for the hypothesis that changes in the histone methylation pattern could represent the persistence detector of an early insult that is transformed to an adverse effect at later time-points in life.

Evaluation of developmental toxicants and signaling pathways in a functional test based on the migration of human neural crest cells.

BACKGROUND: Information on the potential developmental toxicity (DT) of the majority of chemicals is scarce, and test capacities for further animal-based testing are limited. Therefore, new approaches with higher throughput are required. A screening strategy based on the use of relevant human cell types has been proposed by the U.S. Environmental Protection Agency and others. Because impaired neural crest (NC) function is one of the known causes for teratologic effects, testing of toxicant effects on NC cells is desirable for a DT test battery. OBJECTIVE: We developed a robust and widely applicable human-relevant NC function assay that would allow for sensitive screening of environmental toxicants and defining toxicity pathways. METHODS: We generated NC cells from human embryonic stem cells, and after establishing a migration assay of NC cells (MINC assay), we tested environmental toxicants as well as inhibitors of physiological signal transduction pathways. RESULTS: Methylmercury (50 nM), valproic acid (> 10 µM), and lead-acetate [Pb(CH3CO2)4] (1 µM) affected the migration of NC cells more potently than migration of other cell types. The MINC assay correctly identified the NC toxicants triadimefon and triadimenol. Additionally, it showed different sensitivities to various organic and inorganic mercury compounds. Using the MINC assay and applying classic pharmacologic inhibitors and large-scale microarray gene expression profiling, we found several signaling pathways that are relevant for the migration of NC cells. CONCLUSIONS: The MINC assay faithfully models human NC cell migration, and it reveals impairment of this function by developmental toxicants with good sensitivity and specificity.