Test systems of developmental toxicity: state-of-the art and future perspectives
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Reproductive toxicity: need for improved testing systems
Avoiding compounds that cause reproductive toxicity is of fundamental importance for human safety. However, reproductive toxicity testing is also one of the most challenging and expensive fields of toxicology (Wobus and Löser 2011; van Thriel 2011; Hengstler 2011). A large fraction of the animals required in drug development and in the context of REACH will be used in the area of reproductive toxicity to fulfill the respective testing requirements (Seiler et al. 2011; Krug et al. 2013; Stewart and Marchan 2012; Egbowon and Mustapha 2011; Uddin et al. 2013). Hundreds of animals are needed for testing of a single compound. Reproductive toxicity testing includes evaluation of effects on the fertilization process, spermatogenesis, oogenesis but also compromised embryo-fetal development. Currently, animal tests for developmental toxicity follow OECD guidelines 414 (2-generation study), 426 (developmental neurotoxicity) or others. These tests analyze, for example, the numbers of embryo-fetal deaths, altered total and organ weight and anatomical and behavioral abnormalities. They require exposure and analysis of animals over long periods. For example, according to OECD 426, exposure is performed during gestation and lactation and the offspring has to be analyzed for neurological, histological, neurochemical and behavioral alterations. These complex in vivo tests are too laborious and expensive to allow the required testing for thousands of chemicals (Krug et al. 2013), and might also not well reflect the human situation because of inter-species variation. Therefore, there is a general agreement that reliable, faster and more accurate in vitro tests of developmental toxicity are urgently needed (Krause et al. 2013; Leist et al. 2012; van Thriel et al. 2012).
The novel FP7 ESNATS test systems for developmental toxicity
UKN2 uses neural crest cells generated from human embryonic stem cells (hESC) and examines their functional properties (Zimmer et al. 2012).
Standard operation procedures (SOPs) of all test systems are available (Krug et al. 2013). To consider metabolism, the in vitro systems have been combined with cultivated human hepatocytes. It has been demonstrated that inclusion of hepatocytes may enhance toxicity by more than 100-fold or strongly reduce toxic effects in the target cells depending on the type of test compound. To identify in vivo relevant test compound concentrations, techniques of modeling have been improved by integrating metabolic, PBPK and spatio-temporal tissue models (Hoehme et al. 2010; Zeigerer et al. 2012). All test systems have been established in close cooperation with pharmaceutical companies and with regulatory authorities. The starting cells of the novel FP7 ESNATS test systems are either human embryonic stem cells (hESC) or neuronal precursor cells abbreviated above hESC. As far as hESC are involved, pilot experiments have been successfully performed to establish test systems also on the basis of induced pluripotency stem cells (iPSC).
Specific signatures identify DNT compounds
This success encouraged the ESNATS consortium to perform a blinded classification study using six compounds acting either by ‘valproic acid like mechanisms’ (histone deacetylase inhibitors) or by mechanisms similar to methylmercury. Classifiers could be established that clearly differentiate the DNT compounds from their solvent controls. This is remarkable, considering that simpler cell systems, such as fibroblasts or even neuronal cell lines do not allow a sufficient distinction. Genome-wide analyses also made clear that our current categories of DNT, e.g., histone deacetylase (HDAC) inhibitors, mercurials, kinase inhibitors, etc., may not be sufficient to correctly describe the influence of chemicals on the developing central nervous system. Most probably, extended analyses will lead to novel categories and classification systems. The ESNATS proof-of-concept study clearly demonstrates the importance of cell systems that recapitulate critical processes of human development. Exposure to test compounds in vitro must be performed exactly during time windows when such developmental steps take place. In this case, stress response pathways and adverse outcome pathways (AOPs) have been derived from the deregulated genes. For both compound classes AOPs associated with disturbed neuronal development are now available.
Deeper understanding of the test systems
One of the reasons for the success of the ESNATS test systems is that a relatively high effort has been invested to guarantee that the in vitro systems recapitulate relevant processes of human central nervous system development. Should the consortium have chosen an approach with easier already available cell systems and a screening of hundreds of compounds, this approach would most probably have failed. Nevertheless, an even deeper understanding of the established test systems is urgently needed. For example, neuronal differentiation in the ESNATS test systems is characterized by tightly coordinated waves of gene expression (Schulz et al. 2009; Zimmer et al. 2011; Gaspar et al. 2012). This feature of the differentiating stem cells recapitulates expression waves of the developing central nervous systems in vivo. Complex modeling and systems biology approaches will be needed to understand how such ‘waves of development’ are coordinated and how they can be perturbed by toxic compounds. It is also critical to understand how these perturbations are linked to adverse effects in vivo. This leads to a critical aspect of EU-funding policy. In previous projects, funding has been limited to human in vitro cell systems. However, to achieve a better understanding of the in vivo relevance of ‘developmental waves’ in vitro, it should be possible to compare them with the in vivo situation. In vivo data are also required to understand how disturbance of ‘developmental waves’ are linked to adverse effects. Such an understanding could be achieved by comparing developing mouse in vitro systems with mouse in vivo data. This would help to better interpret data of the corresponding human in vitro systems, such as those established by ESNATS. Therefore, future research programs aimed at improving human safety assessment and replacing animal experiments would benefit from inclusion of well-justified supplementary research in rodents and rodent cells, besides human cell systems, in order to guarantee that the in vitro systems indeed recapitulate the most critical steps in vivo.
Reducing complexity and modeling
A central result of ESNATS is that DNT compounds cause specific patterns of gene expression alterations in the novel FP7 ESNATS test systems of developmental toxicity (Krug et al. 2013). To interpret these patterns, software for identification of over-represented biological motifs is usually applied. One result of the ESNATS project is that identification of the transcription factors responsible for the compound-induced gene expression alterations is an efficient strategy to reduce complexity. While some transcription factors indicate a general stress response, others seem to be linked to more specific toxic processes. In future, a close cooperation between experimentalists, biostatisticians and modelers is required to decipher the complex expression patterns and understand their relationship to adverse effects in vivo.
Compound screening and validation studies
Achievements of ESNATS and future directions
Achievements of the ESNATS consortium
Novel in vitro systems have been established that recapitulate critical processes of human central nervous system development; standardization of test systems and sufficient reproducibility have been accomplished
Analyses of broader sets of test compounds must show if all in vivo relevant processes of DNT are represented; eventually optimizations will be required; hESC-based systems may be replaced by iPSC technologies
Procedures of handling of genome-wide complex data have been optimized and standardized: normalization based on optimized frozen RMA, cluster identification, recognition of biological motifs, stability analyses and identification of over-represented transcription factors
Future studies will have to identify the most efficient and accurate techniques of complexity reduction; e.g., are transcription factor-based classification systems superior over gene-based classifiers?
Classifiers for identification of DNT compounds are available; a blinded classification study correctly differentiated DNT compounds from negative controls
Current textbooks do not adequately categorize DNT compounds. Novel more accurate classification systems of DNT and DT have to be developed
The human hepatic metabolism has been included by cultures of primary human hepatocytes and culture medium transfer. Improved techniques of metabolic modeling are available.
Besides the available ‘medium transfer techniques’ more direct technologies of metabolite transfer to the target cells are needed, eventually based on the ‘body-on-a-chip’ principle
PBPK-based techniques for analyses of in vivo relevant concentration are available; in vitro–in vivo extrapolation to the prenatal situation is possible. PBPK modeling has been integrated into spatio-temporal models.
The precision of in vitro-in vivo extrapolation of test compound concentrations must be improved and confirmed, including in vivo analyses of test compound and metabolite concentrations as well as the possibility to predict in vivo concentrations by modeling
The basic principles of concentration and time-resolved compound effects are understood; e.g., unspecific toxicity associated signatures (such as downregulation of metabolic functions) can be differentiated from specific events of dysregulated neuronal development. ‘Waves of development’ in vitro show a high degree of similarity to the in vivo situation.
Control mechanisms of ‘waves of development’ and their susceptibility to chemicals still have to be understood and modeled; a causal understanding of disturbed expression waves and adverse effects in vivo still has to be established.
Human stem cell-based in vitro test systems have been established in ESNATS that recapitulate relevant processes of the developing human central nervous system. A proof-of-concept study demonstrated that compounds causing developmental neurotoxicity can be identified in these systems. Further, projects should be initiated to study a broader range of chemicals and to optimize the test systems. It has become clear that stem cell-based in vitro systems will become an accurate, fast and cost-effective tool for identification of toxic compounds in the broad field of developmental toxicity. This will be a major contribution to human safety.
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