Apr 4, 2025 | 2025, Altex, Journal publications
Abstract
Toxicological test methods generate raw data and provide instructions on how to use these to determine a final outcome such as a classification of test compounds as hits or non-hits. The data processing pipeline provided in the test method description is often highly complex. Usually, multiple layers of data, ranging from a machine-generated output to the final hit definition, are considered. Transition between each of these layers often requires several data processing steps. As changes in any of these processing steps can impact the final output of new approach methods (NAMs), the processing pipeline is an essential part of a NAM description and should be included in reporting templates such as the ToxTemp. The same raw data, processed in different ways, may result in different final outcomes that may affect the readiness status and regulatory acceptance of the NAM, as an altered output can affect robustness, performance, and relevance. Data management, processing, and interpretation are therefore important elements of a comprehensive NAM definition. We aim to give an overview of the most important data levels to be considered during the development and application of a NAM. In addition, we illustrate data processing and evaluation steps between these data levels. As NAMs are increasingly standard components of the spectrum of toxicological test methods used for risk assessment, awareness of the significance of data processing steps in NAMs is crucial for building trust, ensuring acceptance, and fostering the reproducibility of NAM outcomes.
Apr 4, 2025 | 2025, Environmental Toxicology and Chemistry, Journal publications
Abstract
Since the release of the U.S. National Academy’s report calling for toxicology to evolve from an observation-based to a mechanism-based science (National Research Council, 2007), scientific advances have shown that mechanistic approaches provide a deeper understanding of hazards associated with chemical exposures. New approach methodologies (NAMs) have emerged to assess the hazards and risks associated with exposure to anthropogenic and/or nonanthropogenic stressors within the context of reduce, refine, and replace (the 3Rs). Replacement refers to achieving a research goal without using animals. Reduction means applying methods that allow an investigator to obtain comparable information and precision using fewer animals. Refinement refers to changes in procedures that decrease or eliminate the animals’ pain, stress, and discomfort both during experimental procedures and in their daily social and physical environments (Russell & Burch, 1959). The development, acceptance and implementation of NAMs has become an international priority for human health and that of wildlife and ecosystems. The global commitment to nonanimal research is driven by societal values on animal welfare and the uncertainty of mammalian model species as reliable human surrogates. In addition, NAM-based information can potentially unite the different branches of toxicology by its relevance in protecting human health, wildlife, and ecosystems, thereby contributing to public safety, ecological resilience, and sustainability.
Apr 4, 2025 | 2025, Journal publications
Abstract
The potent dihydroorotate dehydrogenase (DHODH) inhibitor brequinar has been investigated as an anticancer, immunosuppressive, and antiviral pharmaceutical agent. However, its toxicity is still poorly understood. We investigated the cellular responses of primary human hepatocytes (PHH) and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1) after a single 24-h exposure up to 100 μM brequinar. Additionally, RPTEC/TERT1 cells underwent repeated daily exposure for five consecutive days at 0.3, 3, and 20 μM. Transcriptomic analysis revealed that PHH were less sensitive to brequinar treatment than RPTEC/TERT1 cells. Upregulation of various phase I and II drug-metabolising enzymes, particularly Cytochrome P450 (CYP) 1 A and 3 A enzymes, in PHH suggests potential detoxification. Furthermore, brequinar exposure led to a significant upregulation of several stress response pathways in PHH and RPTEC/TERT1 cells, including the unfolded protein response, Nrf2, p53, and inflammatory responses. RPTEC/TERT1 cells exhibited greater sensitivity to brequinar at 0.3 μM with repeated exposure compared to a single exposure. Furthermore, brequinar could impair the mitochondrial respiration of RPTEC/TERT1 cells after 24 h. This study provides new insights into the differential responses of PHH and RPTEC/TERT1 cells in response to brequinar exposure and highlights the biological relevance of implementing repeated dosing regimens in in vitro studies.
Apr 4, 2025 | 2025, Antioxidants, Journal publications
Abstract
Proteasome dysfunctions are observed in many human pathologies. To study their role and potential treatment strategies, models of proteasome inhibition are widely used in biomedical research. One frequently used tool is the proteasome inhibitor MG-132. It triggers the degeneration of human neurons, and several studies show protection from pathological events by glutathione or its precursors. It has therefore been concluded that glutathione protects cells from proteasome dysfunction. However, an alternative explanation is that MG-132, which is a peptide aldehyde, is chemically inactivated by thiols, and the apparent protection by glutathione from proteasome dysfunction is an artefact. To clarify this issue, we examined the chemical inactivation of MG-132 by thiols and the role of such reactions for neuroprotection. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we found that MG-132 reacted with L-cysteine to form a stable end product and with glutathione to form an unstable intermediate. Using a cell-free proteasome inhibition assay, we found that high concentrations of L-cysteine can scavenge a substantial fraction of MG-132 and thus reduce proteasome inhibition. Glutathione (or N-acetyl-cysteine) did not alter proteasome inhibition (even at high concentrations). In a final step, we studied human neuronal cultures. We exposed them to MG-132, supplemented the culture medium with various thiols, and assessed intracellular L-cysteine concentrations. The transcriptome response pattern also indicated an inhibition of the proteasome by MG-132 in the presence of L-cysteine. We conclude that thiol concentrations that can be reached in cells do not inactivate MG-132 in pathological models. They rather act in a cytoprotective way as antioxidants.
Apr 4, 2025 | 2025, Altex, Journal publications
Abstract
On occasion of the DNT5 meeting in Konstanz, Germany (April-2024), participants brainstormed on future challenges concerning a regulatory implementation of the developmental neurotoxicity (DNT) in vitro test battery (DNT-IVB). The five discussion topics below outline some of the key issues, opportunities and research directions for the next several years: (1) How to contextualize DNT hazard with information on potential maternal toxicity or other toxicity domains (non-DNT)? Several approaches on how to use cytotoxicity data from NAMs were discussed. (2) What opportunities exist for an immediate or near-future application of the DNT-IVB, e.g. as a prioritisation step or add-on to other information? Initial examples are already emerging; the data can be used even if the battery is not converted to a defined approach. (3) How to establish data interpretation procedures for multi-dimensional endpoints that reduce dimensionality and are suitable for classification? A decision framework is required on how to use the DNT-IVB in a regulatory context. Machine-learning (AI-approaches) may provide novel classification models. (4) How can a battery of molecular initiating events (MIEs) be smartly linked to the DNT-IVB? At what tier of an overall strategy would MIEs be evaluated, and how would one optimally balance cost vs information yield. (5) What is the way forward to scientific validation of DNT NAMs and the DNT-IVB? A large set of animal data would be required for conventional approaches, while mechanistic information may establish relevance in other ways.
Apr 4, 2025 | 2025, Altex, Journal publications
Abstract
The regular t4 workshops on biology-inspired microphysiological systems (MPS) have become a reliable benchmark for assessing fundamental scientific, industrial, and regulatory trends in the MPS field. The 2023 workshop participants concluded that MPS technology as used in academia has matured significantly, as evidenced by the steadily increasing number of high-quality research publications, but that broad industrial adoption of MPS has been slow. Academic research using MPS is primarily aimed at accurately recapitulating human biology in MPS-based organ models to enable breakthrough discoveries. Examples of these developments are summarized in the report. In addition, we focus on key challenges identified during the previous workshop. Bridging gaps between academia, regulators, and industry is addressed. We also comment on overcoming barriers to trust and acceptance of MPS-derived data – the latter being particularly important in a regulatory environment. The status of implementation of the recommendations detailed in the 2020 report has been reviewed. It was concluded that communication between stakeholders has improved significantly, while the recommendations related to regulatory acceptance still need to be implemented. Participants noted that the remaining challenges for increased translation of these technologies into industrial use and regulatory decision-making will require further efforts on well-defined context of use qualifications, together with increased standardization. This will make MPS data more reliable and ultimately make these novel tools more economically sustainable. The long-term roadmap from the 2015 workshop was critically reviewed and updated. Recommendations for the next period and an outlook conclude the report.
