CORDIS

Executive Summary:
Alternative methods of safety assessment, replacing animal testing, are urgently needed in view of the European regulatory changes in the field of cosmetic products. The SEURAT-1 research initiative is a step to addressing the long term strategic target of "Safety Evaluation Ultimately Replacing Animal Testing (SEURAT)". It aimed to develop in vitro strategies towards the replacement of repeated dose toxicity testing in experimental animals, particularly as applicable to cosmetic ingredients. SEURAT-1 is composed of six research projects and a coordination action aiming to develop a novel “human safety assessment strategy” targeting repeated dose toxicity. As one of the building blocks, the goal of DETECTIVE was to identify robust, sensitive and specific, human relevant in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting compounds.
To this aim, the DETECTIVE project has set up a screening pipeline composed of functional and “-omics” technologies, including high content and high throughput screening platforms. Multiple data streams derived from “-omics” readouts evaluated along with traditional toxicological and histopathological endpoints were analysed using integrative statistical analysis, systematic verification and correlation with in vivo data. The identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitutes a major step in this integrated approach towards the replacement of animal testing in human safety assessment.
The work undertaken in DETECTIVE covered liver toxicity, renal toxicity and cardiotoxicity, as well as cross-organ strategies. The “Biomarker Repository” is one of the major outputs of DETECTIVE, which in turn is a substantial input for the ToxBank database (http://toxbank.net/) into which all SEURAT-1 projects feed their data. The “Biomarker Repository” is a database detailing biomarkers (functional and “-omics”) together with the experimental details (in vitro models from which they have been retrieved, compounds and relevant concentrations). This is one of the major achievements of DETECTIVE as it is a concise database based both on literature searches and contributed to by all DETECTIVE partners with their experimental results. Integrated analysis of “-omics datasets from multiple platforms have been conducted identifying both organ-specific and generic biomarkers.

Biomarkers selected for further validation were commonly associated with metabolism, development, protein degradation, stress response and energy metabolism as well as with few other biological functions.
Other significant achievements of DETECTIVE include amongst others a newly developed liver-based in vitro system, namely hepatic cells differentiated from human skin-derived precursors, source for in vitro screening of compounds that induce liver steatosis, a GFP-BAC toxicity pathway reporter platform for the imaging-based chemical safety assessment, as well as the accomplishment of three SEURAT-1 case studies:
- Study 1: “Omics-based in vitro verification of an adverse outcome pathway (AOP) of cholestatic liver injury”, which not only achieved a proof of concept of a previously proposed AOP of cholestasis, but also allowed to identify new key events.
- Study 2: “Valproic acid case study: Detection and verification of biomarkers by using a read across approach”, which addressed the increasing need to advance the integration of animal free alternative methods, such as read across.
- Study 3: “Common stereotypic and non-stereotypic transcriptome biomarkers for verification of toxicant compounds” that has identified and further classified toxicity biomarkers.
The successful completion of the DETECTIVE project advances our understanding of repeated dose toxicity testing methods. This will lay the foundation for subsequent efforts in follow-up activities at the completion of the SEURAT-1 Research initiative. Indeed, many of the collaborations established between DETECTIVE members are ongoing and will continue to advance the topics covered by the DETECTIVE project, including in the new EU-project “EU-ToxRisk”. Furthermore, development of the DETECTIVE cell systems into more physiologically relevant models, including complex cell systems, 3D, advanced micro-bioreactors for cultivation under flow with real time monitoring of essential physico-chemical parameters and organ-on-a-chip technologies are anticipated (Jiang et al. 2015). This expansion will be highly relevant to establishing a solid and reliable basis on which the future in vitro test systems, employed by industry, can be based.

Project Context and Objectives:
As one of the building blocks of the SEURAT-1 Research Initiative (“Safety Evaluation Ultimately Replacing Animal Testing”), DETECTIVE focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessment of chronically acting toxicants relevant for humans.
Emphasis was given to systematic exploitation of a battery of complementary functional and “-omics” readouts, including high content and high throughput screening platforms to identify and investigate human biomarkers in cellular models for repeated dose in vitro testing. While functional parameters give more insights into the effects of toxicants on specific cell functions of interest, “-omics” techniques deliver data on the entire cellular situation at molecular level. Importantly, DETECTIVE has performed for the first time an in-depth investigation of repeated dose effects on epigenetics and microRNA (miRNA) expression, thus exploring whether such analyses deepen our understanding of toxic modes of action. In the last years, these two parameters have been identified as critical for cell behaviour and it has been a challenging task to determine whether long-term application of chemicals will affect cells at this level.
Upon combination and subsequent integration of the various readouts, biomarkers for predicting human long-term toxicity in vitro have been obtained. Based on integrative statistical analysis, systematic verification and correlation with in vivo data, relevant, specific, sensitive and predictive biomarkers have been selected.

DETECTIVE concentrated on hepatotoxic, cardiotoxic and nephrotoxic effects representing three target organs of repeated dose toxicity. Ultimately, developed concepts will also be applicable to other organs or organ systems affected by systemic toxicants such as the nervous system. Furthermore, DETECTIVE was able to define both generic and organ-specific human toxicity pathways.
DETECTIVE, as a multidisciplinary collaboration of European researchers, has achieved its key scientific objectives by
• Establishing a successful private-public partnership addressing an important societal need
• Developing new tools and technologies together with a smart integration approach represents a substantial step forward to animal free safety testing
• Creating a screening pipeline for identifying biomarkers and surrogate endpoints relevant for assessing human long term organ toxicity and the data derived from which has been regularly uploaded to the Quretec (64 GB of data) and TOXBANK data repository (98 GB of data), which made available to research community.
• Compiling a repository of
o verified, stable and easy-to-measure functional and “-omics” biomarkers of different organs, including GLP-compliant SOPs for selected, most relevant biomarkers
o human toxicity pathways relevant for different organs (in cooperation with TOXBANK and NOTOX)
o concepts for translating the knowledge gained to toxicity of the entire organism
o adverse outcome pathways (AOPs)
• Construct from Bile Salt Export Pump Inhibition to Cholestasis
• Cardiotoxicity
• Novel public toxicogenomics directory of chemically exposed human hepatocytes (146 compounds)
• New live cell imaging pathway-based toxicity reporters for identification of hepatotoxicant-induced cellular stress responses
• Human skin-derived precursors (hSKP) as a novel cell source for in vitro screening of compounds that induce liver steatosis
• Completion of 3 case studies at SEURAT-1 level

Project Results:
Summary

The primary goal of DETECTIVE during the project duration (2011-2015) was the identification of robust, sensitive and specific, human relevant, in vitro biomarkers and surrogate endpoints that can be used for safety assessment of chronically acting toxicants. During this period, the objective has been realized through a series of defined milestones. To recapitulate, initially the target organs involved in repeated dose toxicity studies for 154 cosmetic ingredients were analysed and the most frequently affected organs by cosmetic ingredients were identified. The analysis revealed that liver and kidney were the primary target organs. Furthermore, as the mandate of SEURAT-1 was not restricted to cosmetics, it was also relevant to examine common toxicity effects relating to, for example, pharmaceuticals. In this context, the cardiovascular system is also one of the most commonly affected targets associated with attrition during drug development, representing a common cause of drug withdrawal from the market. In the following years, experimental work was pursued bringing together complementary research in the fields of alternative testing for toxicity in liver, heart and kidney cell models using various resources: functional technologies (imaging, impedance measurement, electrical activity), “-omics” technologies (metabolomics, transcriptomic, epigenetics, proteomics), statistics (modelling), bioinformatics and project management. Integrated analysis of “-omics datasets from multiple platforms have been conducted identifying both organ-specific and generic biomarkers. Biomarkers selected for further validation were commonly associated with metabolism, development, protein degradation, stress response and energy metabolism as well as with few other biological functions. The scientific and technical outcome of DETECTIVE has been detailed in the following sections.

Organ based repeat dose toxicity testing models: DETECTIVE

LIVER

Assessment of repeated dose toxicity of valproic acid, aflatoxin B1 and cyclosporine A in the human liver using integrative “-omics” data analyses (Simone van Breda, UM)

This strategy focused on delivering biomarker data based on challenging liver models with valproic acid (VPA), aflatoxin B1 (AFB1) and Cyclosporin A (CsA). The study proceeded with the hypothesis that the most interesting biomarkers would be the toxicant-induced changes in molecular networks which persist after terminating repeated dosing in vitro. This was revealed through cross-omics analysis after the repeated dose exposures on primary human hepatocytes (PHH), pooled from 3 donors, had been washed out for three days. Firstly, in response to VPA, a widely used drug in the treatment of epilepsy which is known to induce liver steatosis (Silva et al. 2008), the generated data led to insights that could be mapped on the AOP construct of steatosis in order to provide information on the mode of action of VPA-induced steatosis. This will lead to identification of new biomarkers and provide insight into the molecular mechanisms of VPA on the level of the epigenome and transcriptome. Next AFB1, a naturally occurring, but highly hepatotoxic and carcinogenic mycotoxin, is produced by several Aspergillus fungi strains (Squire 1981) was examined. Cross-omics analysis identified modulated genes in response to two doses of AFB1 revealing persistent, reversible and newly expressed differentially expressed miRNAs (DE-miRs). A whole series of new genes were identified, which in general, related to pathways of cell cycle and DNA response, transcriptional regulation. Specific pathways within these AFB1-induced biological processes appeared involved in signal transduction cascades for liver toxicity. A final aspect was the assessment of repeated dose toxicity of CsA, an immunosuppressant drug widely used in organ transplantation to prevent rejection. Adverse side effects of CsA include cholestasis (de Mattos et al. 2000). Transcriptome and the miRNA analysis found the persistence of differentially expressed genes (DEGs) and DE-miRs after CsA exposure. Overall, by applying integrative cross-omics analyses to an innovative cell model in a repeated dose regime, the molecular networks persistently affected by prototypical toxicants – VPA, AFB1, and CsA in the liver have been unravelled. In the course of this work promising biomarkers for repeated dose toxicity in humans have been identified. Discussion highlighted that follow-up studies are required that take into account larger numbers of chemicals for training and validating the predictive models. It will also be necessary to use more physiologically relevant doses and to better explore the transition to human disease signatures.

Biomarker study to predict hepatotoxic blood concentrations (Regina Stöber, IFADO)

The aim of this case study was to identify biomarkers which predict human hepatotoxic blood concentrations from publically available genome wide expression data of 150 compounds tested in primary human hepatocytes (PHH). The genes selected were altered by several compounds, overlapped with genes deregulated in human liver disease and covered the most relevant toxic mechanisms. Statistical analysis determined that a large set of compounds could be ‘captured’ by a relatively small set of genes ultimately represented by a list of the top seven potential biomarker genes that fit all the criteria. These genes are all upregulated in liver disease and reflect metabolic, cell cycle, cytoskeleton and protein degradation processes. To evaluate the ability of the biomarker genes to predict hepatotoxicity, a set of compounds that do or do not cause hepatotoxicity were identified and applied to HepG2 or PHH cells and analysed for biomarker RNA induction and cytotoxicity. The work carried out in this strategy has established a human-derived in vitro model based on biomarkers which predict human blood concentrations which cause hepatotoxicity. The novel prediction system might provide a promising tool to identify hazardous compounds during early screening processes in drug development.

A High Content Imaging-based Toxicity Pathway Reporter Platform for Chemical Safety Assessment (Steven Wink, UL)

DILI remains a major concern for drug development and in clinical practice. At the moment PHH are regarded as the gold standard for DILI toxicity testing. However, problems with the availability, inter-donor variability and stability remain critical issues. A BAC-GFP reporter platform has been developed, in which the activation of maladaptive stress pathways, which are typically activated by chemical-induced cellular injury, is monitored (Wink et al. 2014). This system has enabled the establishment and characterization of reporters for oxidative stress, ER stress, and DNA damage, allowing single cell time-resolved and quantitative analysis of the toxicity pathway activation. Exposure of these individual reporters to a library of more than 150 DILI compounds was followed by mapping of the dynamic activation of these toxicity pathways in a 24 h time period at a range of concentrations. By applying bioinformatics tools to cluster the entire time-concentration HepG2-BAC-GFP reporter response profiles of all compounds could be generated. Using this approach allows the clustering of similar mode-of-action compounds. Moreover, this screening strategy enriches for compounds with severe DILI drug labelling. It is anticipated that the cellular stress response reporters may play a key role in future safety assessment of DILI as well as other toxicity liabilities.

KIDNEY

Transcriptomics, epigenomics, miRNA and metabolomic profiling to identify novel renal biomarkers (Alice Limonciel, IMU)

The mycotoxin ochratoxin A (OTA), a contaminant in foods and beverages, a renal carcinogen in rats (Mantle et al. 2005) and a suspected carcinogen in humans, was investigated for its previously reported impact on epigenetic mechanisms such as histone acetylation. The effects of OTA on the renal proximal tubule cell line RPTEC/TERT1 was examined using a repeated exposure protocol over five days followed by a three-day washout recovery. Potassium bromate (KBrO3) was used as positive control for oxidative injury (Scholpa et al. 2014). This study revealed a strong impact of OTA on the transcriptome that has been previously described (Jennings et al. 2012). The contributions of epigenetic modifications and miRNA expression to the modulation of several protective stress responses, notably the Nrf2 response to oxidative stress (Limonciel and Jennings 2014) were also investigated. The alterations identified by metabolomics were particularly interesting, especially as they have high potential for translation as clinical urinary biomarkers.

Integrated “-omics” analyses in the kidney model (Simone van Breda, UM)

This strategy integrated multiple “-omics” analysis for toxicological mechanisms of two renal carcinogens, KBrO3 and OTA. Following three repeated doses over five days, cells were recovered after wash out and processed via multiple “-omics”. Using iCluster+ software, comprehensive maps of regulated networks in response to KBrO3 and OTA were made whereby top key genes, and their dynamic changes over the course of repeated dose treatment and recovery wash out, could be described. In summary, this strategy has provided a global and comprehensive view of toxicological mechanisms for KBrO3 and OTA. Moreover potential new biomarkers for KBrO3 and OTA have been discovered in the context of renal toxicity.

Repeat dose after extended recovery (Paul Jennings, IMU)

Repeat dose investigations are complicated and not necessarily continuous. Few studies have investigated the effect of an extended recovery after an initial repeat dose exposure. In this study the effect of a second repeat dose exposure, to see whether cells truly recover or if there is a “cellular memory” of the first exposure, was investigated. RPTEC/TERT1 kidney cells were exposed to a high, but non cytotoxic concentration of CsA every 24 h for 5 days. Cells were allowed to recover for 8 days (24 h feeding) and treated again every 24 h for 5 days. Experimental readouts included indicators of primary pharmacology, metabolomics, transcriptomics and epigenomics. The results are currently being analysed, but hint to the possibility that indeed there is a “cellular memory” of the first exposure.

HEART

Identification of toxicity biomarkers for anthracyclines in human induced pluripotent stem cell-derived cardiomyocytes (Umesh Chaudhari, Harshal Nemade and Agapios Sachinidis, UKK)

Cardiotoxicity is a well-known side effect of several cytotoxic drugs, especially of the anthracyclines in cancer patients. Anthracyclines are anti-cancer agents with a dose dependent cardiotoxicity that has strong impact on the quality of life and patient survival. This cardiac related side effect limits its use in cancer patients. In this context, a biomarker identification initiative focused on the identification of cardiotoxicity biomarkers in in vitro systems using different “-omics” technologies. The human induced pluripotent stem cell-derived cardiomyocytes ( hiPSC-CMs) have already shown their applicability in various in vitro drug screening tools (Mathur et al. 2015). The purpose of this study was to develop an in vitro repeated exposure toxicity methodology allowing the identification of predictive genomics biomarkers of functional relevance for drug-induced cardiotoxicity in hiPSC-CMs. The cells were incubated with doxorubicin (DOX), a well-characterized cardiotoxicant (Albini et al. 2010), followed by washout of the test compound with further incubation in compound-free culture medium. A panel of 35 genes was deregulated by all three anthracycline family members and can therefore be expected to predict the cardiotoxicity of compounds acting by a similar mechanism as DOX, daunorubicin or mitoxantrone (Chaudhari et al. 2015). The identified gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity. This study has demonstrated that DOX-induced adverse effects on cardiac function can be detected at the genomic level, even before cytotoxicity and arrhythmia are observed. The developed methodology can allow for first-line in vitro preclinical tests and, reduce animal usage in drug safety studies and the costs of safety evaluations.

Effect of cosmetic ingredients and a cholestasis inducing compound on expression levels of proposed potential genomic cardiac specific and non-cardiac specific biomarkers (Umesh Chaudhari, UKK)

Following the use of anthracyclines to identify biomarkers (Chaudhari et al. 2015), the next step was to validate these potential biomarkers with different cosmetic ingredients and one known liver toxicant, bosentan. The cosmetic ingredients chosen were kojic acid, triclosan and 2,7-naphthalenediol which are used in commercial cosmetics products. All three cosmetic ingredients and bosentan at low and middle concentrations showed no significant effect on the expression of the 35 key predictive biomarkers. From these results it was concluded that the panel of 35 genomic biomarkers is suitable to predict cardiotoxicity in humans and, could also be applied in the safety evaluation of drug candidates and cosmetic ingredients. Some further studies are needed to understand the precision of those biomarkers and further streamline the set of biomarker genes.

Predicting Drug-Induced Cardiotoxicity: From Electrophysiological Perspective (Filomain Nguemo and Christoph Schäfer, UKK)

Drug-induced cardiotoxicity takes two primary forms: electrophysiological (electrical activity) and biochemical. Electrophysiological toxicities arise when compounds interact with ion channels or transporters to create a pro-arrhythmic condition in which patients are at increased risk for developing arrhythmias including life-threatening ones such as torsade de pointes. iCell cardiomyocytes from CDI (Cellular Dynamics) were exposed to repeated doses of reference compounds such as Doxorubicin, Lidocaine, E4031 and Quinidine at different concentrations for short (2 days) and long period up to 14 days. Data were recorded using xCELLigence impedance system (for 2D monolayer approach), Multi-electrode array (for 3D approach) and patch clamp (for single cell experiment) technologies. The results revealed that long term exposure of cells to those compounds induced beating abnormalities in dose-dependent manner. It is clearly known that any alterations in ionic currents through ion channels (which contribute to the cardiac action potential) of the cell membrane is the main cause of beating abnormality of the cardiac cell. This study demonstrated that cells do not all respond at the same time or at the same dose of a pharmacologic agent due to cell-to-cell variability. Understanding the determinants of this variability will aid the development of multi-target treatment strategies for many diseases. Data generated in this study provides evidence that a pharmacologic agent may disrupt the expression level of genes and proteins, potentially by molecular and/or functional alterations.

CROSS-ORGAN STRATEGIES

Proteomics (Laxmikanth Kollipara, René P.Zahedi Albert Sickmann, ISAS)

The proteomic approach focused on early/immediate biological responses due to phosphorylation of proteins, which are not detectable by transcriptomics technologies. Protein phosphorylation has a direct impact on enzyme activities and protein-protein interactions. Proteomics has the potential to identify early molecular events following exposure to toxic model substances and to provide kinetic details of affected pathways (Titz et al. 2014). Usually the first response after stimulation of cells is the phosphorylation of heat shock proteins and other components of stress responses (Fulda et al. 2010). ISAS delivered comprehensive relative proteome and phospho-proteome data for human in vitro culture models of heart, liver and kidney cells exposed to sub cytotoxic concentrations of relevant selected compounds using an iTRAQ-based LC-MS approach. Furthermore, ISAS developed an LC-MS/MS-based targeted proteomics assay for the verification and validation of potential renal biomarker candidates in the chemical-induced RPTEC/TERT1 cells provided by IMU.

Metabolomic responses to toxicity in vitro – extracellular versus intracellular measurements (Hector Keun, IC)

An objective of DETECTIVE was to explore the relationship between the metabolome of human in vitro cell systems and exposure to chemicals that cause repeated dose organ toxicity. In this strategy, a number of protocols (NMR, GC-MS and LC-MS) for in vitro models of toxicity including RPTEC, hiPS-CMs, HepaRGTM, primary hepatocytes were developed. Firstly, in co-operation with UKK, the response of hiPS-CMs to DOX was analysed. Secondly, together with IMU, the metabolic response of RPTEC-TERT1 kidney epithelial cells to OTA and KBrO3 was assessed. Finally, in collaboration with VUB, the exposure of cholestatic toxicant bosentan to HepaRGTM liver model cells indicated that low dose exposure to bosentan can induce mitochondrial dysfunction.

Common renal/hepatic strategy to identify cell-specific and generic transcriptomic signatures (Paul Jennings, IMU)

While renal and hepatic in vitro systems are often run within the same project umbrella, they are not usually challenged with the same compounds at the same concentrations. To this end we conducted a focused study to challenge RPTEC/TERT1 and HepaRGTM to the same six compounds at the same concentrations, measuring the same end-points (impedance, glycolysis rate and targeted transcriptomics). The data is currently being analysed, with a view to identify tissue specific sensitivities, tissue specific biomarkers and common mechanistic biomarkers.

Delineation of the Nrf2 pathway in transcriptomic datasets (Alice Limonciel, IMU)

Oxidative stress is a major factor in the development of chemical-induced injury and associated diseases. The identification of the up-regulation of Nrf2 associated genes in in vitro and in vivo systems is becoming an attractive method for classifying compounds with oxidative potential (Wilmes et al. 2011). However, there is still a gap of knowledge regarding the time course of events, key pathway signatures and overlaps with other pathways. In this strategy large transcriptomic data sets were examined with a focus to the Nrf2 pathway and associated pathways in liver and kidney toxicological contexts. The analysis indicates that certain associated pathways closely correlate with Nrf2 hubs, while others do not. The establishment of robust and rigorously selected transcriptomic signatures for these and other transcription-driven mechanisms is a promising avenue to provide deep mechanistic information from transcriptomic data (Limonciel et al. 2015). Beyond hazard identification, the study of concentration- and time-ranges of activation could also provide a means to quantify the activation of these responses for implementation as key events in quantitative AOPs. This topic will be addressed in further EU level and international projects.

Integration of Biomarker Identification Strategies (Mathieu Vinken, VUB)

For the selection of biomarkers, a number of preparative activities were performed. Firstly the establishment of the biomarker repository, including both functional and “-omics” based readouts. Based on literature searches, and contributed to by all DETECTIVE partners, the repository is a database detailing biomarkers together with experimental details (the in vitro models in which they have been retrieved, compounds and relevant concentrations). This is considered one of the major outputs of DETECTIVE, which in turn is a substantial input for the ToxBank database (http://toxbank.net/). In addition, some DETECTIVE partners (IFADO, IMU, UL and VUB) have contributed to the generation of gene lists, specifically of transcriptomics biomarkers. This served as the basis for the generic biomarker identification strategy which allowed the identification of potential biomarkers that are liver, kidney and heart specific, as well as those that appear more generic and are detectable in multiple tissues in cases of toxicity. It is important to highlight that at the start of the project, there was no single specific strategy in place. Instead, several biomarker identification strategies progressed in parallel. Based on the expertise of the different partners, these strategies comprised different target organs (liver, kidney and heart), different set-ups of in vitro models and compounds and different functional and “-omics”-based read-outs. The scope of these strategies also varied with some designed to run only within the context of DETECTIVE while some have relevance to other SEURAT-1 projects or even extend beyond the borders and time constraints of the SEURAT-1 consortium. Following the generation of biomarker data, a number of steps were required in order to make a final selection of biomarkers. Firstly statistical analysis was highlighted as the primary criterion for a putative biomarker to be selected, in collaboration with DKFZ. Groups were also required to provide data to QURE for data storage. From here a compendium of biomarkers (from all partners) will be prepared in the form of the final report for DETECTIVE. The output of this work will be the publication of scientific papers and, where possible, the dissemination and sharing of this work with ToxBank.

Data storage and management (Raivo Kolde, Hedi Peterson, QURE)

The data storage solution for the DETECTIVE consortium was provided by QURE who implemented it using the Qure Data Management Platform. QURE’s data storage solution provided a central platform that stored all raw data from “-omics” experiments, their accompanying metadata and protocols produced by different partners within the DETECTIVE project. The DETECTIVE database (http://detective.quretec.com/) holds data for more than 500 datasets across all the “-omics” readouts and target organs. The database has been available for all the project partners for querying, comparing and receiving the conducted experiments throughout the DETECTIVE project. The SEURAT-1 central warehouse ToxBank requires all the data obtained across the data producing projects, like DETECTIVE, to be uploaded in a specific ISA-Tab format. For all the data uploaded to the DETECTIVE database, QURE automatically formatted the metadata to the ISA-Tab format and uploaded the data to the ToxBank for long-time archiving. This ensures that the valuable datasets produced in this consortium will be available in a structured manner to the broader research community.

Statistics in DETECTIVE: Can we get by with t-tests? (Annette Kopp-Schneider, DKFZ)

Following the identification of a set of biomarkers, extensive statistical analysis was conducted to evaluate their inclusion in the compendium as a toxicity biomarker. DKFZ´s objectives within DETECTIVE have been to perform statistical analysis of derived endpoints and to identify and analyse biomarkers for toxicity in humans. An overview of some of the different statistical techniques and methods employed to answer complex questions and extend the statistical strength of the identification of biomarkers, was presented. To extend the analysis beyond Student’s t-tests required the evaluation of all time points and dose levels in one Analysis of Variance (ANOVA) tests. In addition information can be borrowed about variation across features using Linear Models for Microarray Data (LIMMA, Smyth 2004). A critical aspect in the identification of biomarkers for treatment effects was the joint evaluation of “-omics” technologies. iCluster+ (Mo et al. 2013) was used to find common patterns in data from multiple “-omics” technologies and discriminant features. Integrative visualizations were generated, including heat maps, which show correlations between ordinal and quantitative values, and correlations as annotated scatterplots. Another aspect was data mining for biological information. Here, interactions between transcriptomic features by Gene Network Analysis (Schäfer and Strimmer 2005) and identified tissue-specific transcriptomic features in data from TG-Gates and the European projects carcinoGENOMICS and Predict-IV were investigated. Using Functional Data Analysis (Ramsay et al. 2009) for time course data, it was determined how different a response was in comparison to control, and patterns in the compound space were discovered. Overall, advanced statistical methods were successfully employed and the visualization of statistical results was often the key to interpretation.

SEURAT-1 PROOF-OF-CONCEPTS CASE STUDIES

Challenging the predictive power and robustness of an adverse outcome pathway construct from bile salt export pump inhibition to cholestatic injury (Robim Marcelino Rodrigues, VUB)

This case study was focused on the in vitro verification of an adverse outcome pathway (AOP) construct, from bile salt export pump inhibition to cholestatic injury, in order to confirm established key events and identify new ones. Cholestasis accounts for about half of the cases of Drug-Induced Liver Injury (DILI) and is caused by an accumulation of bile in the liver due to inhibition of the bile salt export pump (BSEP). BSEP inhibition leads to several severe effects which are depicted in the AOP of cholestasis (Vinken et al. 2013). An in vitro model of cholestatic liver injury was developed by exposure of HepaRGTM cells to bosentan, a known inhibitor of BSEP. A collaborative “-omics” approach generated transcriptomic, proteomic, metabolomic and epigenomic experimental readouts. Preliminary data suggest the identification of some key events of the AOP. Furthermore, transcriptomics and proteomics analysis identified genes and proteins that could represent potential new cholestatic biomarkers. The newly identified biomarkers could potentially contribute to the refinement of the AOP, either as individual biomarkers or as general key events.

VPA RAX case study: Detection and verification of biomarkers by using a read across (RAX) approach (Sylvia Escher, ITEM; Jan Hengstler, IFADO and Bob van de Water, UL)

A data-rich lead compound, VPA, was selected given the established knowledge regarding gene changes and a critical steatosis effect. Ten structurally similar branched and unbranched carboxylic acids were selected. Five of them induced steatosis in repeated dose toxicity studies in rodents (termed “in vivo positive”) while the remainder did not affect the liver (termed “in vivo negative”). The aim of this study was to predict systemic toxicity of VPA by using biomarkers and to show that the identified biomarkers discriminate “in vivo positive” from “in vivo negative” VPA analogues. From a list of the 150 highest up-regulated genes by VPA, ten candidate biomarker genes, representing seven typical cellular reactions, were selected. Key toxicity pathways were investigated: oxidative stress, endoplasmic reticulum (ER) stress and DNA damage. Three genes were identified that discriminated between in vitro positive and negative compounds while, in contrast, genes associated with energy and lipid metabolism were not able to discriminate the partly active from inactive compounds. In conclusion, the RAX approach is a promising concept for biomarker detection and validation.

Screening of Perturbed Toxicity Pathways by Transcriptomics Fingerprinting of Data Poor Substances (Agapios Sachinidis, Jan Hengstler)

The study “Common stereotypic and non-stereotypic transcriptome biomarkers for verification of toxicant compounds” has identified and further classified toxicity biomarkers and has established a toxicogenomics directory of chemically exposed human hepatocytes on a basis of the gene array data for 151 compounds available from TG-Gates and other publically available resources. The highest number of deregulated genes has been revealed at the highest tested compound concentration after 24 hours of exposure. Approximately one third of the compounds were responsible for the strongest effects while a large fraction of compounds had a weak effect independent of the time and concentration. In follow-up studies a number of up-regulated genes per compound at highest dose and at 24 hours were studied. Only 32 of the analysed compounds were responsible for the strongest (100-fold) up-regulations within the transcriptome. Furthermore, the SV20 parameter was introduced which represented genes that were significantly modulated by at least 20 compounds. Based on this criterion, all deregulated genes could be separated as providing stereotypic and more compound-specific gene expression responses. Therefore, stereotypic genes could be more relevant when selecting for liver toxicity biomarkers. SV20 genes were commonly associated with metabolism, development, protein degradation, stress response and energy metabolism as well as a few other biological functions. Down-regulated SV20 genes were associated with cell-cycle control, DNA synthesis and repair and immune response as well as a few other biological functions. A more broad transcriptomics analysis revealed a significant number of SV20 genes that were strongly modulated, including in liver samples from patients with non-alcoholic steatosis, hepatocellular carcinoma and other liver diseases. Therefore, a range of the biomarkers that were revealed based on the current study of hepatotoxicity could also be potentially applied to the detection of liver diseases. Furthermore, a comparison of SV20 genes with genes modulated in human cardiomyocytes under exposure with cardiotoxicants has revealed both tissue-specific and generic transcriptomic responses. Additionally, 35 transcriptomic biomarkers established in the initial DETECTIVE cardiac in vitro toxicity study were selected for prediction of toxicity of 3 cosmetic ingredients and bosentan. The cosmetic ingredients were selected from 220 ingredients described in the SCC(NF)P opinions issued between 2000 and 2009 as those of potential cardiotoxicity concern based on repeat dose toxicity animal studies (Vinken et al. 2012), while bosentan was chosen as cholestasis inducing compound. The working concentrations chosen were at least 10-100 fold in excess of the one reported in the animal plasma. No cardiotoxicity was observed under any compound concentrations tested in vitro. Importantly neither cosmetic ingredients tested nor bosentan show any effect on the expression of 35 predictive biomarkers at low and middle compound concentrations. While at highest concentrations of the tested compounds an expression of sub-fraction of tested genes was affected, these highest compound concentrations were not achievable in vivo.

Conclusion

The output of the DETECTIVE project is considerable. The groups of the consortium have published over 50 peer reviewed scientific papers, related to DETECTIVE studies, since the inception of the consortium in 2011. This number will substantially increase once the final analyses are completed and results are collated for publication in the following year. Overall, the DETECTIVE consortium can list a number of achievements as contributing to the legacy of the consortium. Major advancements have been made in the field of integration of in silico methods with in vitro toxicity cell systems for compound hazard and risk assessment. In addition, the screening of toxic compounds in in vitro human cell systems combined with mechanistically relevant biomarkers of toxicity, under either acute or chronic exposure scenarios, will contribute to better safety assessment in humans. The AOP on cholestasis was further completed and can be used for risk assessment, tiered testing approaches, prioritization, test development and for chemical categorization (Vinken et al., 2013; Vinken, 2015). Another major advance is the use of human skin-derived precursors as a cell source for in vitro screening of compounds that induce liver steatosis (Rodrigues et al. 2015). Technology including fluorescent cell sensors for pathway-specific toxicity screening has been developed, suitable for adversity detection in both 2D cell cultures and in 3D spheroid systems (Wink et al. 2014). The data output has been handled in the form of a toxicogenomics directory, with a database of global transcriptomics data for 146 hepato-toxicants with a new biomarker classification (Grinberg et al. 2014). These new biomarker classifiers can be used for hazard prediction and toxicity risk assessment based solely on in vitro data. DETECTIVE has also identified organ-specific and generic toxicity biomarkers. These diverse datasets, acquired during the study, will be stored with ToxBank and be publically accessible. These are considerable resources that provide an invaluable depth and breadth of knowledge in the area of repeated dose toxicology.

Potential Impact:
The successful completion of the DETECTIVE project advances our understanding of repeated dose toxicity testing methods. This will lay the foundation for subsequent efforts in follow-up activities at the completion of the SEURAT-1 Research initiative. Indeed, many of the collaborations established between DETECTIVE members are ongoing and will continue to advance the topics discussed in this report. Future activities are envisaged to address the limited scope of DETECTIVE/SEURAT-1 which focused on the use of a limited number of human cellular systems and test compounds. The knowledge generated through the detection of endpoints and biomarkers of repeated dose toxicity, by the DETECTIVE project, will also contribute to the foundation of future research initiatives, such as for example the recently started research consortium EU-ToxRisk (http://www.eu-toxrisk.eu/). This program will focus on the integration of new concepts for regulatory chemical safety assessment with the ultimate goal to develop reliable, animal-free hazard and risk assessment strategies.
Furthermore, development of the DETECTIVE cell systems into more physiologically relevant models, including complex cell systems, 3D, advanced micro-bioreactors for cultivation under flow with real time monitoring of essential physico-chemical parameters and organ-on-a-chip technologies are anticipated. This expansion will be highly relevant to establishing a solid and reliable basis on which the future in vitro test systems, employed by industry, can be based.
Ultimately, DETECTIVE results will contribute to the reduction of animal experiments and a more efficient and reliable safety assessment.

Specific plans for further use of the results obtained by the individual partners are detailed below.

UKK:
Integrated analysis of multi-platform “-omics” cardiotoxicity data revealed specific expression signatures of genes involved in formation of sarcomeric structures, regulation of ion homeostasis and induction of apoptosis. Eighty-four significantly deregulated genes related to cardiac functions, stress and apoptosis were validated using real-time PCR. Furthermore a limited panel of 35 genes was established and can therefore be expected to predict the cardiotoxicity. The gene panel can be applied in the safety assessment of novel drug candidates as well as available therapeutics to identify compounds that may cause cardiotoxicity.

JRC:
• The results obtained by JRC from the work carried out in DETECTIVE are the basis for further development of AOPs in the area of cardiotoxicity. In particular, contacts have been initiated with the UK National Centre for the Replacement, Refinement and Reduction of animals in research (NC3Rs), which has started a project to develop an AOP for cardiotoxicity. A group of experts are engaged in this activity.
• As indicated above, JRC is collaborating with NC3Rs to further elucidate AOPs leading to heart failure. This activity will further lead to collaboration with OECD. Future connections with the University of Wageningen (The Netherlands) are under consideration.
The expected impact is reduction of animals used for heart failure investigations, which currently is high.

UM:
• Partner UM has successfully accomplished the generation and integration of cross-omics data (on transcriptomics, microRNAs and epigenomics) from a range of organotypical cell models (liver, heart, kidney) exposed to a series of prototypical toxicants. By this, multiple new biomarker candidates for repeated dose toxicity have been identified. In particular, using an innovative study design, microRNAs and DNA methylations have been found which appeared persistently modified, thus maintaining differentially expressed after terminating toxicant treatment. These gene candidates may provide high impact candidates for assessing repeated dose organ toxicity in vitro.

VUB:
• The newly developed liver-based in vitro system, namely hepatic cells differentiated from human skin-derived precursors (hSKP-HPC), will be further tested in a number of national projects for its power to predict specific types of chemical-induced liver injury. Recently, industry has expressed interest in the hSKP-HPC model, which may lead to valorization in future.
• The newly established adverse outcome pathway for cholestatic liver injury will be further tested for its predictivity, reliability and robustness in a number of recently started national and European research projects.
• As such, the VUB work performed in the context of the DETECTIVE project has yielded 2 major deliverables, which may have a direct socio-economic impact:
- A novel liver-based in vitro system was developed, namely hepatic cells differentiated from human skin-derived precursors (hSKP-HPC). This model has been show very useful for the screening of chemicals that induce hepatotoxicity, including steatosis and acute liver failure. Moreover, its predictivity seems better compared to other commonly used hepatic in vitro systems for testing liver toxicity. Recently, industry has expressed interest in the hSKP-HPC model, which may lead to valorization in future. This novel in vitro system will further contribute to the implementation of the 3Rs concept and thus will reduce the number of animal tests.
- A new adverse outcome pathway (AOP) has been established, specifically for cholestatic liver injury. AOPs have been introduced in the fields of toxicology and risk assessment in recent years and have many applications, including serving as the basis for innovative in vitro and in silico tests, elaboration of prioritization and tiered testing approaches and chemical categorization. Following generation of the cholestasis AOP, this conceptual tool has been tested for its predictivity, reliability and robustness in one of the SEURAT-1 case studies. The results confirmed the relevance of the existing elements of the AOP and identified potentially new ones, which in turn may lead to novel biomarkers of cholestatic injury. This AOP will now be further tested in a number of recently started national and European research projects. This will contribute to the implementation of the 3Rs concept and thus will reduce the number of animal tests.

IFADO:
• In about 7 months, we are planning a local symposium in Dusseldorf with North-Rhine Westphalia (NRW) Ministers focusing on in vitro techniques to replace animal testing.
• The new techniques will become part of NRW network of in vitro systems for toxicology (Jürgen Hescheler, Agapios Sachinidis involved).
• Follow up publication on directory of hepatocyte toxicity is planned where DETECTIVE biomarkers will be used to predict blood concentrations (non-protein bound) of chemicals that cause an increased risk of hepatotoxicity (November 2016).

IC:
• Our work in DETECTIVE has helped to establish novel data and methods for interpreting and integrating metabolic data in vitro into a systems biology approach. This will enhance our capacity and that of the wider European Research community to deliver novel biomarkers for chemical safety assessment, for disease diagnosis and prognosis and potentially facilitate the development of preventative interventions to reduce the burden of human disease.

DKFZ:
• During the engagement in DETECTIVE the Biostatistics group has developed methods for the evaluation of molecular data of various origins. A major focus was the integrative evaluation of these data and the methods developed in DETECTIVE will be used for future evaluations in research projects of the German Cancer Research Center. In addition, we have used methods for the analysis of gene networks which is also a topic in collaboration with scientists from the German Cancer Research Center, hence we will explore this topic more thoroughly in the future. We have gained knowledge about the application of functional data analysis for the analysis of high-content high-throughput imaging data. We will pursue this line of research in the future with the goal of establishing an algorithm for the classification of compounds according to their toxicological properties based on their functional data.

ARTTIC:
• We will build on our links with DETECTIVE partners and the experience gained in the management of a toxicology project for future projects, including the recently started H2020 EU-ToxRisk project, coordinated by Bob van de Water (UL).

QURE:
• The data management platform that has been used for this project can be with modifications used for next potential projects where metadata handling needs to take place. It is very valuable to have the pipeline ready for transferring the large data files and data annotations, to convert to relevant formats and forward for central databases like ToxBank.
• The DETECTIVE project allowed us to work together with partners from previous project (ESNATS) but moreover to collaborate with new partners across the Europe. It enabled Quretec tools and bioinformatics services to be more widely known across the partner institutions and laid a foundation for future collaboration in the field of toxicology studies or more general biological data handling.

IMU:
• Studies designed and initiated by IMU and conducted at a cross cluster level will be used for publication in peer-reviewed journals in order to disseminate the approach, results and outcomes and reduce duplicity. The biomarkers identified will be examined and verified in further experiments. The large “-omics” datasets produced will be used for further biomarker identification and integration with other datasets.
• The experiments and results elaborated by IMU have the potential to be exploited for the development of both in vitro and in vivo tissue and urinary biomarkers. Such markers will be very valuable in the future to bridge the gap from human in vitro to first in man studies.

ISAS:
• The quantitative proteomic and phosphoproteomic data generated at ISAS in the course of DETECTIVE are still comprehensively analysed in order to include them in publications together with the partners of the consortium. From the experiments a set of potential biomarkers of renal failure have been identified and targeted mass spectrometry assays have been developed to quantify the respective proteins from urine samples (only few mL required). For these assays, stable isotope-labelled peptides have been synthesized, which will allow the absolute quantification of protein concentrations in urine, which is essential to obtain a good differentiation between healthy donors and patients of different stages of renal failure. It is planned, together with Paul Jennings’s group, IMU (Medical University of Innsbruck, Austria), to further validate this assay by analysing dozens of clinically characterized urine samples. It is planned to jointly apply for third party money in order to considerably extend these efforts in the future.
• In the course of the project essential steps of sample preparation and LC-MS analysis have been optimized, particularly with regard to quantitative proteome and phosphoproteome analyses of small sample amounts, such as the case for induced pluripotent stem cell-derived cardiomyocytes, for which only few micrograms of protein were available. Besides, the project opened new avenues for long-term collaboration, particularly with the group of Paul Jennings and Jan Hengstler (IFADO, Germany). Here, personal discussions at the DETECTIVE meetings led to new hypotheses and ideas that may be the foundation for future projects and joint grant applications. Further collaborative efforts with other DETECTIVE partners as well as planned invitations as external speakers to our institute to foster future collaborations have been discussed in the course of the DETECTIVE meetings. Notably, the close collaboration with Paul Jennings also led to a joint grant proposal for a recent Horizon2020 call.

ITEM:
• Expected impact on research field:
In DETECTIVE biomarker were searched being predictive for systemic toxicity in certain organs. Based on these results we developed a read across concept in DETECTIVE to learn more about the integration of mechanistic knowledge into regulatory risk assessment. This concept has to be further validated and explored in follow up activities. It is one aspect which is now undertaken in the recently started EU Tox Risk project, led by Bob van de Water (University of Leiden) in which ITEM is one out of 39 partners.

Further, in SEURAT we learned, that beside toxicodynamic and hazard characterisation, one key building block for data interpretation and integration are toxicokinetics. This aspect will be further explored at ITEM in the coming years.

• Further connection:
Successful networking on European level and application to follow up EU project with DETECTIVE partners and other institutes.

UL:
• The UL results from DETECTIVE involve the development of a GFP-BAC toxicity pathway reporter platform for the imaging-based chemical safety assessment. At this stage these reporters have undergone there applicability analysis in high content screening settings and found there application in a research setting. The future work will involve the integration of the platform in the EU-ToxRisk project. When possible we will try to integrate this platform in a CRO setting in a NewCo. This is under consideration.
• The impact of our DETECTIVE work involves the establishment of the H2020 EU-ToxRisk project. Prof. van de Water is the coordinator of the EU-ToxRisk project. As such this will allow the integration of the DETECTIVE work within this new project, but also continue collaborations with other DETECTIVE partners as well as SEURAT-1 partners in this new project. This e.g. involves the integration of these reporters in hiPSC in collaboration with Prof. Verfaillie.

List of Websites:
DETECTIVE consortium:

The DETECTIVE consortium is composed of the following organisations:
• Klinikum der Universität zu Köln (UKK)
• JRC - Joint Research Centre - European Communities (JRC)
• Universiteit Maastricht (UM)
• Roche Diagnostics GmbH (ROCHE)
• Vrije Universiteit Brussel (VUB)
• ProteoSys AG (PSY) – withdrawn as of May 2013
• Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. (IFADO)
• Imperial College of Science, Technology and Medicine (IC)
• Deutsches Krebsforschungszentrum (DKFZ)
• ARTTIC (ART)
• OÜ Quretec (QURE)
• Medizinische Universität Innsbruck (IMU)
• Leibniz-Institut für Analytische Wissenschaften – ISAS – e.V. (ISAS)
• Fraunhofer-Gesellschaft zur Förderung der Angewandten Forschung E.V. (ITEM)
• Universiteit Leiden (UL)

Contact details:
Prof. Dr. Jürgen Hescheler
Institute for Neurophysiology
University of Cologne
Robert-Koch-Str. 39
50931 Köln, Germany
Phone: +49-221-478 6960
Email: J.Hescheler@uni-koeln.de

DETECTIVE public website: www.detect-iv-e.eu