The work has been divided in four sub-projects (SPs), each composed of several workpackages (WPs), as follows:
- SP1: Central work packages:
- WP1: Overall strategy and coordination with the other Alternative Testing building blocks
- WP2: Supporting cell systems, quality control and applicability of cell systems
- WP3: Project management
- WP4: Training
- WP5: Dissemination and exploitation
DETECTIVE has defined a set of central WPs that address transversal topics in the project. These are grouped in SP1.
The objective of WP1 is to coordinate the research, identify any possible overlaps and agree on an overall time table for inputs and outputs in the different building blocks of the SEURAT-1 cluster. This WP also includes the overall scientific coordination and strategy.
Quality control and verification of the applicability to readout technologies, stability and reproducibility of all cell systems will be carried out in WP2. As harmonised exposure protocols are key requirements to identify changes at cellular level that are indicative for long-term toxicity following repeated dose administration, various exposure protocols will be compared to select the most appropriate treatment scheme which will be used consistently throughout the project.
Three WPs will address project management (WP3), Training (WP4) and Dissemination and exploitation (WP5).
- SP2: Functional readouts:
The overall objective of SP2 is to develop functional readouts for the identification of biomarkers of repeated dose toxicity for multiple target organs in vitro.
The dose-response curves obtained in SP2 will provide a better understanding of thresholds of concern leading to functional failures of various cell types. The technologies used for the functional readouts, and how they can lead to the development of novel toxicity biomarkers, are described hereafter:
WP6: Electric activity
The contractile properties as well as the electrophysiological characteristics of electrically active cells such as cardiomyocytes can be monitored in real-time using multi-electrode arrays (MEA). This approach will be applicable also to other excitable cells such as neurons.
WP7: Impedance measurements
Impedance measurements allow continuous, multi-parametric monitoring of cell function and integrity in situ. The consortium will apply the xCELLigence impedance measurement system provided by DETECTIVE partner Roche. This will provide a simple and efficient assay for measuring side effects of compounds on hepatocytes and cardiomyocytes.
WP8: (High throughput) Imaging
Thanks to the enormous progress made in imaging techniques during recent years and the possibilities of automated image analysis, imaging technology represents an attractive alternative or an important complement to other techniques, such as the biochemical and “-omics” readout techniques (High content automated imaging, Time-lapse microscopy, and Automated high resolution imaging using transgenic fluorescent reporter cell lines).
WP9: Cell type specific readouts
Cell type specific functional assays as classical reference assays will be used to study effects of toxicants on hepatic, renal or cardiac function. Selected relevant test substances with known toxicity selected by ToxBank will be applied to compare the predictive power of well-established functional tests to novel biomarkers identified in DETECTIVE. Since some of these assays are more time-consuming and less applicable to HTS compared to reporter assays and assays established in WP6, 7 and 8, we will in cooperation with these WPs investigate which of the more laborious conventional assays can be replaced by less time-consuming innovative assays without losing predictive power.
A second contribution will be cell type specific reporter assays. In the second phase of DETECTIVE we will evaluate which reporter assays reliably identify certain classes of compounds and specific toxic mechanisms in order to establish a strategy how to include them into the pipeline for high throughput in vitro screening.
- SP3: “-omics” readouts:
The overall objective targeted by SP3 is to improve traditional biomarkers of toxicity by gathering and integrating data on transcriptomic, proteomic, metabonomic and epigenomic responses to exposure in human in vitro models, which will culminate in a novel set of mechanism-based intermediate biomarkers of repeated dose toxicity applicable to evaluate the safety of different substances.
WP10: Gene expression profiling
In the initial phase of the project, gene expression profiling will be used to investigate several fundamental questions, such as the link of genes to toxicological versus adaptative processes in order to establish a robust and consistent gene expression model for toxicological approaches based on human ESCs.
WP11: Epigenetics and miRNA pofiling
Clear evidence shows that epigenetic inheritance of disease states for generations after the initial exposure . In this context, it is of major relevance to study epigenomic alterations as mechanism underlying repeated dose toxicity. It is of particular relevance to test the hypothesis that epigenomic changes induced by model compounds for repeated dose toxicity persist in in vitro models upon ending exposure. Therefore, the DETECTIVE project will study epigenomics at the level of DNA methylation and histone acetylation.
DNA methylation analysis
DNA methylation analysis may contribute to biomarker development for the prediction of chemical toxicity, particularly in combination with gene expression analysis and other markers of epigenetic changes.
Whole genome histone acetylation analysis
Together with DNA methylation they are often referred to as the epigenome. Genomewide disruption of histone acetylation in response to an environmental carcinogen has been demonstrated. Changes in the histone modifications may affect DNA methylation which has been correlated with deacetylation of histones 3 and 4, along with shifts in histone methylation patterns. It is the dynamic nature of these histone modifications that renders them particularly susceptible to environmental influences.
An interesting possibility is that miRNAs regulate the levels of expression of genes involved in drug metabolism and response to xenobiotics. On the other hand, both drug and xenobiotics exposure might alter the repertoire of miRNA expression. Moreover, miRNA expression is itself regulated by epigenetic factors such as DNA methylation and chromatin structure and thus could mediate the impact of epigenetic reprogramming in response to environmental exposure on a panel of other genes.
The proposed proteomic approach focuses on early/immediate biological responses such as e.g. phosphorylation and oxidation of proteins, which are not detectable by transcriptomics technologies. These posttranslational modifications have a direct impact on enzyme activities and protein-protein interactions. Only at downstream stages gene transcription is activated. The proteomics task has the potential to identify very initial molecular events following exposure to toxic model substances (repeated doses, dose dependent) and provide kinetic details of affected pathways. Usually the first reponse after stimulation of cells can be seen in phosphorylation of heat shock proteins and other components of stress responses. Besides the 2D-PAGE approach we will apply quantitative SRM/MRM strategies to provide unbiased information about phosphorylation cascades. With these data it will be possible to obtain information on the activated pathways and in combination with quantitative technologies also the degree of activation (normalisation to protein amount). The identification of related kinase/phospatase pathways will provide novel biomarker content for high throughput methods. In a further step the first verification of these biomarkers by independent methods (antibodies) will provide the basis for larger scale cross-validation and subsequent application of statistical methods.
This project will extend our current knowledge in that it will explore for the first time, in a systematic way, the relationship between the metabolome of human in vitro cell systems and exposure to chemicals that cause repeated dose organ toxicity. DETECTIVE is particularly timely since metabonomic protocols for in vitro carcinogenicity testing have been developed as part of other consortia (Carcinogenomics) and are ready to be optimised for other cell systems. The consistency of a metabolite structure across cell types and species offers clear advantages to the translation of analytical protocols between models.
- SP4: Integration of biomarkers:
The general objective of SP4 is to evaluate the significance of putative in vitro biomarkers for heart, liver and kidney toxicity by integration of data obtained from all partners involved in this project.
WP14: Bioinformatic and statistical analysis of candidate biomarkers
The work within this WP includes the generation of an electronic data base for raw data, which will be done in close collaboration with the “Integrated data analysis and servicing” project ToxBank. Extensions for storage of results of statistical analyses will be implemented. Together with the partners from data-generating WPs in SP2 and SP3, endpoints will be defined and relevant covariates will be identified. Based on such considerations, a database will be set up and interfaces for raw data collection will be developed.
In addition, interrelationships between established biomarkers and functional readouts will be investigated. Well-defined genomic pathways (SP3) will hence be connected to functional data on heart, liver toxicity and kidney as supplied by SP2.
WP15: Verification, stabilisation and selection of final biomarkers
Prior to the final selection of functional and “-omics” readouts as in vitro biomarkers for repeated dose toxicity in liver, heart and kidney, a number of selection criteria will be defined, such as the sensitivity of the assay, specificity, predictive power, and relevance. The acceptance and use of biomarkers for regulatory purposes requires indeed a set of quality evaluations to determine the scientific validity of the proposed biomarkers, such as information on the predictivity of the biomarker itself but also the methodologies by which it can be assessed. In this context the sensitivity of a method is crucial. Standardised protocols and standardised reference substances (in collaboration with ToxBank) will be identified for selected technologies allowing an effective quality assessment of biomarker methodologies.
All relevant in vivo data available for the reference compounds, as selected by the project ToxBank, will be gathered and compared with the in vitro datasets produced under DETECTIVE in order to come to the most relevant in vitro genes and/or functional readouts that are highly predictive for in vivo repeated dose liver, kidney or heart toxicity.