Ecotoxicogenomic New Approach Methods for Screening Development in Danio rerio
Creators: name: Essfeld, Fabian
Publisher: name: Universität Bielefeld
Year: 2025
Subjects: subject: 570

description: Mankind increasingly relies on chemical compounds, with a significant role they play in various sectors such as technology, medicine, and agriculture. However, this widespread use also raises substantial environmental concerns, particularly regarding unknown adverse effects chemicals such as plant protection products, biocides, pharmaceuticals and industrial chemicals could induce when introduced into ecosystems. Environmental risk assessment (ERA) is therefore crucial both before market authorization and retrospectively, to identify and mitigate potential adverse effects on the environment. Though ERA follows individual regulations for different chemical classes, substances of very high concern (SVHC) play a central role in each of them, as SVHC are more stringently regulated and emphasize to develop safer alternatives. <br>
Traditional ERA methods, which are resource-intensive and often involve extensive animal testing, are not feasible for assessing the vast number of chemicals in use today. This includes not only new chemicals submitted for authorization, but also chemicals that require re-evaluation. This thesis contributes to addressing the need for more efficient and effective risk assessment strategies by emphasizing the development and application of New Approach Methods (NAMs). These methods include high-throughput screening, computational modeling, in vitro assays, and omic techniques, which align with the 3R principles — Replacement, Reduction, and Refinement of animal experiments. Among these NAMs, transcriptome analysis is highlighted for its potential to provide comprehensive and mechanistic insights into chemicals toxicity. <br>
This research concentrates on two primary areas: the mode of action-based screening of thyroid active and immunotoxic compounds, and the application of the transcriptomic point of departure (tPOD) method as an alternative for chronic ecotoxicity endpoints. All experiments conducted within this thesis utilized a modified zebrafish embryo toxicity test (FET) in accordance with OECD test guideline 236. The FET is recognized as an alternative to animal testing and shows potential to support or replace higher-tier tests in the future, aligning with the goal of reducing reliance on animal testing. <br>
Thyroid activity was studied using substances classified into three primary modes of action (MoA): thyroid hormone activation, deiodinase inhibition, and thyroid peroxidase inhibition. The resulting transcriptomic fingerprints were distinct for each MoA, allowing for the identification of biomarkers that can differentiate between the various thyroidal MoA. <br>
Furthermore, immunotoxicity was evaluated using an innovative method that involved eliciting an innate immune response either through Pathogen-Associated Molecular Patterns (PAMPs) or imiquimod. The immune responses were thoroughly analyzed at the transcriptomic level, both with and without clobetasol propionate-induced immunosuppression, allowing for the identification of key biomarkers of immunosuppression. Additionally, the imiquimod-induced immune response can simulate psoriasis, offering a non-animal model to study the molecular mechanisms of this condition and explore potential therapies. <br>
The tPOD approach was evaluated using the prohormone androstenedione. In the chronic Fish Sexual Development Test (FSDT; OECD TG 234), androstenedione induced complete masculinization at the lowest test concentration, preventing the determination of the No Observed Effect Concentration (NOEC). To avoid repeating the test with lower concentrations, which would be both resource-intensive and ethically challenging, a Fish Embryo Toxicity test (FET) was employed to predict the chronic NOEC. Concentration-effect relationships at the gene level were analyzed to identify the concentration resulting in significant changes in gene expression. The lowest tPOD value determined was 24 ng/L, and this value has great potential to be accepted by regulatory authorities as an alternative to the chronic NOEC in the future. <br>
The key findings of this thesis demonstrate that transcriptomic approaches are proficient in identifying changes in gene expression resulting from chemical exposure. This process effectively highlights potential toxicity and the associated biological signaling pathways. These capabilities are important for identifying chemicals that may pose significant risks, particularly those classified as SVHC due to their endocrine-disrupting and immunotoxic properties. <br>
The feasibility and applicability of these methodologies have been demonstrated by the experimental results of this thesis, further supported through a review of numerous studies, indicating their broad spectrum of applications across different chemicals, exposure scenarios, and species. The continued development, adoption and standardization of next generation risk assessment strategies are essential for addressing the complex challenges posed by modern chemical use and ensuring safer environmental and human health outcomes. <br>
In conclusion, this thesis underscores the potential of integrating advanced transcriptomic techniques into ERA to enhance the assessment and regulation of chemicals. By focusing on the prioritization of high-risk compounds and the implementation of NAMs, it aims to contribute to a more efficient, ethical, and comprehensive risk assessment process, ultimately promoting the safer use of chemicals in our society.